THE IMPACT OF VITAMIN B6 DEFICIENCY ON THE - T-Space

THE IMPACT OF VITAMIN B6 DEFICIENCY ON THE ANGIOGENIC RESPONSE TO ISCHEMIA IN VIVO AND IN VITRO

by

Nicole Yuen

A thesis submitted in conformity with the requirements for the degree of Masters of Science

Graduate Department of Nutrition Sciences University of Toronto

© by Nicole Yuen (2012)

The Impact of Vitamin B6 Deficiency on the Angiogenic Response to Ischemia In Vivo and In Vitro

Master of Science 2012 Nicole Yuen Graduate Department of Nutritional Sciences University of Toronto

ABSTRACT

B vitamins are of interest in preventative and protective strategies in cardiovascular disease. However, the safety and efficacy of B vitamins has been questioned. Previous research from this group has demonstrated that B6 supplementation alone or in combination with folic acid and B12 reduces angiogenic response. This study determined the effect of vitamin B6 deficiency on the angiogenic response after ischemia in vivo and in vitro using a rodent model. Results indicated that vitamin B6 deficiency enhanced the early angiogenic response by increasing blood flow in vivo after an ischemic event. In vitro measurements demonstrated that vitamin B6 deficiency influenced endothelial progenitor cell (EPC) function and angiogenic growth factor release early after ischemia. In conclusion, B6 deficiency appears to have a modest effect on increasing blood flow and angiogenic markers after ischemia. Additional research is needed to further characterize the impact of lowered vitamin B6 on angiogenesis and its mechanisms.

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ACKNOWLEDGEMENTS During the past two years, I have undergone great personal and academic growth with so many contributing in more ways than words can describe. First, I would like to thank my supervisor, Dr. Mary Keith, who has provided immense support, feedback, and guidance throughout the entire process. I would like to express my deepest gratitude to her for making this experience enjoyable and educational. Also, thank you to Dr. Howard Leong-Poi and Dr. Elena Comelli, members of my committee, for their feedback and support. I would like to extend my appreciation to members of the Keith lab, both past and present. Thank you to Mavra Ahmed, Parastoo Azizi-Namini, Paul Lee, Sarah Stone and all of the students/volunteers who have contributed to this project. Each of you has played an integral role from feeding rats and collecting data to teaching me the techniques needed and providing moral support. Thank you also to Louisa Ho for assistance with gene expression work and Helen Luck for analysis of tube formation data. My gratitude also extends to the Leong-Poi lab, especially Michael Kuliszewski, Christine Liao, and Dmitriy Rudenko, who have helped with numerous tasks including hindlimb ligation, perfusion imaging and analysis, gene expression work, and providing support. As well, thank you to Dr. James House and his research group in assisting with blood analysis for this project. I would also like to thank all the staff involved with this research from St. Michael’s Hospital and the Li Ka Shing Knowledge Institute vivarium and core research facilities. A sincere thank you extends to Chris Spring who has gone above and beyond the call of duty to teach me needed techniques and troubleshoot problems. Last, but not least, I would like to thank my parents, family, and friends for their encouragement and guidance throughout the entire process. Without your continual support and advice, I would not be where I am today. iii

TABLE OF CONTENTS LIST OF TABLES ...............................................................................................................ix LIST OF FIGURES .............................................................................................................x LIST OF ABBREVIATIONS .............................................................................................xii 1.0 INTRODUCTION .......................................................................................................1 2.0 LITERATURE REVIEW ...........................................................................................4 2.1 Vitamin B6 .............................................................................................................4 2.1.1 Vitamin B6 in Humans ......................................................................................4 2.1.2 Vitamin B6 in Animals .....................................................................................7 2.2 Vitamin B9 and B12 ...............................................................................................8 2.3 B Vitamins, Homocysteine (Hcy) and Cardiovascular Disease (CVD) .................9 2.3.1 B Vitamins & Homocysteine Metabolism ........................................................9 2.3.2 Homocysteine and CVD....................................................................................11 2.3.3 B Vitamins and Homocysteine ..........................................................................12 2.3.4 B Vitamin Supplementation and CVD ..............................................................13 2.4 Vitamin B6 and Angiogenesis ................................................................................15 2.5 Angiogenesis and Endothelial Progenitor Cells (EPCs) ........................................19 3.0 RATIONALE ...............................................................................................................26 4.0 OBJECTIVES ..............................................................................................................26 4.1 Primary Objective ...................................................................................................26 4.2 Secondary Objective ...............................................................................................26 iv

5.0 HYPOTHESIS .............................................................................................................27 5.1 Primary Hypothesis ................................................................................................27 5.2 Secondary Hypothesis ............................................................................................27 6.0 MATERIALS AND METHODS ................................................................................28 6.1 Animal Characteristics and Care ............................................................................28 6.2 Assessment of B Vitamin Status ............................................................................30 6.2.1 Confirmation of B6 Deficiency, Homocysteine, and Cysteine .........................30 6.2.2 B12, Serum and RBC Folate, Homocysteine and Cysteine Status ...................31 6.3 Hindlimb Ischemia Model ......................................................................................31 6.4 In vivo Measurements of Angiogenic Response.....................................................32 6.4.1 Methods of Assessing Angiogenesis .................................................................32 6.4.2 Contrast Enhanced Ultrasound (CEU) ..............................................................33 6.4.3 Laser Doppler Perfusion Imaging (LDPI) .........................................................35 6.4.4 Exercise Capacity Test ......................................................................................36 6.4.5 EPC Quantification Using Fluorescence Activated Cell Sorting (FACS) .......37 6.5 In vitro Measurements of Angiogenic Response....................................................39 6.5.1 Sacrifice and Specimen Collection ...................................................................39 6.5.2 Bone Marrow Cell Isolation ..............................................................................39 6.5.3 Isolation of Cultured Bone Marrow EPCs ........................................................40 6.5.4 Measurement of Apoptosis (TUNEL Assay) ....................................................41 6.5.5 Measurement of Differentiation (Lectin Staining Assay) .................................42 v

6.5.6 Determination of Migration Potential ...............................................................43 6.5.7 Determination of Tube Formation and Length .................................................44 6.6 Gene Expression of VEGF and eNOS ....................................................................44 6.7 Statistical Analysis .................................................................................................46 7.0 RESULTS .....................................................................................................................47 7.1 Rodent Characteristics..........................................................................................47 7.1.1 Experiment One: Day 35 (Late) Endpoint Rodent Weight ...............................47 7.1.2 Experiment Two: Day 3 (Early) Endpoint Rodent Weight ...............................49 7.1.3 Experiment One: Day 35 (Late) Endpoint Rodent Intake .................................51 7.1.4 Experiment Two: Day 3 (Early) Endpoint Rodent Intake.................................53 7.2 Confirmation of Vitamin B6 Status and Associated Cysteine and Hcy Status ......55 7.3 In Vivo Angiogenic Response Measurements ........................................................58 7.3.1 Experiment One (Late): Contrast Enhanced Ultrasound (CEU) .......................58 7.3.2 Experiment One (Late): Laser Doppler Perfusion Imaging (LDPI) .................60 7.3.3 Experiment One (Late): Exercise Capacity Test ...............................................62 7.4 In Vivo Endothelial Progenitor Cell (EPC) Characterization .................................63 7.4.1 Experiment One (Late): EPC Quantification ......................................................63 7.4.2 Experiment Two (Early): EPC Quantification ....................................................65 7.5 In vitro Endothelial Progenitor Cell (EPC) Characterization .................................66 7.5.1 Experiment One (Late): Tube Formation ..........................................................66 7.5.2 Experiment Two (Early): Tube Formation........................................................68 vi

7.5.3 Experiment One (Late): Tube Length ...............................................................71 7.5.4 Experiment Two (Early): Tube Length .............................................................73 7.5.5 Experiment One (Late) and Two (Early): Migration Potential of EPCs ...........74 7.5.6 Experiment One (Late) and Two (Early): Differentiation of EPCs ..................76 7.5.7 Experiment One (Late) and Two (Early): Cell Apoptosis ................................78 7.6 Experiment One (Late) and Two (Early): Expression of VEGF & eNOS .............81 7.7 Experiment One (Late) and Two (Early): End Point Vitamin B6 Status ...............83 7.8 Experiment One and Two: Vitamin B12 and Serum and RBC Folate Status ........84 7.9 Experiment One: Day 35 (Late) Homocysteine and Cysteine Levels ....................88 8.0 DISCUSSION ...............................................................................................................90 8.1 In Vivo Angiogenic Response Measurements ........................................................90 8.1.1 Experiment One (Late): Contrast Enhanced Ultrasound (CEU) .......................90 8.1.2 Experiment One (Late): Laser Doppler Perfusion Imaging (LDPI) .................94 8.1.3 Experiment One (Late): Exercise Capacity Test ...............................................96 8.2 Experiment One (Late) and Two (Early): In vivo EPC Quantification ..................97 8.3 Experiment One (Late) and Two (Early): Expression of VEGF and eNOS ..........99 8.4 In vitro Endothelial Progenitor Cell (EPC) Characterization .................................101 8.4.1 Experiment One (Late) and Two (Early): Migration Potential of EPCs ...........101 8.4.2 Experiment One (Late) and Two (Early): Differentiation of EPCs ..................103 8.4.3 Experiment One (Late) and Two (Early): Tube Formation and Length ...........104 8.4.4 Experiment One (Late) and Two (Early): Cell Apoptosis ................................105 vii

8.5 Experiment One (Late) and Two (Early): Rodent Weight .....................................107 8.6 Experiment One (Late) and Two (Early): Rodent Diet Intake ...............................110 8.7 Vitamin B6 Status Confirmation and Associated Cysteine and Hcy .....................111 8.8 Experiment One (Late) and Two (Early): End Point Vitamin B6 Status ...............114 8.9 Experiment One and Two: Additional Plasma Vitamin Status and Measures .......115 9.0 LIMITATIONS ............................................................................................................117 10.0 CONCLUSION AND FUTURE RESEARCH ........................................................118 11.0 REFERENCES ..........................................................................................................122

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LIST OF TABLES Table 6.1 Vitamin and mineral content in rodent diets .......................................................30 Table 6.2 Diet composition and nutrient requirement of B12, B6 and Folic acid ..............30 Table 6.3Treadmill speed progression for rodent treadmill test ..........................................37 Table 6.4 Standardized treadmill test quality ratings ..........................................................37 Table 7.1 Results and performance rating of rodent exercise capacity test .........................52 Table 8.1 Plasma vitamin B6 values seen in the literature ..................................................117

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LIST OF FIGURES Figure 1.1 Metabolism pathways of homocysteine .............................................................11 Figure 6.1 Experimental timeline and design for rodent B6 deficiency study ....................29 Figure 6.2 CEU determination of blood flow ......................................................................34 Figure 7.20 Experiment one (late/day 35): rodent weight ...................................................74 Figure 7.21 Experiment two (early/day 3): rodent weight ..................................................76 Figure 7.22 Experiment one (late/day 35): rodent diet intake .............................................78 Figure 7.23 Experiment two (early/day 3): rodent diet intake.............................................80 Figure 7.24 Rodent plasma vitamin B6 status .....................................................................82 Figure 7.25 Plasma cysteine levels during feeding period ..................................................83 Figure 7.26 Plasma homocysteine levels during feeding period .........................................83 Figure 7.1 Experiment one (late): CEU of rodent blood flow .............................................48 Figure 7.2 Experiment one (late): LDPI bloodflow images ................................................50 Figure 7.3 Experiment one (late): LDPI of rodent blood flow ............................................51 Figure 7.4 Experiment one (late): FACS analysis post-surgery ..........................................53 Figure 7.5 Experiment one (late): EPC quantification ........................................................54 Figure 7.6 Experiment two (early): EPC quantification ......................................................55 Figure 7.7 Experiment one (late): EPC tube formation .......................................................57 Figure 7.8 Experiment one (late): EPC tube formation images...........................................57 Figure 7.9 Experiment two (early): EPC tube formation ....................................................59 Figure 7.10 Experiment two (early): EPC tube formation images ......................................60 x

Figure 7.11 Experiment one (late): tube length of EPCs .....................................................62 Figure 7.12 Experiment two (early): tube length of EPCs ..................................................63 Figure 7.13 Experiment one (late) and two (early): EPC migration potential.....................65 Figure 7.14 Experiment one (late) and two (early): EPC cell differentiation .....................66 Figure 7.15 Experiment one (late) and two (early): images of cell differentiation .............67 Figure 7.16 Experiment one (late) and two (early): images of cell apoptosis .....................69 Figure 7.17 Experiment one (late) and two (early): cell apoptosis.....................................70 Figure 7.18 Experiment one (late) and two (early): percentage of eNOS expression .........72 Figure 7.19 Experiment one (late) and two (early): percentage of VEGF expression ........72 Figure 7.27 Experiment one (late) and two (early): end point plasma B6 status ................84 Figure 7.28 Experiment one (late) and two (early): rodent plasma vitamin B12 levels ......86 Figure 7.29 Experiment one (late) and two (early): rodent serum folate levels ..................87 Figure 7.30 Experiment one and two: rodent red blood cell (RBC) folate levels ...............88 Figure 7.31 Experiment one (late): rodent plasma cysteine levels ......................................89 Figure 7.32 Experiment one (late): rodent plasma homocysteine levels .............................90

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LIST OF ABBREVIATIONS CAD

Coronary Artery Disease

CEU

Contrast Enhanced Ultrasound

CHD

Coronary Heart Disease

CON

Control Diet (Rodents)

CRC

Colorectal Cancer

CVD

Cardiovascular Disease

DEF

B6 Deficient Diet (Rodents)

EAR

Estimated Average Requirements

eNOS

Endothelial Nitric Oxide Synthase

EPC

Endothelial Progenitor Cell

FA

Folic Acid

FACS

Fluorescence Activated Cell Sorting

G-CSF

Granulocyte Colony Stimulating Factor

Hcy

Homocysteine

HPLC

High Performance Liquid Chromatography

iNOS

Inducible Nitric Oxide Synthase

LDPI

Laser Doppler Perfusion Imaging

MAT

Methionine Adenosyltransferase

MI

Myocardial Infarction

MTHF/ 5-methylTHF

Methyltetrahydrofolate/5-methyltetrahydrofolate

NO

Nitric Oxide

PA/4-PA

Pyridoxic Acid/4-Pyridoxic Acid xii

PBS

Phosphate Buffer Solution

PF

Pair-fed (Rodents)

PFA

Paraformaldehyde

PI

Propidium Iodide

PL

Pyridoxal

PLP

Pyridoxal 5’Phosphate

PN

Pyridoxine

PNP

Pyridoxine 5’Phosphate

PM

Pyridoxamine

PMP

Pyridoxamine 5’Phosphate

RBC

Red Blood Cell

RDA

Recommended Dietary Allowance

SAM

S-adenosylmethionine

SAH

S- adenosylhomocysteine

SAHH

S- adenosylhomocysteine Hydrolase

SDF-1α

Stromal Cell-Derived Factor 1α

SHMT

Serine Hydroxymethyltransferase

THF

Tetrahydrofolate

TUNEL

Tdt-dUTP Terminal Nick End Label

VEGF

Vascular Endothelial Growth Factor

VEGFR

Vascular Endothelial Growth Factor Receptor

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1.0 INTRODUCTION Cardiovascular disease (CVD) is one of the leading causes of mortality in both Canada and the world (Milan, 2011; World Health Organization, 2010). In the past 10 years, a decrease in risk factors such as smoking together with an advancement in medical care have contributed to the decline in cardiovascular disease (Tu et al., 2009). However, the burden of CVD is still substantial on both the individual and families as well as economically on the healthcare sector (Tu et al., 2009). Risk for CVD is influenced by a variety of factors include smoking, age, gender, lack of exercise, diets low in fruits and vegetables and high in fat, as well as inadequacy in vitamin intake (Gaby, 2010; Wierzbicki, 2007). Treatments such as the use of more effective and efficient pharmaceuticals and incorporation of routine physical activity have played a role in the management of CVD but they are not the only options for reducing risk in populations (Capewell & O'Flaherty, 2009; Lee, Hong, Chang, & Saver, 2010; Tu et al., 2009). The field of nutrition and vitamins and their role in CVD have been a major focus in research as it is one of the factors that can be controlled through diet. With nutrition being an alterable risk factor for cardiovascular disease, research has focused on the role of vitamins. Targeted nutrient therapy holds promise as a preventative and protective strategy against CVD. Of interest is the role of B vitamins as they are involved in physiological processes including cell growth, function, and metabolism (Otten, Hellwig, & Meyers, 2006). Attention has been focused on the function of folic acid (FA), vitamin B6, and vitamin B12 as these vitamins are necessary in the metabolism of methionine to homocysteine (Hcy), which has been found to be an independent predictor of CVD risk (Arnesen et al., 1995; Clarke et al., 1991; Friso, Lotto, Corrocher, & Choi, 2012; Nygard et al., 1995;Homocysteine Studies Collaboration, 2002; Wierzbicki, 2007; Study of 1

the Effectiveness of Additional Reductions in Cholesterol and Homocysteine (SEARCH) Collaborative Group et al., 2010) Various studies have reported a link between Hcy levels and cardiovascular disease risk where individuals with CVD have higher total Hcy compared to healthy populations (Arnesen et al., 1995; Clarke et al., 1991; Friso et al., 2012; Graham et al., 1997; Nygard et al., 1997). Similarly, researchers have also investigated the relationship between Hcy and B vitamins, with specific focus on vitamin B6. This research has demonstrated that individuals who had high homocysteine levels were found to also have lower plasma B6 levels (Arnesen et al., 1995; Boushey, Beresford, Omenn, & Motulsky, 1995; Dalery et al., 1995; Verhoef et al., 1996). Thus, these findings have prompted researchers to focus on the potential benefit of B vitamin supplementation in lowering Hcy and associated mortality and CVD risk (Bonaa et al., 2006; Lee et al., 2010; Lonn et al., 2006; Toole et al., 2004). Several studies have demonstrated that B vitamins effectively lower plasma total homocysteine. However, this lowering has not been linked with any changes in mortality (Bonaa et al., 2006; Lonn et al., 2006; Toole et al., 2004). Interestingly, a recent clinical trial demonstrated that supplementation with B6, B12 and folic acid in high risk CVD patients immediately following a myocardial infarction (MI) resulted in a trend towards increased adverse cardiovascular events (Bonaa et al., 2006). One proposed hypothesis for these unexpected findings was that B6 vitamin supplementation might impair the endogenous angiogenic response to ischemia in vivo. Vitamin B6 has not been extensively studied with respect to ischemic events and cardiovascular disease; however, both epidemiological and animals studies have shown an inverse relationship between cancer risk and vitamin B6 dietary intake and plasma levels 2

(Chou et al., 2011; Harris, Cramer, Vitonis, Depari, & Terry, 2012; Key et al., 2012; Larsson, Orsini, & Wolk, 2010; Le Marchand et al., 2011; Theodoratou et al., 2008). Studies by a Japanese research group shed insight into the mechanism on which vitamin B6 acts in suppressing tumourigenesis (Komatsu et al., 2001; Matsubara, Mori, Matsuura, & Kato, 2001; Matsubara, Komatsu, Oka, & Kato, 2003). This research reported that mice fed diets with high doses of vitamin B6 had decreased tumour incidence and numbers (Komatsu et al., 2001). Thus, it was suggested that vitamin B6 impaired tumourigenesis by decreasing cell proliferation, nitric oxide production, oxidative stress and subsequently, angiogenesis (Komatsu et al., 2001; Matsubara et al., 2003). An in vitro study also confirmed that increasing doses of vitamin B6 as pyridoxal 5’ phosphate inhibited the growth of vessels from a mounted aortic ring (Matsubara et al., 2001). Although supplementation of the diet with high doses of B6 may adversely affect vascular remodelling and impair angiogenesis in situations of tumourigenesis, this effect may prove beneficial in ischemic events where neovascularization is important. Although there are studies investigating the role of FA and B12 on CVD risk and mortality, minimal research has been conducted on the impact of B6 supplementation alone or in combination with B vitamins on angiogenic response following ischemia or mechanisms of angiogenesis influenced. However, this research group has investigated vitamin B6 supplementation in combination with FA and B12 on angiogenic response after ischemia using a rodent model and found increased supplementation decreased angiogenesis. Thus, to further understand the influence of vitamin B6 on angiogenesis, this research group proposes to conduct investigations aimed at determining the role of B6 deficiency on the endogenous angiogenic response to ischemia and its related mechanisms. 3

2.0 LITERATURE REVIEW 2.1 Vitamin B6 2.1.1Vitamin B6 in Humans Vitamin B6 is a water soluble vitamin that consists of seven compounds: pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), and their respective 5’phosphate esters as well as pyridoxic acid (PA) (Bowling, 2011; Bowman & Russell, 2006; National Research Council, 1998). Plant sources of B6 exist predominantly as pyridoxine and pyridoxine 5’ phosphate (PNP) while animal sources, including human tissue, contain mainly pyridoxal 5’phosphate (PLP) and in smaller quantities, pyridoxamine 5’phosphate (PMP) (Bowling, 2011; Bowman & Russell, 2006; National Research Council, 1998). PLP is of major importance in the human body as it acts as a co-enzyme to numerous biological processes including amino acid, lipid, and carbohydrate metabolism, neurotransmitter development, immune function, heme biosynthesis and homocysteine homeostasis via conversion of homocysteine to cysteine using a trans-sulfuration pathway (Bowling, 2011; Bowman & Russell, 2006; National Research Council, 1998; Wierzbicki, 2007). Vitamin B6 is found in a variety of food which includes fish, organ meats, and starchy vegetables and fruits as the richest sources. Vitamin B6 is commonly obtained through beef, poultry, fortified cereals, whole grains, vegetables and nuts and has a 75% bioavailability from a mixed diet (National Research Council, 1998). In humans, dietary vitamin B6 enters the digestive tract as PN, PL and PM and is taken up in the jejunum (National Research Council, 1998). Subsequently, most vitamin B6 is then absorbed via passive diffusion and transported to the liver for metabolism into its phosphorylated forms (Bowling, 2011; National Research Council, 1998). PLP is bound and stored mainly in 4

muscle but can also be found in plasma bound to albumin and in erythrocytes (Bowman & Russell, 2006; National Research Council, 1998). The half-life of vitamin B6 in studies is variable but has been estimated to be between 18 to 38 days with a study by Shane (1978) determining half-life of vitamin B6 body stores to be approximately 25 days (S. Johansson, Lindstedt, Register, & Wadstrom, 1966; Shane, 1978) . Vitamin B6 is excreted in the urine most often in the form of 4-pyridoxic acid (4-PA) (National Research Council, 1998). A study by Johansson et al. (1966) investigated the metabolism of tritium-labeled pyroxidine excreted as 4-PA in a small sample size and proposed a compartmental model where pyridoxine was stored in equilibrium between a larger compartment with a small turnover and a smaller compartment with a large turnover. This model approximated a human body store of about 40 to 150mg of vitamin B6 of which the average elimination from the reservoir was 2-3% translating to 1.7 to 3.6mg per day (S. Johansson et al., 1966). However, the estimates for vitamin B6 within the body have been variable with estimates ranging between 107-725µmol (Coburn et al., 1988). Coburn et al. (1988) conducted research analyzing vitamin B6 in muscle to directly estimate the human vitamin B6 pool and determined vitamin B6 levels in muscle to be closer to 900µmol. Vitamin B6 status is often measured directly by assessing the concentration of PLP in plasma, erythrocytes, or urine or indirectly using erythrocyte aminotransferase saturation (Bowman & Russell, 2006; National Research Council, 1998). Evidence suggests that more than one measure should be taken to properly assess vitamin B6 status (Leklem, 1990). However, plasma PLP, comprising of 70-90% of total vitamin B6 in plasma, is the most common measure of vitamin B6 status as it is the most accurate reflection of liver and tissue stores (Bowman & Russell, 2006; Leklem, 1990; National Research Council, 1998) 5

The recommended dietary allowance (RDA) for vitamin B6 in humans varies depending on age. In adults aged 19-50, the RDA is 1.3mg/day for both males and females in both the United States and Canada (Otten et al., 2006).Various studies have evaluated and proposed a plasma PLP concentration of 20-25 nmol/L as an indicator of adequacy (Bowman & Russell, 2006; National Research Council, 1998; Shane, 1978; Simpson, Bailey, Pietrzik, Shane & Holzgreve, 2010). For example, a study by Kretsch et al. (1991) examined the effects of vitamin B6 deficiency in eight healthy young women by measuring electroencephalography (EEG) tracings. Within 12 days of consuming the B6 deficient diet, subjects had PLP levels averaging 9.78nmol/L and two subjects demonstrated abnormalities in EEG recordings (Kretsch, Sauberlich, & Newbrun, 1991). These abnormalities resolved during the first week of vitamin B6 repletion indicating deficiency and functional impairment occurred rapidly with the potential, had the deficient diet continued, to present in the other subjects. These results consequently suggest that a plasma PLP level of 10nmol/L is considered suboptimal (Kretsch et al., 1991; National Research Council, 1998). Similarly, research by Leklem (1990) suggested that plasma PLP levels greater than 30 nmol/L is considered adequate. However, the estimated average requirements (EAR) of 1.1.mg/day for males and non-pregnant females was selected with the understanding that it may be an overestimate of B6 requirements needed for adequate health (National Research Council, 1998) Deficiencies may be pharmacologically induced or due to malnutrition. The clinical symptoms of B6 deficiency include seborrheic dermatitis, microcytic anemia, epileptiform convulsions, decreased growth, depression and confusion (Bowman & Russell, 2006; National Research Council, 1998). Conversely, large oral supplemental B6 doses of 6

2000mg/day or more have been associated with development of sensory neuropathy and dermatological lesions and thus an upper tolerable limit has been set for 100mg/day (Otten et al., 2006; National Research Council, 1998).

2.1.2 Vitamin B6 in Animals In animals, specifically laboratory rats, nutritional requirements vary according to age and developmental state and therefore, assessment of vitamin B6 requirements remains difficult (Coburn, 1994; National Research Council, 1995). Coburn (1994) reports evidence suggesting that numerous factors such as protein and carbohydrate intake, exercise, as well as natural and synthetic agents can affect B6 metabolism and subsequent requirements. Additionally, the varying forms of vitamin B6 also make it difficult to accurately measure and quantify (Coburn, 1994). Despite numerous studies investigating optimal B6 intake for rodents, definitive data on adequate B6 requirements remains inconclusive with various investigations reporting different values at which B6 status is maintained (Coburn, 1994; National Research Council, 1995). For example, Coburn (1994) summarized various studies that look at B6 requirements for growth and maintenance. These studies generally concluded that a level equivalent to 1-2 mg/kg diet of vitamin B6 is needed to maintain minimal growth; however, as rodents continue to grow and increase in weight, the amount of vitamin B6 per kg diet needed may also increase (Coburn, 1994). Additionally, a greater amount closer to about 6mg of vitamin B6/kg diet is necessary to maintain normal liver serum and erythrocyte aminotransferase activity (Coburn, 1994). Moreover, Coburn (1994) also discussed that in an unstressed adult,

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minimal maintenance requirement is about 0.2 mg/kg diet although greater investigation for long term requirements are needed (Coburn, 1994). Conventionally, the accepted estimated nutrient requirement for vitamin B6 in rats for maintenance and growth is approximately 6mg/kg diet, the standard level of supplemented vitamin in rodent diet (National Research Council, 1995). Rodents fed diets deficient in vitamin B6 exhibit physical symptoms such as scaling dermatitis on the tail, face, feet and ears, decreased growth, microcytic anemia, hyperexcitability and convulsions (National Research Council, 1995; Coburn, 1994; Nishijima et al., 2006). Some studies have also shown B6 deficient rats to demonstrate deficits in active and passive avoidance learning, decreased liver enzymes, and taste preference to sodium chloride (National Research Council, 1995). Similar to human populations, vitamin B6 in rats is predominantly found in muscle and stored as a coenzyme for glycogen phosphorylase. Consequently, plasma PLP can be used to measure vitamin B6 status (Coburn, 1994; National Research Council, 1995). In fact, a study conducted by Lumeng et al. (1978) validated use of plasma PLP as a measure of vitamin B6 status in rats. They concluded that plasma PLP concentration is a valid and sensitive indicator of vitamin B6 status where plasma levels increased with pyridoxine intake and with PLP content in muscle (Lumeng, Ryan, & Li, 1978).

2.2 Vitamin B9 and B12 Vitamin B9, also known as folate in its natural form or folic acid in its synthetic form, and B12, or cobalamin, are also water soluble B-complex vitamins that work closely in metabolic pathways that utilize vitamin B6 (Bowman & Russell, 2006; Friso et al., 2012; 8

Wierzbicki, 2007). The recommended dietary allowance for folate and B12 for adult humans are 400 µg/day and 2.4 µg/day respectively. For rodents, specifically a rat, the estimated requirements for folate and B12 respectively are 1mg/kg diet and 50 µg/kg diet. Folate, which can be found in dietary sources such as fortified grain products, dark green vegetables, beans and legumes is used in many reactions involving nucleic and amino acids, including various steps in the production of methionine to homocysteine (Bowman & Russell, 2006; Otten et al., 2006). For instance, folate is metabolised into methyltetrahydrofolate (MTHF) which is an important functional and regulatory component for the production of methionine from homocysteine. Moreover, folate plays a vital role in the production of s-adenosylmethionine (SAM) which in turn is used in the conversion of methionine to homocysteine. Similarly, B12, found mainly in meat products and fortified cereals and grains, plays an important role as a co-enzyme in the same reaction as folate converting homocysteine into methionine to help regulate levels in the body (National Research Council, 1998; Otten et al., 2006). Currently, recommendations from the American Heart Association (AHA) do not promote the use of folic acid and B12 vitamin supplementation to reduce CVD risk (American Heart Association, January 2012).

2.3 B Vitamins, Homocysteine (Hcy), and Cardiovascular Disease (CVD) 2.3.1 B Vitamins & Homocysteine Metabolism. In humans, vitamin B6, along with vitamin B12 and folic acid, play an indispensable role in the metabolism of homocysteine (Figure 1.1) (Bowman & Russell, 2006; Friso et al., 2012; Selhub, 1999; Wierzbicki, 2007; ). Homocysteine is a sulphur containing amino acid that can be affected by numerous factors including renal and hepatic impairment, diabetes, 9

dyslipidemia, elevated blood pressure and smoking ( Friso et al., 2012; Wierzbicki, 2007). It is produced by the conversion of dietary methionine which uses a pathway requiring the B vitamins, FA, B6 and B12 (Wierzbicki, 2007). Homocysteine is produced through a series of steps beginning with the conversion of methionine into s-adenosylmethionine (SAM) via methionine adenosyltransferase (MAT). SAM is then metabolized into S-adenosylhomocysteine (SAH) which is then, using SAH hydrolase (SAHH), subsequently converted into Hcy (Bowman & Russell, 2006; Friso et al., 2012; Wierzbicki, 2007). Vitamin B6 plays an indirect role during excess Hcy and depleted methionine. Specifically, B6 acts as a co-enzyme for serine hydroxymethyltransferase (SHMT) to convert tetrahydrofolate (THF) into methyltetralhydrofolate (MTHF) for remethylation of Hcy into methionine (Friso et al., 2012; Wierzbicki, 2007). Homocysteine can also be metabolized via a trans-sulfuration pathway when excess methionine is present. This process, activated by SAM, uses vitamin B6 as a cofactor to cystathionine-β-synthase to convert Hcy and serine to cystathionine and again as a cofactor to cystathionase to further produce α-ketobutyrate and cysteine. Cysteine is then oxidized to taurine or sulphate and eliminated from the body (Friso et al., 2012; Selhub, 1999; Wierzbicki, 2007;). When methionine levels are depleted, Hcy metabolism involves both FA and vitamin B12 (cobalamin) (Bowman & Russell, 2006; Friso et al., 2012; Wierzbicki, 2007).

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Dietary Protein

FA

METHIONINE THF MAT

Serine

SAM

Additional Metabolism Pathways

Methionine synthase with B12

SAH

SHMT with Vitamin B6

SAHH MethylTHF

Glycine

MethyleneTHF

HOMOCYSTEINE

Cystathionine-β-Synthase and PLP

Activated by SAM Serine

CYSTATHIONINE Water Cystathionase and PLP

α-ketobutyrate

CYSTEINE

Taurine

Figure 1.1 Metabolism pathways of homocysteine (Created using information from Bowman & Russell, 2006; Friso et al., 2012; Selhub, 1999; Wierzbicki, 2007)

2.3.2 Homocysteine and CVD Homocysteine has been extensively studied in relation to CVD. Numerous studies spanning several decades have been conducted to establish the relationship of Hcy as a predictor of vascular disease in both populations with heart disease and healthy individuals (Arnesen et al., 1995; Clarke et al., 1991; Clarke, Halsey, Bennett, & Lewington, 2011; Friso et al., 2012; Graham et al., 1997; Nygard et al., 1995; Wierzbicki, 2007). In the 1960s, observations in individuals with homocysteinuria led to the hypothesis that elevated Hcy may be related to elevated vascular disease (McCully, 1969). Since the 11

1960s, with the association between Hcy and CVD being established, researchers have investigated the relationship between Hcy and mortality (Clarke et al., 1991; Nygard et al., 1995; Nygard et al., 1997; Stampfer et al., 1992; Verhoef & Stampfer, 1995). Further evidence conducted in healthy individuals with confirmed vascular disease, including metaanalyses, has strengthened the correlation between elevated plasma total Hcy and CVD and provided evidence of the role of Hcy as an independent predictor of CVD risk and mortality (Clarke et al., 1991; Clarke et al., 2010; Clarke et al., 2011; Homocysteine Studies Collaboration, 2002; McNulty, Pentieva, Hoey, & Ward, 2008; Nygard et al., 1995; Stampfer et al., 1992).

2.3.3 B Vitamins and Homocysteine With evidence demonstrating that Hcy levels are an indicator of CVD, many researchers have investigated methods of lowering Hcy in attempts to lower CVD risk. Of particular interest is the role of B vitamin status in individuals with CVD as it reveals clues to the important role of these vitamins in the metabolism of homocysteine. Specifically, epidemiological studies have shown that lowered vitamin B6 status is associated with cardiovascular disease risk including atherosclerosis compared to adequate levels (Arnesen et al., 1995; Boushey et al., 1995; Dalery et al., 1995; Graham et al., 1997; Spinneker et al., 2007; Verhoef et al., 1996). Among the first studies to examine the relationship between B vitamins, Hcy and coronary artery disease was one conducted by Dalery et al. (1995). Mean homocysteine levels were higher in the bottom quartile for folate, vitamin B12 and B6 (p