A candidate gene analysis of vitamin B12 deficiency in elderly women and the nursing implications of personalized medicine

Functional vitamin B12 (cobalamin) deficiency is a subtle, progressive clinical disorder affecting 6-23% of elderly adults. Its symptoms, including fatigue, mood disturbances, and decreased strength, are vague and erroneously attributed to aging. Detection of cobalamin deficiency in elderly adults is confounded by clinical heterogeneity and lack of standardization in metabolic tests. Whereas some patients are asymptomatic with slightly altered metabolite profiles, others develop severe clinical outcomes. Better understanding of biologic factors contributing to cobalamin deficiency heterogeneity in older adults is needed. This is a candidate gene association study evaluating the relationship between genetic variation in the cobalamin-transport molecules (transcobalamin II and its receptor) with cobalamin-related outcome parameters in 795 research participants of the Women's Health and Aging 1 and 2 Studies. Research participant DNA was whole genome amplified and genotyped using the iPLEX Sequenom mass spectroscopy platform. Relationships between genotypes and clinical parameters were assessed using two-way analysis of variance and two-way analysis of covariance, on the fixed factors, race and Single Nucleotide Polymorphism genotype. Results of the dissertation research generated several genetic associations that are useful for further hypothesis testing and clinical validation research. In the transcobalamin II gene, two missense variants were associated with homocysteine and methylmalonic acid levels (rs9621049, rs35838082), two intronic variants were associated with serum cobalamin and homocysteine levels (rs4820888, rs4820887), and one missense variant was associated with mean corpuscular volume (rs11801198). A cluster of SNPs in the promoter region of the transcobalamin II gene was associated with the physical performance parameters, hand grip strength, and walking speed. In the transcobalamin II-receptor gene, a missense coding SNP (rs2336573) was associated with mean serum cobalamin concentrations. Scientific advances responsible for the technology used in this dissertation are being incorporated into healthcare. The tailoring of treatment to an individual's genetic make-up is termed Personalized Medicine. To assist nursing professionals in understanding and preparing for use of these technologies, four elements of Personalized Medicine are reviewed, including 1) discovery of novel biology that guides clinical translation mechanisms, 2) genetic risk assessment, 3) molecular diagnostic technology, and 4) pharmacogenetics and pharmacogenomics. Opportunities for nursing profession engagement are addressed.

A CANDIDATE GENE ANALYSIS OF VITAMIN B12 DEFICIENCY IN ELDERLY WOMEN AND THE NURSING IMPLICATIONS OF PERSONALIZED MEDICINE by Emma Louise Kurnat-Thoma A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Nursing The University of Utah December 2010 Copyright Emma Louise Kurnat-Thoma 2010 All Rights Reserved The Graduate School THE UNIVERSITY OF UTAH STATEMENT OF DISSERTATION APPROVAL The dissertation of_________________Emma Louise Kurnat-Thoma____________ has been approved by the following supervisory committee members: _____Ginette A. Pepper______ , Co-Chair _____11/4/2010____________ Date Approved _____Lawrence C. Brody_____ , Co-Chair _____10/13/2010___________ Date Approved _____Jack M. Guralnik______ , Member _____10/15/2010___________ Date Approved _____Diane L. Kelly________ , Member _____10/22/2010___________ Date Approved _____Patricia A. Murphy_____ , Member _____10/26/2010___________ Date Approved _____Bob Wong____________ , Member _____11/2/2010____________ Date Approved and by____Maureen Keefe___________, Chair of the Department of___Nursing____ _______________________________ Charles A. Wight, Dean of The Graduate School ABSTRACT Functional vitamin B12 (cobalamin) deficiency is a subtle, progressive clinical disorder affecting 6-23% of elderly adults. Its symptoms, including fatigue, mood disturbances, and decreased strength, are vague and erroneously attributed to aging. Detection of cobalamin deficiency in elderly adults is confounded by clinical heterogeneity and lack of standardization in metabolic tests. Whereas some patients are asymptomatic with slightly altered metabolite profiles, others develop severe clinical outcomes. Better understanding of biologic factors contributing to cobalamin deficiency heterogeneity in older adults is needed. This is a candidate gene association study evaluating the relationship between genetic variation in the cobalamin-transport molecules (transcobalamin II and its receptor) with cobalamin-related outcome parameters in 795 research participants of the Women's Health and Aging 1 and 2 Studies. Research participant DNA was whole genome amplified and genotyped using the iPLEX Sequenom mass spectroscopy platform. Relationships between genotypes and clinical parameters were assessed using two-way analysis of variance and two-way analysis of covariance, on the fixed factors, race and Single Nucleotide Polymorphism genotype. Results of the dissertation research generated several genetic associations that are useful for further hypothesis testing and clinical validation research. In the transcobalamin II gene, two missense variants were associated with homocysteine and methylmalonic acid levels (rs9621049, rs35838082), two intronic variants were associated with serum cobalamin and homocysteine levels (rs4820888, rs4820887), and one missense variant was associated with mean corpuscular volume (rs11801198). A cluster of SNPs in the promoter region of the transcobalamin II gene was associated with the physical performance parameters, hand grip strength, and walking speed. In the transcobalamin II-receptor gene, a missense coding SNP (rs2336573) was associated with mean serum cobalamin concentrations. Scientific advances responsible for the technology used in this dissertation are being incorporated into healthcare. The tailoring of treatment to an individual's genetic make-up is termed Personalized Medicine. To assist nursing professionals in understanding and preparing for use of these technologies, four elements of Personalized Medicine are reviewed, including 1) discovery of novel biology that guides clinical translation mechanisms, 2) genetic risk assessment, 3) molecular diagnostic technology, and 4) pharmacogenetics and pharmacogenomics. Opportunities for nursing profession engagement are addressed. iv For Andrew James Thoma Shaka and Sheba "If we're serious about preventive medicine, and using personalized genomics to inform that, we're not going to change the genome. It's the environment we're going to want to change." -Francis S. Collins, MD, PhD Personalized Medicine Coalition Address, 2009: Washington, D.C. TABLE OF CONTENTS ABSTRACT………………………………...……………………………………………iii LIST OF FIGURES.……...……………………………………..………………………...x ACKNOWLEDGEMENTS…….……………………………………………………...…xi Chapters 1 OVERVIEW OF DISSERTATION RESEARCH…………………………………....1 Statement of Clinical Research Problem………………………………………….1 Dissertation Project Scientific Aims and Research Questions…………………....3 References………………………………………………………………………...7 2 THE MOLECULAR, PHYSIOLOGIC, GENETIC, AND CLINICAL BASIS OF VITAMIN B12 METABOLISM IN AGING INDIVIDUALS……..............………..8 History and Origins………………………………………………………………..8 Chemical Properties……………………………………………………………...11 Structure………………………………………………………………………….12 Synthesis…………………………………………………………………………13 Food Sources and Daily Requirements…………………………………………..14 Physiologic Properties…………………………………………………………...15 Cobalamin Transport Genetics and Molecular Biology…………………………21 Pathophysiology of Cobalamin Deficiency……………………………………...26 Gerontologic Research Factors…………………………………………………..37 The Dissertation's Clinical Measurements of Altered Cobalamin Status……….42 References………………………………………………………………………..52 3 STUDY DESIGN, MEASUREMENT, METHODS, AND ANALYSIS…….…….77 Introduction………………………………………………………………………77 Study Design……………………………………………………………………..77 Outcome Variable Measurement………………………………………………...84 Covariate Variable Measurement………………………………………………..89 Genotyping Methods……………………………………………………………..90 Protection of Human Subjects………………………………………………….101 Analysis………………………………………………………………………...101 References………………………………………………………………………111 4 ASSOCIATION OF TRANSCOBALAMIN II AND TRANSCOBALAMIN II-RECEPTOR GENETIC VARIATION WITH COBALAMIN METABOLITE LEVELS IN ELDERLY WOMEN…………………………………………………119 Abstract………………………………………………………………………....119 Introduction……………………………………………………………………..120 Methods…………………………………………………………………………122 Results…………………………………………………………………………..130 Discussion………………………………………………………………………136 Acknowledgements……………………………………………………………..142 References………………………………………………………………………143 5 ASSOCIATION OF TRANSCOBALAMIN II AND TRANSCOBALAMIN II-RECEPTOR GENETIC VARIATION WITH CLINICAL FEATURES OF VITAMIN B12 DEFICIENCY IN ELDERLY WOMEN…………………………155 Abstract…………………………………………………………………………155 Introduction……………………………………………………………………..156 Methods…………………………………………………………………………158 Results…………………………………………………………………………..168 Discussion………………………………………………………………………175 Acknowledgements……………………………………………………………..180 References………………………………………………………………………181 6 TRANSLATION OF GENETICS AND GENOMICS FOR NURSING- PERSONALIZED MEDICINE……………………………………………………195 Abstract…………………………………………………………………………195 Introduction……………………………………………………………………..196 Clinical Application of Genetics and Genomics: Personalized Medicine……..198 Implications and Opportunities for Nursing in Personalized Medicine………...211 Concluding Remarks……………………………………………………………216 References………………………………………………………………………218 7 CONCLUSIONS…………………………………………………………………..242 Summary of Dissertation Research and Its Scientific Context…………………242 Dissertation Research Limitations and Future Research……………………….253 Implications for Leadership and Policy………………………………………...256 References………………………………………………………………………258 viii Appendices A WHOLE GENOME AMPLIFICATION FOR DISSERTATION………………...261 B MANUAL GENOTYPE CALLING OF WHOLE GENOME AMPLIFIED MATERIAL…………………………………………………………263 C HUMAN SUBJECTS CLASSIFICATION FOR DISSERTATION RESEARCH…………………………………………………...265 D DISSERTATION DATA………………………………………………………….266 ix LIST OF FIGURES Figure Page 1 The Biochemical Role of Cobalamin (Vitamin B12) in the Cell……………….…...74 2 The Metabolic Pathway Overlap of Cobalamin and Folate in the Cell….………….76 3 Race by SNP Genotype Interaction for SNP rs4820887 on Mean Homocysteine…………………………………………………………….153 4 Transcobalamin II-Receptor SNP rs2336573 and Mean Serum Cobalamin……....154 5 Transcobalamin II SNP rs1801198 and Mean Corpuscular Volume…………........194 6 Four Elements of Personalized Medicine……………………………….…….…...238 7 Basic Foundation of Genome Wide Association Studies………………….….…...239 8 Age-Related Macular Degeneration: From HapMap to Clinical Trial in <5 Years……...……………………………………………….240 9 The Pharmacogenomic and Policy Implications of CYP 2C9 and VKORCI Genetic Testing………………………………………………………………..…………241 ACKNOWLEDGEMENTS With a grateful and humble heart, I first and foremost joyfully acknowledge God and the dear Lord and Savior, Jesus Christ, who provided me with countless graces and gifts-among them courage, persistence, patience, wisdom, and love, to fulfill the requirements of this work. "For our heart shall rejoice in Him, because we have trusted in His holy name,"-Psalm 33:21. One of His greatest blessings to me in realizing this achievement is my dear husband Andrew, whose enormous heart provided faithful love and courageous support during every step of this work. Andy, there are not adequate enough words to express the depth and magnitude of my appreciation and love for you. Thank you always and forever. With Andy, are my pups Shaka and Sheba, whose mountain, ocean, and backyard shenanigans always kept me joyfully laughing. My family-mother Marian, father John, sisters Rebecca and Mary, grandmothers Dorothy and Josephine, always kept me going through their love and pride in the work I aspired to perform. It's meant the world to me. Another great blessing is the profound happiness I received from many dear friends: Travis and Cameron for cheerful support; Peggy and Nick for constant love and teaching me meaningful prayer; Pete and Liz for support; Anne you were always my ear; Katy and Phil my dear blessings in faithful living; Taura and Bob my ever present shoulder for strength; Erica my constant ray of sunshine; Erin my great source of joy and comfort; Lauri and Gwen my dear colleagues; Claire my unending source of laughter; Dawn, my always fired-up cheerleader; Rev. Lawana Sowell, my relief of frustration and renewal of Spirit in Him; Patricia and Prit, my always-present source of kindness; Praveen and Rebecca, my shining example of gratefulness for life's blessings and loving acceptance of trial; Kim, Kate, Stephanie, Ashley and Sandra, my NIH "sisters"; the loving community and parish staff at St. Jane Frances de Chantal church; and my dear neighbors, Allen, Nancy and Van, Melanie and Matthias, Laura and Lawrence-all sources of comfort, laughter, and advice. Professionally, this project has been financially supported by a National Institutes of Health (NIH) predoctoral Intramural Research Training Award through the National Institute of Nursing Research (NINR). In addition to completion of academic requirements at University of Utah's College of Nursing under the direction of Dr. Ginette Pepper, the dissertation was performed in the laboratory of my primary mentor, Dr. Lawrence Brody, at the National Human Genome Research Institute (NHGRI) in Bethesda, MD, through NIH's Graduate Partnerships Program. Gratitude is extended to members of my supervisory committee for their warm and collegial support: Dr. Jack Guralnik, Dr. Diane Kelly, Dr. Bob Wong, and Dr. Patricia Murphy. I am also appreciative for the efforts of many collaborators on the Women's Health and Aging Studies (WHAS), including Dr. Jack Guralnik, Dr. Sally Stabler, and Dr. Amy Matteini. Recognition and appreciation is also extended to Dr. Francis Collins, Dr. Sharon Milgram, and Dr. Pat Sokolove for their kind and thoughtful mentorship throughout my years at NIH. Warm thanks are also extended to the members of the Lawrence Brody and Francis Collins laboratories, and the NHGRI administrative support staff, for their scientific and technical expertise in conducting the dissertation research. xii Lawrence Brody Laboratory David Bernard, Manjit Kaur, Kristine Krebs, Marina Lee, Faith Pangilinan, Patricia Porter-Gill, Reid Prentice, Nicole Stone. Francis Collins Laboratory Lori Bonnycastle, Peter Chines, Michael Erdos, Mario Morken, Narisu Narisu, Praveen Sethupathy, Amy Swift, Urraca Tavarez. NHGRI Andy Baxevanis, Jay Latman, Jack Moore. xiii CHAPTER 1 OVERVIEW OF DISSERTATION RESEARCH Statement of Clinical Research Problem Prevalence of metabolically confirmed cobalamin (vitamin B12) deficiency among community-dwelling elderly is between 6% and 23%, and depending upon definition criteria used-as high as 40.5% (Allen, 2009; Baik & Russell, 1999; Johnson et al., 2003; Lindenbaum, Rosenberg, Wilson, Stabler, & Allen, 1994; Pennypacker et al., 1992). Classic hematological and neurological manifestations include megaloblastic anemia, psycho-cognitive decline, and functional impairment. Less recognized is sub-clinical deficiency, involving subtle biochemical and clinical changes, resulting in unrecognized or misattributed diagnosis. Manifestations of cobalamin deficiency are vague and include fatigue, decreased cognition, malaise, peripheral insensitivity, decreased strength, sleep, and mood disturbances, which present prior to grossly elevated metabolite profiles and hallmark presence of megaloblastic anemia. Long-term consequences of cobalamin deficiency in older adult individuals may increase the disability trajectory, resulting in increased frequency of hospital admissions, lengthier and more severe hospitalizations, and greater degrees of chronic disablement that significantly effect mobility and quality of life (Bartali et al., 2006). 2 Numerous challenges in detection, diagnosis, and treatment of cobalamin deficiencies in older adults exist secondary to lack of accurate laboratory assays and vast clinical heterogeneity. While some individuals are asymptomatic with low-normal cobalamin levels and slightly altered metabolite screening panels, others with similar profiles develop severe, permanent clinical outcomes (Carmel & Sarrai, 2006). For these reasons, relatively little progress has been made in the identification of cobalamin deficient individuals who would benefit most from pre-emptive supplementation of the nutrient. Better understanding of the genetic factors contributing to clinical heterogeneity surrounding cobalamin deficiency, subclinical deficiency states, and treatment responses could enhance clinical care of elderly individuals. To identify possible factors contributing to the clinical heterogeneity in cobalamin deficiency, this study used a candidate gene approach to perform a secondary analysis of data and banked biologic samples from the Women's Health and Aging Studies. Because of their roles in cobalamin physiology and metabolism, the candidate genes for the dissertation research included the cobalamin carrier protein (transcobalamin II) and the cobalamin carrier protein receptor (transcobalamin II-receptor). There are different forms of cobalamin; for the purposes of this work, the terms cobalamin and vitamin B12 will be used interchangeably to denote all chemical forms of the nutrient unless otherwise specified through more exact terminology (methyl-, 5' deoxyadenosyl-, cyano-, etc.). 3 Dissertation Project Scientific Aims and Research Questions The goal of this project was to determine if genetic variants, Single Nucleotide Polymorphisms (SNPs), in two candidate cobalamin metabolic genes were associated with clinical and biochemical parameters in a cohort of community-dwelling elderly women. Secondary scholarly aims were to orient the scientific data within a broader translation framework for the field of professional nursing, and thus relatable to the context of Personalized Medicine. Aim 1 Accounting for folate status, Aim 1 was to determine if there are differences in the hematological vitamin B12 indicators, hemoglobin concentration and mean corpuscular volume (MCV) level, by race and SNP genetic variation in the transcobalamin II (vitamin B12 carrier molecule) and transcobalamin II-receptor (vitamin B12 carrier molecule receptor) genes. Research Question 1.1 Do hemoglobin concentrations differ by race and SNPs in the transcobalamin II and transcobalamin II-receptor genes? Research Question 1.2 Do MCV levels differ by race and SNPs in the transcobalamin II and transcobalamin II-receptor genes? 4 Aim 2 Accounting for renal function (questions 2.1-2.3), folate (question 2.2), and cobalamin status (questions 2.2-2.3), Aim 2 was to determine if there are concentration differences in the biochemical vitamin B12 indicators, serum cobalamin, homocysteine, and serum methylmalonic acid, by race and SNP genetic variation in the transcobalamin II and transcobalamin II-receptor genes. Research Question 2.1 Accounting for renal function, are there differences in serum cobalamin concentrations by race and SNPs in the transcobalamin II and transcobalamin II-receptor genes? Research Question 2.2 Accounting for renal function, folate, and cobalamin status, are there differences in homocysteine levels by race and SNPs in the transcobalamin II and transcobalamin II-receptor genes? Research Question 2.3 Accounting for renal function and cobalamin status, are there differences in serum methylmalonic acid levels by race and SNPs in the transcobalamin II and transcobalamin II-receptor genes? 5 Aim 3 Aim 3 was to determine if there are differences in the neurologic vitamin B12 indicators, depression score and peripheral vibration sensitivity, by race and genetic variation within the transcobalamin II and transcobalamin II-receptor genes. Research Question 3.1 Do depression scores differ by race and SNPs in the transcobalamin II and transcobalamin II-receptor genes? Research Question 3.2 Do peripheral extremity vibratory sensation scores differ by race and SNPs in the transcobalamin II and transcobalamin II-receptor genes? Aim 4 Aim 4 was to determine if there are differences in the functional performance vitamin B12 indicators, hand grip strength, and walking speed, by race and genetic variation within the transcobalamin II and transcobalamin II-receptor genes. Research Question 4.1 Does hand grip strength differ by race and SNPs in the transcobalamin II and transcobalamin II-receptor genes? 6 Research Question 4.2 Accounting for standing height, does walking speed differ by race and SNPs in the transcobalamin II and transcobalamin II-receptor genes? Aim 5 Aim 5 was to identify opportunities for the field of professional nursing in the area of genetics and genomics, or Personalized Medicine. Research Question 5.1 What is the current state of genetic/genomic science as relevant for professional nurses in research, education, and practice settings? 7 References Allen, L. H. (2009). How common is vitamin B-12 deficiency? American Journal of Clinical Nutrition, 89(2), 693S-696S. doi: ajcn.2008.26947A [pii] 10.3945/ajcn.2008.26947A [doi] Baik, H. W., & Russell, R. M. (1999). Vitamin B12 deficiency in the elderly. Annual Review of Nutrition, 19, 357-377. doi: 10.1146/annurev.nutr.19.1.357 [doi] Bartali, B., Semba, R. D., Frongillo, E. A., Varadhan, R., Ricks, M. O., Blaum, C. S., et al. (2006). Low micronutrient levels as a predictor of incident disability in older women. Archives of Internal Medicine, 166(21), 2335-2340. Carmel, R., & Sarrai, M. (2006). Diagnosis and management of clinical and subclinical cobalamin deficiency: Advances and controversies. Current Hematology Reports, 5(1), 23-33. Johnson, M. A., Hawthorne, N. A., Brackett, W. R., Fischer, J. G., Gunter, E. W., Allen, R. H., et al. (2003). Hyperhomocysteinemia and vitamin B-12 deficiency in elderly using Title IIIc nutrition services. American Journal of Clinical Nutrition, 77(1), 211-220. Lindenbaum, J., Rosenberg, I. H., Wilson, P. W., Stabler, S. P., & Allen, R. H. (1994). Prevalence of cobalamin deficiency in the Framingham elderly population. American Journal of Clinical Nutrition, 60(1), 2-11. Pennypacker, L. C., Allen, R. H., Kelly, J. P., Matthews, L. M., Grigsby, J., Kaye, K., et al. (1992). High prevalence of cobalamin deficiency in elderly outpatients. Journal of the American Geriatrics Society, 40(12), 1197-1204. CHAPTER 2 THE MOLECULAR, PHYSIOLOGIC, GENETIC, AND CLINICAL BASIS OF VITAMIN B12 METABOLISM IN AGING INDIVIDUALS History and Origins The critical importance of vitamin B12 to human physiology has a rich history steeped in vigilant patient observation. The initial discovery of what is now known to be pernicious anemia can be traced to 1824 when James Scarfe Combe, a civil practice physician, described an anecdotal account of an individual whose symptoms included severe pallor, thirst, diarrhea, and excessive urination (Combe, 1824). The patient, Mr. Alexander Haynes, initially presented to Combe in July of 1821 at which point the symptoms progressively became worse until his death in February 1822. Dr. Combe's report did not gain recognition as being significant until 1849, when English physician Thomas Addison submitted a description of anemia "commencing insidiously, and proceeding very slowly, so as to occupy a period of several weeks, or even months, before any serious alarm is taken by either the patient or by the patient's friends" (Addison, 1849). Addison's clinical observations outlined physiological characteristics unlike any other previous classification of anemia, most striking of which was the subtle and progressive clinical course of symptom development. A historical 9 account cites that in 1872, a German physician from Zurich, Dr. Biermer, noted the severe intractable progressive characteristics of the Addisonian anemia in 15 subjects and issued the title "pernicious anemia" (Sinclair, 2008). Patients suffered over decades from pernicious anemia and its unsuccessful treatment; common therapeutic interventions for the condition included dietary modifications, exposure to sunlight, and even use of an arsenic supplementation known as "Fowler's Solution" (Sinclair, 2008). Things would not change until 1925 when a team of three physicians, Drs. Whipple, Hooper, and Robscheit, researched hematopoeisis associated with chronic blood loss. Among the treatment interventions studied was the administration of beef liver to canines and significant findings were obtained; dogs who were bled and fed the uncooked liver were noted to experience rapid resolution of anemic symptoms (Whipple, 1925). Two prominent Boston-area physicians in the United States who learned of the work developed a protocol for human patients with pernicious anemia, and fed 45 patients a high protein daily diet incorporating 120-240 grams of raw liver (Minot, 1926). Within days of starting treatment, patients' jaundice began to resolve, reticulocyte counts increased, and hemoglobin values normalized. This discovery went on to be confirmed by many practicing physicians and in 1934, Drs. Whipple, Minot, and Murphy were the first Americans to receive the Nobel Prize in Medicine and Physiology. In 1948, the fraction responsible for this physiologic, dubbed vitamin B12, was purified from liver and kidney and shortly after, daily dietary intake requirements were established (Rickes, Brink, Koniuszy, Wood, & Folkers, 1948; E. Smith, 1948). As researchers identified cobalt as a key component of vitamin B12, its name was changed from vitamin B12 to cobalamin, and in 1955-1956, a scientific team 10 led by Dorothy Hodgkin used x-ray crystallography to identify and elucidate its crystalline 3-dimensional structure. Using electron density measurements, Hodgkin's landmark effort identified atomic positions of elements surrounding the central cobalt atom (Hodgkin et al., 1956; Hodgkin, Pickworth, Robertson, Trueblood, & Prosen, 1955). The discovery led to another Nobel Prize, this time in Chemistry, and was issued to Dr. Hodgkin in 1964. Scientific progress from the late 1950s through the 1960s was notable for the identification of variable chemical isoforms of cobalamin and the key enzymes directing molecular rearrangements in metabolic reduction/oxidation reactions. In 1958, Barker, Weissbach, and colleagues discovered a key biologic role for vitamin B12 in the bacterial model system Clostridium tetanomorphum; the conversion of L-methylmalonyl- Coenzyme A required adenosylcobalamin to formulate succinyl-Coenzyme A (Barker, Smyth, Wawszkiewicz, Lee, & Wilson, 1958; Barker, Smyth, Wilson, & Weissbach, 1959; Barker, Weissbach, & Smyth, 1958). In 1962, Smith and colleagues created carbon-enriched methylcobalamin and found that it could serve as a cofactor for methionine synthase (Guest, Friedman, Woods, & Smith, 1962). Shortly after, Weissbach deducted the chemical reaction responsible for methionine synthesis, and proved methylcobalamin serves as the necessary cofactor for conversion of homocysteine to methionine (Weissbach & Taylor, 1966). In the 1970s, scientists produced the total chemical synthesis of cobalamin, a monumental achievement. The effort was notable for 11 years' worth of chemical reaction calculations and required the skills of over 100 collaborating scientists (Woodward, 1973). Heading the project at Harvard University's Department of 11 Chemistry was Dr. Robert Woodward, who, in 1965, also received the Nobel Prize for Chemistry. In the 1980s-1990s, scientists identified the stereochemistry and functional mechanisms responsible for cobalamin-dependent reduction/oxidation rearrangements. The piecing together of all the intricate steps of aerobic microbial cobalamin biosynthesis in 1993 was a capstone achievement spanning over 25 years of scientific research (Battersby, 1994). Elucidation of three-dimensional structures of the methionine synthase and methylmalonyl CoA enzymes provided understanding of how vitamin B12 reactions occur in both mammalian and microbial species (Dixon, Huang, Matthews, & Ludwig, 1996; Drennan, Huang, Drummond, Matthews, & Lidwig, 1994; Mancia et al., 1996). Recently, techniques and knowledge derived from fields such as genetics, molecular biology, and recombinant engineering are permitting not just discovery, but purposeful manipulation of both the aerobic and anaerobic cobalamin pathways to understand intermediate steps and biosynthetic processes across all life forms (Battersby, 1994; Warren, Raux, Schubert, & Escalante-Semerena, 2002). Chemical Properties The chemical activities of vitamin B12 vary temporally, spatially, and across numerous life forms. Their overall function can be broken down into three distinct categories where they can serve as 1) mutases, facilitating electron exchange between hydrogen and other atoms nested between two carbon atoms; 2) ribonucleotide reductases, reducing ribonucleotide triphosphate to 2'-deoxyribonucleotide phosphate via adenosylcobalamin; and 3) intermolecular methyl group transfers, shuttling of methyl 12 groups across chemical bonds and intermediaries as catalyzed by methylcobalamin (Green & Miller, 2007; Martens, Barg, Warren, & Jahn, 2002). In the animal kingdom, cobalamin is used for only two enzymatic reactions, which will be discussed in a later section. Structure The organometallic cobalamin molecule (chemical formula C83H88O14N14PCo; molecular weight 1355 daltons) is among the most structurally complex found in all of nature. There are two primary features in addition to its central cobalt atom, including (1) a planar corrin ring and (2) a nucleotide that lies perpendicular to the planar group. Comprising the nucleotide is a base, 5,6-dimethylbenzimidazole, and a phosphorylated sugar, ribose-3-phosphate. The 5,6-dimethylbenzimidazole base is exclusive to the cobalamin molecule in nature, and the ribose is unusually phosphorylated at carbon position 3. The corrin ring contains a group of four pyrroles (5-member rings of C4H4NCH3) with each N atom affixed and coordinated to the central cobalt atom. A fifth ligand extends from the central cobalt atom, where various functional (R) groups may attach and yield various biologic forms of cobalamin. The four primary groups in mammals include a 5'-deoxyadenosyl group (adenosylcobalamin), a hydroxyl group (hydroxocobalamin), a methyl group (methylcobalamin), and a glutathione group (glutathionylcobalamin). The two co-enzyme forms of cobalamin directly relevant to humans are adenosylcobalamin, found in mitochondrial membranes, and methylcobalamin in the cytosol, which is 13 clinically measurable in human plasma. Other forms of cobalamin exist in plants and bacteria. Variable oxidation and reduction states of the cobalt atom yields greater complexity, where arrangement of electrons can result in varying forms of hydroxo-, adenosyl-, and methylcobalamin. For example, trivalent cob(III)alamin represents full oxidation capacity in the hydroxocobalamin form, but adenosylcobalamin and methylcobalamin contain divalent cob(II)alamin and monovalent cob(I)alamin states of the cobalt atom. As the C-Co chemical bonds in cobalamin are extremely sensitive to degradation, in the presence of light and a cyanide source, all cobalamin forms are converted to cyanocobalamin. This is cobalamin's most stable form, and subsequently is the commercial preparation used for the majority of pharmaceutical and therapeutic applications in the U.S. (National Academy of Sciences, 1998). Regardless of the form, the cobalamin that is delivered to mammalian cells is enzymatically activated to either methylcobalamin or 5'-deoxyadenosylcobalamin (Scott, 1999). Synthesis Vitamin B12 is the most chemically complex vitamin; in biologic systems that produce it, the coordinated and functional integration of over 30 genes is required. Although cobalamin is required by humans and mammals, synthesis of vitamin B12 is exclusive to microorganisms, and even then-limited to members of the Archea and Eubacteria families (Raux, Schubert, & Warren, 2000). Plants and fungi are understood not to produce or use cobalamin (Benner, Ellington, & Tauer, 1989). 14 Microbial synthesis of vitamin B12 can be either aerobic or anaerobic, and begins along a complex branched pathway starting with uroporphyrinogen III. The formation of adenosylcobalamin from uroporphyrinogen III has three distinct steps including 1) synthesis of the corrin ring, 2) construction of the nucleotide base (right-angle) ligand, and 3) the piecing together of the corrin ring with the base ligand to produce the final coenzyme (Roth, Lawrence, Rubenfield, Kieffer-Higgins, & Church, 1993). Both aerobic and anaerobic pathways begin with uroporphyrinogen III, with precorrin biosynthetic intermediates of cobalamin successively carrying methyl groups across varying numbered carbon units (i.e., precorrin 1, precorrin 2, precorrin 3A, etc.) (Battersby, 1994). However, the most significant difference between the two biosynthetic pathways is the point of cobalt insertion; aerobic production features a late insertion whereas anaerobic production is characterized by early incorporation (Warren et al., 2002). Metabolism of cobalamin in mammalian cells will be discussed later in this chapter. Food Sources and Daily Requirements As cobalamin is produced exclusively by certain bacteria, natural sources of its production are found in microorganisms from soil, sewage, water, human and other mammalian intestines, and animal rumens (first stomach of plant-eating mammals such as cattle, sheep, and goats). Thus, human dependency on cobalamin is rooted in dietary intake of the animals ingesting microbially synthesized vitamin B12. Human food sources rich in vitamin B12 are animal-based protein, including liver, meat, seafood, shellfish, eggs, and dairy products. 15 Vitamin B12 content for standard Western diets varies between 5-30 micrograms per day and fulfills average daily intake requirements of 7-8 micrograms per day for men, 4-5 micrograms per day for women, and 3-4 micrograms per day for children under age five (Beck, 2001). For adolescents, the recommended daily intake requirement for cobalamin is approximately 2 micrograms per day (The Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Subcomittee on Upper Reference Levels of Nutrients, Food and Nutrition Board, 1998). Intestinal microbes represent an additional nondietary source of vitamin B12 for humans, and account for absorption of 1- 5 micrograms per day (Heyssel, Bozian, Darby, & Bell, 1966). Obligatory losses of the nutrient have been established at a rate of 0.1% of the body's circulatory cobalamin pool per day, and occurs independently from other factors such as total body storage amounts in tissues and the liver (Heyssel et al., 1966). Physiologic Properties Absorption The normal absorption of vitamin B12 in mammals demonstrates both passive and active mechanisms (Rosenblatt & Fenton, 2001). Passive absorption accounts for 1- 2% of oral intake, occurs rapidly throughout the entire gastrointestinal tract, and is extremely inefficient (Green & Miller, 2007). Active absorption is dependent upon the coordinated actions of binding proteins that attach to ingested dietary protein in the stomach and intestine, and facilitate its entry into the plasma (Herrmann, Obeid, Schorr, & Geisel, 2003; Rosenblatt & Fenton, 2001). Age-related changes in humans related to these processes are discussed in a later section in this chapter. 16 Intrinsic Factor After food containing vitamin B12 enters the stomach, gastric parietal cells in the fundus and body of the stomach release pepsin and intrinsic factor (IF). In the acidic environment of the stomach, pepsin breaks down food/protein particles to release vitamin B12, which attaches to salivary R-binder proteins (haptocorrin family). Salivary R-binder/ B12 complexes then pass through the duodenum, where the pancreatic enzyme trypsin splits them apart. This frees vitamin B12 to bind to IF. Binding of vitamin B12 to IF is dependent on specific folding interactions with the 5,6-dimethylbenzimidazole base and the corrin ring, after which the complex shrinks to close around the cobalamin molecule (Lien, Ellenbogen, Law, & Wood, 1974). This binding process protects both IF and vitamin B12; free IF is particularly susceptible to rapid degradation by pancreatic enzymes, and similarly in acid, free vitamin B12 is susceptible to side chain modifications of the corrin ring and removal of axial ligands (Kondo et al., 1982). In mammals, all forms of vitamin B12 (methylcobalamin, adenosylcobalamin, cyanacobalamin) are absorbed by the IF-dependent mechanism. Cubulin and Megalin IF/B12 complexes travel the small intestine until they come into contact with IF receptors embedded on the outer surface of the terminal ileum, and are endocytosed. Ileal mucosal receptors preferentially accept IF/B12 over free IF, and binding takes place in villous cells as opposed to crypts (Kapadia & Essandoh, 1988; Mathan, Babior, & Donaldson, 1974). There are two structural subcomponents to the IF receptors-cubulin and megalin. Cubulin is a peripherally attached glycoprotein on the intestinal brush 17 border, and megalin is a large, endocytotic transmembrane glycoprotein (Alpers, 2005; Birn et al., 1997; Christensen & Birn, 2002). On the external border of the enterocyte, cubulin uses calcium ions to recognize and bind the IF/B12 complex (Barth & Argraves, 2001; Birn, 2006). Upon cubulin recognition and ligand binding of the IF/B12 complex, megalin facilitates entry of IF/B12 into the ileal enterocyte via receptor-mediated endocytosis (Green & Miller, 2007). Cubulin and megalin have broader physiologic functions than just the binding of IF/B12 complexes in the small intestine. Located in the plasma membranes and endocytoplasmic surfaces of cells across various types of epithelial tissue, cubulin and megalin colocalize for ligand binding in renal epithelium, visceral yolk sacs, and in the placental cytotrophoblast (Birn et al., 1997; Moestrup et al., 1998). Other ligands bound by the cubulin and megalin dual-receptor complex include albumin, vitamin-D binding protein, and hemoglobin (Birn et al., 2000; Cui, Verroust, Moestrup, & Christensen, 1996; Gburek et al., 2002; Nykjaer et al., 1999; Nykjaer et al., 2001). Categorized as part of the low-density lipoprotein family, megalin also binds ligands singularly (without cubulin) in a wider array of epithelial tissues. Present in lung alveoli, epididymis, endometrium, oviduct, inner ear, thryocytes, eye cilia, choroid plexus in cerebral ventricles, kidney, and parathyroid, megalin binds ligands, including retinol binding protein, lactoferrin, apolipoproteins (B, E, J, H), hormones, drugs, toxins, enzymes, immune, stress-response-related proteins, and B12 transcobalamin II complex during enterohepatic recirculation (Christensen & Birn, 2002). 18 Transport Transcobalamin II After an ileal enterocyte absorbs the IF/B12 complex, vitamin B12 is dissociated from IF in lysosomal compartments. It is then paired with the cobalamin transport protein transcobalamin II, released into the portal circulation, and carried to cells in target tissues throughout the body. After ingestion of food, the time required for absorption and binding to transcobalamin II is approximately 3-4 hours (Carkeet et al., 2006). Under normal physiologic conditions, transcobalamin II is 20-30% saturated, and represents 10- 20% of the total circulatory cobalamin pool (Refsum, Johnston, Guttormsen, & Nexo, 2006). Also called holotranscobalamin (holoTC), it is the biologically active form of vitamin B12 in the body and has a half-life of approximately 2 hours (Lindgren, Kilander, Bagge, & Nexo, 1999). Upon arrival at target cells, the B12/transcobalamin II binds to the transcobalamin II-receptor embedded in a cell's plasma membrane, and vitamin B12 is endocytosed into an intracellular lysosome (Christensen & Birn, 2002). Transcobalamin I (Haptocorrin) In contrast to transcobalamin II-mediated transport of cobalamin (10-20%), the remaining 80-90% of circulating B12 in blood is bound to plasma transcobalamin I (Carmel, 1985). Transcobalamin I has a second isoform known as transcobalamin III. Both are encoded by a single gene. Transcobalamin III contains the same protein core as transcobalamin I but is glycosylated differently. These sugar moieties change the biophysical properties of the molecule but do not significantly alter vitamin B12 binding. For the purposes of this work, transcobalamin I will be used to denote both isoforms. 19 Part of the haptocorrin family, transcobalamin I belongs to a group of cobalamin-binding glyoproteins that are also found in saliva and gastric mucosa (R-binders), (Russell-Jones & Alpers, 1999). In the blood, transcobalamin I does not facilitate direct B12 uptake from a receptor-mediated mechanism; it binds to an asialoglycoprotein receptor in the liver and transports released hepatic cobalamin stores, recycling some into bile. Hypothesized functions of transcobalamin I include the removal of cobalamin analogs from the bloodstream (Burger, Schneider, Mehlman, & Allen, 1975; Hardlei & Nexo, 2009). Vitamin B12 analogs bound to the transcobalamin I protein turn over extremely slowly and demonstrate a half-life of approximately 10 days (Finkler & Hall, 1967). There is significant unsaturated binding capacity and up to 47.1% of transcobalamin I in plasma can be free of cobalamin analogs (Beck, 2001). Transcobalamin I receptors are not ubiquitously expressed in tissues; thus, in the blood, transcobalamin I's role as an effective transport protein is unclear. Metabolism: Vitamin B12 Biochemistry Vitamin B12 is required in all mammalian cells for one-carbon metabolism and cellular mitosis (Refsum et al., 2006). It plays important roles in two essential reactions, one mitochondrial and the other cytoplasmic (Figure 1). In the mitochondria, vitamin B12 (5'-deoxyadenosylcobalamin) is required for the enzyme methylmalonyl CoA mutase, which catalyzes conversion of methylmalonyl CoA to succinyl CoA. This conversion is critical for odd-chain fatty acid oxidation and ketogenic amino acid catabolism (Green & Miller, 2007). 20 In the cytoplasm, vitamin B12 (methylcobalamin) is used in the conversion of homocysteine to methionine, and simultaneously overlaps with folate-dependent methylation and carbon exchange (Allen, Stabler, Savage, & Lindenbaum, 1993). Methionine is necessary for methylation, proper protein synthesis, and DNA formation. Methylcobalamin catalyzes a two-substrate two-product reaction. The conversion of homocysteine to methionine and folate-dependent reactions co-occur, the latter being the conversion of 5-methylenetetrahydrofolate (5-methyl THF) to tetrahydrofolate (THF). The end result of cobalamin-folate one-carbon metabolism mechanism is DNA precursor formation, deoxythymidine monophosphate (dTMP) (Beck, 2001). Recycling Mechanisms Storage Half of ingested B12 is delivered immediately to tissues by transcobalamin II while the other half is taken up by the liver (National Academy of Sciences, 1998). In healthy adults, total body cobalamin stores are between 2 and 4 milligrams, with total hepatic content between 1 and 1.5 milligrams (Grasbeck, 1959). The large majority of hepatic cobalamin stores (up to 70%) are comprised of adenosylcobalamin. Mobilization and liberation of vitamin B12 stores is hypothesized to occur via hepatic cell haptocorrin surface receptors (Burger et al., 1975). Reabsorption B12 undergoes enterohepatic recycling and between 0.5-9.0 micrograms per day is released into the gastrointestinal tract from biliary content (Grasbeck, 1959; Grasbeck, 21 Nyberg, & Reizenstein, 1958). Of the 0.5-9.0 micrograms, 65-70% is intestinally reabsorbed through the actions of intrinsic factor (Booth & Spray, 1960). Also classified as part of the enterohepatic circulatory process, vitamin B12 from sloughed intestinal epithelial cells is absorbed. It has been hypothesized that presence of bile may enhance cobalamin absorption from the intestine (Green, Jacobsen, Van Tonder, Kew, & Metz, 1982). Excretion Vitamin B12 bound to carrier proteins filters through renal glomeruli with tubular reabsorption to prevent excessive losses. In the renal tubular epithelium, colocalized cubulin and megalin absorb cobalamin/transcobalamin II via receptor-mediated endocytosis (Birn, 2006). Normal renal filtration uptake of the cobalamin/transcobalamin II complex is estimated at 1.5 micrograms (Lindemans, van Kapel, & Abels, 1986). Because very small amounts of vitamin B12 binding proteins are measurable in human urine, it is recognized that tubular reabsorption is very effective (Hall, 1964; Wahlstedt & Grasbeck, 1985). When circulating levels of the vitamin B12/transcobalamin II complex exceeds the rate-limited binding ability of megalin and cubulin, the excess is excreted into urine. Cobalamin Transport Genetics and Molecular Biology Individual Genes in Transport The cobalamin transport system is categorized into three main groups of ligands and receptors and includes intrinsic factor (IF) and the IF receptor, transcobalamin II and 22 the transcobalamin II-receptor, and haptocorrin and the haptocorrin receptor (Seetharam & Yammani, 2003). This section provides an overview of key genetic and molecular biology principles of the genes studied in this dissertation, transcobalamin II and the transcobalamin II-receptor. Also highlighted is the significance of the biologic overlap of transcobalamin II with the other cobalamin transporters, as demonstrated by shared exonic sequences, interspecies conservation, and amino-acid homology (Russell-Jones & Alpers, 1999). Transcobalamin II Gene The transcobalamin II gene, located at 22q12.2, has nine exons, eight introns, and a total length of 19,887 base pairs. The final protein product is non-glycosylated, comprises 427 amino acid residues, and yields a molecular mass of 43 kDa (Seetharam & Li, 2000). In humans, expression occurs across many different tissue types, but at varying levels. Li and associates (1994) reported a single 1.9kb 32P-labelled cDNA band present across heart, brain, placenta, lung, liver, muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and leukocyte tissue samples using Northern blot analysis (Li, Seetharam, Rosenblatt, & Seetharam, 1994). Quadros and associates (1989) identified a similar band, but analyses identified amino acid differences at three codon positions, leading to the hypothesis that there are multiple isoforms of transcobalamin II (Quadros, Rothenberg, & Jaffe, 1989). Kidney tissue expresses highest levels of transcobalamin II mRNA and compared to this kidney baseline (100%), heart and pancreas tissues were at 15%, placenta, lung, liver, muscle, prostate, and ovary tissues at 7-9%, and all remaining tissues were at between 2-5% of kidney mRNA 23 expression. Regulation of gene expression occurs through a promoter demonstrating features including a 1kb 5'-flanking region that is GC-rich, absence of a TATA start-transcription motif, and presence of multiple transcription start sites such as Sp1, CF1, HIP1, Ets-1, and MED-1 elements (Li, Seetharam, & Seetharam, 1995, 1998). Transcobalamin II expression occurs through binding to a TGGTCC (5'-3') hexameric sequence that is located 121bp upstream from the transcription start site (Regec, Quadros, & Rothenberg, 2002). Transcobalamin II-Receptor Gene The transcobalamin II-receptor gene, located at 19p13.3, has five exons, four introns, and is 6,229 base pairs in length. The final protein product is heavily glycosylated with a molecular mass of 58,000 atomic mass units (Quadros, Sai, & Rothenberg, 1994). Expression of the transcobalamin II-receptor gene has been identified in many human tissues. Bose and associates (1995) used immunoblotting experiments to ascertain presence of a noncovalent homodimerized 124 kDa protein band (Bose, Seetharam, & Seetharam, 1995). Quantitative evaluation showed expression in human kidney was the greatest (100%) followed by placenta at 28%, intestine at 18%, and liver at 2%. Subsequent monomeric purification of the 124 kDa fragment yielded a 62 kDa band bound to a phospholipid bilayer. Study of the purified 62 kDa fragment identified a single polypeptide with 27% carbohydrate content and four intermolecular disulfide bonds; these characteristics were thought to indicate that transcobalamin II-receptor's functional 24 importance extended beyond ligand binding to include Golgi-trafficking across plasma membranes (Bose & Seetharam, 1997). Despite these novel purification findings, they proved incomplete and irreproducible until 2005, when the binding and functional properties of transcobalamin II were described by Quadros and associates (Quadros, Nakayama, & Sequeira, 2005). Results differed significantly from previously published work, where a 58 kDa band was observed and demonstrated binding specificity lacking in previous reports. Full purification and definitive identification of transcobalamin II-receptor's primary structure and gene sequence was published in 2009 (Quadros, Nakayama, & Sequeira, 2009). Because transcobalamin II-receptor is highly glycosylated, the true size and the conformations of the attached sugars were difficult to identify and likely contributed to ambiguous results between various laboratory efforts. Quadros and associates (2009) used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate a single homogenous band of 58-60 kDa that was 252 amino acid residues in length. Comprising the 252-amino acid sequence is an extracellular domain of 199 residues, a transmembrane domain of 21 residues, and a cytoplasmic domain of 32 residues. Within the molecule are 18 residues that comprise two low-density lipoprotein receptor-class A domains, indicating that disulfide bonding is likely. Relationships Between Cobalamin Transport Genes Since cobalamin is a highly polar and complex molecule, it cannot easily cross plasma membranes in physiologically significant amounts. All higher animals, including humans, use hydrophobic cobalamin transport proteins to bind dietary vitamin B12, and 25 facilitate its movement from the stomach and intestine (haptocorrin R-binders and intrinsic factor) into the plasma and cells (transcobalamin II), and across circulatory storage pools (transcobalamin I in the haptocorrin family). Sequence Alignment Although proteins across the three groups are distinct and function through specific receptors, they share biologic similarity in their capacity to bind cobalamin with high affinity (Seetharam, 1999). This insight was first appreciated when Hoedemaker and associates described presence of IF in the stomachs of multiple species (Hoedemaeker, Abels, Wachters, Arends, & Nieweg, 1966). Several decades later, molecular biologic approaches significantly advanced this understanding. Through techniques such as cloning, genetic sequence from multiple organisms was compared in genes across the three groups. A 1988 analysis of genetic sequence (cDNA) for rat IF determined primary structural domains for cobalamin binding and outlined preliminary homologies to other biochemical proteins (Dieckgraefe, Seetharam, Banaszak, Leykam, & Alpers, 1988). Comparison of these genetic rat IF features to human transcobalamin II and transcobalamin I (haptocorrin) sequences yielded identification of homologous regions across the groups; transcobalamin II had 20% amino acid identity with both human transcobalamin I and rat IF (Platica et al., 1991). Simultaneous progress in porcine model systems produced additional alignments between rat IF, porcine haptocorrin, and human transcobalamin I (Hewitt, Seetharam, Leykam, & Alpers, 1990; Johnston, Bollekens, Allen, & Berliner, 1989). Collectively from these studies, overall protein alignment was 26 at 33% and found within them were six regions of high structural sharing, at 60-90% similarity in amino acid sequence. The most highly conserved of these six regions extends across 15 residues, from position 174 to 188 (174SVDTAAMAGLAFTCL188). Ancestral Origins The overlap across species for the cobalamin binding protein genes is significant because it indicated a likely common ancestral gene of evolutionary origin. Recent mammalian (rat, mouse, porcine, bovine, human) phylogenetic analysis of the highly conserved 15-amino acid residue showed an evolutionary relationship suggesting that transcobalamin II evolved earlier than other cobalamin binding proteins (Kalra, Li, Yammani, Seetharam, & Seetharam, 2004). The radial tree that was generated from this work pictured a relationship where transcobalamin II evolved independently and earlier than that of IF and haptocorrin, which co-evolved in a more dependent manner. The analysis was also significant for the convergence of mouse and rat transcobalamin II, with 95-98% sequence homology compared to 71-74% for human and bovine counterparts. Pathophysiology of Cobalamin Deficiency Since total-body stores of cobalamin are between 2 and 5 milligrams and daily requirements are at several micrograms per day, abrupt cessation of cobalamin dietary intake does not yield immediately clinically observable effects. It can take many years for classical cobalamin deficiency clinical symptoms to appear as tissues slowly release needed minute daily requirements. There are numerous causes of clinical cobalamin 27 deficiency that result from 1) insufficient intake from poor diet or mal-absorptive pathology, 2) increased metabolic needs (i.e., pregnancy), and 3) impaired use or vitamin activation in tissues such as that which occurs in genetic inborn errors of metabolism. The majority of documented clinical deficiencies arise from the first category stemming from varying states of impaired gastrointestinal absorption. Traditional and common clinical definitions of the cobalamin deficiency include 1) presence of megaloblastic anemia and/or neuropsychiatric alterations that respond to supplemental cobalamin therapy, or 2) decreased total serum cobalamin with or without altered biochemical metabolites (Miller et al., 2006; Savage, Lindenbaum, Stabler, & Allen, 1994). Megaloblastic Anemia When there is decreased availability of dTMP coming from conversion of homocysteine to methionine, deoxyuridine monophosphate (dUMP) is erroneously incorporated into DNA. DNA repair enzymes subsequently recognize this misincorporated uracil and cleave out the base. Incomplete DNA repair leads to frequent gaps and breaks in DNA sequence (Goulian, Bleile, & Tseng, 1980). Improper DNA synthesis results in cellular derangement and premature cell death. Although required in all cells, vitamin B12 is needed most by dividing cells, such as those in the bone marrow. In absence of adequate dTMP, all hematopoietic precursors undergo abnormal DNA synthesis and yield delayed or halted cell division events observable via variable cell morphologies (Aster, 2005). As a result, up to 90% of an affected patient's red blood cell 28 precursors may be destroyed prior to their release into the blood, compared to 10-15% under normal circumstances (Babior & Franklin Bunn, 2005). Destruction of red blood cell precursors on this scale will result in decreased hemoglobin concentration and elevated mean corpuscular volume (MCV); presence of both are classic hallmark hematologic indicators of megaloblastic changes (Andres et al., 2006). Normal hemoglobin values in adult women according to the World Health Organization are 12-16 g/dL, and levels under 12 g/dL are considered indicative of anemia (Aster, 2005; Chaves, Ashar, Guralnik, & Fried, 2002). MCV is used in anemia classification discerning microcytic (MCV below 83 fL), normocytic (MCV 83 to 103 fL), and macrocytic (MCV greater than 103 fL) categories (Williamson et al., 1995). Thus, macrocytic anemia is characterized by a low red blood cell count, decreased hemoglobin concentration, and abnormally large (macrocytic) red blood cells, and indicates presence of cobalamin or folate deficiency (Aster, 2005). Cobalamin's overlap with folate will be discussed later in this section. In cobalamin deficiency, rapidly dividing granulocytic precursors demonstrate nuclear immaturity while protein assembly continues normally. Although cytoplasmic features are intact, nuclei demonstrate visible chromatin clumping, become abnormally large for the cell's size, and develop numerous, hyper-segmented granules (Chui et al., 2001). Malformed platelets, thrombocytopenia, hypercellular bone marrow, and decreased white blood cell counts can also occur (Allen et al., 1993). Accompanying these cellular changes, symptoms commonly reported by patients include vertigo, light-headedness, palpitations, and chest pain. Physical examination may yield pallor, jaundice (secondary to red blood cell destruction), rapid heart rate, evidence 29 of cardiomegaly, and a holosystolic murmur. Insidiously, anemia impacts a person's perception and understanding of their own health, resulting in feelings of fatigue, decreased strength, and a poor sense of well-being (Eisenstaedt, Penninx, & Woodman, 2006; Guralnik, Eisenstaedt, Ferrucci, Klein, & Woodman, 2004; Penninx et al., 2004; Woodman, Ferrucci, & Guralnik, 2005). Life threatening hematological changes can also occur, including symptomatic pancytopenia, pseudo-thrombotic microangiopathy, and hemolytic anemia (Andres et al., 2006). Neurologic Changes Although anemic symptoms usually occur first in cobalamin deficiency, neurological symptoms can present prior to, in concordance with, or separately from hematological alterations (Carmel, 2000; Carmel, Green, Rosenblatt, & Watkins, 2003; Lindenbaum et al., 1988). The central nervous system and neural cells are dependent upon continuous supply of nutrients (Selhub, Bagley, Miller, & Rosenberg, 2000). For example, myelin sheaths experience frequent turnover and are dependent upon methylations of precursor proteins and essential fatty acid oxidations (Scott, 1999). When altered, the primary feature of neurologic pathology resulting from decreased cobalamin is demyelination that affects both central and peripheral neurons (Green & Kinsella, 1995). There are two hypotheses on the pathophysiology that leads to demyelination: 1) in the absence of cobalamin, there is decreased synthesis of the methyl group donor S-adenosylmethionine, which prevents precursor myelin basic protein from being formed, (the S-adenosylmethionine hypothesis); and 2) in the absence of cobalamin, 30 mitochondrial precursor methylmalonyl CoA accumulates, becomes toxic, and disrupts odd-chain fatty acid metabolism in neurons, (the adenosylcobalamin hypothesis). The first of these hypotheses, the S-adenosylmethionine hypothesis, is favored to the second, adenosylcobalamin hypothesis. The S-Adenosylmethionine Hypothesis In the cytoplasm, methylcobalamin serves as a cofactor for the conversion of homocysteine to methionine. This enzymatic conversion feeds two cycles, the methylation cycle and the DNA replication cycle (Figure 2), (Scott, 1999). The universal methyl donor S-adenosylmethionine donates a methyl group to S-adenosylhomocysteine for which to use in methylation of proteins, DNA, lipids, and other needed substrates (Dinn et al., 1980). Without proper methylation, required substrates are not produced properly, including neurotransmitters, membrane phospholipids, and precursor proteins used in nerve conduction, such as myelin basic protein (Metz, 1992). Multiple nervous system components become affected by these derangements, including long tracts of white matter in the posterior and lateral columns of the spinal cord, sensory fibers responsible for vibration sensitivity and position sense, and motor fibers controlling movement. The Adenosylcobalamin Hypothesis The alternative hypothesis to neurodegenerative changes observed in cobalamin deficiency postulates that neurologic symptoms arise from insufficient conversion of methylmalonyl CoA to succinyl CoA in the mitochondria. There is evidence that suggests 31 that accumulated methylmalonic acid disrupts odd-chain fatty acid metabolism, leading to neurological damage (Frenkel, 1973). Although there are numerous studies that identify quantitative abnormalities of odd-chain and branched-chain fatty acids in vitamin B12-deficient spinal cord and peripheral nerve tissue, there is not a clear relationship between these measurements and clinical development of cobalamin neuropathy (Kishimoto, Williams, Moser, Hignite, & Biermann, 1973; Levy, Mudd, Schulman, Dreyfus, & Abeles, 1970; Ramsey, Scott, & Banik, 1977). Further evidence against the adenosylcobalamin hypothesis comes from children with inherited disorders yielding high methylmalonic acid levels; despite having extraordinarily high levels, the patients' neurologic features are inclusive of mental retardation and muscular hypotonia and not those of cobalamin deficiency neuropathy (Rosenblatt & Cooper, 1987). Clinical Progression and Effects When there is insufficient methylation for normal neurologic homeostasis, small vacuoles in the myelin sheath result in focal swelling of individual neuronal fibers (Pant, Asbury, & Richardson, 1968). The focal swellings expand in scope to develop larger foci; beginning at the cervicothoracic junction of the spinal cord, posterior columns are usually the first to be affected before spreading up and down the cord and into anterior segments. On magnetic resonant imaging, increased T2-weighted signal, decreased T1-weighted signal, and contrast enhancement of the posterior and lateral spinal cord columns in cervical and upper thoracic segments are observed (Locatelli, Laureno, Ballard, & Mark, 1999). 32 Clinical presentation of the focal neuronal swelling is initially mild and measurable only by electrophysiological methods (Carmel & Sarrai, 2006). Neuropsychiatric symptoms, including parasthesias, ataxia, memory loss, mood alterations, and extremity weakness, are common initial presentations. If untreated, these symptoms become more severe and progress to numbness and tingling of extremities, clonus, weakness, spasticity of extremities, ataxia, abnormal reflexes, and gait and visual disturbances (Babior & Franklin Bunn, 2005). Underlying these worsening symptoms is progressive demyelination that affects peripheral nerves, posterior and lateral columns of the spinal cord, and the cerebrum (Allen et al., 1993). If continuing uncorrected, permanent pathology such as axonal degeneration and neuronal death occur (Babior & Franklin Bunn, 2005). Cerebellar involvement is inclusive of urinary and/or fecal incontinence, and cranial nerve decompensation, including visual disturbances and optic neuritis (Allen et al., 1993). Decreases in cognitive function, development of dementia, personality changes, and occurrence of depression and Parkinsonian symptoms have all been documented as part of the clinical neurologic vitamin B12 deficiency profile (Carmel, 2000; Carmel & Sarrai, 2006; Clarke et al., 2003). Metabolite Abnormalities Prior to development of hallmark anemic or neurologic symptoms, metabolite assays are a valuable tool in providing an indication of a patient's vitamin B12 status. In deficiency states, the cobalamin metabolic reaction precursors methylmalonic acid (mitochondrial indicator) and homocysteine (cytosolic indicator) are not metabolized and 33 accumulate in cells and tissues (Lindgren et al., 1999). Increased serum methylmalonic acid and total homocysteine correlate with hematological and neurologic symptoms of clinical vitamin B12 deficiency and decrease responsively with supplemental cobalamin therapy (Henning, Tepel, Riezler, & Naurath, 2001; Naurath et al., 1995; Rajan et al., 2002). In clinical practice, normal vitamin B12 metabolic profiles include serum cobalamin at >258 pmol/liter, total homocysteine at 5.4- methylmalonic acid (MMA) at <280 nmol/liter (Carmel, 2000; Carmel et al., 2003; Carmel & Sarrai, 2006). Clinical vitamin B12 deficiency is diagnosed when serum !!" nmol/liter. Less restrictive parameters are used for ascertaining presence of subclinical vitamin B12 deficiency, which is present when serum cobalamin is 185-258 pmol/liter, total homocysteine is 15-# !!" # -999 nmol/liter. Altered metabolite levels in subclinical deficiency can be present even if hemoglobin concentration and MCV are normal. In addition to reflecting altered vitamin B12 status, abnormal metabolite concentrations have been hypothesized to affect risk for development of comorbid pathology, such as homocysteine and cardiac disease. For some time, elevated serum homocysteine was recognized as a risk factor for the occurrence of cardiovascular disease and thrombosis (Refsum, Ueland, Nygard, & Vollset, 1998). However this role is no longer clear, as a recent randomized evaluation of folic acid and vitamin B12 versus placebo on blood homocysteine failed to demonstrate beneficial effects in preventing myocardial infarction (Study of the Effectiveness of Additional Reductions in Cholesterol and Homocysteine Collaborative Group, 2010). 34 Elevated methylmalonic acid and homocysteine concentrations occur in over 90% of patients with vitamin B12 deficiency, are increased prior to development of overt clinical symptoms, and often accompany normal serum cobalamin levels (Herrmann et al., 2003; Stabler, Allen, Savage, & Lindenbaum, 1990). Both methylmalonic acid and homocysteine concentrations can be altered in other disease states, such as inborn errors of metabolism, folate deficiency, and renal insufficiency (Allen, Lindenbaum, & Stabler, 1996; Bostom & Lathrop, 1997; Herrmann et al., 2000; Manns et al., 1999; Metz et al., 1996; D. S. Rosenblatt & Cooper, 1987). Serum homocysteine can be elevated in folic acid deficiencies, but methylmalonic acid elevations are specific to cobalamin deficiency (Stabler, Lindenbaum, & Allen, 1997). To better define subtle features of early clinical development of cobalamin deficiency, four stages of progressive metabolite alterations and clinical characteristics have been suggested: (1 and 2) depletion of plasma and cell stores; (3) incidence of functional imbalances as measured by decreased biologically active cobalamin in serum, increased homocysteine and/or methylmalonic acid; and (4) appearance of megaloblastic and neuropsychiatric clinical symptoms (Herrmann et al., 2003). Measurement Challenges Even in individuals with known cobalamin deficiency, normal circulating levels of vitamin B12 can be maintained at the cost of tissues for several years (Lindenbaum, Savage, Stabler, & Allen, 1990). Total serum cobalamin demonstrates poor sensitivity and specificity for ascertainment of when the body is "low" in cobalamin from a tissue perspective (Miller et al., 2006). Serum holotranscobalamin, the biologically active 35 cobalamin in serum that is bound to transcobalamin II, is recognized for providing improved detection of cobalamin status in individuals (Green, 2008; Lindgren et al., 1999). Although more precise measurement alternatives such as holotranscobalamin can provide clinicians better proof of deficiency, it is generally accepted that interpreting multiple testing analytes with clinical presentation symptoms yields accurate detection (Green & Miller, 2007; Herrmann et al., 2003; Herrmann et al., 2000; Obeid, Schorr, Eckert, & Herrmann, 2004). Measurement of cobalamin metabolites can be challenging, as sophisticated techniques require specialized equipment and training. This is primarily because methylmalonic acid and homocysteine levels accumulating in cobalamin deficiency states are typically in the nanomolar to micromolar range, and accurately detected by gas chromatography-mass spectrometry (GC-MS) and high-pressure liquid chromatography (HPLC) in both serum and urine (Green, 1995; Miller et al., 2006). Due to their cost, many laboratories cannot afford the technology required for these specialized assays. For laboratories that do perform them, accurate interpretation can be affected by absence of standardized laboratory procedures, assay reagents, and testing approaches. For serum cobalamin, concentrations reported by laboratories often vary from those used in international measurement standards, contributing to clinician misinterpretation. Coupled with absence of consensus on what is considered ‘elevated' or ‘decreased' due to poor correlation with actual tissue level-clinicians can erroneously underestimate the complexity of commonly used cobalamin laboratory tests (Carmel et al., 2003). 36 Cobalamin Overlap with Folate and the Methyl-Folate Trap As mentioned, megaloblastic anemia and elevated homocysteine clinical features can also be caused by decreased folate. This is because cobalamin metabolism overlaps with folate metabolism in the conversion of homocysteine to methionine (Figure 3). In the absence of adequate cobalamin, an intracellular backlog of 5- methylenetetrahydrofolate occurs. Since the 5,10-methylenetetrahydrofolate enzyme strongly favors one direction, it does not go backwards in the pathway; thus, the cell has plenty of folate but it is "trapped" in an unusable form for DNA synthesis (Scott & Weir, 1981; Scott & Weir, 1994). The resulting clinical presentation is identical to that of megaloblastic anemia caused by true deficiency of folate. Synthetic folate (folic acid) enters downstream of the methionine synthase conversion, and as it is converted to tetrahydrofolate from dihydrofolate, can rescue DNA synthesis in cobalamin deficiency. However, it does not resolve elevated homocysteine levels, as cobalamin is required for conversion of homocysteine to methionine. If a well-meaning health care provider aims to treat megaloblastic anemia with folic acid without fully reviewing metabolic parameters, the patient experiences resolution of megaloblastic anemia as cobalamin deficiency continues, masked by this treatment (Pfeiffer, Caudill, Gunter, Osterloh, & Sampson, 2005). In such cases, it is not until progression of neurologic symptoms becomes significantly severe that cobalamin deficiency is suspected and more detailed metabolite profiles are checked. In the advent of widespread folate fortification, research into vitamin B12 and folate status has identified that low-cobalamin levels exist with high folate levels, and can compound risk of neurologic decline (Morris et al., 2005; Selhub, Morris, & Jacques, 2007). 37 Gerontologic Research Factors Cobalamin Deficiency Nutritional deficiencies in the elderly population are well characterized as functions of expected age-related physiological changes (Saltzman & Russell, 1998). At worst, 20-50% of the aging population experience some form of vitamin B12 deficit (Lindenbaum, Rosenberg, Wilson, Stabler, & Allen, 1994; Selhub et al., 2000; Stabler et al., 1997). Pernicious anemia (Type A atrophic gastritis) is the end-stage presentation of autoimmune gastritis and is the primary cause of frankly overt clinical vitamin B12 deficiency in North American populations (Baik & Russell, 1999). For every elderly adult female diagnosed with pernicious anemia, approximately five males are affected, and Caucasian and African American elderly adults shoulder increased prevalence of pernicious anemia compared to other racial demographic groups (Baik & Russell, 1999). Antibodies that attack H+/K+ ATPase pumps progressively destroy parietal cells, causing continued decline of vitamin B12 absorption and extraction from protein (Morris, Jacques, Rosenberg, & Selhub, 2007). In contrast, Type B atrophic gastritis is a naturally occurring phenomenon of aging, stemming from decreased secretion of stomach acid, pepsin, and intrinsic factor (Saltzman & Russell, 1998). Concomitant with achlorydia stemming from all forms of gastric atrophy, microorganism overgrowth fostered by decreased gastric acid production can competitively consume ingested vitamin B12 in aging individuals (Suter, Golner, Goldin, Morrow, & Russell, 1991). Although more rare in elderly populations, poor diet, decreased dietary intake of foods containing vitamin B12, and strict vegetarianism can also result in dietary deficiency for aging adults. 38 Additional causative factors include side effects from medications and comorbid conditions that increase nutritional need for vitamin B12. Medications interfering with vitamin B12 absorption commonly prescribed in elderly individuals include antiepileptic agents, proton pump inhibitors, histamine receptor antagonists, the antidiabetic drug metformin, antibiotics, and cholestyramine (Wolters, Strohle, & Hahn, 2004). Comorbid conditions contributing to malabsorption or increasing nutritional requirements include intestinal diseases (Crohn's), gastric or ileal resections, alcohol intake, smoking, renal insufficiency, diabetes mellitus, and lymphoma (Wolters et al., 2004). Subclinical Cobalamin Deficiency Subclinical B12 deficiency is a burden largely shouldered by older adults because symptoms indicating its presence are often interpreted by clinicians to be associated with other disease states or nonspecific effects of aging (Andres et al., 2004). The subtle nature of weakness, fatigue, headache, depression, shortness of breath, malaise, vertigo, early dementia, and sensory parasthesias is common in aging patients and often improperly attributed to other causative mechanisms (Baik & Russell, 1999; Clarke et al., 2003). Unrecognized deficiency or misattribution of symptoms to folate increases chance of progression to irreversible neurological changes. For elderly adults, permanent neurological changes commonly experienced by the elderly stemming from unrecognized cobalamin deficiencies include taste alterations, memory loss, parasthesias, spinal cord subacute degeneration, neuropathy, gait ataxia, dementia, anosmia, incontinence, impotence, decreased visual acuity, and psychosis (Baik & Russell, 1999; Carmel, 2000). For elderly individuals, the best primary defense against permanent neuropsychiatric 39 injury is maintaining a healthy awareness for subclinical cobalamin deficiency in the event of nonspecific symptom presentations. Although measuring cobalamin metabolite levels helps greatly in ascertaining presence of deficiency in older adults, several clinical factors are known to affect accurate detection. Similar to trends in other age groups, erroneous attribution of megaloblastic anemia to folate in the absence of a full metabolic workup can obscure diagnosis (Allen & Casterline, 1994; Pfeiffer et al., 2005). Common in elderly individuals, renal insufficiency is known to cause increases in both homocysteine and methylmalonic acid (Herrmann, Obeid, Schorr, & Geisel, 2005; Herrmann, Schorr, Geisel, & Riegel, 2001; Obeid, Kuhlmann, Kirsch, & Herrmann, 2005). Molecular etiology for accumulations of homocysteine and methylmalonic acid stemming from renal dysfunction are largely unknown. However, elevations in either serum homocysteine or methylmalonic acid from renal insufficiency are modest compared to those that occur in true cobalamin deficiency states (Savage et al., 1994; Stabler, Lindenbaum, & Allen, 1996). Renal Function Assessment In clinical practice, serum creatinine is a common measure of renal function and is the most widely used method of assessing an individual's renal status. However, it is recognized that relying on serum creatinine measurement alone is limiting, as it does not correlate with actual physiologic glomerular filtration rates (Shemesh, Golbetz, Kriss, & Myers, 1985; Stevens, Coresh, Greene, & Levey, 2006). In older adults, renal function slowly declines and tubular secretion of creatinine increases; thus, elevations in serum 40 creatinine are not seen until more than half of total glomerular filtration is lost (Giannelli et al., 2007). In addition, decreased muscle mass in older adults is associated with decreased creatinine production. If kidney problems in older adults are not diagnosed, renal disease can progress and result in additional costly comorbidities, clinical complications, and drug toxicities. In a recent study of 660 elderly adults with normal serum creatinine values, alternative renal-estimation equations identified that up to 39% of them could be classified as having renal impairment (Giannelli et al., 2007). A common measure of renal-function estimation used in elderly individuals that employs age, weight, and serum creatinine, is the Cockcroft-Gault estimation equation (Cockcroft & Gault, 1976). For females, the Cockcroft-Gault formula incorporates a constant of 0.85 to account for 15% less muscle mass as compared to males. The Cockcroft-Gault has been used in many studies of older adults, and although reported to underestimate creatinine clearance, is considered by many experts a more valid and reliable method than if using serum creatinine alone (Froissart, Rossert, Jacquot, Paillard, & Houillier, 2005; Lamb et al., 2005). Role of Cobalamin in Cognition There is a significant and storied history of the suspected role of cobalamin in cognition, with much clinician speculation focused on delineating cause and effect between altered homocysteine levels and dementia in Alzheimer's disease (McCaddon, 2006). However, the concept that deficits in nutritional status would precipitate neuropsychiatric decline was not fully acknowledged until 1990, when it was reported that low-normal concentrations of cobalamin were associated with cognitive impairment 41 (Bell et al., 1990). The chart review by Bell et al. of 102 geriatric inpatients found that the 3.7% of patients who were vitamin B12 deficient were more likely to have significantly lower Mini-Mental State Examination scores. Shortly later, Rosenberg and Miller's review of dietary factors and neuro-cognitive health concluded that elderly individuals were extremely susceptible to cognitive decline from subtle and progressive forms of subclinical nutritive deficiencies (Rosenberg & Miller, 1992). An unintended consequence of this position was the assumption that vitamin B12 indeed contributed to pathologic progression of cognitive decline in elderly patients, without there being presence of strong prospective controlled clinical evidence to confirm it. Since that time, numerous research studies have reported variable efficacy of vitamin B12 on the protection and maintenance of cognitive function (Smith & Refsum, 2009). Total homocysteine and cognitive measures were inversely related in 2,096 elderly adult dementia and stroke-free participants (>60 years) of the Framingham Offspring Study (Elias et al., 2005). A 3-year prospective evaluation of the association between dietary B-vitamin intake and cognitive decline in a subset of 321 men in the Veterans Affairs Normative Aging Study revealed that the association of poor cognitive status with low vitamin B12 and high homocysteine was only applicable to performance on a construction praxis spatial copying score (Tucker, Qiao, Scott, Rosenberg, & Spiro, 2005). A 6-year prospective observational study of 3,718 community-dwelling elderly individuals over 65 indicated that high dietary B12 intake was associated with slower cognitive decline, but only in the oldest individuals (Morris et al., 2005). Authors of a recent Cochrane Collaboration meta-analysis concluded that there is no evidence that folic acid with or without vitamin B12 improves cognitive function of elderly individuals 42 with or without dementia; however, long-term supplementation may be protective for healthy elders with high homocysteine levels (Malouf & Grimley Evans, 2008). This is likely due to the great clinical and research heterogeneity across studies in the literature. Variability in cognitive assessment tools, clinical outcome parameters, quasi-experimental study designs, laboratory metabolite measurements, and deficiency threshold cutoffs contribute to poor-quality evidence from which to draw clinical conclusions (Raman et al., 2007). The Dissertation's Clinical Measurements of Altered Cobalamin Status The dissertation research uses multiple measures of cobalamin status for elderly adult women subjects. Measures previously discussed as related to pathophysiology, diagnosis, and clinical management of vitamin B12 deficiency include hemoglobin, mean corpuscular volume, serum cobalamin, and the biochemical metabolites homocysteine and methylmalonic acid. However, the dissertation also explores relationships to other clinical outcomes, including depression, peripheral neuropathy, and functional decreases in strength and speed of ambulation. Depression Patients experiencing alterations in mood, primarily depression, are commonly reported to also have abnormal cobalamin and folate laboratory values (Bell et al., 1990; Carney et al., 1990; Lindenbaum et al., 1988; Savage & Lindenbaum, 1995). Previous work in the Women's Health and Aging Study found a twofold increase in risk of severe 43 depression in patients who experienced clinical cobalamin deficiency as measured by serum cobalamin, homocysteine, and methylmalonic acid levels (Penninx et al., 2000). A longitudinal association study of depressive symptoms in 3,503 older adults over 7 years identified that for each 10 additional micrograms of vitamin B12 intake, there was a 2% lower odds of depressive symptoms per year (Skarupski, Tangeny, Li, Ouyang, Evans & Morris, 2010). Although the association between vitamin B12 intake and depression in older adults is reported in multiple observation studies, causation of its presence has not been able to be determined. Hypothesized molecular origins of the connection may stem from insufficient cobalamin and folate to drive the conversion of homocysteine to methionine, preventing methylation of key neurotransmitters in the brain, including the monoamines dopamine and serotonin (Bottiglieri et al., 2000; Weir & Scott, 1999). However, many behavioral factors that accompany depressive symptomology in aging individuals, such as decreased appetite and socioeconomic status, likely contribute to this association as well (Donini, Savina, & Cannella, 2003). Depression is a challenging phenomenon to measure; it displays lack of agreement between clinicians and researchers on the concepts that comprise it, and classification mechanisms across numerous settings and groups vary widely according to patient subpopulation and clinical care specialty (Pasacreta, 2004). Depression contains both affective and somatic components, which results in significant overlap with comorbid symptomology (i.e., fatigue, decreased appetite) obscuring accurate research measurement. Common to elderly populations, dementia is a common confounder where 44 psychomotor retardation and passive responses to examiner questions are misinterpreted as depressive pathology. The Geriatric Depression Scale (GDS) was derived for use in geriatric populations so that somatic depression manifestations and dementia presence do not threaten validity of accurate detection (Feher, Larrabee, & Crook, 1992; Yesavage et al., 1982). Comprised of 30 items requiring a yes/no answer, higher GDS scores indicate depression severity. Generally accepted cutoffs are no depression (scores less than or equal to 9), mild depression (scores 10-13), and severe depression (scores greater than or equal to 14) (Ferrucci, Kittner, Corti, & Guralnik, 1995; Lyness et al., 1997; Norris, Gallagher, Wilson, & Winograd, 1987). Criterion validity using psychiatric evaluation of DSM III diagnostic standards for mild depression on GDS was 89%, and for severe symptoms was 78%. Specificity values were similar at 73% for mild depression and 86% for severe depression (Norris et al., 1987). Although GDS measurements are reliable and valid in older persons with respect to research diagnostic criteria and DSM-IV criteria, presence of mild or severe depression using the screening scale is not necessarily indicative of a clinical diagnosis. Peripheral Neuropathy Abnormal sensation is a widely reported indicator of presence or development of vitamin B12 deficiency pathology (Carmel et al., 2003). Abnormal sensation is also considered a key subtle clinical manifestation indicative of subclinical vitamin B12 deficiency (Carmel & Sarrai, 2006). As discussed in previous pathophysiology sections, aberrated methylation for myelin basic protein precursors from altered conversion of 45 homocysteine to methionine disrupts neuronal signaling. The neurologic syndrome of cobalamin deficiency commonly starts with peripheral paresthesias in the feet before progressing to more severe pathology (Beck, 2001; Green & Miller, 2007). In a study of neurologic aspects of cobalamin deficiency in 369 cobalamin deficient patients, it was found that paresthesia was the most common neurologic finding at the time of diagnosis on physical examination (Healton, Savage, Brust, Garrett, & Lindenbaum, 1991). Comprising 87.7% of the paresthesia symptom profiles, the most common abnormality was found to be significant diminishment of vibration sensitivity in the feet, or feet and legs up to the knees. Less commonly, diminished abnormality also extended up from the feet to include the iliac crest, lower thoracic area, midthoracic area, hands and elbows, and shoulders. Alterations in vibration sensitivity stemming from large-fiber peripheral nerve function provide an objective measurement for declining nerve function (Ferrucci, Kittner, et al., 1995). Vibratory perception sensory thresholds in older adults are reproducible and reliable indicators of polyneuropathy (de Neeling, Beks, Bertelsmann, Heine, & Bouter, 1994). Vibration perception testing (VPT) protocols involve serial application of quantified vibration and assessing if subjects are able to feel it or not. Depending upon a patient's response, the strength of stimulus is incrementally adjusted until an individual can no longer sense vibration. VPT can be administered via electronic or mechanical clinical measurement tools, such as vibrometers and tuning forks. Although reliability is well established for varying measurement modalities individually, measurement methods are not interchangeable due to slight physiological differences in how sensory neurons transmit mechanical stimuli (tuning fork) as compared to energy- 46 based stimuli (voltage or current) (Temlett, 2009). Key advantages to using voltage- and current-based modalities include the removal of variability coming from inconsistent techniques of generating and applying the tuning fork's blade intensity. VPT in the Women's Health and Aging Studies (WHAS) 1 and 2 were assessed using different techniques, WHAS 1 with a vibrometer modality and WHAS 2 with a tuning fork. In WHAS 1, the VPT used was modeled after a diabetic neuropathy protocol, where vibration measures are considered valid if 18 or less stimulation attempts are made, and not more than one error occurs in the first eight attempts (Ferrucci, Kittner, et al., 1995; Maser et al., 1989). The amplitude of vibration stimulus is converted to a micron unit measurement, with higher values indicating that a stronger stimulus was required to elicit a correct sensory response from the patient (Resnick et al., 2000; Volpato et al., 2003). Accepted neuropathic functional micron unit cutoffs include normal function at less than 3.43 units, mild dysfunction at 3.44-4.87 units, moderate dysfunction at 4.88-6.31 units, and severe dysfunction at over 6.31 units (Resnick, Vinik, Heimovitz, Brancati, & Guralnik, 2001; Volpato, Leveille, Blaum, Fried, & Guralnik, 2005). Functional Indicators As reviewed in earlier sections, 5' adenosylcobalamin serves as the coenzyme for methylmalonyl-coenzyme A mutase, which facilitates conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A in the mitochondria. This conversion is necessary for catabolism of odd-chain fatty acids and some amino acids, and results in maintenance of normal energy metabolism. 47 In the absence of this conversion, methylmalonyl-coenzyme A and its precursor, propionic acid, accumulates and diffuses out of the mitochondria into the cytoplasm and disrupts normal metabolism (Brass, Tahiliani, Allen, & Stabler, 1990). The cellular accumulation of propionic acid and methylmalonic acid (acyl-coenzyme A thioesters) has been shown to inhibit gluconeogenesis from pyruvate, pyruvate oxidation, fatty acid oxidation, and ureogenesis (Brass, 1986; Glasgow & Chase, 1976; Walajtys-Rode, Coll, & Williamson, 1979; Walajtys-Rode & Williamson, 1980). In cobalamin deficiency, the combined effects of deranged energy production processes in addition to accumulated methylmalonic, propionic acids, disrupts energy homeostasis and impairs the action of multiple critical enzymes (Depeint, Bruce, Shangari, Mehta, & O'Brien, 2006; Kolker & Okun, 2005). The clinical effects of these metabolic impairments can be severe, such as that which is demonstrated by individuals diagnosed with methylmalonic acidurias. As a diagnostic class, methylmalonic acidurias are a group of autosomal recessive genetic disorders that offer valuable biologic insight to the broader mechanisms of cobalamin deficiency. Methylmalonic acidurias are caused by autosomal recessive mutations in genes that code for methylmalonyl-coenzyme A mutase or the cobalamin complementation groups, which synthesize 5' adenosylcobalamin for use in the mitochondria (Chandler et al., 2007; Coelho et al., 2008; Rosenblatt & Fenton, 2001). Inherited methylmalonic acidurias produce severe clinical symptoms that are often fatal. Because of their severity, clinical study of organ-system derangements from drastically excessive methylmalonic acid levels is limited to animal studies, observations of affected newborn infants, postmortem analyses, and children with less severe defects who have matured into adolescence and young adulthood. Common symptoms experienced by 48 individuals with methylmalonic acidurias include lactic acidosis, decreased muscle strength, muscular hypotonia, lethargy, and failure to thrive (Coelho et al., 2008; Rosenblatt & Cooper, 1987; Tanpaiboon, 2005). In a recent characterization of the methylmalonic aciduria phenotype, investigators examined correlations of murine and human disease characteristics and found that altered metabolism in skeletal muscle was a significant source of pathology (Chandler et al., 2007). Although the autosomal recessive methylmalonic acidurias do not present in older adults and thus cannot be generalized to the geriatric population, they may offer biologic insight into concomitant metabolic processes when methylmalonic acid concentrations are significantly elevated. For example, methylmalonic acidurias in neonates and children produce drastically elevated methylmalonic acid levels at approximately 1000 nmol/L, a level which is similar to that of severe cobalamin deficiency in older adults. Although these metabolic concentrations may be similar, a key limitation in understanding the relationship between elevated methylmalonic acid concentration and poor functional status in elderly individuals is that it is relatively unexplored in the area of cobalamin metabolism. Available data is limited to several separate investigations of neuromuscular effects of cobalamin deficiency and its possible genetic influences. In a focused observational study of 153 cases of cobalamin deficiency in older adults, 16 individuals experienced weakness in limbs and 28 individuals experienced difficulties walking (Healton et al., 1991). Descriptive data on the neurophysiologic profiles of older individuals with cobalamin deficiency identify ataxia as a common clinical presentation, with confirmatory decreases in motor neuron action potentials (Fine, Soria, Paroski, 49 Petryk, & Thomasula, 1990). Recent investigations of the Women's Health and Aging Study cohorts have identified that genetic variation in cobalamin metabolism genes influences methylmalonic acid concentrations and functional status (Matteini et al., 2008; Matteini et al., 2010). The dissertation study more fully explored these relationships by examining the functional performance measures hand grip strength and 4-meter walking speed. Hand Grip Strength Hand grip strength measurements are an indication of basic upper extremity function and measure total force of upper limb muscles (Rantanen, Era, & Heikkinen, 1994). Although it is a measurement of isometric strength in the upper body, hand grip strength has been found to also correlate with other skeletal muscle groups in the body (Rantanen, Pertti, Kauppinen, & Heikkinen, 1994). For this reason, hand grip strength is commonly used as an estimate of overall body strength. Hand grip strength has consistently demonstrated value in clinical and research settings as a reliable and valid measurement technique. It is a sensitive predictor of progressive disability, and morbidity, mortality in elderly adults (Blake et al., 1988; Kerr et al., 2006; Phillips, 1986; Rantanen, Era et al., 1994; Rantanen et al., 1999). Additionally, it has been found to be a useful clinical assessment parameter in screening individuals for nutritional deficiencies (Klidjian, Archer, Foster, & Karran, 1982; Matos, Tavares, & Amaral, 2007). Crucial for work with elderly patient populations, the hand dynamometers that measure grip strength force are portable, inexpensive, rapid, simple to use, and ideal for clinical assessments in home and community health settings. 50 Various studies identify use of grip strength as a valuable measurement technique, since it demonstrates both low intra- and interobserver variability and high clinical reproducibility (Bohannon, 2006; Schaubert & Bohannon, 2005; Windsor & Hill, 1988). However, it is recommended that use of dynamometers in research and clinical settings is consistent with respect to device manufacturer (Guerra & Amaral, 2009). A recent comparison report identified that although accuracy of dynamometers is high in elderly adults, measurements do not correlate well across manufacturers. Walking Speed An indication of strength, mobility, coordination, proprioception, reflex control, and balance, the ability to walk provides significant information about a patient across numerous functional parameters (Ferrucci, Guralnik, et al., 1995). One of these parameters, lower extremity muscle strength, is determined by the speed at which an individual is able to walk (Holloszy, 1995; Schwartz, 1997). Walking speed, or gait velocity, in combination with other key lower extremity functional health assessments can predict disability, mortality, and nursing home admission across diverse elderly populations (Fried, Bandeen-Roche, Chaves, & Johnson, 2000; Guralnik, Ferrucci, Simonsick, Salive, & Wallace, 1995; Guralnik et al., 1994; Ostir, Markides, Black, & Goodwin, 1998; Seeman et al., 1994). When used singularly, gait speed is a relatively accurate proxy for full lower extremity performance battery examination, and predicts incident disability up to 6 years (Guralnik et al., 2000). Furthermore, longitudinal evaluations identify that changes in lower extremity strength over time are linearly 51 associated with meaningful changes in gait speed for sedentary older adults (Purser, Pieper, Poole, & Morey, 2003). In addition to their predictive capacity for adverse outcomes, walking speed measurements are simple, reliable, and inexpensive to obtain. Commonly identified factors known to affect their accurate measurement include height and gender (Samson et al., 2001). Presence of dual tasking, simultaneously performing another task while an individual is walking, has also been recognized to affect gait measurement and its interference correlates to the difficulty of the concurrent task (Ble et al., 2005; Springer et al., 2006). Environmental and contextual factors, such as familiarity with surroundings, lighting, smooth walking surface, and use of assistive medical equipment, are also recognized to modify walking speed assessments (Hoenig et al., 2006; Richardson, Thies, DeMott, & Ashton-Miller, 2004a, 2004b). 52 References Addison, T. (1849). On anaemia: Disease of the suprarenal capsules. London Medical Gazette, 43, 517-518. Allen, L. H., & Casterline, J. (1994). Vitamin B-12 deficiency in elderly individuals: Diagnosis and requirements. American Journal of Clinical Nutrition, 60(1), 12- 14. Allen, R. H., Lindenbaum, J., & Stabler, S. P. (1996). High prevalence of cobalamin deficiency in the elderly. Transactions of the American Clinical and Climatological Association, 107, 37-45; discussion 45-37. Allen, R. H., Stabler, S. P., Savage, D. G., & Lindenbaum, J. (1993). Metabolic abnormalities in cobalamin (vitamin B12) and folate deficiency. The FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology, 7(14), 1344-1353. Alpers, D. H. (2005). What is new in vitamin B(12)? Current Opinion in Gastroenterology, 21(2), 183-186. doi: 00001574-200503000-00009 [pii] Andres, E., Affenberger, S., Zimmer, J., Vinzio, S., Grosu, D., Pistol, G., et al. (2006). Current hematological findings in cobalamin deficiency. A study of 201 consecutive patients with documented cobalamin deficiency. Clinical and Laboratory Haematology, 28(1), 50-56. doi: CLH755 [pii] 10.1111/j.1365- 2257.2006.00755.x [doi] Andres, E., Loukili, N. H., Noel, E., Kaltenbach, G., Abdelgheni, M. B., Perrin, A. E., et al. (2004). Vitamin B12 (cobalamin) deficiency in elderly patients. Canadian Medical Association Journal, 171(3), 251-259. Aster, J. (2005). Red blood cell bleeding disorders: Anemias of diminished erythropoesis. In V. Kumar, A. Abbas, & N. Fausto (Eds.), Robbins and cotran pathologic basis of disease (7th ed., pp. 638-642). Philadelphia, PA: Elsevier Saunders. Babior, B., & Franklin Bunn, H. (2005). Megaloblastic anemias. In D. Kasper, E. Braunwald, A. Fauci, S. Hauser, D. Longo, & J. L. Jameson (Eds.), Harrison's principles of internal medicine (16th ed., pp. 601-607). New York City, NY: McGraw-Hill Medical Publishing Division. Baik, H. W., & Russell, R. M. (1999). Vitamin B12 deficiency in the elderly. Annual Review of Nutrition, 19, 357-377. doi: 10.1146/annurev.nutr.19.1.357 [doi] 53 Barker, H. A., Smyth, R. D., Wawszkiewicz, E. J., Lee, M. N., & Wilson, R. M. (1958). Enzymic preparation and characterization of an alpha-L-beta-methylaspartic acid. Archives of Biochemistry and Biophysics, 78(2), 468-476. doi: 0003- 9861(58)90371-0 [pii] Barker, H. A., Smyth, R. D., Wilson, R. M., & Weissbach, H. (1959). The purification and properties of beta-methylaspartase. The Journal of Biological Chemistry, 234(2), 320-328. Barker, H. A., Weissbach, H., & Smyth, R. D. (1958). A coenzyme containing pseudovitamin B(12). Proceedings of the National Academy of Sciences of the United States of America, 44(11), 1093-1097. Barth, J. L., & Argraves, W. S. (2001). Cubilin and megalin: Partners in lipoprotein and vitamin metabolism. Trends in Cardiovascular Medicine, 11(1), 26-31. doi: S1050-1738(01)00080-9 [pii] Battersby, A. R. (1994). How nature builds the pigments of life: The conquest of vitamin B12. Science, 264(5165), 1551-1557. Beck, W. (2001). Cobalamin (Vitamin B12). In R. Rucker, D. McCormick, J. Suttie, & L. Machlin (Eds.), Handbook of vitamins (3rd ed., pp. 463-512). New York City, NY: Marcel Dekker, Inc. Bell, I. R., Edman, J. S., Marby, D. W., Satlin, A., Dreier, T., Liptzin, B., et al. (1990). Vitamin B12 and folate status in acute geropsychiatric inpatients: Affective and cognitive characteristics of a vitamin nondeficient population. Biological Psychiatry, 27(2), 125-137. doi: 0006-3223(90)90642-F [pii] Benner, S. A., Ellington, A. D., & Tauer, A. (1989). Modern metabolism as a palimpsest of the RNA world. Proceedings of the National Academy of Sciences of the United States of America, 86(18), 7054-7058. Birn, H. (2006). The kidney in vitamin B12 and folate homeostasis: Characterization of receptors for tubular uptake of vitamins and carrier proteins. American Journal of Physiology. Renal Physiology, 291(1), F22-36. doi: 291/1/F22 [pii] 10.1152/ajprenal.00385.2005 [doi] Birn, H., Fyfe, J. C., Jacobsen, C., Mounier, F., Verroust, P. J., Orskov, H., et al. (2000). Cubilin is an albumin binding protein important for renal tubular albumin reabsorption. The Journal of Clinical Investigation, 105(10), 1353-1361. doi: 10.1172/JCI8862 [doi] 54 Birn, H., Verroust, P. J., Nexo, E., Hager, H., Jacobsen, C., Christensen, E. I., et al. (1997). Characterization of an epithelial approximately 460-kDa protein that facilitates endocytosis of intrinsic factor-vitamin B12 and binds receptor-associated protein. The Journal of Biological Chemistry, 272(42), 26497-26504. Blake, A. J., Morgan, K., Bendall, M. J., Dallosso, H., Ebrahim, S. B., Arie, T. H., et al. (1988). Falls by elderly people at home: Prevalence and associated factors. Age and Ageing, 17(6), 365-372. Ble, A., Volpato, S., Zuliani, G., Guralnik, J. M., Bandinelli, S., Lauretani, F., et al. (2005). Executive function correlates with walking speed in older persons: The InCHIANTI study. Journal of the American Geriatrics Society, 53(3), 410-415. doi: JGS53157 [pii]10.1111/j.1532-5415.2005.53157.x [doi] Bohannon, R. W. (2006). Grip strength predicts outcome. Age and Ageing, 35(3), 320; author reply 320. doi: afj061 [pii] 10.1093/ageing/afj061 [doi] Booth, M., & Spray, G. (1960). Vitamin B12 activity in the serum and liver of rats after total gastrectomy. British Journal of Haematology, 6, 288-295. Bose, S., & Seetharam, B. (1997). Effect of disulfide bonds of transcobalamin II receptor on its activity and basolateral targeting in human intestinal epithelial Caco-2 cells. The Journal of Biological Chemistry, 272(33), 20920-20928. Bose, S., Seetharam, S., & Seetharam, B. (1995). Membrane expression and interactions of human transcobalamin II receptor. The Journal of Biological Chemistry, 270(14), 8152-8157. Bostom, A. G., & Lathrop, L. (1997). Hyperhomocysteinemia in end-stage renal disease: Prevalence, etiology, and potential relationship to arteriosclerotic outcomes. Kidney International, 52(1), 10-20. Bottiglieri, T., Laundy, M., Crellin, R., Toone, B. K., Carney, M. W., & Reynolds, E. H. (2000). Homocysteine, folate, methylation, and monoamine metabolism in depression. Journal of Neurology, Neurosurgery and Psychiatry, 69(2), 228-232. Brass, E. P. (1986). Effect of alpha-ketobutyrate on palmitic acid and pyruvate metabolism in isolated rat hepatocytes. Biochimica et Biophysica Acta, 888(1), 18-24. doi: 0167-4889(86)90065-0 [pii] Brass, E. P., Tahiliani, A. G., Allen, R. H., & Stabler, S. P. (1990). Coenzyme A metabolism in vitamin B-12-deficient rats. Journal of Nutrition, 120(3), 290-297. 55 Burger, R. L., Schneider, R. J., Mehlman, C. S., & Allen, R. H. (1975). Human plasma R-type vitamin B12-binding proteins: The role of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B12-binding protein in the plasma transport of vitamin B12. The Journal of Biological Chemistry, 250(19), 7707- 7713. Carkeet, C., Dueker, S. R., Lango, J., Buchholz, B. A., Miller, J. W., Green, R., et al. (2006). Human vitamin B12 absorption measurement by accelerator mass spectrometry using specifically labeled (14)C-cobalamin. Proceedings of the National Academy of Sciences of the United States of America, 103(15), 5694- 5699. doi: 0601251103 [pii]10.1073/pnas.0601251103 [doi] Carmel, R. (1985). The distribution of endogenous cobalamin among cobalamin-binding proteins in the blood in normal and abnormal states. American Journal of Clinical Nutrition, 41(4), 713-719. Carmel, R. (2000). Current concepts in cobalamin deficiency. Annual Review of Medicine, 51, 357-375. doi: 10.1146/annurev.med.51.1.357 [doi] Carmel, R., Green, R., Rosenblatt, D. S., & Watkins, D. (2003). Update on cobalamin, folate, and homocysteine. Hematology/ the Education Program of the American Society of Hematology, 1, 62-81. Carmel, R., & Sarrai, M. (2006). Diagnosis and management of clinical and subclinical cobalamin deficiency: Advances and controversies. Current Hematology Reports, 5(1), 23-33. Carney, M. W., Chary, T. K., Laundy, M., Bottiglieri, T., Chanarin, I., Reynolds, E. H., et al. (1990). Red cell folate concentrations in psychiatric patients. Journal of Affective Disorders, 19(3), 207-213. doi: 0165-0327(90)90093-N [pii] Chandler, R. J., Sloan, J., Fu, H., Tsai, M., Stabler, S., Allen, R., et al. (2007). Metabolic phenotype of methylmalonic acidemia in mice and humans: The role of skeletal muscle. BMC Medical Genetics, 8, 64. doi: 1471-2350-8-64 [pii] 10.1186/1471- 2350-8-64 [doi] Chaves, P. H., Ashar, B., Guralnik, J. M., & Fried, L. P. (2002). Looking at the relationship between hemoglobin concentration and prevalent mobility difficulty in older women. Should the criteria currently used to define anemia in older people be reevaluated? Journal of the American Geriatrics Society, 50(7), 1257- 1264. doi: jgs50313 [pii] Christensen, E. I., & Birn, H. (2002). Megalin and cubilin: Multifunctional endocytic receptors. Nature Reviews. Molecular Cell Biology, 3(4), 256-266. doi: 10.1038/nrm778 [doi] nrm778 [pii] 56 Chui, C. H., Lau, F. Y., Wong, R., Soo, O. Y., Lam, C. K., Lee, P. W., et al. (2001). Vitamin B12 deficiency--need for a new guideline. Nutrition, 17(11-12), 917-920. doi: S0899900701006669 [pii] Clarke, R., Refsum, H., Birks, J., Evans, J. G., Johnston, C., Sherliker, P., et al. (2003). Screening for vitamin B-12 and folate deficiency in older persons. American Journal of Clinical Nutrition, 77(5), 1241-1247. Cockcroft, D. W., & Gault, M. H. (1976). Prediction of creatinine clearance from serum creatinine. Nephron, 16(1), 31-41. Coelho, D., Suormala, T., Stucki, M., Lerner-Ellis, J. P., Rosenblatt, D. S., Newbold, R., et al. (2008). Gene identification for the cblD defect of vitamin B12 metabolism. New England Journal of Medicine, 358(14), 1454-1464. doi: 358/14/1454 [pii]10.1056/NEJMoa072200 [doi] Combe, J. (1824). History of a case of anemia. Transactions. Medico-Chirurgical Society of Edinburgh, 1, 194-204. Cui, S., Verroust, P. J., Moestrup, S. K., & Christensen, E. I. (1996). Megalin/gp330 mediates uptake of albumin in renal proximal tubule. American Journal of Physiology, 271(4 Pt 2), F900-907. de Neeling, J. N., Beks, P. J., Bertelsmann, F. W., Heine, R. J., & Bouter, L. M. (1994). Sensory thresholds in older adults: Reproducibility and reference values. Muscle and Nerve, 17(4), 454-461. doi: 10.1002/mus.880170414 [doi] Depeint, F., Bruce, W. R., Shangari, N., Mehta, R., & O'Brien, P. J. (2006). Mitochondrial function and toxicity: Role of B vitamins on the one-carbon transfer pathways. Chemico-Biological Interactions, 163(1-2), 113-132. doi: S0009-2797(06)00133-5 [pii] 10.1016/j.cbi.2006.05.010 [doi] Dieckgraefe, B. K., Seetharam, B., Banaszak, L., Leykam, J. F., & Alpers, D. H. (1988). Isolation and structural characterization of a cDNA clone encoding rat gastric intrinsic factor. Proceedings of the National Academy of Sciences of the United States of America, 85(1), 46-50. Dinn, J. J., Weir, D. G., McCann, S., Reed, B., Wilson, P., & Scott, J. M. (1980). Methyl group deficiency in nerve tissue: A hypothesis to explain the lesion of subacute combined degeneration. Irish Journal of Medical Science, 149(1), 1-4. Dixon, M. M., Huang, S., Matthews, R. G., & Ludwig, M. (1996). The structure of the C-terminal domain of methionine synthase: Presenting S-adenosylmethionine for reductive methylation of B12. Structure, 4(11), 1263-1275. 57 Donini, L., Savina, C., & Cannella, C. (2003). Eating habits and appetite control in the elderly: The anorexia of aging. International Psychogeriatrics, 15(1), 73-87. Drennan, C. L., Huang, S., Drummond, J. T., Matthews, R. G., & Lidwig, M. L. (1994). How a protein binds B12: A 3.0 A X-ray structure of B12-binding domains of methionine synthase. Science, 266(5191), 1669-1674. Eisenstaedt, R., Penninx, B. W., & Woodman, R. C. (2006). Anemia in the elderly: Current understanding and emerging concepts. Blood Reviews, 20(4), 213-226. doi: S0268-960X(05)00070-6 [pii]10.1016/j.blre.2005.12.002 [doi] Elias, M. F., Sullivan, L. M., D'Agostino, R. B., Elias, P. K., Jacques, P. F., Selhub, J., et al. (2005). Homocysteine and cognitive performance in the Framingham offspring study: Age is important. American Journal of Epidemiology, 162(7), 644-653. doi: kwi259 [pii]10.1093/aje/kwi259 [doi] Feher, E. P., Larrabee, G. J., & Crook, T. H. (1992). Factors attenuating the validity of the Geriatric Depression Scale in a dementia population. Journal of the American Geriatrics Society, 40(9), 906-909. Ferrucci, L., Guralnik, J., Bandeen-Roche, K., Lafferty, M. E., Pahor, M., & Fried, L. P. (1995). Physical performance measures. In J. M. Guralnik, L. P. Fried, E. M. Simonsick, J. D. Kasper, & M. E. Lafferty (Eds.), The Women's Health and Aging Study: Health and social characteristics of older women with disability, (pp. 35- 49). Bethesda, MD: National Institutes of Health, National Institute on Aging. Ferrucci, L., Kittner, S. J., Corti, C., & Guralnik, J. M. (1995). Neurological conditions. In J. M. Guralnik, L. P. Fried, E. M. Simonsick, J. D. Kasper, & M. E. Lafferty (Eds.), The Women's Health and Aging Study: Health and social characteristics of older women with disability, (pp. 140-151). Bethesda, MD: National Institutes of Health, National Institute on Aging. Fine, E. J., Soria, E., Paroski, M. W., Petryk, D., & Thomasula, L. (1990). The neurophysiological profile of vitamin B12 deficiency. Muscle and Nerve, 13(2), 158-164. doi: 10.1002/mus.880130213 [doi] Finkler, A. E., & Hall, C. A. (1967). Nature of the relationship between vitamin B12 binding and cell uptake. Archives of Biochemistry and Biophysics, 120(1), 79-85. Frenkel, E. P. (1973). Abnormal fatty acid metabolism in peripheral nerves of patients with pernicious anemia. Journal of Clinical Investigation, 52(5), 1237-1245. doi: 10.1172/JCI107291 [doi] 58 Fried, L. P., Bandeen-Roche, K., Chaves, P. H., & Johnson, B. A. (2000). Preclinical mobility disability predicts incident mobility disability in older women. Journals of Gerontology. Series A, Biological Sciences and Medical Sciences, 55(1), M43- 52. Froissart, M., Rossert, J., Jacquot, C., Paillard, M., & Houillier, P. (2005). Predictive performance of the modification of diet in renal disease and Cockcroft-Gault equations for estimating renal function. Journal of the American Society of Nephrology, 16(3), 763-773. doi: ASN.2004070549 [pii] 10.1681/ASN.2004070549 [doi] Gburek, J., Verroust, P. J., Willnow, T. E., Fyfe, J. C., Nowacki, W., Jacobsen, C., et al. (2002). Megalin and cubilin are endocytic receptors involved in renal clearance of hemoglobin. Journal of the American Society of Nephrology, 13(2), 423-430. Giannelli, S. V., Patel, K. V., Windham, B. G., Pizzarelli, F., Ferrucci, L., & Guralnik, J. M. (2007). Magnitude of underascertainment of impaired kidney function in older adults with normal serum creatinine. Journal of the American Geriatrics Society, 55(6), 816-823. doi: JGS1196 [pii]10.1111/j.1532-5415.2007.01196.x [doi] Glasgow, A. M., & Chase, H. P. (1976). Effect of propionic acid on fatty acid oxidation and ureagenesis. Pediatric Research, 10(7), 683-686. Goulian, M., Bleile, B., & Tseng, B. Y. (1980). Methotrexate-induced misincorporation of uracil into DNA. Proceedings of the National Academy of Sciences of the United States of America, 77(4), 1956-1960. Grasbeck, R. (1959). Calculations on vitamin B12 turnover in man. With a note on the maintenance treatment in pernicious anemia and the radiation dose received by patients ingesting radiovitamin B12. Scandinavian Journal of Clinical and Laboratory Investigation, 11, 250-258. Grasbeck, R., Nyberg, W., & Reizenstein, P. (1958). Biliary and fecal vit. B12 excretion in man: An isotope study. Proceedings of the Society for Experimental Biology and Medicine, 97(4), 780-784. Green, R. (1995). Metabolite assays in cobalamin and folate deficiency. Baillieres Clinical Haematology, 8(3), 533-566. Green, R. (2008). Indicators for assessing folate and vitamin B12 status and for monitoring the efficacy of intervention strategies. Food and Nutrition Bulletin, 29(2 Suppl), S52-63; discussion S64-56. Green, R., Jacobsen, D. W., Van Tonder, S. V., Kew, M. C., & Metz, J. (1982). Absorption of biliary cobalamin in baboons following total gastrectomy. Journal of Laboratory and Clinical Medicine, 100(5), 771-777. 59 Green, R., & Kinsella, L. J. (1995). Current concepts in the diagnosis of cobalamin deficiency. Neurology, 45(8), 1435-1440. Green, R., & Miller, J. (2007). Vitamin B12. In J. Zempleni, R. Rucker, D. McCormick, & J. Suttie (Eds.), Handbook of vitamins (4th ed., pp. 413-457). Boca Raton, FL: CRC Press, Taylor & Francis Group. Guerra, R. S., & Amaral, T. F. (2009). Comparison of hand dynamometers in elderly people. The Journal of Nutrition, Health, and Aging, 13(10), 907-912. Guest, J. R., Friedman, S., Woods, D. D., & Smith, E. L. (1962). A methyl analogue of cobamide coenzyme in relation to methionine synthesis by bacteria. Nature, 195, 340-342. Guralnik, J. M., Eisenstaedt, R. S., Ferrucci, L., Klein, H. G., & Woodman, R. C. (2004). Prevalence of anemia in persons 65 years and older in the United States: Evidence for a high rate of unexplained anemia. Blood, 104(8), 2263-2268. doi: 10.1182/blood-2004-05-1812 [doi] 2004-05-1812 [pii] Guralnik, J. M., Ferrucci, L., Pieper, C. F., Leveille, S. G., Markides, K. S., Ostir, G. V., et al. (2000). Lower extremity function and subsequent disability: Consistency across studies, predictive models, and value of gait speed alone compared with the short physical performance battery. Journals of Gerontology. Series A, Biological Sciences and Medical Sciences, 55(4), M221-231. Guralnik, J. M., Ferrucci, L., Simonsick, E. M., Salive, M. E., & Wallace, R. B. (1995). Lower-extremity function in persons over the age of 70 years as a predictor of subsequent disability. New England Journal of Medicine, 332(9), 556-561. Guralnik, J. M., Simonsick, E. M., Ferrucci, L., Glynn, R. J., Berkman, L. F., Blazer, D. G., et al. (1994). A short physical performance battery assessing lower extremity function: association with self-reported disability and prediction of mortality and nursing home admission. Journal of Gerontology, 49(2), M85-94. Hall, C. A. (1964). Long-term excretion of Co-57-Vitamin B12 and turnover within the plasma. American Journal of Clinical Nutrition, 14, 156-162. Hardlei, T. F., & Nexo, E. (2009). A new principle for measurement of cobalamin and corrinoids, used for studies of cobalamin analogs on serum haptocorrin. Clinical Chemistry, 55(5), 1002-1010. doi: clinchem.2008.114132 [pii] 10.1373/clinchem.2008.114132 [doi] Healton, E. B., Savage, D. G., Brust, J. C., Garrett, T. J., & Lindenbaum, J. (1991). Neurologic aspects of cobalamin deficiency. Medicine, 70(4), 229-245. 60 Henning, B. F., Tepel, M., Riezler, R., & Naurath, H. J. (2001). Long-term effects of vitamin B(12), folate, and vitamin B(6) supplements in elderly people with normal serum vitamin B(12) concentrations. Gerontology, 47(1), 30-35. doi: ger47030 [pii] Herrmann, W., Obeid, R., Schorr, H., & Geisel, J. (2003). Functional vitamin B12 deficiency and determination of holotranscobalamin in populations at risk. Clinical Chemistry and Laboratory Medicine, 41(11), 1478-1488. Herrmann, W., Obeid, R., Schorr, H., & Geisel, J. (2005). The usefulness of holotranscobalamin in predicting vitamin B12 status in different clinical settings. Curr Drug Metab, 6(1), 47-53. Herrmann, W., Schorr, H., Bodis, M., Knapp, J. P., Muller, A., Stein, G., et al. (2000). Role of homocysteine, cystathionine and methylmalonic acid measurement for diagnosis of vitamin deficiency in high-aged subjects. European Journal of Clinical Investigation, 30(12), 1083-1089. doi: eci746 [pii] Herrmann, W., Schorr, H., Geisel, J., & Riegel, W. (2001). Homocysteine, cystathionine, methylmalonic acid and B-vitamins in patients with renal disease. Clinical Chemistry and Laboratory Medicine, 39(8), 739-746. doi: 10.1515/CCLM.2001.123 [doi] Hewitt, J. E., Seetharam, B., Leykam, J., & Alpers, D. H. (1990). Isolation and characterization of a cDNA encoding porcine gastric haptocorrin. European Journal of Biochemistry, 189(1), 125-130. Heyssel, R. M., Bozian, R. C., Darby, W. J., & Bell, M. C. (1966). Vitamin B12 turnover in man. The assimilation of vitamin B12 from natural foodstuff by man and estimates of minimal daily dietary requirements. American Journal of Clinical Nutrition, 18(3), 176-184. Hodgkin, D., Kamper, J., Mackay, M., Pickworth, J., Trueblood, K. N., & White, J. G. (1956). Structure of vitamin B12. Nature, 178(4524), 64-66. Hodgkin, D., Pickworth, J., Robertson, J., Trueblood, K., & Prosen, R. (1955). The crystal structure of the hexacarboxylic acid derived from B12 and the molecular structure for the vitamin. Nature, 176(4477), 325-328. Hoedemaeker, P. J., Abels, J., Wachters, J. J., Arends, A., & Nieweg, H. O. (1966). Further investigations about the site of production of Castle's gastric intrinsic factor. Laboratory Investigation, 15(7), 1163-1173. 61 Hoenig, H., Ganesh, S. P., Taylor, D. H., Jr., Pieper, C., Guralnik, J., & Fried, L. P. (2006). Lower extremity physical performance and use of compensatory strategies for mobility. Journal of the American Geriatrics Society, 54(2), 262-269. doi: JGS588 [pii] 10.1111/j.1532-5415.2005.00588.x [doi] Holloszy, J. (1995). Workshop on sarcopenia: Muscle atrophy in old age. Journal of Gerontology, 50A, 1-157. Johnston, J., Bollekens, J., Allen, R. H., & Berliner, N. (1989). Structure of the cDNA encoding transcobalamin I, a neutrophil granule protein. Journal of Biological Chemistry, 264(27), 15754-15757. Kalra, S., Li, N., Yammani, R. R., Seetharam, S., & Seetharam, B. (2004). Cobalamin (vitamin B12) binding, phylogeny, and synteny of human transcobalamin. Archives of Biochemistry and Biophysics, 431(2), 189-196. doi: S0003- 9861(04)00464-3 [pii] 10.1016/j.abb.2004.08.011 [doi] Kapadia, C. R., & Essandoh, L. K. (1988). Active absorption of vitamin B12 and conjugated bile salts by guinea pig ileum occurs in villous and not crypt cells. Digestive Diseases and Sciences, 33(11), 1377-1382. Kerr, A., Syddall, H. E., Cooper, C., Turner, G. F., Briggs, R. S., & Sayer, A. A. (2006). Does admission grip strength predict length of stay in hospitalised older patients? Age and Ageing, 35(1), 82-84. doi: 35/1/82 [pii] 10.1093/ageing/afj010 [doi] Kishimoto, Y., Williams, M., Moser, H. W., Hignite, C., & Biermann, K. (1973). Branched-chain and odd-numbered fatty acids and aldehydes in the nervous system of a patient with deranged vitamin B12 metabolism. Journal of Lipid Research, 14(1), 69-77. Klidjian, A. M., Archer, T. J., Foster, K. J., & Karran, S. J. (1982). Detection of dangerous malnutrition. JPEN. Journal of Parenteral and Enteral Nutrition, 6(2), 119-121. Kolker, S., & Okun, J. G. (2005). Methylmalonic acid--an endogenous toxin? Cellular and Molecular Life Sciences, 62(6), 621-624. doi: 10.1007/s00018-005-4463-2 [doi] Kondo, H., Binder, M. J., Kolhouse, J. F., Smythe, W. R., Podell, E. R., & Allen, R. H. (1982). Presence and formation of cobalamin analogues in multivitamin-mineral pills. Journal of Clinical Investigation, 70(4), 889-898. Lamb, E. J., Wood, J., Stowe, H. J., O'Riordan, S. E., Webb, M. C., & Dalton, R. N. (2005). Susceptibility of glomerular filtration rate estimations to variations in creatinine methodology: A study in older patients. Annals of Clinical Biochemistry, 42(Pt 1), 11-18. doi: 10.1258/0004563053026899 [doi] 62 Levy, H. L., Mudd, S. H., Schulman, J. D., Dreyfus, P. M., & Abeles, R. H. (1970). A derangement in B12 metabolism associated with homocystinemia, cystathioninemia, hypomethioninemia and methylmalonic aciduria. American Journal of Medicine, 48(3), 390-397. doi: 0002-9343(70)90070-7 [pii] Li, N., Seetharam, S., Rosenblatt, D. S., & Seetharam, B. (1994). Expression of transcobalamin II mRNA in human tissues and cultured fibroblasts from normal and transcobalamin II-deficient patients. Biochemical Journal, 301 (Pt 2), 585- 590. Li, N., Seetharam, S., & Seetharam, B. (1995). Genomic structure of human transcobalamin II: Comparison to human intrinsic factor and transcobalamin I. Biochemical and Biophysical Research Communications, 208(2), 756-764. doi: S0006-291X(85)71402-7 [pii] 10.1006/bbrc.1995.1402 [doi] Li, N., Seetharam, S., & Seetharam, B. (1998). Characterization of the human transcobalamin II promoter. A proximal GC/GT box is a dominant negative element. Journal of Biological Chemistry, 273(26), 16104-16111. Lien, E. L., Ellenbogen, L., Law, P. Y., & Wood, J. M. (1974). Studies on the mechanism of cobalamin binding to hog intrinsic factor. Journal of Biological Chemistry, 249(3), 890-894. Lindemans, J., van Kapel, J., & Abels, J. (1986). Uptake of transcobalamin II-bound cobalamin by isolated rat kidney tubule cells. Scandinavian Journal of Clinical and Laboratory Investigation, 46(3), 223-232. Lindenbaum, J., Healton, E. B., Savage, D. G., Brust, J. C., Garrett, T. J., Podell, E. R., et al. (1988). Neuropsychiatric disorders caused by cobalamin deficiency in the absence of anemia or macrocytosis. New England Journal of Medicine, 318(26), 1720-1728. Lindenbaum, J., Rosenberg, I. H., Wilson, P. W., Stabler, S. P., & Allen, R. H. (1994). Prevalence of cobalamin deficiency in the Framingham elderly population. American Journal of Clinical Nutrition, 60(1), 2-11. Lindenbaum, J., Savage, D. G., Stabler, S. P., & Allen, R. H. (1990). Diagnosis of cobalamin deficiency: Relative sensitivities of serum cobalamin, methylmalonic acid, and total homocysteine concentrations. American Journal of Hematology, 34(2), 99-107. Lindgren, A., Kilander, A., Bagge, E., & Nexo, E. (1999). Holotranscobalamin - a sensitive marker of cobalamin malabsorption. European Journal of Clinical Investigation, 29(4), 321-329. 63 Locatelli, E. R., Laureno, R., Ballard, P., & Mark, A. S. (1999). MRI in vitamin B12 deficiency myelopathy. Canadian Journal of Neurological Sciences, 26(1), 60-63. Lyness, J. M., Noel, T. K., Cox, C., King, D. A., Conwell, Y., & Caine, E. D. (1997). Screening for depression in elderly primary care patients. A comparison of the Center for Epidemiologic Studies-Depression Scale and the Geriatric Depression Scale. Archives of Internal Medicine, 157(4), 449-454. Malouf, R., & Grimley Evans, J. (2008). Folic acid with or without vitamin B12 for the prevention and treatment of healthy elderly and demented people. Cochrane Database of Systematic Reviews(4), CD004514. doi: 10.1002/14651858.CD004514.pub2 [doi] Mancia, F., Keep, N. H., Nakagawa, A., Leadlay, P. F., McSweeney, S., Rasmussen, B., & Evans, P. R. (1996). How coenzyme B12 radicals are generated: The crystal structure of methylmalonyl-coenzyme A mutase at 2 A resolution. Structure, 4(3), 339-350. Manns, B. J., Burgess, E. D., Parsons, H. G., Schaefer, J. P., Hyndman, M. E., & Scott- Douglas, N. W. (1999). Hyperhomocysteinemia, anticardiolipin antibody status, and risk for vascular access thrombosis in hemodialysis patients. Kidney International, 55(1), 315-320. doi: 10.1046/j.1523-1755.1999.00258.x [doi] Martens, J. H., Barg, H., Warren, M. J., & Jahn, D. (2002). Microbial production of vitamin B12. Applied Microbiology and Biotechnology, 58(3), 275-285. doi: 10.1007/s00253-001-0902-7 [doi] Maser, R. E., Nielsen, V. K., Bass, E. B., Manjoo, Q., Dorman, J. S., Kelsey, S. F., et al. (1989). Measuring diabetic neuropathy. Assessment and comparison of clinical examination and quantitative sensory testing. Diabetes Care, 12(4), 270-275. Mathan, V. I., Babior, B. M., & Donaldson, R. M., Jr. (1974). Kinetics of the attachment of intrinsic factor-bound cobamides to ileal receptors. Journal of Clinical Investigation, 54(3), 598-608. doi: 10.1172/JCI107797 [doi] Matos, L. C., Tavares, M. M., & Amaral, T. F. (2007). Handgrip strength as a hospital admission nutritional risk screening method. European Journal of Clinical Nutrition, 61(9), 1128-1135. doi: 1602627 [pii] 10.1038/sj.ejcn.1602627 [doi] Matteini, A., Walston, J., Bandeen-Roche, K., Arking, D., Allen, R., Fried, L., et al. (2008). Markers of B-vitamin deficiency and frailty in older women. The Journal of Nutrition, Health, and Aging, 12(5), 303-308. 64 Matteini, A., Walston, J., Bandeen-Roche, K., Arking, D., Allen, R., Fried, L., et al. (2010). Transcobalamin II variants, decreased vitamin B12 availability and increased risk of frailty. The Journal of Nutrition, Health, and Aging, 14(1), 73- 77. McCaddon, A. (2006). Homocysteine and cognition--a historical perspective. Journal of Alzheimer's Disease: JAD, 9(4), 361-380. Metz, J. (1992). Cobalamin deficiency and the pathogenesis of nervous system disease. Annual Review of Nutrition, 12, 59-79. doi10.1146/annurev.nu.12.070192.000423 [doi] Metz, J., Bell, A. H., Flicker, L., Bottiglieri, T., Ibrahim, J., Seal, E., & McGrath, K. M. (1996). The significance of subnormal serum vitamin B12 concentration in older people: A case control study. Journal of the American Geriatrics Society, 44(11), 1355-1361. Miller, J. W.,