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The enzymatic hydrolysis of pyridoxine-5'-beta-D-glucoside in the mammalian small intestine

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Title:
The enzymatic hydrolysis of pyridoxine-5'-beta-D-glucoside in the mammalian small intestine
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Mackey, Amy Dene, 1972-
Publication Date:
Language:
English
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viii, 89 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Caco 2 cells ( jstor )
Enzymes ( jstor )
Glucosides ( jstor )
Hydrolysis ( jstor )
Intestines ( jstor )
Liver ( jstor )
Metabolism ( jstor )
Microvilli ( jstor )
Rats ( jstor )
Small intestine ( jstor )
Dissertations, Academic -- Food Science and Human Nutrition -- UF ( lcsh )
Food Science and Human Nutrition thesis, Ph. D ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 2002.
Bibliography:
Includes bibliographical references.
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Printout.
General Note:
Vita.
Statement of Responsibility:
by Amy Dene Mackey.

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THE ENZYMATIC HYDROLYSIS OF PYRIDOXINE-5'-BETA-D-GLUCOSIDE IN THE
MAMMALIAN SMALL INTESTINE














By

AMY DENE MACKEY


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY
OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
































would like to dedicate this work to the memory of my grandmother


who contributed significan my academic career. Her learning (especially spellir knowledge to others as s[


Marjorie J. Mackey,


t financial support and unending encouragement throughout devotion to the educational process and enthusiasm for


was an inspiration


can only hope to pass on


as much


te did in and out of the classroom for over 25 years.


g














ACKNOWLEDGEMENTS

would like to thank members of my Ph.D. advisory committee including Lynn B.


Bailey, Ph.D.,


Robert J


. Co


usins


Ph.D.


J


esse


F. Gregory Ill,


Ph.D., and Donald


muelson, Ph.D.


A special thank you is extended to Jesse F


Gregory Ill,


Ph.D


for all


of his patience,


guidance,


and opportunity that he has


offered to me ove


r the


past y


ears.


It would be diffi


cult to find a better scientist


or kinder


person than Dr. Gregory


would


to ackn


owledge Robert M


C


Mahon, Ph.D. for his


nvaluable scientific expertise and


assistance


throughout my research


would like to thank George


Henderson


Ph.D.


his collaboration on the pyridoxine


acknowledge all


of


the


disaccharide project.


would also like to


wonderful graduate students and post-doctoral fellows


that


have


been hon


ored to know and work with


over


the years,


especially Brandon Lewis


and Sara


Rathman.


Withou


these two remarkable


individuals


,graduate


school wo


uld not have


been nearly


as educational or


enjoyable.


would like to express my gratitude to


Catherine


Carman and Justin Townsend for their assistance


nthe


experi m


ents


conducted


in the final year of my research project.


would like


to thank my family.


My parents,


who always


expected m


e


to be a


professional student, provided me


with the unconditional financial and


emotional


support


that only parents could give.


My younger sister


Barbara


has been a continuous model


of strength and perseverance,


as well as a reliable comedic relief


would also like to


thank mu nran nna rents for en


nt-uirnann mc


and listening to me


even when they did not


Sa


like


for









like to thank Steven Davis.


I thank him for his unlimited generosity, encouragement, and


patience.














TABLE OF CONTENTS Page






CH APT ERS

11 . INT% JTF DOC TLIONC...............................a..............a..........a.*a....S..........a...a.a..a..a.a... aaaa*aaaaaaI a a a. a.a..---.-a-a-a-a-a-a-- 1


Review of the Literature..


2


.. .......... 22


HYDROLYTIC ACTIVITY TOWARD PYRIDOXINE-5'-3-D-GLUCOSIDE IN RAT


INTESTINAL MUCOSA IS NOT INCREASE BY VITAMIN B-6 DEFICIENCY OF BASAL DIET COMPOSITION AND PYRIDOXINE INTAKE ..........I.........


Initriiduction ..........
Materials and Methods.


Results .... Discussion..


EFFECT


....a.......a...... ..I....*. . . .. . . ...............****............ .a.....*..........* ...................a
*. .........I ...........* * ...........* ..* ........*ll ..........*aa ....................... * al ll.. .. ........... a


.....................................................................aa.a.aa..........*......*..*......*................ I * * * * * a .


23

23 25 28 33


3 .


ENZYMATIC HYDROLYSIS OF PYRIDOXINE-5'-{-D-GLUCOSIDE IS CATALYZED


BY INTESTINAL LACTASE-PHLORIZIN HYDROLASE ...


Introduction...


Materials and Methods..


Results.....
Discussion ..


................................ .........................a......a......4.................a.......a.a.a..*.a....a.a.............a.
......................a...... ........... ............ a..............a........................a.......................... a a al


39

39 41 44 50


4. THE INTESTINAL ABSORPTION, HYDROLYSIS, AND METABOLISM OF PYRIDOXINE-5'-13-D-GLUCOSIDE IN A CELL CULTURE MODEL USING CACO-2


HUMAN INTESTINAL EPITHELIAL CELLS.

Inr tr1d ucti in . .... a.... ....... ~aaaa ... ..................... a
Materials and Methods...... ....... ........a........
R e s ls .. . . . .. . . . .. . . .. . . . .. . . . .. . . .


....... ...... ...... ...... ...... ...........aaaaaaaaa *aaaaaa*............... .aaa..


2.


.57

.57 .59 61


















BIOGRAPH ICAL SKETCH ...................................................---.------.----------------------....... 89














Abstract of Dissertation Presented to the Graduate School


of the Uni


versity of Florida in Partial


Fulfillment


of the


Requirements for the Degree of Doctor of Philoso


phy


THE ENZYMATIC HYDROLYSIS OF PYRIDOX


MAMMALIAN


SMALL


INE-5'-BETA-D-GLUGOSIDE IN THE


NTESTINE


By


Amy D. Deceml


Chair: Major D


Mackey


ber 2002


Jesse F. Gregory, Ill )epartment: Food Science and Human Nutrition


An importan


source


of dietary vitamin B-6 is provided by pyridoxine-5'-%3-D-


glucoside (PNG),


a form of vitamin B-6 found in foods of plan


origin.


While this


glycos


B-6


ylated form of vitamin B-6 contributes a significan


ntake


,PNG is not completely bioavailable.


utilization of PNG is its


intestinal hydrolysis b


y


C


portion


1


5


An obligatory step ir


ytosolic PNG hydrolase


%) of daily vitamin n the metabolic


PNGH


and


lactase-phiorizin hydrolase (LPH).


This research was


focused on the hydrolytic pr


ocess


PNG


in the mammalian small


intestine.


Using a rat model


the effect of vitamin B-6


status


on PNG hydrolytic activity was examined by feeding eith


er AIN


-76A or AIN-93G


diets with graded concentrations


pyridoxine


PN


).


Vitamin B-6 def


cieno


y did not


increase PNG hydroly


greater in rats


sis by PNGH or LPH.


fed AIN-93G than rats fed AIN-76A


Rat growth and PNG hydroly


, regardless o


ss


were


dietary PN


of









LPH exhibited a Km of 1 .0 mmol/L toward PNG and a Km of 16 mmol/L toward lactose.


Lactose was a hydrolyzed at ti


competitive


e


same


nhibitor of PNG hydrolysis


active site.


as both substrates were


n addition to the novel PNG hydrolytic function


of


LPH


,we observed rat LPH to exhibit transf erase activity in vitro.


Formation of a PN-


disaccharide was detected in reaction mixtures containing LPH, PNG,


and lactose.


Caco-2


cells were also used to examin


e


PNG hydrolysis,


absorption, and metabolism.


Concentrations


of PNG that were


_<50 pimol/L were absorbed by a saturable


mechanism


,potentially through a carrier-mediated pr


ocess.


At concentrations


pimol/L,


PNG was absorbed by passive


diffusion.


ntracellular concentrations


of PNG


signif


C


antly


increased


as PNG increased in the media.


ncreasing


concentrations o


lactose in the


media did not signify


C


antly


change th


e


intracellular con


centrations


of


PNG;


how


ever


ntracellular


concentration of pyridoxal (PL) significantly decreased.


Collectively


these


results


ndicate that 1) vitamin B-6 defi


ciency does not


consistently


increase


intestinal PNG hydrolytic activity


2)


PNG is hydrolyzed


in the small


ntestine by


PNGH and LPH


,with most of the hydrolysis catalyzed by LPH, and 3) food selection,


i.e., dairy products eaten with fruits and vegetables and


individuals with lactose


intolerance or lactase insufficiency may reduce PNG bioavailability.


>50















CHAPTER 1 INTRODUCTION Twenty-five years ago, a glycosylated form of vitamin B-6 isolated from rice bran


was identified


as 5'


-o -(f-D-glucopyranosyl)-pyridoxine,


or pyridoxine-5'-]3-D-glucoside


Pyridoxine-5'


-p-D-glucoside


PNG) is an abundant


source of vitamin B-6


n plan


oods


and nearly absen


in animal-derived foods.


PNG


contributes a signif


cant


proportion of our daily


intake o


vitamin B-6; how


ever.


its nutritional valu


e


is limited by its


intestinal hydrolysis.


needs to be


For the


complete metabolic utilization of PNG


hydrolyzed to release free pyridoxine,


which is one


the glycosidic bond


form of vitamin B-6


that


can be converted to the coenzymatic forms of pyridoxal-5'-


phosphat


e


PLP


pyridoxamin


e-5'-phosphate


PMP


PLP and PMP are


esse


ntial to the


normal function of


over 100 enzymes that participate


in metabolic pathways


of nutrients and other


biological molecules.


Since the identification of the vitamer


PNG-related researc


h has


focused on the


nutritional properties a


PNG


ncluding its


bioavailability and metabolism as well as


analyti


cal techniques used to measure its concentration in food and other biological


materials.


Critical to the


nutrit


ional significance of PNG is the accurate determination of


concentration in the food supply


Improvements in the


anal


yses


of glycos


ylated and


non-glycos


ylated forms of vitamin B-6 have


led to better measurements of PNG


concentration


n food (2-4


).


Previous research done by this laboratory has


significantly


1


(


).


and


it


s






2


shown that the metabolic utilization of PNG is not equivalent to that of PN and that there


are species differences in the efficiency of metabolic utilization.


The metabolic utilization


of PNG is largely dete


rmined by its partial hydrolysis and not by the exten


of absorption


in the


small


intestine.


PNG is hydrolyzed by intestinal I5


-glucosidases that were


reported to exhibit greater activity with decreasing concentrations of dietary pyridoxine


,13).


The


-glucosidases,


purification and


broad specifi


city B3


nitial characterization of the


cytosolic


-glucosidase and pyridoxine-5'-3


-D-glucoside


hydrolase (PNG hydrolase


from pig intestine provided in vitro


evidence


of


specific


ntestinal catalys


is of PNG hydrolysis by the novel PNG hydrolase, and not BS3G


These investigations


14).


served as a foundation for further examination of intestinal


PNG hydrolysis and its


mplications


on PNG bioavailability.


This laboratory has since


conducted


nvestigations that have


examined the


absorption and hydrolysis


of


PNG,


ncluding th


e


s


uboellular localization of PNG hydrolytic activities, the effect a


dietary PN


and other


dietary components on


intestinal PNG hydrolysis,


and the


hydrolysis of PNG


by the brush border membrane enzyme


lactase-phiori


ZI


n hydrolase


LPH


).


The


researc


described in th


e


subsequent chapters has broadened our perspective


on th


e


absorption


and metabolic processing of PNG


in the


mammalian small


ntestine.


Review of the Literature


History


Vitamin B-6 is an


esse


ntial nutrient to mammalian spe


cies as it functions


as a


coenzyme


in several metabolic pathways.


established when the


deficien


vitamin was described


cy in rats known as acrodynia


15).


The properties of vitamin B-6 were


as a factor that alleviated and preve


Acrodynia,


nitially


nted a


a B-vitamin deficiency, is a


PN)


1


(


2


B3


h


,






3


Chemistry

Vitamin B-6 is a term used to describe a family of related compounds having


pyridoxin


e-Iike activity with the


core structure of 2-methyl,


3-hydroxy,


5-hydroxymethyl


pyridine.


Naturally occurring form


carbon 4 of the pyridine


ring


s


Figure 1


of vitamin B-6 differ


1


),


which yields th


in the substituent present


e


on


three primary vitamers:


pyridoxin


e


PN),


an alcohol


pyridoxal


PL


an aid


ehyde; and pyridoxamine


amine.


Phosphorylation at the


5'-hydroxymethyl of the


pyridine ring forms the


respective


phosphoric esters:


PNP


PLP


and PMP


.Pyridoxal


-5'-


phosphate (PLP


pyridoxamine-5'-


phosphate


PMP


are th


e


predominant forms of the


coenzyme


found


animal tissues.


Vitamin B-6 that


is


catabolized is


conve


rted


into 4-pyridoxic a


Ci


d


4-PA


and excreted in the urine.


Vitamin B-6 also


exists


as a giy


cosylated molecule that is


synthesized by plants.


Natu rally


occurring


arms


of


glycosylated vitamin B-6 consist of a pyridoxine mol


ecule


with one or more mon


osaco


haride or other


complex


OH> H3C


~CH tO-R2


oligosacoharide units attached at the 4' or


via B-glycosidic linkage.


.


N>


9-


The


5' position


most abundant


H-


glycosyl


lated form of dietary vitamin B-6 is pyridoxine-


5'-1-D-glucoside, which is


comprised of a pyridoxin


PN = CH2OH PL= CHO PM= CHNH,


PN, PL, PM= H


PNP= PG3 PLP= P03 PMP= P03


molecule with one


position o


pyrido


glu


cosyl


g


roup f3-linked to th


xine.


Vitamin B-6 is soluble


in water and stable


Figure 1 structures


-1


Oh


emical


of vitamin B-6.


under acidic pH; however,


in alkaline


solution


vitamin


PM


),


an


and


in


R


R,


R,


e


5',


e






4


for this reaction since the pyridine ring provides an electron sink for the free electron pair


left on th


e


a-carbon after the bond between a proton or carboxyl group to the u-carbon is


broken.


The negative charge is stabilized by the


PLP ring.


Thi


s


carbonyl-amine


reaction


is th


e


mechanism by which PLP-depe


nden1


enzymes


function.


Functions of Vitamin B-6


Vitamin B-6


most often as PLP


s


involved


n a host of metabolic reaction


ncluding the metabolism of amino acids,


lipids,


and one


carbon units


and the


pathways


of gluconeogenesis and heme biosynthesis.


Vitamin B-6 is widely recognized for its role


in amino acid metabolism.


PLP is a


coenzyme


for aminotransf erases, decarboxylases,


racemases


and dehydratases.


The


aminotransferases use PLP in the interconversion o


amino acids and their respective a-


keto acids by forming a stable


Sc


hiff base intermediate wh


e


re PLP becomes


an electron


sink for negatively charged catalytic


ntermediates.


The role of vitamin B-6


in


ipid metabolism is not clearly defined.


Compositional


changes in phos


pholipid were reported to occur in response to changes in dietary


concentrations


of vitamin B-6


in rats


19,20).


More recentl


y


Tsuge and colleagues


reported that Wistar rats fed a diet deficient


in vitamin B-6 had a 64%


reduction


n AG-


desaturase and 80% reduction


n acyl CoA oxidase activity compared to the control


group.


The decreased activity caused an alteration


in polyunsaturated fatty acid


metabolism


,most notably with a reduction in docosahexaenoic a


cid (DHA


These


effects are likely to due to an


impaired methylation cycle via 5-adenosylmethionine,


which decreases phosphatidy


cholin


e


and carnitine synth


esis.


s


(21









methylene tetrahydrofolate tram tetrahydrofolate.


A metabolic intermediate


in the


carbon cycle is the


amino acid homocysteine.


Homacysteine


concentration is


maintained


, in part,


by PLP-dependent enzymatic pathways.


The


catabolism of


homocysteine by the


transsulfuration pathway is dri


yen by two PLP-dependen


enzymes.


The first is cystathionin


e


B3-synthase, the enzyme


that catal


yzes


th


e


condensation of


serine


with homocysteine to form cystathionine.


Th


e


second enzyme,


y-cystathion


ase,


hydrolyzes


C


ystathionin


e


to release cysteine and c-ketobutyrate


coneo


genesis is the metabolic pathway that provides a


source


of


glu


cose


novo from non-glu


case


precursors.


Certain amino acid


s


serve


as non-glu


cose


precursors to help maintain concentration


s


of glucose.


The first step in producing


case


f


rom gluconeogenic amino acids is th


e


transamination of the


amin


a


acid to form


its respective


enzyme


ct-keto acid, which is a PLP-dependent process.


Another PLP-dependent


involved in carbohydrate metabolism is glycogen phosphorylase.


Glycogen


phospharylase catalyzes


the


cleavage a1


glycog


en to sequentially release glu


case-1 -


phosphate residues


that may be cony


e


rted to the


glycolytic


intermediate


, glu


cose-6-


phosphate.


PLP associated with glycogen phosphorylase accounts for a large pooi of


whole body vitamin B-6.


It is estimated that up to 80-90%


of body PLP is bound to


glycogen phosphorylase


The biosynth


esis


ofh


eme


also requ


ires


vitamin B-6


as a coenzyme.


Delta-


aminolevulinate


sy


nth


ase,


a PLP-dependen1


enzyme,


catal


yzes


condensation o


succinyl


CoA and glycine to form 6-aminolevulinate,


which is the


precursor for the porphyrin ring


used to


synthesize hemoglobin


Def icien


cy


in vitamin B-6 would therefore precipitate a


5


one


Glu


22).


giu


de


2


3).






6


reduced ALAS activity is often overcome with pyridoxine supplementation,


if the disorder


is clinically classified


as pyridoxine-responsive.


Mutations


in th


e


ALAS2 gen


e


migh


alter


PLP binding sites on the


enzy


me (24,25).


There is


evidence that vitamin B-6 also function


s


on a molecular level


as a


modulator of gene expression.


Evidence


of transcriptional regulation was provided by


Oka and coworkers


in rats with vitamin B-6 defi


C


lency


26).


Vitamin B-6 deficient rats


had a greater abundance


of hepatic total poly


A)+ RNA than vitamin B-6 adequate rats;


howeve


Irthe


increase


was nonspecific and


control B3-actin levels,


which would


expectedly remain constant


well standardized,


,were similarly


Oka and coworkers


ncreased.


Although mRNA levels we


subsequently reported 5-


and 7-fold


re not


noreases


in


hepatic glycogen phosphorylase mRNA (2


7


) and albumin mRNA


28),


respectively


vitamin B-6 defi


cient rats compared to control rats.


Sato et al.


(29)


si


mila


ri


y reported that


vitamin B-6 def


ciency in rats increased the rate of cystathionase


sy


nth


esis,


which was


explained by a se deficient rats had


kveral-fo essenti


Id


increase


in


ally the same


C


ystathionase mRNA.


amount of immunoreac


However, vitamin B-6 live cystathionase protein


as control rats.


The authors concluded that vitamin B-6 deficiency upregulated


cystathionase


5


ynth


esis


but


also decreased the


rate of degradation by ly


s0s0


enzymes. Vitamin B-6 Requirements


The Food and Nut


ri


tion Board recently revised th


e


requirem


ents f


or B vitamins,


ncluding vitamin B-6.


An Estimated Average Requirement


EAR) of 1.1 mg/d and a


RDA of 1


.3


mg/d for men and w


omen


19-50 y) was establis


hed after th


orough review of


the


in


mal






7


As reviewed in the mast recent report by the Food and Nutrition Board (1998),


vitamin B-


6 requirements were ty~


protocols with dietary


pically based on 'ood) vitamin B-6


nvestigation


s


in combination with


utilizing depletion-repletion


sy


nthetic pyridoxine.


rage vitamin B-6 intake


as estimated by nationally representative


nutrient intake


surv


eys


is approximately


.5


mg/d for women and


2


mg/d for men


Food and Nutrition


Board 1998).


Although an


insuffi


cien


dietary intake of vitamin B-6 is not prevalent


in the


eral population,


the


re are significant portions of the population at risk for sub-optimal


vitamin B-6 intakes such


as pregnant and lactating worn


e


n (30


,31)


and th


e


elderly


Vitamin B-6 in Foods and Supplements


Vitamin B-6 is fairly well distributed throughout the food


supply


Foods of animal


orngin


such


as meat


fish


,eggs,


and dairy products are rich


in vitamin B-6.


Vitamin B-6


exists mostly


as PL and PM and their phosphorylated form


s


(PLP and PMP) in foods of


animal origin.


Additionally, fortified breakfast


cereals and vitamin


suppleme


nts provi


de a


significant amount of vitamin B-6,


as PN.


to th


e


typical Ame


rican diet.


Many vegetables


and whole grain


cereal products are good sources a


the vitamin


as well.


The


predominant forms found in foods of plan


origin are usually glycosy


lated.


Plant


tissues


may


contain up to


75%


of


their vitamin B-.6


as PNG (2).


Vitamin B-6 loss during cooking and th


ermal processing is


si


gnif


cant; however


exhibits remarkable


stability during storage and handling.


The


nutritional


mpact a1


reduction


n vitamin B-6


content by heating was


observed


in the 1950s


in


infants that


were formula fed.


Coursin reported that infants receiving formula that was


processed without subsequent fortification had convulsive


I *
fllg Ira*, * i flfl t' C~tN C'~ ~


seiz


th


ermally


ures along with other


fl I ICfl ',+t~A k~ /


Ave


gen


32).


It


a





8


Gregory


the


1


7), the thermal reaction products found in milk were


bioavailability o


the


naturally occurring B-6 vitamers; however,


nitially thought to reduce


re-evaluation 0


th


e


evidence


found that the


thermal processing o


the


formula destroyed the


native


vitamin


B-6.


Similar to infant formulas,


dietary


s


upplements contain pyridoxine hydrochloride


as the added form of vitamin B-6.


Pyridoxine exhibits the greatest stability among


various B-.6 vitamers


as it is relatively unreactive


with oth


er compounds and is


inexpensive to synthesize. Pyridoxine Glucosides


Pyridoxine


glucosides are


ust one


example of the many vitamin glucosides that


occur in nature.


Other nutritionally significant glycosylated vitamin


s


such as vitamin D,


niacin


,pantothe


nic acid


,and riboflavin have been identif led (34


Glyc


osylated forms


of vitamin B-6 are abundan


in foods


of plan-


origin,


absent in foods


of


animal origin.


The


biological sign


If


icance


of


the glycosylation oi


vitamin B-6


n plants is not completely understood,


but it is presumed to be a storage


form a


the vitamin


34).


A


s


originally


Id


entifiled in the bran of rice as 5'


-O-(13-D-


glucopyran


osy


pyridoxine


'1


Figure 1


-2


),


glycosylated vitamin 8-6 accounts for a


mean 15%


of


total vitamin B-6


intake


n a typical mixed diet (10,30).


This percentage


could vary depending on food selection and en


ergy


intake.


Other giucosides of


pyridoxine have since been identified.


isomers of pyridoxin


Tadera and colleagues


e -Bj-D-glucoside, pyridoxin


isolated the


e- 5'-I3-D-cellobioside,


4'and


and other


conjugated oligosaccharides


35),


as well as pyridoxine-5'-


j3-D-glucoside esterified with


the


).


yet


5',






9


CH2OH

~CH0 OH HONH O HH


Figure 1


-2


Structure o


pyrid


ox'


ne-5'


-B-D-glucoside.


glucosides has been reported (39).


These a-D-glucoside conjugates are more


bioavailable than r3-linked PN glucoside,


as th


e


y are absorbed


intact


and rapidly taken


up and hydrolyzed by the


liver


40,41).


Analysis


and quantification of pyridoxin


e


glucosides have


been reported by a


number of investigators.


Total glycosy


difference in growth responses


lated vitamin B-6 can be determined by the


of yeast before and after treatment


with B-glucosidase


Alternatively


liquid


chromatographic


separation methods have


been established to


meas


ure not only th


e


various


forms


of


vitamin B-6


bu1


also glycos


ylated variants


4.42


,43).


Bioavailability and Absorption of Vitamin B-6


The bioavailability a


vitamin B-6 in humans


cons


uming a mixed diet is


approximately


bioavailability


75%


is


44).


As


recently reviewed by Gregory


likely to be reduced by food matrix


trapping,


1


7) ,


vitamin B-6


nondigestible residue,


and


only partial utilization o


glycosy


Fated forms of vitamin B-6.


A gly


cosy


Fated form of


vitamin B-6


specifici


C


ally pyridoxine-5'


-j3-D-glucoside,


was first though-


to be a


completely


bioavailable form of vitamin B-6


in


rats (4


5).


How


ever


later


studies found a much


ower


2).






0


vitamin


This is consistent with other


investigation


s


examining the bioavailability of PNG


n different rodent species (6-9) and humans


10.11


The


bioavailability of PNG is mu


greater in human


s


50-60


than


n rats (25-


30%


),


which is thought to be


determined by


the extent of enzymatic hydrolysis in the


small


ntestine.


While PNG can be hydrolyzed


in the


enterocyte,


it also appears


in the


urine unchanged (absorbed as the


intact


glucoside)


It was estimated that


~35%


of dietary PNG appears in the urine


in human


as determined from diet composite analysis o


PNG and urinary excretion of PNG


There is evidence that PNG is not hydrolyzed exclusive


y


in the small


intestine.


n a comparison of oral [2H


53


PN and intravenous


(L~v.


[2H2] PNG doses,


it was reported


that the


the


i.


V


dose of PNG


oral PN dose.


,which bypassed the intestine, was utilized at 28%


This observation


indicated that


relative


to


some hydrolysis of PNG occurred


outside of the intestine


10).


n rats


less than 5%


of an intraperitoneal (i.p


dose of


[VHIPNG was metabolically utilized relative


to an


.p. dose of [14C]PN


6).


How


ever.


both


PN and PNG we


re readily absorbed and 93%


of the [3H] isotope was excreted


in the


urine


as PNG and its


hydrolytic product, PN.


Analysis o


liver vitamin B-6 revealed a


lower degree of metabolism of PNG compared to PN with no retention of [3H]PNG.


These


observations indicated that PNG was adequately absorbed in the


ntestine,


poorly utilized,


and rapidly excreted into the urine


6).


The bioavailability of PNG


in other


rodent models was also evaluated to


assess


species relatedness


to humans.


nan


nvestigation using [3H]PNG and


14C]JPN, Banks and Gregory


9)


reported a 10-34


bioavailability of PNG


in rats


69%


n mice.


70%


in hamsters


and 92%


n guinea pigs.


These


results suggested that mice and hamsters might be better


suited for bioavailability


ch


10).


but


%/









mostly conducted in intact rat


intestine


-46


-5


).


There is general agreement that the


phosphate group of dietary PLP is hydrolyzed by luminal alkaline


phosphatases,


releasing PL that is readily absorbed.


PLP can be


absorbed across the


enterocyte


apical membrane


ntact


,although to a lesser exten-


than its dephosphorylated form


(50)


Once


absorbed


,vitamin B-6 can be phosphorylated by pyridoxal kinase


for the


purpose of metabolic trapping.


While th


e


intestinal absorption of PLP is described


passive


and non-saturable


, the metabolic ph


osph


orylation o


absorbed vitamin B-6 is


known to be saturable


alkaline


(52


,53)


Similar to PLP


,PMP also is first acted on by intestinal


phosphatases to yield PM that is absorbed; however


saturating concentrations,


Collectively,


it too will be absorbed with the


data from vitamin B-6 absorption studies indi


5


when PMP is presen


at


'-phosphate attached (54).


cate that the rates o


absorption for B-6 vitamers are PL>PN>PM.


phosphorylated forms


of vitamin B-6 undergo int


phosphatase prior to absorption. At PLP and PMP are directly absorbed,


At physio


ogical con


estinal hydrolysis


concentrations we!


with their


centrations by alkaline


above a physiologi


5'-phosphate group,


cal


dose,


although at a rate


seve


ral fold lower than their respective


dephosphorylated compounds


(47,


54


To cross


basolateral membran


e


and


enter into portal circulation,


B-6 vitam


ers are in a


nonphosphorylated form.


The


intestine does not appear to provide phosphorylat


ed B-6


vitamers to the rest of the organs; rath


er the liv


e


r is responsible fo


r ph


osphorylation o


vitamin B-6 for the distribution to other


organs.


Transport and Metabolism


Vitamin B-6 that is absorbed and metabolized by the


ntestine


enters th


e


portal


- ~r:... ~ -- ii II L. -. ..~J .. ...IL. .~ -- ~L. Irrrx J


5',


PL)


as


the









by extrahepatic organs.


Nonphosphorylated forms of vitamin B-6 are phosphorylated


liver


by pyridoxal kinase.


Pyridoxal kinase,


using ATP-Zn+


as a chelated


cosubstrate


catal


yzes


the phosphorylation of the


5'


position of PN,


PL, and PM to form


PNP.


PLP


and PMP


, respectively (Figure 1-3) (57


,58).


In addition to the


enzymatic


phosphorylation function o


the


liver


,FMN-.dependent pyridoxamine (pyridoxin


phosphate oxidase is an abundant


enzyme


in the liver


that is responsible for


conve


rsion of PNP and PMP to PLP (58).


This reaction is criti


cal f


0


r the metabolism o1


dietary PN since


most tissues


have


an insuff


dient amount of oxidase activity to convert


PN toPL


59


).


This enzyme


also is


of importance because it is tightly regulated by


product inhibition.


High concentrations of PLP slow the


rate of conve


rsion o


PNP to


PLP


,which pre


vents an


excessive


accumulation o1


intracellular PLP


Catabolites of vitamin B-6 are gen


e


rated in the


liver.


Pyridoxamine


pyrido


phosphate oxidase catalyzes


the formation of PLP from PNP and PMP


wh


ch


, in turn,


is dephosphorylated to PL.


PL in


excess


is oxidized to the metabolic excretion product,


4- pyridoxic acid (4-PA) by aldehyde oxidase


FAD) or pyridoxal dehydrogenase


(NAD),


enzy


mes that are present


in the liver and kidney.


PLP and PL are the predominan


circulating forms


of vitamin 8-6


comprising


approximately


75-80%


of


the


total body B-6


18).


As reviewed by Coburn


turnover


of


vitamin B-.6


as PLP


,may be described


as a five


compartment model


consisting of muscle,


liver


,plasma,


erythro


cytes,


and all other pools,


which are grouped


together into one additional


compartmen


t.


Evidence


from tracer studies calculated total


body concentration of vitamin B-6 to be 1


5


nmol/g and th


e


total pooi


size


in human


the


2


in


e)


5'


th


e


5'-


xi


ne)


two


23


the


s


is
















PNP


-o
a
Co
-D
-4.
C') CD


'1


-D

0 'K


FMN
dependent pyridoxamine (pyridoxine) 5'-phosphate
oxidase


A


n 0)
Co
CD


FMN
dependent pyridoxamin


e


(pyridoxine)


5'-


# PLP


-c
a
C))
V


0)
C,,
CD


L


7;
0)
Cl) CD


PL


PN


Co
C
C
a U, 5: 0)
CD CD
Co


V


a
CD
Sc
a
B Co
CD
0)
Co
CD


-q


phosphate xiasge


PMP


transamin


ases


-c
- .
C
0
x 0)


-c
0
CD
V
r 03
Co CD


A


0)
Cl) CD


~0
a
C 0)


PM


-C
Sc
-9
a
0 'K 0.)


PNG


4-PA


Figure 1


-3.


Interconversion and metabolic


sm of vitamin B-6.


Abbreviations


used:;


PNG (pyridoxine


phosphate)


5'-J-D-glucoside),


PLP (pyridoxal 5'-phosphate),


PN


pyrido


xi


PL (pyridoxal),


ne), PNP (pyrido


4-PA


xine


4-pyridox


5'-


ic


acid),


PMP


pyridoxamine


5


'-phosphate),


PM


pyridoxamine).


is localized to the muscle (61


).


The remaining vitamin B-6 is con


centrate


d


in the


liver


but also distributed to the brain


,heart, adrenal glands,


kidn


ey


and p


ancreas


Metabolic utilization of PNG and effects of dietary PNG on the


normal


metabolism of non-giycos


ylated B-6 vitamers were examined previously.


In rats.


comparison of bioavailability between purified [VHJPNG and alfalfa


sprouts that were


ntrinsicallv


enriched with r3H1PN


.


formina [3H]PNG. was done in relation to


r3Hl-


3


23).


a


and






1


4


form that would naturally occur in a plant-derived food in relation to pyridoxine. Rats that


were fed the purified PNG received 88%


of


their vitamin B-6


as PNG.


Rats that we


re fed


the alginate gel with the alfalfa sprout homogenate added consumed ~45%


of their


vitamin B-6 as PNG.


Control rats consumed >80


o /


of their vitamin B-6


as PN.


Isotop


ratios of [3H


/


14C]


dose


n the


liver


,carcass and plasma were significantly low


e


r in the


rats fed any form of PNG than control rats that were fed PN


.Conve


rsely, th


e


relative


isotopic ratios in the urine and feces were significantly higher than controls.


indicated that the absorption and metabolic utilization of PNG was


These


incomplete.


data


Dietary


PNG was not retained in the


live


r as PMP, PLP, or PL.


Significantly more PNG was


excreted


in the urine


and feces than PN.


There was no f3H]PNG detected


in the livers


rats fed the glucoside


8) .


Furthe


r evaluation PNG bioavailability was done


in rats either


PN or PNG as their sole


source o


vitamin B-6.


Changes


in vitamin B-6 status were


assessed using erythrocyte aspartate transaminase (EAST) stimulation,


plasma PLP


concentration


,and urinary 4-PA excretion in response


to the form of dietary vitamin B-6


Rats con


suming their vitamin B-6 as PNG had an elevated stimulation of EAST with


PLP


, lower plasma PLP concentrations,


and a diminished 4-PA excretion compared to


rats consuming their dietary vitamin B-6 as PN.


This same group of


nvestigators also


examined the metabolic utilization o


PNG compared to PN by using isotopically labeled


[3H]PNG and


14C]PN given either as an oral or intrape


ritoneal dose to vitamin B-6


adequate or defic


ient


rats (


6).


Rats


njected with both [3H]PNG and


14CJPN


excreted


twice the amount of [14C-labeled 4-PA when fed a diet adequate


in vitamin B-6


compared to a diet deficient


in vitamin B-6.


In contrast


, rats fed a diet containing no


* .2. .. A *- .3


a. *, .. * -


Ic


of


7) .









hepatic vitamin B-6 metabolites detected as [3H] or [140], were not signif


cantly diff


erent


between adequate and deficient


rats, regardless of route of administration.


Although th


retention of [3H


-labeled B-6 vitam


ers was sign


icantly less


in the liver and carcass as


compared to


4vi-aelvtamers,


PNG appeared to undergo partial hydrolysis and act


as PN


in the metabolic


interconversion of hepatic vitamin B-6


(


6


).


Th


e


results from these


studies


showed that PNG is not as eff


ciently metabolized as PN and that vitamin B-6


cienc


y reduces vitamin B-6 catabolite clearance,


i.e.. a reduction


in 4-PA


excretion.


Gilbert and Gregory


5) investigated the effect of dietary PNG on the metabolic


utilization a


co-ingested PN


n rats using radiolabeled


14C]PN and unlabeled PNG.


Administration of PNG concurrently with PN antagonized the utilization o


PN as


observed through the altered distribution of hepatic vitamers and reduced retention o1


labeled PN


Of particular interest was the


direct linear relationship found between the


dose a-


PNG and th


e


appearance


of [4CJPNP, which was


later


supported by Nakano


and Gregory


13),


who also reported that as dietary PNG


increases


in rats,


lI


ver PLP


decreases.


Since


dietary PNG


interfered with the normal production of hepatic PLP


Gilbert and Gregory (5) hypothesized that PNG had an


nhibitory effect on pyridoxamine


(pyridoxine


5'-phosphate oxidase.


Howeve


r. furth


er investigation of the


inhibitory


effects a1


PNG on purified pyridoxamine (pyridoxine) 5'-phosphate oxidase and found no


effect on the


activity in vitro


62


These results w


ere corroborated by Zhang et al.


(63).


In live


r cell homogenates, t3H]PN


incubated


in th


e


presence of PNG at either 0.5 or 5.0


ptmol/L did not significantly alter


the specific activity of pyridoxal kinase or pyridoxamine


(pyridoxine


5' -phosphate oxidase.


The authors concluded that an increased liver


PNP


*1 *. I' flk . j * r.~ r r1in.u :1t.....I 11....L..


5


e


def









cellular uptake and metabolism o


PNG relativ


e


to PN, which was


20 and 0.2


respectively


Effects of dietary PNG on the metabolism of PN also have


been


investigated


humans.


Hansen and


coworkers monitored vitamin B-6 status in response


to feeding


women diets with varying concentrations


diets that were designated


of PNG


64


).


Nine healthy women were f


as either high or low in PNG for 18d each in a


ed


crossover


design.


Vitamin B-6 nutritional status was


assessed


by using plasma and erythroc


PLP


erythrocyte aspartate and alanine aminotransaminase


(EAST and EALT)


stimulation


,urinary 4-PA and total B-6 fecal


excretion


as indicators.


Th


e


diet providing


27%


of total vitamin B-6


as PNG


high PNG diet) yielded a 10%


lower urinary 4-PA,


lower fecai vitamin B-6


excretion


and 18


% 0


andi1


7%/


lowe


r plasma and erythrocyt


e


PLP.


respective


y, than the diet providing


9% total vitamin B-6


as PNG (low PNG diet


. Th


EAST and EALT


stimulation tests were similar between the two diets.


Similarly, Nakano


et al.


1


observed a de


cline


in 4-PA


excretion in human subjects giv


en increasing


doses of [2H2]PNG.


Rats are less


susceptible than humans


to th


e


antagonistic effects o1


PNG on PN.


Cytosolic Pyridoxine-5'


Prior to the purification and identification of the


cytosolic PNG hydrofase from pig


intestinal mu


cosa


,PNG was presumed to be hydrolyzed


in th


e


small


ntestine by another


soluble j-glucosidase,


broad specificity p-glucosidase (BSI3G) (6,8,1


2


,13).


To better


understand the hydrolysis


of PNG by BSI3G, MoMahon and


coworkers purified and


partially characterized not only


ntestinal BS3G


but also a novel


cytosolic B3-glucosidase,


63).


16


in


yte


1


2%


e


-f-D-glucoside Hydrolase (PNG Hydrolase)


%,









from the guinea pig live


r (68).


While BSJ3G does not hydrolyze PNG


14) or other


nutritionally substrates,


significan


oligosaccharides,


with particular spe


citficity to ary


displays ac I aglycones


tivity for a wide range


(69


of


,70).


Cytosolic PNG hydrolase is a solub


le


j3-glucosidase with an


acidic pH optimum


(5.5) and a molecular mass


of 130 kD (denatured) and 160 kD


native


In


contrast to


BSI3G, PNG hydrolase does not hydrolyze


ary


glucosides but exhibits


hydrolytic affinity


for PNG


cellobiose,


and lactose.


PNG hydrolase


was


C


haracterized to have


a Km of


0.88


+0.12 mmol/L and a Vmax of 13.2 0.8 pmol/htmg protein for PNG


nhibition


experim


e


nts found that PNG hydrolase, like other


3- glucosidases,


was


inhibited by


conduritol B epoxide and glucono-S-Iactone.


Hydrolytic activities


of


both PNG hydrolase


and BSI3G are affected by


s


ulfhydry


reagents,


which


mplicated


C


ysteine


residues


in the


catalysis


at the


active


site.


There is remarkable amin


0


acid homology between


short amino


acid sequences


obtained from


cytosolic PNG hydrolase


~95-99


%)


purified from pig


intestine and the


brush border en


zy


me, lactase phiorizin hydrolase


LPH


(Tseung,


et al.


,unpublished)


analyzed by the


BLAST alignmen


procedure


(71


).


All of the short sequences


aligned


with rat


rabbit, and human LPH


Five of these


sequences


(total of


51 amino


aol


ds) also


aligned with regions


of the precursor sequence


of LPH (aa 20-866) that are not presen


in the mature LPH


enzyme.


However


th


ere is a region in the precur


sor sequence


LPH


aa 458-806) whe


re no aligning sequences


from


cytosoli


C


PNG hydrolase were


found.


This might indicate that cytosolic PNG hydrolase is a splice variant derived from


LPH gene.


It is also well known that substantial sequ


ence


homology exists


among


17


as


th


e


of








Lactase Phiorizin Hydrolase


The


digestion of dietary carbohydrates relies on the disacoharidases present in


small


intestine.


Perhaps the most important carbohydrate-digesting enzyme to the


developing mammalian species nourished via a milk-based diet is the


intestinal bru


border en


zyme


lactase phiori


zi


n hydrolase (lactase).


Lactase phlorizin hydrolase (EC


.23, 62, and 108)


LPH) is a membrane bound glycoprotein


n the intestine


that is


primarily responsible for th


e


hydrolysis o


dietary lactose


nto its constituent


monosaccharides giu


case


and galactose.


LPH was previously pur


ifiled and


characterized by


several laboratories from the


s


mall


intestine


of differen


Lactase phiorizin hydrolase is named for its


two distinct hydrolytic activities:


one for the


hydrolysis


is of lactose and oth


er hydrophilic 5-glucosides (lactase),


and th


e


other for the


hydroly


ss


of aryl- or alkyl


-glucosides,


such


as j3-glu


coce


ramides (phlorizin hydrolase


-75).


Wacker and colleagues assigned the lactase activity to glutamate1273


and the


phlorizin hydrolase activity to glutamate1749


75).


The


LPH gene


LOT) was


localized to


hromosome


2 and oDNA cloning led to the


translation of th


e


LPH sequence to 192


amino acids


72).


The


enzyme is


synthesized


as a single polypeptide gly


cosylated


precursor of 21


5


kDa (pro-LPH


),


which undergoes folding to form a homodimer; a step


that is


C


ritical for membrane targeting and enzymatic activity


76


,77).


Transport of th


homodimer through the Golgi apparatus


yields a complex glycosylated form of the


protein (230 kDa)


78).


Subsequ


e


ntly


the protein undergoes two proteolytic cleavages.


The


first occurs


ntracellularly at Arg734,


which yields a transport-competent form of the


enzyme.


The


seco


nd cleavage,


occurring after in


sertion


in to the


microvillar m


embrane,


the


1


8


3.2.


1


S


h


spe


C


les


72).


73


C


7


e









revealed that LPH expression increases from crypt to mid-villu


s,


and declines


at th


villus


ti


p


(77,


81,82).


The control o


LPH expression has been studied extensively


n light of the


connection with the post-weaning decline


n lactase activity that is observed in maturing


mammals.


Current opinion on the decline


of lactase activity focuses on tran


scriptional


control of the LPH protein


(77


.This is supported by data that positively correlate levels


mRNA and LPH activity


in rats (81 ,83,84),


pigs


(85),


and


sheep (86).


In spite of


compelling evidence of transcriptional control translational control of LPH expression. Usir


phenylalanin


e


the


re are reports of secondary,


ig ['3C3- or [VHJ-leucine


or post-


and [3C]-


,Dudley and coworkers have measured the brush border


fractional


synthesis rate


FSR) a


intestinal disacoharidases (87


),


noluding isoforms of LPH (88) to


assess


dietary effects on intracellular processing of LPH (85,89,90


).


The


ratio oi


isotopic enrichment of an LPH glycosylated precursor and the


enrichment of a mature


LPH isofarm yields th


e


FSR for LPH (8


7


).


Changes


in LPH isoform labeling,


FSR),


response to a dietary treatment,


e.g. protein deficien


diet


85),


i


ndicativ


e


of changes


in post-translational modification and further


the


regulation


of mature LPH expression.


This procedure is validated by a strong relationship betwee


the


LPH isoforms


n the


and developmental differences in LPH activity (91


relative abundance


).


of


Using this


methodology, post-translational control of LPH expression was reported to be


protein malnutrition (85,90) and increased by administration o


reduced


nsulin-like growth factor-I


(IGF-1) (92,93) and colostrum (89


A reduced expression and activity of LPH


in humans


most often occurs during


I' I * .3 .3, I I a a 3'..a *..t r..........I I


1


9


e


of


the


th


e


in


In






20


usually recognized by the variety of abdominal and intestinal related symptoms that are


caused by the osmotic effect of undigested lactose presen


in the


umen.


Although


nearly all mammals are born with lactase,


some


nfants are barn with a rare congenital


lactase deficiency.


nitial clinical characterization of intestinal disaccharidases,


ncluding D3-


galactosidases,


as well as their defi


ciencies were reported in a series of papers by Arne


Dahlqvist and coworkers (95).


and accurate determination o


Dahlqvist is also credited with the developmen


lactase activity as measured by the release 0


of


glu


a rapid


cose


using glu


cose


oxidase and a colorimetric agen


96) .


The condition of lactose


ntolerance can be diagnosed by assessing the extent of lactose digestion.


Lactase


activity can be directly m


easu red by intestinal mucosa biopsy or by an


intestinal


perfusion method that pumps lactose into the intestine and measures glu


case


release by


sampling the contents of the


ume


n (97


).


Although these methods are considered to be


most accurate


, they are used


infrequ


ently and are the


most


invasive.


More


convenient tests used in the routine


diagn


osis of lactose intolerance


include breath tests,


blood sampling,


and urine


analyses


(98


).


Traditionally


breath hydrogen concentrations


are used to detect lactase deficiency.


This test measures the


release of H


2


in the breath


after


a dose of lactose.


Undigested lactose is fermented by colonic microflora,


which


produces


hydrog


en gas


that


is absorbed into portal circulation and later


expired


in th


breath (99


).


Simila


ri


y


breath


13002 can be measured after


ngestion of


13C]lactose.


Determination of


blood glucose or galactose concentrations


following a dose of lactose


can also be used to diagnose lactose


intolerance.


More recently,


it was


suggested that


Jr~'-I 0 0


the


e






21


The prevalence of lactose intolerance


differs globally.


Worldwide


it is estimated


75%


of


the


adult population is lactase deficient.


In the United States, the lowest


prevalence is among whites of Western European descent


1


5%) and increases to 80%


in the


African-Am


eri


C


an and 90%


in the Asian-American population.


In Asian


countries,


percentage


of lactose


intolerance


is at least 50%


and varies amo


ng ethnicities


It is proposed that the persistence of lactase activity,


rather than the


loss


s


abnormal.


As reviewed by Vesa,


hypotheses to explain lactase persistence were derived from


cultural practices and natural genetic selection


98


).


At the


adv


ent of


dairy farming,


pulations that had higher levels a1


lactase activity would exhibit an


evolutionary


advantage for survival


in times of dietary


scarcity


.Today,


th


e


highest prevalence


lactase persistence


is observed


in individuals that are descendents from areas with a


long history o


dairy farming,


as in those from Western Europe.


Genetic markers of


physiological decline


n lactase activity were recently identified by examining variants in


lactase


LOT) gen


e


(102).


Seve


n polymorphic microsatellite markers flanking the


LOT gene


n nine Finnish families (n=


196) with adult-onset hypolactasia we


re analyzed.


ndividual family members we


re biochemically ye


rified to be lactose


intolerant


tolerant.


Two DNA variants that we


G/A-2201 8 with the


re upstream of LOT were identified:


C and G alleles associated with lactase def


cien


cy.


C/T-1 3910 and


ndividuals that


were diagnosed with lactase def


ciency


n-59) we


re all homozygous (CC


with respect to


CIT variant and 6 were heterozygous


(GA) and 53 homozygous


GG


with respect to


0/A variant.


None


of


the


lactase persistence


subjects (n=


1


37) were homozygous


with respect to the C or 0 alleles.


Subjects (n


=74) were TT with respect to the C/T


I * A a a a' -'I- a . . I. I. I


that


the


101


pa


of


th


e


Th


e


or


th


e


the






22


was also examined.


White North Americans had the lowest prevalence


followed


by the


Fren


ch (41


%0)


and black North Am


ericans


(79


%0).


These data are not


only


consistent with


C


urrent epidemiological estimates of prevalence


of


lactose


intoleran


also propose anoth


er potentia


method of


screening


individuals for th


e


diagn


osis


lactose intolerance.


In summary, the pre


ceding review of th


e


literature discussed the


nutritional


significance


and bioavailability of PNG


as an important form of dietary vitamin B-6.


ntestinal B-glucosidases catal


yze


the


ntestinal hydrolysis


of


PNG


,which is the limiting


factor of PNG bioavailability


. Th


e


following research sought to


examine


bioavailability o


PNG


in mammals with particular focus on its


ntestinal hydrolys


Hypotheses


The researc


h described


in the subsequent chapters was


based on th


e


existing


literature and preliminary studies done to describe the


hydrolysis


and metabolism of


PNG.


The


presen-


studies w


ere done to test the hypoth


eses:


Dietary vitamin B-6 deficien


cy in


creases


the


PNG hydrolytic activity


n the


mammalian


small intestine.


The brush border membrane


enzyme,


lactase phiorizin hydrolase,


catalyzes


the hydrolysis


is of pyridoxine-5'-j3-D-glucoside


bu


ce,


of


the


is.


1


2


7.6%














CHAPTER


HYDROLYTIC ACTIVITY TOWARD PYRIDOXINE-D-D-GLUCOSIDE


IN RAT


INTESTINAL MUCOSA


S


NOT INCREASED BY VITAMIN B-6 DEFICIENCY


OF BASAL DIET COMPOSITION AND PYRIDOXINE Introduction


EFFECT


INTAKE


ne-5'


-43-D-glucoside


PNG),


a gly


cosylated form of pyrid


oxine


provides


sign


ificant


dietary source


of


vitamin B-6.


This form o1


vitamin B-6 was


first isolated from


rice bran


1


and can be found in foods of plan


origin (2,18,42).


PNG accounts for


75%


of


the vitamin B-6 p


resent


n plan


tissues.


n a m


xed Am


e


C


an diet.


it is estimated


that half of the dietary vitamin B-6 intake is from plant


sou roes


(103)


Although PNG


provides approximately 1


5%


of the


total vitamin B-6 in typical diets in


the United States


(30,64


some patterns


of food selection could lead to a much larg


er proportion o


dietary


vitamin B-6


as PNG.


The bioavailability of PNG


in humans


is 50-60


o /


relative


to pyridoxine,


10.11


which is greater than that estimated for rats (25-30


%0) (-9).


The


rate-limiting factor in


utilization of PNG


as a source


of vitamin B-6 is the


enzy


matic hydroly


glucosidic bond,


rather than the


intestinal absorption of intact PNG or free PN derived


from PNG hydroly


ss


(4,8


.The variation


s


n PNG bioavailability within and among


species may be explained by differences


in


intestinal en


zy


matic activities toward PNG.


Cytosolic broad


spec


ificity j-glucosidase (BSI3G) (66) was


nitially thought to be


- - - . * - . .


2


Py


rid


ox'


a


5-


the


ss


of


th


e


ri









cytosolic D-gucosidase,


designated pyridoxine-5'-43-D-glucoside hydrolase


PNG


hydrolase),


that catalyzed th


e


hydrolysis


of PNG


Recent data from this laboratory


indicate that PNG


can be hydrolyzed in both the


cytosolic and membrane


subcellular


ractions


of


rat


s


mall


ntestinal mucosa.


n an


nvestigation 0


the age-dependent


enzymatic hydrolysis


of


lactose and PNG


Armada et al.


105)


measured PNG hydrolysis


in the


cytosolic and brush border membrane suboellular fractions of intestinal mu


cosa


nursing,


weaned


and adult rats.


A concurrent publication by Mackey et al.


106)


reported the


kinetic analysis


of PNG hydroly


ss


as catalyzed by rat brush border


membrane


and purified lactase-phiori


zi


n hydrolase


LPH


),


a f-glucosidase


in the


intestinal brush border membrane.


hydrolysis to be catalyzed in both


s


Although these


ubcellular


investigation


S


reported PNG


compartments of the intestinal mu


cosa,


contribution of these cytosolic and brush border


membrane


enzymes


toward PNG


hydrolysis


in vivo is not fully known.


Previous


nvestigations


of the


effects


0:


vitamin B-6 nutritional status


on cytosolic


PNG hydrolase activity in the


hydrolase activity was


s


mall


ntestine of guinea pigs and rats


nversely related to vitamin B-6 status


(


1


2


s


,13).


howed that PNG


Subsequen


studies


Mackey et al.,


unpublish


ed) examined more closely the effect of vitamin B-6


nutritional status on the activity of


cytosolic PNG hydrolase


n rat intestine and showed


that vitamin B-6 status had little or no reproducible effect on this enzymatic activity


.This


raised the question of whether


the vitamin B-6 status of the


differences


in the basal diet may have had an effect on


rodents and perhaps


intestinal B3-glucosidase activity.


preliminary rat studies that did not find an effect a-


dietary vitamin B-6 on


ntestinal B3-


24


In


the


Our









mostly cornstarch,


is a more fermentable carbohydrate for the microflora of th


e


cecum


and large


intestine


than


sucrose


,which is the major carbohydrate component of the


AIN-


76A formulation.


In the presence


of fermentable carbohydrate,


the


gut microflora


actively


synth


esize


protein,


growth factors,


and other nutrients,


including vitamin B-6


108),


which may


enhance the vitamin B-6 nutritional status


of the rat.


The purpose of the


present study was


to 1) determin


e


the


subcellular distribution


enzymatic activ


ities toward PNG


in rat


ntestinal mucosa


as a function of vitamin B-6


status


and


, 2)


examine the


differences


betwee


n AIN


-76A and AIN-93G


pur


ifiled diets on


PNG hydrolase activity in the


cytosolic and me


mbrane-associated


suboellular


compartments.


Material


s


and Methods


Animals and diet.


Weanling male rats


Hsd:Sprague


Dawley SD


Harlan


Laboratories


Indianapolis,


N),


wei


ghing ~50 g were used


in both study 1 and 2.


Rats


were housed in hanging wire m


esh stain


less


steel cages and maintained at


constant


temperature with 1


2h


Ii


ght:dark


cycle. All procedures


for animal care and treatment


were approved by


the


Institutional Animal Care and Use


Committee at the


Univ


e


rsity of


Florida.


n study 1


the rats


(n


=29


were randomly


assi


gned to


one


of


f


our AIN-93G


purified diets


1


0


7


from Dyets,


nc.


(Bethlehem,


PA) that differed


only in the


concentration of pyridoxine.


Rats were given free


access


to diets that provided


7


mg/kg


n=5),


1 mg/kg


0


.5 mg/kg


and 0 mg/kg


n=6


pyridoxine-HCI.


Another


group of rats


n-6) was


pair-fed a diet containing*


7 mg/kg pyridoxine


7


mg/kg,


A


25


of


(


pf)


n=6),


n=6),









n=4),


0.5


mg/kg (n


-5), 0.1 mg/kg


n=5), or 0 mg/kg


(n


=5


PN-HCI,


or AIN-93G with 2


mg/kg


1 mg/kg


0


.5


mg/kg


n=5) ,.


0.1 mg/kg


orO0 mg/kg (n


-5) PN-HCI.


Study


2 was conducted at a time separate from study 1.


After


5


weeks


the


rats in both studies were anesthetized by Halothane


Alocarbon Laboratories


,River Edge,


NJ


nhalation and exsanguinated by cardiac


puncture.


Blood was collected with heparinized needles and


sy


ringes and


transferred to


evacuated tubes containing EDTA


as an anticoagulan


t.


Plasma was obtained by


centrifugation at


2.000


x g for 20 minutes and stored at -80


C0


until analysis.


Small


intestine was harvested,


ushed with 9 g/L NaCI at 4


~C to remove


intraluminal contents,


cut longitudinally


and mu


cosa


was


obtained by


scraping with a glass slide,


all of which


were done on ice or at 4 00.


All procedures were done


under gold fluo


rescent


li


g


ht to minimize photoch


emical


degradation o-


vitamin B-6.


HPLC analysis of vitamin B-6 concentrations.


Plasma pyridoxal


5'


-phosphate


PLP


was measured


as the


semic


arbazone derivative by rev


e


rse-phase


fluorometri


HPLC using a modification o


the method of Ubbink et al.


109


PN concentration of


both AIN-76A and AIN-93G diets was


meas


ured using reve


rse-phase


fluorometric HPLC


110)


after extraction


as described previously


4


).


This analysis


was


not perfo


rmed on


diets used


in study 1.


Tissue preparation for enzyme activity assays.


ntestinal mu


cosa


was


homogenized using a Potter-El


vehjem tissue homogenizer


in 5 volumes of


homog


enization buffer containing


25 mmol/L sodium phosphate,


pH


7.4


,containing 50


I If I * - -


26


C


the


n=5),


n=5),


n=5),









resuspended in homogenization buffer using a Potter-Elvehjem homogenizer.


Brush


border


membrane was isolated according to the method of Kessler et al.


111) with


some modification


112).


Briefly


non-brush border m


embranes were precipitated by the


addition a


solid MgCl


2 (to yield a final concentration of 10 mmol/L) to a portion of


mucosal crude horn


ogenate, followed by centrifugation at


3,


000


x g for 10 mn.


The


supernatant was


centrifuged at 40,000 x g for 20 min to obtain a brush border pellet.


pellet was


resuspended using a Potter-Elvehjem homogenizer


again at 40,000 x g for 20 mn.


resuspended


The


and centrifuged


final brush border membrane pellet was


in homogenization buffer with a Potter-Elvehjem homogenizer


Using


specific activity of th


e


brush border me


mbrane


enzyme sucrase,


brush border


membrane


isolation


consistently yielded an


enrichmen


factor of ~10-14, which is


consistent with other published values


111


,113).


Using PNG


as the substrate, the


crude


homogenate


and cytosolic


total m


e


mbran


and brush border


membrane


suboeliular


fractions of intestinal mu


cosa


were assayed for hydrolytic activity in study 1 and cytosolic


and total membrane subcellular fractions were


assa


yed for activity


n study


Measurement of PNG hydrolytic activity.


In vitro


assay


s


of enzyme activity


were don


e


on th


e


diff


e


rent suboellular fractions of intestinal mu


cosa


using PNG


as the


ubstrate.


PNG was prepared by biological


sy


nthesis using alfalfa seeds and purified


described by Gregory and Nakano


110).


All activity


assays


were done


under


conditions


that allowed the m


easurement of


initial rate.


The


assay


for the


hydrolysis


of PNG was


done by a minor modification


of the


method of


Nakano and Gregory


1


3).


Reaction mixtures contained 80 mmol/L sodium


-,......,... -.. - ..., U d


-. ,'


.............................r


I I, 1~~* In n
rt t'r .1.


27


Th


e


the


s


2.


as






28


Statistical analyses.


In study 1


differences in plasma PLP and PNG hydrolytic


enzyme activities among the


rats fed the diets containing different concentrations a1


pyridoxine we


re analyzed using one-way analysis


0


variance


(11 5) and Student-


Newman-Keuls pairwise


comparison test with SigmaStat


software (Jandel Corporation,


n Rafael


,CA).


n study


2


, linear regression was


used to analyze


raw o


r log


transformed data.


This was done to account for


small differences


in measured PN


concentration


n the


AIN-76A and AIN-93G diets.


Rat weigh


gain,


plasma and liv


er PLP


concentrations regression line


,and enzymatic activities were analyzed by a t-test comparison o1


slopes and y-intercepts between th


e


lI


near


AIN-76A and AIN-.93G diet groups


116) using Prism


conce


software (GraphPad Software,


ntration was log transformed to normalize the


Inc., San Diego


CA


).


Plasma PLP


distribution and variance.


Dietary


PN concentrations were log transformed to linearize the


liver PLP concentrations.


valu


e


of


less than 0.05 was


considered to be statistically


Si


g


ni


ican


t.


Data are presented


as means


SEM.


Results


Diet and nutritional status.


n study 1


rat growth


signify


canti


y


noreased


0.0001) as dietary PN concentration


increased, with final body wei


ghts of 255


4 g


mg/kg,


ad fibitum),


197 5


g (7 mg/kg,


pair-fed,


pl


),


229


+3


g


1 mg/kg),


mg/kg


),


and 164


+4g (0 mg/kg).


Th


e


AIN-76A and AIN-9


3G


diets from study


were analyzed for pyridoxine


PN) concentrations


at the conclusion of the study.


each


intended level of


fortification


,pyridoxine


concentrations we


re consistently high


er in


AIN-76A diets


than


in the AIN-93G diets.


The PN content of AIN-76A and AIN-93G


A I* A -a A


a


/!r ,AI


a


-A L/


-A 2/I


II7' . JLfl *J fl f'It LJ , O2 2 ri Ii . rw *j* ,,~r% Id


Sa


A P


(P<


7


0


.5


222


the


2


At


Aic


O


s


ama


n


-Jin+n /;., .sam.,+R


/I \


-


- 4 ~~ I-~ , 4 4 ~~ -~ s


6 g


~


nan.





29


significantly increased with


increasing concentrations of dietary RN in rats


fed either AIN-


76A or -93G diets


P<0.o001


Th


e


regression lines


describing the


weight gain of rats


fed AIN-76A or AIN-93G


as a function of dietary RN did not have


significantly duff


erent


slopes


Figure


2-


),


but did


exhibit differen


y-intercepts.


The y-intercept of the


regression line


describing weight gain was significantly higher


P<0.o01


for rats


fed AIN-


93G than the


y-intercept of the regression line of the rats fed the AIN-76A diet


53.5


.7 g).


300


200100



0-


0


A AIN-76A


A


1


2


Al N-93G


3


Dietary PN (mg/kg)


Figure 2-1 and AIN-9


Lin


ear regression analyses


of weigh


gain o


3G with different concentrations of pyridoxin


e


rats PN)


fed AIN-76A


after


5


wk.


The


apes


op


of the


e=36.4.


lines


The


were not


significan


y-intercepts a


the


ti


y differen


regression


(P= 0.7322);:


lines


were signif


pooled


C


antly


differen


(P<


0.001).


n study 1


increases


n dietary PN con


centration


mproved vitamin B-6 nutritional


status


among the different groups of rats.


Con


centrations of PLP


in liver


and plasma


study 1 w


ere normally distributed with equal variance.


PLP concentrations in p


asma


'1


35


vs.


0)
C
*
(U 0) .0-a
SC 0)
*
ci)


si sI


in






30


Table 2-1.


Indicators of vitamin B-6 nutritional status


toward PNG


n th


e


sma


ntestine


of rats after


and mucosal hydrolytic activity


cons


uming pyridox


ne


defined


diets for


Dietary
Vitamin B-6 (mg PN/kg
diet)2


7 mg/kg
7 mg/kg pf1
1 mg/kg 0.5 mg/kg 0 mg/kg Values are m


Vitamin B-6 Nutritional Status


Plasma PLP


nmol/L


1


352


726 229 165


eans


+


59 a


Liver PLP


nmol/g


6
5


+92a
-34'


+


45 12bc
SEM. M


.3


0.3a


.6 1


13.0


1


9.7 1


9.2 1.


eans


within a


C


.5ab .2"


PNG hydrolytic activity


Brus


Cytoso


nmo


PN/h*mg


protein)


1.1 0.3


0.7 0.5


a


h border


membrane


nmo


PN/h*mg


protein)


.2


+0.2 +0.2


0.4 0


.2


0.9 0.4


olumn with unlike


20.7 17.8 16.7


a


13.2


letters were


+0.6a
1.2 2.+
+0.7ab


1.4a


found to be


statistically differe


Abbreviations used:


nt at P

PNG, pyrid


oxine


glucoside; PN,


pyridoxine;


pi


,pair fed;n PLP


pyridoxal


-5


'-phosphate


both AIN-76A and AIN-93G purified diets significantly in


C


reased


P

in response


to increasing concentrations of PN


in the diets.


Plasma PLP concentrations


fed the AIN-.76A or A concentration as the


hIN-93G diet


slopes of the


increased similarly with


increasing dietary PN


lines were found to not be statisti


C


ally differen


-0.54).


Similarly


the y-intercepts of th


e


regression lines for plasma PLP


concentrations


were also not statistically different (P


were not statistically diff


from th


e


e


rent between the


parallel slopes of the regression lines


regression line for liver


PLP concentrations of


=0.75).


Liver PLP concentrations


groups fed the two basal diets


(P


=0.6793).


Th


e


as interpreted


y-intercept for the


the rats fed AIN-76A was


significantly


highe


ri(P


-0.009) than the y-intercept for rats fed the AIN-93G diet


Figure


5


wk.


2


(P


of


th


e


rats


2


-2


).









the brush border


membrane fraction was significantly greater


in rats that were pair-fed


the 7 mg/kg PN than rats fed the


7 mg/kg and 0 mg/kg diets


ad libitum


Th


ere was


effect


0


dietary PN concentration or pair-feeding on PNG hydrolytic specific activity


meas


ured in the


0


ytosolic fraction (P


=0.35).


Enzymatic activity data from study 1 w


e


re used to make further


comparisons


among the


intestinal mucosa


subcellular fractions


(crude homogenate,


cytoso


total and


brush border membrane) within the different


dietary PN con


centrations


Figure


2-3).


With PNG hydrolyt


ic


activity expressed on the


basis of mucosal weight,


th


e


s


uboellular


distribution a1


activity


could be


assessed.


Although dietary PN concentration did not


affect the


subcellular distribution of PNG hydrolysis,


the activity measured in the total


and brush border membrane


fraction


s


was


sign


icantly greater than that measured


cytosolic fractions


(P

Recovery of brush border


membran


e


from the


crude


homogenate was ~45%


as measured by lactase activity


After adjusting PNG hydrolytic


activity for its respective


recovery, brush border membrane


acco


unted for 50-60% o


total PNG hydrolytic activity m


easu red


in intestinal mu


cosa.


while


C


ytosolic activity


contributed 10


%/


of


the


total PNG hydrolytic activity.


n study


2,


PNG hydrolytic activity


in the


cytosolic fraction sign


ican


ti


y


ncreased


with


increasing dietary PN


in rats


fed th


e


AIN


-76A


P=0.02), but not the AIN-93G


O .6547).


PNG hydrolytic activity m


easured


in the total membrane


fractions was


influe


nced by dietary PN in rats


significantly different from


zero


).


fed either basal diet (regression line


The


slopes that were not


slopes of the regression lines describing the PNG


hydrolytic activity in the


cytosol


ic


and total membran


e


fractions


were not


significantly


*'rv . r I A I** ~YflA A *** flflr~ ~ 1r r% A'


3


1


no


In


the


(P=


diet


not














0~
-J
0~
0
0)
0
-J


-J
0
2
C


3-


2-


B


-
0)
0
E
C 0~
-J
a-


6


T


0


15fO1


1







0


I


3


6O-


Deday PN (makg)


0
E
C


a
-J
a-


5040-


A


Ak


1


2


30


3


I


I


20t 100-


-15


Figure


2-2.


Linear regression analys


es of pyridoxal-5'-phosphate (PLP


concentrations


in rats fed AIN-


76A and AIN-93G.


(A


Plasma PLP concentrations were log transformed (in


set) for analysis.


Regression lines for plasma PLP concentrations did not have signif


icanti


y differen


si


ope


(P=O


.5397)


y-intercept (P=O.74


53).


B)


Dietary PN concentrations were


log transformed (in


set)


for analy


Regression


lines


fo


r liver PLP concentrations did not have


differen


slopes (P=0.6793),


but significantly


different y-intercepts (P=O.009).


A


-J
0
2
C a
-J
a-


-1,0


LOgFc
(I


Dietary PN (mg/kg)


0


2


Dietar y PN (mg/lkg)


C


sis.










(U


E 0)
4C
z.


600500 400300 2CC0-


-:
*

* -


Crude


Cvtoso


Tota


membrane


Brush border


homogenate


membrane


Figure mucos


2


-.3


a) in th


Suboellular dist


e


small


concentrations of PN.


ribution of PNG hydrolytic activity


intestinal mucos


nmo


PN/h*g


a from rats fed AIN-93G with different


Suboellular fractions tested included:?


crud


e homog


enate


cyto


sal


, total membrane, and brush border membrane.


within a dietary con


irrespective 0 but significan


centration of PN


dietary PN concentration.


tly differen


Bars with different letters


ndicate significance at P

y -intercepts of the regression lines describing these data.


When PNG was used


as a substrate, there was a trend toward higher


specific activit


n the


total membrane fraction than in the


cytosolic fraction,


regardless of diet


formulation


AIN-76A or AIN-93G


or PN con


centration


which


is


consisten


with th


e


data


n study 1


Discussion


results reported h


ere extend our understanding o


with focus on the partial hydroly


sis


of PNG.


We


the


bioavailability o


were particularly


vitamin B-6


interested in the


subcellular localization of PNG hydrolysis and the


effect of vitamin B-6 nutritional status


on the


hydrolytic enzyme activities toward PNG


in the rat


small intestine.


a


-r


33


0 2100
C


C
,-


b


C


les


The











A


U,
>0
4-0

0
0-c 00..
a)
025
C


100-


C


A AIN-76A SAIN-93G


I -


= I
I
A


2


3


Dietary PN (mg/kg)


B


(U U,
>0 ~0

0 CUD) or
U
a) 025
C


750









C-


C


AAIN-76A AIN-93G


1


2


3


Dietary PN (mg/kg)


Figure 2-4


Reg ressi


on analyses


for PNG hydrolyti


C


activity (nmol PN/h*g


mucosa) in rats fed AIN-76A and AIN-93G.


(A)


Regression


lines


f


or PNG


hydrolytic activity


in the


cytosol


ic


fraction did not have signif


cantly different


slope


(P=0.2368),


but did exhibit signif


cantly different y-intercepts (P


<0.00 1).


Regression lin


es for PNG hydrolytic


activity in the total membrane traction


did not have


different


signifi


canti


y-intercepts


(P


y differen


slope


P=0.63


7


=0.007).


),


bu


did


exhibit


significantly


34


(B)






35


The absence of a robust effect of dietary PN on cytosolic PNG hydrolytic activity


was contrary to earlier


work done


in this laboratory which showed an in


verse


relation


ship


between dietary PN and


rats (


1


3


).


cytosolic PNG hydrolase spec


n that study (13), mucosal


ific activity toward PNG in adu


It


cytosolic PNG hydrolytic activity in rats fed a


purified diet


AIN-76A


providing 0 mg/kg RN was


twice that measured


in rats fed


mg/kg PN.


Similarly


Banks


et al.


12) reported th


e


same magnitude


of


change in


mucosal


cytosolic PNG hydrolase activity,


using PNG


as a substrat


e


in guinea


pigs fed


diets providing 0 and


3 mg/kg dietary PN.


Rats


in the presen


investigation were


chronically fed purified diets


eith


e


rAIN-93G or AIN


-76A


providing a wide range


dietary PN concentrations,


but


exhibited no


sign


ficant


ncrease


in


in vitro


cytosolic PNG


hydrolase


specific activity with decreasing dietary RN concentration.


were pair-fed an adequate


concentration of PN


7


However


rats


that


mg/kg) exhibited a mean PNG


hydrolytic activity in the brush border membrane that was


twice


that measured in rats fed


mg/kg RN ad libitum


n study 1


.This was likely to be a PN-independent effect of food


intake that might have


altered ave


rail rates of protein


synth


esis


or degradation.


The


combined data from the


two studies


indicate


a trend for in


C


reasing dietary PN


concentration to


increase PNG hydrolytic activity in not only the cytosolic fraction,


also the


brush border membrane


s


ubcellular fraction of intestinal mucosa.


Basal diet composition was speculated to have


an effect on the vitamin B-6


nutritional statu


s


and possibly


intestinal en


zy


matic activities


,which led to the comparison


AIN-76A and AIN-93G purified diets.


The predominantly starch-based carbohydrate


component o1


AIN-93G may be a more favorable energy


source for metabolically active


* a


-


p


- a .t r a ~ a a * &~.. a a n 4 L~ a a a .. 4 a .~ t) IS a a % n n *~.. a 4 a a a a 4 a a4 a - a


2


of


7


but


of









AIN-93G diets had a signi


fi


cantly high


er final body weigh


gain than rats fed AIN-76A,


regardless of PN concentration.


This


suggested that there might be an effect of basal


diet composition on the nutritional status


of


growing rats.


A recent comparison of the


AIN-76A and AIN-93G diets found only gastric hisotpathologi


the


purified diets,


weigh


cal differences


with more pathologies occurring in rats fed the AIN-76A diet


differences between rat


s


in rats fed


120).


No


fed either the AIN-93G or AIN-76A diets were detected;


how


ever


, rats examined by Lien et al.


120


were at least


3 times


larger and more


mature than the


growing (w


weaning rats used in our study.


eaning) rats respond differently to the


There is a possibility that rapidly two basal diet formulations.


Results from study


2


also


indicated that


0


hanges in mucosal PNG hydrolytic


activity in the


rat small


ntestine


are i


uenced by basal diet composition and not only


dietary PN.


Rats


fed the


AIN-93G diet had significantly greater


cytosolic and total


membrane


hydrolytic activity than rats


fed AIN-76A, regardless o


RN concentration.


How


ever.


rats


n study


2


that we


re fed AIN-76A exhibited a positive


relation


ship between


cytosolic PNG hydrolase activity and dietary PN concentration.


In


contrast. this effect


was absent in the


rats fed th


e


AIN-93G.


These results


indicate that the


in vitro


hydrolysis o


PNG by rat intestinal mu


cosa


is actually increased with in


C


reasing dietary


PN concentration and may be further influenced by other


dietary components that affect


mall


intestin


e


in gen


eral (e.g.


protein


s


ynthesis,


processing,


or degradation).


n the presen


study, we


were interested


in determining the


subcellular


distribution of PNG hydrolytic a composition on this distribution


activity in


intestinal mucosa and potential effects of diet


Our laboratory has recently discove


red that the


- - * I I I I . S -a a * I i I ~ I * ,


36


the


s









relative


to mucosal weight.


The greatest PNG hydrolytic activity was detected in the


crude homogenate,


which is comprised a-


all


suboellular fractions.


Activity m


easured


total and brush border membrane fractions was


greater than the


activity measured


cytosolic regardless o


dietary PN concentration.


Since our isolation of brush border


membrane


was


incomplete,


we adjusted the PNG hydrolytic activity in the


brush border


membrane


to reflect 100%


recove


ry


e


found that 50-60% hydrolytic activity toward


PNG


n th


e


intestinal mucosa


was localized to the


brush border


membrane of rat


intestinal mucosa.


membran


e


Si


milarly, we found that nearly all of the activity measured in the


fraction could be attributed to the brush border membrane.


primary function of LPH is to catal


yze


the


hydroly


ss


of


total


Although the


dietary lactose, this laboratory


found it to hydrolyze


PNG, and further


evidence


of


seco


ndary


substrates for LPH


been recently reported (1


The


results from the


presen


study


along with our recen


kinetic analysis


of LPH-


catalyzed PNG hydrolysis


106) and partial characterization of th


e


cytosolic PNG


hydrolase


14),


we hay


e


constructed a model to


explain th


e


ntestinal absorption and


processing of dietary PNG


not be absorbed at all


and


A fraction of the


conseque


ntly


PNG that enters the


may be excreted


intestinal


in the feces.


umen may


PNG also may


be absorbed


as the intact glucoside and


excreted in the urine


unchanged or undergo


ntracellular hydrolysis by cytosolic PNG hydrolase, which would release free PN.


Alternatively


PNG could be hydrolyzed at the brush border membrane by LPH and


absorbed


as free PN and glu


case.


In addition to the partial hydrolysis


of PNG that


occurs


in the intestine


there is


evidence


of hydrolysis


in othe


r organs.


An i


nvestigation


the


37


th


e


In


In


has


has


21).


. W









vitro


assay


s of PNG hydrolytic activity found that rat kidney, along with the intestinal


mucosa


,was able to partially hydrol


yze


PNG.


Overall, these data have advanced our


understanding of the absorption and metabolism of PNG.


38














CHAPTER 3


ENZYMATIC HYDROLYSIS OF PYRIDO


XINE-5'-J3-D-GLUCOSIDE IS CATALYZED BY


INTESTINAL LACTASE-PHLORIZIN HYDROLASE Introduction


A significan


dietary


source of vitamin B-6 for humans is provided by


glycos


ylated form of the vitamin, pyridoxine-5'


-j-D-glucoside


PNG).


PNG, found


only in


foods of plan


origin,


provides a mean o1


1


5%


of


total vitamin B-6 in a mixed diet,


depending on food selection


3


0,64).


PNG


exhibits an approximate 50% bioavailability


in humans


(10,11


and


25-30%


in rats


(6-9) relative


to pyridoxine, the


metabolically


usable form


of


vitamin B-6.


The rate-limiting step


in the


utilization


of


PNG is not its


intestinal absorption,


but rather the


enzy


matic


hydrolysis


of


th


e


5-glucosidic linkage to


release glucose and pyridoxine in the


intact


intestine (6-8).


, this form is not metabolized or retained by the


Although PNG can be absorbed liver (6-8) and can antagonize the


metabolism of nonglycosy


ated forms


of vitamin B-6


5


,63).


The


remainder a


PNG that


is not absorbed is likely to be accounted for through fe


cal


asses.


This laboratory has


examined th


e


intracellular


.e.


cytosolic,


hydrolysis


of PNG


mammalian


small


ntestinal mu


cosa.


Intestinal cytosolic PNG hydroly


sis was


nitially


thought to be catalyzed by a broad specificity j-glucosidase


BS


f3G) (EC


3.2.1


a cytosolic enzyme found


i


n the


intestine and liver


12,1


3,


104).


Cytosolic PNG


hydroly


ss


was


also reported to be inve


rsely related to vitamin B-6 nutritional status


a


in


.21)


66),


in









hydrolase (PNG hydrolase)(14).


It was found that PNG hydrolase, not BSB3G, was


responsible for the cytosolic


cleavage of PNG.


Partial characterization of cytosolic PNG


hydrolase revealed its


ability to hydrolyze


PNG


as well


as lactose and cellobiose


but not


sucrose


1


4) .


Partial amino acid analys


is revealed


substantial sequ


ence


homology


ignificant differences


between PNG hydrolase and the brush border m


embran


e


enzyme


lactase phlori


zi


n hydrolase (LPH) (EC


3.2.1


.23,


62


,and 108)(74,1


22),


th


e


enzy


primarily responsible for the hydrolysis


of


dietary lactose (C.W


Tseung, L.G. M


cMahon.


and J.


F. Gregory, unpublished).


A recent reassessment of mucosal PNG hydrolase activity in the


rat small


intestine


indicated that ~50%


of the


activity


asso


ciated with the total membrane fraction


could be attributed to enzymatic activity in the brush border


membrane


(A.


D. Mackey,


.unpublished).


Hydrolysis o1


glucosides and oligosaccharides by enzymes


present


intestinal brush border often precedes the


absorption o


individual monosaccharides


or the


aglycone of glycos


ylated molecules,


such as flavonoid and isoflavon


e


glucosides.


Day and colleagues


2


1) found that quercetin,


genistein,


and daidzen p-glucosides


were hydrolyzed by purified sheep LPH prior to absorption.


brush border f-glucosidase; thus,


LPH is the


we hypothesize that LPH is responsible


only mammalian ble for the


hydroly


ss


of PNG, a p-gluooside.


LPH has two distinct catalytic active


sites:


one for the


hydrolysis


of


lactose and flavonoid glucosides and another,


phiori


zi


n hydrolase, for the


hydrolysis o1


phiori


zi


n and B3-glucosylceramides


(75,


123).


As reported by Day et al.


site o


catalysis


fo


r flavonoid and isoflavonoid glucoside hydroly


ss


was


experimentally


assigned to the


lactase active


site. not the


phlorizin hydrolase site.


40


S


but


me


al


the


et


In


1


21),


the






41


catalytic properties.


We also report the in vitro formation of a P N-disaccharide, which is


evidence


of a transfe


rase activity for mammalian LPH.


Materia


Is


and Methods


Materials.


Pyridoxin


e


PN)


hydrochloride,


lactose


, glu


cose


,phioridizin


(phiorizin


),


phloretin,


Sephacry


S400, Sephacry


S200


2'


,4'-dinitropheny


1-2


-f Iuoro-2-


deoxy-1-D-glucopyranoside (2F-DNPGlc),


D -glucal,


protease


nhibitor cocktail, goat-anti-


rabbit IgG hors


eradish peroxidase antibody,


and a glucose detection kit were obtained


from


Sigma chemical Co.


(St.


Louis


,MO).


Prestained protein molecular mass


markers


were obtained from


nvitrog


en (Carlsbad,


CA


).


DEAE cellulose was obtained from


Whatman


Clifton


NJ


),


and Simply Blu


e


SafeStain was


purchased from


nvitrogen


(Carlsbad,


from Pierce


CA).


Maleimide-activated keyhole limpet hemocyanin


(Rockford,


IL) .


Polyvinyldifluoride (PVDF


KLH


I mmobilon-P


was obtained


was obtained from


Millipore Corporation


Bedford


,MA).


ECL Plus


chemiluminescent reagen


was


purchased from Amersham-Pharmacia Biotech (Buckinghamshire,


England).


Pyridoxine-5'-j3-D-glucoside was


prepared by biological


s


ynth


esis from pyridoxin


e


using


germinating alfalfa seeds and purified chromatographically (1

Purification of lactase phiorizin hydrolase (LPH).


3


,11 0).


LPH was purified according


to the


Triton


X-1 00 solubilization method of Wacker et al.


7


5) with the exceptions


phacryl S200 substituted for Sephadex G-200 and omission of the


final anti-lactase


affinity purify


cation column.


During this purification,


all materials were kept on ice or at 4


Brush border membrane


was isolated from rat


ntestine for the purification of LPH


according to the method Kessler


et al.


111) with


some modifications


12).


W


eanling


Se


of


C.








total mucosa.


Electrophoretic characterization of the purified enzyme was done


under denaturing conditions using


8%


w/v) polyacrylamide gels according to Laemmli


using a mmn


-gel electrophoresis apparatus


from Novex (Invitrogen;


Carlsbad


,CA).


A commercially available prestained protein molecular mass standard ranging from 10-


173 kD was used.


Gels were stain


ed with Simply Blue


Sa


feStain for 1 h followed by


destaining overnight in distilled,


deionized water.


Antibody production and Western blot analysis.


Th


e


peptide


CTLFHFDLPQALEDQG was


sy


nthesized with an additional


cysteine


at th


e


carboxyl-


terminus


and veri


ified by mass


spectrometry amino acid analy


ss


by Research


Ge


netics


(Huntsville,


AL).


This pe


ptide was conjugated at the


terminally added cysteine residue


to maleimide-activated keyhole limpet hemocyanin


KLH) according to manufacturer


instructions.


KLH-conjugated peptide was injected


(Cocalico Biologicals,


mmunoblotting,


Reamstown


or Western analyses,


into N


() for production o1 were done as de


Jew Zealand White Rabbits polyclonal antiserum.


scribed by Harlow and Lane


(125).


Purified LPH protein was resolved on an 8%


The gel was


w/v


electroblotted to polyvinyldifluoride (PVDF) for


SDS-PAGE at 130 V for


2


2


.5 h at 12V in a transfer


buffer


containing 192 mmol/L glyc


ine.


25 mmol/L Tris/ 0.05% SDS/1 0%


methanol.


The


blot was


stain


ed with Amido black and destained.


For the


protein detection,


the stained


blot was


cut i


nto strips for different antisera treatm


ents.


Primary antibody treatments


included whole immune


serum


pre-immune


serum


and peptide-competed serum.


The


peptide-competed


serum was prepared by


incubating the


whole immune


serum


with 10 jg of peptide at room temperature for 1 h prior to application to the


blot.


The


- I......rI...........I I.... 'r-%I-~n--' ,jn ,,' a. am'~


go01


1


(


24)


42


h.


0


.5


mL)






43


addition of the secondary antibody (goat anti-rabbit IgG horseradish peroxidase


conjugate


at 1:5000 dilution.


Unbound


seco


ndary antibody was removed by washing


with PBST


no NFDM).


A chemiluminescent/fluorescent substrate (ECL-Plu


s)


was


then


applied and incubated at room temperature for


5


mn.


The


excess


substrate was


blotted


away and protein bands were visualized by fluores


cence


emission (Storm 840


rescent


optical scanner;


Am


e


rsham-Pharmacia Biotech;


Sunnyvale,


CA).


Enzyme activity assays.


The


standard assay for PNG hydrolytic activity was


erformed according to Nakano and Gregory


13).


Standard activity


assays


for PNG


hydrolysis we


re performed in a reaction mixture containing 10 tg purified LPH, 0.25


mmol/L PNG in 80 mmol/L sodium ph


osphate, pH 6.0 that was


incubated at


37


C0


fori1


Pyridoxin


e


release was measured u


sing reve


rse-phase HPLC with fluorometric


detection (110).


Lactase activity was measured according to Dahlqvist (96) with


modification of the 50 mmol/L maleate buffer to a 50 mmol/L sodium phosphate,


pH 6.0


assay buffer.


Standard activity


assays


for lactose and other


disaccharide hydrolys


were done


using


25 mmol/L disaccharide.


Glucose release was quantified using a


reagent kit based on the glu


cose


oxidase reaction with colorimetric


detection.


Phlor


hydrolase activity was


meas


ured by reverse


phase


HPLC with UV detection of the


aglycone,


phioretin


126).


Standard


assa


y mixtures contained 100 mmol/L phiorizin


50 mmol/L sodium


citrate, pH


5.9.


All activity assays


were conducted under


conditions


that allowed measuremen


of


nitial rate.


For kinetic studies.


reactions


nvolving LPH-catalyzed hydrolys


is of


PNG and


lactose were conducted at various con


centrations


as specified


in text or tables.


fiuo


p


h.


is


zin


in






44


Vmax, and K,) were calculated by nonlinear regression using SigmaPlot Enzym


e


Kinetics


Module


,v 1.0 (SP


SS5


no.


San Rafael.


CA


software.


Protein concentration was determined by a colorimetric method (114


using


bovine


serum albumin


as the standard.


Liquid chromatography-mass spectrometry (LC-MS).


mass


To obtain molecular


and fragmentation information regarding a novel reaction product formed during


incubation of purified LPH with PNG and lactose,


LC-MS


and LC-


MS/MS


anai


yses


were


performed in atmospheric pressure chemical ionization (APCI) mode (LC:


Hewlett


Packard model 1100; Agilent; Palo Alto,


CA and MS:


Thermo Finnigan model TSQ7000;


n Jose


,CA).


The


HPLC separation was


done isocratically with 5 mmol/L ammonium


acetate with 0.1


v/v


acetic a


old as th


e


mobile phase


at a flow rate of 0.6 mL/min with


a reverse-phase


column (018 Ph


e


nosphere,


50


x 4.6 mm


5 jm column


Ph


enomenex;


Torrance.


CA


).


The


analy


s


is was conducted by monitoring ions from 75-600 m/z.


the MS-MS


analys


is.


a collision ene


rgy of 30 V was


used to fragm


ent


th


e


parent mass


(M+1


of m/e


494.


Results


Lactase phiorizin hydrolase (LPH) purification.


LPH from rat


small


ntestinal


mucosa


was


purified 300-fold relative


to the


lactase activity measured in mucosal


crude


homogenate.


The enzyme was


purified to near homogeneity and the purification


repeated with similar results.


The approximate molecular mass o


purified LPH by SDS-


polyacrylamide gei electrophoresis was


35-


140 kDa.


ln th


e


published method used for


purif


cation


,an anti-lactase antibody affinity column was used to further separate LPH


Sa


For






45


reported in the purification method


75). Staining of the SDS-PAGE revealed two major


bands o1


protein:


one at


~135-140 kD and another at ~200-220 kD (Figure


band of protein at 1


35-


140 kD corresponds to mature LPH.


The band at 200-220 kD is


consistent


with th


e


MW of


a precursor form of LPH (91).


West


ern analyses


were done


confirm furth


e


r the relative


purity of the enzym


e


preparation


Figure


3-1B).


Th


e


peptide


used for antibody production had amino acid sequence


homology with regions


precursor and mature forms of LPH, but no other mammalian


intestinal


enzy


mes


catalog


ed


in


Swiss-Prot and GenBank databases.


No protein bands were detected


in the


blot strips


incubated with PBST-NFDM alone and the secondary antibody alon


e


(lanes


respectively).


The


whole immune


serum-treated strip revealed three


immunogenic protein bands (lane


3) ,


which were not visible


n the peptide-competed


antibody treatment


(lane


The


fourth visible band


~-60 kD) was


found to be an artifact


the antibody under the condition


s


of this analysis.


The


band at -80 kD detected by


whole immune


serum was


also detected by the


pre-immune serum,


which


indicates


that the


recognition of the band by the whole immune


serum was not sequence specif


protein bands at


35


-140 kD and 220-220 kD that were recognized by this


polyclonal antibody appeared to be the mature and precursor forms of LPH.


closer inspection of the Coomassie-stained gel,


Upon


trace amounts of additional high


molecular mass bands were observed.


Further assays


for disacoharidase activity of


purified LPH preparation revealed residual maltase-giucoamylase (EC


3


.2.


1.20 and 3) at


a specific activity of 3.8 pmol/min*mg protein.


This activity was controlled for


in later


kinetic characterization studies.


3 -


1


A).


Th


e


to


n th


e


and


2


of


*1


th


e


(Ian


e


5).


Th


e


ic


the









Kinetic characterization.


Purified LPH from rat small


intestinal mucosa


catalyzed the hydrolysis o-


PNG and followed Miohaelis-Menten kinetics.


Values


for Kin,


Vmnax, and kcat were lower


when PNG was used as the substrate as compared to lactose.


Values for kcat and Vmax/Km indicated that lactose hydrolysis was catalyzed more


efficiently than the hydrolysi


s


of


PNG


Table 3-1).


20 jg LPH


marker lane


173 1 11 80
___61


Figure 3-1. preparation.


Purification of LPH from rat intestine.


Gel


(8%


w/v acrylamide


has two


A)


anes:


SDS-PAGE of purified LPH


one


arge lane containing


enzyme preparation and another for the molecular mass


marker.


Purified enzyme (20


pig) was loaded into one large well.


V for 2 hours.


Enzyme preparation was electrophoresed at 130


Gel was stained with commercially available formulation of Coomassie


blue stain (SimplyBlue SafeStain,


10-173 kDa.


nvitrogen).


Molecular weight markers ranged from


Protein band at 135-140 kDa corresponds to LPH.


of purified LPH preparation.


Protein


(B


Western analysis


20 pig) was separated by SDS-PAGE (8%


w/v


acrylamide) and transferred to PVDF membrane.


Membrane was cut into strips and


subjected to various


antibody treatments.


Lane 1


No antibody applied: Lane


Secondary antibody (goat-anti-rabbit HRP) only; Lane 3:


secondary antibody; Lane 4:


Whole immune serum and


Peptide-competed serum and secondary antibody;


Lane 5:


Pre-immune sera and secondary antibody.


Application of


- --


46


A


l


B


1I


2


3


4


5


4<


V


173 11


80


61


>'<


$7
K$> <4
K


4/


2 .






47


Table 3-1


Kinetic parameters of purified rat lactase phiorizin hydro


lase


(LPH) using


pyridoxine-5'-fS-D-glucoside


PNG


and lactose as substrates.


Km


mmol/L


V max
(pmol/min *mg LPH)

kcat (s"1)


kcat/Km


mmo


I/


I


L


-1


s>4)


Vmax/Km


Substrate


PNG


1 .0 0.1


0.11


Lactose


1


5.0


+0.01


1.o


1.0


0.11


6


1


+0.1


47


2


.9


0.3


1


Inhibition studies.


The substrate PNG


exhibited


s


ubstrate inhibition at


concentrations greater than


3


mmol/L


Figure


3-2).


Due to limited availability of PNG,


only concentrations ranging from 0.01 Km to 5Km we


re tested.


The


reaction products


(giu


cose


properties.


and pyridoxine),


Pyridoxine


(0


lactose


and other


.5-10 mmol/L


did not


disaccharides were also tested for inhibitory


nhibit LPH-catalyzed PNG hydroly


sis,


but the monosaccharide


, glu


cose


inhibited the


reaction at concentrations


25 mmol/L


equal to 64


+5


mmol/L.


The


primary


substrate for LPH, lactose


25


-100 mmol/L),


nhibited PNG hydroly


sis


competitively with a Ki equal to 56


8 mmol/L (Figure


This


suggests that the two substrates


PNG and


actose) are hydrolyzed at the


same


active


site.


Cellobiose


maltose


lactose


sucrose


,and trehalose (2


5-


100 mmol/L


were


3


-3).


; Ki











U PURIFIED LACTASE


U


U


O MUCOSAL BRUSH BORDER

U


0


5


6


[Pyridoxi ne-5'-3-D-gI ucoside3, (mM)


Figure


3-2.


Effect o1


substrate concentration on the activity of LPH-catalyzed PNG


hydrolysis. is shown.


Comparison of rat small intestin


maltose was reduced by 35-40%


in the


e


brush border m


presence o1


embrane and purified LPH


2.0 gmol/L 1 -deoxynojirimycin,


an


nhibitor o


maltase-glucoamylase activity


Identification of site of PNG hydrolys


is


by LPH.


The


nhibitor


2F-DNPGlc


binds to both the lactase and phior


zi


n hydrolase


active


sites


inhibiting both hydrolytic


activities


*1


28


,129).


The


lactase activity was


preserved by pre-inoubation with D-glucal,


which yields


rever


sible blockage o


the


lactase active


site


1


23).


Subsequ


e


nt addition of


2F-DNPGlc to th


e


glucal-modified LPH leads to the


selective


nhibition of the


phiori


hydrolase active


s


ite


123).


This experim


e


ntal protocol allowed the


contribution


of


each


activity from the respective activ


e


sites to be calculated


1


2


3)


Hydrolysis


of


PNG and


lactose was


reduced


00-fold in the presence


of 2F-DNPGlc without


glucal


as compared


to the uninhibited


enzyme.


Phioretin release was reduced by 85-90%


in the


p


resence


250


48


U


U


U


C
0
L...
a


0~

C


200 150 100 50


U


U


0


0


0


0


0


1


2


3


4


1


27).


zin


of






49


10



8



6


10


20


30


lactose lactose lactose lactose lactose


= 0mrM = 25nmM =50mM =75mrM = 100 mM


40


1/[Pyridoozdne-5'-B-D-glucosidej,


(rrM)


Figure


3


catalyze


Figure


3-4


-. q


Lineweaver-Burk and Michael


d hydrolysis of PNG with


).


These findings strongly


is


-Menten


nset


increasing amounts a


lad


plots of LPHlose as inhibitor.


suggest that PNG was hydrolyzed at the


lactase


active


site of LPH.


Formation of other pyridoxine glycosides.


HPLC analyses


of


enzyme


reaction mixtures containing LPH, PNG


s


ubstrat


e


),


and lactose (inhibitor) detected not


only the product, PN, and the


s


ubstrate


PNG.


but also anothe


r fluorescent compound


- * -L


r t, 1r:.....


,-t r\


'AITrl ~z2 rLaTt3rflI~~r1 Tirno fAr ~ 4 mm 1-mnE *rn .a.-%' T kn a .-nnn mt rS tkc' r'r\rnnr,,u irirJ fr~rrrrirJ tAa r


300
U
ase


C


4


2 -


-10


0






50


1.0


0.8 0.6 0.4 0.2


0.0


inhibitor


I


glucal+inhibitor


no inhibitor


Figure 3-4.


Identi


ication of


site of PNG hydrolys


is


by LPH.


Protection of


active site with D-glucal prior to addition of 2F-DNPGlc (inhibitor


ectively inhibited phiori


zin hydrolase


active site.


Completely


nhibited


enzyme


with 2F-DNPGlc


exhibited 10%


the PNG hydrolyti


C


activity o


glucal-protected enzyme.


further supported by an MS/MS


experiment


on the


M+1 ion using a collision


ene


rgyo0


30V


In addition to the residual M+1 ion of th


observed at m/z and pyridoxine.


e


disaccharide, major fragments were


332 and 170, corresponding to the M+1 ions


The LC-MS and MS/MS


of pyridoxine glucoside


experiments produced the same results when


HPLC purified disaccharide from the enzyme reaction mixture was utilized.


This


pyridoxine disaccharide has a pyridoxine


molecule with the


5'- 5-D-gucoside and another


case


or galactose moiety.


Si


milar to the observation of the pyridoxine disaccharide


formed


in the presence


of lactose, two other disaccharides tested in the LPH


nhibition


experiments, cellobiose and maltose,


also formed fluorescent compounds which


exhibited retention times different from that formed with lactose.


These


compounds,


while detectable


were minor in relation to the amount of the PN-disaccharide formed


iL.-----------------------


I----1--~- ----A ------


I - -- - -- -I - -- ---------------.2 -t - - - --


I rio F' roL'or1~n rn 'Z2 flTflESfl 'z, flr ~Rffl ro I flflfl.flflflflfl.nT rn ci in'z~ nr'n 'T~ rmcin nnnnnnTrflTtnn


r
0
E
C
z a-


sel


th


e


the


glu


in


-




















10


Minutes


PN-disaccharide


10


Minutes


Figu


re 3-5.


Sa


mp


e


chromatogram


s


of


enzym


e


reac


tion mixtures of LPH-


catalyze hyd rolys hydrolys


d PNG hydrolysis.


is without added


is with added


min; pyridoxine-


5'-


A), Chromatogram from LPH


actose.


actose


B3-D-gluco


s


(25 ide (


-catalyzed PNG


(B), Chromatogram from LPH-cataly


mM).
PNG),


Retention times:


pyridoxine


zed PNG


PN),


4.3 min; pyridoxine disaccharide


3.3
PN-


disa


ocharide),


5.3


mn.


Assays


were conducted using purified rat LPH


growing evidence that LPH catalyzes


the


hydrolysis of not only lactose,


but oth


glucosides,


such


as plan


J-glucans


and flavonoid and isoflavonoid glucosides


21,130).


Dietary PNG


exhibits


only 50-60%


of


the


bioavailability of free PN


as a


source


of


vitamin B-6 for humans


10,11), which is due to incomplete hydrolysis


of the


glycosidic linkage of PNG.


The results reported


n the present study


suggest that the


51


PNG


A


1000 -


4,,
S


0-


0


PN


5


B


1woo


a
S


0


PNG


PN


0


5


1


(


e


r1p-











RT 5 37


is3


0 12 34 56 7 81 011


C)
0
C Co
-o
C
n
~0
C)
Co
0)


100;

90852 80~ 752 701 651 601552 502 45i 40 35~ 30-


332


170


314


1 52


494


251


2W
15< 108 10-, 136
5 296
O


476


100 150 200 250 300 350 400 450 500


Time


(mn)


LC-MS/MS


on the ion at m/e 494;


analysis


collision


in the


ene


rgy,


APCO 30 V


positive ion mode


),


during the incubation of LPH with PNG and lactose.


(MS/MS


experiment


of the PN-disaccharide product formed


The predominant ions are


paren other


mol


s


ecular


ignificant


on (M+1


Ions


at m/e


at m/e


476,


332,


314


and M+1 ion of pyridoxine at m/e 170.


andi1


52


are produced by the


loss


of


water


from the


respective M+1 ions.


absorption into the cytosolic compartment by PNG hydrolase.


This LPH-catalyzed


hydrolysis may be an


important determinant of the bioavailability of PNG


n populations


with lactase def i


cieno


y, the hydrolysis,


and h


ence


,nutritional utilization of PNG may be


less efficien


than


n population


s


that exhibit lactase persistence.


LPH was purified from 20d old rats to adequate amount of starting tissue. The rec


insure reasonable lactase activity with an


Ignition of both the mature and precursor


forms of LPH by our antibody in the


Western analysis allnwnrl us


tn xinrifv th


52


Figure 3-6


m/z


The


p


nualitation








(lactose


calculated for purified rat LPH were consistent with published values


73


126)(Table


representative


3-


).


Th


e


kinetic parameters


or lactose and PNG) appeared to be


of the concentrations of substrates that would be


encountered


physiologically by LPH.


However.


under


typical dietary conditions,


the amount


of PNG


digesta


entering the small


ntestine


would be in the micromolar


concentration range,


while the concentration of lactose


millimolar range) would greatly exceed that of PNG.


The


relatively high Km value


for PNG (1.0 mmol/L


suggests


that LPH has th


e


capacity to


hydrolyze


physiological concentrations of PNG, but fractional saturation of substrate of


enzyme


would be low.


Similarly


kinetic ratios


also


indicated that LPH favored th


hydrolys


s


of


lactose over PNG.


Although LPH-catalyzed PNG hydrolysis was


nhibited by one


of its


products,


cose


the


concentration at which


nhibition occurred may not be


physiologically


relevant (


25


mmol/L


).


n accounting for the stoichiometry of the hydrolytic reaction of


PNG


the


amount of glu


case


released (micromolar con


centration) would not be


to cause


product


inhibition.


However, the glucose released from the hydrolysis


of


other


disaccharides


such


as lactose, may contribute to the observed inhibition by glu


case.


The observed competitive


nhibition of lactose on LPH-catalyzed PNG hydrolysis is


consistent


The


with the


fact that PNG and lactose are hydrolyzed at the


nhibition may not have


a dramatic effect on the


same active


s


bioavailability of PNG since


;ite. foods


that contain PNG are of plant origin,


which are devoid of lactose.


Unless a dairy product


and plant-derived food were


consumed together


PNG


should be at least partially


hydrolyzed by LPH.


The Ki for lactose (56 8 mmol/L


did not agree with the


cal


culated


It i,-~r l.~n+t%1rr% (IC -1- 1 rnrr%,%I/I % I~' n r.


53


in


the


glu


e


s


uff


C


ient






54


liberate PN, which may undergo enzymatic modification to form PNG or PN-


disaccharide.


The m


echanism o-


the


hydrolysis/tran


s-glycos


ylation was


not examined


further


in this study.


Cellobiose


,while not present


in apprec


able amounts in the


human diet.


also is a


substrate for LPH (74,1


31); th


eref ore


, it was not unexpected that this 1-linked


disaccharide would also


nhibit PNG hydrolysis.


The 1-disaccharide,


maltose also


nhibited the release of PN by LPH, which was


not anticipated.


The subsequent


detection o


maltase-glucoamylase activity (EC


3.2.


1 .20 and 3) (80)


as a contaminant


the LPH preparation is consistent with the presence


of minor high molecular mass


protein bands


(200-220 kDa) faintly stained


n the


SDS-PAGE.


Western analy


ss


using


our antibody that was sequence specific to LPH, but not maltase-glucoamy


lase


s


howed


that the more prominently stained high molecular mass band was


Ii


kely to be a precursor


form of LPH and not maltase-glucoamylase.


Despite th


e


relatively low abundance of the


maltase-glucoamylase contaminant,


its hydrolytic activity toward maltose had an effect


on LPH-cataly


zed PNG hydrolysis.


What appeared to be


the


inhibition o1


PN release,


may actually have


been the


LPH-catalyzed formation of another maltose-


or glu


cose-


derived pyridoxine oligosaccharide from PN or PNG,


or both.


Alternatively, th


hydrolysis


of


maltose releases monosaccharide units


(D-glucose) which could


inhibit


PNG hydrolysis


as catalyzed by LPH.


Maltase-glucoamylase activity was reduced by


63%


in the


presence of 2.0 pM 1 -deoxynojirimycin without affecting lactose hydrolys


In reaction mixtures


containing LPH,


PNG, maltose


( 25


mmol/L


),


and deoxynojirimycin,


release of PN was increased by an average of 35%


and the formation o1


other


of


e


the


is.






55


competitive


nhibitor of lactase


75).


Although maltase-glucoamylase was present in th


purified LPH preparation,


maltase-glucoamylase appears not to alter the


kinetics of PNG


hydrolysis


in the absence


nhibition experim


e


of f3-linked disaccharides.


nt with B3-glucosidase


nhibited with this fluoroglucoside,


This is further supported by the


nhibitor 2F-DNPGlo.


the release o


PN is negligible


When LPH is completely le, indicating that maltase-


glucoamylase alon


been


e


is not capable of hydrolyzing PNG.


Maltase-glucoamylase that had


co-purified with lactase by papain solubilization from 1 5d old rat intestine


was


reported to have


wh


a final specific activity of 36 pImoI/min*mg protein in another study


ch is approximately 10 times


th


e


specific activity measured in the current study.


Fluoroglucosides are widely used


as general


nhibitors o1


B-glucosidases


129).


,4'-dintrophenyl-2-deoxy-2-fluoro-J3-D-gluoopyranoside (2F-DNPGlc) is a mec


hanism-


based


nhibitor of j-glucosidases,


linkage of the


and


exerts its effect by forming a stable covalen1


2' position of the fluoroglucoside to glutamic acid residues at the


active


sites of LPH.


The use of glucal in conjunction with the inhibitor


in the


protocol allows


ndividual examination of the


lactase and phlori


zi


n hydrolase active


sites


on LPH (1


The d same


leglycos


ylation of flavonoid and isoflavonoid glucosides was


experimental procedure


1


21).


studied using this


nhibition of the phlorizin hydrolase site with


2F-


DNPGlc did not diminish the


hydrolysis of the quercetin glucosides.


Similar


to the


results reported for the


flavonoid and isoflavonoid gluco


s


ides


1


21


),


we concluded that


PNG is hydrolyzed at th


e


lactase active


site of LPH.


Variou


derivatives


s


pyridoxine glucosides,


,are known to exist widely


ncluding


in nature


seve


1


32);


ral pyridoxi however,


ne


oligosaccharide


the formation of


e


2'


73),


23).






56


PNG, with particular attention to the vitamin in human LPH expression and activity and the inhibition of PNG hydrolysis by lactose.














CHAPTER 4


UPTAKE, HYDROLYSIS, AND METABOLISM OF PYRIDO


Xl


N


IN A CELL CULTURE MODEL USING CACO-2 Introduction


E-5'-3 -D-GLUCOSIDE CELLS


An important


source


vitamin B-6 in the human diet is pyridoxine-


5'-3-D-


coside(


PNG) wh


ch is fo


und in foods of plan


origin.


This gly


cosy


lated form provides


approximately 1


5%


of


total vitamin B-6 intake


n a typical mixed diet; howeve


r. this


percentage can vary depending on food selection


30).


The metabolic utilization o


form of vitamin B-6 is limited by its partial hydrolysis


by B3-glucosidases in the


small


ntestine.


Hydrolysis


of PNG yields the products glu


cose


and PN.


PN can be


metaboli


zed to the coenzyme PLP


.Relative


to PN


PNG


exhibits a 50%


bioavailability in


humans


10,11) and 25-30%


n rats (7,8).


The


in


complete


bioavailability of PNG is due


to its


limited hydrolysis


in the small


ntestine.


This laboratory previously reported the


ntestinal hydroly


ss


of PNG to be catalyzed by a novel


ntracellular f-glucosidase


The en


zyme


spe


cifically hydrolyzed PNG and was


later designated cytosolic pyridoxine-


5'-j3-D-gfucoside hydrolase (PNG hydrolase


).


More rece


ntly


this laboratory discovered


that the


brush border membrane B3-glucosidase,


lactase phiorizin hydrolase (LPH


),


also


catalyzed the


hydrolysis


of PNG


106).


LPH is the intestinal en


lactose


zyme that is responsible for the hydrolysis


,a disaccharide that is important for energy derivation for dev


of dietary


eloping mammals.


glu


this


14).






58


PNG hydrolysis and therefore, the two substrates are hydrolyzed at the same active site.


spec


ulated that the


inhibition by lactose observed


in vitro would have implications on


in vivo hydrolytic and absorptive


processes


of PNG.


ntestinal hydrolysis


of


PNG


vivo might be reduced


in the presence


of lactose.


which would reduce


the bioavailability


of PNG.


decrease the


Simultaneous consumption of plant-derived foods with dairy products could


hydrolytic release of free PN and consequently, affect the vitamin B-6


nutritional status a1


the


individual


There is also the


potential for a reduced PNG


bioavailability


n lactose


intolerant


or lactase deficien


individuals.


Subcellular


fractionation of


ntestinal mu


cosa


revealed that 50-60% a


the


hydrolysis


of


PNG was


localized to the brush border membrane


in the rat small


intestine


which was


catalyzed by LPH


Mackey et al.,


unpublished).


This accounts for a large proportion of


PNG hydrolytic activity present


in the small


intestine and thus


lactase


nsufficien


migh1


also reduce


PNG bioavailability through the


reduction or complete loss a


LPH


hydrolytic activity toward PNG.


in vivo ab


rats.


sorption of vitamin B-6 has


been extensively examined


ntestinal absorption of PN occurs by a non-saturable,


passive


in th


e


intestin


diffusion pr


e


ocess.


PN is rapidly absorbed where it is


subject to phosphorylation,


forming pyridoxine


phosphate (PNP


which can be converted to pyridoxal


5


'-ph


osphate


PLP) by PNP


oxidase


33


,134).


Following th


e


oxidation of PNP to PLP


the


intestine


dephosphorylates PLP to release PL


into portal circulation (134


).


PNG hydroly


ss


occurs


mostly in the small


ntestine to release PN, which is passively absorbed;


how


ever


,PNG,


as the


glucoside,


can be absorbed and


excreted


intact in the


urine (10,13).


We


th


e


in


the


li


k


el


y


cy


of


S'






59


While these cells hydrolase activity


express LPH,


(M


they were previously found not to exhibit cytosolic PNG


oMahon, L.G & Gregory, J.F., unpublished data).


Hydrolytic activity


toward PNG m


easu red


brush border LPH.


in these cells could therefore be attributed solely to the


The purpose of the present study was to


examine


the


activity a1


uptake and


metabolism of PNG in the absence


and presence of


lactose


n acell


culture model using


established


Caco-2


human colon carcinoma cell line.


Materials and Methods


Materials.


Vitamin B-6 compounds (except PNG


),


lactose


HEPES


,D-glu


case,


and L-glutamine were purchased from Sigma Chemical Co.


(St.


Louis


,MO).


Cell


culture


grade


NaC


CaCl


2, Mg


504,


KOI


LICI


,hydroxymethyl


aminomethane


hydrochloride


Tris-


HCI),


2


-(-4-morphiolino


-ethan


esul


fon


Ic


acid


MES),


and plastic cell


culture


supplies


were purchased from Fisher


Scientific


Atlanta,


GA


).


Cell


culture media, media


supplements,


and trypsin-EDTA were purchased from


nvitrog


en (Carlsbad,


CA


PNG


was


biologically


sy


nthesized and purified


chromatographically


110).


culture.


Caco-2


human colon carcinoma cells


,passage 18,


were obtained


from American


Type Culture Collection (Rockville,


MD


).


Cells were propagated and


maintained at 370C0


95%


air


5% CO2 atmosphe


re) in DMEM containing 4


.5 g/L glu


case,


25


mmol/L HEPES,


growth medium was


44 mmol/L sodium bicarbonate


supplemented with


and 4 mmol/L glutamine.


mmol/L sodium pyruvate,


The


00 pmol/L non-


esse


ntial amino acids


and 20% fetal bovine


serum plus


00 U/L pen


icillin


100 U/L


streptomycin,


and 50 pg/L gentamycin


136).


For routin


e


s


ubculturing,


cells were


washed with


Ca.2


- and Mg


2- free pho


sphate buffered saline


PBS


and deta


ched with


the


Cel






60


performed on monolayers 7-9 days post-confluency, a time when lactase activity peaks


in Caco-2 cells (137).


Cells from passages


23-35


were used in th


e


experiments.


Uptake experiments.


Uptake experiments we


re don


e


by adding treatment


media containing different concentrations of PNG to the top of the cell monolaye


r with


and without lactose to th


buffer containing 1


23


e


cells.


Treatm


mmol/L NaC


ent medium was prepared with Krebs-.Ringer


4.93 mmol/L KOI.


1


.23 mmol/L MgSO4, 0.85 mmol/L


CaC[2 at pH


,, 5 mmol/L glu


7.4.


case


5


mmol/L glutamine,


Monolayers were washed


3 times with


10 mmol/L HEPES, and 10 mmol/L MES


2-3 mL Krebs-Ringer buffer


(37


00)


prior to th


e


addition of treatment media containing different concentrations of PNG and


lactose.


Treatment m


edia were added to monolaye


rs and


incubated at


37


C0


1n 5%


002


atmosph


e


re for times


as indicated in the


text.


Prior to the termination o1


incubation


sample of treatment medium was


desired time by addition of 3


collected.


mL of Krebs-Ringer


Incubation was


buffer


(4


then terminated at the


C0),


followed by two additional


was


hes.


Cell m


onolayers were mechanically lifted from the


plates with a sterile plastic


spatula


nto 0


.5 mL PBS and homogenized using a Polytron homogeni


zer at medium


speed for 1


5


sec.


An aliquot of crud


e homogenate was


collected for protein


measurement and the remaining crude cellular homogenate was


centrifuged at 200,000


x g for 30 min to obtain a cytoso


1ic


suboellular fraction.


Protein concentration was


meas


ured spectrophotometrically


114)


using bovine


serum albumin


as the standard.


Measurement of hydrolys


s


and uptake.


Ratios of PN:PNG were calculated


for the


treatment media prior to and after each inc


ubation period.


Detection of PNG in


cytosolic compartment of the


Caco-2 cells above that measured in cells receiving no


- n.mr~


-i-I- -- -i .1


I.


I


OflflITIflfl flU L.J "U I - In Tflfl Trnnrrr~nnr ~ nnUUI rv~ a ar~ V. *nrt' rr% rttt% 1.,.. ~ n *1 nnrI


a


the









of the assay buffer


106).


Lactase activity was measured by a colorimetric assay


described by Dahlqvist (96) with


some


modification.


Vitamin B-6 analyses.


Treatment media were analyzed for PN and PNG both


before and after incubation period using reverse-phase


fluorometric HPLC


110).


Intracellular


concentrations


of PN, PNG, PM, and PMP were measured using


on-pair


reverse-phase HPLC with fluorometric detection and PLP were measured by reverse-phase HPL


4) .


Intracellular concentration


C with fluorometric detection o


3Of PL PL- and


PLP-se


micarbazones


109).


Statistica


analyses.


Data are presented


as means


+SEM of multiple


experiments done at separate times and are expressed


as pmol vitamin B-6


/mg protein


Differences


between PN:PNG ratios in treatment medium from before and after


incubation periods we


re detected using a one-way analysis


of variance


115).


Differences in intracellular


way analysis


of


concentrations of vitamin B-6 were examined using a one-


variance with Student-Newman-Keuls pairwise comparison test.


Differences


in PNG uptake and metabolism


in the


presence


of lactose we


re analyzed by


a two-way analysis


of variance using concentrations of PNG and lactose


as factors.


Pairwise comparisons were performed using the Student-Newman Keuls test.


aforementioned statistical anal


yses


were performed using


Si


gmaStat statistical software


Jandel Corporation,


Sa


n Rafael


,CA).


Results


Uptake of PNG as a function of concentration.


PNG uptake


in Caco-


cells was examined at concentrations ranging from 0.5 pimol/L to 500 pimol/L (Figure 4-


a - - . * -- -' ~. . - ~. - - - . - . .. .


6


1


as


Th


e


2









concentrations


of PNG in the incubation media over 30 min. PNG


concentrations


betwee


n 0-50 pmo/L were taken up in a saturable p


rocess


Figure 4-1A),


while uptake of


A


CD
z 0.


*3




E
.4'


.8
.6

.2


1.0
0.8 0.6 0.4 0.2 0.0


0


10


20


30


40


50


60


PNG (pmol/L)


B


a
z a-


.
.0
E
-:

E a.


16 14 12 10
8
6
4
2


0


0


100


200


300


400


PNG


(pmol/L)


Figure 4-1. function of


Pyridoxin


C


e-5'


-f3-D-gluco


oncentration after


side (


PNG


abs


30 min incubation.


orp


ti


on by C


aco-2 cells


Absorption of PNG is


as a


saturable


at c 50 pmol/L


A)


and non-saturable at


represents a mean of at least two replicate


>50 pmol/L


S


from


(


B).


E


ach data point


3 independen


expenim


ents.


Data are presented


as means +


SEM.


62


500


600


-









concentration that yielded a high intracellular concentration o


PNG after a 30 min


incubation


Figure 4


-2


)PNG uptake


ncreased linearly up to 1


5


minutes and after that


500 400 300 200

100

0


0


-F


20


40


60


80


100


120


140


Time (min)


Figure 4-2.


Abs


orp


by Oaco-2 cells a


s


ti


on o


pyridoxin


a function of time.


3- D-glu


coside


PNG


at 200 pmol/L


Each data point represents at


east two


replicates from three ind


ependen


experiments.


Data are expre


ssed as


means


+SEM.


appeared to plateau.


Distribution of intracellular B-6 vitamers


n Caco-2 cells.


The


determination


ntracellular concentrations of vitamin B-6 in Caco-2 cells withou


added PNG


revealed that most of the vitamin B-6


nside the cell was in the form of pyridoxine


Figure


4-3)


Increasing concentrations of PNG


in the treatment media did no


si


gnifican


change the


intracellular concentrations of PN, PLP


,PM, or PMP.


PNG hydrolysis by Caco-2 cells.


Activity assays


for lactase we


re don


e


on th


total m


embrane


fraction isolated from ultracentrifugation (200,000


x g,


30 min


of Caco-2


cell crude homog


enates.


Lactase activity for Caco-2 cells between 7-9 days


post-


63


C
4..
0
1~
a 0,
E
0
E
a
0
z
0.


of


ti


y


e


T


e-5'-






64


to post-incubation in experiments using 25, 50, or 100 gmol/L PNG in the treatment media.


140


120 100


80 60


40


*



0.

.

E


-m


PLP


PL


PM


PMP


Figure 4-


3 .


Distribution of


intrace


Ilular concentrations


of vitamin B-6


n Ca


co-2


without


added PNG.


Abbreviations:


pyridoxine


PN


pyridoxal-5'-.pho


s


phate (PLP


pyridoxal


PL)


,pyridoxamine


PM),


and pyridoxamin


e-5'-phosphate


PMP).


Bars


represent means


SEM from at


east


3


replicates fro


m 4


independent experiments.


Effect of lactose on PNG uptake and metabolism.


The effect o1


lactose on


PNG uptake and metabolism was examined by incubating cell monolayers with 0


100, and 200 pmol/L PNG with 0,


75,'


or 150 mmol/L lactose for 30 min


ntracellular PNG concentration significantly increased


as the concentrations


Figure 4-4).


of PNG


20


0


PN


m


cell


s


50


25,


11









Table 4-1.


Pyridoxine to pyridoxine-5'-


g-D-glucoside


PN:PNG


ratios


in treatment


media before and


noubation period.


PN:PNGPN:PNG PN:PNG
PNG (pmoI/L) pre-incubation1 post-incubation Mean change

25 0.0020 0.0003 0.0025 0.0005 0.0006 0.0006

50 0.0018 0.0001 0.0020 0.0002 0.0462 0.0460

100 0.0017 0.0001 0.0017 0.0003 0.0004 0.0002


noubations


were done for 30 min at


37


C0.


Values within column were not sign


ifcantly


different and the post-incubation ratio was not significantly diffe incubation ratio.


0


1~


r


50


r


1~


r


100


.-r-


rent from the pre-


nO mM lactose


0 75


mM lactose


50 mM lactose


200


PNG (gmoI/L)


Figure 4-4.


Effect of


lacto


se on the intracellular concentrations of pyridoxal


(PL).


ncreasing concentrations of lactose significantly decreased the


intracellular concentration of PL.


Bars represent means


+SEM of at


east


2 replicates from 2 independent experiments.


2:LL - - - - -


rr1rr1t~ ''I It3flTr~C2fl Tr,.z2r, nnhIcz' Irgnsa~ori ~n,,.r. v1'i *tz~'r~1tzn . ~1r3rn 'R'c..~nrA r.,, ~,,r1*TIr'r2r1T ,.,TTnr,-~r. nn C'


65


45 40


35 30 25 20


-'


1


5


10
5

0


--






66


250


-II


i-I


Sodium


Potassium


Lithium


Tris


Figure 4


-5.


Effect o


sodium and other


monovalent cations on the


b


s


orption of pyridoxine-


min incubation.


5'-B


-glucoside


PNG


in Caco-2


cells after


a 30


Bars represent means SEM of three replicates from


2


independent experiments.


hydrophobic


li


pid bilayer o


the


ntestinal epithelial cell plasma membrane.


This


suggests that the absorption of PNG could be fac


ilitated by a carrier.


To determine


wheth


e


r PNG uptake was mediated through a sodium-dependent transporter


sodium


n the Krebs-Ringer buffer was replaced by potassium,


lithium


and Tris


hydroxymethyl) aminomethane-HCI to create a


solution of


equivalent ionic strength


(Figure 4-5).


PNG was added to each of the treatment media at 100 pmol/L and uptake


was measured over


30 mn.


Intracellular concentration


s


of


PNG were not significantly


different (P


=0.39) among th


e


cells incubated


in the sodium


,potassium,


lithium


or Tris


treatment media.


Discussion


200


* I.
af
E)




a.


50


100 50


0


a


the


I


--






67


to enterocytes in the small


intestine


,which permit the


close


examination of PNG uptake


and metabolism.


PNG was taken up by the Caco-2 cell monolayers


in a pattern that is consisten


with passiv


e


diffusion.


However


at low concentrations of PNG (5-50 pmol/L


),


th


e


uptake


of PNG displayed a saturable component.


linearly as the concentration of PNG


ntracellular concentration


s


>50 pimol/L) provided to the cells


of PNG increased increased. The


range of PNG added to the cells encompasses an amount of PNG that would be


cons


umed daily in th


e


diet.


Average dietary intake of vitamin B-6 by th


e


U.S. population


is 1.7 mg/d (NHANES III


).


Although food


selection ultimately determines


the percentage


vitamin B-6 de


rived from PNG, PNG contributes approximately 1


5%


of


total


vitamin B-


6 intake


,which corresponds to ~200 pig PNG daily.


This


amount


~200 [pg) divided


equally over


three meals yields the con


s


umption of


~65 pg for each meal.


Caco-2 cells


incubated with 100 pmol/L


-66 pg) PNG took up this amount by passive diffusion.


suggests that absorption of dietary PNG


Absorption of smaller quantities o1


n humans is mostly by passive


PNG may be facilitated by a carrier.


This


diffusion. This pattern of


absorption is similar to that observed


nutrients.


in the intestinal absorption of folate and many other


At low physiological concentrations of folate, the vitamin is absorbed by a pH-


dependen


,Na -independent carrier mediated transport system,


while pharmacological


concentrations


of folate are absorbed by a diffusion process


1


38,139).


The


exchange


of sodium for other monovalent cation


s


did not change PNG uptake by Caco-2 cells;


however, the concentration of PNG used in these experiments was


in the range where


passive diffusion, and not carrier-mediated absorption would predominate.


of









is also consistent with a recent report of PN uptake by cultured opposum kidn


ey


OK)


cells


141).


Howeve


r. we cannot reconcile the


difficulty that PNG,


as a very hydrophil


molecule


,would encounter in crossing the


highly


charged and hydrophobic pla


sma


membrane.


There is also recent


evidence


that other gly


cosylated compounds derived


from the diet


such


as quercetin D-glucosides either interact with or are transported by a


sodium-dependen


glu


case


transporter (SGLT)


142-1 44).


Although the absence of


sodium did not cause


a significan


decrease in PNG uptake,


we cannot


rule out the


possibility that PNG is taken up by a carrier mediated process.


Th


e


transport


mec


hanism is further confounded by the dual role ascribed to LPH.


n a recent


letter to


editor


,Arts and colleagues


briefly discussed the observation that LPH hydrolyzes


glucosidic bonds and is thought to transport th


e


aglycones


n a Na -independent process


(145


).


While this may explain the


transport of PN from PNG


into the cell.


the


p


rocess


which the


ntact glucoside,


PNG


enters into the


cell remains


unclear


It is


nteresting to


note that th


e


detection of the


ntact glucoside


inside the Caco-2 cells furth


er supports


previous


observations


made by this laboratory that PNG can be absorbed intact (


7


,10).


As


indicated


cells is pyridoxine.


n Figure 4-3,


This was


the most abundant form of vitamin B-6 inside Caco-2


not wholly unexpected since these cells were maintained


in


DMEM that contained 4 mg/L of pyridoxine-


HCIl


as the


source of vitamin B-6.


Consequently


it was difficult to measure th


e


s


mall changes that likely were


occurring


intracellular PN concentration


s


in response


to i


ncreasing concentrations of PNG.


However.


even the smallest


c


hange in


ntracellular PNG concentrations was easily


detected because


PNG is not presen


in DMEM, and there is no in vitro


sy


nthesis a1


68


Ic


th


e


f3-


b


y


in









affect this measurement.


Once hydrolyzed, PN could be taken up by the cell and


metabolized or released into the treatmen


medium.


Alternatively, over


the


incubation


period,


intracellular PN might


efflux out of the cell into the treatment medium.


The


measured in the treatment medium could contain PN originating from the hydrolysis


PNG and that released from


intracellular pools.


w


e


did however


obtain


indirect


evidence


of PNG hydrolysis.


Intracellular concentrations


of PL significantly


increased as


PNG concentration


increased in the treatment medium


Figure 4-4).


PN taken up from


the hydrolysis o


reported to th


e


PNG would be metabolized


major metabolic product o1


inside the


the small


cell to form PL and PLP.


ntestine released


PL is


nto portal blood


134).


Since intracellular PL was presen


n relatively low concentrations


in the Caco-2


cells without added PNG


small increases in PL were easily detected.


w


e


did not


measure the


concentration of PL in the treatment


media after the


30 min


noubation


we suspect that


intracellular PL would elfflux into the media.


The addition of lactose to the treatm


e


nt media did not appear to have a direct


effect on the uptake of PNG.


However


we have evidence that intracellular vitamin B-6


metabolism was changed,


perhaps as a consequence of a reduction in PNG hydrolysis


at the cellular surface.


n the presence of lactose, LPH is


nhibited with regard to PNG


hydrolytic activity.


Intracellular concentrations of PL significantly declined with


ncreasing lactose concentrations.


The con


centration of 150 mmol/L was chosen since


is the approximate concentration o


lactose


in milk.


At 150 mmol/L and hal


concentration,


75 mmol/L


, intracellular PL was significantly reduced


in cells


incubated


with PNG.


One possible explanation for this observation is that since PL is the greatest


~A+.,rnn D 2 nn+nkal4n rntannnA ..~4.-% rnr4rd rjrn, ~ /40 A


69


PN


of


but


It


this








Under well-nourished or saturated conditions


(high PN concentrations> PLP


concentrations did not increase in the rat intestine, likely due to potent product inhibition


by PLP on pyridoxine~pyridoxamine 5'-phosphate oxidase that


s


observed


in the


liver


147).


summary, this study provides in vitro


e


vidence that PNG is taken up


ntact by


ntestinal epithelial cells


n a Na-independent process that is saturable at low


concentrations and passively absorbed at concentrations >50 pmol/L,


which is similar to


the absorption of PN.


Lactose diminished the metabolic utilization of PNG


in Caco-2


cells, which might translate into a reduction of the in vivo bioavailability of PNG.


70


In














CHAPTER 5
SUMMARY AND CONCLUSIONS


The preceding research describes novel observation


understanding of pyridoxine-5'-f3-D-glucoside bioavailability


s that extend our with particular focus on its


absorption,


hydro


ysIs,


and metabol


ism


in the


s


mall


intestine.


The


following discu


ssIon


integrates the data presented


in the three previou


s


chapters


to provide a comprehensive


view o


the absorptive


process


of PNG and how it relates to


ts bioavailability.


Based on previous results from this laboratory, a decrease in dietary vitamin B-6


conce


ntration


significantly increased the


ntestinal hydrolysis


of PNG by cytosolic


PNG


hydrolase


-1


2


1


3


.This inverse relationship between an


induced vitamin B-6 defi


cien


intestinal cytosolic PNG hydrolase activity was observed


in both rats


13) and


guinea pigs


12).


Chapter


2


reports the


results of two studies that were designed to


examine


this observation more closely.


Although a vitamin B-6 deficiency was achieved,


as assessed by a significan


reduction


n p


asma and liver PLP concentrations, the


effect


of vitamin B-6 def


ciency on cytosolic PNG hydrolase activity was


not reproducible.


There was a trend toward an


increase


in in vitro PNG hydrolytic activity


as catalyzed by


cytosof ic PNG hydrolase and brush border


membrane LPH


as dietary vitamin B-6


increased.


n this respect, it appears that there is not a strong compensatory


upregulation of the hydrolytic activity that could


enhance


th


e


metabolic utilizati


ono01


PNG


in a time


of dietary vitamin B-6 def


ciency.


and


cy









studies done by this laboratory in the past and present.


The investigation don


Nakano et al.


Preliminary studies


(13) used older rats


that were used


weighing ~200g and fed PN-defined diets for


as a foundation for the


2 wk.


present work


examined the effects of age and duration of diet f


eeding on intestinal PNG hydrolytic


activity and found no


significant effects of rat age


or length of study period.


One other notabi


e


difference


between the


p


rese


nt studies and previous


studies


done by this laboratory is in the diet that was fed to the rats.


w


e


speculated there to be


an effect o1


basal diet fo


rmulation


AIN-93G or AIN-76A


on PNG hydrolytic activity


. The


present studies consistently fed the


AIN-93G diet with different


concentrations


pyridoxine


to rats over a


5 wk period to


change their vitamin B-6 nutritional status.


AIN-93G diet was formulated to reduce


sy


mptoms


such


as tooth decay and kidney


ification that were associated with


chronically feeding the AIN-76A diet to rats


As discussed


in Chapter


2,


the carbohydrate source in the


AIN


-76A diet is


sucrose,


which was


replaced with starch in the AIN-93G diet.


The starch based AIN


-93


G diet


theoretically could


support the


energy requirements of the


gut microflora.


Starches


oligosaccharides that are not completely hydrolyzed and absorbed in the small


ntestine


travel to th


e


large intestine


where the


residen-


colonic microflora ferment th


e


undigested


starch to salvage metabolic energy


A thriving population of gut microflora


sy


nthesizes


short


chain fatty acids,


proteins,


growth factors,


and vitamins


such as vitamins B-6 and


K, which could be


absorbed by the colon


108


,148).


AIN-93G also is


supplemented with


trace


mine


rals that are absent


in the


AIN-76A diet


107) and although these differences


exist. the


diets are intended to be nutritionally equivalent.


In spite of this, we observed


72


e


by


cal


C


of


Th


e


1


07).


and


initially









present, it is unclear what specific dietary


compone


nt(s)


niluence these


outcome


measures.


The purification and partial kinetic characterization of LPH,


presented


in Chapter


revealed that LPH was the


enzyme


in the brush border


that was able to catalyze


hydrolysis of PNG.


The primary function of LPH is to catal


yze


th


e


hydrolysis


of dietary


lactose and the hydrolytic activity toward PNG had not been previously reported.


valu


e


calculated for lactose was


16 times


greater than that calculated for PNG.


considering the quantity of PNG that is introduced to the


intestine daily (~


1I


5%


of the


total


vitamin B-6


ntake of 1


.7


mg/d),


th


e


enzyme is well-suited to catalyze


PNG hydroly


How


ever, the en


zy


me


is not likely to be working efficiently and would catalytically prefer


hydrolysis


of


lactose to PNG.


Not surprisingly


lactose was found to be a


competitive


inhibitor of PNG hydrolysis.


This leads u


s


to the


questions of whether PNG


bioavailability is reduced by the


containing lactose


composition of a mixed meal,


i.e.


consumed concurrently with fruits or vegetables


dairy products


containing PNG


PNG bioavailability is diminished


in


individuals with lactose


intolerance


or lactase


nsufficiency.


n addition to the


PNG hydrolytic function of LPH


we also observed yet another


novel


unction of mammalian LPH.


In the


presence


of both PNG and lactose, LPH


exhibits glu


cosyl-transf erase activity


in vitro.


We


measured not only th


e


hydrolytic


product of PNG, PN, but also a


seco


ndary product, PN-.disaccharid


e.


The


significance


this reaction in vivo is not completely understood,


but may further


contribute to a


reduction


in PNG bioavailability.


* p. p ., . * .. S *~%*


73


3


th


e


Th


e


Km


In


the


S's.


or if


Of









intestinal mucosa was just


as importan


as the hydrolysis measured in the cytosolic


fraction.


Chapter


2


also described the calculation of the relative


contribution of LPH and


cytosolic PNG hydrolase to total PNG hydrolysis measured


in the


S


mall


intestine.


LPH is


only 1-glucosidase in the


brush border membrane and any hydrolysis


measured


brush border membrane is due


to the activity of LPH.


It was estimated


n the rat that


50-60%


of the


PNG hydrolysis


in the


small


intestinal mucosa was catalyzed by brush


border LPH.


n comparison,


cytosolic PNG hydrolase was found to catal


yze


only 10%


the hydrolysis


0-


PNG


in the rat intestin


e.


It was


nitially thought that the hydrolysis a1


PNG was


exclusively catalyzed by cytosolic PNG hydrolase,


but we


now have


strong


evidence to support that PNG is hydrolyzed by extracellular (LPH


hydrolase


and


ntracellular


enzymes and that LPH is responsible for most of this hydrolysis.


PNG


Therefore,


based on th


e


results obtained from the present studies in rats and


Caco-2


cells.


PNG


bioavailability is greatly dependent on a functionally active LPH.


Results from the study of PNG absorption and hydrolysis


by Caco-2


cells


presented in Chapter


4 support previous data gen


erated by this


aboratory.


While


PNG


can be hydrolyzed


in the


small


ntestine


to form PN and glu


case


, it is not a complete


hydrolytic process and PNG can be absorbed


as the


intact glucoside


7.


10.11


).


PNG


provided to


Caco-2


cells was


rapidly taken up in a saturable process at con


centrations


less than 50 pmol/L,


and taken up in a non-saturable, passive


diffusion p


rocess


concentrations greater than 50 pmol/L.


We


also collected preliminary data that


uggested that there is little paracellular absorption of PNG (data not


shown


).


Pilot


experiments were done with the Caco-2 cells grown on permeable membrane


nserts


74


th


e


th


e


In


of


S


at









monolayer.


PNG has a MW that is very simila


to ph


enol red


MW=331).


Therefore


believe


that PNG absorption proceeds mostly via transcellular route; however


absorption by the paracellular route


cannot be excluded.


Application of these data to


human dietary intake of PNG


indicates that PNG derived from the diet is mostly


absorbed by p


assive


diffusion:


however


, absorption of PNG


in


individuals consuming a


diet limiting


n foods of plan


origin might occur through carrier-mediated transport.


n summary,


the results from the


studies discussed in the previous


three chapters


indicate that 1


PNG can be absorbed intact,


as it was detected


intracellularly


in the


Caco-2


cells


, 2)


PNG


can be en


zymatically hydrolyzed to release free PN at the


border membrane


by LPH, or by cytosolic PNG hydrolase,


and 3)


n vitro hydrolysis


markedly reduced


in the


presence


of


lactose.


We


have constructed a model of PNG


absorption based on the results from our laboratory


Figure


5 -


).


This model


incorporates many years of PNG bioavailability research and p


understanding of PNG


resents our current


ntestinal hydrolysis and absorption.


Future research should add


ress


the possible reduction


n PNG bioavailability in


individuals with lactose intolerance.


Lactose intolerance


or lactase


nsuff


cien


C


y, affects


75%


of the population worldwide


(101


and this


ntestinal condition could compromise


vitamin B-6 nutritional status


n populations


consuming a vegetarian based diet.


Con


versely,


in


individuals with


actase persistence, PNG bioavailbility may be


increased.


How


ever


, lactose co-ingested with foods of plan


origin providing PNG might decrease


the amount of vitamin B-6 derived from PNG.


These questions


remain to be answered


and further investigation is warranted.


75


we


bru


S


h


is






Limited
hydrolysis by other tissues


A


PNG -


PNG


(


. PNG


PN


r


PN


t


other 6-6 vitamers


u nabsorbed


Ti


B-E


2


Figure 5-1


Illustration of PNG hydrolysis, absorption, and metabolism in the small intestine.


Abbreviations used: PNG, pyridoxine-5'-3-D-glucoside, PN, pyridoxine, LPH, lactase-phorizin hydrolase, PNGH, PNG hydrolase.


>


y






77


LITERATURE CITED


Yasumoto, K.,


Tsuj


H.. !


wam


K


bran of a bound form of vitamin B-6 and its


pyridoxine.


Agric.


Biol.


Chem.


41: 1061


.& Mitsuda, H.


identification as


1


5'


9


77)


-o


-1067.


-(


solation from rice


J3-D-glu


cospyrano


Kabir


H.


Le


klem


J


E


& Miller


L.


T. (1983) M


easurement


of


glycos


ylated


vitamin B-6 in foods.


J. Food


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48: 1422-1425.


Greg


ory,


J.


1988)


Methods for determination of vitamin B-6


n foods and


other biolo


gical materials:


a critical review. J. Food. Comp. Anal.


1: 105-1


Gregory


J.


F


& Sa


rtan


,D. B. (1991) Improved chromatographic


determination of free and glycosyl


lated forms of vitamin B-6 in foods. J. Agric.


Food.


Chem. 39


:899-905.


Gilbert.


J


A


.& Gregory


J


F. (1992) Pyridoxine-5'-p-D-glucoside affects


metabolic


utilizati


0


n of pyridoxine in rats. J. Nutr.


122: 1029-1035.


Trumbo


,RP R., &


Gregory


J.F


1988) Metabol


Ic


utilization of pyridoxine-BS-


coside in rats:


influ


ence


of


vitamin B6 status and route of administration.


J.


Nutr.


118:


1336-1 342.


7. Trumbo


,P. R., Greg


ory,


J.


F.


& Sa


rtain


1988)


incomplete utilization of


pyridox


ne -Bf-glucosi


ide


as vitamin B-6 in the


rat. J. Nutr.


118: 170-175.


S.L.


,Gregory,


J. F.


& Sa


rtain


1986) Determination of pyrido


xine


-glucosi


ide


bioavailability us


ng


ntri


nsic and


extrins


C


abeling


in th


e


rat. J.


Agric.


Food.


Chem. 34: 857-862.


9. Banks


M.


A


.& Gregory,


J.


1994) M


ice


hamsters


,and gumn


e


a pigs differ


in effi


ci


ency of pyrid


oxine-5'


4- -D-glucoside


utilization. J. Nutr.


124: 406-414.


Gregory,


J. F


Trumbo


P.R


.Bailey, L. B.,


Toth


J.


P


.Baumgartner,


T.G


& Cerda.


J.


1991


Bioavailability of pyridoxin


e-5' -


3-.D-glucos


ide determined in humans


by stable-isotopic methods. J. Nutr.


121: 177-186.


Nakano


H


McMahon


L.G.


, &Greg


oryJ


J


Ft(


1997) Pyridoxin


2.


3 .


sy


l)


4.


5 .


23.


6.


g'u


the


8.


nk


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BIOGRAPHICAL SKETCH


Amy Dene Mackey was born on January 6,


1972


in


Sc


henectady, New York.


She grew up in Beliwood, Pennsylvania,


where sh


e


graduated from Beliwood-Anti


s


High


School


n1990


She receiv


ed her Bachelor of


Sc


ience degree


in biology from Juniata


in Huntingdon,


Penn


sylvania,


n 1994.


In Decemb


e


r ot 1997, she was


awarded


er Master of Science degree


in nutrition from the Pennsylvania


State


Unive


rsity


January 1998,


she entered into the University of Florida to pursue a doctoral degree


nutritional sciences.


Coll


ege


h


In


In









certify that


have read this study and that


in my opinion it conforms to


acceptable standards of scholarly presentation and is fully adequate,


in scope and


quality, as


a dis


sertation for


the degree of Doct of Philoso hy.

Je e -r r ' h i


I .. gt y us, t oi
Professor of Food Science


and


Human Nutrition


I certify that I acceptable standard


have read th


s


is


of scholarly pre


study and that in my opinion it conforms to


sentation and


s


fully adequate,


in scope and


quality


as ad


ssertation for the degree of Docr of Philosophy.


Lynn SNBailey


Profemtor of Food Science and
Human Nutrition


certify that


have read th


is


study and that in my opinion it conforms to


acceptable standards of scholarly presentation and is fully adequate, ip tcope and quality, as a dissertation for the degree of Dopier /tiisp


Robert J. Cousi
Boston Family Professor of Human Nutrition


I certify that I acceptable standard


quality


as a di


have read this study and that in my opinion it conforms to


s


of scholarly ore


sentation and is fully adequate,


ssertation for the degree of Doctor o Ppilos hy.


in scope and


Donald Samuelson Professor of Veterinary Medicine


This di


ssertation was


submitted to the Graduate Faculty of the College of


Agricultural and Life Sciences


and to the Graduate School and was accepted as


partial fulfillment of the requirements for the degree of Doctor of Philosophy.


December 2002


V


Dean, College of Agricu tural nd
Life Sciences


C


7






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PAGE 1

THE ENZYMATIC HYDROLYSIS OF PYRIDOXINE-5'-BETA-D-GLUCOSIDE IN THE MAMMALIAN SMALL INTESTINE By AMY DENE MACKEY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2002

PAGE 2

I would like to dedicate this work to the memory of my grandmother, Marjorie J. Mackey, who contributed significant financial support and unending encouragement throughout my academic career. Her devotion to the educational process and enthusiasm for learning (especially spelling) was an inspiration. I can only hope to pass on as much knowledge to others as she did in and out of the classroom for over 25 years.

PAGE 3

ACKNOWLEDGEMENTS I would like to thank members of my Ph.D. advisory committee including Lynn B. Bailey, Ph.D., Robert J. Cousins, Ph.D., Jesse F. Gregory III, Ph.D., and Donald Samuelson, Ph.D. A special thank you is extended to Jesse F. Gregory III, Ph.D., for all of his patience, guidance, and opportunity that he has offered to me over the past years. It would be difficult to find a better scientist or kinder person than Dr. Gregory. I would like to acknowledge Robert McMahon, Ph.D. for his invaluable scientific expertise and assistance throughout my research. I would like to thank George Henderson, Ph.D. for his collaboration on the pyridoxine disaccharide project. I would also like to acknowledge all of the wonderful graduate students and post-doctoral fellows that I have been honored to know and work with over the years, especially Brandon Lewis and Sara Rathman. Without these two remarkable individuals, graduate school would not have been nearly as educational or enjoyable. I would like to express my gratitude to Catherine Carman and Justin Townsend for their assistance in the experiments conducted in the final year of my research project. I would like to thank my family. My parents, who always expected me to be a professional student, provided me with the unconditional financial and emotional support that only parents could give. My younger sister, Barbara, has been a continuous model of strength and perseverance, as well as a reliable comedic relief. I would also like to thank my grandparents for encouraging me and listening to me even when they did not understand. I would also like to thank my dog, Sydney, for her faithful companionship and exuberant disposition on days that I needed it the most. Last, but not least, I would iii

PAGE 4

like to thank Steven Davis. I thank him for his unlimited generosity, encouragement, and patience. iv

PAGE 5

TABLE OF CONTENTS Page ACKNOWLEDGMENTS i" ABSTRACT vii CHAPTERS 1. INTRODUCTION 1 Review of the Literature 2 Hypotheses 22 2. HYDROLYTIC ACTIVITY TOWARD PYRIDOXINE-5'-(3-D-GLUCOSIDE IN RAT INTESTINAL MUCOSA IS NOT INCREASE BY VITAMIN B-6 DEFICIENCY: EFFECT OF BASAL DIET COMPOSITION AND PYRIDOXINE INTAKE 23 Introduction 23 Materials and Methods 25 Results 28 Discussion 33 3. ENZYMATIC HYDROLYSIS OF PYRIDOXINE-5'-(3-D-GLUCOSIDE IS CATALYZED BY INTESTINAL LACTASE-PHLORIZIN HYDROLASE 39 Introduction 39 Materials and Methods 41 Results 44 Discussion 50 4. THE INTESTINAL ABSORPTION, HYDROLYSIS, AND METABOLISM OF PYRIDOXINE-5'-P-D-GLUCOSIDE IN A CELL CULTURE MODEL USING CACO-2 HUMAN INTESTINAL EPITHELIAL CELLS 57 Introduction 57 Materials and Methods 59 Results 61 Discussion 66 5. SUMMARY AND CONCLUSIONS 71 LITERATURE CITED 77 v

PAGE 6

BIOGRAPHICAL SKETCH

PAGE 7

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE ENZYMATIC HYDROLYSIS OF PYRIDOXINE-5'-BETA-D-GLUCOSIDE IN THE MAMMALIAN SMALL INTESTINE By Amy D. Mackey December 2002 Chair: Jesse F. Gregory, III Major Department: Food Science and Human Nutrition An important source of dietary vitamin B-6 is provided by pyridoxine-5'-(3-Dglucoside (PNG), a form of vitamin B-6 found in foods of plant origin. While this glycosylated form of vitamin B-6 contributes a significant portion (15%) of daily vitamin B-6 intake, PNG is not completely bioavailable. An obligatory step in the metabolic utilization of PNG is its intestinal hydrolysis by cytosolic PNG hydrolase (PNGH) and lactase-phlorizin hydrolase (LPH). This research was focused on the hydrolytic process of PNG in the mammalian small intestine. Using a rat model, the effect of vitamin B-6 status on PNG hydrolytic activity was examined by feeding either AIN-76A or AIN-93G diets with graded concentrations pyridoxine (PN). Vitamin B-6 deficiency did not increase PNG hydrolysis by PNGH or LPH. Rat growth and PNG hydrolysis were greater in rats fed AIN-93G than rats fed AIN-76A, regardless of dietary PN concentration. LPH catalyzed 50-60% of the total PNG hydrolysis occurring in the small intestine, while PNGH catalyzes 10%. Purification of LPH from rat small intestine confirmed that it was the brush border enzyme that catalyzed the hydrolysis of PNG. vii

PAGE 8

LPH exhibited a Km of 1 .0 mmol/L toward PNG and a Km of 16 mmol/L toward lactose. Lactose was a competitive inhibitor of PNG hydrolysis as both substrates were hydrolyzed at the same active site. In addition to the novel PNG hydrolytic function of LPH, we observed rat LPH to exhibit transferase activity in vitro. Formation of a PNdisaccharide was detected in reaction mixtures containing LPH, PNG, and lactose. Caco-2 cells were also used to examine PNG hydrolysis, absorption, and metabolism. Concentrations of PNG that were < 50 jimol/L were absorbed by a saturable mechanism, potentially through a carrier-mediated process. At concentrations >50 |imol/L, PNG was absorbed by passive diffusion. Intracellular concentrations of PNG significantly increased as PNG increased in the media. Increasing concentrations of lactose in the media did not significantly change the intracellular concentrations of PNG; however, intracellular concentration of pyridoxal (PL) significantly decreased. Collectively, these results indicate that 1) vitamin B-6 deficiency does not consistently increase intestinal PNG hydrolytic activity, 2) PNG is hydrolyzed in the small intestine by PNGH and LPH, with most of the hydrolysis catalyzed by LPH, and 3) food selection, i.e., dairy products eaten with fruits and vegetables and individuals with lactose intolerance or lactase insufficiency may reduce PNG bioavailability. viii

PAGE 9

CHAPTER 1 INTRODUCTION Twenty-five years ago, a glycosylated form of vitamin B-6 isolated from rice bran was identified as 5'-0-((3-D-glucopyranosyl)-pyridoxine, or pyridoxine-5'-p-D-glucoside (1). Pyridoxine-5'-p-D-glucoside (PNG) is an abundant source of vitamin B-6 in plant foods and nearly absent in animal-derived foods. PNG contributes a significant proportion of our daily intake of vitamin B-6; however, its nutritional value is limited by its intestinal hydrolysis. For the complete metabolic utilization of PNG, the glycosidic bond needs to be hydrolyzed to release free pyridoxine, which is one form of vitamin B-6 that can be converted to the coenzymatic forms of pyridoxal-5'-phosphate (PLP) and pyridoxamine-5'-phosphate (PMP). PLP and PMP are essential to the normal function of over 100 enzymes that participate in metabolic pathways of nutrients and other biological molecules. Since the identification of the vitamer, PNG-related research has focused on the nutritional properties of PNG including its bioavailability and metabolism as well as analytical techniques used to measure its concentration in food and other biological materials. Critical to the nutritional significance of PNG is the accurate determination of its concentration in the food supply. Improvements in the analyses of glycosylated and non-glycosylated forms of vitamin B-6 have led to better measurements of PNG concentration in food (2-4). Previous research done by this laboratory has significantly advanced the understanding of PNG bioavailability. In vivo studies of PNG bioavailability in rats (5-8), mice, hamsters, guinea pigs (9) and humans (10,1 1) have 1

PAGE 10

2 shown that the metabolic utilization of PNG is not equivalent to that of PN and that there are species differences in the efficiency of metabolic utilization. The metabolic utilization of PNG is largely determined by its partial hydrolysis and not by the extent of absorption in the small intestine. PNG is hydrolyzed by intestinal p-glucosidases that were reported to exhibit greater activity with decreasing concentrations of dietary pyridoxine (PN) (12,13). The purification and initial characterization of the cytosolic (3-glucosidases, broad specificity p-glucosidase and pyridoxine-5'-(3-D-glucoside hydrolase (PNG hydrolase), from pig intestine provided in vitro evidence of specific intestinal catalysis of PNG hydrolysis by the novel PNG hydrolase, and not BSPG (14). These investigations served as a foundation for further examination of intestinal PNG hydrolysis and its implications on PNG bioavailability. This laboratory has since conducted investigations that have examined the absorption and hydrolysis of PNG, including the subcellular localization of PNG hydrolytic activities, the effect of dietary PN and other dietary components on intestinal PNG hydrolysis, and the hydrolysis of PNG by the brush border membrane enzyme lactase-phlorizin hydrolase (LPH). The research described in the subsequent chapters has broadened our perspective on the absorption and metabolic processing of PNG in the mammalian small intestine. Review of the Literature History Vitamin B-6 is an essential nutrient to mammalian species as it functions as a coenzyme in several metabolic pathways. The properties of vitamin B-6 were initially established when the vitamin was described as a factor that alleviated and prevented a deficiency in rats known as acrodynia (15). Acrodynia, a B-vitamin deficiency, is a condition diagnosed in rats with symptoms of dermatological lesions and edematous paws and ears (16). Vitamin B-6 was identified from rice bran by Gyorgy in 1 935 and further isolated and crystallized three years later by five independent laboratories (17).

PAGE 11

3 Chemistry Vitamin B-6 is a term used to describe a family of related compounds having pyridoxine-like activity with the core structure of 2-methyl, 3-hydroxy, 5-hydroxy methyl pyridine. Naturally occurring forms of vitamin B-6 differ in the substituent present on carbon 4 of the pyridine ring (Figure 1-1), which yields the three primary vitamers: pyridoxine (PN), an alcohol; pyridoxal (PL), an aldehyde; and pyridoxamine (PM), an amine. Phosphorylation at the 5'-hydroxymethyl of the pyridine ring forms the respective phosphoric esters: PNP, PLP, and PMP. Pyridoxal-5'-phosphate (PLP) and pyridoxamine-5'-phosphate (PMP) are the predominant forms of the coenzyme found in animal tissues. Vitamin B-6 that is catabolized is converted into 4-pyridoxic acid (4-PA) and excreted in the urine. Vitamin B-6 also exists as a glycosylated molecule that is synthesized by plants. Naturally occurring forms of glycosylated vitamin B-6 consist of a pyridoxine molecule with one or more monosaccharide or other complex oligosaccharide units attached at the 4' or 5' position via p-glycosidic linkage. The most abundant glycosylated form of dietary vitamin B-6 is pyridoxine5'-P-D-glucoside, which is comprised of a pyridoxine molecule with one glucosyl group (3-linked to the 5' position of pyridoxine. Vitamin B-6 is soluble in water and stable under acidic pH; however, in alkaline solution, vitamin B-6 is susceptible to degradation by photo-oxidation and heat (18). Both PL and PLP will react with free amino groups, such as aor e-amino groups found on amino acids, to form a Schiff base. PLP is well suited as a coenzyme CHoO-R, H,C^N-^H PN= CH 2 OH PL= CHO PM= CH 2 NH 2 PN, PL, PM= H PNP= P0 3 ' PLP= P0 3 PMP= PO,' Figure 1-1 . Chemical structures of vitamin B-6.

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4 for this reaction since the pyridine ring provides an electron sink for the free electron pair left on the a-carbon after the bond between a proton or carboxyl group to the a-carbon is broken. The negative charge is stabilized by the PLP ring. This carbonyl-amine reaction is the mechanism by which PLP-dependent enzymes function. Functions of Vitamin B-6 Vitamin B-6, most often as PLP, is involved in a host of metabolic reactions including the metabolism of amino acids, lipids, and one carbon units, and the pathways of gluconeogenesis and heme biosynthesis. Vitamin B-6 is widely recognized for its role in amino acid metabolism. PLP is a coenzyme for aminotransferases, decarboxylases, racemases, and dehydratases. The aminotransferases use PLP in the interconversion of amino acids and their respective aketo acids by forming a stable Schiff base intermediate where PLP becomes an electron sink for negatively charged catalytic intermediates. The role of vitamin B-6 in lipid metabolism is not clearly defined. Compositional changes in phospholipid were reported to occur in response to changes in dietary concentrations of vitamin B-6 in rats (19,20). More recently, Tsuge and colleagues (21) reported that Wistar rats fed a diet deficient in vitamin B-6 had a 64% reduction in A6desaturase and 80% reduction in acyl CoA oxidase activity compared to the control group. The decreased activity caused an alteration in polyunsaturated fatty acid metabolism, most notably with a reduction in docosahexaenoic acid (DHA). These effects are likely to due to an impaired methylation cycle via S-adenosylmethionine, which decreases phosphatidylcholine and carnitine synthesis. As reviewed by Selhub (22), vitamin B-6 plays a large role in one-carbon metabolism. PLP serves as a coenzyme for serine hydroxymethyltransferase (SHMT), which catalyzes the interconversion of glycine and serine as well as the formation of

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5 methylene tetrahydrofolate from tetrahydrofolate. A metabolic intermediate in the one carbon cycle is the amino acid homocysteine. Homocysteine concentration is maintained, in part, by PLP-dependent enzymatic pathways. The catabolism of homocysteine by the transsudation pathway is driven by two PLP-dependent enzymes. The first is cystathionine (3-synthase, the enzyme that catalyzes the condensation of serine with homocysteine to form cystathionine. The second enzyme, y-cystathionase, hydrolyzes cystathionine to release cysteine and a-ketobutyrate (22). Gluconeogenesis is the metabolic pathway that provides a source of glucose de novo from non-glucose precursors. Certain amino acids serve as non-glucose precursors to help maintain concentrations of glucose. The first step in producing glucose from gluconeogenic amino acids is the transamination of the amino acid to form its respective a-keto acid, which is a PLP-dependent process. Another PLP-dependent enzyme involved in carbohydrate metabolism is glycogen phosphorylase. Glycogen phosphorylase catalyzes the cleavage of glycogen to sequentially release glucose-1phosphate residues that may be converted to the glycolytic intermediate, glucose-6phosphate. PLP associated with glycogen phosphorylase accounts for a large pool of whole body vitamin B-6. It is estimated that up to 80-90% of body PLP is bound to glycogen phosphorylase (23). The biosynthesis of heme also requires vitamin B-6 as a coenzyme. Deltaaminolevulinate synthase, a PLP-dependent enzyme, catalyzes condensation of succinyl CoA and glycine to form 8-aminolevulinate, which is the precursor for the porphyrin ring used to synthesize hemoglobin. Deficiency in vitamin B-6 would therefore precipitate a microcytic hypochromic anemia in which erythrocytes have a reduced concentration of hemoglobin. The effects from inherited forms of sideroblastic anemia can be caused by deficiencies in 8-aminolevulinate-synthase (ALAS) activity. The anemia that results form

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6 reduced ALAS activity is often overcome with pyridoxine supplementation, if the disorder is clinically classified as pyridoxine-responsive. Mutations in the ALAS2 gene might alter the PLP binding sites on the enzyme (24,25). There is evidence that vitamin B-6 also functions on a molecular level as a modulator of gene expression. Evidence of transcriptional regulation was provided by Oka and coworkers in rats with vitamin B-6 deficiency (26). Vitamin B-6 deficient rats had a greater abundance of hepatic total poly (A)+ RNA than vitamin B-6 adequate rats; however, the increase was nonspecific and control (3-actin levels, which would expectedly remain constant, were similarly increased. Although mRNA levels were not well standardized, Oka and coworkers subsequently reported 5and 7-fold increases in hepatic glycogen phosphorylase mRNA (27) and albumin mRNA (28), respectively, in vitamin B-6 deficient rats compared to control rats. Sato et al. (29) similarly reported that vitamin B-6 deficiency in rats increased the rate of cystathionase synthesis, which was explained by a several-fold increase in cystathionase mRNA. However, vitamin B-6 deficient rats had essentially the same amount of immunoreactive cystathionase protein as control rats. The authors concluded that vitamin B-6 deficiency upregulated cystathionase synthesis, but also decreased the rate of degradation by lysosomal enzymes. Vitamin B-6 Requirements The Food and Nutrition Board recently revised the requirements for B vitamins, including vitamin B-6. An Estimated Average Requirement (EAR) of 1.1 mg/d and a RDA of 1 .3 mg/d for men and women (19-50 y) was established after thorough review of the available literature. These values have been reduced from previous recommendations set in 1989 (Food and Nutrition Board 1989). To determine the requirement, plasma PLP concentration (<20 nmol/L) was used as the major vitamin B-6 status indicator since it best represents tissue stores (Food and Nutrition Board 1998).

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7 As reviewed in the most recent report by the Food and Nutrition Board (1998), vitamin B6 requirements were typically based on investigations utilizing depletion-repletion protocols with dietary (food) vitamin B-6 in combination with synthetic pyridoxine. Average vitamin B-6 intake as estimated by nationally representative nutrient intake surveys is approximately 1 .5 mg/d for women and 2 mg/d for men (Food and Nutrition Board 1998). Although an insufficient dietary intake of vitamin B-6 is not prevalent in the general population, there are significant portions of the population at risk for sub-optimal vitamin B-6 intakes such as pregnant and lactating women (30,31 ) and the elderly (32). Vitamin B-6 in Foods and Supplements Vitamin B-6 is fairly well distributed throughout the food supply. Foods of animal origin such as meat, fish, eggs, and dairy products are rich in vitamin B-6. Vitamin B-6 exists mostly as PL and PM and their phosphorylated forms (PLP and PMP) in foods of animal origin. Additionally, fortified breakfast cereals and vitamin supplements provide a significant amount of vitamin B-6, as PN, to the typical American diet. Many vegetables and whole grain cereal products are good sources of the vitamin as well. The predominant forms found in foods of plant origin are usually glycosylated. Plant tissues may contain up to 75% of their vitamin B-6 as PNG (2). Vitamin B-6 loss during cooking and thermal processing is significant; however, it exhibits remarkable stability during storage and handling. The nutritional impact of a reduction in vitamin B-6 content by heating was observed in the 1950s in infants that were formula fed. Coursin reported that infants receiving formula that was thermally processed without subsequent fortification had convulsive seizures along with other neurological abnormalities (33). Neurological based symptoms were alleviated by administration of synthetic pyridoxine to the infants, which prompted the routine fortification of formulas with pyridoxine. The incident led to a number of investigations that determined how the loss of vitamin B-6 by heating occurred. As reviewed by

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8 Gregory (17), the thermal reaction products found in milk were initially thought to reduce the bioavailability of the naturally occurring B-6 vitamers; however, re-evaluation of the evidence found that the thermal processing of the formula destroyed the native vitamin B-6. Similar to infant formulas, dietary supplements contain pyridoxine hydrochloride as the added form of vitamin B-6. Pyridoxine exhibits the greatest stability among the various B-6 vitamers as it is relatively unreactive with other compounds and is inexpensive to synthesize. Pyridoxine Glucosides Pyridoxine glucosides are just one example of the many vitamin glucosides that occur in nature. Other nutritionally significant glycosylated vitamins, such as vitamin D, niacin, pantothenic acid, and riboflavin have been identified (34). Glycosylated forms of vitamin B-6 are abundant in foods of plant origin, yet absent in foods of animal origin. The biological significance of the glycosylation of vitamin B-6 in plants is not completely understood, but it is presumed to be a storage form of the vitamin (34). As originally identified in the bran of rice as 5'-0-(P-Dglucopyranosyl) pyridoxine (1) (Figure 1-2), glycosylated vitamin B-6 accounts for a mean 15% of total vitamin B-6 intake in a typical mixed diet (10,30). This percentage could vary depending on food selection and energy intake. Other glucosides of pyridoxine have since been identified. Tadera and colleagues isolated the 4' and 5' isomers of pyridoxine-(3-D-glucoside, pyridoxine-5'-P-D-cellobioside, and other conjugated oligosaccharides (35), as well as pyridoxine-5'-p-D-glucoside esterified with malonic or 3-hydroxy-3-methylglutaric acid (36,37), or cellobiosyl-indoleacetate (38). Only beta configurations of pyridoxine glucosides appear to occur naturally in mammalian and plant tissues; however, microbial formation of pyridoxine-a-D-

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9 CH 2 OH OH CH 2 OH HO. cHr-° H 3 C N H Figure 1-2. Structure of pyridoxine-5'-(3-D-glucoside. glucosides has been reported (39). These a-D-glucoside conjugates are more bioavailable than p-linked PN glucoside, as they are absorbed intact and rapidly taken up and hydrolyzed by the liver (40,41 ). Analysis and quantification of pyridoxine glucosides have been reported by a number of investigators. Total glycosylated vitamin B-6 can be determined by the difference in growth responses of yeast before and after treatment with p-glucosidase (2). Alternatively, liquid chromatographic separation methods have been established to measure not only the various forms of vitamin B-6, but also glycosylated variants (4,42,43). Bioavailability and Absorption of Vitamin B-6 The bioavailability of vitamin B-6 in humans consuming a mixed diet is approximately 75% (44). As recently reviewed by Gregory (17), vitamin B-6 bioavailability is likely to be reduced by food matrix trapping, nondigestible residue, and only partial utilization of glycosylated forms of vitamin B-6. A glycosylated form of vitamin B-6, specifically pyridoxine-5'-|3-D-glucoside, was first thought to be a completely bioavailable form of vitamin B-6 in rats (45). However, later studies found a much lower percentage of PNG (25%) to be effectively absorbed and metabolized in rats (6,8). In everted sacs of rat intestine, PNG was not completely metabolized to the coenzymatic form of PLP (41), indicating that intact PNG was not a metabolically usable form of the

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10 vitamin. This is consistent with other investigations examining the bioavailability of PNG in different rodent species (6-9) and humans (10,1 1). The bioavailability of PNG is much greater in humans (50-60%) than in rats (25-30%), which is thought to be determined by the extent of enzymatic hydrolysis in the small intestine. While PNG can be hydrolyzed in the enterocyte, it also appears in the urine unchanged (absorbed as the intact glucoside). It was estimated that -35% of dietary PNG appears in the urine in humans, as determined from diet composite analysis of PNG and urinary excretion of PNG (10). There is evidence that PNG is not hydrolyzed exclusively in the small intestine. In a comparison of oral [ 2 H 5 ] PN and intravenous (i.v.) [ 2 H 2 ] PNG doses, it was reported that the i.v. dose of PNG, which bypassed the intestine, was utilized at 28% relative to the oral PN dose. This observation indicated that some hydrolysis of PNG occurred outside of the intestine (10). In rats, less than 5% of an intraperitoneal (i.p.) dose of [ 3 H]PNG was metabolically utilized relative to an i.p. dose of [ 14 C]PN (6). However, both PN and PNG were readily absorbed and 93% of the [ 3 H] isotope was excreted in the urine as PNG and its hydrolytic product, PN. Analysis of liver vitamin B-6 revealed a lower degree of metabolism of PNG compared to PN with no retention of [ 3 H]PNG. These observations indicated that PNG was adequately absorbed in the intestine, but poorly utilized, and rapidly excreted into the urine (6). The bioavailability of PNG in other rodent models was also evaluated to assess species relatedness to humans. In an investigation using [ 3 H]PNG and [ 14 C]PN, Banks and Gregory (9) reported a 10-34% bioavailability of PNG in rats, 69% in mice, 70% in hamsters, and 92% in guinea pigs. These results suggested that mice and hamsters might be better suited for bioavailability studies applicable to human nutrition. Intestinal absorption of vitamin B-6 occurs by a nonsaturable, passive process (18). The majority of dietary vitamin B-6 derived from animal sources is in the forms of PLP and PMP. Investigations of PLP absorption, transport, and metabolism have been

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11 mostly conducted in intact rat intestine (46-51). There is general agreement that the 5'phosphate group of dietary PLP is hydrolyzed by luminal alkaline phosphatases, releasing PL that is readily absorbed. PLP can be absorbed across the enterocyte apical membrane intact, although to a lesser extent than its dephosphorylated form (PL) (50). Once absorbed, vitamin B-6 can be phosphorylated by pyridoxal kinase for the purpose of metabolic trapping. While the intestinal absorption of PLP is described as passive and non-saturable, the metabolic phosphorylation of absorbed vitamin B-6 is known to be saturable (52,53). Similar to PLP, PMP also is first acted on by intestinal alkaline phosphatases to yield PM that is absorbed; however, when PMP is present at saturating concentrations, it too will be absorbed with the 5'-phosphate attached (54). Collectively, data from vitamin B-6 absorption studies indicate that the rates of absorption for B-6 vitamers are PL>PN>PM. At physiological concentrations, phosphorylated forms of vitamin B-6 undergo intestinal hydrolysis by alkaline phosphatase prior to absorption. At concentrations well above a physiological dose, PLP and PMP are directly absorbed, with their 5'-phosphate group, although at a rate several fold lower than their respective dephosphorylated compounds (47,54). To cross the basolateral membrane and enter into portal circulation, B-6 vitamers are in a nonphosphorylated form. The intestine does not appear to provide phosphorylated B-6 vitamers to the rest of the organs; rather the liver is responsible for phosphorylation of vitamin B-6 for the distribution to other organs. Transport and Metabolism Vitamin B-6 that is absorbed and metabolized by the intestine enters the portal circulation, predominantly as PL, and is bound to albumin or erythrocytes (55) and is transported to the liver. Although PL and PLP constitute 75-80% of the circulating forms of vitamin B-6 (18), pyridoxine can also be bound and transported by erythrocytes (56). The primary site of vitamin B-6 metabolism is the liver where PLP is generated for use

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12 by extrahepatic organs. Nonphosphorylated forms of vitamin B-6 are phosphorylated in the liver by pyridoxal kinase. Pyridoxal kinase, using ATP-Zn+ as a chelated cosubstrate, catalyzes the phosphorylation of the 5' position of PN, PL, and PM to form PNP, PLP, and PMP, respectively (Figure 1-3) (57,58). In addition to the enzymatic phosphorylation function of the liver, FMN-dependent pyridoxamine (pyridoxine) 5'phosphate oxidase is an abundant enzyme in the liver that is responsible for the conversion of PNP and PMP to PLP (58). This reaction is critical for the metabolism of dietary PN since most tissues have an insufficient amount of oxidase activity to convert PN to PL (59). This enzyme also is of importance because it is tightly regulated by product inhibition. High concentrations of PLP slow the rate of conversion of PNP to PLP, which prevents an excessive accumulation of intracellular PLP. Catabolites of vitamin B-6 are generated in the liver. Pyridoxamine (pyridoxine) 5'-phosphate oxidase catalyzes the formation of PLP from PNP and PMP, which, in turn, is dephosphorylated to PL. PL in excess is oxidized to the metabolic excretion product, 4-pyridoxic acid (4-PA) by aldehyde oxidase (FAD) or pyridoxal dehydrogenase (NAD), two enzymes that are present in the liver and kidney. PLP and PL are the predominant circulating forms of vitamin B-6, comprising approximately 75-80% of the total body B-6 (18). As reviewed by Coburn (23), the turnover of vitamin B-6, as PLP, may be described as a five compartment model consisting of muscle, liver, plasma, erythrocytes, and all other pools, which are grouped together into one additional compartment. Evidence from tracer studies calculated total body concentration of vitamin B-6 to be 15 nmol/g and the total pool size in humans is approximately 1000 (xmoles (23). This estimation was further supported by data from muscle biopsies from 12 human subjects (60). As reviewed earlier, one of the functions of vitamin B-6 is to serve as a coenzyme for glycogen phosphorylase. Up to 80% of the vitamin B-6 pool, as measured in rats, is associated with glycogen phosphorylase, which

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13 PNP o w T3 =r 0) w• 0) os CD T3 P & w 9 CD x lu J!) PN CO c o o 03 "CD CL 1 03 c/> 0 03 PNG FMNdependent pyridoxamine (pyridoxine) 5'-phosphate oxidase FMNdependent pyridoxamine (pyridoxine) 5'-phosphate oxidase > PLP PMP T3 =r o os T3 ZT 0) i— 03 CD transaminases T3 03 2 t PL CL CD O CO CD =5 03 03 CD CL O X 03 4-PA o 03 0) oT 03 CD 03 g PM Figure 1-3. Interconversion and metabolism of vitamin B-6. Abbreviations used: PNG (pyridoxine 5'-|3-D-glucoside), PN (pyridoxine), PNP (pyridoxine 5'phosphate), PLP (pyridoxal 5'-phosphate), PL (pyridoxal), 4-PA (4-pyridoxic acid), PMP (pyridoxamine 5'-phosphate), PM (pyridoxamine). is localized to the muscle (61). The remaining vitamin B-6 is concentrated in the liver, but also distributed to the brain, heart, adrenal glands, kidney, and pancreas (23). Metabolic utilization of PNG and effects of dietary PNG on the normal metabolism of non-glycosylated B-6 vitamers were examined previously. In rats, a comparison of bioavailability between purified [ 3 H]PNG and alfalfa sprouts that were intrinsically enriched with [ 3 H]PN, forming [ 3 H]PNG, was done in relation to [ 3 H]and [ 14 C]-PN (8). Rats were fed an alginate gel that was extrinsically labeled with [ 14 C]PN and purified [ 3 H]PNG or [ 3 H]PN-enriched alfalfa sprouts. This experimental protocol allowed the observation of the metabolic utilization of a purified form of PNG as well as a

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14 form that would naturally occur in a plant-derived food in relation to pyridoxine. Rats that were fed the purified PNG received 88% of their vitamin B-6 as PNG. Rats that were fed the alginate gel with the alfalfa sprout homogenate added consumed -45% of their vitamin B-6 as PNG. Control rats consumed >80% of their vitamin B-6 as PN. Isotopic ratios of [ 3 H] / [ 14 C] dose in the liver, carcass and plasma were significantly lower in the rats fed any form of PNG than control rats that were fed PN. Conversely, the relative isotopic ratios in the urine and feces were significantly higher than controls. These data indicated that the absorption and metabolic utilization of PNG was incomplete. Dietary PNG was not retained in the liver as PMP, PLP, or PL. Significantly more PNG was excreted in the urine and feces than PN. There was no [ 3 H]PNG detected in the livers of rats fed the glucoside (8). Further evaluation PNG bioavailability was done in rats either PN or PNG as their sole source of vitamin B-6. Changes in vitamin B-6 status were assessed using erythrocyte aspartate transaminase (EAST) stimulation, plasma PLP concentration, and urinary 4-PA excretion in response to the form of dietary vitamin B-6 (7). Rats consuming their vitamin B-6 as PNG had an elevated stimulation of EAST with PLP, lower plasma PLP concentrations, and a diminished 4-PA excretion compared to rats consuming their dietary vitamin B-6 as PN. This same group of investigators also examined the metabolic utilization of PNG compared to PN by using isotopically labeled [ 3 H]PNG and [ 14 C]PN given either as an oral or intraperitoneal dose to vitamin B-6 adequate or deficient rats (6). Rats injected with both [ 3 H]PNG and [ 14 C]PN excreted twice the amount of [ 14 C]-labeled 4-PA when fed a diet adequate in vitamin B-6 compared to a diet deficient in vitamin B-6. In contrast, rats fed a diet containing no vitamin B-6 excreted the same amount of [ 3 H]-labeled 4-PA as the rats fed the vitamin B6 adequate diet, with 93% of the label excreted as PN and PNG. The increased urinary clearance of [ 3 H] indicated the reduced utilization of PNG relative to PN and that a deficiency in vitamin B-6 did not improve the metabolic processing of PNG. Additionally,

PAGE 23

15 hepatic vitamin B-6 metabolites detected as [ 3 H] or [ 14 C], were not significantly different between adequate and deficient rats, regardless of route of administration. Although the retention of [ 3 H]-labeled B-6 vitamers was significantly less in the liver and carcass as compared to [ 14 C]-labeled vitamers, PNG appeared to undergo partial hydrolysis and act as PN in the metabolic interconversion of hepatic vitamin B-6 (6). The results from these studies showed that PNG is not as efficiently metabolized as PN and that vitamin B-6 deficiency reduces vitamin B-6 catabolite clearance, i.e., a reduction in 4-PA excretion. Gilbert and Gregory (5) investigated the effect of dietary PNG on the metabolic utilization of co-ingested PN in rats using radiolabeled [ 14 C]PN and unlabeled PNG. Administration of PNG concurrently with PN antagonized the utilization of PN as observed through the altered distribution of hepatic vitamers and reduced retention of labeled PN. Of particular interest was the direct linear relationship found between the dose of PNG and the appearance of [ 14 C]PNP, which was later supported by Nakano and Gregory (13), who also reported that as dietary PNG increases in rats, liver PLP decreases. Since dietary PNG interfered with the normal production of hepatic PLP, Gilbert and Gregory (5) hypothesized that PNG had an inhibitory effect on pyridoxamine (pyridoxine) 5'-phosphate oxidase. However, further investigation of the inhibitory effects of PNG on purified pyridoxamine (pyridoxine) 5'-phosphate oxidase and found no effect on the activity in vitro (62). These results were corroborated by Zhang et al. (63). In liver cell homogenates, [ 3 H]PN incubated in the presence of PNG at either 0.5 or 5.0 nmol/L did not significantly alter the specific activity of pyridoxal kinase or pyridoxamine (pyridoxine) 5'-phosphate oxidase. The authors concluded that an increased liver PNP was the result of PNG competitively inhibiting the uptake of PN in isolated hepatocytes. The formation of PLP from PNP proceeds through the requisite phosphorylation of PN to PNP, which is then oxidized to PLP. Without an adequate supply of PN to the liver, the amount of PLP formed from PNP is reduced. Zhang and coworkers also calculated the

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16 cellular uptake and metabolism of PNG relative to PN, which was 20 and 0.2%, respectively (63). Effects of dietary PNG on the metabolism of PN also have been investigated in humans. Hansen and coworkers monitored vitamin B-6 status in response to feeding women diets with varying concentrations of PNG (64). Nine healthy women were fed diets that were designated as either high or low in PNG for 18d each in a crossover design. Vitamin B-6 nutritional status was assessed by using plasma and erythrocyte PLP, erythrocyte aspartate and alanine aminotransaminase (EAST and EALT) stimulation, urinary 4-PA and total B-6 fecal excretion as indicators. The diet providing 27% of total vitamin B-6 as PNG (high PNG diet) yielded a 10% lower urinary 4-PA, 12% lower fecal vitamin B-6 excretion, and 18% and 17% lower plasma and erythrocyte PLP, respectively, than the diet providing 9% total vitamin B-6 as PNG (low PNG diet). The EAST and EALT stimulation tests were similar between the two diets. Similarly, Nakano et al. (11) observed a decline in 4-PA excretion in human subjects given increasing doses of [ 2 H 2 ]PNG. Rats are less susceptible than humans to the antagonistic effects of PNG on PN. Cytosolic Pyridoxine-5'-p-D-glucoside Hydrolase (PNG Hydrolase) Prior to the purification and identification of the cytosolic PNG hydrolase from pig intestinal mucosa, PNG was presumed to be hydrolyzed in the small intestine by another soluble p-glucosidase, broad specificity P-glucosidase (BSPG) (6,8,12,13). To better understand the hydrolysis of PNG by BSPG, McMahon and coworkers purified and partially characterized not only intestinal BSPG, but also a novel cytosolic p-glucosidase, pyridoxine-5'-P-D-glucoside hydrolase from pig jejunum (14). BSPG is a soluble enzyme purified from guinea pig and human liver that has an approximate molecular weight of 50-60 kD (65-67). The cDNA encoding this protein has been cloned and sequenced

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17 from the guinea pig liver (68). While BSpG does not hydrolyze PNG (14) or other nutritionally significant oligosaccharides, it displays activity for a wide range of substrates, with particular specificity to aryl aglycones (69,70). Cytosolic PNG hydrolase is a soluble p-glucosidase with an acidic pH optimum (5.5) and a molecular mass of 130 kD (denatured) and 160 kD (native). In contrast to BSPG, PNG hydrolase does not hydrolyze aryl glucosides but exhibits hydrolytic affinity for PNG, cellobiose, and lactose. PNG hydrolase was characterized to have a K m of 0.88 ± 0.12 mmol/L and a V max of 13.2 ± 0.8 nmol/h*mg protein for PNG. Inhibition experiments found that PNG hydrolase, like other (3-glucosidases, was inhibited by conduritol B epoxide and glucono-5-lactone. Hydrolytic activities of both PNG hydrolase and BS(3G are affected by sulfhydryl reagents, which implicated cysteine residues in the catalysis at the active site. There is remarkable amino acid homology between short amino acid sequences obtained from cytosolic PNG hydrolase (-95-99%) purified from pig intestine and the brush border enzyme, lactase phlorizin hydrolase (LPH) (Tseung, et al., unpublished) as analyzed by the BLAST alignment procedure (71 ). All of the short sequences aligned with rat, rabbit, and human LPH. Five of these sequences (total of 51 amino acids) also aligned with regions of the precursor sequence of LPH (aa 20-866) that are not present in the mature LPH enzyme. However, there is a region in the precursor sequence of LPH (aa 458-806) where no aligning sequences from cytosolic PNG hydrolase were found. This might indicate that cytosolic PNG hydrolase is a splice variant derived from the LPH gene. It is also well known that substantial sequence homology exists among 3-glucosidases, so LPH and PNG hydrolase might be products of related, but distinct genes.

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18 Lactase Phlorizin Hydrolase The digestion of dietary carbohydrates relies on the disaccharidases present in the small intestine. Perhaps the most important carbohydrate-digesting enzyme to the developing mammalian species nourished via a milk-based diet is the intestinal brush border enzyme lactase phlorizin hydrolase (lactase). Lactase phlorizin hydrolase (EC 3.2.1.23, 62, and 108) (LPH) is a membrane bound glycoprotein in the intestine that is primarily responsible for the hydrolysis of dietary lactose into its constituent monosaccharides glucose and galactose. LPH was previously purified and characterized by several laboratories from the small intestine of different species (72). Lactase phlorizin hydrolase is named for its two distinct hydrolytic activities: one for the hydrolysis of lactose and other hydrophilic p-glucosides (lactase), and the other for the hydrolysis of arylor alkyl-glucosides, such as (3-glucoceramides (phlorizin hydrolase) (73-75). Wacker and colleagues assigned the lactase activity to glutamatei 2 73 and the phlorizin hydrolase activity to glutamatemg (75). The LPH gene (LCT) was localized to chromosome 2 and cDNA cloning led to the translation of the LPH sequence to 1927 amino acids (72). The enzyme is synthesized as a single polypeptide glycosylated precursor of 215 kDa (pro-LPH), which undergoes folding to form a homodimer; a step that is critical for membrane targeting and enzymatic activity (76,77). Transport of the homodimer through the Golgi apparatus yields a complex glycosylated form of the protein (230 kDa) (78). Subsequently, the protein undergoes two proteolytic cleavages. The first occurs intracellular^ at Arg 734 , which yields a transport-competent form of the enzyme. The second cleavage, occurring after insertion in to the microvillar membrane, is catalyzed by pancreatic trypsin at Arg 868 , which generates the mature form of LPH (130-160 kDa) (79,80). The expression of LPH is greatest in the jejunum. Immunohistochemical and in situ hybridization detection methods in several species

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19 revealed that LPH expression increases from crypt to mid-villus, and declines at the villus tip (77,81,82). The control of LPH expression has been studied extensively in light of the connection with the post-weaning decline in lactase activity that is observed in maturing mammals. Current opinion on the decline of lactase activity focuses on transcriptional control of the LPH protein (77). This is supported by data that positively correlate levels of mRNA and LPH activity in rats (81 ,83,84), pigs (85), and sheep (86). In spite of the compelling evidence of transcriptional control, there are reports of secondary, or posttranslational control of LPH expression. Using [ 13 C]or [ 3 H]-leucine and [ 13 C]phenylalanine, Dudley and coworkers have measured the brush border fractional synthesis rate (FSR) of intestinal disaccharidases (87), including isoforms of LPH (88) to assess dietary effects on intracellular processing of LPH (85,89,90). The ratio of the isotopic enrichment of an LPH glycosylated precursor and the enrichment of a mature LPH isoform yields the FSR for LPH (87). Changes in LPH isoform labeling, (FSR), in response to a dietary treatment, e.g. protein deficient diet (85), is indicative of changes in post-translational modification and further, the regulation, of mature LPH expression. This procedure is validated by a strong relationship between the relative abundance of the LPH isoforms and developmental differences in LPH activity (91 ). Using this methodology, post-translational control of LPH expression was reported to be reduced in protein malnutrition (85,90) and increased by administration of insulin-like growth factor-l (IGF-1) (92,93) and colostrum (89) A reduced expression and activity of LPH in humans most often occurs during the transition from a predominantly milk-based diet to a diet of solid food. Lactase decline occurs somewhere between the ages of 5-7, but as late as 20 years (94). In humans, the normal physiological decline in LPH activity is termed adult-onset lactase decline and is more commonly known as lactase deficiency or lactose intolerance. It is

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20 usually recognized by the variety of abdominal and intestinal related symptoms that are caused by the osmotic effect of undigested lactose present in the lumen. Although nearly all mammals are born with lactase, some infants are born with a rare congenital lactase deficiency. Initial clinical characterization of intestinal disaccharidases, including (3galactosidases, as well as their deficiencies were reported in a series of papers by Arne Dahlqvist and coworkers (95). Dahlqvist is also credited with the development of a rapid and accurate determination of lactase activity as measured by the release of glucose using glucose oxidase and a colorimetric agent (96). The condition of lactose intolerance can be diagnosed by assessing the extent of lactose digestion. Lactase activity can be directly measured by intestinal mucosa biopsy or by an intestinal perfusion method that pumps lactose into the intestine and measures glucose release by sampling the contents of the lumen (97). Although these methods are considered to be the most accurate, they are used infrequently and are the most invasive. More convenient tests used in the routine diagnosis of lactose intolerance include breath tests, blood sampling, and urine analyses (98). Traditionally, breath hydrogen concentrations are used to detect lactase deficiency. This test measures the release of H 2 in the breath after a dose of lactose. Undigested lactose is fermented by colonic microflora, which produces hydrogen gas that is absorbed into portal circulation and later expired in the breath (99). Similarly, breath 13 C0 2 can be measured after ingestion of [ 13 C]lactose. Determination of blood glucose or galactose concentrations following a dose of lactose can also be used to diagnose lactose intolerance. More recently, it was suggested that the ratio of [ 13 C]/[ 2 H]-glucose in blood after ingestion of [ 13 C] lactose and [ 2 H] glucose was a more accurate way to assess lactose digestion (1 00). Low concentrations of galactose in the urine following a dose of lactose may also diagnose lactose intolerance.

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21 The prevalence of lactose intolerance differs globally. Worldwide, it is estimated that 75% of the adult population is lactase deficient. In the United States, the lowest prevalence is among whites of Western European descent (15%) and increases to 80% in the African-American and 90% in the Asian-American population. In Asian countries, the percentage of lactose intolerance is at least 50% and varies among ethnicities (101). It is proposed that the persistence of lactase activity, rather than the loss, is abnormal. As reviewed by Vesa, hypotheses to explain lactase persistence were derived from cultural practices and natural genetic selection (98). At the advent of dairy farming, populations that had higher levels of lactase activity would exhibit an evolutionary advantage for survival in times of dietary scarcity. Today, the highest prevalence of lactase persistence is observed in individuals that are descendents from areas with a long history of dairy farming, as in those from Western Europe. Genetic markers of physiological decline in lactase activity were recently identified by examining variants in the lactase (LCT) gene (102). Seven polymorphic microsatellite markers flanking the LCT gene in nine Finnish families (n=196) with adult-onset hypolactasia were analyzed. The individual family members were biochemically verified to be lactose intolerant or tolerant. Two DNA variants that were upstream of LCT were identified: C/T-13910 and G/A-22018 with the C and G alleles associated with lactase deficiency. Individuals that were diagnosed with lactase deficiency (n=59) were all homozygous (CC) with respect to the C/T variant and 6 were heterozygous (GA) and 53 homozygous (GG) with respect to the G/A variant. None of the lactase persistence subjects (n=137) were homozygous with respect to the C or G alleles. Subjects (n=74) were TT with respect to the C/T variant and AA with respect to the G/A variant. The remaining 63 were heterozygous at both positions. The authors speculated that the variant identified in all of the lactose intolerant individuals is the original form of the gene and that it mutated to acquire the ability to hydrolyze lactose. The prevalence of CC genotype among different populations

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22 was also examined. White North Americans had the lowest prevalence (7.6%), followed by the French (41%) and black North Americans (79%). These data are not only consistent with current epidemiological estimates of prevalence of lactose intolerance, but also propose another potential method of screening individuals for the diagnosis of lactose intolerance. In summary, the preceding review of the literature discussed the nutritional significance and bioavailability of PNG as an important form of dietary vitamin B-6. Intestinal p-glucosidases catalyze the intestinal hydrolysis of PNG, which is the limiting factor of PNG bioavailability. The following research sought to examine the bioavailability of PNG in mammals with particular focus on its intestinal hydrolysis. Hypotheses The research described in the subsequent chapters was based on the existing literature and preliminary studies done to describe the hydrolysis and metabolism of PNG. The present studies were done to test the hypotheses: 1 ) Dietary vitamin B-6 deficiency increases the PNG hydrolytic activity in the mammalian small intestine. 2) The brush border membrane enzyme, lactase phlorizin hydrolase, catalyzes the hydrolysis of pyridoxine-5'-(3-D-glucoside

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CHAPTER 2 HYDROLYTIC ACTIVITY TOWARD PYRIDOXINE-|3-D-GLUCOSIDE IN RAT INTESTINAL MUCOSA IS NOT INCREASED BY VITAMIN B-6 DEFICIENCY: EFFECT OF BASAL DIET COMPOSITION AND PYRIDOXINE INTAKE Introduction Pyridoxine-5'-|3-D-glucoside (PNG), a glycosylated form of pyridoxine provides a significant dietary source of vitamin B-6. This form of vitamin B-6 was first isolated from rice bran (1 ) and can be found in foods of plant origin (2,1 8,42). PNG accounts for 575% of the vitamin B-6 present in plant tissues. In a mixed American diet, it is estimated that half of the dietary vitamin B-6 intake is from plant sources (103). Although PNG provides approximately 15% of the total vitamin B-6 in typical diets in the United States (30,64), some patterns of food selection could lead to a much larger proportion of dietary vitamin B-6 as PNG. The bioavailability of PNG in humans is 50-60% relative to pyridoxine, (10,1 1) which is greater than that estimated for rats (25-30%) (6-9). The rate-limiting factor in the utilization of PNG as a source of vitamin B-6 is the enzymatic hydrolysis of the |3glucosidic bond, rather than the intestinal absorption of intact PNG or free PN derived from PNG hydrolysis (4,8). The variations in PNG bioavailability within and among species may be explained by differences in intestinal enzymatic activities toward PNG. Cytosolic broad specificity (3-glucosidase (BS(3G) (66) was initially thought to be responsible for the hydrolysis of dietary PNG (12,13,104). However, purification and kinetic analysis of BS(3G from pig intestine revealed that this enzyme does not hydrolyze PNG (14). This observation led to the detection and purification of a novel intestinal 23

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24 cytosolic p-glucosidase, designated pyridoxine-5'-(3-D-glucoside hydrolase (PNG hydrolase), that catalyzed the hydrolysis of PNG. Recent data from this laboratory indicate that PNG can be hydrolyzed in both the cytosolic and membrane subcellular fractions of rat small intestinal mucosa. In an investigation of the age-dependent enzymatic hydrolysis of lactose and PNG, Armada et al. (105) measured PNG hydrolysis in the cytosolic and brush border membrane subcellular fractions of intestinal mucosa in nursing, weaned, and adult rats. A concurrent publication by Mackey et al. (106) reported the kinetic analysis of PNG hydrolysis as catalyzed by rat brush border membrane and purified lactase-phlorizin hydrolase (LPH), a p-glucosidase in the intestinal brush border membrane. Although these investigations reported PNG hydrolysis to be catalyzed in both subcellular compartments of the intestinal mucosa, the contribution of these cytosolic and brush border membrane enzymes toward PNG hydrolysis in vivo is not fully known. Previous investigations of the effects of vitamin B-6 nutritional status on cytosolic PNG hydrolase activity in the small intestine of guinea pigs and rats showed that PNG hydrolase activity was inversely related to vitamin B-6 status (12,13). Subsequent studies (Mackey et al., unpublished) examined more closely the effect of vitamin B-6 nutritional status on the activity of cytosolic PNG hydrolase in rat intestine and showed that vitamin B-6 status had little or no reproducible effect on this enzymatic activity. This raised the question of whether differences in the basal diet may have had an effect on the vitamin B-6 status of the rodents and perhaps intestinal p-glucosidase activity. Our preliminary rat studies that did not find an effect of dietary vitamin B-6 on intestinal Pglucosidase activity used AIN-93G purified diet instead of the AIN-76A diet that was used in previous studies (12,13). Although there are several differences between the two diet formulations (107), we hypothesize that the carbohydrate in the AIN-93G,

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25 mostly cornstarch, is a more fermentable carbohydrate for the microflora of the cecum and large intestine than sucrose, which is the major carbohydrate component of the AIN76A formulation. In the presence of fermentable carbohydrate, the gut microflora actively synthesize protein, growth factors, and other nutrients, including vitamin B-6 (108), which may enhance the vitamin B-6 nutritional status of the rat. The purpose of the present study was to 1 ) determine the subcellular distribution of enzymatic activities toward PNG in rat intestinal mucosa as a function of vitamin B-6 status and, 2) examine the differences between AIN-76A and AIN-93G purified diets on PNG hydrolase activity in the cytosolic and membrane-associated subcellular compartments. Materials and Methods Animals and diet. Weanling male rats (Hsd:Sprague Dawley SD; Harlan Laboratories, Indianapolis, IN), weighing -50 g were used in both study 1 and 2. Rats were housed in hanging wire mesh stainless steel cages and maintained at constant temperature with 12h light:dark cycle. All procedures for animal care and treatment were approved by the Institutional Animal Care and Use Committee at the University of Florida. In study 1 , the rats (n=29) were randomly assigned to one of four AIN-93G purified diets (107) from Dyets, Inc. (Bethlehem, PA) that differed only in the concentration of pyridoxine. Rats were given free access to diets that provided 7 mg/kg (n=5), 1 mg/kg (n=6), 0.5 mg/kg (n=6), and 0 mg/kg (n=6) pyridoxine-HCI. Another group of rats (n=6) was pair-fed a diet containing 7 mg/kg pyridoxine (7 mg/kg, pf) matched to the intake of rats fed the 0 mg/kg diet. Animal weight and food intake were monitored daily over the study period. In study 2, rats (n=49) were fed one of 10 AINformulated purified diets (107) from Dyets, Inc. (Bethlehem, PA). Rats were randomly assigned to either basal diet AIN-76A or AIN-93G: AIN-76A with 2 mg/kg (n=5), 1 mg/kg

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26 (n=4), 0.5 mg/kg (n=5), 0.1 mg/kg (n=5), or 0 mg/kg (n=5) PN-HCI, or AIN-93G with 2 mg/kg (n=5), 1 mg/kg (n=5), 0.5 mg/kg (n=5), 0.1 mg/kg (n=5), or 0 mg/kg (n=5) PN-HCI. Study 2 was conducted at a time separate from study 1 . After 5 weeks, the rats in both studies were anesthetized by Halothane (Alocarbon Laboratories, River Edge, NJ) inhalation and exsanguinated by cardiac puncture. Blood was collected with heparinized needles and syringes and transferred to evacuated tubes containing EDTA as an anticoagulant. Plasma was obtained by centrifugation at 2,000 x g for 20 minutes and stored at -80 °C until analysis. Small intestine was harvested, flushed with 9 g/L NaCI at 4 °C to remove intraluminal contents, cut longitudinally, and mucosa was obtained by scraping with a glass slide, all of which were done on ice or at 4 °C. All procedures were done under gold fluorescent light to minimize photochemical degradation of vitamin B-6. HPLC analysis of vitamin B-6 concentrations. Plasma pyridoxal 5'-phosphate (PLP) was measured as the semicarbazone derivative by reverse-phase fluorometric HPLC using a modification of the method of Ubbink et al. (109). PN concentration of both AIN-76A and AIN-93G diets was measured using reverse-phase fluorometric HPLC (110) after extraction as described previously (4). This analysis was not performed on the diets used in study 1 . Tissue preparation for enzyme activity assays. Intestinal mucosa was homogenized using a Potter-Elvehjem tissue homogenizer in 5 volumes of homogenization buffer containing 25 mmol/L sodium phosphate, pH 7.4, containing 50 mmol/L mannitol, 1 mmol/L EDTA, and a general use protease inhibitor cocktail from Sigma Chemical Co. (St. Louis, MO). The cytosolic and total membrane fractions were obtained by centrifugation of the mucosal crude homogenate at 200,000 x g for 30 min at 4°C. Cytosolic supernatant was removed and the resulting membrane pellet was

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27 resuspended in homogenization buffer using a Potter-Elvehjem homogenizer. Brush border membrane was isolated according to the method of Kessler et al. (111) with some modification (112). Briefly, non-brush border membranes were precipitated by the addition of solid MgCI 2 (to yield a final concentration of 1 0 mmol/L) to a portion of mucosal crude homogenate, followed by centrifugation at 3,000 x g for 10 min. The supernatant was centrifuged at 40,000 x g for 20 min to obtain a brush border pellet. The pellet was resuspended using a Potter-Elvehjem homogenizer and centrifuged again at 40,000 x g for 20 min. The final brush border membrane pellet was resuspended in homogenization buffer with a Potter-Elvehjem homogenizer. Using the specific activity of the brush border membrane enzyme sucrase, brush border membrane isolation consistently yielded an enrichment factor of -10-14, which is consistent with other published values (111,113). Using PNG as the substrate, the crude homogenate and cytosolic, total membrane, and brush border membrane subcellular fractions of intestinal mucosa were assayed for hydrolytic activity in study 1 and cytosolic and total membrane subcellular fractions were assayed for activity in study 2. Measurement of PNG hydrolytic activity. In vitro assays of enzyme activity were done on the different subcellular fractions of intestinal mucosa using PNG as the substrate. PNG was prepared by biological synthesis using alfalfa seeds and purified as described by Gregory and Nakano (110). All activity assays were done under conditions that allowed the measurement of initial rate. The assay for the hydrolysis of PNG was done by a minor modification of the method of Nakano and Gregory (13). Reaction mixtures contained 80 mmol/L sodium phosphate, pH 6.0, and 0.25 mmol/L PNG. Reaction mixtures were incubated for 60 min at 37 °C and terminated by incubation in 100 °C water bath for 5 min. The amount of pyridoxine (PN) released was measured by reverse-phase HPLC with fluorometric detection (13). Protein concentration was determined using a colorimetric assay (114).

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28 Statistical analyses. In study 1 , differences in plasma PLP and PNG hydrolytic enzyme activities among the rats fed the diets containing different concentrations of pyridoxine were analyzed using one-way analysis of variance (115) and StudentNewman-Keuls pairwise comparison test with SigmaStat software (Jandel Corporation, San Rafael, CA). In study 2, linear regression was used to analyze raw or log transformed data. This was done to account for small differences in measured PN concentration in the AIN-76A and AIN-93G diets. Rat weight gain, plasma and liver PLP concentrations, and enzymatic activities were analyzed by a t-test comparison of linear regression line slopes and y-intercepts between the AIN-76A and AIN-93G diet groups (116) using Prism software (GraphPad Software, Inc., San Diego, CA). Plasma PLP concentration was log transformed to normalize the distribution and variance. Dietary PN concentrations were log transformed to linearize the liver PLP concentrations. A P value of less than 0.05 was considered to be statistically significant. Data are presented as means ± SEM. Results Diet and nutritional status. In study 1 , rat growth significantly increased (P<0.0001) as dietary PN concentration increased, with final body weights of 255 ± 4 g (7 mg/kg, ad libitum), 197 ± 5 g (7 mg/kg, pair-fed, pf), 229 ± 3 g (1 mg/kg), 222 ± 6 g (0.5 mg/kg), and 164 ± 4 g (0 mg/kg). The AIN-76A and AIN-93G diets from study 2 were analyzed for pyridoxine (PN) concentrations at the conclusion of the study. At each intended level of fortification, pyridoxine concentrations were consistently higher in the AIN-76A diets than in the AIN-93G diets. The PN content of AIN-76A and AIN-93G diets (in parentheses) was: AIN-76A; 2 mg/kg PN (2.2 mg/kg), 1 mg/kg (1.1 mg/kg) 0.5 mg/kg (0.52 mg/kg), 0.1 mg/kg (0.12 mg/kg), 0 mg/kg (0.05 mg/kg), and AIN-93G; 2 mg/kg (1.8 mg/kg), 1 mg/kg (0.88 mg/kg), 0.5 mg/kg (0.42), 0.1 mg/kg (0.09 mg/kg), 0 mg/kg (0.03 mg/kg). In study 2, weight gain (final weight (g) initial weight (g))

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29 significantly increased with increasing concentrations of dietary PN in rats fed either AIN76A or -93G diets (P<0.0001 ). The regression lines describing the weight gain of rats fed AIN-76A or AIN-93G as a function of dietary PN did not have significantly different slopes (Figure 2-1 ), but did exhibit different y-intercepts. The y-intercept of the regression line describing weight gain was significantly higher (P<0.001 ) for rats fed AIN93G than the y-intercept of the regression line of the rats fed the AIN-76A diet (153.5 vs. 135.7 g). 300 n 3 .E 200' (0 OB CO |j 100' a AIN-76A a AIN-93G 0 12 3 Dietary PN (mg/kg) Figure 2-1 . Linear regression analyses of weight gain of rats fed AIN-76A and AIN-93G with different concentrations of pyridoxine (PN) after 5 wk. The slopes of the lines were not significantly different (P= 0.7322); pooled slope=36.4. The y-intercepts of the regression lines were significantly different (P<0.001). In study 1, increases in dietary PN concentration improved vitamin B-6 nutritional status among the different groups of rats. Concentrations of PLP in liver and plasma in study 1 were normally distributed with equal variance. PLP concentrations in plasma and liver significantly increased (P<0.0001) with increasing dietary PN (Table 2-1). In study 2, plasma PLP concentrations were log transformed to meet the assumptions of normal distribution and equal variance. Plasma and liver PLP concentrations for rats fed

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30 Table 2-1 . Indicators of vitamin B-6 nutritional status and mucosal hydrolytic activity toward PNG in the small intestine of rats after consuming pyridoxine defined diets for 5 wk. Vitamin B-6 Nutritional Status 1 PNG hydrolytic activity Dietary Vitamin B-6 (mg PN/kg diet) 2 Plasma PLP (nmol/L) Liver PLP (nmol/g) Cytosol (nmol PN/h*mg protein) Brush border membrane (nmol PN/h*mg protein) 7 mg/kg 7 mg/kg pf 1 mg/kg 0.5 mg/kg 0 mg/kg 1352 ±59 a 726 ± 92 a 229 ± 34 b 165 ± 9 b 45±12 bc 16.3±0.3 a 15.6 ± 1.5 a 13.0 ± 1.2 ab 9.7±1.2 b 9.2 ± 1.1 b 1.1 ±0.3 a 0.7 ± 0.2 a 0.5±0.2 a 0.4 ± 0.2 a 0.9 ± 0.4 a 1 1 .2 ± 0.6 a 20.7 ± 1 .2 b 17.8±2.1 ab 16.7±0.7 ab 13.2±1.4 a 1 Values are means ± SEM. Means within a column with unlike letters were found to be statistically different at P<0.05. 2 Abbreviations used: PNG, pyridoxine glucoside; PN, pyridoxine; pf, pair fed; PLP, pyridoxal-5'-phosphate both AIN-76A and AIN-93G purified diets significantly increased (P<0.001) in response to increasing concentrations of PN in the diets. Plasma PLP concentrations of the rats fed the AIN-76A or AIN-93G diet increased similarly with increasing dietary PN concentration as the slopes of the lines were found to not be statistically different (P=0.54). Similarly, the y-intercepts of the regression lines for plasma PLP concentrations were also not statistically different (P=0.75). Liver PLP concentrations were not statistically different between the groups fed the two basal diets as interpreted from the parallel slopes of the regression lines (P=0.6793). The y-intercept for the regression line for liver PLP concentrations of the rats fed AIN-76A was significantly higher (P=0.009) than the y-intercept for rats fed the AIN-93G diet (Figure 2-2). PNG hydrolytic activities. In study 1 , PNG hydrolytic specific activities of the small intestinal mucosa were determined in the cytosolic and brush border membrane subcellular fractions using PNG as the substrate (Table 2-1 ). The activity measured in

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31 the brush border membrane fraction was significantly greater in rats that were pair-fed the 7 mg/kg PN than rats fed the 7 mg/kg and 0 mg/kg diets (ad libitum). There was no effect of dietary PN concentration or pair-feeding on PNG hydrolytic specific activity measured in the cytosolic fraction (P=0.35). Enzymatic activity data from study 1 were used to make further comparisons among the intestinal mucosa subcellular fractions (crude homogenate, cytosol, total and brush border membrane) within the different dietary PN concentrations (Figure 2-3). With PNG hydrolytic activity expressed on the basis of mucosal weight, the subcellular distribution of activity could be assessed. Although dietary PN concentration did not affect the subcellular distribution of PNG hydrolysis, the activity measured in the total and brush border membrane fractions was significantly greater than that measured in cytosolic fractions (P<0.001 ). Recovery of brush border membrane from the crude homogenate was -45% as measured by lactase activity. After adjusting PNG hydrolytic activity for its respective recovery, brush border membrane accounted for 50-60% of the total PNG hydrolytic activity measured in intestinal mucosa, while cytosolic activity contributed 1 0% of the total PNG hydrolytic activity. In study 2, PNG hydrolytic activity in the cytosolic fraction significantly increased with increasing dietary PN in rats fed the AIN-76A (P=0.02), but not the AIN-93G diet (P=0.6547). PNG hydrolytic activity measured in the total membrane fractions was not influenced by dietary PN in rats fed either basal diet (regression line slopes that were not significantly different from zero). The slopes of the regression lines describing the PNG hydrolytic activity in the cytosolic and total membrane fractions were not significantly different between groups fed the AIN-76A or AIN-93G diets (Figure 2-4). However, rats fed the AIN-93G diet had greater hydrolytic activity toward PNG in the cytosolic (P<0.0001) and total membrane (P=0.006) fractions than rats fed the AIN-76A diet,

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32 (B/iouiu) did < CD CD CO fCD < < r co I — < — I o CD O l — * — r o tSH. I CL Is Q o CO 00 (B/IOUJU) did 6j ("l/IOUJU) did 0l Bon r CO * < -I Q. (0 Q CD CO d " 11 is CL C f o 2 k w ^ IS Q £.1 0 (0 — CO co c C CO O i_ ° CO C 0 | E 8^ ~L 0 6? E q! I — co •S CO Q. CD co o I o CL 0 It "D -a a> >*E" CO co 2 c as co c ^= c CO o 'c cd co co CD co o o s> 0 CO 0 0 CO c o 10 "co o ~ "o co Q0 o g S 8 is 1 « CL CO CO c E o w w J3 CO Q_ 0 o>< 0 t'DO. co 0 Cl O co 0 ^— ID 0 > CO -C *— » o c g T3 CO c o CO i— *-< c 0 o c o o o c g co CD 2 co .E en , CD o o 0 > Si C 0 — *—> — c CO «J co c 0 0 CD 0) 0 5z DC ^ h/|oiuu) did

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33 ^6001 a > O 500x 5 I 400o (0 O) c c o *jz 300" X 1 jE 200» | 100b Crude homogenate Cvtosol Total membrane Brush border membrane Figure 2-3. Subcellular distribution of PNG hydrolytic activity (nmol PN/h*g mucosa) in the small intestinal mucosa from rats fed AIN-93G with different concentrations of PN. Subcellular fractions tested included: crude homogenate, cytosol, total membrane, and brush border membrane. Bars with different letters within a dietary concentration of PN indicate significance at P<0.05. irrespective of dietary PN concentration. This was concluded from the parallel slopes, but significantly different y-intercepts of the regression lines describing these data. When PNG was used as a substrate, there was a trend toward higher specific activities in the total membrane fraction than in the cytosolic fraction, regardless of diet formulation (AIN-76A or AIN-93G) or PN concentration, which is consistent with the data in study 1 . The results reported here extend our understanding of the bioavailability of vitamin B-6 with focus on the partial hydrolysis of PNG. We were particularly interested in the subcellular localization of PNG hydrolysis and the effect of vitamin B-6 nutritional status on the hydrolytic enzyme activities toward PNG in the rat small intestine. We observed pronounced changes in plasma and liver PLP concentrations in response to variations in dietary PN concentration in both studies that were in general agreement with other studies of rats fed PN defined diets (6,1 17-1 19). Both AIN-76A Discussion

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34 A a AIN-76A a AIN-93G Dietary PN (mg/kg) B _ 7501 a AIN-76A ^ AIN-93G 1 2 Dietary PN (mg/kg) Figure 2-4. Regression analyses for PNG hydrolytic activity (nmol PN/h*g mucosa) in rats fed AIN-76A and AIN-93G. (A) Regression lines for PNG hydrolytic activity in the cytosolic fraction did not have significantly different slope (P=0.2368), but did exhibit significantly different y-intercepts (P<0.001). (B) Regression lines for PNG hydrolytic activity in the total membrane fraction did not have significantly different slope (P=0.637), but did exhibit significantly different y-intercepts (P=0.007). and AIN-93G diets yielded increased plasma and liver PLP concentrations as dietary PN increased. Liver, but not plasma, PLP concentrations appeared to be influenced by another dietary component in addition to PN since the y-intercept of the regression line for the AIN-76A diet was significantly higher than the AIN-93G diet.

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35 The absence of a robust effect of dietary PN on cytosolic PNG hydrolytic activity was contrary to earlier work done in this laboratory which showed an inverse relationship between dietary PN and cytosolic PNG hydrolase specific activity toward PNG in adult rats (13). In that study (13), mucosal cytosolic PNG hydrolytic activity in rats fed a purified diet (AIN-76A) providing 0 mg/kg PN was twice that measured in rats fed 2 mg/kg PN. Similarly, Banks et al. (12) reported the same magnitude of change in mucosal cytosolic PNG hydrolase activity, using PNG as a substrate, in guinea pigs fed diets providing 0 and 3 mg/kg dietary PN. Rats in the present investigation were chronically fed purified diets (either AIN-93G or AIN-76A) providing a wide range of dietary PN concentrations, but exhibited no significant increase in in vitro cytosolic PNG hydrolase specific activity with decreasing dietary PN concentration. However, rats that were pair-fed an adequate concentration of PN (7 mg/kg) exhibited a mean PNG hydrolytic activity in the brush border membrane that was twice that measured in rats fed 7 mg/kg PN ad libitum in study 1 . This was likely to be a PN-independent effect of food intake that might have altered overall rates of protein synthesis or degradation. The combined data from the two studies indicate a trend for increasing dietary PN concentration to increase PNG hydrolytic activity in not only the cytosolic fraction, but also the brush border membrane subcellular fraction of intestinal mucosa. Basal diet composition was speculated to have an effect on the vitamin B-6 nutritional status and possibly intestinal enzymatic activities, which led to the comparison of AIN-76A and AIN-93G purified diets. The predominantly starch-based carbohydrate component of AIN-93G may be a more favorable energy source for metabolically active microflora that synthesize vitamin B-6, assuming that some of the starch is not fully hydrolyzed in the rat small intestine. Under these conditions, the microflora could make more vitamin B-6 available for absorption, and further, improve the vitamin B-6 nutritional status of a rat not receiving an adequate amount of dietary vitamin B-6. Rats fed the

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36 AIN-93G diets had a significantly higher final body weight gain than rats fed AIN-76A, regardless of PN concentration. This suggested that there might be an effect of basal diet composition on the nutritional status of growing rats. A recent comparison of the AIN-76A and AIN-93G diets found only gastric hisotpathological differences in rats fed the purified diets, with more pathologies occurring in rats fed the AIN-76A diet (120). No weight differences between rats fed either the AIN-93G or AIN-76A diets were detected; however, rats examined by Lien et al. (120) were at least 3 times larger and more mature than the weaning rats used in our study. There is a possibility that rapidly growing (weaning) rats respond differently to the two basal diet formulations. Results from study 2 also indicated that changes in mucosal PNG hydrolytic activity in the rat small intestine are influenced by basal diet composition and not only dietary PN. Rats fed the AIN-93G diet had significantly greater cytosolic and total membrane hydrolytic activity than rats fed AIN-76A, regardless of PN concentration. However, rats in study 2 that were fed AIN-76A exhibited a positive relationship between cytosolic PNG hydrolase activity and dietary PN concentration. In contrast, this effect was absent in the rats fed the AIN-93G. These results indicate that the in vitro hydrolysis of PNG by rat intestinal mucosa is actually increased with increasing dietary PN concentration and may be further influenced by other dietary components that affect the small intestine in general (e.g. protein synthesis, processing, or degradation). In the present study, we were interested in determining the subcellular distribution of PNG hydrolytic activity in intestinal mucosa and potential effects of diet composition on this distribution. Our laboratory has recently discovered that the intestinal brush border membrane hydrolysis of PNG is catalyzed by LPH (106), which complements the intracellular hydrolysis of cytosolic PNG hydrolase reported previously (14). To make direct comparisons of PNG hydrolytic activities among subcellular fractions obtained from intestinal mucosa, in vitro activity from study 1 was expressed

PAGE 45

37 relative to mucosal weight. The greatest PNG hydrolytic activity was detected in the crude homogenate, which is comprised of all subcellular fractions. Activity measured in the total and brush border membrane fractions was greater than the activity measured in the cytosolic regardless of dietary PN concentration. Since our isolation of brush border membrane was incomplete, we adjusted the PNG hydrolytic activity in the brush border membrane to reflect 100% recovery. We found that 50-60% hydrolytic activity toward PNG in the intestinal mucosa was localized to the brush border membrane of rat intestinal mucosa. Similarly, we found that nearly all of the activity measured in the total membrane fraction could be attributed to the brush border membrane. Although the primary function of LPH is to catalyze the hydrolysis of dietary lactose, this laboratory has found it to hydrolyze PNG, and further evidence of secondary substrates for LPH has been recently reported (121). The results from the present study, along with our recent kinetic analysis of LPHcatalyzed PNG hydrolysis (106) and partial characterization of the cytosolic PNG hydrolase (14), we have constructed a model to explain the intestinal absorption and processing of dietary PNG. A fraction of the PNG that enters the intestinal lumen may not be absorbed at all and, consequently, may be excreted in the feces. PNG also may be absorbed as the intact glucoside and excreted in the urine unchanged or undergo intracellular hydrolysis by cytosolic PNG hydrolase, which would release free PN. Alternatively, PNG could be hydrolyzed at the brush border membrane by LPH and absorbed as free PN and glucose. In addition to the partial hydrolysis of PNG that occurs in the intestine, there is evidence of hydrolysis in other organs. An investigation of PNG bioavailability in humans, using stable isotopically labeled [ 2 H]-PNG, reported the recovery of the label in the vitamin B-6 urinary metabolite, 4-PA, from an intravenous dose, which suggested that some PNG was hydrolyzed outside of the intestine (10). These data are further supported by the observations of Nakano and Gregory (13). In

PAGE 46

38 vitro assays of PNG hydrolytic activity found that rat kidney, along with the intestinal mucosa, was able to partially hydrolyze PNG. Overall, these data have advanced our understanding of the absorption and metabolism of PNG.

PAGE 47

CHAPTER 3 ENZYMATIC HYDROLYSIS OF PYRIDOXINE-5'-p-D-GLUCOSIDE IS CATALYZED BY INTESTINAL LACTASE-PHLORIZIN HYDROLASE Introduction A significant dietary source of vitamin B-6 for humans is provided by a glycosylated form of the vitamin, pyridoxine-5'-p-D-glucoside (PNG). PNG, found only in foods of plant origin, provides a mean of 15% of total vitamin B-6 in a mixed diet, depending on food selection (30,64). PNG exhibits an approximate 50% bioavailability in humans (10,1 1) and 25-30% in rats (6-9) relative to pyridoxine, the metabolically usable form of vitamin B-6. The rate-limiting step in the utilization of PNG is not its intestinal absorption, but rather the enzymatic hydrolysis of the p-glucosidic linkage to release glucose and pyridoxine in the intestine (6-8). Although PNG can be absorbed intact, this form is not metabolized or retained by the liver (6-8) and can antagonize the metabolism of nonglycosylated forms of vitamin B-6 (5,63). The remainder of PNG that is not absorbed is likely to be accounted for through fecal losses. This laboratory has examined the intracellular, i.e. cytosolic, hydrolysis of PNG in mammalian small intestinal mucosa. Intestinal cytosolic PNG hydrolysis was initially thought to be catalyzed by a broad specificity p-glucosidase (BSpG) (EC 3.2.1 .21) (66), a cytosolic enzyme found in the intestine and liver (12,13,104). Cytosolic PNG hydrolysis was also reported to be inversely related to vitamin B-6 nutritional status in rodents (12,13). To further study cytosolic PNG hydrolysis, this laboratory purified BSPG from pig intestinal mucosa, which led to the identification and subsequent purification of a distinct and novel cytosolic p-glucosidase, designated pyridoxine-5'-P-D-glucoside 39

PAGE 48

40 hydrolase (PNG hydrolase)(14). It was found that PNG hydrolase, not BS(3G, was responsible for the cytosolic cleavage of PNG. Partial characterization of cytosolic PNG hydrolase revealed its ability to hydrolyze PNG as well as lactose and cellobiose, but not sucrose (14). Partial amino acid analysis revealed substantial sequence homology, but significant differences between PNG hydrolase and the brush border membrane enzyme lactase phlorizin hydrolase (LPH) (EC 3.2.1.23, 62, and 108)(74,122), the enzyme primarily responsible for the hydrolysis of dietary lactose (C.W. Tseung, L.G. McMahon, and J. F. Gregory, unpublished). A recent reassessment of mucosal PNG hydrolase activity in the rat small intestine indicated that -50% of the activity associated with the total membrane fraction could be attributed to enzymatic activity in the brush border membrane (A. D. Mackey, et al., unpublished). Hydrolysis of glucosides and oligosaccharides by enzymes present in the intestinal brush border often precedes the absorption of individual monosaccharides or the aglycone of glycosylated molecules, such as flavonoid and isoflavone glucosides. Day and colleagues (121) found that quercetin, genistein, and daidzen (3-glucosides were hydrolyzed by purified sheep LPH prior to absorption. LPH is the only mammalian brush border P-glucosidase; thus, we hypothesize that LPH is responsible for the hydrolysis of PNG, a P-glucoside. LPH has two distinct catalytic active sites: one for the hydrolysis of lactose and flavonoid glucosides and another, phlorizin hydrolase, for the hydrolysis of phlorizin and p-glucosylceramides (75,123). As reported by Day et al. (121), the site of catalysis for flavonoid and isoflavonoid glucoside hydrolysis was experimentally assigned to the lactase active site, not the phlorizin hydrolase site. The kinetic characterization of LPH-catalyzed hydrolysis of PNG by purified rat LPH is reported herein including the use of lactose as a substrate for comparison of

PAGE 49

41 catalytic properties. We also report the in vitro formation of a PN-disaccharide, which is evidence of a transferase activity for mammalian LPH. Materials and Methods Materials. Pyridoxine (PN) hydrochloride, lactose, glucose, phloridizin (phlorizin), phloretin, Sephacryl S400, Sephacryl S200, 2',4'-dinitrophenyl-2-fluoro-2deoxy-P-D-glucopyranoside (2F-DNPGIc), D-glucal, protease inhibitor cocktail, goat-antirabbit IgG horseradish peroxidase antibody, and a glucose detection kit were obtained from Sigma Chemical Co. (St. Louis, MO). Prestained protein molecular mass markers were obtained from Invitrogen (Carlsbad, CA). DEAE cellulose was obtained from Whatman (Clifton, NJ), and Simply Blue SafeStain was purchased from Invitrogen (Carlsbad, CA). Maleimide-activated keyhole limpet hemocyanin (KLH) was obtained from Pierce (Rockford, IL). Polyvinyldifluoride (PVDF; Immobilon-P) was obtained from Millipore Corporation (Bedford, MA). ECL Plus chemiluminescent reagent was purchased from Amersham-Pharmacia Biotech (Buckinghamshire, England). Pyridoxine-5'-p-D-glucoside was prepared by biological synthesis from pyridoxine using germinating alfalfa seeds and purified chromatographically (13,1 10). Purification of lactase phlorizin hydrolase (LPH). LPH was purified according to the Triton X-100 solubilization method of Wacker et al. (75) with the exceptions of Sephacryl S200 substituted for Sephadex G-200 and omission of the final anti-lactase affinity purification column. During this purification, all materials were kept on ice or at 4 °C. Brush border membrane was isolated from rat intestine for the purification of LPH according to the method Kessler et al. (1 1 1 ) with some modifications (1 1 2). Weanling male and female rats (Hsd:Sprague-Dawley, Harlan, Indianapolis, IN) (n=45) were used for the purification of LPH. Small intestine was harvested, flushed with 0.9% (w/v) NaCI, cut longitudinally, and mucosa was scraped with a glass slide, yielding approximately 20

PAGE 50

42 g of total mucosa. Electrophoretic characterization of the purified enzyme was done under denaturing conditions using 8% (w/v) polyacrylamide gels according to Laemmli (124) using a mini-gel electrophoresis apparatus from Novex (Invitrogen; Carlsbad, CA). A commercially available prestained protein molecular mass standard ranging from 10173 kD was used. Gels were stained with Simply Blue SafeStain for 1 h followed by destaining overnight in distilled, deionized water. Antibody production and Western blot analysis. The peptide CTLFHFDLPQALEDQG was synthesized with an additional cysteine at the carboxylterminus and verified by mass spectrometry amino acid analysis by Research Genetics (Huntsville, AL). This peptide was conjugated at the terminally added cysteine residue to maleimide-activated keyhole limpet hemocyanin (KLH) according to manufacturer instructions. KLH-conjugated peptide was injected into New Zealand White Rabbits (Cocalico Biologicals, Reamstown, PA) for production of polyclonal antiserum. Immunoblotting, or Western analyses, were done as described by Harlow and Lane (125) . Purified LPH protein was resolved on an 8% (w/v) SDS-PAGE at 130 V for 2 h. The gel was electroblotted to polyvinyldifluoride (PVDF) for 2.5 h at 12V in a transfer buffer containing 192 mmol/L glycine, 25 mmol/L Tris/ 0.05% SDS/10% methanol. The blot was stained with Amido black and destained. For the protein detection, the stained blot was cut into strips for different antisera treatments. Primary antibody treatments included whole immune serum, pre-immune serum, and peptide-competed serum. The peptide-competed serum was prepared by incubating the whole immune serum (0.5 mL) with 10 ng of peptide at room temperature for 1 h prior to application to the blot. The strips were blocked in phosphate buffered saline (PBST) (137 mmol/L NaCI/2.7 mmol/L KCI/5.4 mmol/L Na 2 HP04/1-8 mmol/L KH 2 PO4/0.05% (v/v) Tween-20) containing 5% (w/v) nonfat dry milk (NFDM) for 1 h. Primary antibody treatments were applied at 1 :500 dilution (in PBST-NFDM) followed by washing with PBST without NFDM and subsequent

PAGE 51

43 addition of the secondary antibody (goat anti-rabbit IgG horseradish peroxidase conjugate) at 1 :5000 dilution. Unbound secondary antibody was removed by washing with PBST (no NFDM). A chemiluminescent/fluorescent substrate (ECL-Plus) was then applied and incubated at room temperature for 5 min. The excess substrate was blotted away and protein bands were visualized by fluorescence emission (Storm 840 fluorescent optical scanner; Amersham-Pharmacia Biotech; Sunnyvale, CA). Enzyme activity assays. The standard assay for PNG hydrolytic activity was performed according to Nakano and Gregory (13). Standard activity assays for PNG hydrolysis were performed in a reaction mixture containing 10 |Kj purified LPH, 0.25 mmol/L PNG in 80 mmol/L sodium phosphate, pH 6.0 that was incubated at 37 °C for 1 h. Pyridoxine release was measured using reverse-phase HPLC with fluorometric detection (110). Lactase activity was measured according to Dahlqvist (96) with modification of the 50 mmol/L maleate buffer to a 50 mmol/L sodium phosphate, pH 6.0 assay buffer. Standard activity assays for lactose and other disaccharide hydrolysis were done using 25 mmol/L disaccharide. Glucose release was quantified using a reagent kit based on the glucose oxidase reaction with colorimetric detection. Phlorizin hydrolase activity was measured by reverse phase HPLC with UV detection of the aglycone, phloretin (126). Standard assay mixtures contained 100 mmol/L phlorizin in 50 mmol/L sodium citrate, pH 5.9. All activity assays were conducted under conditions that allowed measurement of initial rate. For kinetic studies, reactions involving LPH-catalyzed hydrolysis of PNG and lactose were conducted at various concentrations as specified in text or tables. Experiments examining active site inhibition, along with selective active site protection using 2F-DNPGIc and a glycal, D-glucal, were conducted as described by Day et al. (121) and Arribas et al. (123). Other potential inhibitors as specified were added to standard reaction mixtures without pre-incubation of enzyme. Kinetic constants (K m ,

PAGE 52

44 V maxi and Ki) were calculated by nonlinear regression using SigmaPlot Enzyme Kinetics Module, v 1.0 (SPSS, Inc., San Rafael, CA) software. Protein concentration was determined by a colorimetric method (114) using bovine serum albumin as the standard. Liquid chromatography-mass spectrometry (LC-MS). To obtain molecular mass and fragmentation information regarding a novel reaction product formed during incubation of purified LPH with PNG and lactose, LC-MS and LC-MS/MS analyses were performed in atmospheric pressure chemical ionization (APCI) mode (LC: Hewlett Packard model 1 100; Agilent; Palo Alto, CA and MS: Thermo Finnigan model TSQ7000; San Jose, CA). The HPLC separation was done isocratically with 5 mmol/L ammonium acetate with 0.1% (v/v) acetic acid as the mobile phase at a flow rate of 0.6 mL/min with a reverse-phase column (C18 Phenosphere, 50 x 4.6 mm, 5 ^m column; Phenomenex; Torrance, CA). The analysis was conducted by monitoring ions from 75-600 m/z. For the MS-MS analysis, a collision energy of 30 V was used to fragment the parent mass (M+1 ) of m/e 494. Results Lactase phlorizin hydrolase (LPH) purification. LPH from rat small intestinal mucosa was purified 300-fold relative to the lactase activity measured in mucosal crude homogenate. The enzyme was purified to near homogeneity and the purification repeated with similar results. The approximate molecular mass of purified LPH by SDSpolyacrylamide gel electrophoresis was 135-140 kDa. In the published method used for purification, an anti-lactase antibody affinity column was used to further separate LPH from aminopeptidase N (MW 145 kD) (75). This investigation did not use an affinity chromatography purification step to remove peptidase activity. Although aminopeptidase N may not have been completely separated from the final purified LPH, the enrichment of lactase activity from the crude homogenate was consistent with that

PAGE 53

45 reported in the purification method (75). Staining of the SDS-PAGE revealed two major bands of protein: one at -135-140 kD and another at -200-220 kD (Figure 3-1 A). The band of protein at 135-140 kD corresponds to mature LPH. The band at 200-220 kD is consistent with the MW of a precursor form of LPH (91 ). Western analyses were done to confirm further the relative purity of the enzyme preparation (Figure 3-1 B). The peptide used for antibody production had amino acid sequence homology with regions in the precursor and mature forms of LPH, but no other mammalian intestinal enzymes cataloged in Swiss-Prot and GenBank databases. No protein bands were detected in the blot strips incubated with PBST-NFDM alone and the secondary antibody alone (lanes 1 and 2, respectively). The whole immune serum-treated strip revealed three immunogenic protein bands (lane 3), which were not visible in the peptide-competed antibody treatment (lane 4). The fourth visible band (-60 kD) was found to be an artifact of the antibody under the conditions of this analysis. The band at -80 kD detected by the whole immune serum was also detected by the pre-immune serum, which indicates that the recognition of the band by the whole immune serum was not sequence specific (lane 5). The protein bands at 135-140 kD and 220-220 kD that were recognized by this polyclonal antibody appeared to be the mature and precursor forms of LPH. Upon closer inspection of the Coomassie-stained gel, trace amounts of additional high molecular mass bands were observed. Further assays for disaccharidase activity of the purified LPH preparation revealed residual maltase-glucoamylase (EC 3.2.1.20 and 3) at a specific activity of 3.8 |imol/min*mg protein. This activity was controlled for in later kinetic characterization studies.

PAGE 54

46 Kinetic characterization. Purified LPH from rat small intestinal mucosa catalyzed the hydrolysis of PNG and followed Michaelis-Menten kinetics. Values for K m , V max , and k cat were lower when PNG was used as the substrate as compared to lactose. Values for kcat and V max /K m indicated that lactose hydrolysis was catalyzed more efficiently than the hydrolysis of PNG (Table 3-1). A B 20 \ig LPH marker lane Figure 3-1 . Purification of LPH from rat intestine. (A) SDS-PAGE of purified LPH preparation. Gel (8% w/v acrylamide) has two lanes: one large lane containing enzyme preparation and another for the molecular mass marker. Purified enzyme (20 l^g) was loaded into one large well. Enzyme preparation was electrophoresed at 130 V for 2 hours. Gel was stained with commercially available formulation of Coomassie blue stain (SimplyBlue SafeStain, Invitrogen). Molecular weight markers ranged from 10-173 kDa. Protein band at 135-140 kDa corresponds to LPH. (B) Western analysis of purified LPH preparation. Protein (20 jag) was separated by SDS-PAGE (8% w/v acrylamide) and transferred to PVDF membrane. Membrane was cut into strips and subjected to various antibody treatments. Lane 1 : No antibody applied; Lane 2. Secondary antibody (goat-anti-rabbit HRP) only; Lane 3: Whole immune serum and secondary antibody; Lane 4: Peptide-competed serum and secondary antibody; Lane 5: Pre-immune sera and secondary antibody. Application of chemiluminescent/fluorescent substrate reagent (ECL Plus) was followed by detection using a fluorescent optical scanner.

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47 Table 3-1 . Kinetic parameters of purified rat lactase phlorizin hydrolase (LPH) using pyridoxine-5'-P-D-glucoside (PNG) and lactose as substrates. Substrate PNG Lactose 1.0 ± 0.1 16 ±1 (mmol/L) ^max 0.11 ±0.01 5.0 ±0.1 (nmol/min*mg LPH) kcat (s1 ) 1.0 47 Koat/Km (mmol/LV) 1.0 2.9 Vmax/K m 0.11 0.31 Inhibition studies. The substrate PNG exhibited substrate inhibition at concentrations greater than 3 mmol/L (Figure 3-2). Due to limited availability of PNG, only concentrations ranging from 0.01 K m to 5K m were tested. The reaction products (glucose and pyridoxine), lactose, and other disaccharides were also tested for inhibitory properties. Pyridoxine (0.5-10 mmol/L) did not inhibit LPH-catalyzed PNG hydrolysis, but the monosaccharide, glucose inhibited the reaction at concentrations £ 25 mmol/L; K, equal to 64 ± 5 mmol/L. The primary substrate for LPH, lactose (25-100 mmol/L), inhibited PNG hydrolysis competitively with a K equal to 56 ± 8 mmol/L (Figure 3-3). This suggests that the two substrates (PNG and lactose) are hydrolyzed at the same active site. Cellobiose, maltose, lactose, sucrose, and trehalose (25-100 mmol/L) were also added individually to standard reaction mixtures containing PNG (0.01-0.25 mmol/L) and purified LPH. While sucrose and trehalose did not affect PNG hydrolysis, cellobiose and maltose inhibited the enzymatic release of PN. The inhibition of PNG hydrolysis by

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48 250 § 200 1 2 Q. 5? 150 E 100 c 50 o > D PURIFIED LACTASE O MUCOSAL BRUSH BORDER 0 0 1 2 3 4 5 6 [Pyridoxine-5'-|3-D-glucoside], (mM) Figure 3-2. Effect of substrate concentration on the activity of LPH-catalyzed PNG hydrolysis. Comparison of rat small intestine brush border membrane and purified LPH is shown. maltose was reduced by 35-40% in the presence of 2.0 |j.mol/L 1-deoxynojirimycin, an inhibitor of maltase-glucoamylase activity (127). Identification of site of PNG hydrolysis by LPH. The inhibitor 2F-DNPGIc binds to both the lactase and phlorizin hydrolase active sites, inhibiting both hydrolytic activities (128,129). The lactase activity was preserved by pre-incubation with D-glucal, which yields reversible blockage of the lactase active site (123). Subsequent addition of 2F-DNPGIc to the glucal-modified LPH leads to the selective inhibition of the phlorizin hydrolase active site (123). This experimental protocol allowed the contribution of each activity from the respective active sites to be calculated (123). Hydrolysis of PNG and lactose was reduced 100-fold in the presence of 2F-DNPGIc without glucal as compared to the uninhibited enzyme. Phloretin release was reduced by 85-90% in the presence of 2F-DNPGIc with or without glucal as compared to the activity of the free enzyme. Incubation of purified LPH with glucal prior to the addition of 2F-DNPGIc maintained the hydrolytic activities toward lactose and PNG to 50-65% of the uninhibited enzyme

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49 i ~ 1 1 1 i -10 0 10 20 30 40 1/[Pyridoxine-5'-(3-D-glucosicle], (rnlvl)" 1 Figure 3-3. Lineweaver-Burk and Michaelis-Menten (inset) plots of LPHcatalyzed hydrolysis of PNG with increasing amounts of lactose as inhibitor. (Figure 3-4). These findings strongly suggest that PNG was hydrolyzed at the lactase active site of LPH. Formation of other pyridoxine glycosides. HPLC analyses of enzyme reaction mixtures containing LPH, PNG (substrate), and lactose (inhibitor) detected not only the product, PN, and the substrate, PNG, but also another fluorescent compound with a retention time of 5.3 min (Figure 3-5). The amount of this compound formed was dependent on the concentration of lactose. Initial LC-MS (APCI positive ion mode) analysis of the enzyme reaction mixture showed the unidentified compound to have M+1 ion of 494 (m/z), corresponding to a disaccharide of pyridoxine (Fig 3-6). This was

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50 1.0 -1 £ 0.8 I — 1 — o E 0.6 c Z 0.4 , I Q. > 0.2 o.o -I 1 1 1 ' ' 1 ' ' 1 inhibitor glucal+inhibitor no inhibitor Figure 3-4. Identification of site of PNG hydrolysis by LPH. Protection of active site with D-glucal prior to addition of 2F-DNPGIc (inhibitor) selectively inhibited phlorizin hydrolase active site. Completely inhibited enzyme (with 2F-DNPGIc) exhibited 10% the PNG hydrolytic activity of the glucal-protected enzyme. further supported by an MS/MS experiment on the M+1 ion using a collision energy of 30V. In addition to the residual M+1 ion of the disaccharide, major fragments were observed at m/z 332 and 170, corresponding to the M+1 ions of pyridoxine glucoside and pyridoxine. The LC-MS and MS/MS experiments produced the same results when the HPLC purified disaccharide from the enzyme reaction mixture was utilized. This pyridoxine disaccharide has a pyridoxine molecule with the 5'-(3-D-glucoside and another glucose or galactose moiety. Similar to the observation of the pyridoxine disaccharide formed in the presence of lactose, two other disaccharides tested in the LPH inhibition experiments, cellobiose and maltose, also formed fluorescent compounds which exhibited retention times different from that formed with lactose. These compounds, while detectable, were minor in relation to the amount of the PN-disaccharide formed in the presence of lactose and were independent of disaccharide concentration. Discussion The finding of PNG as a novel substrate for LPH has extended our understanding of mechanisms governing vitamin B-6 bioavailability and has added to the

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51 1000PNG § E PN AJ 5 Minutes I 10 Figure 3-5. Sample chromatograms of enzyme reaction mixtures of LPHcatalyzed PNG hydrolysis. (A), Chromatogram from LPH-catalyzed PNG hydrolysis without added lactose. (B), Chromatogram from LPH-catalyzed PNG hydrolysis with added lactose (25 mM). Retention times: pyridoxine (PN), 3.3 min; pyridoxine-5'-P-D-glucoside (PNG), 4.3 min; pyridoxine disaccharide (PNdisaccharide), 5.3 min. Assays were conducted using purified rat LPH. growing evidence that LPH catalyzes the hydrolysis of not only lactose, but other |3glucosides, such as plant p-glucans and flavonoid and isoflavonoid glucosides (121,130). Dietary PNG exhibits only 50-60% of the bioavailability of free PN as a source of vitamin B-6 for humans (10,1 1), which is due to incomplete hydrolysis of the glycosidic linkage of PNG. The results reported in the present study suggest that the digestion of PNG may commence at the intestinal brush border where LPH can catalyze the hydrolysis of PNG releasing free PN, which is then passively absorbed. This extends our previous findings of the hydrolysis of PNG that can occur immediately after

PAGE 60

52 100 95^ 90 85 80 75^ 70? 651 ech 5K 5K 45^ 40 : 35 30 : 25 20' 15 10 5RT: 5.37 0 1 t] W < r ii>>: "m i I I 23456789 10 11 Time (min) 10095-90 85 ! 80 i 75: 7tf 651 60| 55: 50: 45: 40i 35 30: 251 20-\S 10; 5332 1,08 136 AW 314 i' V I ' I'I ^ I p V l l'| I ' I ' I I | I N l '| I I 1 1 I | * 1 f i'| I 1 100 150 200 250 300 350 400 450 500 m/z Figure 3-6. LC-MS/MS analysis, in the APCI positive ion mode (MS/MS experiment on the ion at m/e 494; collision energy, 30 V), of the PN-disaccharide product formed during the incubation of LPH with PNG and lactose. The predominant ions are parent molecular ion (M+1) at m/e 332, and M+1 ion of pyridoxine at m/e 170. The other significant ions at m/e 476, 314, and 152 are produced by the loss of water from the respective M+1 ions. absorption into the cytosolic compartment by PNG hydrolase. This LPH-catalyzed hydrolysis may be an important determinant of the bioavailability of PNG. In populations with lactase deficiency, the hydrolysis, and hence, nutritional utilization of PNG may be less efficient than in populations that exhibit lactase persistence. LPH was purified from 20d old rats to insure reasonable lactase activity with an adequate amount of starting tissue. The recognition of both the mature and precursor forms of LPH by our antibody in the Western analysis allowed us to verify the qualitative abundance of our purified enzyme. This recognition was sequence specific since the pre-immune serum and peptide-competed antibody did not detect the high molecular mass bands that were recognized by the whole immune serum. Values of K m and V max

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53 (lactose) calculated for purified rat LPH were consistent with published values (73,126)(Table 3-1). The kinetic parameters (for lactose and PNG) appeared to be representative of the concentrations of substrates that would be encountered physiologically by LPH. However, under typical dietary conditions, the amount of PNG in digesta entering the small intestine would be in the micromolar concentration range, while the concentration of lactose (millimolar range) would greatly exceed that of PNG. The relatively high K m value for PNG (1 .0 mmol/L) suggests that LPH has the capacity to hydrolyze physiological concentrations of PNG, but fractional saturation of substrate of the enzyme would be low. Similarly, kinetic ratios also indicated that LPH favored the hydrolysis of lactose over PNG. Although LPH-catalyzed PNG hydrolysis was inhibited by one of its products, glucose, the concentration at which inhibition occurred may not be physiologically relevant (> 25 mmol/L). In accounting for the stoichiometry of the hydrolytic reaction of PNG, the amount of glucose released (micromolar concentration) would not be sufficient to cause product inhibition. However, the glucose released from the hydrolysis of other disaccharides such as lactose, may contribute to the observed inhibition by glucose. The observed competitive inhibition of lactose on LPH-catalyzed PNG hydrolysis is consistent with the fact that PNG and lactose are hydrolyzed at the same active site. The inhibition may not have a dramatic effect on the bioavailability of PNG since foods that contain PNG are of plant origin, which are devoid of lactose. Unless a dairy product and plant-derived food were consumed together, PNG should be at least partially hydrolyzed by LPH. The Ki for lactose (56 ± 8 mmol/L) did not agree with the calculated K m for lactose (16 ± 1 mmol/L). One possible explanation for this discrepancy may be in the formation of alternative pyridoxine glucosides. Some PNG may be diverted from its P-glucosidic hydrolysis process to a trans-glycosylation reaction that yields a pyridoxine disaccharide and a reduction in free PN. Alternatively, PNG may be hydrolyzed to

PAGE 62

54 liberate PN, which may undergo enzymatic modification to form PNG or PNdisaccharide. The mechanism of the hydrolysis/trans-glycosylation was not examined further in this study. Cellobiose, while not present in appreciable amounts in the human diet, also is a substrate for LPH (74,131); therefore, it was not unexpected that this p-linked disaccharide would also inhibit PNG hydrolysis. The p-disaccharide, maltose also inhibited the release of PN by LPH, which was not anticipated. The subsequent detection of maltase-glucoamylase activity (EC 3.2.1 .20 and 3) (80) as a contaminant of the LPH preparation is consistent with the presence of minor high molecular mass protein bands (200-220 kDa) faintly stained in the SDS-PAGE. Western analysis using our antibody that was sequence specific to LPH, but not maltase-glucoamylase, showed that the more prominently stained high molecular mass band was likely to be a precursor form of LPH and not maltase-glucoamylase. Despite the relatively low abundance of the maltase-glucoamylase contaminant, its hydrolytic activity toward maltose had an effect on LPH-catalyzed PNG hydrolysis. What appeared to be the inhibition of PN release, may actually have been the LPH-catalyzed formation of another maltoseor glucosederived pyridoxine oligosaccharide from PN or PNG, or both. Alternatively, the hydrolysis of maltose releases monosaccharide units (D-glucose) which could inhibit PNG hydrolysis as catalyzed by LPH. Maltase-glucoamylase activity was reduced by 63% in the presence of 2.0 1-deoxynojirimycin without affecting lactose hydrolysis. In reaction mixtures containing LPH, PNG, maltose (>25 mmol/L), and deoxynojirimycin, the release of PN was increased by an average of 35% and the formation of other pyridoxine disaccharides was decreased compared to reactions without deoxynojirimycin. Increasing the concentration of 1-deoxynojirimycin could have likely negated this inhibition; however, at higher concentrations, deoxynojirimycin also is a

PAGE 63

55 competitive inhibitor of lactase (75). Although maltase-glucoamylase was present in the purified LPH preparation, maltase-glucoamylase appears not to alter the kinetics of PNG hydrolysis in the absence of (3-linked disaccharides. This is further supported by the inhibition experiment with p-glucosidase inhibitor 2F-DNPGIc. When LPH is completely inhibited with this fluoroglucoside, the release of PN is negligible, indicating that maltaseglucoamylase alone is not capable of hydrolyzing PNG. Maltase-glucoamylase that had been co-purified with lactase by papain solubilization from 15d old rat intestine was reported to have a final specific activity of 36 |amol/min*mg protein in another study (73), which is approximately 10 times the specific activity measured in the current study. Fluoroglucosides are widely used as general inhibitors of P-glucosidases (129). 2', 4'-dintrophenyl-2-deoxy-2-fluoro-P-D-glucopyranoside (2F-DNPGIc) is a mechanismbased inhibitor of (3-glucosidases, and exerts its effect by forming a stable covalent linkage of the 2' position of the fluoroglucoside to glutamic acid residues at the active sites of LPH. The use of glucal in conjunction with the inhibitor in the protocol allows individual examination of the lactase and phlorizin hydrolase active sites on LPH (123). The deglycosylation of flavonoid and isoflavonoid glucosides was studied using this same experimental procedure (121 ). Inhibition of the phlorizin hydrolase site with 2FDNPGIc did not diminish the hydrolysis of the quercetin glucosides. Similar to the results reported for the flavonoid and isoflavonoid glucosides (121), we concluded that PNG is hydrolyzed at the lactase active site of LPH. Various pyridoxine glucosides, including several pyridoxine oligosaccharide derivatives, are known to exist widely in nature (132); however, the formation of pyridoxine disaccharides catalyzed by enzymes in the small intestine has not been reported. Future research will address the nutritional significance of LPH hydrolysis of

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56 PNG, with particular attention to the vitamin in human LPH expression and activity and the inhibition of PNG hydrolysis by lactose.

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CHAPTER 4 UPTAKE, HYDROLYSIS, AND METABOLISM OF PYRIDOXINE-5'-P -D-GLUCOSIDE IN A CELL CULTURE MODEL USING CACO-2 CELLS Introduction An important source of vitamin B-6 in the human diet is pyridoxine-5'-(3-Dglucoside (PNG) which is found in foods of plant origin. This glycosylated form provides approximately 15% of total vitamin B-6 intake in a typical mixed diet; however, this percentage can vary depending on food selection (30). The metabolic utilization of this form of vitamin B-6 is limited by its partial hydrolysis by P-glucosidases in the small intestine. Hydrolysis of PNG yields the products glucose and PN. PN can be metabolized to the coenzyme PLP. Relative to PN, PNG exhibits a 50% bioavailability in humans (10,1 1) and 25-30% in rats (7,8). The incomplete bioavailability of PNG is due to its limited hydrolysis in the small intestine. This laboratory previously reported the intestinal hydrolysis of PNG to be catalyzed by a novel intracellular p-glucosidase (14). The enzyme specifically hydrolyzed PNG and was later designated cytosolic pyridoxine5'-P-D-glucoside hydrolase (PNG hydrolase). More recently, this laboratory discovered that the brush border membrane P-glucosidase, lactase phlorizin hydrolase (LPH), also catalyzed the hydrolysis of PNG (106). LPH is the intestinal enzyme that is responsible for the hydrolysis of dietary lactose, a disaccharide that is important for energy derivation for developing mammals. Although kinetic analyses of purified rat LPH revealed that lactose was a better substrate than PNG, PNG was a secondary substrate for LPH. Further investigation of LPHcatalyzed PNG hydrolysis in vitro showed that lactose was a competitive inhibitor of 57

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58 PNG hydrolysis and therefore, the two substrates are hydrolyzed at the same active site. We speculated that the inhibition by lactose observed in vitro would have implications on the in vivo hydrolytic and absorptive processes of PNG. Intestinal hydrolysis of PNG in vivo might be reduced in the presence of lactose, which would reduce the bioavailability of PNG. Simultaneous consumption of plant-derived foods with dairy products could decrease the hydrolytic release of free PN and consequently, affect the vitamin B-6 nutritional status of the individual. There is also the potential for a reduced PNG bioavailability in lactose intolerant or lactase deficient individuals. Subcellular fractionation of intestinal mucosa revealed that 50-60% of the hydrolysis of PNG was localized to the brush border membrane in the rat small intestine, which was likely catalyzed by LPH (Mackey et al., unpublished). This accounts for a large proportion of the PNG hydrolytic activity present in the small intestine and thus, lactase insufficiency might also reduce PNG bioavailability through the reduction or complete loss of LPH hydrolytic activity toward PNG. In vivo absorption of vitamin B-6 has been extensively examined in the intestine of rats. Intestinal absorption of PN occurs by a non-saturable, passive diffusion process. PN is rapidly absorbed where it is subject to phosphorylation, forming pyridoxine 5'phosphate (PNP), which can be converted to pyridoxal 5'-phosphate (PLP) by PNP oxidase (133,134). Following the oxidation of PNP to PLP, the intestine dephosphorylates PLP to release PL into portal circulation (134). PNG hydrolysis occurs mostly in the small intestine to release PN, which is passively absorbed; however, PNG, as the glucoside, can be absorbed and excreted intact in the urine (10,13). Caco-2 human colon carcinoma cells were chosen as a model for the present investigation since these cells, when allowed to differentiate, express the morphology and many of the hydrolytic enzymes present in the intestinal brush border (including LPH) (135), which provides an environment that closely resembles the small intestine.

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59 While these cells express LPH, they were previously found not to exhibit cytosolic PNG hydrolase activity (McMahon, L.G & Gregory, J.F., unpublished data). Hydrolytic activity toward PNG measured in these cells could therefore be attributed solely to the activity of brush border LPH. The purpose of the present study was to examine the uptake and metabolism of PNG in the absence and presence of lactose in a cell culture model using the established Caco-2 human colon carcinoma cell line. Materials and Methods Materials. Vitamin B-6 compounds (except PNG), lactose, HEPES, D-glucose, and L-glutamine were purchased from Sigma Chemical Co. (St. Louis, MO). Cell culture grade NaCI, CaCI 2 , MgS0 4 , KCI, LiCI, hydroxymethyl aminomethane hydrochloride (TrisHCI), 2-(-4-morphlolino)-ethanesulfonic acid (MES), and plastic cell culture supplies were purchased from Fisher Scientific (Atlanta, GA). Cell culture media, media supplements, and trypsin-EDTA were purchased from Invitrogen (Carlsbad, CA). PNG was biologically synthesized and purified chromatographically (110). Cell culture. Caco-2 human colon carcinoma cells, passage 1 8, were obtained from American Type Culture Collection (Rockville, MD). Cells were propagated and maintained at 37°C (95% air, 5% C0 2 atmosphere) in DMEM containing 4.5 g/L glucose, 25 mmol/L HEPES, 44 mmol/L sodium bicarbonate, and 4 mmol/L glutamine. The growth medium was supplemented with 1 mmol/L sodium pyruvate, 100 ^mol/L nonessential amino acids, and 20% fetal bovine serum plus 100 U/L penicillin, 100 U/L streptomycin, and 50 ng/L gentamycin (136). For routine subculturing, cells were washed with Ca +2 and Mg +2 free phosphate buffered saline (PBS) and detached with 0.25% (w/v) trypsin with 1 mM EDTA. For uptake experiments, cells were plated on 3section 100 mm plates at 1.5 x 10 5 cells per section. Growth medium was changed every 2-3 days with a change 24 h prior to an uptake experiment. Uptake studies were

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60 performed on monolayers 7-9 days post-confluency, a time when lactase activity peaks in Caco-2 cells (137). Cells from passages 23-35 were used in the experiments. Uptake experiments. Uptake experiments were done by adding treatment media containing different concentrations of PNG to the top of the cell monolayer with and without lactose to the cells. Treatment medium was prepared with Krebs-Ringer buffer containing 123 mmol/L NaCI, 4.93 mmol/L KCI, 1 .23 mmol/L MgS0 4 , 0.85 mmol/L CaCI 2 , 5 mmol/L glucose, 5 mmol/L glutamine, 10 mmol/L HEPES, and 10 mmol/L MES at pH 7.4. Monolayers were washed 3 times with 2-3 mL Krebs-Ringer buffer (37 °C) prior to the addition of treatment media containing different concentrations of PNG and lactose. Treatment media were added to monolayers and incubated at 37 °C in 5% C0 2 atmosphere for times as indicated in the text. Prior to the termination of incubation, a sample of treatment medium was collected. Incubation was then terminated at the desired time by addition of 3 mL of Krebs-Ringer buffer (4 °C), followed by two additional washes. Cell monolayers were mechanically lifted from the plates with a sterile plastic spatula into 0.5 mL PBS and homogenized using a Polytron homogenizer at medium speed for 15 sec. An aliquot of crude homogenate was collected for protein measurement and the remaining crude cellular homogenate was centrifuged at 200,000 x g for 30 min to obtain a cytosolic subcellular fraction. Protein concentration was measured spectrophotometrically (114) using bovine serum albumin as the standard. Measurement of hydrolysis and uptake. Ratios of PN:PNG were calculated for the treatment media prior to and after each incubation period. Detection of PNG in the cytosolic compartment of the Caco-2 cells above that measured in cells receiving no additional PNG in the treatment medium was interpreted to be the amount of PNG taken up by the cells. Enzyme activity assays. PNG hydrolytic activity in the cytosolic fraction of Caco-2 cells was determined according to Nakano and Gregory (110) with modification

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61 of the assay buffer (106). Lactase activity was measured by a colorimetric assay as described by Dahlqvist (96) with some modification. Vitamin B-6 analyses. Treatment media were analyzed for PN and PNG both before and after incubation period using reverse-phase fluorometric HPLC (110). Intracellular concentrations of PN, PNG, PM, and PMP were measured using ion-pair reverse-phase HPLC with fluorometric detection (4). Intracellular concentrations of PL and PLP were measured by reverse-phase HPLC with fluorometric detection of PLand PLP-semicarbazones (109). Statistical analyses. Data are presented as means ± SEM of multiple experiments done at separate times and are expressed as pmol vitamin B-6 /mg protein. Differences between PN:PNG ratios in treatment medium from before and after incubation periods were detected using a one-way analysis of variance (115). Differences in intracellular concentrations of vitamin B-6 were examined using a oneway analysis of variance with Student-Newman-Keuls pairwise comparison test. Differences in PNG uptake and metabolism in the presence of lactose were analyzed by a two-way analysis of variance using concentrations of PNG and lactose as factors. Pairwise comparisons were performed using the Student-Newman Keuls test. The aforementioned statistical analyses were performed using SigmaStat statistical software (Jandel Corporation, San Rafael, CA). Results Uptake of PNG as a function of concentration. PNG uptake in Caco2 cells was examined at concentrations ranging from 0.5 nmol/L to 500 u.mol/L (Figure 41). After 30 min at 37 °C, PNG was first detected in the intracellular compartment of the cells at 5 ^mol/L, whereas no PNG was detected below this concentration. The intracellular concentration of PNG significantly increased (P<0.0001) with increasing

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62 concentrations of PNG in the incubation media over 30 min. PNG concentrations between 0-50 ^mo/L were taken up in a saturable process (Figure 4-1 A), while uptake of A .E 1.8-1 0 10 20 30 40 50 60 PNG Oxmol/L) B ^ 16-1 c 0 100 200 300 400 500 600 PNG (ujnol/L) Figure 4-1 . Pyridoxine-5'-(3-D-glucoside (PNG) absorption by Caco-2 cells as a function of concentration after 30 min incubation. Absorption of PNG is saturable at < 50 nmol/L (A) and non-saturable at >50 nmol/L (B). Each data point represents a mean of at least two replicates from 3 independent experiments. Data are presented as means ± SEM. PNG at concentrations greater than 50 nmol/L was passive (Figure 4-1 B). Uptake of PNG as a function of time. PNG uptake was tested at 1 , 2.5, 3, 5, 10, 15, 30, 60, and 120 min with 200 nmol/L PNG in the treatment media, which was a

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63 concentration that yielded a high intracellular concentration of PNG after a 30 min incubation (Figure 4-2). PNG uptake increased linearly up to 15 minutes and after that ^ 500 1 Time (min) Figure 4-2. Absorption of pyridoxine-5'-|3-D-glucoside (PNG) at 200 u.mol/L by Caco-2 cells as a function of time. Each data point represents at least two replicates from three independent experiments. Data are expressed as means ± SEM. appeared to plateau. Distribution of intracellular B-6 vitamers in Caco-2 cells. The determination of intracellular concentrations of vitamin B-6 in Caco-2 cells without added PNG revealed that most of the vitamin B-6 inside the cell was in the form of pyridoxine (Figure 4-3). Increasing concentrations of PNG in the treatment media did not significantly change the intracellular concentrations of PN, PLP, PM, or PMP. PNG hydrolysis by Caco-2 cells. Activity assays for lactase were done on the total membrane fraction isolated from ultracentrifugation (200,000 x g, 30 min) of Caco-2 cell crude homogenates. Lactase activity for Caco-2 cells between 7-9 days postconfluency was 332 ± 1 1 nmol/h*mg protein. PNG hydrolytic activity was not detected in the cytosolic fraction of Caco-2 cells. PNG hydrolysis by lactase was measured by the comparison of PN:PNG ratios in the treatment media on top of the cells before and after the incubation period (Table 4-1). PN:PNG ratios did not significantly change from pre-

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64 to post-incubation in experiments using 25, 50, or 100 jxmol/L PNG in the treatment media. 140 .E 120 0) o a. 100 O) E 3 80 E a CD i CQ c £ 60 F 40 20 0 PN PLP PL PM PMP Figure 4-3. Distribution of intracellular concentrations of vitamin B-6 in Caco-2 cells without added PNG. Abbreviations: pyridoxine (PN), pyridoxal-5'-phosphate (PLP), pyridoxal (PL), pyridoxamine (PM), and pyridoxamine-5'-phosphate (PMP). Bars represent means ± SEM from at least 3 replicates from 4 independent experiments. Effect of lactose on PNG uptake and metabolism. The effect of lactose on PNG uptake and metabolism was examined by incubating cell monolayers with 0, 25, 50, 100, and 200 nmol/L PNG with 0, 75, or 150 mmol/L lactose for 30 min (Figure 4-4). Intracellular PNG concentration significantly increased as the concentrations of PNG added to the cells increased. The presence of lactose, at any concentration, did not significantly change the intracellular concentration of PNG. However, intracellular concentrations of PL were significantly lower (P=0.01) in cells incubated with 75 and 150

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65 Table 4-1 . Pyridoxine to pyridoxine-5'-|3-D-glucoside (PN:PNG) ratios in treatment media before and incubation period. PN:PNG PN:PNG PN:PNG PNG (umol/L) pre-incubation 1 post-incubation Mean change 25 0.0020 ± 0.0003 0.0025 ± 0.0005 0.0006 ± 0.0006 50 0.0018 ±0.0001 0.0020 ± 0.0002 0.0462 ± 0.0460 100 0.0017 ±0.0001 0.001 7 ±0.0003 0.0004 ± 0.0002 1 1ncubations were done for 30 min at 37 °C. Values within column were not signifcantly different and the post-incubation ratio was not significantly different from the preincubation ratio. 0 mM lactose 75 mM lactose 1 50 mM lactose 0 50 100 PNG (nmol/L) 200 Figure 4-4. Effect of lactose on the intracellular concentrations of pyridoxal (PL). Increasing concentrations of lactose significantly decreased the intracellular concentration of PL. Bars represent means ± SEM of at least 2 replicates from 2 independent experiments. mmol/L lactose than cells treated with no lactose. There were no significant differences in intracellular PLP concentrations in cells treated with lactose. Effect of sodium on PNG uptake. On the basis of the hydrophilic structure of PNG, one would predict that it would be difficult for the molecule to pass through the

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66 250 O 200 o E 150 O 100 E Q. o z 50 Sodium Potassium Lithium Tris Figure 4-5. Effect of sodium and other monovalent cations on the absorption of pyridoxine-5'-P-glucoside (PNG) in Caco-2 cells after a 30 min incubation. Bars represent means ± SEM of three replicates from 2 independent experiments. hydrophobic lipid bilayer of the intestinal epithelial cell plasma membrane. This suggests that the absorption of PNG could be facilitated by a carrier. To determine whether PNG uptake was mediated through a sodium-dependent transporter, the sodium in the Krebs-Ringer buffer was replaced by potassium, lithium, and Tris (hydroxymethyl) aminomethane-HCI to create a solution of equivalent ionic strength (Figure 4-5). PNG was added to each of the treatment media at 100 ^mol/L and uptake was measured over 30 min. Intracellular concentrations of PNG were not significantly different (P=0.39) among the cells incubated in the sodium, potassium, lithium, or Tris treatment media. Discussion The results from this study provide new insight into the intestinal absorption and metabolism of PNG. Vitamin B-6 uptake and metabolism were not examined in any intestinal epithelial cell culture prior to this investigation. Caco-2 cells function similarly

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67 to enterocytes in the small intestine, which permit the close examination of PNG uptake and metabolism. PNG was taken up by the Caco-2 cell monolayers in a pattern that is consistent with passive diffusion. However, at low concentrations of PNG (5-50 ^mol/L), the uptake of PNG displayed a saturable component. Intracellular concentrations of PNG increased linearly as the concentration of PNG (>50 nmol/L) provided to the cells increased. The range of PNG added to the cells encompasses an amount of PNG that would be consumed daily in the diet. Average dietary intake of vitamin B-6 by the U.S. population is 1 .7 mg/d (NHANES III). Although food selection ultimately determines the percentage of vitamin B-6 derived from PNG, PNG contributes approximately 15% of total vitamin B6 intake, which corresponds to -200 \ig PNG daily. This amount (-200 jag) divided equally over three meals yields the consumption of -65 ng for each meal. Caco-2 cells incubated with 100 pimol/L (-66 \ig) PNG took up this amount by passive diffusion. This suggests that absorption of dietary PNG in humans is mostly by passive diffusion. Absorption of smaller quantities of PNG may be facilitated by a carrier. This pattern of absorption is similar to that observed in the intestinal absorption of folate and many other nutrients. At low physiological concentrations of folate, the vitamin is absorbed by a pHdependent, Na + -independent carrier mediated transport system, while pharmacological concentrations of folate are absorbed by a diffusion process (138,139). The exchange of sodium for other monovalent cations did not change PNG uptake by Caco-2 cells; however, the concentration of PNG used in these experiments was in the range where passive diffusion, and not carrier-mediated absorption would predominate. Collectively, the results of these studies suggest that physiological concentrations of PNG are taken up by a Na + -independent process that is similar to the absorption of PN as previously determined rat small intestine (52,140). This observation

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68 is also consistent with a recent report of PN uptake by cultured opposum kidney (OK) cells (141). However, we cannot reconcile the difficulty that PNG, as a very hydrophilic molecule, would encounter in crossing the highly charged and hydrophobic plasma membrane. There is also recent evidence that other glycosylated compounds derived from the diet such as quercetin p-glucosides either interact with or are transported by a sodium-dependent glucose transporter (SGLT) (142-144). Although the absence of sodium did not cause a significant decrease in PNG uptake, we cannot rule out the possibility that PNG is taken up by a carrier mediated process. The transport mechanism is further confounded by the dual role ascribed to LPH. In a recent letter to the editor, Arts and colleagues briefly discussed the observation that LPH hydrolyzes pglucosidic bonds and is thought to transport the aglycones in a Na + -independent process (145). While this may explain the transport of PN from PNG into the cell, the process by which the intact glucoside, PNG, enters into the cell remains unclear. It is interesting to note that the detection of the intact glucoside inside the Caco-2 cells further supports previous observations made by this laboratory that PNG can be absorbed intact (7,10). As indicated in Figure 4-3, the most abundant form of vitamin B-6 inside Caco-2 cells is pyridoxine. This was not wholly unexpected since these cells were maintained in DMEM that contained 4 mg/L of pyridoxine-HCI as the source of vitamin B-6. Consequently, it was difficult to measure the small changes that likely were occurring in intracellular PN concentrations in response to increasing concentrations of PNG. However, even the smallest change in intracellular PNG concentrations was easily detected because PNG is not present in DMEM, and there is no in vitro synthesis of PNG. The ratio of PN:PNG and its change over time provides a rough estimate of the PNG that is hydrolyzed at the apical surface of the cells. We did not observe any significant changes in these ratios and there are several complicating factors that might

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69 affect this measurement. Once hydrolyzed, PN could be taken up by the cell and metabolized or released into the treatment medium. Alternatively, over the incubation period, intracellular PN might efflux out of the cell into the treatment medium. The PN measured in the treatment medium could contain PN originating from the hydrolysis of PNG and that released from intracellular pools. We did however, obtain indirect evidence of PNG hydrolysis. Intracellular concentrations of PL significantly increased as PNG concentration increased in the treatment medium (Figure 4-4). PN taken up from the hydrolysis of PNG would be metabolized inside the cell to form PL and PLP. PL is reported to the major metabolic product of the small intestine released into portal blood (134). Since intracellular PL was present in relatively low concentrations in the Caco-2 cells without added PNG, small increases in PL were easily detected. We did not measure the concentration of PL in the treatment media after the 30 min incubation, but we suspect that intracellular PL would efflux into the media. The addition of lactose to the treatment media did not appear to have a direct effect on the uptake of PNG. However, we have evidence that intracellular vitamin B-6 metabolism was changed, perhaps as a consequence of a reduction in PNG hydrolysis at the cellular surface. In the presence of lactose, LPH is inhibited with regard to PNG hydrolytic activity. Intracellular concentrations of PL significantly declined with increasing lactose concentrations. The concentration of 150 mmol/L was chosen since it is the approximate concentration of lactose in milk. At 150 mmol/L and half this concentration, 75 mmol/L, intracellular PL was significantly reduced in cells incubated with PNG. One possible explanation for this observation is that since PL is the greatest intestinal vitamin B-6 metabolite released into portal circulation (134), its concentration inside the cell might be most dramatically affected if the metabolic flux of PN to PL is reduced. Although PL concentrations declined, PLP concentrations did not significantly change in the present study. Our results are consistent with those of Middleton (146).

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70 Under well-nourished or saturated conditions (high PN concentrations) PLP concentrations did not increase in the rat intestine, likely due to potent product inhibition by PLP on pyridoxine:pyridoxamine 5'-phosphate oxidase that is observed in the liver (147). In summary, this study provides in vitro evidence that PNG is taken up intact by intestinal epithelial cells in a Na-independent process that is saturable at low concentrations and passively absorbed at concentrations >50 nmol/L, which is similar to the absorption of PN. Lactose diminished the metabolic utilization of PNG in Caco-2 cells, which might translate into a reduction of the in vivo bioavailability of PNG.

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CHAPTER 5 SUMMARY AND CONCLUSIONS The preceding research describes novel observations that extend our understanding of pyridoxine-5'-p-D-glucoside bioavailability, with particular focus on its absorption, hydrolysis, and metabolism in the small intestine. The following discussion integrates the data presented in the three previous chapters to provide a comprehensive view of the absorptive process of PNG and how it relates to its bioavailability. Based on previous results from this laboratory, a decrease in dietary vitamin B-6 concentration significantly increased the intestinal hydrolysis of PNG by cytosolic PNG hydrolase (12,13). This inverse relationship between an induced vitamin B-6 deficiency and intestinal cytosolic PNG hydrolase activity was observed in both rats (13) and guinea pigs (12). Chapter 2 reports the results of two studies that were designed to examine this observation more closely. Although a vitamin B-6 deficiency was achieved, as assessed by a significant reduction in plasma and liver PLP concentrations, the effect of vitamin B-6 deficiency on cytosolic PNG hydrolase activity was not reproducible. There was a trend toward an increase in in vitro PNG hydrolytic activity as catalyzed by cytosolic PNG hydrolase and brush border membrane LPH as dietary vitamin B-6 increased. In this respect, it appears that there is not a strong compensatory upregulation of the hydrolytic activity that could enhance the metabolic utilization of PNG in a time of dietary vitamin B-6 deficiency. In an attempt to further study this indirect relationship between dietary vitamin B6 and PNG hydrolytic activity similar conditions, controlled feeding studies using pyridoxine-defined diets were done in rats to explore potential differences between 71

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72 studies done by this laboratory in the past and present. The investigation done by Nakano et al. (13) used older rats weighing ~200g and fed PN-defined diets for 2 wk. Preliminary studies that were used as a foundation for the present work initially examined the effects of age and duration of diet feeding on intestinal PNG hydrolytic activity and found no significant effects of rat age or length of study period. One other notable difference between the present studies and previous studies done by this laboratory is in the diet that was fed to the rats. We speculated there to be an effect of basal diet formulation (AIN-93G or AIN-76A) on PNG hydrolytic activity. The present studies consistently fed the AIN-93G diet with different concentrations of pyridoxine to rats over a 5 wk period to change their vitamin B-6 nutritional status. The AIN-93G diet was formulated to reduce symptoms such as tooth decay and kidney calcification that were associated with chronically feeding the AIN-76A diet to rats (107). As discussed in Chapter 2, the carbohydrate source in the AIN-76A diet is sucrose, which was replaced with starch in the AIN-93G diet. The starch based AIN-93G diet theoretically could support the energy requirements of the gut microflora. Starches and oligosaccharides that are not completely hydrolyzed and absorbed in the small intestine travel to the large intestine where the resident colonic microflora ferment the undigested starch to salvage metabolic energy. A thriving population of gut microflora synthesizes short chain fatty acids, proteins, growth factors, and vitamins such as vitamins B-6 and K, which could be absorbed by the colon (108, 148). AIN-93G also is supplemented with trace minerals that are absent in the AIN-76A diet (107) and although these differences exist, the diets are intended to be nutritionally equivalent. In spite of this, we observed significantly greater rat growth and PNG hydrolytic activity in the cytosolic and total membrane fractions in rats fed the AIN-93G in comparison to rats fed AIN-76A, regardless of dietary PN concentration. There is a lack of information on the effects of feeding AIN-93G and AIN-76A diets side-by-side in controlled nutritional studies. At

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73 present, it is unclear what specific dietary component(s) influence these outcome measures. The purification and partial kinetic characterization of LPH, presented in Chapter 3, revealed that LPH was the enzyme in the brush border that was able to catalyze the hydrolysis of PNG. The primary function of LPH is to catalyze the hydrolysis of dietary lactose and the hydrolytic activity toward PNG had not been previously reported. The K m value calculated for lactose was 16 times greater than that calculated for PNG. In considering the quantity of PNG that is introduced to the intestine daily (-15% of the total vitamin B-6 intake of 1.7 mg/d), the enzyme is well-suited to catalyze PNG hydrolysis. However, the enzyme is not likely to be working efficiently and would catalytically prefer the hydrolysis of lactose to PNG. Not surprisingly, lactose was found to be a competitive inhibitor of PNG hydrolysis. This leads us to the questions of whether PNG bioavailability is reduced by the composition of a mixed meal, i.e. dairy products containing lactose consumed concurrently with fruits or vegetables containing PNG, or if PNG bioavailability is diminished in individuals with lactose intolerance or lactase insufficiency. In addition to the PNG hydrolytic function of LPH, we also observed yet another novel function of mammalian LPH. In the presence of both PNG and lactose, LPH exhibits glucosyl-transferase activity in vitro. We measured not only the hydrolytic product of PNG, PN, but also a secondary product, PN-disaccharide. The significance of this reaction in vivo is not completely understood, but may further contribute to a reduction in PNG bioavailability. Perhaps the most noteworthy finding of this research is that LPH was able to hydrolyze PNG and it is apparently responsible for a large proportion of the intestinal hydrolysis of PNG. After some preliminary feeding studies (data not shown) it soon became evident that the activity measured in the brush border membrane fractions of

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74 intestinal mucosa was just as important as the hydrolysis measured in the cytosolic fraction. Chapter 2 also described the calculation of the relative contribution of LPH and cytosolic PNG hydrolase to total PNG hydrolysis measured in the small intestine. LPH is the only (3-glucosidase in the brush border membrane and any hydrolysis measured in the brush border membrane is due to the activity of LPH. It was estimated in the rat that 50-60% of the PNG hydrolysis in the small intestinal mucosa was catalyzed by brush border LPH. In comparison, cytosolic PNG hydrolase was found to catalyze only 10% of the hydrolysis of PNG in the rat intestine. It was initially thought that the hydrolysis of PNG was exclusively catalyzed by cytosolic PNG hydrolase, but we now have strong evidence to support that PNG is hydrolyzed by extracellular (LPH) and intracellular (PNG hydrolase) enzymes and that LPH is responsible for most of this hydrolysis. Therefore, based on the results obtained from the present studies in rats and Caco-2 cells, PNG bioavailability is greatly dependent on a functionally active LPH. Results from the study of PNG absorption and hydrolysis by Caco-2 cells presented in Chapter 4 support previous data generated by this laboratory. While PNG can be hydrolyzed in the small intestine to form PN and glucose, it is not a complete hydrolytic process and PNG can be absorbed as the intact glucoside (7,1 0,1 1 ). PNG provided to Caco-2 cells was rapidly taken up in a saturable process at concentrations less than 50 nmol/L, and taken up in a non-saturable, passive diffusion process at concentrations greater than 50 pimol/L. We also collected preliminary data that suggested that there is little paracellular absorption of PNG (data not shown). Pilot experiments were done with the Caco-2 cells grown on permeable membrane inserts and intercellular permeability was tested by measuring the passage of phenol red, a marker for paracellular permeability. Trace amounts of phenol red, a low molecular weight compound (MW 352), were detected on the basolateral side of the Caco-2 cell

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75 monolayer. PNG has a MW that is very similar to phenol red (MW=331 ). Therefore, we believe that PNG absorption proceeds mostly via transcellular route; however, absorption by the paracellular route cannot be excluded. Application of these data to human dietary intake of PNG indicates that PNG derived from the diet is mostly absorbed by passive diffusion; however, absorption of PNG in individuals consuming a diet limiting in foods of plant origin might occur through carrier-mediated transport. In summary, the results from the studies discussed in the previous three chapters indicate that 1) PNG can be absorbed intact, as it was detected intracellular^ in the Caco-2 cells, 2) PNG can be enzymatically hydrolyzed to release free PN at the brush border membrane by LPH, or by cytosolic PNG hydrolase, and 3) in vitro hydrolysis is markedly reduced in the presence of lactose. We have constructed a model of PNG absorption based on the results from our laboratory (Figure 5-1). This model incorporates many years of PNG bioavailability research and presents our current understanding of PNG intestinal hydrolysis and absorption. Future research should address the possible reduction in PNG bioavailability in individuals with lactose intolerance. Lactose intolerance, or lactase insufficiency, affects 75% of the population worldwide (101) and this intestinal condition could compromise vitamin B-6 nutritional status in populations consuming a vegetarian based diet. Conversely, in individuals with lactase persistence, PNG bioavailbility may be increased. However, lactose co-ingested with foods of plant origin providing PNG might decrease the amount of vitamin B-6 derived from PNG. These questions remain to be answered and further investigation is warranted.

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76 Q_ CO 03

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BIOGRAPHICAL SKETCH Amy Dene Mackey was born on January 6, 1972, in Schenectady, New York. She grew up in Bellwood, Pennsylvania, where she graduated from Bellwood-Antis High School in 1990. She received her Bachelor of Science degree in biology from Juniata College in Huntingdon, Pennsylvania, in 1994. In December of 1997, she was awarded her Master of Science degree in nutrition from the Pennsylvania State University. In January 1998, she entered into the University of Florida to pursue a doctoral degree in nutritional sciences. 89

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Docto* of Philosophy. Jesse F. Gregory III, Ch£ Professor of Food Science and Human Nutrition I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Lynn B) Bailey Professor of Food Science and Human Nutrition I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate.Jn^cope and quality, as a dissertation for the degree of Doc Robert J. Cousk Boston Family Professor of Human Nutrition I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and js fully adequate, in scope and quality, as a dissertation for the degree of Doctor i Donald Samuelson Professor of Veterinary Medicine This dissertation was submitted to the Graduate Faculty of the College of Agricultural and Life Sciences and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December 2002 Dean, College of Agricultural Life Sciences ind Dean, Graduate School