<%BANNER%>

Investigation into the Role of Serum Vitamin D and its Carrier Protein in Type 1 Diabetes

Permanent Link: http://ufdc.ufl.edu/UFE0024481/00001

Material Information

Title: Investigation into the Role of Serum Vitamin D and its Carrier Protein in Type 1 Diabetes
Physical Description: 1 online resource (36 p.)
Language: english
Creator: Bierschenk, Lindsey
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: 1, 25, autoimmune, binding, calcidiol, d, dependent, diabetes, hydroxycholecalciferol, insulin, juvenile, mellitus, protein, type, vitamin
Medicine -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Previous studies, largely in Northern Europe, have suggested an association between type 1 diabetes (T1D) and reduced serum 25(OH)-vitamin D levels. To ascertain whether this association was present in a solar rich region, we measured serum 25(OH)-vitamin D levels of 415 individuals in Florida, USA. Study subjects included 153 controls, 46 new-onset T1D patients, 110 established T1D patients (onset > 5 months from diagnosis), and 106 first-degree relatives of diabetic patients. UV Index climatological data was obtained to estimate average relative solar exposure for subjects at the time of sample collection. This population was expanded to measure vitamin D binding protein (VDBP) in the serum of 153 controls, 203 T1D subjects, and 116 relatives. Serum 25(OH)-vitamin D (mean ng/mL; 95% CI) levels were similar amongst healthy controls (27.2; 23.3-31.1), new-onset T1D patients (21.8; 18.2-25.3), subjects with established T1D (27.7; 22.4-33.1), and their first-degree relatives (23.6; 21.0-26.3) (p=0.705). Suboptimal vitamin D levels (? 30 ng/mL) were observed in 70.1% of controls, 76.1% of new-onset T1D subjects, 68.5% of established T1D subjects, and 68.8% of relatives. Interestingly, suboptimal vitamin D levels were present in similar proportion for all groups and estimated UV exposure did not significantly impact vitamin D levels (p=0.779). Further analysis of vitamin D status by measurement of its carrier protein, VDBP, revealed a significant difference (p=0.0055) in serum concentrations (mean ?g/mL; 95% CI) between the control (528.2; 467.3-589.0) and T1D groups (424.8; 403.6-446.0). Serum VDBP concentrations were not significantly different for relatives (496.9; 410.3-583.4) compared to control or T1D subjects (p=NS). Linear regression analysis of VDBP levels versus disease duration revealed no association (r=0.0026, p=0.5158). In sum, VDBP serum concentrations appear to be of significance in the presence of T1D while suboptimal 25(OH)-vitamin D levels were characteristic of the entire study population. Future directions would include exploring the relationship of reduced VDBP concentrations in the pre-diabetes state, as well as correlation with genetic variants of the vitamin D pathway. While vitamin D supplementation may still play a role in T1D prevention, a large prospective study would be required to substantiate this claim. Overall, our data support recent calls for increased vitamin D supplementation.
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 Lindsey Bierschenk.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Atkinson, Mark A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024481:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024481/00001

Material Information

Title: Investigation into the Role of Serum Vitamin D and its Carrier Protein in Type 1 Diabetes
Physical Description: 1 online resource (36 p.)
Language: english
Creator: Bierschenk, Lindsey
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: 1, 25, autoimmune, binding, calcidiol, d, dependent, diabetes, hydroxycholecalciferol, insulin, juvenile, mellitus, protein, type, vitamin
Medicine -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Previous studies, largely in Northern Europe, have suggested an association between type 1 diabetes (T1D) and reduced serum 25(OH)-vitamin D levels. To ascertain whether this association was present in a solar rich region, we measured serum 25(OH)-vitamin D levels of 415 individuals in Florida, USA. Study subjects included 153 controls, 46 new-onset T1D patients, 110 established T1D patients (onset > 5 months from diagnosis), and 106 first-degree relatives of diabetic patients. UV Index climatological data was obtained to estimate average relative solar exposure for subjects at the time of sample collection. This population was expanded to measure vitamin D binding protein (VDBP) in the serum of 153 controls, 203 T1D subjects, and 116 relatives. Serum 25(OH)-vitamin D (mean ng/mL; 95% CI) levels were similar amongst healthy controls (27.2; 23.3-31.1), new-onset T1D patients (21.8; 18.2-25.3), subjects with established T1D (27.7; 22.4-33.1), and their first-degree relatives (23.6; 21.0-26.3) (p=0.705). Suboptimal vitamin D levels (? 30 ng/mL) were observed in 70.1% of controls, 76.1% of new-onset T1D subjects, 68.5% of established T1D subjects, and 68.8% of relatives. Interestingly, suboptimal vitamin D levels were present in similar proportion for all groups and estimated UV exposure did not significantly impact vitamin D levels (p=0.779). Further analysis of vitamin D status by measurement of its carrier protein, VDBP, revealed a significant difference (p=0.0055) in serum concentrations (mean ?g/mL; 95% CI) between the control (528.2; 467.3-589.0) and T1D groups (424.8; 403.6-446.0). Serum VDBP concentrations were not significantly different for relatives (496.9; 410.3-583.4) compared to control or T1D subjects (p=NS). Linear regression analysis of VDBP levels versus disease duration revealed no association (r=0.0026, p=0.5158). In sum, VDBP serum concentrations appear to be of significance in the presence of T1D while suboptimal 25(OH)-vitamin D levels were characteristic of the entire study population. Future directions would include exploring the relationship of reduced VDBP concentrations in the pre-diabetes state, as well as correlation with genetic variants of the vitamin D pathway. While vitamin D supplementation may still play a role in T1D prevention, a large prospective study would be required to substantiate this claim. Overall, our data support recent calls for increased vitamin D supplementation.
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 Lindsey Bierschenk.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Atkinson, Mark A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024481:00001


This item has the following downloads:


Full Text

PAGE 1

1 INVESTIGATION INTO THE ROLE OF SERUM VITAMIN D AND ITS CARRIER PROTEIN IN TYPE 1 DIABETES By LINDSEY MARIE BIERSCHENK A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREME NTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

PAGE 2

2 2009 Lindsey Marie Bierschenk

PAGE 3

3 To my family, who taught me t he value of believing in myself

PAGE 4

4 ACKNOWLEDGMENTS First I would like to thank Dr. Mark Atkinson for believing in me enough to accept me into his laboratory and for providing me with a place to learn the true value of scientific research. when you are a team player, use your moral compass, and never give up on even the loftiest of goals. Clive both contagious and inspirational. I have much gratit ud e to Dr. Nancy Denslow and Dr. Sihong Song for serving on my graduate committee and for providing me with excellent advice and support. I would like to thank my parents and sister for supporting me throughout some of the most challenging times in my li fe, from being diagnosed with type 1 diabetes to pursuing my dream of finding a cure for the disease. Most of all, I would like to thank God for guiding my

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 6 LIST OF FIGURES ................................ ................................ ................................ ......................... 7 ABSTRACT ................................ ................................ ................................ ................................ ..... 8 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 10 Type 1 Diabetes Overview ................................ ................................ ................................ ..... 10 Vitamin D and Type 1 Diabetes ................................ ................................ ............................. 11 Vitamin D Metabolite ................................ ................................ ................................ ...... 11 Vitamin D Binding Protein ................................ ................................ .............................. 12 Vitamin D Pathway Associated Polymorphisms ................................ ............................. 13 Protective Effects of Vitamin D ................................ ................................ ...................... 13 Immunomodulatory Role of Vitamin D ................................ ................................ .......... 14 Introduction to Experimental Design ................................ ................................ ...................... 1 4 2 MATERIALS AND METHODS ................................ ................................ ........................... 16 Serum 25(OH) Vitamin D Levels ................................ ................................ .......................... 16 St atistical Analysis ................................ ................................ ................................ .................. 17 3 RESULTS ................................ ................................ ................................ ............................... 19 Serum 25(OH) Vitamin D ................................ ................................ ................................ ...... 19 Vitamin D Binding Protein ................................ ................................ ................................ ..... 20 4 DISCUSSION ................................ ................................ ................................ ......................... 29 Serum 25(OH) Vitamin D Levels in Type 1 Diabetes ................................ ........................... 29 Vitamin D Binding Protein Levels in Type 1 Diabetes ................................ .......................... 30 5 FUTURE RESEARCH DIRECTIONS ................................ ................................ .................. 32 LIST OF REFERENCES ................................ ................................ ................................ ............... 33 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ......... 36

PAGE 6

6 LIST OF TABLES Table page 3 1 Classification of vitamin D status in relation to type 1 diabetes risk ................................ 23

PAGE 7

7 LIST OF FIGURES Figure page 3 1 Serum 25(OH) vitamin D levels among four subgro ups of our study population ........... 22 3 2 Female and male serum 25(OH) vitamin D level s in each experimental cohort ............... 23 3 3 Age versus serum 25(OH) vitamin D l evels ................................ ................................ ..... 24 3 4 Impact of estimated UV B exposure on serum 25(OH) vitamin D levels ........................ 24 3 5 Vitamin D binding protein levels among 3 subgro ups of our study population ............... 25 3 6 Serum 25(O H) vitamin D levels and vitamin D binding protein levels in healthy controls ................................ ................................ ................................ .............................. 25 3 7 Serum 25(OH ) vitamin D levels and vitamin D binding protein levels in subjects with new onset type 1 diabetes. ................................ ................................ ......................... 26 3 8 Serum 25(O H) vitamin D levels and vitamin D binding protein levels in subjects with established type 1 diabetes ................................ ................................ ........................ 26 3 9 Serum 25(OH ) vitamin D levels and vitamin D binding protein levels in first degree rela tives of type 1 diabetics ................................ ................................ ............................... 27 3 10 Vitamin D binding protein levels in female and male gro ups ................................ .......... 27 3 11 Age versus vitamin D bi nding protein levels ................................ ................................ .... 28

PAGE 8

8 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 INVESTIGATION INTO THE ROLE OF SERUM VITAMIN D AND ITS CARRIER PROTEIN IN TYPE 1 DIABETES By Lindsey Marie Bierschenk May 2009 Chair: Mark Atkinson Major: Medical Sciences Previous studies, largely in Northern Europe, have suggested an association between type 1 diabetes (T1D) and reduced serum 25(OH) vitamin D levels. To ascertai n whether this association was present in a solar rich region, we measured serum 25(OH) vitamin D levels of 415 individuals in Florida, USA. Study subjects included 153 controls, 46 new onset T1D patients, 110 established T1D patients (onset > 5 months fr om diagnosis), and 106 first degree relatives of diabetic patients. UV Index climatological data was obtained to estimate average relative solar exposure for subjects at the time of sample collection. This population was expanded to measure vitamin D bin ding protein (VDBP) in the serum of 153 controls, 203 T1D subjects, and 116 relatives. Se rum 25(OH) vitamin D (mean ng/mL ; 95% CI) levels were similar amongst healthy controls (27.2; 23.3 31.1), new onset T1D patients (21.8; 18.2 25.3), subjects with est ablished T1D (27.7; 22.4 33.1), and their first degree relatives (23.6; 21.0 26.3) (p=0.705). Subopti mal ) were observed in 70.1% of controls, 76.1% of new onset T1D subjects, 68.5% of established T1D subjects, and 68.8% of rel atives. Interestingly, suboptimal vitamin D levels were present in similar proportion for all groups and estimated UV exposure did not significantly impact vitamin D levels (p=0.779).

PAGE 9

9 Further analysis of vitamin D status by measurement of its carrier pr otein, VDBP, revealed a significant difference (p=0.0055) in serum concentrations (mean g/mL; 95% CI) between the control (528.2; 467.3 589.0) and T1D groups (424.8; 403.6 446.0). Serum VDBP concentrations were not significantly different for relatives ( 496.9; 410.3 583.4) compared to control or T1D subjects (p=NS). Linear regression analysis of VDBP levels versus disease duration revealed no association (r=0.0026, p=0.5158). In sum, VDBP serum concentrations appear to be of significance in the presen ce of T1D while suboptimal 25(OH) vitamin D levels were characteristic of the entire study population. Future directions would include exploring the relationship of reduced VDBP concentrations in the pre diabetes state, as well as correlation with genetic variants of the vitamin D pathway. While vitamin D supplementation may still play a role in T1D prevention, a large prospective study would be required to substantiate this claim. Overall, our data support recent calls for increased vitamin D supplement ation.

PAGE 10

10 CHAPTER 1 INTRODUCTION Type 1 Diabetes Overview Type 1 diabetes (T1D), also referred to as juvenile, autoimmune, or insulin dependent diabetes, is a form of diabetes mellitus that occurs when the body loses its innate ability to produce the insulin hormone necessary for glucose utilization. T1D affects approximately 1 in every 300 people in the United States, and according to the American Diabetes Association, T1D accounts for 5 10% of all diabetes cases. Approximately 85% of newly diagnosed individuals have no prior family history of disease (1). The majority of diagnoses are made in children and young adults, as well as people of European descent (1). The disease process begins with a yet unknown multifactorial immune insult resulting in autoimmune destruction of the insulin producing beta cells of the pancreatic islets and subsequent inability to maintain euglycemia (2). The immediate effect of insulin deficiency is a state of hyperglycemia, or elevated glucose in the blood, which can easily progress into a life threatening condition known as diab etic ketoacidosis (3). When insulin is absent, the body cannot use glucose for energy, so the liver resorts to fat breakdown. This fat breakdown produces ketone bodies that will continue to accumulate and lower the pH of the body to the point of death by acidosis. Inadequate management of blood glucose levels over time can result in a number of complications, including kidney, eye, cardiovascular, and nerve damage. In addition to the direct health complications associated with T1D, daily life is forever altered due to a routine of constant blood glucose monitoring and insulin dosing in an attempt to achieve euglycemia (4). While enough insulin must be supplied to prevent hyperglycemia, it is just as important to avoid hypoglycemia, or low blood glucose l evels, caused by an insulin overdose. Even with the advances in science and technology that have made living with diabetes

PAGE 11

11 state in the presence of diabetes. Co nsequently, an enormous effort is under way to both understand the causes of, and develop therapies for, T1D. T1D has the classical hallmarks of an autoimmune disease, with both environmental and enesis (5). Genetically, HLA DR, HLA DQ, and HLA DP allelic variants and the diabetes related autoantibodies GADA, IAA, ICA, and IA2A determine the level of risk an individual ma y have for developing T1D (6,7). Twin concordance studies have confirmed tha overall susceptibility to T1D (5), indicative of a powerful environmental influence in T1D etiopathogenesis. Diverse arrays of environmental factors have been associated with the disease, including vi ruses, infant feeding practices, and childhood immunizations, amongst others (8). Although the exact environmental component remains elusive, a number of published reports suggest vitamin D status may play a role (9, 10, 11). Vitamin D and Type 1 Diabe tes The link between type 1 diabetes (T1D) and vitamin D emerged with data suggesting that subjects developing the disease had lower serum concentrations of this metabolite than healthy controls (12, 13, 14). Around the world, disease incidence has been s hown to exhibit seasonality (15). The north south gradient hypothesis suggests the number of T1D cases correspond to distance from the equator. In other words, the amount of UV B exposure determined by geographical location correlates with the frequency o f disease. Published reports of greater incidence of disease at higher latitudes support this phenomenon (16). Vitamin D Metabolite Vitamin D is a fat soluble prohormone that regulates calcium and phosphorus levels in the body. Production of the prima ry source of circulating vitamin D begins via a photochemical

PAGE 12

12 reaction of solar ultraviolet B (UV B) radiation (wavelength 290 315 nm) with 7 dehydrocholesterol in the skin (17). This multi step process, as outlined by Holick, continues with cutaneous syn thesis of vitamin D3 (cholecalciferol) which undergoes hydroxylation in the liver by 25 hydroxylase enzymes to create the circulating metabolite, known as 25(OH) vitamin D, or calcidiol ( 17). Next, calcidiol is hydroxylated hydroxylas e enzymes to produce the biologically active form of vitamin D, known as 1,25(OH)2 vitamin D, or calcitriol. Calcitriol binds the vitamin D plasma carrier protein, known as vitamin D binding protein (VDBP), which transports the metabolite to various tissue s throughout the body. Nuclear v itamin D r eceptors ( n VDRs) are located i n nearly all tissues of the body, including antigen presenting cells and activated T cells of the immune system (17, 18). Consequently, vitamin D regulates, in part, gene expression of cells that possess functional n VDRs. Many factors may influence UV B exposure and the ability to synthesize vitamin D, such as cloud cover, smog, geographical location, season, clothing, sunscreen use, melanin, body mass index, age, and diet. Aside f rom sunlight exposure, dietary vitamin D is available as vitamin D2 (ergocalciferol) and D3 (cholecalciferol), derived from irradiated plant and animal sources respectively. While foods such as butter, milk, and cereal are often fortified with vitamin D, the content is far less than required to meet the recommended daily requirements (18). Vitamin D deficiency leads to the development of rickets and osteomalacia. Vitamin D deficienc 30 ng/mL and suffi ciency as > 30 n g/mL (17, 19). Vitamin D Binding Protein Vitamin D binding protein (VDBP), also known as Group Specific Component (Gc), is a highly polymorphic plasma protein with a molecular weight of 52 59 kDa (20). The polymorphisms give rise to 3 major isotypes, named Gc2, Gc1s, and Gc1f, which differ by

PAGE 13

13 am ino acid substitutions and glycosylation, and their frequencies vary according to geographic location (21). VDBP is synthesized by the liver and released into the blood where it binds and transports vitamin D metabolites, scavenges actin, binds fatty acid s, and has a role in macrophage activation and chemotaxis (21). The published normal VDBP plasma conc entration range is 300 600 g/mL (21). Additionally, VDBP concentrations are markedly higher than its vitamin D ligands (22), and no complete absence of VDBP has ever been detected in humans. Its properties suggest it possesses a unique ability to modulate inflammatory immune responses. Vitamin D Pathway Associated Polymorphisms Vitamin D polymorphisms, such as the promoter polymorphism CYP27B1 1260 a nd the intronic SNP CYP27B1 +2838, have been implicated in T1D (23). Polymorphisms in the CYP27B1 gene are of importance because it encodes the enzyme responsible for converting vitamin D3 into its biologically active form. Polymorphisms in nuclear vitam in D receptors (nVDRs) prese nt in a variety of cells throughout the body may adversely affect receptor binding of vitamin D metabolites, leading to an impaired transcription of genes regulated by vitamin D (18). Protective Effects of Vitamin D In a meta analysis of data from several European studies, children supplemented with vitamin D redu ced their risk of developing type 1 diabetes (T1D) by 29% (24). Investigation of T1D anim al models revealed that removal of vitamin D accelerates onset of diabetes wh ile pharmacological treatment with vitamin D analogues served to prevent or delay disease (25, 26). This protective effect was also noted among individuals at risk for the autoimmune disease multiple sclerosis who maintained sufficient levels of vitamin D (27). Simil ar to T1D studies, numerous reports have shown that the incidence of multiple sclerosis de creases with increased UV B exposure and proximity to the equator (28). Vitamin D may also protect against

PAGE 14

14 autoimmune diseases like T1D that have been l inked to viral infection. Specifically, vitamin D induces cathelicidin, an antimicrobial peptide, increasing the efficacy of fighting infections, thereby reducing risk of disease progression (29). Immunomodulatory Role of Vitamin D The presence of nucl ear vitamin D receptors (nVDRs) i n immune system cells suggests a possible role for vitamin D in regulating the immune response. Activated macrophages and dendritic cells have been shown to contain the enzyme responsible for converting vitamin D into its biologically active form, calcitriol (18). Calcitriol has been shown to promote phagocytosis by macrophages (18) as well as the development of Th2 lymphocytes (30). Additionally, calcitriol is capable of downregulating antigen presentation and production of inflammatory cytokines like IL 2 and IL 12 (30). Vitamin D exerts its effects on hundreds of genes, ultimately influencing cell proliferation, differentiation, and death (17). Introduction to Experimental Design Numerous studies, the majority of wh ich were based out of northern European countries, have reported lower vitam in D serum concentrations in patients with type 1 diabetes (T1D) compared to healthy controls. One study performed in the United States, also suggesting reduced vitamin D levels i n T1D patients, failed to provide values amongst healthy control individuals and hence, did not identify disease specificity. In an attempt to contribute to this model and fill an underlying knowledge void, we addressed the basis for this association in a solar rich environment. Specifically, we sought to measure serum 25(OH) vitamin D levels from T1D patients, first degree relatives of diabetic patients, and healthy controls resident to Florida, USA. Additional support for a role of vitamin D in the a utoimmune process of T1D comes from studies that have shown an a ssociation between specific vitamin D binding protein (VDBP)

PAGE 15

15 polymorphisms and T1D markers (31). Genetic variants of VDBP are of significance because they alter the binding affinity of the ca rrier protein for vitamin D. While genotypic VDBP associations have been observed, phenotypic associations have not been made. For example, serum levels of VDBP have neither been measured nor compared among diabetics and nondiabetics. For this reason, w e sought to measure VDBP levels from T1D patients, their first degree relatives, and healthy controls.

PAGE 16

16 CHAPTER 2 MATERIALS AND METHODS Serum 25(OH) Vitamin D Levels We obtained serum from 415 individuals in Florida, divided into the following co horts: controls (median age 22.0 years; range 5 65 years; females =84, total =153), subjects with new 35.0; females =23; total =46), those with established disease in which presence of T1D extended beyond 5 months (median age 16.0; range 5.1 62.6; females =50; total =110), and relatives of those with T1D (median age 21.0; range 1.0 62.6; females =54; total =106). All samples were collected under informed consent with University of Flori da Institutional Review Board approval. 25(OH) vitamin D levels were quantified in duplicate with a commercial EIA k it (ALPCO; Salem, NH) using 50L of banked serum from each subject; an analyte shown previously as stable under such conditions (32). The levels of 25(OH) vitamin D were interpolated from a standard curve after reading the absorbance on a M5 Spectramax plate reader using Softmax Pro 4.8 software (Molecular Devices, Sunnyvale, CA). The intra and inter assay coefficients of variation for th is assay were 10.7% and 13.2% respectively. 25(OH) vitamin D deficiency was defined a s less than or equal to 20 ng/mL, insufficiency as 21 30 ng/mL, and sufficiency as > 30 ng/mL (17,19). UV Index (UVI) climatological data was obtained from the National Weather Service (NWS) and United States Environmental Protection Agency (EPA) websites ( http://www.cpc.ncep.noaa.gov and http://www.epa.gov ) to determine relative UV exposure. Ba sed on the previous five years worth of data for the proximate city of Jacksonville, FL, we established UV exposure monthly means: January: 3.215, February: 4.08, March: 5.96, April: 7.68, May: 8.238, June: 8.578, July: 8.976, August: 8.254, Septe mber: 6.902, October: 5.11,

PAGE 17

17 November: 3.694, and December: 2.79. The numbers correspond to the UVI scale (1 11+) developed by the NWS and EPA and implemented by the World Health Organization (WHO). Vitamin D Binding Protein Levels We obtained serum from 472 individuals, 386 of which we had previously measured 25(OH) vitamin D levels on. The samples included 153 controls, 203 T1D subjects (new onset and established), and 116 first degree relatives of T1D subjects. All samples were from individuals resident to Florida, USA and collected under informed consent with University of Florida Institutional Review Board approval. VDBP levels were quantified in duplicate with a commercial EIA k it (ALPCO; Salem, NH) using 10L of banked seru m from each subject. The levels of VDBP were interpolated from a standard curve after reading the absorbance on a M5 Spectramax plate reader using Softmax Pro 4.8 software (Molecular Devices, Sunnyvale, CA). The intra and inter assay coefficients of var iation for this assay were 5.0% and 12.7% respectively. The normal reference range for VDBP levels was 300 600 g/mL or 30 60 mg/dL (21). Statistical Analysis For 25(OH) vitamin D data, analysis of multiple, unpaired group comparisons was achieved using the non parametric Kruskal test. The relationship between age and 25(OH) vitamin D levels was analyzed by linear regression. All analyses were done using GraphPad Prism software ver 5.00 (H.J. Motulsky, Analyzing Data with G raphPadPrism, 1999, GraphPad Software Inc., San Diego, CA, www.graphpad.com ). For VDBP data, analysis of multiple, unpaired group comparisons was achieved using the non parametric Kruskal test. The relationship between 25(OH) vitamin D levels and VDBP levels was analyzed by linear regression. Additionally, the association of age and disease duration with VDBP levels was considered using linear regression

PAGE 18

18 analysis. To determine the relati onship between VDBP levels and gender, the non parametric Mann Whitney test was used.

PAGE 19

19 CHAPTER 3 RESULTS Serum 25(OH) V itamin D 25( OH) vitamin D levels (mean ng/mL ; 95% CI) were as follows: healthy controls (27.2; 23.3 31.1), new onset T1D (21.8; 18.2 25.3), established T1D (27.7; 22.4 33.1), and first degree relatives (23.7; 21.0 26.3) (Figure 3 1). The medians did not vary significantly among individuals at varying degrees of disease risk (p =0.705). Subopti ng/mL ) wer e observed in 70.1% of controls, 76.1% of new onset T1D subjects, 68.5% of patients with established T1D, and 68.8% of relatives; values that while low, were not significantly different from each other (Table 3 1; p =NS). Next, previous reports have stat ed that vitamin D deficiency is most prevalent among children, adolescents, and the elderly and that lower vitamin D levels are found in men versus women (33, 34). As such, we compared gender segregated (Figure 3 2) and age segregated (Figure 3 3) 25(OH) vitamin D levels. No significant gender differences were observed (p =0.493). Using linear regression, we compared age and serum 25(OH) vitamin D levels in our overall study population and the individual cohorts. For all groups combined, r =0.004 p =0. 224; healthy controls, r = 0.010 p =0.214; new onset T1D, r =0.0001 p =0.963; established T1D, r =0.013 p =0.238; and relatives, r =0.075 p =0.005. In sum, regression analysis revealed no trend in vitamin D levels as it pertained to age overall (p =NS ) but the relatives subgroup did reveal a significant inverse relationship with age (p =0.005, data not shown). Since sunlight plays a major role in vitamin D synthesis, we then examined vitamin D levels as a function of the month the sample was drawn, a nd therefore examined the influence of UV B exposure. The samples were grouped according to month drawn, and placed into one of four possible 3 month blocks, each block formed on the basis of similar UV B indices (Figure 3

PAGE 20

20 4). The 25(OH) v itamin D (repor ted as mean ng/mL ; 95 % CI) levels for the November/December/January group of 11 2 samples (26.9; 21.6 32.2) had an average estimated UV exposure of 3.23. The October/February/March group of 1 13 samples (24.2; 20.6 27.9) had an average estimated UV exposure of 5.05. The September/April/May group of 8 4 samples (26.6; 21.5 31.7) had an average e stimated UV exposure of 7.61. Finally, t he June/July/August group of 10 6 samples (25.7; 22.6 28.9) had an average estimated UV exposure of 8.60. Comparison of the vi tamin D levels between each three month block showed no significant difference (p =0.779). Further analysis revealed no significant differences on a monthly exposure basis or examining controls versus new onset T1D, established T1D, or first degree relati ves (data not shown). Vitamin D Binding Protein VDBP concentrations were determined for 3 experimental cohorts: 153 healthy controls, 203 T1D subjects, and 116 relatives of T1D subjects (Figur e 3 5). VDBP levels (mean g/mL ; 95% CI) were as follows: he althy controls (528.2; 467.3 589.0), T1D subjects (424.8; 403.6 446.0), and relatives (496.9; 410.3 583.4). Using the non parametric Kruskal Wallis with test, we were able to determine T1D subjects had significantly lower VDBP levels when comp ared to healthy controls (p =0.0028). VDBP levels were also compared with 25(OH) vitamin D levels for 152 controls, 43 new onset T1D subjects, 98 established T1D subjects, and 93 first degree relatives. Linear regression analysis of each group is as fol lows: controls (p =0.9194; r =0.00007) (Figure 3 6), new onset T1D (p =0.2569; r =0.03124) (Figure 3 7), established T1D (p =0.9289; r =0.00008) (Figure 3 8), and relatives (p =0.1459; r =0.02310) (Figure 3 9). Overall, no significant association was detected among any study population (p=NS).

PAGE 21

21 Without knowledge of the differences that may exist in VDBP levels according to gender, we analyzed VDBP levels of 238 female and 233 male samples using the non parametric Mann Whitney method (Figure 3 10). VD BP levels (mean g/mL ; 95% CI) were as follows: females (514.9; 460.8 569.0), and males (435.6; 408.9 462.3). Analysis revealed the means between the two groups were significantly different (p < 0.0001). In an effort to determine the effect of disease d uration on the VDBP levels of T1D subjects, we performed linear regression analysis (data not shown) and determined the duration of disease did not significantly impact VDBP levels (p =0.5158; r =0.0026). Along these same lines, we sought to identify whe ther or not age affected VDBP levels (Figure 3 11). Of 458 samples analyzed by linear regression, no significance was found (p =0.1643; r =0.004238).

PAGE 22

22 Figure 3 1. Serum 25(OH) vitamin D levels among four subgro ups of our study population (n = 415; p= 0.705). Box and whiskers plot (median, whiskers 5 Orange and red lines represent the cutoffs for insufficiency and deficiency respectively. Lef t y axis, 25(OH) vitamin D ng/mL ; ri ght y axis, 25(OH) vitamin D nmol/L

PAGE 23

23 Table 3 1. Classification of vitamin D status in relation to type 1 diabetes risk Normal > 30n g/mL N (%) Insufficient 20 30ng/mL N (%) Deficient < 20ng/mL N (%) Healthy Controls (n=153) 46 (30.1) 31 (20.3) 76 (49.7) New Onset T1D (n=46) 11 (23.9) 13 (28.3) 22 (47.8) Established T1D (n=110) 34 (30.9) 29 (26.4) 47 (42.7) Relatives (n=106) 34 (32.1) 22 (20.8) 50 (47.2) Figure 3 2. Female and male serum 25(OH) vitamin D levels in each experimental cohort (p =0.493). Box and whiskers plot (median, whiskers 5 Orange and red lines represent the cutoffs for insufficiency and deficiency respectively. Lef t y axis, 25(OH) vitamin D ng/mL ; right y axis, 25(OH) vitamin D nmol/L

PAGE 24

24 Figure 3 3. Age vs. serum 25(OH) vitamin D levels. Orange and red lines represent the cutoffs for 25(OH) vitamin D insufficiency and deficiency respectively Blue line indicates linear regression (r 2 = 0 .003; p = 0.224). Figure 3 4. Impact of estimated UV B exposure on serum 25(OH) vitamin D levels. Orange and red lines represent the cutoffs for 25(OH) vitamin D insufficiency and deficiency respectively. Samples categorized by 3 month blocks with the most similar estimated UV exposure rates (p = 0.779).

PAGE 25

25 Figure 3 5. V itamin D b inding p rotein levels (g/mL ) among 3 subgroups of our study population (n = 472 ; p = 0.0028). Bar graph; blue lines represent mean with SEM. Figure 3 6. Serum 25(OH) vitamin D levels (nmol/L) vs. vitamin D binding p rotein levels (g/mL ) in healthy controls. Blue line indicates linear regression (r 2 = 0.00007; p = 0.9194).

PAGE 26

26 Figure 3 7. Serum 25(OH) vitamin D levels (nmol/L ) vs. vitamin D binding p rotein levels (g/mL ) in subjects with new onset type 1 diabetes Blue line indicates linear regression (r 2 = 0.03214; p = 0.2569). Figure 3 8. Serum 25(OH) vitami n D levels ( nmol/L ) vs. vitamin D b in ding p rotein levels (g/mL ) in subjects with established type 1 diabetes Blue line indicates linear regression (r 2 = 0.00008; p = 0.9289).

PAGE 27

27 Figure 3 9. Serum 25(OH) vi tamin D levels (nmo l /L ) vs. vitamin D binding p rotein levels (g/mL ) in first degree relat ives of type 1 diabetics Blue line indicates linear regression (r 2 = 0.0231; p = 0.1459). Figure 3 10. Vitamin D binding p rotein levels (g/mL ) in female (n = 238) and m ale (n = 233) groups (p < 0.0001). Bar graph; blue lines represent mean with SEM.

PAGE 28

28 Figure 3 11. Age vs. vitamin D binding p rotein levels (g/mL ) Blue line indicates linear regression (r2 = 0.004238; p = 0.1643).

PAGE 29

29 CHAPTER 4 DISCUSSION Serum 25(OH) Vitamin D Levels in T ype 1 Diabetes Our study did not find significant differences in vitamin D levels between healthy controls, subjects with T1D and first degree relatives, using samples obtained in a solar rich region of the United States. However, to our surprise, we identified that within each group there exists a high frequency of vitamin D insufficiency despite the sun rich environment of Florida. Specifically, vitamin D levels in more than two thirds of each population wer e below the recommended 30 ng/mL level; placing these subjects in the category of vitamin D insufficient or deficient. While we may have an ecological fallacy bias in assigning UV Index aggregate data to individual subjects, other f actors such as sun avoidance practices to inadequate supplementation may also account for the low 25(OH) vitamin D levels observed in this cross sectional study. dram cutaneous synthesis of vitamin D year round. Given the amount of UV available to Florida residents and the fortification of milk products with vitamin D, the low ser um levels of vitamin D found add credence to the recent recommendation by the American Academy of Pediatrics to double the amount of vitamin D supplementation provided to children (35). Worth noting are recent studies that suggest vitamin D3 is more effic ient at raising serum 25(OH) vitamin D levels than D2 (36). This indicates that supplementing with vitamin D2 alone may prove ineffective at raising serum 25(OH) vitamin D to an optimal level. Overall, vitamin D is an essential prohormone not only necess ary for the usual role in calcium homeostasis, but also vital for immune function.

PAGE 30

30 Vitamin D Binding Protein Levels in Type 1 Diabetes Despite a lack of association of 25(OH) vitamin D levels with healthy controls, T1D subjects, and first degree relative s of T1D subjects, we did find a significant difference in the VDBP levels when comparing control and T1D study populations. Interestingly, mean VDBP levels were highest in controls and lowest in T1D subjects, with VDBP levels of first degree relatives at an intermediate level. The VDBP concentration mean for each group fell within the normal VDBP conc entration range of 300 600 g/mL however, there were a number of values in each study population that fell outside this reference range. This may be expla ined by the daily fluctuations that occur with VDBP concentrations. VDBP levels undergo a decline in the morning, followed by a rapid increase, and finally, a plateau throughout the remainder of the day (20). Investigation into the relationship between 25(OH) vitamin D levels and VDBP levels of healthy controls, new onset T1D subjects, established T1D subjects, and first degree relatives of T1D subjects failed to reveal any significant association. This was not too surprising since VDBP concentrations a re not regulated by vitamin D metabolites, and because VDBP circulates at a much higher concentration than its ligand (22). However, a recent study of healthy women that measured the serum levels of 1,25(OH)2 vitamin D, the active vitamin D metabolite, f ound a positive correlation with VDBP concentrations (37). After further analysis, we did not find a significant relationship between duration of T1D and VDBP levels or between age and VDBP levels. However, we discovered that VDBP levels were significan tly higher in women compared to men, although the VDBP means for females and males were maintained within the normal reference range of 300 600 g/mL This association warrants further investigation.

PAGE 31

31 Conclusion The use of vitamin D analogues for delaying or p reventing the development of type 1 diabetes (T1D) may have potential, but will require additional research to develop a regimen that is safe and timely, as well as applicable to all individuals at an increased risk genetically for developing T1D. Our data supports the need for increasing the current recommendations set for vitamin D supplementation. This recommendation is based on finding suboptimal vitamin D levels in the majority of our entire study population despite an environment with a bundant sunlight. The presence of si gnificantly lower levels of vitamin D binding protein (VDBP) in T1D subjects further supports the notion that the vitamin D pathway may have a role in disease pathogenesis. Also worth considering is the impact low vita ability to fight infection. If sufficient vitamin D levels can be maintained, perhaps the current increasing trends in autoimmune diseases like T1D can be reversed, or at the very least, limited until a cure can be found.

PAGE 32

32 CHAPTER 5 FUTURE RESEARCH DIRECTIONS In light of our findings of low circulating serum 25(OH) vitamin D throughout the entire study population in similar proportion, yet significantly lower concentrations of vitamin D binding protein (VDBP) in subjects with type 1 diabetes (T1D) additional studies will be required to determine a definitive role. A large prospective study to further define the contribution of vitamin D in pathogenesis of T1D is urgently needed as trials using the acti ve form of vitamin D for T1D prevention are undergoing discussion. Another priority includes identifying whether or not the differences we observed in VDBP levels correlate with VDBP allelic frequencies of Gc1f, Gc1s, and Gc2 as other reports have shown ( 38,39). Also of interest hydroxylase enzyme responsible for converting 25(OH) vitamin D into its bioactive form. Previous studies have shown specific CYP27B1 polymorphisms associate with T1D (10), and we seek to determine whether or not these hydroxylase, 25(OH) vitamin D, and VDBP levels.

PAGE 33

33 LIST OF REFERENCES 1. Atkinson MA, Eisenbarth GS. Type 1 diabetes: new perspectives on disease pathogenesis and treatment. Lancet 200 1; 358 (9277): 221 229. 2. Morran MP, McInerney MF, Pietropaolo M. Innate and adaptive autoimmunity in type 1 diabetes. Pediatr Diabetes 2008; 9(2): 152 161. 3. Kitabchi AE, Wall BM. Management of diabetic ketoacidosis. Am Fam Physician 1999; 60(2): 455 464. 4. Scheidegger U, Allemann S, Scheidegger K, et al. Continuous subcutaneous insulin infusion therapy: effects on quality of life. Swiss Med Wkly 2007; 137(33 34): 476 482. 5. Kim MS, Polychronakos C. Immunogenetics of type 1 diabetes. Horm Res 2005; 64(4): 180 188. 6. Alizadeh BZ, Koeleman BP. Genetic polymorphisms in susceptibility to type 1 diabetes. Clin Chim Acta 2008; 387(1 2): 9 17. 7. Mrena S, Virtanen SM, Laippala P, et al. Models for predicting type 1 diabetes in siblings of affecte d children. Diabetes Care 2006; 29(3): 662 667. 8. Gale EA. Spring harvest? Reflections on the rise of type 1 diabetes. Diabetologia 2005; 48(12): 2445 2450. 9. Peechakara SV, Pittas AG. Vitamin D as a potential modifier of diabetes risk. Nat Clin Pra ct Endocrinol Metab 2008; 4(4): 182 183. 10. Bailey R, Cooper JD, Zeitels L, et al. Association of the vitamin D metabolism gene CYP27B1 with type 1 diabetes. Diabetes. 2007; 56(10): 2616 2621. 11. Ramos Lopez E, Jansen T, Ivaskevicius V, et al. Protectio n from type 1 diabetes by vitamin D receptor haplotypes. Ann N Y Acad Sci 2006; 1079: 327 334. 12. Pozzilli P, Manfrini S, Crino A, et al. Low levels of 25 hydroxyvitamin D3 and 1,25 dihydroxyvitamin D3 in patients with newly diagnosed type 1 diabetes. H orm Metab Res 2005; 37(11): 680 683. 13. Littorin B, Blom P, Scholin A, et al. Lower levels of plasma 25 hydroxyvitamin D among young adults at diagnosis of autoimmune type 1 diabetes compared with control subjects: results from the nationwide Diabetes Inc idence Stud in Sweden (DISS). Diabetologia 2006; 49(12): 2847 2852. 14. Svoren BM, Volkening LK, Wood JR, et al. Significant vitamin D deficiency in youth with type 1 diabetes mellitus. J Pediatr 2009; 154(1): 132 134.

PAGE 34

34 15. Mooney JA, Helms PJ, Jolliffe IT, et al. Seasonality of type 1 diabetes mellitus in children and its modifications by weekends and holidays: retrospective observational study. Arch Dis Child 2004; 89: 970 973. 16. Mohr SB, Garland CF, Gorham ED, et al. T he association between ultraviolet B irradiance, vitamin D status and incidence rates of type 1 diabetes in 51 regions worldwide. Diabetologia 2008; 51(8): 1391 1398. 17. Holick, MF. Vitamin D deficiency. N Engl J Med 2007; 357(3): 266 281. 18. Mathieu C, Gysemans C, Giulietti A, et al. Vitamin D and diabetes. Diabetologia 2005; 48(7): 1247 1257. 19. Holick MF. Diabetes and the vitamin D connection. Curr Diab Rep 2008; 8(5): 393 398. 20. Speeckaert M, Huang G, Delanghe JR, et al. Biological and clinica l aspects of the vitamin D binding protein (Gc globulin) and its polymorphism. Clin Chim Acta 2006; 372(1 2): 33 42. 21. Gomme PT, Bertolini J. Therapeutic potential of vitamin D binding protein. Trends Biotechnol 2004; 22(7): 340 345. 22. Cooke NE, Hadda d JG. Vitamin D binding protein (Gc globulin). Endocr Rev 1989; 10(3): 294 307. 23. Bailey R, Cooper JD, Zeitels L, et al. Association of the vitamin D metabolism gene CYP27B1 with type 1 diabetes. Diabetes 2007; 56(10): 2616 2612. 24. Zipitis CS, Akoben g AK. Vitamin D supplementation in early childhood and risk of type 1 diabetes: a systematic review and meta analysis. Arch Dis Child 2008; 93(6): 512 517. 25. Giulietti A, Gysemans C, Stoffels K, et al. Vitamin D deficiency in early life accelerates typ e 1 diabetes in non obese diabetic mice. Diabetologia 2004; 47(3): 451 462. 26. Giarratana N, Penna G, Amuchastequi S, et al. A vitamin D analog down regulates proinflammatory chemokine production by pancreatic islets inhibiting T cell recruitment and type 1 diabetes development. J Immunol 2004; 173(4): 2280 2287. 27. Munger KL, Levi n LI, Hollis BW, et al. Serum 25 hydroxyvitamin D levels and risk of multiple sclerosis. JAMA 2006; 296(23): 2832 2838. 28. Raghuwanshi A, Joshi SS, Christakos S. Vitamin D and multiple sclerosis. J Cell Biochem 2008; 105(2): 338 343.

PAGE 35

35 29. Grant WB. Hypothesis ultraviolet B irradiance and vitamin D reduce the risk of viral infections and their sequelae, including autoimmune diseases and some cancers. Photochem Photobiol 2008; 84(2): 356 365. 30. Alpert PT, Shaikh U. The effects of vitamin D deficiency and insufficiency on the endocrine and paracrine systems. Biol Res Nurs 2007; 9(2): 117 129. 31. Onganga JC, Pinget M, Belcourt A. Vitamin D binding protein gene polymorphism association with IA 2 autoantibodies in type 1 diabetes. Clin Bioc hem 2005; 38(5): 415 419. 32. Bodnar LM, Catov JM, Wisner KL, et al. Racial and seasonal differences in 25 hydroxyvitamin D detected in maternal sera frozen for over 40 years. Br J Nutr 2009; 101(2): 278 284. 33. Holick MF. The influence of vitamin D on b one health across the life cycle. J Nutr 2005; 135(11): 2726 2727. 34. Hagenau T, Vest R, Gissel TN, et al. Global vitamin D levels in relation to age, gender, skin pigmentation and latitude: an ecologic meta regression analysis. Osteoporos Int 2009; 2 0(1): 133 140. 35. Wagner CL, Greer FR. Prevention of rickets and vitamin D deficiency in infants, children, and adolescents. Pediatrics 2008; 122(5): 1142 1152. 36. Houghton LA, Vieth R. The case against ergocalciferol (vitamin D2) as a vitamin supplement. Am J Clin Nutr 2006; 84(4): 695 697. 37. Bouillon R, Van Assche FA, Van Baelen H, et al. Influence of the vitamin D binding protein on the serum concentration of 1,25 dihhydroxyvitamin D3. Significance of the free 1,25 dihydroxyvitamin D3 concentration. J Clin Invest 1981; 67(3): 589 596. 38. Lauridsen AL, Vestergaard P, Heickendorff L, et al. Plasma concentrations of 25 hydroxyvitamin D and 1,25 hydroxyvitamin D are related to the phenotype of Gc (vitamin D binding protein): a cross sectional study on 5 95 early postmenopausal women. Calcif Tissue Int 2005; 77(1): 15 22. 39. Lauridsen AL, Vestergaard P, Nexo E. Mean serum concentration of vitamin D binding protein (Gc globulin) is related to the Gc phenotype in women. Clin Chem 2001; 47(4): 753 756.

PAGE 36

36 BIOGRAPHICAL SKETCH Lindsey Marie Bierschenk was born in St. Louis, Missouri, where she attended Francis Howell High School for grades 9 12. In her senior year of high school, she was diagnosed with type 1 diabetes. She received a Bachelor of Science degree from the University of Missouri Columbia in 2007. Her major was biology with a minor in Spanish. As an undergraduate, Lindsey studied approaches for delaying onset of type 1 diabetes in the NOD mouse model. In 2 007, she joined the laboratory of Dr. Mark Atkinson where she studied the role of vitamin D in individuals with type 1 diabetes. In May of 2009, she received a Master of Science degree in Medical Sciences from the University of Florida.