Investigation of Effects of Restricted Vitamin B6 Supply on the Metabolism of Cultured Hepg2 Cells Using Analysis of Ami...

MISSING IMAGE

Material Information

Title:
Investigation of Effects of Restricted Vitamin B6 Supply on the Metabolism of Cultured Hepg2 Cells Using Analysis of Amino Acid Patterns and the in Vivo Kinetics of the Transsulfuration Pathway
Physical Description:
1 online resource (95 p.)
Language:
english
Creator:
Deratt, Barbara N
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Food Science and Human Nutrition
Committee Chair:
Gregory, Jesse F, Iii
Committee Members:
Wohlgemuth, Stephanie
Sitren, Harry S

Subjects

Subjects / Keywords:
b6 -- hplc -- hydrogen -- sulfide -- vitamin
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre:
Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Pyridoxal phosphate (PLP) functions as a coenzyme in cellular one carbon metabolism and many other roles in amino acid interconversion and catabolism.  The PLP-dependent transsulfuration enzymes, cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE), have been implicated in hydrogen sulfide (H2S) production concurrent with the formation of lanthionine and homolanthionine. Recent research has identified H2S as an endogenously produced gasotransmitter that modulates physiological functions in the cardiovascular and central nervous systems.  My objective was to investigate the effects of restricted vitamin B6 supply on the metabolism of cultured HepG2 cells by analyzing the amino acid patterns and the in vivo kinetics of the transsulfuration pathway.  Cells were cultured for 6 weeks in media containing concentrations of PL that represented severe (10 nM), marginal (50 nM), adequate (200 nM) and supraphysiological (2000 nM) conditions.  Severely deficient cells had elevated concentrations of valine, threonine, glycine, glutamine, asparagine, and alanine compared to marginal deficiency or adequacy, while lanthionine concentration was decreased in deficiency (P<0.002). U-13C5 L-methionine and (3,3 D2) L-cysteine indicated significantly lower fractional synthesis rates of lanthionine and homolanthionine at 10 and 50 nM PL, whereas that of cystathionine was higher in cells cultured in 50 nM PL.  Remethylation rates and homocysteine synthesis were not affected by vitamin B6 restriction.  Overall, these findings suggest CSE was impaired in severe and marginal deficiency while CBS was only impaired in severe deficiency, indicating a decrease in H2S production.  These results and observations of H2S biomarker production suggest a mechanism by which vitamin B6 inadequacy influences cardiovascular disease risk
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Barbara N Deratt.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Gregory, Jesse F, Iii.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-02-28

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0045979:00001


This item is only available as the following downloads:


Full Text

PAGE 1

1 INVESTIGATION OF EFF ECTS OF RESTRICTED V ITAMIN B6 SUPPLY ON THE METABOLISM OF CULTUR ED HEPG2 CELLS USING ANALYSIS OF AMINO AC ID PATTERNS AND THE IN VIVO KINETICS OF THE TRANSSULFURATION PAT HWAY By BARBARA DERATT A THESIS P RESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

PAGE 2

2 2013 Barbara DeRatt

PAGE 3

3 To my grandfath er, James DeRatt Sr.

PAGE 4

4 ACKNOWLEDGMENTS It is with utmost gratitude I acknowledge my major advisor, Dr. Jesse Gregory for his significant contributions throughout research positive criticism and guidance this thesis would not represent the knowledge ire to be a scientist of his caliber in my career. I would also like to thank my committee members, Dr. Harry Sitren and Dr. Stephanie Wohlgemuth for their id eas, s uggestions, and support throughout my project. I have now seen first hand how research does not simply answer a question, it provides avenues for further research This project would not have been possible if not for my colleagues and friends, Maria Ralat Luisa Rios Avila, Vanessa DaSilva and Greg Guthrie. My sincere appreciation goes to the members of my family who have always been a positive influence during my master s research. M y parents, Patricia and James DeRatt and grandparents, Barbara Barham a nd James DeRatt Sr. were always supportive in my decision to continue my education I would also like to thank my sisters Lindsey and Jamie D eRatt for their continuous love and support

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF T ABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 15 2 LITERATURE REVIEW ................................ ................................ .......................... 17 Vitamin B6 ................................ ................................ ................................ .............. 17 History ................................ ................................ ................................ .............. 17 Chemistry and Function ................................ ................................ .................... 17 Food Sources and Bioavailabil ity ................................ ................................ ..... 19 Absorption and Metabolism ................................ ................................ .............. 20 Requirements and Allowances ................................ ................................ ......... 22 Deficiency and Toxicity ................................ ................................ ..................... 22 Status Assessment ................................ ................................ ........................... 24 Vitamin B6 and Cardiovascular Disease Risk ................................ .................. 24 Amino Acids ................................ ................................ ................................ ............ 25 Structure and Function ................................ ................................ ..................... 25 Requirements ................................ ................................ ................................ ... 26 Metabolism ................................ ................................ ................................ ....... 26 Transamination ................................ ................................ ................................ 27 Amino Acids and Vitamin B6 Status ................................ ................................ 28 One Carbon Metabolism ................................ ................................ ......................... 29 Transsulfuration Pathway ................................ ................................ ....................... 30 Hydrogen Sulfide ................................ ................................ ................................ .... 31 Hypotheses and Specific Aims ................................ ................................ ............... 32 Overall Rationale ................................ ................................ .............................. 32 Hypotheses ................................ ................................ ................................ ...... 33 Specific Aims ................................ ................................ ................................ .... 33 3 AMINO ACID METABOLIS M IN HEPG2 CELLS ARE AFFECTED AT VARIOUS CONCENTRATIONS OF VI TAMIN B6 ................................ ................................ ... 38 Materials and Methods ................................ ................................ ............................ 38 Materials ................................ ................................ ................................ ........... 38

PAGE 6

6 Cellular Depletion ................................ ................................ ............................. 38 Sample Preparation ................................ ................................ .......................... 39 PLP Analysis ................................ ................................ ................................ .... 40 Homocysteine Analysis ................................ ................................ .................... 40 Amino Acid Analysis ................................ ................................ ......................... 41 GC/MS Analysis ................................ ................................ ............................... 41 Homolanthionine Synthesis ................................ ................................ .............. 42 Statistical Analysis ................................ ................................ ............................ 43 Results ................................ ................................ ................................ .................... 43 Vitamin B6 HepG2 Cellular Depletion ................................ .............................. 43 Amino Acid Analysis Method Modifications ................................ ...................... 43 Homocysteine Analysis Shows Differences between Amino Acid Concentrations in PL Concentration Groups in Cultur ed Cells and in Extracellular Media ................................ ................................ ........................ 44 Cellular Concentrations of Amino Acids Are Affected by Vitamin B6 Status in HepG2 Cells ................................ ................................ .............................. 45 Discussion ................................ ................................ ................................ .............. 46 4 VITAMIN B6 STATUS IN HEPG2 CELLS AFFECTS THE TRANSSULFURATION AND REMETHYLATION PATHWA YS WHEN ANALYZED BY STABLE I SOTOPE TRACER TIME C OURSE .............................. 57 Materials and Methods ................................ ................................ ............................ 58 Stable Isotope Materials ................................ ................................ ................... 58 Cellular Preparation ................................ ................................ .......................... 58 Kinetic Analysis ................................ ................................ ................................ 59 Statistical Analysis ................................ ................................ ............................ 59 Results ................................ ................................ ................................ .................... 59 Discussion ................................ ................................ ................................ .............. 61 5 CONCLUSIONS ................................ ................................ ................................ ..... 75 APPENDIX: GENERAL METHODS ................................ ................................ ............. 79 Protein Concentration Measurement ................................ ................................ ...... 79 Hydrogen Sulfide Measurement ................................ ................................ ............. 80 REFERENCES ................................ ................................ ................................ .............. 81 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 95

PAGE 7

7 LIST OF TABLES Table page 2 1 Recommended dietary intakes for B6 (mg/day ) by age and gender ................... 34 2 2 Tolerable Upper Intake levels for B6 (mg/day) by age and gender ..................... 34 3 1 Final storage conditions for each flask collected for weekly analysis. ................ 50 3 2 Mobile phase gradients programs for separation of Dns amino acids. ............... 50 3 3 PL co ncentrations in media determined by HPLC. ................................ ............. 50 3 4 Correlation coefficients for each amino acid standard curve. ............................. 51 3 5 Recovery an alysis for each amino acid in cell analysis. ................................ ..... 52 3 6 Intracellular and extracellular amino acid concentrations. ................................ .. 53 3 7 Intracellu lar amino acid concentrations. ................................ ............................. 54 4 1 Enrichment plateaus and methylation cycle kinetics. ................................ .......... 64 4 2 FSR and ASR of amino acids in v arious concentrations of B6. .......................... 65

PAGE 8

8 LIST OF FIGURES Figure page 2 1 (1) pyridoxine, (2) pyridoxamine, (3) pyridoxal chemical structures. Phosphorylate d forms are shown below each respective vitamer. ..................... 34 2 2 This model of vitamin B6 shows cellular trapping, interconversion, and release. The enzymes responsible for each action have also been identified. .. 35 2 3 The generic structure of amino acids. ................................ ............................. 35 2 4 The transamination process produces nonessential amino acids using PLP and transaminase enzyme complex. ................................ ................................ .. 36 2 5 One carbon metabolism and transsulfuration pathway con tain four PLP dependent enzymes ................................ ................................ .......................... 37 2 6 The transsulfuration pathway synthesizes H 2 S as a by product catalyzed by CSE and CBS enzymes. ................................ ................................ ................... 37 3 1 Cellular PLP concentration dur ing 6 week stabilization for each concentration of PL. ................................ ................................ ................................ .................. 55 3 2 A representative chromatogram of amino acid separation. ................................ 56 4 1 Enr ichment time course of precursor [U 13 C] methionine. ................................ .. 66 4 2 Enrichment time course of precursor [D2] cysteine. ................................ ........... 67 4 3 Enrichment time course of product [ 13 C 4 ] methionine. ................................ ........ 68 4 4 Enrichment time course of product [ 13 C 4 ] homocysteine. ................................ ... 69 4 5 Enrichment time course of product [ 13 C 4 ] cystathionine. ................................ ..... 70 4 6 Enrichment time course of product [D2] lanthionine. ................................ .......... 71 4 7 Enrichment time course of product [ 13 C 4 ] homolanthionine. ............................... 72 4 8 Regression curve analysis in SignmaPlot 12.0 determined initial rate (I) and enrichment plateau (Ep) for each amino acid in each sample. ........................... 73 4 9 Hydrogen sulfide production capacity plotted versus lanthionine concentration in HepG2 cells. ................................ ................................ ............. 73 4 10 Hydrogen sulfide production cap acity plotted versus homolanthionine concentration in HepG2 cells. ................................ ................................ ............. 74

PAGE 9

9 A 1 Absorbance of BSA standards versus concentration of standards provides the linear equation needed to quantify total pr otein concentration in cell samples. ................................ ................................ ................................ ............. 79 A 2 Hydrogen sulfide production capacity differs under varying concentrations of B6 (10, 50, 200, and 2000 nmol/L PL), displayed as mean standard devi ation. ................................ ................................ ................................ ............ 80

PAGE 10

10 LIST OF ABBREVIATIONS AA Amino acids ASR Absolute synthesis rate B6 Vitamin B6 CBS synthase CO 2 Carbon dioxide CoA Coenzyme A CSE lyase Csn Cystathionine CVD Cardiovascular disease Cys Cysteine DHF Dihydrofolate DNA Deoxyribonucleic acid EAR Estimated Average Requirement EDTA Ethylenediaminetetraacetic acid Ep Enrichment plateau FSR Fractional synthesis rate GABA Gamma aminobutyric acid GC/MS Gas chromatography/mass s pectroscopy Gly Glycine GSH Glutathione H 2 S Hydrogen sulfide HBSS HCl Hydrochloride Hcy Homocysteine

PAGE 11

11 HEPES 4 (2 hydroxyethyl) 1 piperarzineethanesulfonic acid HFBA heptafluorobutyric anhydride HPLC High p e rformance liquid c hromatography I Initial rate IOM Institute of Medicine LPH Lactase phlorizin hydrolase MEM/EBSS d Salts Met Methionine L microliter mg milligram NH 4 OH Ammonium hydroxide nM nanomolar PL Pyridoxal PLP Pyridoxal p hosphate PM Pyridoxamine PMP Pyridoxamine p hosphate PN Pyridoxine PNG D glucoside PNGH D glucoside hydrolase PNP Pyridoxine p hosphate RDA Recommended d ietary allowance RM Rem e thylation SAH S adenosylhomocysteine SAM S adenosylmethionine Ser Serine

PAGE 12

12 SHMT Serine hydroxylmethyl transferase TCA Trichloroacetic acid TCEP Tris (2 carboxyethyl)phosphine THF Tetrahydrof olate TM Transmethylation TS Transsulfuration pathway UL Upper limit

PAGE 13

13 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INV ESTIGATION OF EFFECT S OF RESTRICTED VITA MIN B6 SUPPLY ON THE METABOLISM OF CULTUR ED HEPG2 CELLS USING ANALYSIS OF AMINO AC ID PATTERNS AND THE IN VIVO KINETICS OF THE TRANSSULFURATION PAT HWAY By Barbara DeRatt August 2013 Chair: Jesse Gregory Major: Nut ritional Sciences Pyridoxal phosphate (PLP) functions as a coenzyme in cellular one carbon metabolism and many other roles in amino acid interconversion and catabolism. The PLP dependent transsulfuration enzymes, cystathionine beta synthase (CBS) and cys tathionine gamma lyase (CSE) have been implicated in hydrogen sulfide (H 2 S) production concurrent with the formation of lanthionine and homolanthionine. Recent research has identified H 2 S as an endogenously produced gasotransmitter that modulates physiolo gical functions in the cardiovascular and central nervous systems. My objective was to investigate the effects of restricted vitamin B6 supply on the metabolism of cultured HepG2 cells by analyzing the amino acid patterns and the in vivo kinetics of the t ranssulfuration pathway. Cells were cultured for 6 weeks in media containing concentrations of PL that represented severe (10 nM), marginal (50 nM), adequate (200 nM) and supraphysiological (2000 nM) conditions. Severely deficient cells had greater conc entrations of valine, threonine, glycine, glutamine, asparagine, and alanine compared to marginal deficiency or adequacy, while lanthionine concentration was lower in deficiency (P<0.002). Extracellular homocysteine and

PAGE 14

14 cysteine concentrations were great er in severely deficient cells (P<0.002). Metabolic flux studies using [U 13 C 5 ] L methionine and (3,3 D2) L cysteine indicated significantly lower fractional synthesis rates of lanthionine and homolanthionine at 10 and 50 nM PL, whereas that of cystathioni ne was higher in cells cultured in 50 nM PL. Remethylation rates and homocysteine synthesis were not affected by vitamin B6 restriction. Overall, these findings suggest CSE was impaired in severe and marginal deficiency while CBS was only impaired in sev ere deficiency indicating a decrease in H 2 S production. These results and observations of H 2 S biomarker production suggest a mechanism by which vitamin B6 inadequacy influences cardiovascular disease risk

PAGE 15

15 CHAPTER 1 INTRODUCTION Overt B6 deficiency is due to high bioavailability in natural and fortified foods. However, B6 insufficiency is widespread in specific sub population s such as the elderly and young women as well as with chronic alcohol abu se Epidemiological evidence suggest s low dietary intake or plasma concentrations of B6 are associated with increased CVD risk, although recent trials demonstrated the ineffectiveness of B6 supplementation on the prevention of cardiovascular incident recu rrence. D etermining specific mechanistic relationships has proven challenging due to the magnitude of critical functions that require B6 and the many stages of CVD. To date, mechanisms of B6 modulation on CVD risk are unknown. Inadequate B6 status has been related to inflammation, immune function, and thrombosis : all pivotal components in the progression of CVD Elevated homocysteine, hypercholesterolemia, and many other risk factors have b een related to CVD, however even in these stud ies, B6 status rem ained an independent risk factor for CVD Due to the necessity of B6 in many metabolic reactions, it is likely B6 is a limiting factor in many reactions affecting CVD risk. The recent discovery of endogenously produced H 2 S which acts as a neuromodulator and smooth muscle relaxant provides another possible mechanism of B6 modulation against CVD H 2 S on c e thought to be toxic, is produced endogenously via the reduction of thiols and thiol containing molecules. The two enzymes responsible for the majority of endogenously produced H 2 S are PLP dependent, thus providing a hypothetical connection between H 2 S production and B6 status.

PAGE 16

16 T his study was designed to investigate biomarkers of H 2 S production as well as other amino acids during restriction of B6 in ad dition to kinetics in the transsulfuration pathway in HepG2 cells The first study sought to examine the effects of B6 concentration, ran ging from severe to marginal deficiency into adequacy, on intracellular and extracellular amino acid concentrations. The second study used s table isotopes, (3,3 D2) L cysteine and [U 13 C 5 ] L methionine, to determine synthesis rates of amino acids in the transsulfuration pathway and H 2 S biomarkers under B6 constraints. The synthesis rates of these biomarkers suggest a pa rallel relationship with H 2 S production. Labeled products of [U 13 C 5 ] L methionine such as homocysteine and cystathioni ne can be used to determine the extracellular concentration in which inhibition of CBS and CSE occurs in cultured HepG2 cells. By deter mining a connection between H 2 S production and B6 status, it will provide preliminary data for a possible mechanism in which B6 is related to CVD risk

PAGE 17

17 CHAPTER 2 LITERATURE REVIEW Vitamin B6 History The first report of a water soluble nutritional fact or later identified as B6 was made in 1934 ( 1 2 ) Purification and crystallization of pyridoxine was reported by five laboratories in 1938 ( 3 8 ) A year later, synthesis confi rmed the proposed structure. In the 1940s, studies of the nutritional requirements of lactic acid bacteria led to the identification and recognition of pyridoxal and pyridoxamine as natural forms of the vitamin, as well as demonstrating the active coenzyme form of B6 was pyridoxal 5 phosphate ( 6 9 12 ) Since its discovery, the understanding of B6 properties, metaboli c function, and role in maintaining health have continuously expanded. Chemistry and Function B6 denotes the family of water soluble vitamins with the shared structure of 2 methyl, 3 hydroxyl, 5 hydroxymethyl pyridines exhibiting the nutritional activity of pyridoxine The three main derivatives of B6 differ in one carbon substituent at position 4 of 2 methyl 3 hydroxy 5 hydroxymethyl pyridines as seen in Figure 2 1 Pyridoxine (PN) contains an alcohol substituent for pyridoxal (PL) it is an aldehyde sub stituent and for pyridoxamine (PM) it is an amine substituent Each can each be phosphorylated at the hydroxymethyl group yielding the additional three members; phosphate ( PNP ) phosphate ( PLP ) phosphate ( PMP ) The structural differences cause pyridoxine to have a hydroxymethyl function, pyridoxamine, an aminomethyl function, a nd pyridoxal, a formyl function ( 13 )

PAGE 18

18 The pyridine ring system of B6 molecules exist mainly as zwitterions at neutral pH due to the basic character of the pyridinium N and the acidic nature of the 3 OH. Thus the net charge of B6 is dependent on environmental pH. T amino group o f phosphate ester of PLP and PM P will contribute to the charge of the vitamin. The complex ionization at several ionic sites explain s the reactivity in enzymatic processes ( 14 ) The coenzymatic action of PLP in most B6 dependent reactions involves a carbonyl amine condensation. PLP readily condenses with the unch arged amino group of amines while PMP will react with aldehydes or ketones to form Schiff bases (aldimines) at the 4 formyl substituent While Schiff bases can exist devoid of metal ions, their stability is greatly increased by covalent bonding with a meta l ion. PL and PM can form Schiff bases however the phosphorylated forms do so more readily because the phosphate group prevents the formation of an internal hemiac eta l bridge between the C 5 hydroxymethyl group and the C 4 aldehyde of PL thereby mainta ining the carbonyl in a reactive form ( 15 ) A lkaline environments offer optimum pH for Schiff base formation and stability ( 16 ) B6 affects nearly all aspects of metabolic function and cellular homeostasis while specifically acting as a coenzyme in processes including amino acid metabolism, one carbon metabolism and nucleotide synthesis, neurotransmitter met abolism, heme synthesis, glucon eogenesis, and glycogenolysis. Though involved in carbohydrate and lipid metabolism, PLP is most extensively linked to amino acid metaboli sm by functioning as a coenzyme in synthesis, degradation, and interconversion of ami no acids. The pathways constituting o n e carbon metabolism contain four enzymes that

PAGE 19

19 require PLP, without the remethylation cycle and DNA synthesis would be affected. Amino acid decarboxylases form hormones and neurotransmitters such as epinephrine, ser otonin carboxyl group from an aminolevulinate, through a reaction with glycine and succinyl CoA G lycogenolysis reactions also require PLP for enzymatic cleavage of glycoge n and general acid catalysis. Food Sources and Bioavailability B6 exists in food in different chemical forms. Plants also contain glycos y lated form s of B6 generally as pyridoxine D glucoside (PNG), altho ugh other glycosylated forms exist. PNG compr ise s 5 75% of total B6 and account s for 15 20% of B6 in mixed diets ( 17 ) The obligatory step in nutritional utilization of PNG is the glucosidic bond which releases PN ( 18 19 ) PLP and PMP make up >80% of the B6 found in animal origin food such as meats, fish, eggs, and dairy products Today, breakfast cereals and beverages are fortified with B6 as PN HCl because of its stability ( 20 ) Dietary PL, PN, PM, PLP, PMP, and PNP if present, are approximately 75% bioavailable in a typical mixed diet ( 21 ) The acidic environment of the stomach dissociates Schiff bases thus fre eing the vitamers prior to absorption The bioavailability of PNG is 50 60% relative to PN, however variation exists among individuals ( 22 23 ) B6 from animal sources is approx imately 10% more digestible than plant sources ( 24 ) Food processing can affect B6 absorption. Non enzymatic inter conversion between B6 compounds occurs when the double bond of the Schiff base migrates causing subsequ ent hydrolysis and dissociation ( 25 ) Foods that contain aldehyde and

PAGE 20

20 amine forms of B6 are especially susceptible to transamination during ther mal processing. For example, c oncentrations of PM and PMP increase d during cooking or thermal processing of meat and dairy products ( 26 27 ) T his exchange between PLP and PMP is not detrimental to the nutritional sta t us of B6 in foods since both are readily absorbed. Degradation of B6 in food is dependent on the form of B6 temperature, pH of the solution, and the presence of other reactive compounds. B6 is susceptible to photochemical oxidation resulting in 4 pyridoxic acid phosphate formation a nutritionally inactive derivative Light induced degradation will cause B6 losses in food processing, storage, preparation, and analysis ( 28 29 ) Ink et al used intrinsic and extrinsic labeling in rats to show thermal processing of food reduces the bioavailability of B6 from animal tissue by 25 30% ( 30 ) PNG co administered with PN antagonistically affects the utilization and metabolism of PN and other non glycosylated forms of B6 in humans and rats ( 22 31 32 ) Absorption and Metabolism Absorptio n of B6 in humans occurs in th e small intestine, primarily in the jejunum, and from bacterial production in the large intestine ( 33 ) Alkaline phosphatase in the small intestine hydrolyzes the phosphorylated forms at the brush border membrane enabling absorption Glycosly ated forms, specifically PNG, were found to be effectively absorbed in humans and rats but not completely hydrolyzed in the intestine by the cy to solic enzymes PNGH or the brush border membrane enzyme LPH ( 19 22 23 30 34 36 ) Many kinetic studies show intestinal absorption of PL, PM, and PN occurs by simple, non satur able diffusion ( 37 39 ) although evidence exists for a carrier mediated

PAGE 21

21 absorption mechanism that is dependent upon pH and concentration of the vitamin ( 33 40 ) Once absorbed into the enter o cyte, PL, PM, and PN are metabolically trapped by phosphorylation catalyzed by p yridoxal kinase. The addition of the negatively charged phosphate group hinders diffusion across the cell membrane while promoting protein binding. D ephosphorylation allows for movement across the basolateral membrane and into portal circulation ( 39 ) Vitamers are metabolized mainly in the liver to the metabolica lly active form, PLP, as shown in Figure 2 2. PL, PN, and PM are phosphorylated by PL kinase using ATP zinc as a cofactor and phosphoryl donor ( 41 ) Hepatic PL kinase activity is ten fold higher than that of phosphatase causing PLP to be the predominate form of B6 PMP and PNP interconversion into PLP i s catalyzed by FMN dependent pyridoxine phosphate oxidase ( 42 43 ) While extrahepatic tissues utilize B6 few contain oxidase activity to yield PLP from other forms. PNP oxidase prevents excess PLP production b y its susceptibility to product inhibition by PLP ( 44 ) If excess PLP is produced, it is catabolized in the liver by FAD dep endent aldehyde oxidase and NAD dependent aldehyde dehydrogenase into 4 PA, then exc reted in quantifiable concentrations in urine ( 45 46 ) Non specific phosphatases dephosphorylate PLP releas ing PL from the liver into circulation where it is bound to albumin in the plasma or hemoglobin in erythrocytes for transport ( 47 50 ) Tissue uptake requires circu lating PLP to be dephosph o rylated by plasma membrane phosphatases, thereby allowing carrier mediated transport across cellular membranes ( 51 52 ) In tissue, B6 is again trapped by phosphorylation and concentrated into the mitochondria and cytosol. Total body concentrations of B6 are

PAGE 22

22 approximately 170 mg (1000 mol) in adults ( 50 ) The largest pool is located in skeletal muscle, constituting 70 80% of the total pool, bound to glycogen phosphorylase ( 50 53 55 ) Research shows muscle tissue is resilient to B6 restriction and supplementation whereas plasma concentration is direc tly affected by intake ( 54 ) Requirement s and Allowances Controlled dietary studies and observation al investigations have concluded that plasma PLP concentration is dependent upon B6 intake and antagonistically affected by dietary protein intake ( 56 58 ) While the 1989 RDA was based on the B6 protein relationship, the current RDA was revised to account for average requirements ( 59 60 ) The cu rrent RDA for adults, 1.3 mg nmol/L ( 61 ) Since the criteria for an adequate dietary intake is based on PLP status, increased intake is recommended for pregnant and lactating women and also elderly as s een in Table 2 1 Current debate exists on the sufficiency of the RDA for B6 R ec ent NHANES data estimated the average B6 intake in the United States to be 1.86 mg/day for individuals not consuming supplements however plasma PLP concentrations were in dicative of deficiency in all subgroups examined ( 61 ) Rimm et al. showed an increased risk of coronary heart disease in people consuming the current RDA (1.3 mg) compared to those consuming greater amounts (>1.7 mg), indicating additional protective effects could be attained at greater intake of B6 ( 62 ) Deficiency and Toxicity B6 d eficiency may be caused by insufficient dietary intake, impai red absorption and interconversi on or unavailability due to drug in teractions ( 41 63 66 ) B6 deficiency can either be severe (<20 nmol/L PLP) or marginal (20 30 nmol/L PLP), each defined by

PAGE 23

23 plasma PLP concentration ranges ( 67 68 ) Adequate B6 status is defined by a PLP concentration of >30 nmol/L. The prevalence of marginally to severely deficien t individuals in the population is between 9 and 30%, with significantly elevated indices of deficiency in women of childbearing age and in elderly ( 61 ) Clinical manifestations of severe B6 deficiency are seizures, skin lesions, and microcytic anemia. S eizures in inf ants fed a commercial formula in which B6 degraded due to processing and storage were most likely a result of impaired neurotransmitter production ( 69 70 ) Neurotransmitter l evels were shown in rat s to be depressed by B6 restriction ( 71 ) Some d octors have exploited the relationship between neurotransmitter production and B6 status by prescribing patients suffering from depression B6 supplements ( 72 ) The results fr om this prescription were varied at best. Other manifestations of B6 deficiency are abnormal red blood cell formation. Without PLP, aminolevulinate cannot be produced, thus limiting heme synthesis Limited hemoglobin synthesis causes red blood cell size to decreased resulting in mircocytic anemia ( 73 ) Skin lesions also appear in severe deficiency but are corrected with B6 supplementation ( 74 ) The upper limit (UL) for B6 was set at 100 mg per day due to reports of sensory neuropathy caused by pyridoxine supplements of over 500 mg per day (Table 2 2) ( 60 75 ) Megadoses of B6 were given to treat premenstrual syndrome, asthma, and c ertain sensory neuropathies until r eports stated neurotoxicity and photosensitivity were occurring at chronic doses of greater than 1 g/day ( 76 ) Toxic effects were not disp layed in individuals consuming 100 mg per day ( 60 ) Toxicity of B6 has not been reported from

PAGE 24

24 dietary intake alone. Currently, most daily vitamins provide approximately 2 mg B6 so pulation. Status Assessment B6 can be measured through direct or indirect means. The most common direct measurement of B6 nutritional status is the quantification of PLP by HPLC or tyrosine decarboxylase PLP concentration reflects tissues stores and is a reliable indicator of long term B6 status ( 77 78 ) Urinary excretion of PLP as its catabolic product, 4 PA, can also quantified to determine B6 concentration from daily intake. Indirect mea surements of B6 status assess the activity of PLP dependent enzymes such as ky n urinase after providing a bolus of precursor, in many cases tryptophan ( 79 ) Inhibition of enzyme activity is then related to B6 status Indirect measurements of B6 are not specific and can often be affected by other unrelated factors. Vitamin B6 and Cardiovascular Disease Risk The pathogenesis of CVD involves the interplay between several genetic, nutritional, and life style factors t hus the final outcome of this disease is due to many different contributory agents M onkeys fed a pyridoxine deficient diet for 5 months developed atherosclerotic lesions lead ing to further inquires of the relationship between B6 and CVD ( 80 81 ) Furthermore human studies have determined B6 deficiency is an independent risk factor for atherothrombotic and cerebrovascular disease ( 82 84 ) Many epidemiological studies reinstate this correlation in pa tients with coronary artery disease risk The magnitude of the association between B6 and CVD is lo wer than other factors such as blo od lipid levels and homocysteine concentration, but it indicate s the coenzymatic function of B6 in total body metabolism plays a co mpounding role in disease development.

PAGE 25

25 There are many hypotheses of the mechanisms by which B6 is associated with CVD The most published is the dependence of B6 in the transsulfuration pathway to degrade homocysteine. Elevated homocysteine in patients is a well known predictor of CVD ( 85 86 ) Robinson et al demonstrated through multivariate analy sis that low PLP concentration is a risk factor of coronary artery disease independent of homocysteine concentration suggesting a potential protective effect of B6 through mechanisms unrelated to homocysteine metabolism ( 87 88 ) In addition to homocysteine elevation, B6 concentration inversely relates to C reactive protein values ( 89 ) C reactive protein is an inflammatory marker and also a predictor of coronary artery disease ( 90 91 ) B6 plays a role in coagulation by inhibiting ADP rece ptors and prolonging bleeding by occupying the glycopro tein IIb/IIIa receptor or down regulating its synthesis ( 92 98 ) B6 may induce hypercholesterolemia by inhibiti on of advanced glycation and lipooxidation of end products ( 99 101 ) EPA and DHA production were hindered by B6 deficiency thus reducing the presence and protective effects of omega 3 fatty acids ( 102 105 ) T lymphocyte, macrophage differentiation, and interleukin production were shown to be affect ed by B6 status thereby possibly impairing immunological functi on ( 106 108 ) As briefly stated above, B6 has many possible mechanisms affecting the development of CVD While these identify a few of t he mechanisms, there a re undoubtedly more unknown mechanisms by which B6 affects diseased states. Amino Acids Structure and Function Amino acids utilized by humans are amino acids have the same core structure consisting of an amino group, carboxyl group, a hydrogen atom and a reactive group (Figure 2 3) The reactive group R group gives each amino acid

PAGE 26

26 its unique physical and chemical propert ies. The simplest amino acid is glycine, carbon bonded to a carboxyl group, an amin o group, and two hydrogen atoms. Larger amino acids that have different substituents from each bond carbon can exist in two different molecular configurations; D a nd L isomers. L amino acids are present in prote ins and biological environments ( 109 ) Amino acids are categorized according to their R groups that dictate their chemical nature ; aliphatic, acidic, basic, aromatic, sulfur containing, and secondary amino acids. Each group has a similar structural compo nent in the R group substitu ent ( 110 ) Peptide bonds are amide bonds between ami no acids leading to the formation of peptides and proteins. The bond is formed between the carboxyl group of the first amino acid and the amino group of the second amino acid. The peptide bond appears to be a single bond but has double bond characteristi cs such as rigidity. Requirements Although there are many amino acids in nature, only about 20 are found in proteins. While forming proteins, amino acids can also control the initiation of mRNA translation and regulate protein synthesis ( 111 ) Essential amino acids are especially important to consume because they cannot be synthesized in sufficient amounts internally. Overloading one amino acid can cause a defi ciency in another so maintaining a balance is essential. Current RDAs are based on nitrogen balance studies from Rose and others in the 1950s ( 112 ) Metabolism Am ino acids can be synthesized internally or must be acquired from dietary intake. This divides them into the categories of essential, nonessential and conditionally essential. The 9 essential amino acids are histidine, isoleucine, leucine, lysine,

PAGE 27

27 methion ine, phenylalanine, th r eonine, tryptophan, and valine. Nonessential amino acids are alanine, asparagine, aspartic acid, and glutamic acid. These can be derived from keto acids in the transamination process Conditional amino acids are only essenti al unde r certain conditions such as illness and stress. For nonessential amino acids to be synthesized in the body, PLP must be present to serve as the coenzyme of transamination reaction If the initial amino acid is acid is n ot available to mak e the final amino acid. For acid so they must be ingested The keto acid intermediates can be used for additional purposes such providing substrates for the citric acid cycle. Excess a min o acids can be converted to pyruvic acid and acetyl CoA t o be used in lipogenesis or synthesized into glucose and glycogen. This conversion to glucose is stimulated by the hormones cortisone, glucagon and cortisol. Nonessential amino acids can be made fr om alternative processes such as cysteine production from methionine or serine and glycine production from phosphoglyceric aci d ( 113 ) Transamination Transamination is the transfer of an amine group from one molecule to another by a transaminase enzyme thereby produc ing nonessential amino acids in vivo A decreas ed rate of transamination occurred in c orrespondence to PLP deficiency ( 114 ) This double replacement reaction begins with an amino acid and a PLP enzyme complex. PLP forms a Schiff base with the enzyme to link to the active site. Then a new Schiff base for ms between the amino group of the amino acid and the PLP which has substituted from the enzyme PLP linkage. This produces the intermediate aldimine. This linkage acts as an electron sink weakening the bond Once there is a break in the double bond, t he intermediate is now called a ketamine. Hydrolysis then occurs and the

PAGE 28

28 final product is an keto acid product and PMP enzyme complex. Since this is a double replacement reaction, dehydrating a new keto acid substrate and the PMP enzyme complex will reverse the reaction T he final result will be a new amino acid and a PLP enzyme complex ( 115 ) a s depicted in Figure 2 4 The main keto acids in this pathway are ketoglutarate and pyruvate needed for glutamate and alanine production Subsequently, glutamate can serve as the source of the amine group to make additional amino acids. Therefore there are many amino acids that can be produced from a few initial ly d igested amino acids ( 113 ) Amino Acids and Vitamin B6 Status Vitamin B6 is an essential coenzyme in amino acid metabolism. Many studies conclude amino acid concentrations vary significantly in B6 deficiency. A human study by Park and Link s wiler in 1970, using a B6 deficient diet for 3 weeks, showed many changes in urinary excretion of free amino acids after a methionine load test. Plasma concentrations of glycine, serine, and threo n ine increased in the fasting and 2 hour postprandial states in marginally B6 deficient subjects There w as also an increase in the urinary excretion of serine and threonine before and after the methionine load. Plasma concentrations of alanine, isoleucine, leucine and valine decreased with B6 depletion ( 116 ) While many other amino acids use B6 as a coenzyme, regulatory pathways prevent amino acid imbalance in deficient states. Studies conducted in this laboratory display ed the effects of B6 deficiency in conjunction with one carbon me tabolism and the transsulfuration pathway. The concentration of glycine, which is an integral component of one carbon metabolism, significantly increased in participants consuming a B6 deficient diet. Human studies have shown mild B6 depletion d id not af fect the concentrations of homocysteine,

PAGE 29

29 methionine, and serine ( 117 118 ) In the methylation cycle, f asting h omocysteine increase d slightly in B6 deficiency ( 119 121 ) however other studies show ed moderate B6 deficiency had little effect on fasting homocysteine concentr ations ( 119 ) The transsulfuration pathway is directly affected by B6 deficiency because of two PLP dependent enzymes responsible for product formation. B6 r estriction studies in humans have show n an increase in plasma cystathionine c oncentration ( 118 121 ) as well as fractional synthesis rates ( 122 ) Glutathione synthesis rate not concentration was affected by B6 status in red blood cells in healthy men and women while rat studies show ed an increase in gl utathione concentration in B6 deficiency ( 123 124 ) One Carbon Metabolism One carbon meta bolism is compris ed of ; the folate cycle which acquires one ca rbon units to support DNA synthesis the methionine cycle which provides for methylation processes, regeneration of methionine and regulation of homocysteine and the thymidylate cycle which synthesizes DNA As seen in Figure 2 5 t here are four PLP depend ent enzymes in one carbon metabolism and the transsulfuration pathway; (1) serine hydroxymethyltransferase (SHMT); (2) glycine decarboxylase of the glycine lyase (CSE). Studies in hum ans and rats have found the PLP dependent enzymes were impaired in B6 deficiency ( 125 ) The folate cycle converts dietary folate in to tetrahydrofolate (THF) form. Folic acid is reduc ed to THF in a two step process by dihyd r ofolate reductase. Throughout the folate c ycle, tetrahydrofolate polyglut amate cof actors are the acceptors and donators of one carbons units differing in the oxidation of N5 or N10 positions ( 126 ) SHMT PLP complex redistribute s a one carbon unit from serine to THF to yield glycine

PAGE 30

30 and 5,10 methylene THF. The glycine cleavage system, also PLP dependent, yields CO 2 NH 3 and a one carbon u nit which is also accepted by THF to form 5,10 methylene TH F 5,10 methyleneTHF can donate a carbon for the synthesis of thymidylate from deoxyuridylate, an initial and rate limiting step in DNA synthesis. 5,10 methyleneTHF also produces 10 formylTHF in which the formyl group provides two carbons that are incorporated into the purine ring for DNA and RNA synthesis. Methylenetetrahydrofolate reductase partially reduces 5,10 methyleneTHF to 5 methylTHF. This enzyme is disposed to a common genetic polymorp hism that reduces its functionality in folate deficiency 5 methylTHF is the primary transpor t form in the body. 5 methyl THF is demethyla ted by methionine synthase using vitamin B12 as a coenzyme to accept th e methyl group producing methyl cobalamin. Th e methyl group is then tr ansferred to homocysteine producing methionine in the methylation cycle. M ethionine is activated by ATP to for m S adenosyl methionine (SAM). SAM is a methyl donor in over 100 methyltransferase react ions such as in DNA methylation neurotransmitter synthesis, and phospholipid synthesis. SAH is formed when SAM is demethylated. The removal of adenosine from SAH produces homocysteine. Homocysteine has two metabolic fates; it is rem eth y l ated by methionine synthase or catab o lized in the transsulfuration pathway. Transsulfuration Pathway The transsulfuration pathway aides in the regulation of homocysteine and controls the production of cys teine, ultimately yielding glut athione. Homocysteine condenses with serine in a reaction catalyz ed by PLP in complex with CBS to produce cystathionine in an irreversible reaction. Cystathionine is then hydrolyzed by CSE to yield cysteine. As seen in the F igure 2 6 the enzymes mentioned can also produce

PAGE 31

31 additional products. H 2 S is produced at many points in this pathway. While measuring H 2 S content in plasma is difficult it has been proposed that measuring specific products associated with H 2 S production such as homolanthionine and lanthionine can give insight on H 2 S production ( 127 ) The transsulfuration pathway has been extensively studied in relation with B6 deficiency due to the d ependent enzymes in the pathway SAM regulates of the transsulfuration pathway by controlling CBS activation such that in restricted conditions the pathway is driven towards remethylatio n rather than transsulfuration. Animal studies show ed that sever e B6 deficiency affected the activity of both enzymes with CSE being more s ensitive ( 125 128 132 ) In human studi es, the transsulfuration flux was impaired in B6 deficiency seen by increased homocysteine and cystathionine concentrations ( 119 122 ) H ydrogen Sulfide H 2 S is best known as a toxic pollutant and has been linked to tissue dama ge and inflammation at high levels. However, recent publications describe H 2 S as a gaseous signaling molecule that modulates physiological functions and is regulated ( 133 134 ) Specifically, in the brain H 2 S appears to function as a neuromodulator by enhancing N methyl D aspa r t ate receptor mediated responses and f acilitating the induction of long term potentiatio n in the hippocampus ( 135 ) H 2 S also function s as an endogenous smooth muscle relaxant in vertebrates ( 136 140 ) Nitric oxide is a well known gaseou s transmitter with many shared functions in vivo Nitric oxide has been more extensively studied than H 2 S in respect to mechanistic functions It is not known if H 2 S and nitric oxide exert their effects in vivo independently or in tandem, however current research

PAGE 32

32 suggests H 2 S is involved in the regulation of nitric oxide mediated signaling events and/or vice versa ( 141 ) Most H 2 S is produced endogenously from desulfuration of transsulfuration amino acids catalyzed by CBS and CSE, which are PLP dependent enzymes ( 142 ) Mice with the CSE enzyme de leted have reduced levels of H 2 S in serum, heart, aorta, and other tissues ( 143 ) H 2 S con centration is difficult t o measure due to rapid oxidation and volatility, and literature shows wide variation in reported H 2 S concentration s ( 144 ) As H 2 S is formed, by produc ts homolanthionine and lanthionine are also formed These products have been proposed as biomarkers of hydrogen sulfide production although their quantitative relationship and function of remain unknown ( 127 ) The concentration of endogenously produced H 2 S in health y humans do es not approach toxicity H 2 S is metabolized by oxidation in the mitochondria or methylation in the cytosol into sulfate ( 141 ) It also can be scavenged by methemoglobin or oxidized glutathione or consumed by endogenous oxidant species in the vasculature such as nitric oxide ( 145 ) The resulting conjugat ed sulfate is excreted by the kidney ( 146 ) Hypotheses and Specific Aims Overall Rationale Previous studies determined the effects of B 6 status on specific amino acid concentrations and kinetics. This study aimed to determine the effect of B6 on a complete intracellular amino acid profile in cultured HepG2 cells as well as specific extracellular amino acids. In additional to amino acid patterns, the transsulfurat ion pathway amino acids were quantified and fractional synthesis rates determined. The focus on the transsulfuration pathway was due to the production of H 2 S and its biomarkers, predominantly produced by the CBS and CSE enzymes. These results

PAGE 33

33 provide pre liminary evidence regarding the effects of B6 status on H 2 S production in an attempt to explain the relationship between B6 deficiency and CVD risk. HepG2 cells were used in this project because previous studi es show this cell line simulated physiological conditions under B6 constraints. Hepatic tissue is also the main site f or B6 interconversion so by using a human liver cell line, the PL administered will be converted into the enzymatically active f orm. Hypotheses 1. The amino acid profile of HepG2 cells will vary according concentration o f B6 with some amino acids more directly affected than others. 2. The transsulfuration pathway in B6 deficient cells will be hindered causing an accumulation of two intermediates due to decreased enzyme activity. H 2 S concen trations, as well as the biomarkers of H 2 S production; lanthionine and h omolanthionine, will increase with PLP concentration in HepG2 cells. 3. The flux of amino acids in the transsulfuration pathway will be hindered in PLP deficient cells due to CBS and CSE inhibition. Specific Aims Quantify various amino acids as well as H 2 S biomarkers in HepG2 cells adapted to specific concentrations o f B6 a) Modify existing HPLC methods to quantify the intracellular and specific extracellular concentrations of 25 amino aci ds b) Use GC/MS to quantify cystathionine, lanthionine, and homolanthionine intracellular concentrations Measure transsulfuration flux and fractional synthesis rates of amino acids associated with the transsulfuration and remethylation pathways in HepG2 cel ls at various concentrations of cellular B6

PAGE 34

34 Table 2 1 Recommended dietary intakes for B6 (mg/day) by age and gender ( 147 ) Age Male Female Pregnanc y Lactation Birth to 6 months 0.1 mg* 0.1 mg* 7 12 months 0.3 mg* 0.3 mg* 1 3 years 0.5 mg 0.5 mg 4 8 years 0.6 mg 0.6 mg 9 13 years 1.0 mg 1.0 mg 14 18 years 1.3 mg 1.2 mg 1.9 mg 2.0 mg 19 50 years 1.3 mg 1.3 mg 1.9 mg 2.0 mg 5 1+ years 1.7 mg 1.5 mg Table 2 2 Tolerable Upper Intake levels for B6 (mg/day) by age and gender ( 147 ) Age Male Female Pregnancy Lactation Bir th to 6 months NA NA 7 12 months NA NA 1 3 years 30 mg 30 mg 4 8 years 40 mg 40 mg 9 13 years 60 mg 60 mg 14 18 years 80 mg 80 mg 80 mg 80 mg 19+ years 100 mg 100 mg 100 mg 100 mg F igure 2 1 (1) p yridoxine (2) pyridoxamine, (3) pyridox al chemical structures. Ph osphorylated forms are shown below each respective vitamer

PAGE 35

35 Figure 2 2. This model of vitamin B6 shows cellular trapping, interconversion, and release. The enzymes responsible for each action have also been identified. Figure 2 3. The generic structure of amino acids.

PAGE 36

36 Figure 2 4. The transamination process produces nonessential amino acids using PLP and transaminase enzyme complex

PAGE 37

37 Figure 2 5. One carbon metabolism and transsulfuration pathway contain four PLP de pendent enzymes: (1) serine hydroxymethyltransferase; (2) glycine synthase; lyase ( 125 ) Figure 2 6. The transsulfuration pathway synthesizes H 2 S as a by product catalyzed by CSE and CBS enzymes Redrawn from ( 148 )

PAGE 38

38 C HAPTER 3 AMINO ACID METABOLISM IN HEPG2 CELLS ARE AFFECTED A T VARIOUS CONCENTRATIONS OF VI TAMIN B6 Metabolic perturbations of amino acid concentrations due to altered B6 nutrition demonstrated B6 essentiality in amino acid metabolism. Lanthionine and hom olanthionine have been proposed to be biomarkers reflecting the extent of H 2 S production These amino acids are produced by B6 dependent enzymes CSE and CBS during the synthesis of H 2 S ( 127 ) Some amino acids are more susceptible to B6 inadequacy du e to reduced enzyme activity. This experiment use d HPLC and GC/MS methods to quantify individual amino acids in HepG2 cells cultivated in media conta ining 10 nmol/L (severe deficiency) 50 nmol/L (marginal deficiency) 200 nmol/L (adequate ) or 2000 nmol/L PL (supraphysiological) B y understanding amino acid metabolism in relation to B6 concentration disease risk can be assessed ( 149 ) Materials and Methods Materials Human hep a toma cell line (HepG 2) was purchased from American T ype Culture Collection (Manassas, V A). Cell culture media and all other medium supplements were purchased from HyClone (Logan, UT) or Cellgro (Mannassas, VA). Bradford assay solution was purchased from Bio Rad (Hercules, CA). All amino acids, used for calibration were purchased from Sigma Aldrich (St. Louis, MO). All other chemicals and solvents were HPLC grade or above and purchased from Fisher Scientific (Pittsburgh, PA) or Sigma Aldrich (St. Louis, MO). Cellular Depletion All media was supplemented with 2 mM L g lutamine 0.1 mM non essential amino acids, 1 mM sodium pyruvate antibiotic/antimycotic solution (1x) and 10% fetal

PAGE 39

39 bovine s erum HepG2 c ells were grown to con fluency in complete media (SH30024.01) for two weeks before transfer in to 75.0 cm 2 flasks. Basal m edia devoid of added B6 ( RRC125193 ) was used to prepare working media with four different concentrations of added pyridoxal; 10 nmol/L PL represented severe d efici ency, 50 nmol/L PL represe nted marginal deficiency, 200 nmol/L represented adequate B6 status, and 20 0 0 nmol/L which was equivalent to the PL concentrations found in commercial media. Cells were passaged every 3 4 days depending on confluency for 6 w eek s until intracellular PLP concentration reached a steady state Cells were passaged by removing the media from each flask and washing with 2 mL of DPBS. Then 1 mL of try p sin was 2 for 4 minutes. Respective media were then added to remove cells from the plate bottom and dilute trypsin. The cells were transferred to new flasks in diluted quantities or collected for analysis. Cel ls were incubated in 5% CO 2 Sample Preparation Each week, 4 flasks used th e above stated cell removal proc ess and were collected in 15mL E ppendorf tubes. The tubes were centrifuged at 50 x g for 10 min and media removed. Cells were then washed t hree times with DPBS, each followed by centrifugation The final pellet of cells was diluted into 1.5 m L of cold distilled water and son i cated with a Sonic Dismembrator for 30 seconds in continuous mode setting 3 The dispersed pellet was immediate ly al iquoted into separate vials for Bradford Assay, homocysteine assay, amino aci d a nalysis, and PLP assay. Table 3 1 shows the amounts and additions made to each vial before storage at

PAGE 40

4 0 PLP Analysis Intrac ellular PLP concentration was analyzed weekly Samples collected in the above conditions were sonicated to di srupt cells then 500 L 10% (w : v ) TCA was added imm ediately for protein precipitation and the mixture was stored until analysis. After samples thawed, the mixture was clarified by centrifugation at 10,600 x g for 10 minutes. A 750 L portion of the supernatant was transferred to a 15 m L Falcon tube and 5 0 L of 0.5 M semicarbazide was added to deriv atize PLP and PL into their sem icarbazone forms for fluorescence detection The samples were mixed thoroughly 15 minutes. Samples returned to room temperature before repetitive extraction with 3 mL diethylether. A single extraction with 3 mL methylene chloride removed TCA and cellular lipids from the sample PLP and PL semicarbazone derivatives were measured by HPLC (Thermo, West Palm Beach, FL) with reverse phase Microsorb MV C18 column ( ) column alkalinization with 4% NaOH enhanc ed fluorescence. Peaks were detected with a fluorescence detector at an excitation wavelength of 350 nm and emission wavelength of 478 nm. Isocratic mobile phase at flow rate 1.1 mL/min (0.05M KH 2 PO 4 with 3% acetonitrile,2.9 pH) was used to achieve PL an d PLP separation ( 150 ) Homocystein e Analysis Before analysis each sample was supplemented with saline and an internal standard. Then 10 L of 10% TCEP was added to reduce disulfide bonds. After 30 minutes of incubation, 100 L of TCA is added to precipitate proteins. This mixture was t hen vortexed and centrifuged for 10 minutes at 13,040 x g A mixture (200 L ) of sodium hydroxide and borate buffer was added to e ach sample vial along with 50 L of sample supernatant. The vials were vortexed

PAGE 41

41 a heating block After returning to room temperature, samples were measured by HPLC using reverse phase Microsorb MV C 8 column ( ) The a utosampler temperature was maintained at Isocratic mobile phase ( 0.05 M acetic acid/acetate buffer pH 5.2 ) achieve d separation of homocysteine, cysteinylglycine, glutathione, and cysteine by fluorescence detection at an excitation and emission wavelength of 385 nm and 515 nm respectively ( 151 ) Amino Acid Analysis Cell samples were stored with 500 L methanol to prevent amino acid degradation. Thawed samples were vortexed the n centr ifuged for 10 minutes at 3000 x g. The supernatant was removed and dried using a Speed Vac S ample s were reconstituted in 1 mL of 40 mM lithium carbonate buffer (pH 9.5, with HCl) Dansyl chloride dissolved in acetonitrile 5.56 mM, served as the derivatization reagent D ns Cl solution (500 L ) was incubated with the samples for 45 m inutes in darkness at room temperature to ensure Dns amino acid binding. The reaction was quenched by 100 L of 2% (v:v) ent h anolamine Each sample was filtered (0.45 m, Fisherbrand) before quantification by HPLC ( 152 ) Previously publicized methods did not provide complete separation, so a novel gradient method and column constructio n was used ( 152 153 ) Mobile phase gradient programs and solvent compositions used are listed in Table 3 2 enomenex) and Ultrasphere IP column (5 m x 4.6mm x 25cm) were used in succession to separate all 21 amino acids GC/MS Analysis A previously validated GC/MS method was modified to quantify lanthionin e, homolanthionine and cystathionine Dowex pipette columns (50WX8 200) packed with

PAGE 42

42 glass wool and containing 0.8 mL of resin (25% (w : v ) in 1M NaOH), were equilibrated by acid/base washes. S ample s containing 5 00 L of the initial cell suspension were ac idified with 50 L of 66% TCA (v:v) The internal standard norleucine, was added to each sample and standard. This mixture was vortex ed applied to the column and washed with 20 mL of deioni zed water. The bound amino acids were eluted with 3 m L of NH 4 O H into 4 mL reaction vials. Eluate s were dried in a Speed Vac dried, the samples were reconstituted with 10 L ethanethiol for sulfur c ontaining amino acids and 500 L of an esterification reagent containing acetyl chloride and 1 propanol. Each sample was After heating, samples were samples dried, they were reconstituted with 100 L heptafluorobutyric anhydride (HFBA) s olution and 10 L ethanethiol. Samples were capped with nitrogen, vortexed completing amino acid derivatization Samples were then dried once more before reconstitution with 200 L ethyl acetate. Samples were an alyzed using GC/MS ( 154 156 ) Homolanthionine Synthesis To determine the retention time of homolanthionine for HPLC and GC/MS methods, homolanthionine was produced enzymatically Approximately 7 mM of homocysteine was dissolved 100 mM HEPES buffer, pH 7.4 The sample was preincubated fo of CSE (2.5 mg/m L in HEPES buffer, provided by Dr. Ruma Banerjee University of Michigan Medical School ) was added and incubated 45 minutes. The reaction was terminated by the addition of 1 mL of methanol The sample was centrifuged for 10 minutes at 10 000 x g and the supernatant was transferred in to amber vials for storage at ( 127 )

PAGE 43

43 Statistical Analysis Each concentration of B6 had four replicates. Duplicate injections per sample were performed by HPLC or GC/MS and a complete calibration curve was analyzed after every 10 injections. Amino acid concentrations were identified using Chromeleon sof tware (Thermo Scientific). Concentrations were statistically analyzed by Dr. Chi at the University of Florida. All data w ere log transformed to pass the Gaussian assumption. One way ANOVA with pairwise comparisons was performed for each sample, P<0.002 The software used to obtain these results was SAS 9.3. Results Vitamin B6 HepG2 Cellular Depletion Analysis of fetal bovi ne serum showed that it contained approximately 17 nmol/L PL and negligible PLP forcing adjustments to the addition of PL. The m edia targeted to be 10, 50, 200, and 2000 nmol/L PL respectively, were experimentally determined to be 17.7, 45.3, 239, and 1709 nmol/L PL as shown in T able 3 3 Four samples per concentration of PL were used to determine weekly PLP status Concentrati ons over the 6 week time period are shown as mean s (Figure 3 1) Amino acid concentration experiments and stable isotope tracer study began after week 6, when a steady PLP concentration of each B6 concentration was maintained for 3 week s prior. Final PLP concentrations (nmol/mg protein ) were 52.5, 105, 123, and 143 for 10, 50, 200, and 200 nmol/L PL respectively at the time o f experimentation. Amino Acid Analysis Method Modifications M odifications to published amino acid methods allowed 21 amino acids to b e separated and determined by HPLC with fluorescence detection. A representative

PAGE 44

44 c hromatogram is shown in Figure 3 2 After establishing this method, standard curves were developed for each amino acid with the exception of homolanthionine. The correlatio n coefficients for each amino aci d are listed in Table 3 4 After the standard curve was deemed adequate, cell and plasma samples were supplemented with approximately 20 g/mL of specific amino acids to determine the recovery of this assay. The percent ag e recovery of each a mino acid is listed in Table 3 5 After validation of this method, four independent samples of HepG2 cells from each B6 concentration were tested from the week 6 time point. Homocysteine Analysis Shows Differences between Amino Acid Concentrations in PL Concentration Groups in Cultured Cells and in Extracellular Media Homocysteine, glutathione (GSH), cysteinylgly cine (c ysgly) and cyst e ine were measured in media and cell samples. Each amino acid responded differently to B6 status. Se vere (10 nmol/L PL) and marginally (50 nmol/L PL) deficient media contained significant ly greater homocysteine concentration s compared to adequate concentrations (200 and 2000 nmol/L PL) of B6 Cellular concentrations of homocysteine did not differ signif icantly among the various concentrations of PL. GSH concentrations in media also remained approximately constant for all B6 concentrations. In culture d cells GSH was significantly lower in 10 nmol/L PL (severe B6 deficiency ) than in other concentrations of B6 Similar trends were observed for c ysgly and cysteine in media; both were significantly greater in 10 nmol/L PL while other B6 concentrations were equivalent No significant differences were observed for c ysgly or cysteine concentration s in cellul ar analysis. These data for amin othiols are presented in Table 3 6

PAGE 45

45 Cellular Concentrations of Amino A cids Are Affected by V itamin B6 Status in HepG2 C ells The concentration of intracellular amino acids, including lanthionine, cystathionine, and homolan t hionine are presented in Table 3 7 GC/MS was used to quantify l anthionine, cystathionine and homolanthionine due to lower detection limits and greater specificity. Homolanthionine was quantified using the cystathionine calibration curve in GC/MS in vie w of the similarity of these two amino acids and the lack of a commercially available source of pure homolanthionine. Small quantities of h omolanthionine w ere produced spontaneously in the derivatization of homocysteine standards The amount produced in creased in a linear relationship with the concentration of homocysteine. From these amounts, on e can conclude endogenous production of homolanthionine was much greater than non enzymatic production due to derivatization. Lanthionine, homolanthionine, and cystathionine were three amino acids produced in the transsulfuration pathway by B6 dependent enzymes measured by GC/MS. Homolanthioni ne and lanthionine concentration s were greater in cells with adequate B6 status. Lanthionine concentration was signific antly lower in 10 nmol/L PL and 50 nmol/L PL (P<0.002) while homolanthionine trended toward a lower concentration in B6 deficient cells (P<0.02 ). Cystathionine concentration was greater in marginally deficient cells compared to severely deficient cells P <0.06 There was no significant difference in cystathionine concentration between adequate (200 nmol/L PL) and supraphysiological (2000 nmol/L PL) B6 concentrations. Amino acid analysis by HPLC was used to quantify a ll other amino acids in T able 3 7 Whi le m ost amino acids are dependent on B6 for synthesis and/ or degradation, not

PAGE 46

46 all showed significant differences in concentration according to B6 restriction Alanine concentra tions were significantly greater in severe (10 nmo l/L PL) deficiency compared to marginal (50 nmol/L PL) deficiency and adequate (200 and 2000 nmol/L PL) B6 status. Asparagine, threonine, and valine concentrations were greater in deficient cells compared to other concentrations of B6 (P<0.002) C oncentrations of leucine (P<0.01) l ysine (P<0.008) and serine (P<0.03) trended toward an increase in severe deficiency compared to marginal deficiency Glycine was significantly greater in severe B6 deficiency compared to marginal deficiency and supraphysiological (2000 nmol/L PL) B6 conc entrations. Glutamine concentration was significantly greater in severe and adequate B6 status compared to marginal B6 deficiency and supraphysiological B6 status Asparagine to aspartate ratios were not statistically different between various concentrati ons of B6 The ratio of glutamine to glutamate was significantly greater in 10 nmol/L PL compared to 2000 nmol/L PL. Discussion This study sought to determine the impact of B6 restriction on metabolism in HepG2 cells by assessing the full profile of amin o acids, including the H 2 S biomarkers, lanthionine and homolanthionine. Previous research has identified PLP as an essential coenzyme in amino acid metabolism but there has not been a relationship established between B6 status and H 2 S production. In addi tion to quantifying H 2 S biomarkers in relation to B6 status, this study also displayed the differences in amino acid concentrations between severe deficiency, marginal deficiency, and adequate B6 status. In ag reement with earlier studies, changes in concen tration of amino acids consistent with B6 deficiency were observed Glycine elevation induced by B6 deficient

PAGE 47

47 media was reported in this study as well as in investigations of both animals and humans demonstrating a function al change in PLP dependent meta bolism ( 116 ) Glycine elevation could be a result of decreased SHMT or glycine decarbox y lase activity as both enzymes are sensitive to a loss of activity at varying degrees of B6 ( 157 158 ) Serine elevation in B6 deficient cells may also be explained by impaired SHMT activity although the re are many enzymes that are PLP dependent that catabolize serine. Alanine, asparagine glutamine, threonine and valine metabolism were affected by B6 status in this study While the mechanisms responsible for these significant differences were not stud ied, one can infer changes were due to the many PLP dependent enzymes that synthesize or catabolize these amino acids. The ra tio of glutamine to glutamate was greater in severely deficient cells (10 nmol/L PL) compared to supr aphysiological B6 concentratio n (2000 nmol/L PL). This is consistent with previous findings in B6 restriction studies in humans ( 159 ) although the mechanism is unknown. Transsulfuration metabolites have been studied extensively in relation to B6 status due to the PLP dependent enzymes, CBS and CSE H omocysteine concentration was shown to be inve rsely associated with PLP concentration in humans ( 160 ) but changes are often minor due to tight regulation of one carbon metabolism. T his simple cell model showed a significantly greater concentration of homocysteine in B6 deficient cells suggesting an imbalance bet ween the transsulfuration pathway and the remethylation cycle The greater concentration of homocysteine in conjunction with a lower cystathionine concentration (P<0.06) could indicate decreased CBS activity in 10 nmol/L PL ( 120 124 ) CBS activity is not affected until severe B6 deficiency

PAGE 48

48 de ducing that 10 nmol/L PL was a model of severely deficient cells. C yst athionine elevation is a strong indicator of marginal B6 deficiency (P<0.09) ( 118 121 122 ) which lead to the determination that 50 nmol/L PL was representative of marginal deficiency Cysteine elevation i n media samples in severe B6 deficiency indicate d t hat although CBS and CSE activities decreased the e levated concentrations of homocysteine force d product formation through the transsulfuration pathway ( 161 ) However the breakdown of cysteine after the transsul furation pathway also require d PLP dependent enzymes which may have contribute d to the elevation in cysteine pools that remain ed in deficiency. GSH is a final product from the formation of cysteine in the transsulfur ation pathway used in many protective r oles in the body Previous research from this laboratory show ed contrasting results. Studies by Lima et al and Davis et al show ed significant increases in GSH concentrations in human plasma and rat liver when B6 was restricted ( 124 162 ) Lamers et al observed a decrease in GSH synthesis in B6 deficiency and concluded that the effects of marginal deficiency o n GSH synthesis were not caused by altered precursor concentrations ( 123 ) In the pre sent study, GSH was significantly decre ased in severely deficient media Due to the elevation of cysteine but decrease in GSH concentration it is suggested that cysteine concentration was not a limiting factor in GSH synthesis. H 2 S and its biomarkers, lanthionine and homolanthionine are by products of the transsulfuration pathway Previous publications have reported a range of values for H 2 S concentration s in tissues and biological fluids, which indicates that methods of direct quantification of H 2 S m ay be unreliable Therefore the quantification of lanthionine and homolanthionine could be used as indirect measurements for H 2 S

PAGE 49

49 production ( 144 ) This study demonstrate d th at lanthionine and homolanthionine were readily measurable by GC/MS in cell extracts. Lanthionine and homolanthionine concentration s were lower in B6 deficient cells, suggesting H 2 S production and concentration may have also been lower In conclusion, B6 deficiency has been linked to increased disease risk, specifically CVD ( 149 ) While there are many indicators of CVD there is no mechanistic understanding of the relationship between B6 deficiency and CVD ( 149 ) This s tudy show ed that H 2 S biomarker concentrations which may be indicative of H 2 S production were lower in B6 deficiency. Mouse model studies have shown CSE depletion decrease s H 2 S levels in the brain, serum, and heart ( 143 ) Because H 2 S is a vaso relaxant and essential in smooth muscle function, it demonstrates a possible explanation of how B6 deficiency increases CVD risk ( 133 135 141 143 ) This preliminary study allows for mechanistic effect s to be further investig ated

PAGE 50

50 Table 3 1. Final storage conditions for each flask collected for weekly analysis. Table 3 2 Mobile phase gradients programs for separation of Dns amino acids. Mobile Phases : A. 0.6% acetic acid, 0.08% triethylamine (v:v) in DiH 2 O B. 0.6% acetic acid, 0.08% triethylamine (v:v) in 80/20 acetonitrile/ DiH 2 O. Gradient Shapes: 0, no slope; 5, lin ear. Time Acetonitrile (%) Mobile Phase A Mobile Phase B Gradient Shape 0 26.4 67 33 5 33 28.0 65 35 5 37 40.0 50 50 5 50 42.4 47 53 0 54 42.4 47 53 5 72 44.8 44 56 5 86 56.0 30 70 5 97 80.0 0 100 0 102 80.0 0 100 5 104 26.4 67 33 0 114 26.4 67 33 0 Table 3 3 PL concentrations in media determined by HPLC Media Concentration (nM) Actual PL (nM) 10 17.7 50 45.3 200 239 2000 1709 Assay Amount of Cells (L) Sample additions Homocysteine 50 Bradford 100 300 L H 2 O PLP 500 500 L 10% TCA Amino Acid 500 500 L Methanol

PAGE 51

51 Table 3 4. Correlation coeffic ients for each amino acid standard curve. Amino Acid Correlation Coefficient Histidine 0.982 Arginine 0.993 Asparagine 0.990 Glutamine 0.987 Serine 0.986 Glutamate 0.979 Hydroxyp roline 1.00 Aspartate 0.965 Threonine 0.989 Methionine Sulfone 0.975 Glycine 1.00 Alanine 0.992 Proline 1.00 Methi onine 0.998 Valine 0.999 Tryptophan 0.995 Isol eucine 0.988 Phenylalanine 0.965 Leucine 1.00 Lanthionine 0.888 Cystathionine 0.97 0 Cysteine/Cystine 0.996 Lysine 1.00 Tyrosine 1.00 Data is expressed in 3 points in the standar d curve and replicated before each run.

PAGE 52

52 Table 3 5. Recovery analysis for each amino acid in cell analysis. Amino Acids Percent age Recovery Histidine 102 Arginine 116 Asparagine 105 Glutamine 107 Serine 79.5 Hydroxyp roline 93.1 Threonine 101 Methionine Sulfone 86.4 Glycine 99.8 Alanine 102 Proline 104 Methionine 100 Valine 99.7 Tryptophan 100 Iso leucine/Phenylalanine 104 Leucine 93.5 Lanthionine 90.8 Cystathionine 91.5 Lysine 93.2 Data exp ressed as mean, n=3.

PAGE 53

53 Table 3 6 Intracellular and extracellular amino acid concentrations. Pydridoxal Concentration 10 nM 50 nM 200 nM 2000 nM Media (nmol/mg protein) Homocysteine 13.21.56 a 9.780.432 a 5.130.572 b 5.930.505 b GSH 1.020.222 1.060.249 1.070.140 1.170.099 CysGly 6.660.693 a 5.340.190 b 5.460.341 b 4.580.389 b Cyst e ine 59.16.23 a 35.71.99 b 29.93.63 b 33.62.56 b Cell (nmol/mg protein ) Homocysteine 0.2170.128 0.2090.075 0.1210.036 0.1420.052 GSH 12914.1 b 17415.8 a 16812.1 a 1534.60 a,b CysGly 1.770.039 2.010.394 3.511.29 1.890.110 Cyst e ine 61.93.44 50.33.63 55.32.92 52.32.35 Values are means SD, n=4. Da ta analyzed by ANOVA. Means with unlike superscripts in the same row are different, p<0.00 2

PAGE 54

54 Table 3 7 Intracellular amino acid concentrations. Pyridoxal Concentration 10 nM 50 nM 200 nM 2000 nM nmol/mg protein Alanine 64.98.96 a 29.54.54 b 57.5 2.64 a 46.23.22 a Arginine 6.580.30 2.871.58 7.071.2 6.160.974 Asparagine 11.90.873 a 8.790.730 a 10.071.08 a 7.710.625 b Aspartate 39.77.08 35.616.34 53.113.3 40.910.71 Glutamate 12230.1 1199.80 18917.1 13224.3 Glutamine 11111.3 a 63.66.4 4 b 1313.46 a 72.82.65 b Glycine 30.10.935 a 19.81.92 b 25.21.12 a 19.80.914 b Histidine 18.53.56 20.25.31 11.81.86 10.83.28 Leucine 12.80.36 8.071.92 11.40.959 9.020.778 Lysine 11.91.40 8.850.968 9.860.454 9.030.663 Methionine 2.970.392 1 .710.274 3.350.264 1.430.269 Proline 30.74.34 30.41.68 29.01.62 28.22.28 Serine 13.30.867 4.092.25 10.72.61 8.543.22 Threonine 19.51.81 a 6.322.33 b 21.23.36 a 10.42.28 a,b Tryptophan 2.300.342 2.290.187 2.310.059 2.060.125 Tyrosine 4. 880.846 4.340.879 5.040.463 4.780.647 Valine 9.850.386 a 6.090.717 b 8.760.508 a 6.740.448 b Cystathionine 4.240.215 5.500.385 4.710.567 4.380.793 pmol/mg protein Lanthionine 0. 6981.06 b 1.380.179 b 1.560.181 a 1.700.594 a Homolanthionine 93 615 8 119 16 8 125 26 2 177 66 0 Values are means SD, n=4. Data analyzed by ANOVA. Means with unlike superscripts in the same row are different p<0.00 2

PAGE 55

55 Figure 3 1 Cellular PLP concentration during 6 week stabilization for each concentratio n of PL.

PAGE 56

56 Figure 3 2. A representative chromatogram of amino acid separation.

PAGE 57

57 CHAPTER 4 VITAMIN B6 STATUS IN HEPG2 CELLS AFFECTS THE TRANSSULFURATION AND REMETHYLATION PATHWA YS WHEN ANALYZED BY STABLE ISOTOPE TRACE R TIME COURSE Stable isotope tracer experiments in cell culture and human subjects allow scientists to understand metabolite kinetics over specific time periods T his study focused on quantifying the formation of transsulfuration products. [U 13 C] L methionine and (3,3 D2) L cysteine were used as precursors of labeled amino acids and H 2 S biomarkers in cultured HepG2 cells. Methionine is a precursor of S adenosylmethionine (SAM) in one carbon metabolism. SAM is the main one carbon donor used in over 100 transmethylation reactions ; it is al so an allosteric activator of CBS, an enzyme in the transsulfuration pathway Homocysteine is produced from SAM after which it can either be remethylated for recycling of methionine or shunted into the transsulfuration pathway to form cystathionine and la ter cysteine. T he transsulfuration enzymes catalyze side reactions which produce H 2 S and its biomarkers. Condensation of two homocysteine molecules produces homolanthionine while the condensation of two cysteine molecules produces lanthionine. This study was conducted to determine if the labeling of lanthionine and homolanthionine was quantifiable in in vivo stable isotope experiments and also if the kinetics of the transsulfuration pathway were affected in B6 restriction Previous research in this lab h as shown [U 13 C 5 ] methionine resulted in [ 13 C 4 ] homocysteine which in turn labeled cys tathionine. Fractional synthesis rates were determined and the effect on amino acid metabolism based on B6 status was assessed.

PAGE 58

58 Materials and Methods Stable Isotope Mate rials [U 13 C 5 ] L Methionine 97 98% purity, and (3,3 D2) L Cysteine 98% purity, were purchased from Cambridge Isotopes (Andover, MA) Complete cell culture media was enriched by 20% with [U 13 C 5 ] L Methionine and (3,3 D2) L Cysteine based upon calculatio ns of methionine and cysteine present in media. Each concentration of B6 (10, 50, 200, and 2000 nmol/L PL) was enriched as stated above. All other chemicals used were HPLC grade of higher and purchased from Fisher Scientific (Pittsburgh, PA) or Sigma Ald rich (St. Louis, MO). Cellular Preparation HepG2 cells were maintained as stated previously After final passage, cells were grown to ~90% confluency and fresh media was added to cell flasks 24 hours prior to experimentation. On the day of the experiment, the media were removed and the cells were washed with D PBS. Enriched media, 15 mL, was added to each 75 cm 2 flask. The time points of this study were 0, 0.5, 1, 2, 4, and 6 ho urs. At each time point, 500 L of medium was removed and the cells washed wi th D PBS. The cells were scra ped from the bottoms of the flasks by a rubber spatula and collected in 500 L D PBS. Cell and medium aliquots (500 L) were immediately acidified with 50 L of 66% TCA, vortexed and stored at 80 until analysis. The day of analysis, cells were thawed and 100 L of the internal standard norleucine, was added. Then samples were homogenized by the ultrasonic h omogenizer and both sample sets (cell and media) were centrifuged at 6,100 x g for 15 m in. The supernatants were isolated and analysis followed according to GC/MS methods reported previously ( 154 156 )

PAGE 59

59 Kinetic Analysis [U 13 C 5 ] Methionine pr oduced [ 13 C 4 ] homocysteine through the remethylation pathway in one carbon metabolism. From this point, [ 13 C 4 ] homocysteine c ould enter the transsu lfuration pathway to produce [ 13 C 4 ] cystathionine and ketobutyrate or cycle back into [ 13 C 4 ] methionine Due to homocysteine condensation which produced homolanthionine; the possible products were [ 13 C 4 ] or [ 13 C 8 ] homolanthionine. (3,3 D2) Cysteine produce d [D2] or [D4] lanthionine in condensation reactio ns Time points of each amino acid were plotted using SigmaPlot 12.0. Enrichment plateaus (Ep) were determined for each precursor and product that reached steady state within the first 30 minutes. Regression curves from y = a (1 e bx ) were applied to each graph to determine Ep and initial rate (I) as represented in Figure 4 8 FSR and ASR were the determined using the followi ng equations. Methionine remethylation and homocysteine production were determined by subsequent equations. FSR=I/Ep Remethylation = Ep M+4 methionine /Ep (M+4)+(M+5) methionine ASR=([AA])(FSR) Hcy Production= Ep M+4 homocysteine /Ep (M+4)+(M+5) methionine Statistical Analysis All data are presented as mean standard deviation. Data were log transformed to pass the Gaussian assumption. One way ANOVA with pairwise multiple comparisons was used to determine statistical significance between each group of B6 concentrations o f F SR and ASR P<0.05. All statistical analyses were performed by SigmaPlot 12.0 software. Results The stable isotope tracer experiment produced quantifiable labeled products f rom [U 13 C 5 ] L methionine and (3,3 D2) L c ysteine Ratios of labeled to unlabeled amino

PAGE 60

60 acids wer e plotted versus time (Figures (4 1) (4 7) ). The enrichment plateau f or methionine (Table 4 1) in t he 10, 50, 200, and 2000 nM PL concen trations indicated ~13% media enrichment Methionine enrichment plateaus were approximately equivalent in each concentration of B6 The cysteine enrichment was much lower and did not plateau as quickly as methionine ( Figure 4 2 ) T he enrichment ratios o f cysteine over the six hour time course f o r 10, 50, 200, and 2000 nmol/L PL were ~1.3% and 50 nmol/L PL was significantly greater than all other enrichment ratios. [ 13 C 4 ] M ethionine reached enrichment plateau within 3 0 minutes ( Figure 4 3 ) indicating remethylation While r emethylation was significantly different between marginal B6 deficiency (50 nmol/L PL) and adequate B6 status (200 and 2000 nmol/L PL) there did not seem to be a great change in remethylation due t o B6 status. [ 13 C 4 ] Homocysteine reached enrichment plateau after 2 hours. The initial rate of product formation corresponds to the steepest slope in the enrichment curve. The FSR was determined f rom the enrichment plateau o f the precursor directly prece ding that amino acid in f ormation. The apparent ASR was determined by multiplying the F SR with the initial concentration o f the amino acid normalized per mg/ protein. The F SR and ASR values f or each PL concentration and each label ed product are listed in Table 4 2. FSR of homocysteine was not stati sti cally different between concentration s of B6 FSR of cystathionine was significantly greater in marginal B6 deficiency compared to all other concentration s of B6 [ D2 ] Lanthionine FSR was significantly gre ater in adequate and supraphysiological concentrations of B6 compared to severe and marginal deficiency [ 13 C 4 ] ho molanthionine FSR was

PAGE 61

61 significantly greater in adequate B6 cells (200 and 2000 nmol/L PL) compared to deficient cells (10 and 50 nmol/L). D iscussion Stable isotopes are a valuable tool in metabolic kinetic studies. The target enrichment of each precursor in this study was approximately 20%, however [ U 13 C 5 ] methionine was 13% and [ D2 ] c ysteine was 1. 3 % enriched. The low enrichment of cystei ne differed from prelimi nary experimentation leading to the belief that the stock solution became insoluble or there was human error The low labeling of cysteine did produce a sufficient ly label ed la nthionine produ ct enabling FSR determination. [U 13 C 5 ] M ethionine reached an enrichment plateau within the first 30 minutes of the 6 hour time course whereas (3,3 D2) cysteine did not most likely due to its ability to disulfide bond reversibly Remethylation of methionine determined by [ 13 C 4 ] methionine ind icated very small changes due to B6 status By labeling amino acids, the fractional synthesis rates of products via the transsulfuration pathway were determined also determining the synthesis of H 2 S biomarkers Homocysteine FSR as well as homocysteine pr oduction rate did not change between B6 concentrations; suppo rting previous studies ( 117 125 ) Elevations of homocysteine concentration were seen in media samples in deficient cells yet there was no ch ange in FSR. This suggest s that the homocysteine pool was greater due to the decreased activity of the transsulfuration enzymes not because of greater synthesis from the meth ionine precursor. While Lamers et al showed an increase in the FSR of cystathionine in B6 restriction in human patients, Davis et al observed no effects of B6 restriction on cystathionine FSR ( 117 122 ) Our study showed severely deficient cells (10 n mol/L PL )

PAGE 62

62 had a lower c ystathionine FSR whereas cells with marginal deficiency (50 nmol/L PL) had a higher cystathionine FSR compared to cells in adequate (200 and 2000 nmol/L PL) B6 concentrations The concentration of cystathionine in severely deficient cells is a lso lower than in 50 nmol/L PL concentration This could be due to decreased CBS activi ty or dysregulation of SAM since the pool of homocysteine was elevated but the synthesis and concentration of its catabolic product, cystathionine, was depressed. Martinez et al showed SAM liver concentration was de creased in rats fed a B6 deficient diet ( 125 ) C ystathionine FSR and concentration w ere greater in marginally deficient cells compared to other B6 concentratio ns. Cystathionine concentration elevation has been shown to be an indicator of marginal B6 deficiency thereby supporting o ur conclusion that 50 nmol/L PL represents physiological marginal deficiency ( 121 124 ) In addition to the tr aditional products of the transsulfuration pathway (homocysteine, cystathionine, and cysteine) lanthionine and homolanthionine were also measured for enrichment and FSR [ 13 C 4 ] H omolanthionine and lanthionine FSR s were significantly greater in cells of a dequate (200 and 200 nmol/L PL) B6 status compared to cells deficient (10 and 50 nmol/L PL) in B6. Greater synthesis of homolanthionine and lanthionine in B6 adequacy suggests the production of H 2 S was also greater in cells of adequate B6 status ( 127 ) Dually labeled homolanthionine and lanthionine were visibly present in the spectrum but too low to reliably quantify for this experiment. There was, however, a r ise in presence of these products throughout the time course. In conjunction with the conclusion that greater H 2 S biomarker production was associated with higher B6 concentration s d ata from Appendix showed the H 2 S production capacity was greater in cell s of adequate B6 status compared to deficient

PAGE 63

63 cell s Dr s Ruma Banerjee and Omer Kabil University of Michigan Medical School, quantified by GC the capacity at which cells in each concentration of B6 could produce H 2 S Cell lysates placed in buffers cont aining high concentrations of cysteine and homocysteine (>>K m ) allow ed CSE and CBS to produce H 2 S at V max These in vitro conditions allowed assessment of the influence of cellular B6 availability on the capacity to produce H 2 S. The ability to produce H 2 S and the increased production of H 2 S biomarkers observed in this study showed that H 2 S production was affected by B6 status in HepG2 cells. F igure 4 9 and 4 10 display ed the relationship between lanthionine and homolanthionine concentration in accordance to H 2 S production capacity.

PAGE 64

64 Table 4 1. Enrichment plateaus and methylation cycle kinetics. 10 nM 50 nM 200 nM 2000 nM Ep [U 13 C 5 ] Methionine 0 .130 0 .00253 0 .131 0 .000523 0 .128 0 .00143 0 .137 0 .00771 [D2]Cysteine 0.01130.00103 b 0.01920.000676 a 0 .01220.00238 b 0.01010.000830 b [ 13 C 4 ]Methionine 0.0187.000483 a 0.01070.000231 b 0.01980.00138 a 0.02130.00253 a Methionine Remethylation 0.162 0. 00 3 42 a, b 0.145 0 .00156 b 0.174 0 .0110 a 0.1 7 70.0160 a Homocysteine Production 0.9720.0608 1.030.0248 0.96 40.0393 0.9800.0827 Values are means SD, n=4. Data analyzed by ANOVA. Symbols indicate statistically difference p<0.0 5

PAGE 65

65 Ta ble 4 2. F SR and ASR o f amino acids in various concentrations of B6 10 nM 50 nM 200 nM 2000 nM FSR (hr 1 ) ASR (p m ol/mg protein/hr) FSR (hr 1 ) ASR (p mol/mg protein/hr) FSR (hr 1 ) ASR (p mol/mg protein/hr) FSR (hr 1 ) ASR (nmol/mg protein/hr) [ 13 C 4 ] Cystathionine 0 .135 0 0134 c 35435 1 a 0.243 0 .00488 a 1 10 0 222 b 0.145 0 .00552 c 75628 8 c 0.173 .00719 b 1 31 0 54 7 d [ 13 C 4 ] Homocysteine 0.941 0 .137 20429 6 a 0 .816 0 .173 17036 2 a 0 .733 0 .0195 88 72 36 b 0 .708 0 .0816 10011 6 b [D2] Lanthionin e 0.245 0 .0788 b 0 1710 0551 b 0 .106 0 .0140 b 0 1480 0195 b 0 .298 0 .119 a,b 0 4770 191 b 0 .697 0 .307 a 1 190 522 a [ 13 C 4 ] Homolanthionine 0 .00662 0 .00221 b 0 62 0 0 206 b 0 .00487 0 .000971 b 0 5790 116 b 0 .00999 0 .00364 a 1 250 455 a,b 0 .0287 0 .0224 a 5 093 97 a Values are means SD, n=4. Data analyzed by ANOVA. Symbols indicate statistically difference p<0.0 5

PAGE 66

66 Figure 4 1. Enric hment t ime course of precursor [U 13 C] m ethionine.

PAGE 67

67 Figure 4 2. Enrichment time course of precursor [D2] cysteine.

PAGE 68

68 Figure 4 3. Enr ichment time course of product [ 13 C 4 ] m ethionine.

PAGE 69

69 Figure 4 4. Enr ichment time course of product [ 13 C 4 ] h omocysteine.

PAGE 70

70 Figure 4 5. Enrichment time course of product [ 13 C 4 ] cystathionine

PAGE 71

71 Figure 4 6 Enr ichment time course of product [D2] l anthionine

PAGE 72

72 Figure 4 7. Enrichment time course of product [ 13 C 4 ] h omolanthionine.

PAGE 73

73 Figure 4 8. Regression curve analysis in SignmaPlot 12.0 determined initial rate (I) and enrichment plateau (Ep) for each amino acid in each sample. Figure 4 9. H y d rogen sulfide production capac it y plotted versus lanthionine concentration in HepG2 cells. 10 nM 50 nM 200 nM 2000 nM 0.00E+00 2.00E-04 4.00E-04 6.00E-04 8.00E-04 1.00E-03 1.20E-03 1.40E-03 1.60E-03 1.80E-03 0 0.5 1 1.5 2 2.5 3 3.5 4 Lanthionine Concentration (nmol/ mg protein) Hydrogen Sulfide Production Capacity (nmol/g cells/hr) Lanthionine I Ep

PAGE 74

74 Figure 4 10. Hy d roge n sulfide production capacity plotted versus homolanthionine concentration in HepG2 cells. 10 nM 50 nM 2000 nM 200 nM 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 0 0.5 1 1.5 2 2.5 3 3.5 4 Homolanthionine Concentration (nmol/ mg protein) Hydrogen Sulfide Production Capacity (nmol/g cells/hr) Homolanthionine

PAGE 75

75 CHAPTER 5 CONCLUSIONS The vast number of reactions that require B6 demonstrates its essentiality in bodily functions. From neurotransmitter synthesis, to amino acid metabolism, to disease, B6 is involved in at least one mechanism needed for each process. While most of these mechanisms are well understood, the mechanism in which B6 relates to CVD is unknown. Several hypotheses have been developed with experiment al support but more possibilities exist. This research project so ught to determine the relationship between B6 s tatus H 2 S production to provide preliminary data for anot her such mechanism to relate B6 and CVD. H 2 S once thought to be solely a toxic chemic al, has now been shown to be produced and regulated in the body. Endogenously produced H 2 S acts as a gas signaling molecule as well as a physiologic vasodilator and regulator of blood pressure ( 143 ) CSE an d CBS are the main producers of H 2 S, both requiring PLP as a coenzyme. Therefore, H 2 S biogen e sis is PLP dependent. S ince PLP could possibly regulate H 2 S production and tha t production affects vasodilation and blood pressure, both impact factors for CVD one can theoretically define a relationship between B6 and H 2 S modulating CVD risk The initial experiment quantified amino acid concentrations in cells of various concentrations of B6 ranging from severely deficient to adequate. Biomarkers of H 2 S produc tion, lanthionine and homolanth ionine, could indirectly quantify the production of H 2 S in the cells since direct quantification was unavailable and most likely unreliable. Data from this experiment showed that severe deficiency (10 nmol/L PL) and marginal deficiency (50 nmol/L PL) impaired the metabolism of amino acids: g lycine, alanine,

PAGE 76

76 aspar a gine, glutamate, leucine, and valine The ratio of glutamine to glutamate was also affected by B6 status although the mechanism of this result is unknown. The trans sulfuration products; homocysteine, cystathionine, glutathione, and cysteine were also affected in deficiency due to decreased activity of CSE and CBS. The concentration of lanthionine and homolanthionine decreased in B6 deficient cells. Most of these da ta were in agree ment with previous studies in humans, cells, or rats on the effects of B6 status on amino acid concentration. This novel determination of H 2 S biomarker concentrations gives an initial view into connection between H 2 S concentration and B6 s tatus. The second experiment sought to determine the kinetics of the transsulfuration pathway as well as the methionine cycle by the use of isotopic labeling techniques. H 2 S biomarker s production were determined and quantified indicating endogenous prod uction was substantial Lanthionine and homolanthionine FSRs were lower in B6 deficiency compared to adequate B6 status. The methylation cycle was not affected by B6 restriction but the activity of the transsulfuration enzymes were, causing significant d ifferences in synthesis rates of transsulfuration amino acids. The inhibition of CBS and CSE as well as the lower FSRs of lanthionine and homolanthionine suggest that H 2 S production was inhibited by B6 restriction Collaboration with Dr. Ruma Banerjee, University of Michigan Medical School, reinforced the association of H 2 S with B6 concentration. In vitro experiments of HepG2 cell lysates place d in buffers with high concentrations of homocysteine and cysteine induce d H 2 S production at maximum rate. Thu s the limiting factor was the concentration of B6 which influences the activities of CBS and CSE This experiment

PAGE 77

77 studied the production capacity of H 2 S in each concentration of B6 A correlation between B6 concentration and H 2 S production cap acity was s een among replicates, show ing production capacity was decreased in B6 deficiency. These data also showed lanthionine and homolanthionine were accurate biomarkers of H 2 S production because their concentrations in cells generally paralleled the production c apacity. However, homolanthionine is likely to be a better biomarker due to its resistance to degradation ( 163 ) HepG2 cells responded to B6 deficiency similar to previously published data in rat and hu man studies. Therefore, HepG2 cells can be used to model physiological results of B6 restriction in humans Since this is a reliable model, the effect of B6 concentration on lanthionine and homolanthionine concentratio n should be seen in other studies These experiments also relate d the production and concentration of H 2 S biomarkers to the production capacity of H 2 S Therefore, by quantifying the concentration of lanthionine and homolanthionine one can infer the production of H 2 S is directly related, co ncluding that B6 status affected H 2 S production in HepG2 cells. This project provides preliminary data to support another mechanism in which B6 status is related to CVD Presently, H 2 S is known as a neuromodulator and vaso relaxant in many species and i ts regulation is necessary to maintain normal cardiovascular function. T here is a link between B6 deficiency and CVD although the mechanism is uncertain. F uture studies in rats and humans could determine if this correlation remains. Furthermore, studies in humans with CVD may identify if these mechanistic predictions

PAGE 78

78 exist in diseased state s as well as in the excretory pathways Continuing research in H 2 S production could allow for a reliable method of determining CVD risk.

PAGE 79

79 APPENDIX A GENERAL METHODS Protein Concentration Measurement The Bradford assay was used to determine p rotein concentration in cells in order to normalize data. BioRad Protein Assay Solution was diluted 1:5 in HPLC grade water. The dye solution was then filtered through Whatman # 1 filter paper. Bovine serum albumin (BSA) diluted i n to 1, 0.8, 0.4, 0.2, 0.1 mg/mL by serial dilutions to act as a calibration curve. 50 L of each sample of standard was added to 2.5 mL working dye solution. Samples were vortexed and allowed to sit f or 5 minutes before analysis. Samples were read at 595 nm absorbance using a spectrophotometer (Beckman DU 640) ( 164 ) Absorbance was recorded for all samples. A graph of standard concentrations versus absorbance allowed for the con centration of samples to be determined. A representative graph from standards is shown below. Figure A 1. Absorbance of BSA standards versus concentration of standards provides the linear equation needed to quantify total protein concentration in cell samples y = 0.9859x 0.0172 R = 0.9985 0 0.2 0.4 0.6 0.8 1 1.2 0 0.2 0.4 0.6 0.8 1 1.2 Absorbance BSA Concentration (mg/ml) Bradford Assay

PAGE 80

80 Hydrogen Sulfide Measurement Collaboration with Dr. Ruma Banerjee and Dr. Omer Kabil at the University of Michigan resulted in the quantification of H 2 S production capacity in the four concentra tions of B6 In vitro experiments with cell lysate s of each concentration of PL were analyzed by GC for H 2 S concentration. Cell l ysates were added to a buffer containing 10 mM cysteine and 10 mM homocysteine (>> K m ) Concentration of substrates greater than K m enabled the enzymes producing hydrogen sulf ide to operate at V max with B6 concentration acting as the limiting factor. These experiments were replicated 5 times on separate days. Figure A 2 show s H 2 S production capacity was decreased in cells under B6 restriction. This data supplements results fo und from quantifying H 2 S biomarkers as indicators of H 2 S production. Figure A 2 H ydrogen sulfide production capacity differs under varying concentrations of B6 (10, 50, 200, and 2000 nmol/L PL) displayed as mean standard deviation

PAGE 81

81 REFERENCES 1. Birch TW, Gyrgy P. A study of the chemical nature of vitamin B(6) and methods for its preparation in a concentrated state. Biochem J. 1936;30:304 10. 2. Gyorgy P. Vitamin B2 and the pellagra like dermatitis in rats. Nature. 1934;133:4 48 49. 3. Gyrgy P. Crystalline vitamin B6. J Am Chem Soc. 1938;60:983 84. 4. Kuhn R, Wendt G. ber das antidermatitische Vitamin der Hefe. Berichte der deutschen chemischen Gesellschaft (A and B Series). 1938;71:780 82. 5. Ichiba A, Michi K. Cristallin e vitamin B6. Sci Pap Inst Phys Chem Res. 1938;34:623 26. 6. Snell EG, BM Williams, RJ. Occurence in natural products of a physiologically active metabolite of pyridoxine. J Biol Chem. 1942;143:519 30. 7. Keresztesy JC, Stevens JR. Crystalline vitamin B 6. Exp Biol Med. 1938;38:64 65. 8. Lepkovsky SS. Crystalline Factor 1. Science. 1938;87:169 70. 9. Snell EE. The vitamin activities of "pyridoxal" and "pyridoxamine". J Biol Chem. 1944;154:313 14. 10. Snell E. The vitamin B6 group: 1. Formation of addi tional members from pyridoxine and evidence concerning their structure. J Am Chem Soc. 1944;66:2082 88. 11. Harris SA, Heyl D, Folkers K. The structure and synthesis of pyridoxamine and pyridoxal. J Biol Chem. 1944;154:315 16. 12. Gunsalus IC, Bellamy WD Umbreit WW. A phosphorylated derivative of pyridoxal as the coenzyme of tyrosine decarboxylase. J Biol Chem. 1944;155:685 86. 13. McCormick D. Present Knowledge in Nutrition 9 edn. Washington (DC): International Life Sciences Institute; 2006. 14. Snel l EE. Vitamin B6. Compr Biochem. 1963;2:48 58. 15. Wiesinger H, Hinz H J. Kinetic and thermodynamic parameters for Schiff base formation of vitamin B6 derivatives with amino acids. Arch Biochem Biophys. 1984;235:34 40.

PAGE 82

82 16. Matsuo Y. Formation of Schiff b ases of pyridoxal phosphate. Reaction with metal ions. J Am Chem Soc. 1957;79:2011 15. 17. Andon MB, Reynolds RD, Moser Veillon PB, Howard MP. Dietary intake of total and glycosylated vitamin B 6 and the vitamin B 6 nutritional status of unsupplemented la ctating women and their infants. Am J Clin Nutr. 1989;50:1050 8. 18. Nakano H, Gregory JF. Pyridoxine and pyridoxine d glucoside exert different effects on tissue B glucosidase activity in rats. J Nutr. 1995;125:27 51 62. 19. Mackey AD, Henderson GN, Gregory JF. Enzymatic hydrolysis of pyridoxine d glucoside Is catalyzed by intestinal lactase phlorizin hydrolase. J Biol Chem. 2002;277:26858 64. 20. Gregory J, Ink SL. Identification and quantification of pyrido xine beta glucoside as a major form of vitamin B6 in plant derived foods. J Agric Food Chem. 1987;35:76 82. 21. Tarr J, Tamura T. Availability of vitamin B 6 and pantothenate in average american diet. J Clin Nutr. 1981;34:1328 37. 22. Nakano H, McMahon L G, Gregory JF. Pyridoxine d glucoside exhibits incomplete bioavailability as a source of vitamin B 6 and partially inhibits the utilization of co ingested pyridoxine in humans. J Nutr. 1997;127:1508 13. 23. Gregory J, Trumbo P, Bailey L. Bioavailabil ity of pyridoxine 5' B D glucoside determined in humans by stable isotope methods. J Nutr. 1991;121:177 86. 24. Roth Maier DA, Kettler SI, Kirchgessner M. Availability of vitamin B 6 from different food sources. Int J Food Sci Nutr. 2002;53:171 79. 25. G regory JF. Bioavailability of vitamin B6. Eur J Clin Nutr. 1997;51(suppl):S43 S48. 26. Bowers JA, Craig J. Components of vitamin B6 in turkey breast muscle. J Food Sci. 1978;43:1619 19. 27. Gregory JF, Ink SL, Sartain DB. Degradation and binding to food proteins of vitamin B 6 compounds during thermal processing. J Food Sci. 1986;51:1345 51. 28. Reiber H. Photochemical reactions of vitamin B6 compounds, isolation and properties of products. Acta Biochim Biophysica. 1972;279:310 15.

PAGE 83

83 29. Saidi B, Warthese n JJ. Influence of pH and light on the kinetics of vitamin B6 degradation. J Agric Food Chem. 1983;31:876 80. 30. Ink SL, Gregory JF, Sartain DB. Determination of vitamin B6 bioavailability in animal tissues using intrinsic and extrinsic labeling in the r at. J Agric Food Chem. 1986;34:998 1004. 31. Nakano H, Gregory JF. Pyridoxine d glucoside influences the short term metabolic utilization of pyridoxine in rats. J Nutr. 1995;125:926 32. 32. Gilbert JA, Gregory JF. Pyridoxine d glucoside affects the metabolic utilization of pyridoxine in rats. J Nutr. 1992;122:1029 35. 33. Said ZM, Subramanian VS, Vaziri ND, Said HM. Pyridoxine uptake by colonocytes: a specific and regulated carrier mediated process. Am J Physiol Cell Physiol. 2008;294:C1192 C97 34. Armada LJ, Mackey AD, Gregory JF. Intestinal brush border membrane catalyzes hydrolysis of pyridoxine d glucoside and exhibits parallel developmental changes of hydrolytic activities toward pyridoxine d glucoside and lactose in rats. J Nutr. 2002;132:2695 99. 35. Mackey AD, Lieu SO, Carman C, Gregory JF. Hydrolytic activity toward pyridoxine d glucoside in rat intestinal mucosa is not increased by vitamin B 6 deficiency: Effect of basal diet composition and pyridoxine intake. J Nutr. 200 3;133:1362 67. 36. Mackey AD, McMahon RJ, Townsend JH, Gregory JF. Uptake, hydrolysis, and metabolism of pyridoxine d glucoside in Caco 2 cells. J Nutr. 2004;134:842 46. 37. Buss DD, Hamm MW, Mehansho H, Henderson LM. Transport and metabolism of pyr idoxine in the perfused small intestine and the hind limb of the rat. J Nutr. 1980;110:1655 63. 38. Hamm MW, Mehansho H, Henderson LM. Transport and metabolism of pyridoxamine and pyridoxamine phosphate in the small intestine of the rat. J Nutr. 1979;109: 1552 59. 39. Mehansho H, Hamm MW, Henderson LM. Transport and metabolism of pyridoxal and pyridoxal phosphate in the small intestine of the rat. J Nutr. 1979;109:1542 51. 40. Kozik A, McCormick DB. Mechanism of pyridoxine uptake by isolated rat liver cel ls. Arch Biochem Biophys. 1984;229:187 93.

PAGE 84

84 41. Merrill AHH, JM Wang, E Mcdonald, BW Millikan, WJ. Metabolism of vitamin B6 by human liver. J Nutr. 1984;114:1664 74. 42. Kazarinoff MN, McCormick DB. Rabbit liver pyridoxamine (pyridoxine) 5' phosphate oxid ase. Purification and properties. J Biol Chem. 1975;250:3436 42. 43. McCormick DB, Chen H. Update on interconversions of vitamin B 6 with its coenzyme. J Nutr. 1999;129:325 27. 44. Merrill AH, Henderson JM. Vitamin B6 metabolism by human livera. Ann N Y Acad Sci. 1990;585:110 17. 45. Gregory JF, Kirk JR. Determination of urinary 4 pyridoxic acid using high performance liquid chromatography. Am J Clin Nutr. 1979;32:879 83. 46. Van Hoof VO, De Broe ME. Interpretation and clinical significance of alkaline phosphatase isoenzyme patterns. Crit Rev Clin Lab Sci. 1994;31:197 293. 47. Ink SL, Mehansho H, Henderson LM. The binding of pyridoxal to hemoglobin. J Biol Chem. 1982;257:4753 57. 48. Lumeng L, Brashear RE, Li TK. Pyridoxal 5' phosphate in plasma: sourc e, protein binding, and cellular transport. J Lab Clin Med. 1974;84:334 43. 49. Mehansho H, Henderson LM. Transport and accumulation of pyridoxine and pyridoxal by erythrocytes. J Biol Chem. 1980;255:11901 07. 50. Coburn SP, Lewis DL, Fink WJ, Mahuren JD Schaltenbrand WE, Costill DL. Human vitamin B 6 pools estimated through muscle biopsies. Am J Clin Nutr. 1988;48:291 4. 51. Said HM, Ortiz A, Vaziri ND. Mechanism and regulation of vitamin B6 uptake by renal tubular epithelia: studies with cultured OK c ells. American Journal of Physiology: Renal Physiology. 2002;282:F465 F71. 52. Zhang Z, Gregory JF, McCormick DB. Pyridoxine d glucoside competitively inhibits uptake of vitamin B 6 into isolated rat liver cells. J Nutr. 1993;123:85 89. 53. Bender D A. Novel functions of vitamin B6. Proc Nutr Soc. 1994;53:625 30. 54. Coburn SP. Location and turnover of vitamin B6 pools and vitamin B6 requirements of humans. Ann N Y Acad Sci. 1990;585:76 85. 55. Krebs EG, Fischer EH. Phosphorylase and related enzymes of glycogen metabolism. Vitam Horm. 1964;22:399 410.

PAGE 85

85 56. Miller LL, JE Shultz, ED The effect of dietary protein on the metabolism of vitamin B6 in humans J Nutr. 1985;83:1663 72. 57. Hansen CL, JE Miller, LT. Vitamin B 6 status of women with a constant intake of vitamin B 6 changes with three levels of dietary protein. J Nutr. 1996;126:1891 901. 58. Pannemans DVDB, H Westerterp, KR. The influence of protein intake on vitamin B6 metabolism differs in young and elderly humans. J Nutr. 1994;124. 59. Food and Nutrition Board. Recommended dietary allowances. National Academy of Sciences/National Research Council Report and Circular Series. 1989. 60. Panel on Vitamins, National Academy of Sciences, Institute of Medicine of the National Academies. Dietary ref erence intakes for thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. Washington (DC): Institute of Medicine of the National Academies. The National Academies Press; 1998. 61. Morris MS, Picciano MF, Jacqu es PF, Selhub J. Plasma pyridoxal 5' phosphate in the US population: the National Health and Nutrition Examination Survey, 2003 2004. Am J Clin Nutr. 2008;87:1446 54. 62. Rimm EB, Willett WC, Hu FB, Sampson L, Colditz GA, Manson JE, Hennekens C, Stampfer MJ. Folate and vitamin B6 from diet and supplements in relation to risk of coronary heart disease among women. J Am Med Assoc. 1998;279:359 64. 63. Cilliers K, Labadarios D, Schaaf HS, Willemse M, Maritz JS, Werely CJ, Hussey G, Donald PR. Pyridoxal 5 pho sphate plasma concentrations in children receiving tuberculosis chemotherapy including isoniazid. Acta Pdiatrica. 2010;99:705 10. 64. Stewart JW, Harrison W, Quitkin F, Liebowitz MR. Phenelzine induced pyridoxine deficiency. J Clin Psychopharm. 1984;4:22 5 26. 65. Weir MR, Keniston RC, Enriquez JI, Sr., McNamee GA. Depression of vitamin B6 levels due to gentamicin. Vet Hum Toxicol. 1990;32:235 8. 66. Weir MR, Keniston RC, Enriquez JI, Sr., McNamee GA. Depression of vitamin B6 levels due to dopamine. Vet Hum Toxicol. 1991;33:118 21. 67. Leklem J. Vitamin B6 In: Shils MO, J Shike, M, editors. Modern Nutrition in health and disease 9th edition. Philadelphia (PA): Lea & Febinger; 1999. p. 413.

PAGE 86

86 68. Gregory III JF. Vitamin B6 deficiency In: Carmel RJ, DW, editors. Homcysteine in Health and Disease Cambridge (UK): Cambridge University Press; 2001. 69. Bessey OA, Adam DJD, Hansen AE. Intake of vitamin B6 and infantile convulsion: A first approximation of requirements of pyridoxine in in fants. Pediatrics. 1957;20:33 44. 70. Nelson EM. Association of vitamin B6 deficiency with convulsions in infants. Public health reports (1974). 1956. 71. Paulose CS, Dakshinamurti K, Packer S, Stephens NL. Sympathetic stimulation and hypertension in the pyridoxine deficient adult rat. Hypertension. 1988;11:387 91. 72. Veninga KS. Effects of oral contraceptives on vitamins B6, B12, C, and folacin. J Nurse Midwifery. 1984;29:386 90. 73. Verloop MC, Rademaker W. Anaemia due to pyridoxine deficiency in man Br J Haematol. 1960;6:66 80. 74. Bendich A, Cohen M. Vitamin B6 safety issues. Ann N Y Acad Sci. 1990;585:321 30. 75. Berger A, Schaumburg H, Schroeder C, Apfel S, Reynolds H. Dose response, coasting, and differential fiber vulnerability in human toxic neuropathy. A prospective study of pyridoxine neurotoxicity. Neurology. 1992;42:1367 67. 76. Bernstein AL. Vitamin B6 in clinical neurology. Ann N Y Acad Sci. 1990;585:250 60. 77. Lumeng L, Ryan MP, Li T K. Validation of the diagnostic value of plasma p phosphate measurements in vitamin B6 nutrition of the rat. J Nutr. 1978;108:545 53. 78. Lui A, Lumeng L, Aronoff G, Ting Kai L. Relationship between body store of vitamin B6 and plasma pyridoxal P clearance: metabolic balance studies in humans J Lab Clin Med. 1985;106:491 97. 79. Leklem J. Vitamin B6 A status report. J Nutr. 1990;120:1503 07. 80. Rinehart JF, Greenberg LD. Arteriosclerotic lesions in pyridoxine deficient monkeys. Am J Pathol. 1949;25:481. 81. Rinehart J, Greenberg L. Vitami n B6 deficiency in the Rhesus monkey: With particular reference to the occurence of atherosclerosis, dental caries, and hepatic cirrhosis. Am J Clin Nutr. 1956;4:318 28.

PAGE 87

87 82. Kok FJ, Schrijver J, Hofman A, Witteman JCM, Kruyssen DACM, Remme WJ, Valkenburg HA. Low vitamin B6 in patients with acute myocardial infarction. Am J Cardio. 1989;63:513 16. 83. Kelly PJ, Kistler JP, Shih VE, Mandell R, Atassi N, Barron M, Lee H, Silveira S, Furie KL. Inflammation, homocysteine, and vitamin B6 status after ischemic s troke. Stroke. 2004;35:12 15. 84. Kelly PJ, Shih VE, Kistler JP, Barron M, Lee H, Mandell R, Furie KL. Low vitamin B6 but not homocyst(e)ine is associated with increased risk of stroke and transient ischemic attack in the era of folic acid grain fortifica tion. Stroke. 2003;34:51 54. 85. Eikelboom JW, Lonn E, Genest JJ, Hankey G, Yusuf S. Homocyst(e)ine and cardiovascular disease: A critical review of the epidemiologic evidence. ann Intern Med. 1999;131:363 75. 86. Refsum H, Ueland PM. Recent data are not in conflict with homocysteine as a cardiovascular risk factor. Curr Opin Lipidol. 1998;9:533 39. 87. Robinson K, Mayer EL, Miller DP, Green R, van Lente F, Gupta A, Kottke Marchant K, Savon SR, Selhub J, Nissen SE, et al. Hyperhomocysteinemia and low pyr idoxal phosphate: common and independent reversible risk factors for coronary artery disease. Circulation. 1995;92:2825 30. 88. Friso S, Girelli D, Martinelli N, Olivieri O, Lotto V, Bozzini C, Pizzolo F, Faccini G, Beltrame F, Corrocher R. Low plasma vit amin B 6 concentrations and modulation of coronary artery disease risk. Am J Clin Nutr. 2004;79:992 98. 89. Friso S, Jacques PF, Wilson PWF, Rosenberg IH, Selhub J. Low circulating vitamin B6 is associated with elevation of the inflammation marker C react ive protein independently of plasma homocysteine levels. Circulation. 2001;103:2788 91. 90. Liuzzo G, Biasucci LM, Gallimore JR, Grillo RL, Rebuzzi AG, Pepys MB, Maseri A. The prognostic value of C reactive protein and serum amyloid A protein in severe un stable angina. New Engl J Med. 1994;331:417 24. 91. Ridker PM, Hennekens CH, Buring JE, Rifai N. C reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. New Engl J Med. 2000;342:836 43.

PAGE 88

88 92. Brattstrm L, Stavenow L, Galvard H, Nilsson ehle P, Berntorp E, Jerntorp P, Elmsthl S, Pessah rasmussen H. Pyridoxine reduces cholesterol and low density lipoprotein and increases antithrombin III activity in 80 year old men with low plasma pyridoxal 5 phosphate. Sca ndinavian J Clin Lab Invest. 1990;50:873 77. 93. Brownlee M, Vlassara H, Cerami A. Inhibition of heparin catalyzed human antithrombin III activity by nonenzymatic glycosylation: possible role in fibrin deposition in diabetes. Diabetes. 1984;33:532 35. 94 Cattaneo M, Lombardi R, Lecchi A, Bucciarelli P, Mannucci PM. Low plasma levels of vitamin B6 are independently associated with a heightened risk of deep vein thrombosis. Circulation. 2001;104:2442 46. 95. Packham MA, Lam SC, Mustard JF. Vitamin B6 as a n antithrombotic agent. Lancet. 1981;2:809 10. 96. Khatami M, Suldan Z, David I, Li W, Rockey JH. Inhibitory effects of pyridoxal phosphate, ascorbate and aminoguanidine on nonenzymatic glycosylation. Life Sci. 1988;43:1725 31. 97. Palareti G, Salardi S, Piazzi S, Legnani C, Poggi M, Grauso F, Caniato A, Coccheri S, Cacciari E. Blood coagulation changes in homocystinuria: Effects of pyridoxine and other specific therapy. J Pediatr. 1986;109:1001 06. 98. Schoene NW, Chanmugam P, Reynolds RD. Effect of ora l vitamin B6 supplementation on in vitro platelet aggregation. Am J Clin Nutr. 1986;43:825 30. 99. Degenhardt TP, Alderson NL, Arrington DD, Beattie RJ, Basgen JM, Steffes MW, Thorpe SR, Baynes JW. Pyridoxamine inhibits early renal disease and dyslipidemi a in the streptozotocin diabetic rat. Kidney Int. 2002;61:939 50. 100. Yamada K. Treatment of arteriosclerosis vitamins and sulfuric esters of polysaccharides. Jap Heart J. 1961;2:281 96. 101. Vijayammal P, Kurup P. Pyridoxine and atherosclerosis: Role o f pyridoxine in the metabolism of lipids and glycosaminoglycans in rats fed normal and high fat, high cholesterol diets containing 16 % casein. Aus J Biol Sci. 1978;31:7 20. 102. Cunnane SC, Manku MS, Horrobin DF. Accumulation of linoleic and gamma linole nic acids in tissue lipids of pyridoxine deficient rats. J Nutr. 1984;114:1754 61. 103. Kirschman JC, Coniglio JG. The role of pyridoxine in the metabolism of polyunsaturated fatty acids in rats. J Biol Chem. 1961;236:2200 03.

PAGE 89

89 104. Bordoni A, Hrelia S, L orenzini A, Bergami R, Cabrini L, Biagi PL, Tolomelli B. Dual influence of aging and vitamin B6 deficiency on delta 6 desaturation of essential fatty acids in rat liver microsomes. Prostaglandins Leukotrienes Essen Fatty Acids. 1998;58:417 20. 105. Zhao M Ralat MA, da Silva V, Garrett TJ, Melnyk S, James SJ, Gregory JF. Vitamin B 6 restriction impairs fatty acid synthesis in cultured human hepatoma (HepG2) cells. Am J Physiol Endocrinol Metabol. 2013;304:E342 E51. 106. Meydani SN, Hayek M, Coleman L. Inf luence of vitamins E and B6 on immune response. Ann N Y Acad Sci. 1992;669:125 39. 107. Rail LC, Meydani SN. Vitamin B6 and immune competence. Nutr Rev. 1993;51:217 25. 108. Roubenoff R, Roubenoff RA, Selhub J, Nadeau MR, Cannon JG, Freeman LM, Dinarello CA, Rosenberg IH. Abnormal vitamin b6 status in rheumatoid cachexia inflammation. Arthritis Rheum. 1995;38:105 09. 109. Davies JS, B. Elementary biochemistry: An introduction to the chemistry of living cells Englewood Cliffs (NJ): Prentice Hall, Inc.; 1979. 110. Spallholz J. Nutrition: Chemistry and Biology Englewood (NJ): Prentice Hall; 1989. 111. Pencharez PY, V. Present Knowledge in Nutrition 9 edn. Washington (DC): Int ernational Life Sciences Institue; 2006. 112. Rose WC, Lambert GF, Coon MJ. The amino acid requirements of man: VII. General procedure; the tryptophan requirement. J Biol Chem. 1954;211:815 27. 113. Ophardt C. Virtual Chembook. 2003. [cited 2103 July]. Available from: [ http://www.elmhurst.edu/~chm/vchembook/631transam.html] 114. Lichstein HC. Function of the vitamin B6 group: pyridoxal phosphate (codecarboxylase) in transaminati on. J Biol Chem. 1945;161:311. 115. Metzler DE, Olivard J, Snell EE. Transamination of pyridoxamine and amino acids with glyoxylic acid. J Am Chem Soc. 1954;76:644 48. 116. Park YK, Linkswiler H. Effect of vitamin B6 depletion in adult man on the plasma concentration and the urinary excretion of free amino acids. J Nutr. 1971;101:185 91.

PAGE 90

90 117. Davis SR, Scheer JB, Quinlivan EP, Coats BS, Stacpoole PW, Gregory JF. Dietary vitamin B 6 restriction does not alter rates of homocysteine remethylation or synthes is in healthy young women and men. Am J Clin Nutr. 2005;81:648 55. 118. Lamers Y, Williamson J, Ralat M, Quinlivan EP, Gilbert LR, Keeling C, Stevens RD, Newgard CB, Ueland PM, Meyer K, et al. Moderate dietary vitamin B 6 restriction raises plasma gycine and cystathionine concentrations while minimally affecting the rates of glycine turnover and glycine cleavage in healthy men and women. J Nutr. 2009;139:452 60. 119. Miller JW, Ribaya Mercado JD, Russell RM, Shepard DC, Morrow FD, Cochary EF, Sadowski JA, Gershoff SN, Selhub J. Effect of vitamin B 6 deficiency on fasting plasma homocysteine concentrations. Am J Clin Nutr. 1992;55:1154 60. 120. Smolin LA, Benevenga NJ. Accumulation of homocyst(e)ine in vitamin B 6 deficiency: a model for the study of cysta synthase deficiency. J Nutr. 1982;112:1264 72. 121. Ubbink JB, van der Merwe A, Delport R, Allen RH, Stabler SP, Riezler R, Vermaak W. The effect of a subnormal vitamin B 6 status on homocysteine metabolism. J Clin Invest. 1996;98:177. 122. La mers Y, Coats B, Ralat M, Quinlivan EP, Stacpoole PW, Gregory JF. Moderate vitamin B 6 restriction does not alter postprandial methionine cycle rates of remethylation, transmethylation, and total transsulfuration but increases the fractional synthesis rate of cystathionine in healthy young men and women. J Nutr. 2011;141:835 42. 123. Lamers Y, O'Rourke B, Gilbert LR, Keeling C, Matthews DE, Stacpoole PW, Gregory JF. Vitamin B 6 restriction tends to reduce the red blood cell glutathione synthesis rate witho ut affecting red blood cell or plasma glutathione concentrations in healthy men and women. Am J Clin Nutr. 2009;90:336 43. 124. Lima CP, Davis SR, Mackey AD, Scheer JB, Williamson J, Gregory JF. Vitamin B 6 deficiency suppresses the hepatic transsulfurati on pathway but increases glutathione concentration in rats fed AIN 76A or AIN 93G diets. J Nutr. 2006;136:2141 47. 125. Martinez M, Cuskelly GJ, Williamson J, Toth JP, Gregory JF. Vitamin B 6 deficiency in rats reduces hepatic serine hydroxymethyltransfer ase and synthase activities and rates of in vivo protein turnover, homocysteine remethylation and transsulfuration. J Nutr. 2000;130:1115 23. 126. Wagner C. Biochemical role of folate in cellular metabolism In: Bailey L, editors. Folate in health and disease 2 edition. Boca Raton (FL): CRC Press; 1994. p. 23 42.

PAGE 91

91 127. Chiku T, Padovani D, Zhu W, Singh S, Vitvitsky V, Banerjee R. H2S biogenesis lyase leads to the novel sulfur metabolites lanthionine and homolanth ionine and is responsive to the grade of hyperhomocysteinemia. J Biol Chem. 2009;284:11601 12. 128. Sturman JA, Rassin DK, Gaull GE. Distribution of transsulphuration enzymes in various organs and species. Int J Biochem. 1970;1:251 53. 129. Smolin LA, Be nevenga NJ. Factors affecting the accumulation of homocyst(e)ine in rats deficient in vitamin B 6. J Nutr. 1984;114:103 11. 130. Takeuchi F, Izuta S, Tsubouchi R, Shibata Y. Glutathione levels and related enzyme activities in vitamin B 6 deficient rats fe d a high methionine and low cystine diet. J Nutr. 1991;121:1366 73. 131. Finkelstein JD, Chalmers FT. Pyridoxine effects on cystathionine synthase in rat liver. J Nutr. 1970;100:467 69. 132. Brown FC, Gordon P. Cystathionine synthase from rat liver: part ial purification and properties. Can J Biochem. 1971;49:484 91. 133. Li L, Bhatia M, Zhu YZ, Zhu YC, Ramnath RD, Wang ZJ, Anuar FBM, Whiteman M, Salto Tellez M, Moore PK. Hydrogen sulfide is a novel mediator of lipopolysaccharide induced inflammation in t he mouse. FASEB J. 2005. 134. Dominy JE, Stipanuk MH. New roles for cysteine and transsulfuration enzymes: production of H2S, a neuromodulator and smooth Muscle Relaxant. Nutr Rev. 2004;62:348 53. 135. Abe K, Kimura H. The possible role of hydrogen sulfi de as an endogenous neuromodulator. J Neurosci. 1996;16:1066 71. 136. Zhao W, Wang R. H2S induced vasorelaxation and underlying cellular and molecular mechanisms. Am J Physiol Heart Circ Physiol. 2002;283:H474 H80. 137. Zhao W, Zhang J, Lu Y, Wang R. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener. EMBO J. 2001;20:6008 16. 138. Teague B, Asiedu S, Moore PK. The smooth muscle relaxant effect of hydrogen sulphide in vitro: evidence for a physiological role to control intest inal contractility. Br J Pharmacol. 2002;137:139 45. 139. Sidhu R, Singh M, Samir G, Carson RJ. L cysteine and sodium hydrosulphide inhibit spontaneous contractility in isolated pregnant rat uterine strips in vitro. Pharmacol Toxicol. 2001;88:198 203.

PAGE 92

92 14 0. Dombkowski RA, Russell MJ, Olson KR. Hydrogen sulfide as an endogenous regulator of vascular smooth muscle tone in trout. Am J Physiol Regul Integr Comp Physiol. 2004;286:R678 R85. 141. Whiteman M, Moore PK. Hydrogen sulfide and the vasculature: a nove l vasculoprotective entity and regulator of nitric oxide bioavailability? J Cell Mol Med. 2009;13:488 507. 142. Stipanuk MH, Beck PW. Characterization of the enzymic capacity for cysteine desulphhydration in liver and kidney of the rat. Biochem J. 1982;20 6:267 77. 143. Yang G, Wu L, Jiang B, Yang W, Qi J, Cao K, Meng Q, Mustafa AK, Mu W, Zhang S, et al. H2S as a physiologic vasorelaxant: hypertension in mice with lyase. Science. 2008;322:587 90. 144. Furne J, Saeed A, Levitt M D. Whole tissue hydrogen sulfide concentrations are orders of magnitude lower than presently accepted values. Am J Physiol Regul Integr Comp Physiol. 2008;295:R1479 R85. 145. Whiteman M, Li L, Kostetski I, Chu SH, Siau JL, Bhatia M, Moore PK. Evidence for the formation of a novel nitrosothiol from the gaseous mediators nitric oxide and hydrogen sulphide. Biochem Biophys Res Commun. 2006;343:303 10. 146. Fiorucci S, Distrutti E, Cirino G, Wallace JL. The emerging roles of hydrogen sulfide in the gastrointe stinal tract and liver. Gastroenterology. 2006;131:259 71. 147. IOM. Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline The National Academies Press; 1998. 148. Singh S, Bane rjee R. PLP dependent H2S biogenesis. Biochimica et Biophysica Acta (BBA) Proteins & Proteomics. 2011;1814:1518 27. 149. Friso S, Lotto V, Corrocher R, Choi S. Vitamin B6 and cardiovascular disease In: Stanger O, editors. Water Soluble Vitamins N etherlands: Springer 2012. p. 265 90. 150. Ubbink JB, Serfontein WJ, De Villiers LS. Stability of pyridoxal 5 phosphate semicarbazone: Applications in plasma vitamin B6 analysis and population surveys of vitamin B6 nutritional status. J Chroma B. 1985;342 :277 84. 151. Pfeiffer CM, Huff DL, Gunter EW. Rapid and accurate HPLC assay for plasma total homocysteine and cysteine in a clinical laboratory setting. Clin Chem. 1999;45:290 92.

PAGE 93

93 152. Tapuhi Y, Schmidt DE, Lindner W, Karger BL. Dansylation of amino aci ds for high performance liquid chromatography analysis. Anal Biochem. 1981;115:123 29. 153. Tyopponen JT. Rapid and sensitive determination of Dns amino acids in plasma using high speed octadecyl liquid chromatographic columns. J Chromatogr. 1987;413:25 3 1. 154. Lichtenstein AH, Cohn JS, Hachey DL, Millar JS, Ordovas JM, Schaefer EJ. Comparison of deuterated leucine, valine, and lysine in the measurement of human apolipoprotein A I and B 100 kinetics. J Lipid Res. 1990;31:1693 701. 155. Liu SM, Figliomen i S. Gas chromatography/mass spectrometry analyses of [2,3,3 d3]Serine, [2,3,3 d3]cysteine and [3 13C]cysteine in plasma and skin protein: measurement of transsulphuration in young sheep. Rapid Commun Mass Spectrom. 1998;12:1199 203. 156. Liu SM, Mata G, Figliomeni S, Powell BC, Nesci A, Masters DG. Transsulfuration, protein synthesis rate and follicle mRNA in the skin of young Merino lambs in response to infusions of methionine and serine. Br J Nutr. 2000;83:401 09. 157. Nijhout HF, Gregory JF, Fitzpatri ck C, Cho E, Lamers KY, Ulrich CM, Reed MC. A mathematical model gives insights into the effects of vitamin B 6 deficiency on 1 carbon and glutathione metabolism. J Nutr. 2009;139:784 91. 158. Scheer JB, Mackey AD, Gregory JF. Activities of hepatic cytoso lic and mitochondrial forms of serine hydroxymethyltransferase and hepatic glycine concentration are affected by vitamin B 6 intake in rats. J Nutr. 2005;135:233 38. 159. Gregory JF, III, Park Y, Lamers Y, Bandyopadhyay N, Chi Y Y, Lee K, Kim S, da Silva V, Hove N, Ranka S, et al. Metabolomic analysis reveals extended metabolic consequences of marginal vitamin B 6 deficiency in healthy human subjects. PLoS ONE. 2013;8:635 44. 160. Selhub J, Jacques PF, Wilson PF, Rush D, Rosenberg IH. VItamin status and i ntake as primary determinants of homocysteinemia in an elderly population. J Am Med Assoc. 1993;270:2693 98. 161. Stipanuk MH. Sulfur amino acid metabolism: pathways for production and removal of homocysteine and cysteine. Annu Rev Nutr. 2004;24:539 77. 162. Davis SR, Quinlivan EP, Stacpoole PW, Gregory JF. Plasma glutathione and cystathionine concentrations are elevated but cysteine flux Is unchanged by dietary vitamin B 6 restriction in young men and women. J Nutr. 2006;136:373 78.

PAGE 94

94 163. Lipton SH, Bodw ell CE, Coleman AH. Amino acid analyzer studies of the products of peroxide oxidation of cystine, lanthionine, and homocystine. J Agric Food Chem. 1977;25:624 28. 164. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem. 1976;72:248 54.

PAGE 95

95 BIOGRAPHICAL SKETCH Barbara N. DeRatt is from Wilson, North Carolina. She attended Beddingfield High School and graduated in 2007 as valedictorian. She then at tended North Carolina Wesleyan College and received a Bachelor of Science in pre medicine and b iology in 2011. After graduation, s he received a graduate assistantship from the D epartment of Food Science and Human Nutrition at the University of Florida to pursue her degree After graduation she will pursue her Ph.D. under her current advisor, Dr. Jesse Gregory.