Facultad De Ciencias De La Actividad Física Y Del Deporte - Inef

UNIVERSIDAD POLITÉCNICA DE MADRID

FACULTAD DE CIENCIAS DE LA ACTIVIDAD FÍSICA Y DEL DEPORTE - INEF

Evolution and treatment of vitamin B12 deﬁciency as a risk factor for (cognitive and functional) neurodegenerative diseases in institutionalized elderly

Evolución y tratamiento de la deﬁciencia de vitamina B12 como factor de riesgo de enfermedades neurodegenerativas (cognitivas y funcionales) en las personas mayores institucionalizadas

TESIS DOCTORAL

ULRIKE ALBERS

Licenciada en Ciencia y Tecnología de los Alimentos

2012

DEPARTAMENTO DE SALUD Y RENDIMIENTO HUMANO

FACULTAD DE CIENCIAS DE LA ACTIVIDAD FÍSICA Y DEL DEPORTE - INEF

Evolution and treatment of vitamin B12 deﬁciency as a risk factor for (cognitive and functional) neurodegenerative diseases in institutionalized elderly

Evolución y tratamiento de la deﬁciencia de vitamina B12 como factor de riesgo de enfermedades neurodegenerativas (cognitivas y funcionales) en las personas mayores institucionalizadas

Ulrike Albers

Licenciada en Ciencia y Tecnología de los Alimentos

Directores:

María Marcela González Gross, Doctora en Farmacia, Profesora Titular de la Universidad Politécnica de Madrid

Agustín Meléndez Ortega, Doctor en Ciencias de la Actividad Física y del Deporte, Profesor “Ad honorem” de la Universidad Politécnica de Madrid

Madrid, 2012

Photo: Foto Krummen, Lingen

Ulrike Albers, 2012 Edita: Universidad Politécnica, Av. Ramiro Maeztu 7, 28040 Madrid Imprime: MuzArt, Impresión Digital; c/ Gaztambide, 56; 28015 Madrid ISBN-10: 84-96398-59-5 ISBN-13: 978-84-96398-59-7

Tribunal nombrado por el Magfco. y Excmo. Sr. Rector de la Universidad Politécnica de Madrid, el día 20 de junio de 2012.

PRESIDENTE: D. Luis A. Moreno Aznar, PhD

VOCAL: Dña Mary Ward, PhD

VOCAL: Dña Ángeles Carbajal Azcona, PhD

VOCAL: Dña Christina Breidenassel, PhD

SECRETARIA: Dña Ma del Carmen González Chamorro, PhD

Realizado el acto de defensa y lectura de la Tesis el día 20 de junio de 2012 en Madrid.

Caliﬁcación:

EL PRESIDENTE LOS VOCALES

LA SECRETARIA

On ne reçoit pas la sagesse, il faut la découvrir soi-même après un trajet que personne ne peut faire pour nous, ne peut nous épargner, car elle est un point de vue sur les choses.

A la recherche du temps perdu, Marcel Proust

A Pilar Carazo Salmerón

This PhD Thesis has been possible thanks to the following grants and funding:

Project I Project title Vitamin B12 deﬁciency as a risk factor for neurodegenerative diseases. Longitudi- nal study for the evaluation of changes in the vitamin B12 status in Spanish elderly. (P061100509); funded by Axis-Shield Diagnostics Ltd. and Abbott Diagnostics. S.A. Project II Project title Vitamin B12 deﬁciency as a risk factor for neurodegenerative diseases. Effect of a cobalamin supplement during one month. (P051100649); supported by Whitehall Much GmbH and Abbott Diagnostics. S.A.

Ulrike Albers has been ﬁnancially supported by the University Politécnica de Madrid with a predoctoral scholarship (2008-2011). Grants from the Universidad Politécnica de Madrid to do internships in the following institutions.

• Instituto de Medicina Molecular, Lisbon, Portugal. 20th of September-20th De- cember 2009.

• Centre d’Investigations Cliniques CHU-Inserm, CIC 9301. Lille, France. 2nd October-31st of December 2010.

• School of Biomedical Sciences, University of Ulster, Coleraine, UK. 18th June- 18th September 2011.

PUBLICATONS AND PRESENTATIONS FROM THE THESIS

Refereed Conference Proceedings and Abstracts

Albers U, Pedrero R, Moran P, Cañada D, Meléndez A, González-Gross M. One year follow-up of biochemical and cobalamin status among Spanish institutionalized elderly. 11th European Nutrition Conference (FENS). Madrid, Spain. 26-29.10.2011. Ann Nutr Metab 2011, 58(suppl 3): 48. González-Gross M, Pedrero-Chamizo R, Albers U. Interrelación entre la condición física, el estado nutricional vitamínico y el estado cognitivo y su influencia sobre la calidad de vida en mayores. 4° Congreso internacional de Actividad Física Deportiva para Mayores. Málaga, Spain. 4-6.03.2011. Proceedings ISBN 978-84-777858980. pp. 71-86. Albers U, Palacios G, Pedrero-Chamizo R, Meléndez A, Pietrzik K, González-Gross M. Plurimedication among Spanish institutionalized elderly, a fact that should be considered when examining their nutritional status. II World Congress of Public Health Nutrition and I Latin American Congress of Community Nutrition. Porto, Portu- gal. 23-25.09.2010. Public Health Nutr 2010, 13(9A): 248. Pedrero-Chamizo R, Albers U, Valtueña J, Casajús JA, Castillo MJ, González -Gross M. Strength training is essential in elderly women. 5th World Conference on Women and Sport. Sydney, Australia. 20-23.05.2010. Abstract Book ISBN 978-0-9806738- 0-7, 2010. Pedrero-Chamizo R, Albers U, Valtueña J, Moreno S, Pietrzik K, Castillo MJ González- Gross M. Relation Between Physical Strength and Cognitive Function in Spanish In- stitutionalized Elderly. 3rd International Congress on Physical Activity and Public Health (ICPAPH). Toronto, Canada. 5-8.05.2010. Pedrero Chamizo R, Albers U, Morencos E, Pastor JC, Meléndez A, Castillo MJ, González–Gross M. Valoración del estado nutricional en mayores institucionalizados a través de diferentes marcadores. II Congreso de la FESNAD. Barcelona. Spain. 3-5.03.2010. Albers U, Pedrero-Chamizo R, Meléndez A, Castillo MJ, Pietrzik K, González-Gross M. How effective can a 500 µg oral supplement of vitamin B12 for just 28 days be for institutionalized elderly? XIXth IAGG World Congress of Gerontology and Geriatrics. Paris, France. 5-9.07.2009 Pedrero-Chamizo R, Albers U, Valtueña J, Cañada D, Jathe R, Meléndez A, Castillo MJ, González-Gross M: Evolución de los niveles de fuerza en población mayor institucionalizada de la Comunidad de Madrid. III Jornadas Nacionales de Medicina del Deporte. Zaragoza, Spain. 29-30.05.2009. Pedrero-Chamizo R, Albers U, Valtueña J, Jiménez-Pavón D, Meléndez A, González- Gross M. Valores de referencia en fuerza de prensión manual en población mayor institucionalizada y de vida independiente. III Congreso Internacional de Actividad Físico Deportiva para Mayores. Málaga, Spain. 12-14.03.2009 Pedrero-Chamizo R, Albers U, Piera M, Jiménez-Pavón D, Meléndez A, Castillo MJ, González-Gross M. Gender, age and cognitive score effects on strength in Spanish institutionalized elderly. 13th Annual Congress of the European College of Sport Sciences. Estoril, Portugal. 9-12.07.2008 Pedrero-Chamizo R, Albers U, Valtueña J, Pietrzik K, Meléndez A, Castillo MJ, González-Gross M. Vitamin B12 deficiency. Effects on physical strength and cognitive health in Spanish institutionalized elderly. 6th international Conference on Nutrition and Fitness. Athens, Greece. 15-17.05.2008 Albers, U, Pedrero R, Díaz V, Tobaruela JL, Pietrzik K, Castillo MJ, Meléndez A, González-Gross M. Gender differences in vitamin B12 status and functional fitness parameters in Spanish institutionalized elderly. The 6th World Congress on The Aging Male. Tampa, Florida, United States, 21-24.02.2008. Pedrero R, Albers U, Jiménez-Pavón D, Cupeiro R, Meléndez A, Castillo MJ, Gutiér- rez A, González-Gross M. Valoración funcional en personas mayores institucionalizadas. XII Congreso Nacional de la Federación Española de Medicina del De- porte. Sevilla, Spain. 24-27.10.2007. Albers U, Meléndez A, Pietrzik K, Tobaruela JL, Castillo MJ, González-Gross M. Effectiveness of oral Vitamin B12 supplementation in Spanish institutionalized elderly. 7th International Conference VITAMINS - Nutrition and Diagnostics. Prague, Czech Republic. 19-21.09.2007. Albers U, Díaz V, Peinado A, Alvarez M, Sola R, Pietrzik K, Caballero J, Meléndez A, Castillo MJ, González-Gross M. Physical strength and Vitamin B12 in relation to cognitive score in Spanish institutionalized elderly. 10th European Nutrition Con- ference Paris, France. 10-13.07.2007. Ann Nutr Metab 2007; 51 (suppl 1): 119-120.

Refereed Journal Publications

Albers U, Palacios G, Pedrero-Chamizo R, Meléndez A, Pietrzik K, González-Gross M. La polimedicación en mayores institucionalizados. Su efecto sobre los niveles de vitamina B12, folato y homocisteína. Nutr Hosp. 2012;27(1): 296-300. Albers U, Pedrero R, Meléndez A, Pietrzik K, Castillo MJ, González-Gross M. Ef- fectiveness of a 28 days oral cyanocobalamin supplementation on vitamin B status in Spanish institutionalized elderly. (accepted in Int J Vitam Nutr Res) Contents

Contents I

List of Tables V

List of Figures VII

Nomenclature IX

Glossary XI

Abstract of the Thesis XIII

Resumen de la Tesis XVII

1 INTRODUCTION 1 1.1 Introduction to the Thesis ...... 1 1.2 Signiﬁcance of the Thesis ...... 5

2 REVIEW OF RELATED LITERATURE 9

2.1 Deﬁnition of Vitamin B12 and Food Sources ...... 9 2.2 Dietary Sources and Availability ...... 10 2.3 Requirements for the Daily Intake of Cobalamin in the Elderly . . 11

2.4 Absorption, Storage and Excretion of Vitamin B12 ...... 12 2.4.1 Absorption and Storage ...... 12

2.4.2 Enterohepatic Circulation and Excretion of Vitamin B12 . 14

2.5 Functions of Vitamin B12 ...... 15

2.6 Prevalence of Vitamin B12 Deﬁciency in the Elderly ...... 16

2.6.1 Factors Contributing to Poor Vitamin B12 Status with Ageing 16

I 2.6.2 Other Causes and Effects of Vitamin B12 Deﬁciency in the Elderly ...... 18

2.7 Clinical and Subclinical Manifestations of Vitamin B12 Deficiency in the Elderly ...... 19 2.7.1 Neurologic Effects of Vitamin B12 Deficiency ...... 19 2.7.2 Vitamin B12 and Cognitive Impairment ...... 20 2.7.3 Haematologic Effects of Deficiency ...... 21 2.7.4 Cardiovascular Effects of Deficiency ...... 22 2.7.5 Cognitive Impairment and the Function of the Neuromus- cular System ...... 23 2.8 Laboratory Diagnosis of Vitamin B Deficiency ...... 25 2.8.1 Indicators of Haematologic Status ...... 28 2.8.2 Serum or Plasma Cobalamin Levels ...... 28 2.8.3 Holotranscobalamin (HoloTC, Active B12)...... 28 2.8.4 Serum Methylmalonic Acid ...... 29 2.8.5 Serum Homocysteine Concentration ...... 29 2.9 Oral Supplements as Alternatives to Conventional Vitamin B12 Treatment...... 29

3 HYPOTHESESAND OBJECTIVES 33 3.1 Hypotheses of Study I ...... 33 3.2 Objectives of Study I ...... 33 3.3 Hypotheses of Study II ...... 33 3.4 Objectives of Study II ...... 34 3.5 Hypotheses of Study III ...... 34 3.6 Objectives of Study III ...... 34

4 METHODOLOGY 37 4.1 Study Designs ...... 38 4.1.1 Study I (Longitudinal) ...... 38 4.1.2 Study II (Intervention) ...... 38

II 4.1.3 Study III (Polypharmacy) ...... 38 4.2 Ethical Aspects ...... 39 4.3 Materials and Equipment ...... 39 4.3.1 Equipment ...... 39 4.3.2 Procedures ...... 39 Phlebotomy and Blood Distribution ...... 39 4.3.2.1 Blood analyses ...... 39 Vitamin B12 Status ...... 41 Haematological Parameters ...... 41 Biochemical Status ...... 41 4.3.2.2 Anthropometric Measurements ...... 41 4.3.2.3 Cognitive Status ...... 43 4.3.2.4 Functional Tests...... 43 4.4 Delimitations of the Studies...... 45 4.5 Justiﬁcation of the Chosen Tests...... 46 4.6 Limitations of the Studies ...... 48 4.7 Statistical Analysis ...... 49

5 RESULTS 53 5.1 Study I (Longitudinal) ...... 53 5.2 Study II (Intervention) ...... 64 5.3 Study III (Polypharmacy) ...... 71

6 DISCUSSION 77 6.1 Study I (Longitudinal) ...... 77 6.2 Study II (Intervention) ...... 82 6.3 Study III (Polypharmacy) ...... 83

7 CONCLUSIONS 87

References 89

III 8 Appendices 117 8.1 Case Report Form Longitudinal Study ...... 117 8.2 Case Report Form Intervention Study ...... 117 8.3 MMSE approved form (version date September 6, 2002) ...... 117

Agradecimientos 149

IV List of Tables

1 Sources of vitamin B12 in foods (modified from [61])...... 11 2 Clinical Manifestiations of Vitamin B12 Deficiency ...... 19 3 Overview of different cut-offs used to define vitamin B12 and folate deficiency in selected Studies, sorted by vitamin B12 ...... 26 4 (Continuation) Overview of different cut-offs used to define vitamin B12 and folate deficiency in selected Studies, sorted by vitamin B12 ...... 27 5 Equipment used for analyses ...... 40 6 Analysed variables and methods used ...... 42 7 Characteristics of the study population...... 53 8 Evolution of parameters related to vitamin B status over a one year period ...... 55 9 Evolution of related haematologic parameters over a one year period ...... 56 10 Evolution of lipid profile over a one year period ...... 57 11 Evolution of anthropometric parameters over a one year period . 59 12 Evolution of functional tests over a one year period ...... 60 13 Evolution of MMSE scores over a one year period ...... 61 14 Partial Pearsons Correlations (with time as control) coefficients and signficance level for selected blood parameters ...... 62 15 Partial Pearsons Correlations (with time as control) coefficients and signficance level between anthropometric parameters and lipid profile ...... 63 16 Characteristics of the study population ...... 64 17 Wilcoxon test (z) for Pre and Post mean values for sCbl and its absolute and relative change ...... 66 18 t-test for Pre and Post mean values (geometric and arithmetic) for holoTC, serum and RBC folate, tHcy, MCV, HCT, Hb and erythrocytes and their absolute and relative changes . . . . . 67

V 19 Summary table One-Way ANOVA with repeated measures and one ﬁxed factor (serum cobalamin cluster) for mean serum cobalamin (sCbl), holo transcobalamin (holoTC) and total homocysteine (tHcy) concentrations ...... 69 20 Number and percentage of elderly people who take one of the ATC group of medication ...... 72 21 Concentrations for the biochemical parameters by population . . 73

VI List of Figures

1 Chemical structure of vitamin B12 ...... 9 2 Absorption, transport and cellular uptake of cobalamins in mammals modiﬁed after [204] ...... 14 3 Biochemical pathway of homocysteine ...... 15 4 Methylmalonyl CoA mutase ...... 16 5 Study design for longitudinal study and distribution of drop outs in the course of follow-up ...... 38 6 Study design for intervention study ...... 38 7 Individual changes of serum cobalamin and HoloTC values after a 28 day supplementation with 500 µg oral cyanocabalamin ...... 65 8 Individual changes of serum tHcy values after a 28 day supplementation with 500 µg oral cyanocabalamin ...... 68 9 Change from pre to posttreatment mean serum cobalamin (sCbl) concentrations by clusters of initial sCbl concentrations ...... 69 10 Change from pre to posttreatment mean holo transcobalamin (holoTC) concentrations by clusters of initial serum cobalamin (sCbl) concentrations ...... 70 11 Change from pre to posttreatment mean total homocysteine (tHcy) concentrations by clusters of initial serum cobalamin (sCbl) concentrations...... 70 12 Percentages of participants outside and inside of the reference interval of tHcy, B12, HoloTC and folate separated by taking or not-taking medications ...... 74

VII

Nomenclature

AD Alzheimer’s Disease

ADL Activity of Daily Living

ATC Anatomical, Therapeutic, Chemical classiﬁcation system

CBC Complete Blood Count

CNS Central Nervous System

COM Centre of Mass

EDAD Encuesta sobre Discapacidad, Autonomía personal y Situaciones de Depen- dencia

Exernet Study Estudio multi-centrico para la Evaluación de los Niveles de Condición Física y su relación con estilos de vida saludables en población mayor Española no institucionalizada

FFB Fat Free Body Mass

HCT Haematocrit

IBP International Biological Programme

INE National Institute for Statistics (in Spanish, Instituto Nacional de Estadística)

ISAK International Society for Advancement of Kinanthropometry

MCV Mean Corpuscular Volume

MMSE Mini Mental State Examination

NHP Nursing Home Placement

PA Pernicious Anaemia

PAR Psychological Assessment Resources

VD Vascular Dementia

W-H ratio Waist-to-Hip ratio

WHO World Health Organisation

GLOSSARY

Ageing in place Having the mental and physical capability of living in one’s own home in old age

Arm strength Number of performed repetitions of the arm curl test

Elderly Term used according to the WHO deﬁnition for a person aged over 65 years

Grip strength Results of the dinamometry of the movement of the hand grip strength

Leg strength Number of performed repetitions of the “30 second chair stand” test

Person with disability Person with problems with one of the 44 activities of daily living according to the Encuesta sobre Discapacidad, Autonomía personal y Situa- ciones de Dependencia (EDAD) of the year 2008

ABSTRACTOFTHETHESIS

Prevalence of vitamin B12 deficiency is very common in elderly people and can reach values as high as 40.5% of the population. It can be the result of the interaction among several factors. Vitamin B12 deficiencies have been associated with neurological, cognitive deterioration, haematological abnormalities and cardiovascular diseases that have an important influence on the health of the elderly and their quality of life. It is necessary to approach the problems arisen from the lack of data relative to them. The main objective of this thesis was to analyse the evolution of vitamin B12 status and related parameters, lipid and haematological profiles and their relationship to health risk factors, and to functional and cognitive status over one year and to determine the effect of an oral supplementation of 500 µg of cyanocobalamin for a short period of 28 days. An additional objective was to analyze the possible effects of medicine intakes on vitamin B status. Three studies were performed: a) a one year longitudinal follow-up with four measure points; b) an intervention study providing an oral liquid supplement of 500 µg of cyanocobalamin for a 28 days period; and c) analysis of the possible effect of medication intake on vitamin B status using the ATC classification of medicines. The participants for these studies were recruited from nursing homes for the elderly in the Region of Madrid. Sixty elders (mean age 84 ± 7y, 19 men and 41 women) were recruited for Study I and 64 elders (mean age 82 ± 7y, 24 men and 40 women) for Study II. For Study III, baseline data from the initially recruited participants of the first two studies were used. An informed consent was obtained from all participants or their mentors. The studies were approved by the Ethical Committee of the University of Granada. Blood samples were obtained at each examination date and were analyzed for serum cobalamin, holoTC, serum and RBC folate and total homocysteine according to laboratory standard procedures. The haematological parameters analyzed were haematocrit, haemoglobin and MCV. For the lipid profile TG, total cholesterol, LDL- and HDL- cholesterol were analyzed. Anthropometric measures (BMI, skinfolds [triceps and subscapular], waist girth and waist to hip ratio), functional tests (hand grip, arm and leg strength tests, static balance) and MMSE were obtained or administered by trained personal. The vitamin B12 supplement of Study II was administered with breakfast and the medication intake was taken from the residents’ anamnesis. Data were analyzed by parametric and non-parametric statistics depending on the obtained data. Comparisons were done using the appropriate ANOVAs or non-parametric tests. Pearsons’ partial correlations with the variable “time” as control were used to define the association of the analyzed parameters.

XIII The results showed that: A) Over one year, in relationship to vitamin B status, serum cobalamin decreased, serum folate and mean corpuscular volumen increased significantly and total homocysteine concentrations were stable. Regarding blood lipid profile, triglycerides increased and HDL-cholesterol decreased significantly. Regarding selected anthropometric measurements, waist circumference increased significantly. No significant changes were observed for the rest of parameters. B) Prevalence of hyperhomocysteinemia was high in the elderly studied, ranging from 60% to 90 % over the year depending on the cut-off used for the classification. LDL-cholesterol values were high, especially among women, and showed a tendency to increase over the year. Results of the balance test showed a deficiency and a tendency to decrease; this indicates that the population studied is at high risk for falls. Lower extremity muscular function was deficient and showed a tendency to decrease. A highly significant relationship was observed between the skinfold of the triceps and blood lipid profile. C) Low cobalamin concentrations correlated significantly with low MMSE scores in the elderly studied. No correlations were observed between vitamin B12 status and functional parameters. D) Regarding vitamin B12 status, holo-transcobalamin seems to be more sensitive for diagnosis; 5-10% of the elderly had a deficiency using serum cobalamin as a criterion, and 45-52% had a deficiency when using serum holotranscobalamin as a criterion. E) 500 µg of cyanocobalamin administered orally during 28 days significantly improved vitamin B12 status and significantly decreased total homocysteine concentrations in institutionalized elderly. No effect of the intervention was observed on functional and cognitive parameters. F) The relative change (%) of improvement of vitamin B12 status was higher when using serum holo-transcobalamin as a criterion than serum cobalamin. G) Antiaenemic drug intake normalized cobalamin, urologic drugs and corticosteroids serum folate, and psychoanaleptics holo-transcobalamin levels. Drugs treating pulmonary obstruction increased total homocysteine concentration significantly. H) The daily mean drug intake was 5.1. Fifty- nine percent of the elderly took medication belonging to 5 or more different ATC groups. The most prevalent were psycholeptic (53%), antiacid (53%) and antithrombotic (47%) drugs.

XIV WHAT IS KNOWN ON THE TOPIC? WHAT DOES THIS PHD THESIS ADD? Data unknown about the evolution of Serum cobalamin decreased, serum folate vitamin B status over a one year period in and mean corpuscular volume increased institutionalized elderly. significantly and total homocysteine concentrations were stable over a one year period in institutionalized elderly. Data unknown about the evolution of upper Results of the balance test showed a and lower extremity functions and balance deficiency and a tendency to decrease capacity in institutionalized elderly over a indicating that the population studied is at one year period. high risk for falls. Lower extremity muscular function was deficient and showed a tendency to decrease and no correlations were found between vitamin B12 status and the functional tests. High oral administered doses of 1 mg and 500 µg of cyanocobalamin administered above and a dose of 500 µg for a treatment orally during four weeks significantly period of eight weeks are effective for improved vitamin B12 status and improving vitamin B status. significantly decreased total homocysteine concentrations. No effect of the intervention was observed on functional and cognitive parameters. Polypharmacy is common in the elderly, but The studied group of elderly people took a information is scarce about the type and total of 858 medications of 292 different quantities taken. brands, which is equivalent to a daily average of 5 medications per person. It is known that certain medications (such as Antianaemic drug intake normalized metformin, H2-antagonists and proton pump cobalamin, urologic drug and corticosteroids inhibitors) affect vitamin B status. serum folate, and psychoanaleptics holotranscobalamin concentrations. Drugs treating pulmonary obstruction increased total homocysteine concentration significantly. Holo-transcobalamin is discussed in the The relative change of improvement of literature as being a more sensitive marker vitamin B12 status was higher when using for diagnosing vitamin B12 deficiency serum holo-transcobalamin as the main compared to serum cobalamin levels. outcome. When choosing it as the criterion for deficiency, a higher prevalence (45-52%) compared to serum cobalamin (5-10%) was observed.

RESUMENDELATESIS

La prevalencia de deficiencia de la vitamina B12 es muy común en las personas mayores y el porcentaje puede llegar a ser de hasta un 40,5% de la población. La deficiencia de vitamina B12 ha sido asociada con el deterioro cognitivo y neurológico, anormal- idades hematológicas, y enfermedades cardiovasculares que tienen una influencia im- portante sobre la salud del anciano y su calidad de la vida. Es necesario abordar los problemas que surgen debido a la escasez de datos relativos a ellos, especialmente en ancianos institucionalizados. El objetivo principal de esta tesis fue describir la evolu- ción del estado de vitamina B12 y sus parámetros relacionados, de los perfiles lipídicos y hematológicos y sus relaciones con factores de riesgo para la salud, y con su estado funcional y cognitivo a lo largo de un año, y el efecto de una suplementación oral de 500 µg de cianocobalamina durante un periodo de 28 días sobre estos parámetros y marcadores de riesgo. Un objetivo adicional fue analizar los posibles efectos de la toma de medicamentos sobre el estado de las vitaminas B12 y folato. Se realizaron tres estudios: a) un seguimiento longitudinal a lo largo de un año con cuatro puntos de medición; b) un estudio de intervención suministrando un suplemento oral líquido de 500 µg de cianocobalamina durante un período de 28 días y c) un análisis del posible efecto de la ingesta de medicación sobre el estado de vitamina B utilizando el sistema ATC de clasificación. Los participantes de los tres estudios fueron reclutados en residencias para personas mayores de la Comunidad Autónoma de Madrid. Sesenta personas (edad media 84 ± 7a, 19 hombres y 41 mujeres) mayores fueron reclutados para el estudio I y 64 personas mayores (edad media 82 ± 7a, 24 hombres y 40 mujeres) para el estudio II. El estudio III, se realizó con los datos de los participantes reclutados inicialmente para el primer punto de los exámenes del estudio I y II. Se obtuvo un consentimiento informado de todos los participantes o de sus tutores. Fue aprobado por el Comité Ético de la Universidad de Granada. Las muestras sanguíneas se obtuvieron en cada punto de los exámenes y fueron anal- izadas para la cobalamina, HoloTC, homocisteina y folato (todos en suero), RBC folato siguiendo procedimientos estándares de laboratorio. Los parámetros hematológi- cos analizados fueron el hematocrito, la hemoglobina y el VCM. Para el nivel lipídico se examinaron los TG, el colesterol total, y el LDL y HDL-colesterol. Las medidas antropométricas (IMC, panículos [tríceps y subescapular], la circunferencia de la cintura, y la ratio cintura/cadera), las pruebas funcionales (prensión manual, fuerza de los brazos y de las piernas, equilibrio estático) y el MMSE de Folstein fueron obtenidas o administradas por un personal previamente entrenado. El suplemento de vitamina B12 del estudio II fue suministrado con el desayuno y el historial sobre la ingesta de

XVII medicación fue recogido de las anamnesis de los residentes. Los datos fueron analizados por pruebas parámetricas y no-paramétricas dependiendo de los datos obtenidos. Las comparaciones se hicieron utilizando los ANOVAs o pruebas no paramétricas. Se calcularon las correlaciones parciales de Pearson con la variable “tiempo” como control para describir las asociaciones entre los parámetros analizados. Los resultados mostraron que: A) A lo largo de un año, relativo al estado de la vitamina B, los valores de la cobalamina sérica disminuyeron, el folato sérico y el volumen corpuscular medio aumentaron de forma significativa y las concentraciones de homocisteína permanecieron estables. B) La prevalencia de hiperhomocisteinemia fue elevada en los ancianos estudiados, entre el 60% y el 90% a lo largo del año dependiendo del punto de corte utilizado para la clasificación. Los valores de LDL-colesterol fueron elevados, especialmente en las mujeres, y mostraron una tendencia a aumentar a lo largo de un año. Los resultados de la prueba del equilibrio estático mostraron una deficiencia y tendencia a disminuir; esto es indicativo de que la población estudiada presentaba un alto riesgo de caídas. La función muscular de las extremidades inferiores fue deficiente y mostró tendencia a empeorar. Se observó una relación altamente significativa entre los panículos del tríceps y el perfil lipídico. C) Las concentraciones bajas de cobalamina se correlacionaron de forma significativa con una menor pun- tuación del MMSE en los ancianos estudiados. No se observó ninguna correlación entre el estado de la vitamina B12 y los parámetros funcionales. D) En relación con el estado de la vitamina B12, la holo-transcobalamina parece ser más sensible para el diagnóstico: 5-10% de los ancianos presentaban deficiencia utilizando la cobalamina en suero como criterio, y 45-52% presentaban deficiencia cuando se utilizaba la holo- transcobalamina como criterio. E) La administración de 500 µg de cianocobalamina durante 28 días mejoró significativamente el estado de la vitamina B12 y disminuyó significativamente las concentraciones de la homocisteína total en los ancianos. No se observaron efectos de la intervención sobre los parámetros funcionales y cognitivos. F) El cambio relativo de mejora en el estado de la vitamina B12 fue superior cuando se utilizaba la holo-transcobalamina como criterio en vez de utilizar la cobalamina. G) La toma de medicamentos antianémicos normalizó los niveles de cobalamina, los cor- ticoesteroides y los farmácos urológicos normalizaron los niveles de folato sérico y los psicoanalépticos los niveles de holo-transcobalamina. Los fármacos contra la obstruc- ción pulmonar aumentaron de forma significativa los niveles de homocisteína. H) La toma media diaria fue de 5.1 medicamentos. Cincuenta y nueve por ciento de los ancianos tomaban medicación perteneciente a 5 o más grupos ATC diferentes. Los más utilizados fueron los psicolépticos (53%), antiácidos (53%) y antitrombóticos (47%).

XVIII ¿QUE SE CONOCE DE ESTE TEMA? ¿QUE AÑADE ESTA TESIS? Se desconocen los datos de la evolución de la La cobalamina sérica disminuyó, el folato vitamina B a lo largo de un período de un año sérico y el volumen corpuscular medio en personas mayores institucionalizadas. aumentaron significativamente y las concentraciones totales de homocisteína se mantuvieron estables a lo largo de un año en personas mayores institucionalizadas. Se desconocen los datos relativos a la Los resultados de la prueba de equilibrio evolución de la función de las extremidades mostraron una deficiencia y una tendencia a superiores e inferiores y la capacidad de disminuir lo que indica que la población equilibrio a lo largo de un año en las personas estudiada tiene un alto riesgo de caídas. La mayores institucionalizadas. función muscular de las extremidades inferiores resultó ser deficiente y mostró una tendencia a disminuir y no se encontraron correlaciones entre el estatus de la vitamina B12 y las pruebas funcionales. La administración oral de altas dosis de 1 mg 500 µg de cianocobalamina administrados o más, y una dosis de 500 µg durante un oralmente durante cuatro semanas mejoraron período de tratamiento de ocho semanas es significativamente el estatus de la vitamina efectivo para mejorar el estatus de la B12 y disminuyeron de forma significativa la vitamina B. concentración de homocisteína total. No se observó ningún efecto de la intervención sobre los parámetros funcionales y cognitivos. La polifarmacia es común en las personas El grupo estudiado de personas mayores mayores, pero la información relativa a los tomaba un total de 858 medicamentos de 292 tipos de medicamentos tomados es escasa. marcas diferentes, lo que es equivalente a un promedio de 5 medicamentos diarios por persona. Se conoce que ciertas medicinas (como la La toma de medicamentos antianémicos metformina, los antagonistas-H2, y los normalizaba la cobalamina, los urológicos y inhibidores de la bomba protónica) afectan al los corticoides el folato sérico, y los estatus de la vitamina B. psicoanalépticos las concentraciones de holo- transcobalamina. Los medicamentos para la obstrucción pulmonar aumentaban significativamente la concentración de homocisteína total. La literatura discute si la holo- El cambio relativo de la mejora del estatus de transcobalamina es un marcador más sensible la vitamina B12 fue mayor utilizando holo- para diagnosticar la deficiencia de la vitamina transcobalamina como resultado principal. B12 en comparación a los niveles séricos de Cuando se escogió este criterio para la cobalamina. deficiencia, se observó una prevalencia mayor (45-52%) que con la cobalamina sérica (5-10%).

INTRODUCTION

1 INTRODUCTION

1.1 Introduction to the Thesis

Worldwide, the proportion of people aged 60+ is increasing. As mortality and fertility decrease, median ages are increasing in nearly all countries, although the rate and pace of this rise vary considerably among the world regions [170]. Europe is considered to be one of the continents that will age fast over future decades. Specifically, Spain is among the top 20 oldest countries today with 22.2% ranking 18 on the list of those with the greatest proportion of the elderly over 60y [249]. The highest number of elderly people can be found in the autonomous regions of Andalucía, Cataluña and Madrid. At present, Europe’s baby boom generation of the 50s and 60s are already reaching age 50 and 60; in the near future, this cohort will enter the elderly group. In 2001, for the first time the proportion of elderly 65y+ exceeded (inflection point) the population younger than 15; today, in Spain, the proportions are 17% and 15%, respectively [3] [2] reflecting the present demographic shift to an old-age structure of the population. Spanish projections estimate the population of 65y+ to be 31.9% of the total population [3] in the year 2049 which corresponds to an absolute number of 15 million of elderly people. Since 1950, the ongoing increase in life expectancy can be attributed largely to improvements in survival after age 60 [254]. Old people, and even very old people, are a more and more important part of reality in Spain. Among various other European countries and Japan, Spain leads the list of countries with the highest life expectancies: Spanish women live 85 years on average and male life expectancy is currently set at 79 years [2]. Among the EU27 countries, life expectancies for Spanish women and men over 65y are with 22.4y and 18.3y, respectively, the second highest after France [3]. The National Institute for Statistics (INE) predicts 87y for Spanish women and 81 for Spanish men in 2030 and although calculated with slower declines in mortality than in the past, the United Nations predicts a life expectancy of 84.5y for the Western European and 81y for the total European population in 2050 [250]. According to some demographic projections, women and men in Europe will survive to become nonage- narians and centenarians. In the countries with the top levels of life expectancy, female life expectancy has increased for 160 years at a constant rate of nearly three months per year [195]. On average, women live longer than men; however, life expectancy has also risen linearly for men, although less rapidly. Improvements in survival that have led to increasing life expectancy are mainly attributed to the complex interaction of progress in income, nutrition, education, better living standards, sanitation, medicine and public health efforts [216] [195]. The possi-

1 bility of reaching an older age is raising the issues of whether longer lives will be lived in good health or disability. It raises questions such as how healthy will one be during the gained years and will one have to face longer periods of disability and frailty. There is some evidence that one is living longer because one is staying healthy longer. Increasing life expectancy does not necessarily mean that the period of disability will be longer. In effect, it appears that this period at the end of life is becoming somewhat shorter [104]. The proportion of people aged over 80 (considered as the threshold value for disability) will increment rapidly in all European countries over the coming decades. Age is the main factor that raises the risk of needing long-term care, which is of special importance to the age groups of 80+ or 85+. In Spain, in 2008, 7,632,925 people in total (4,394,624 women and 3,238,301 men) were over 65 years and 2,123,785 people in total (1,377,425 women and 746,360 men) were over 80 years [146]. At the beginning of 2011 the population of elderly people consisted of 8,092,853 people of whom the octogenarians were the group with the highest growth rate [3]. The rate of disability increases with age. According to the Survey on Disability, Personal Autonomy and Situtions of Dependency (Encuesta sobre Discapacidad, Au- tonomía personal y Situaciones de Dependencia (EDAD) [148]) from the year 2008; at age 80, more than half of the Spanish population experienced problems performing daily living activities [3]. But over the last decade, the age group of 80+ has shown a different tendency than the people between 65-75y. Whereas the percentage of people suffering some kind of disability in the age group of 65-75y decreased from 37.4% in 1999 to 31.2% in 2008 the percentage for the age group 80+ increased from 21.3% in 1999 to 26.7% of the total number of people suffering some kind of disability [148]. From a quantitative point of view, problems of mobility are the basis of the most frequent problems of elderly dependent people reﬂected in difﬁculties performing daily living activities (in terms of household managing) followed by tasks of personal hy- giene [83]. Increasing age does not have to result in a commensurate increase in care demand or long-term care expenditure, but a considerable increase in the numbers of those who need care over the following decades. Following the approach of Doblhammer and Ziegler [74] from the Rostock Center for the Study of Demographic Change there are two scenarios: an optimistic healthy- years-of-life scenario, which assumes that the years gained in life expectancy will be years without disability; and a pessimistic constant-caring scenario, which assumes that the years added will be years of disability. In any case, the greater number of oldest-old will imply a greater need for long term care but supposedly not reach the high values forecast under the pessimistic constant-caring scenario. At the same time, the proportion of middle-aged people who can provide care will decrease.

2 Today, nearly two-thirds of elderly Spanish people in need of care have some kind of family support, whereas about 22% live in a single household [83]. The number of elderly who will move to an institution is likely to rise over the coming years, partly as a result of individual preferences and partly because of a diminishing supply of private care, especially after 2030. At that time, the baby boomers (born between 1957 and 1977) will begin to need long-term care. Spain being a Southern European country will confront particular challenges [74]. At present, the rate of institutionalization is not very high because the family tradition is strong and the old are cared for by younger family members. The ratio of the number of people 85y+ relative to 100 of the people between 45-65y (ratio of family support) has increased progressively and is estimated to keep on increasing so that family resources available for old people’s care to secure adequate service in response to changing needs are decreasing [3]. Most elderly people prefer to remain in their homes because they are able to maintain the integrity of their social network, to preserve their environmental landmarks and to enjoy a higher quality of life [169]. Findings of a recent systematic review by Luppa at al. [169] suggested that predictors of nursing home placement (NHP) in the general population of developed countries are mainly based on underlying cognitive and/or functional impairment, and associated lack of support and assistance in daily living. Also, a meta-analysis of 2007 by Gaugler et al. [108] showed that the strongest predictors of admission to a nursing home in the U.S. were three or more Activities of Daily Living (ADLs) dependencies, cognitive impairment, and prior nursing home use. In fact, two of the most common diseases affecting the elderly are cardiovascular diseases and cognitive impairments. Dementia is the most common cause of cognitive impairment [37]. Two pathologically distinct subtypes of dementia, vascular dementia (VD) and Alzheimer’s disease (AD), constitute the vast majority of cases [1]. AD is the most common cause of dementia in the aged. The age-adjusted prevalence of AD increases exponentially after age 65 and the estimation is that 50% of women will be affected after 85 years [24]. VD is the second most frequent cause of dementia in the elderly after AD [88]. Stroke is a major cause of VD [51] [182]. Cognitive impairment has in most cases a multifactorial origin. Among the nutrition- related risk factors there are hypercholesterolaemia and its association with hypertension, low levels of antioxidants, amino acids, folate, vitamin B6 and vitamin B12 and high levels of homocysteine [110]. Hyperhomocysteinaemia is a risk factor for vascular disease and is associated with AD. Low levels of methionine, an essential amino acid that depends on a normal functioning of the one-carbon metabolism in which homocysteine is an intermediate product have been observed in plasma samples in AD patients in comparison to control subjects [89].

3 Cigolle et al. [55] found that about 32% of individuals aged 90+ had reported cognitive impairment in the U.S.A. The prevalence and proportion of severe stages of cognitive impairment increase with age [127]. Since the disease has a great impact on the quality of life of patient and caregiver alike, the need not only for adequate treatment but also for prevention is high. As pyridoxine, cobalamin and folate play an important role in the nervous system by interacting as coenzymes in the metabolism of methionine [231][186] research on the status of these vitamins is required to give further insights. The elderly represent a vulnerable group in terms of vitamin B supply, especially with regard to cobalamin deficiency. Age-related declines in vitamin absorption and impairment of liberating cobalamin from proteins, as well as an age-related increase in autoimmunity against intrinsic factor or gastric parietal cells are causes of deficiency which are common in the elderly [186]. Elevated homocysteine values as a consequence of vitamin B12 and/or folate deficiencies are also considered as an independent risk factor for cardiovascular diseases, another leading cause of morbidity and mortality in the elderly. Aging-related motor disability is another major health concern for the elderly. The mechanisms underlying impaired motor performance in old age are complex as the central and peripheral nervous systems as well as the muscle tissue itself are involved [162]. There is general consensus that the loss of muscle mass and strength due to aging has a negative impact on motor performance and on the ability to recuperate from falls, leading to a higher risk of fractures and dependency [162]. Thus, preventing falls and gait instability is a very important safety concern, and various programs to improve motor performance among the elderly are commonly built around measuring and addressing specific impairments, such as impaired balance capacity and reduced strength of arms and legs. It is widely accepted that keeping a good level of physical activity is a powerful means of obtaining long term benefits on muscle function, to reduce the frequency of falls, and to maintain independence and a high quality of life in older persons. To be able to perform ADLs keeps the elderly person independent, a very important issue, not only concerning “ageing in place” but also once transferred to a residential setting. In order to perform ADLs sufficient arm strength is needed to move things around and open cans, jars, etc., and leg strength for moving around and transfers. A high percentage of elderly people have to utilize all their muscle power in order to rise from a chair so that a small additional impairment in muscle function may dramatically change their lives from independent to dependent [90][139]. Power handgrip strength is recorded by clinicians as a quick and viable measure of impairment and function which can serve as a useful evaluation of hand strength status [69]. Hand grip strength was highly predictive of functional limitations and disability

4 25 years later among healthy 45 to 68y old men concluding that good muscle strength in midlife may protect people from old age disability by providing a greater safety margin above the threshold of disability [205].

1.2 Signiﬁcance of the Thesis

The present thesis addresses different aspects of the set of problems which have emerged as a consequence of the huge increase in the elderly population. Although ageing is a biological process and not a disease, it is recognized that the elderly present a greater facility for getting ill and for suffering disabilities; with advancing age people are more likely to develop chronic diseases that require medical care and sometimes reach a high degree of frailty and multiple diseases. Lack of specific data related to the functional, haematological, biochemical and cognitive status and medication intake of the elderly and the way in which this population group responds to treatments and medication make it necessary to address these aspects for their clarification. The functional importance of the group of B vitamins in relation to the problems of arteriosclerosis and anaemia, and the combined factors in relation to the deterioration of the nervous system and even more precisely in relation to cognitive impairment and dementias has to be stressed. The problems which have emerged as a consequence of the possible interplay are characteristic of this new situation. Bringing insight is essential for Public Health Policies. The future of Europe is reliant on its ageing population which among other aspects will have impact on health care costs. The rising proportion of old and very old people will have major long-term impact at an individual level as well as at society level [255]. Ageing populations are a challenge, but at the same time they provide an opportunity to think about how to live a longer, healthier life [255]. More people reach advanced age in which they could suffer some kind of difficulties. The situation is even more serious for the elderly who have been institutionalized in most of the cases due to their delicate health status or because of the need for special assistance and in some cases because of lack of direct family support. Health problems are important especially in advanced age and are forecast to increase due to the higher longevity. The significance of the present thesis which is presented in three sections is therefore to study these problems to get a better understanding of the ageing process. Study I focusses on the evolution of vitamin B12 status, lipid profile, anthropometric, functional and cognitive status over a one year study period. Study II describes a 28 day intervention with a 500 µg cyanocobalamin supplementation and Study III centres

5 on the effects of polypharmacy on the vitamin B12, folate and homocysteine status; all in relation to an institutionalized elderly Spanish population.

6 REVIEW OF RELATED LITERATURE

2 REVIEW OF RELATED LITERATURE

As was presented in the previous chapter, there is certain evidence that deficiencies of the vitamins of the vitamin B complex are risk factors for cardiovascular diseases and cognitive impairment especially affecting the population of elderly people. Despite the impressive ways vitamins function individually, oftentimes it is difficult to know which vitamin is responsible for a given effect because the nutrients are inter- connected; the presence or absence of one influence the absorption, metabolism and excretion of another. In this context the interaction between folate and vitamin B12 and/or the relationship between riboflavin and B6 could be mentioned. Deficiency of one nutrient can alter the action or create a deficiency of another [263]. Within the vitamins of the vitamin B complex, vitamin B12 is the vitamin whose absorption is most negatively affected as a consequence of different pathologies, a characteristic especially of the elderly. The main focus of the present review is on the functions, metabolism, problems of detection of deficiency and the consequences of their deficiencies. Maintaining functional integrity of the upper and lower extremities is necessary for performing a series of labours and tasks of everyday life and this integrity might be affected by the above mentioned deficiencies. The following review focusses as well on the effects of these deficiencies on the function of balance and the upper and lower extremities.

Figure 1: Chemical structure of vitamin B12

2.1 Deﬁnition of Vitamin B12 and Food Sources

Vitamin B12 is part of the vitamin B complex, a group of water-soluble nutrients. It is the biggest and has a molecular weight of 1355.4 Da. The term B12 is the generic

9 descriptor for all corrinoids; those are compounds having a corrin ring surrounding a central cobalt (Co) that is linked to a lower as well as an upper ligand. The lower ligand is a benzimidazole group which is bound to the corrin ring by means of a ribose- phosphatase group. The upper ligand is what distinguishes one chemical form from another. In the two different coenzymatically active cobalamin derivatives, the upper ligand is either a methyl or a 5-deoxyadenosyl group (Figure 1)[201]. Both are built in separated cell compartments; methylcobalamin in the cytosol and adenosylcobalamin in the mitochondria. In nature, two other forms of vitamin B12 also exist: hydroxycobalamin and aqua- cobalamin, where hydroxyl and water are bound respectively to the cobalt. Cyanocobal- amin, the synthetic form of vitamin B12, has cyanide bound to the cobalt and is commonly used for supplements and fortiﬁed foods. Hydroxy-, aqua- and cyanocobalamin are enzymatically activated to the methyl- or deoxyadenosylcobalamins in all mammalian cells. Corrinoids other than cobalamins, i.e. cobinamide have been shown to be present in stools and in the circulation, but no physiological functions in humans have been demonstrated so far [7][125].

2.2 Dietary Sources and Availability

Vitamin B12 cannot be synthesized by humans and therefore must be supplied via the diet. It is produced by most microorganisms [225], including bacteria and algae. The vitamin B12 produced by microorganisms gains access to the human food chain via incorporation into food of animal origin. Gastrointestinal fermentation aids the growth of these microorganisms. The vitamin is absorbed and concentrated mainly in animal tissues especially in the liver, the main store of this vitamin. Therefore, animal-origin food is the only natural dietary source for providing large amounts of vitamin B12 (see Table 1) [272]. Products derived from herbivorous animals, such as milk, meat, and eggs, are also important dietary sources except if the animal came from geochemically deﬁcient cobalt regions [181]. Vitamin B12 intake from eggs is generally high, because they are a highly consumed food item [102] although it is generally poorly absorbed (<9%) compared to other animal-origin foods [76]. Plant foods usually do not contain vitamin B12 unless they were in contact with vitamin B12-containing substances, exposed to vitamin B12 synthesizing bacteria, or fortiﬁed with vitamin B12 [18]. Certain plant foods such as dried green and purple lavers (nori) contain substantial amounts of vitamin B12, whereas other consumable algae contain none or only traces of vitamin B12. However, most of the consumable blue-green algae mainly contain pseudovitamin B12, which is inactive in humans [262][261].

10 Table 1: Sources of vitamin B12 in foods (modiﬁed from [61]) Food Vitamin B12 (µg/100g) Meats Beef 1.94-3.64 Beef brain 7.83 Beef kidney 38.3 Beef liver 69-122 Chicken 0.32 Chicken liver 24.1 Ham 0.8 Pork 0.55 Turkey 0.379 Dairy products Milk 0.36 Cheeses 0.36-1.71 Yogurt 0.06-0.62 Fish and sea food Herring 4.3 Salmon 3.2 Trout 7.8 Tuna 2.8 Clams 19.1 Oysters 21.2 Lobster 1.28 Shrimp 1.9 Other Eggs, whole 1.26 Eggs, whites 0.09 Egg yolk 9.26 Vegetables, grains, fruits None contain vitamin B12

2.3 Requirements for the Daily Intake of Cobalamin in the Elderly

The vitamin B12 requirements have been determined via different methodic approaches. On the one hand, by means of curative tests, the amount of vitamin B12, necessary to reverse an already existing megaloblastic anaemia, on the other hand the requirement was calculated by means of the storage quantity and the turnover rate. Apart from that, the vitamin B12 values of healthy and deﬁcient subjects provide an indication of the preferable quantity of supply. Under the condition of entire bioavailability, the minimial requirements for humans can be achieved with a daily intake of less than 1 µg vitamin B12. As with higher intake the utilisation rate of vitamin B12 decreases (IF-dependent reaction), the recommended vitamin B12 intakes vary between 1.4 and 3.0 µg/d due to the selection of different CV (10-20%) and health indicators (maintenance of haematological status or basal losses) [75].

11 Spain participated as one of thirteen countries in the European Nutrition and Health Report 2004 and also in 2009; the assessment of the nutritional average cobalamin intake of European elderly showed that the intake was 6.2 and 7.4 µg for Spanish women and men between 65 and 75 years respectively in 2004 and was the highest cobalamin intake from all participating countries [87]. In the European Nutrition and Health Re- port of 2009 the intake was 3.5 ± 0.5 µg for the elderly population over 64 years. Thus, the intake lies above the reference value of 1.4 µg/day recommended by the Scienitific Committee on Food [62] and also above 3 µg/day recommended by DACH 2000.A study which analyzed the dietary intake of an elderly institutionalized population in León (Spain) revealed that the average intakes of folate and vitamin B12 exceeded RDA, (103% and 144%) [106]. However, 45.83% of males and 5.97% of females showed deficiencies in vitamin B12 and 53.91% of the subjects showed deficiencies in folic acid intake [106]. Because of the usual dietary habits in South Europe, vitamin B12 is generally ingested in amounts exceeding the requirements. In line with other Southern European Countries, in Spain, the proportion of animal products in total energy supply greatly increased over the past four decades (from 13% in the year 1961 to 28% in 2003, respectively) whereas the proportion of vegetable products in total energy supply decreased (from 87% in 1961 to 72% in 2003, respectively) [78]. Although there is agreement upon the fact that vitamin B12 is not one of the critical nutrients in terms of requirement coverage via dietary intake, a longtime coverage of requirements through the depletion of human stores is not possible when suffering certain diseases such as gastrointestinal alterations.

2.4 Absorption, Storage and Excretion of Vitamin B12

2.4.1 Absorption and Storage

Cobalamins cannot be produced by higher organisms so that humans depend on the dietary intake. Mammals possess a complex pathway for absorption, transportation and cellular uptake of cobalamin (Figure 2). There are two pathways for vitamin B12 absorption; an intrinsic factor (IF) dependent absorption and passive diffusion.

IF Dependent Absorption

IF-dependent absorption is an active process, which needs a normally functioning stomach, presence of IF, pancreatic enzymes, and an intact terminal ileum. Vitamin B12 in food is attached to protein and is liberated from dietary protein in the stomach via the acid milieu (high concentration of hydrochloric acid) and pepsin. Once released, free vitamin B12 binds to R protein in the stomach. R protein (or R-binder) is a

12 haptocorrin present in saliva, gastric juice, bile, intestinal juice, and serum which protect vitamin B12 from denaturation in the stomach. In the alkaline milieu of the small intestine, pancreatic enzymes degrade the R protein liberating vitamin B12 which then builds a complex with IF (60-kDa glycoprotein produced by gastric parietal cells after stimulation by food). The vitamin B12-IF complex is stable and enters the ileum. For the B12-IF complex to attach to the receptor site, ionic calcium and a pH greater than 6.0 are needed. Vitamin B12 is transported into the mucosal enterocytes against a concentration gradient, so it is an active process and saturable as well. Various studies indicate that the normal capacity of this system is to absorb about 1.5-2.0 µg of vitamin B12 per meal due to saturation of the ileal receptors [226]. After a few hours of recuperation, the system can absorb an alike amount of vitamin B12 as a subsequent event [226]. Inside the enterocytes, the IF is degraded and the liberated Cbl links to transcobalamin (TC) [202] [188]. The TC-Cbl complex is then released into the plasma, where it is endocytosed via membrane receptors of the target cells. Passive Absorption Vitamin B12 can also be absorbed passively via diffusion in a non-IF mediated man- ner. This has been demonstrated by the fact that patients who suffered from Pernicious Anaemia and therefore had no IF function absorbed a proportion of an administered dose. The rate of the passive diffusion is apparently directly correlated to the quantity administered. Chanarin [43] [44] concluded that once the saturation of the IF-mediated active absorption system at about 1.5-2.0 µg per meal or test dose is reached further cobalamin is absorbed by passive diffusion. Urinary excretion tests have been used to determine the quantity absorbed by diffusion and calculated the rate to be approximately 1%.

Vitamin B12 Binding Proteins Three circulating plasma vitamin B12 binding proteins exist: namely transcobalamin (TC) I, TC II, and TC III. TC I links to around 80% of the circulating vitamin B12, while TC II links to less than 20%. Nevertheless, vitamin B12 enters cells throughout the body mainly linked to TC II. TC II links to 7%–20% of the endogenous cobalamin

[81] [121] and mediates 33%–99% of the total plasma vitamin B12 clearance [6] [120]. Although TC I links 80%–90% of the endogenous cobalamin, TC I mediates less than 1% of the total cellular uptake of vitamin B12 from plasma [5] [6] [95] [114] [123] [149] [229] [230].

Estimates of total-body vitamin B12 stores vary from 2.0 to 5.0 mg [4] [115] [210], of which approximately 60% are stored in the liver, 30% in the muscles and the rest in other tissues. The mean biological half time of the storage are 485 days. Each day

0.143% are eliminated. For the maintenance of high vitamin B12 stores of 5 mg, a

13 Figure 2: Absorption, transport and cellular uptake of cobalamins in mammals modiﬁed after [204] daily intake of 7.2 µg B12 and for the maintenance of stores of 2 mg, a daily intake of around 2.9 µg is needed.

2.4.2 Enterohepatic Circulation and Excretion of Vitamin B12

In hepatocytes, vitamin B12 is added to bile that flows to the small intestine. As indicated before, vitamin B12 attaches to IF and thereafter two thirds are reabsorbed [115] and returned to the liver in the portal blood. Although both Green et al. [116] and Teo et al. [244] suggested that bile enhances vitamin B12 absorption, the enterohepatic circulation of vitamin B12 is dependent on the presence of intrinsic factor. In the absence of intrinsic factor, all the vitamin B12 from the bile is excreted into the stool instead of being recirculated. Individuals with pernicious anaemia, with total absence of IF, develop vitamin B12 deficiency rapidly, in approximately 1–3 years, compared with having vitamin B12 deficiency due to other causes [9] [95] [155]. The vitamin B12 which is not absorbed from the bile is excreted in the feces (average loss about 0.4 µg/day), thus making bile the principal way of eliminating the body of excessive vitamin B12 and removing potentially hazardous vitamin B12 analogues [14] [119]. Vitamin B12 in the feces comes from non-absorbed dietary vitamin B12 or vitamin B12 from bile, desquamated cells, gastric and intestinal secretions, and vitamin B12 synthesized by intestinal bacteria. After high dose supplementation amounts exceed plasma vitamin B12 binding capacity, vitamin B12 is also excreted through urine. Other routes of vitamin B12 loss are through the skin and other body secretions. The amount of vitamin B12 excreted from the body (turnover rate) is fixed at 0.1%–0.2% of total body stores daily, regardless of the pool size [9] [28] [29] [133] [211] [210].

14 Figure 3: Biochemical pathway of homocysteine

Analyses by Heinrich et al. in the late 60s [128] of the last century showed that the oral intake of 0.1-500 µg amount of 60Co-cyanocobalamin was retained quantitatively in the whole body and was exactly the same amount of the intestinal resorbed 60Co- cyanocobalamin quantity. When the retention capacity is exceeded, the proportion of vitamin B12 eliminated with urine increases with rising application rates.

2.5 Functions of Vitamin B12

Vitamin B12 functions as a cofactor for two enzymes: methionine synthase (EC 2.1.1.13), which is involved in methionine biosynthesis, and L-methylmalonyl coenzyme A (CoA) mutase (EC 5.4.99.2), which is involved in amino acid and odd-chain fatty acid metabolism in mammalian cells [46] [92]. Function of Methylcobalamin After having bound to a speciﬁc receptor in the presence of Ca2+ cobalamin enters the cells as a complex with transcobalamin via endocytosis. There, transcobalamin is decomposed proteolytically and cobalamin is liberated into the cytosol as trivalent Cbl (Cbl3+) and afterwards it is reduced to Cbl2+. The latter is bound to the apomethion- insynthase, begins its enzymatical activity and functions as a methyl-transmitter in the synthesis of methionine from homocysteine, in which methyl-THF is the real methyl donor (Figure 3). Function of Adenosylcobalamin Adenosylcobalamin is formed in the mitochondria from cobalamin after reduction of Cbl2+ to Cbl1+(reductive system within the mitochondria) through transfer of adenosyl

15 Figure 4: Methylmalonyl CoA mutase from ATP with seperation of triphosphate (see Eq 1).

Cbl1+ + ATP → Adenosylcobalamin + PPP (1)

Adenosylcobalamin participates in two intramolecular transmission processes:

1. Transfer of methymalonyl-CoA to Succinyl-CoA by methylmalonyl CoA mutase (Figure 4). With the help of this reaction propionic acid, which is formed during the degradation of odd-numbered fatty acids or the amino acids methionine, threonine and isoleucine, and methylmalonyl-CoA, which is formed during the degradation of valine, gain access to the citric acid cycle.

2. Reversible transfer of leucine to 2-aminoisocapronacid (ß-leucine) by the L-α leucine mutase. In this reaction, the aminoacid group moves from C-atom 2 to C-atom 3 in exchange with a hydrogen atom.

2.6 Prevalence of Vitamin B12 Deﬁciency in the Elderly

Vitamin B12 concentrations decline with age, whereas metabolite levels, namely homocysteine and methylmalonic acid increase indicating an impairment of vitamin B12 status. The increased prevalence of vitamin B12 deﬁciency in this population group is due to different factors which include the presence of pernicious anemia (type A atrophic gastritis) and type B atrophic gastritis. The prevalence of both conditions increases with age. The prevalence of subdeﬁcient vitamin B12 levels in the elderly which can be found in the literature differs from 3.0% to 40.5%, depending on the diagnostic criteria chosen [20] [21] [27] [31] [39] [48] [65] [79] [80] [47] [107] [118] [124] [154] [173] [177] [192] [199] [270] [269].

2.6.1 Factors Contributing to Poor Vitamin B12 Status with Ageing

Pernicious Anaemia Pernicious anaemia (PA) is the most common cause of clinically apparent vitamin

B12 deﬁciency in North American and European populations. PA is the ﬁnal stage of

16 autoimmune gastritis, type A chronic atrophic gastritis or gastric atrophy, in which the fundus as well as the body of the stomach are involved. These are the places where the acid-secreting parietal cells and pepsinogen-secreting zymogenic cells are located. When suffering pernicious anaemia, autoantibodies against parietal cells cause loss of gastric parietal cells whose continuous destruction from the gastric mucosa results in impaired IF secretion. Also, blocking antibodies in the gastric juice can link to the vitamin B12 binding site of IF to prevent the formation of the vitamin B12–IF complex. In another less common form of PA, the antibodies allow vitamin B12 to bind to the intrinsic factor but prevent the absorption of the intrinsic factor–vitamin B12 complex by the ileal receptors. Thus, in pernicious anemia, vitamin B12 deﬁciency develops by several mechanisms [247]. Achlorhydria, low serum pepsinogen I concentrations, and high serum gastrin concentrations caused by hyperplasia of gastrin-producing cells are found in type A gastritis. In addition to causing malabsorption of vitamin B12 from the diet, PA also results in an inability to reabsorb the vitamin B12 secreted into the bile (~ 0.3 and 0.5 mg/day) within the enterohepatic circulation of vitamin B12. This results in an even more signiﬁcant negative balance for the vitamin. Like most autoimmune diseases, the incidence of PA increases markedly with age. The mean age at diagnosis of pernicious anemia is 60 years. The female-to-male ratio is approximately 1:5. In Caucasians, the prevalence of the disease rises with increasing age, peaking after age 65 [44].

Atrophic Gastritis and Food-Bound Vitamin B12 Malabsorption Type B chronic atrophic gastritis mainly involves the gastric antrum and is associated to Helicobacter pylori infection. The gastric antrum is initially affected, but later on the gastritis extends to the body of the stomach. Subclinical vitamin B12 deficiency with aging is caused primarily by type B atrophic gastritis accompanied by low acid-pepsin production and food-bound vitamin B12 malabsorption. In addition, hypochlorhydria leads to intestinal bacterial overgrowth, which interferes with vitamin B12 absorption. Therefore, malabsorption of protein-bound vitamin B12 is caused by both mechanisms in individuals with atrophic gastritis and leads to a decreased vitamin B12 status [159] [222] [242]. However, the absorption rate of crystalline vitamin B12 does not decrease in type B atrophic gastritis, as intrinsic factor continues to be produced in sufficient amounts [77] [179]. Alteration with ageing in the functional and structural integrity of the vitamin B12 binding proteins resulting in compromised TC II–B12 delivery system has also been suggested to be a factor in reducing vitamin B12 status in the body [173]. There are only a few cases known of an inborn deficiency of the transport protein Transcobal- amin II. This disease manifests itself in the first weeks of life and leads to – despite

17 often physiological serum cobalamin levels - severe megaloblastic anaemia. Very high administrations of hydroxycobalamin result in the relief of symptoms.

2.6.2 Other Causes and Effects of Vitamin B12 Deﬁciency in the Elderly

Poor vitamin B12 dietary intake is not very common in the elderly. Because of the high vitamin B12 stores and the low turn-over rate, vitamin B12-hypo- or rather avi- taminosis only occur after longterm malnutriton and/or undernourishment or when the gastrintestinal tract absorption is disturbed for a long time (food-bound vitamin B12 malabsorption) and therefore the stores are depleted. Impaired gastric acid production caused by type B atrophic gastritis accompanied by bacterial overgrowth is the underlying mechanism of food-bound vitamin B12 malabsorption in this population. Type A atrophic gastritis (pernicious anemia) and total or rather partial gastrectomy cause deficient intrinsic factor, leading to vitamin B12 malabsorption. Other, infre- quent causes of vitamin B12 malabsorption in the elderly are malabsorptive status after intestinal bypass, exocrinic pancreatic insufficiency, terminal ileal disease i.e. in Morbus Crohn, disturbed cleavage of the vitamin B12-haptocorrin-complex in the duodenum, lymphoma, radiation enteritis, intestinal tuberculosis, infestation with Di- phyllobothrium latum, severe celiac disease, and tropical sprue, Blind-Loop syndrome, extensive parasite decay (for example fish tape worm), as well as a selective inherent B12 resorption disturbance (Imerslund-Gnasbeck-syndrome). Vitamin-Pharmaka-Interactions Beside the mentioned changes of the stomach physiology, a series of drugs (such as colchicine), alcohol or dependence-causing substances impair vitamin B12 metabolism. The set of problems of the vitamin-pharmaceutical-interactions are often neglected, usually discovered by chance, and often long after the drug entered the market. These interactions are even more complex in polypharmacy therapy, often found among old, multimorbid people. Drugs which increase the gastric pH may reduce the absorption of vitamin B12 and therefore produce deficiency by impairing the release of protein-bound vitamin B12 [221] [239]; this is well-reported for the proton pump inhibitor omeprazole [172] [33] and also for histamine-2 receptor antagonists [103] [220]. However, whereas the absorption of protein-bound vitamin B12 is decreased, the absoprtion of cristalline vitamin B12 remains uninfluenced, a fact that is of practical relevance. Other drugs, which negatively influence vitamin B12 absorption are the lipid-lowering cholestyramine, the antibiotika chloramphenicol, clioquinol and neomycin as well as the antidiabeticum metformin [70]. In addition, nitrous oxide rapidly oxidises cobalt

18 bound to methylcobalamin and therefore inactivates methionine synthase resulting in higher homoysteine levels. Elderly people with impaired vitamin B12 and/or folate levels may be notably vulnerable to nitrous anaestesia, which is reported in several case reports.

2.7 Clinical and Subclinical Manifestations of Vitamin B12 Deﬁciency in the El- derly

Vitamin B12 deﬁciency is characterized by the following clinical picture.

Table 2: Clinical Manifestiations of Vitamin B12 Deﬁciency Hematologic Megaloblastic anemia Pancytopenia (leucopenia, thrombocytopenia) Neurologic Paresthesias Peripheral neuropathy Combined systems disease (demyelination of dorsal columns and corticospinal tract) Psychiatric Irritability, personality change Mild memory impairment, dementia Depression Psychosis Cardiovascular Possible increased risk of myocardial infarction and stroke

2.7.1 Neurologic Effects of Vitamin B12 Deﬁciency

Neurologic complications are found in 75%–90% of individuals with clinically apparent vitamin B12 deficiency. The presence of neurologic findings caused by vitamin B12 deficiency is negatively correlated with the degree of anaemia, which means that individuals presenting severe anaemia showed fewer or no neurologic manifestations and vice versa [126][223]. In contrast to what was previously thought that neurological as well as the psychiatric complications only occur at a later stage of vitamin B12 deficiency, these disturbances can predate haematological anomolies by several months or even years or even occur in the absence of hematologic complications. The spinal cord, brain, optic nerves and peripheral nerves may all be affected by vitamin B12 (cobalamin) deficiency, giving rise to one of the classic neurologic syndromes. The neuro-psychological symptoms can be differentiated into subacute combined degeneration of spinal cord, encephalopathy (perniziosa psychosis) and polyneuropathies.

The spinal cord is usually affected ﬁrst and often exclusively. The term subacute combined degenereation (SCD) is customarily reserved for the spinal cord lesion of

19 vitamin B12 deficiency and serves to distinguish it from other types of spinal cord diseases that happen to involve the posterior and lateral columns (loosely referred to as combined system disease). Whether the peripheral neuropathy is a primary component of the disease or is secondary to damage of the fibers of entry in the spinal cord has been debated, but the available pathologic evidence favors the former. The symptoms are symmetric parestisias, acrodistal disturbances, sensory disturbances in the extremities (paresthesia or numbness), motor disturbances alone, especially gait ataxia as well as elevated reflexes and spasticity. The nervous system involvement on subacute combined degeneration is roughly symmetrical and sensory disturbances precede the motor ones; predominantly motor involvement from the beginning and a definite asymmetry of motor or sensory findings maintained over a period of weeks or months should always cast doubt on the diagnosis. Mental signs are said to be frequent, ranging from irriability, apathy, somnolence, suspiciousness and emotional instability to a marked confusional or depressive psychosis or intellectual deterioration. The psychiatric symptoms are acute disorientation in combination with apathy, hallucinations, paranoia and frank psychosis, violent behavior and changes in personality are not common, but vitamin B12 deficiency should be considered as a possible cause of these symptoms [117] [241][126] [223][273] . Anosmia, fecal and urinary in- continence, leg weakness, impaired manual dexterity, and impotence are less frequent symptoms. Rare symptoms are orthostatic lightheadedness, diminished taste, paranoid psychosis, and diminished visual acuity [126]. In 0.5% of cases, visual impairment was found, which might be related to optic atrophy and retrobulbar neuritis or pseudo- tumor cerebri [238].

2.7.2 Vitamin B12 and Cognitive Impairment

Cognitive decline may occur, ranging from concentration impairment to loss of memory, disorientation, and frank dementia, including or not mood changes. Dementia increases at an remarkably rate and becoming a public health problem impacting health care expenditure. It is well known that age is a risk factor for dementia [42]; and the prevalence increases with advancing age [37]. In Europe, prevalence of dementia among elders of 65+ is approximately around 10% [110]. Preservation of cognitive ability well into old age is important not only in terms of promoting adequate health status but also regarding the postponement of the onset of dementia and decreasing its progress. Among the non-genetic and thus modiﬁable risk factors, increased homocysteine levels in addition to poor folate, vitamin B12 and B6 status, all involved in the control of homocysteine, have been found to correlate with impaired cognitive performance [212][259]. The prevalence of high total serum homocysteine depends on age and hyperhomocysteinemia is common in the elderly [113]. Poor status of

20 B vitamins (folate, vitamin B6 and vitamin B12) may play a role in pathogenesis of cognitive impairment in the elderly via hyperhomocysteinemia. Both folate and vitamin B12 are needed for the methylation of homocysteine to methionine and in the re-methylation and synthesis of S-adenosylmethionine, a major methyl donor in the central nervous system. Thus current research suggests that hyperhomocysteinemia caused by an impaired monocarbon metabolism may contribute to cognitive impairment and Alzheimer’s disease [59] and can be considered a sensitive marker of cognitive impairment. On the other hand, raised plasma homocysteine concentrations are a sensitive marker for cobalamin and folate deficiency. Findings suggest that decreased vitamin B12-dependent transmethylation reactions might be involved in the pathogenesis of dementia [243]. Hence, increased homocysteine levels in association with low levels of folate, vitamin B6 and vitamin B12, show significant correlation with decreased performance on cognitive tests [212][259]. For these reasons, B vitamin supplementation has been shown to prevent or reverse cognitive decline. However, the effect of vitamin B12 oral supplementation shows heterogeneous results and is found to be related to the dose, route of administration and duration of treatment. A few trials have been conducted to assess the effect of vitamin B12 supplementation on cognitive function in humans [142][84], with duration of supplementation varying from 4 wk to 6 months. For most of the cognitive tests performed, no improvement was observed for vitamin B12. Only a few cognitive tests showed statistically significant improvement [142]. At least two studies are available in the literature showing statistically significant worsening of cognitive functions after vitamin B12 supplementation [142][84]. Cognitive impairment is multifactorial in origin sharing modifiable and non-modifiable risk factors. As there are no curative treatments for it, the only possibility is that treatment may delay or slow down disease progression. It is highly appropriate to detect such modifiable risk factors like hyperhomocysteinemia, vitamin B12 and folate deficiency, etc. In the absence of curative treatment for dementia, vitamin B12 and folate may be relevant to the clinical course of dementia and should be considered for therapeutic intervention. However, more therapeutic research is needed. Clinical trials must be initiated in high risk populations to determine whether, lowering of blood homocysteine levels by vitamin B supplementation reduces the risk of cognitive impairment, the clinical onset of dementia. Longitudinal studies should also be undertaken to explore the association between nutrition status and cognitive impairment.

2.7.3 Haematologic Effects of Deﬁciency

Vitamin B12 deﬁciency leads inevitably to disturbances of vitamin B12 metabolism processes because of insufﬁcient amounts of active coenzymes (adenosyl- and methylcobalamin) which again results in impaired transmethylation processes by what the

21 close-linked metabolic pathways of folate, methionine and purinenucleotides can be affected (methyltrap-hypothesis). Main symptoms are specific morphologic changes in blood and bone marrow. Erythropoiesis reacts quickly and sensitively to the blocked nucleic acid metabolism because of its high turnover rate. The disturbed DNS replication influences the nucleus maduration, whereas the development of the cytoplasma nearly takes course normally resulting in oversized forms (megalocytes or rather pre-stages of erythrocytes [mega- loblasts]) as well as the hyperchromic macrocytic megaloblastic anaemia (pernicious anaemia, Morbus Addison, Morbus Biermer). In the periphery more or less changed cells can be verified. The mean corpuscular volume (MCV > 110 fl; macrocytosis) and the mean haemoglobin level of erythrocytes (MCH > 40 pg; hyperchromasy) are elevated. The megaloblastic changes of erythropoiesis in the bone marrow can be caused by cobalamin and folate deficiency. A morphologic differential diagnosis is not possible. Granulopoiesis and thromboisis can be affected as well. Megaloblastic anaemia can be rarely traced to inadequate dietary intake. Atrophy of parietal cells of the stomach mucosa is common with a consequent achlorhydria. Auto- antibodies against the parietal cells and against the intrinsic factor (IF) as well as IF deficiency due to a gastrectomy are the main causes for pernicious anaemia, the classsi- cal vitamin B12 deficiency disease. Serum cobalamin levels decrease under 200 pg/ml in the manifest state. The clinical symptoms of pernicious anaemia are skin paleness and mucosa, Hunter’s Glossitis, frailty, fatigue, lack of drive and vertigo.

2.7.4 Cardiovascular Effects of Deﬁciency

Cardiovascular diseases are a problem in the elderly and one of the most important causes for mortality. Among the risk factors, high levels of total cholesterol LDL- cholesterol and triglycerides and low levels of HDL-cholesterol, hypertension and more recently high levels of homocysteine have to be mentioned. Regarding the latter, studies have shown that elevated homocysteine is a risk factor for cardiovascular, cerebrovascular and peripheral arterial disease [141][30][17]. Wald et al. [258] found that for every 5-µmol/L increase in serum homocysteine concentration, the risk of ischemic heart disease increased 20% to 30%. Among the causes of elevated plasma homocysteine we can point out the deﬁciency of the B vitamins and renal impairment. Several possible mechanisms for the association between homocysteine and atherosclerosis have been demonstrated in experimental models. These include stimulation of smooth muscle growth, reduction in endothelial cell growth, impaired endothelial cell relaxation, decreased synthesis of high-density lipoprotein, promotion of autoimmune response, and accumulation of inﬂammatory monocytes in atherosclerotic plaques

22 [17][271][268]. Hcy is considered as a risk factor but recently in the literature there has been a divergence of opinion. Studies were performed for investigating if the risk for cardiovascular diseases onset can be reduced by lowering elevated homocysteine levels. Although the studies suggest that higher B-vitamin intake correlates with less risk for vascular disease and its sequelae, there is uncertainty as to whether it is folic acid, vitamin B6, or vitamin B12 that is responsible, and also whether supplements would provide the same protective beneﬁt as the presence of these nutrients in a varied diet. Those studies, of primary and secondary prevention nature, have had mixed results. Some trials of homocysteine-lowering therapy taking folic acid or other B vitamins showed positive results whereas other randomized controlled trials of supplementation showed no evidence. In addition, a recent Cochrane Database review of eight randomized controlled trials in patients at low risk receiving B-complex vitamins did not ﬁnd a lower risk of myocardial infarction (fatal or nonfatal), stroke, or death from any cause in patients [174]. However, most patients for these trials were recruited regardless of homocysteine levels; most of them had normal baseline plasma homocysteine levels.

2.7.5 Cognitive Impairment and the Function of the Neuromuscular System

The cognitive system plays an important role in helping to appropriately interpret the incoming sensations and plan the subsequent motor response. This system, which comprises the processes of attention, memory storage, and intelligence, provides us with the collective ability to anticipate or adapt our actions in answer to changing task demands and the environment. Age-related impairment of the sensory and motor systems negatively affects older people’s balance and mobility. The same effect was observed for changes in the cognitive system. In fact, as has been stated above at least 10% of all people over the age of 65 and 50% of those older than 80 have some form of cognitive impairment, ranging from mild deﬁcits to dementia. Adverse alterations of the processes of attention, memory, and intelligence are most likely to affect in older people’s ability to anticipate and adapt to changes in the environment. Any decline in cognition or attention will severely compromise one’s ability to ac- curately perceive what type of response is needed and implement the response, or responses, once selected. Not only has it been well documented that older adults with cognitive impairment showed an accelerated impairment of function following an acute illness or hospitalization, but they also experience many more falls than their not-cognitively impaired peers.

23 Balance can be deﬁned as the process by which one controls the body’s center of mass (COM) in terms of the base of support independently if it is stationary or moving. Although one often considers that standing upright in space is a static balance task and leaning through space or walking are dynamic balance tasks, it has to be remembered that keeping a stable upright position involves as well the active contraction of different muscle groups to control the position of the COM against the destabilizing energy of gravity. There is evidence to indicate that the ability to maintain balance easily, no matter whether static or dynamic depends upon the function of the mechanisms in the semicircular canals; the kinesthetic sensations in the muscles, tendons, and joints; visual perception while the body is in motion; and the ability to coordinate these three sources of stimuli [150]. Balance is an important ability which one needs during daily activities (i.e. walking and standing, as well as in most games and sports). Multiple systems contribute to our ability to maintain balance in standing and moving environments. First, the various sensory systems (i.e. vision, somatosensory and vestibular) give one information arising from the surrounding environment and resulting from one’s own actions. This information is of special interest for successful goal- directed action planning as well as the subconscious or automatic adjustments needed to maintain a given position in space or respond rapidly to a change in task or environmental demands. Between the ages of 50 and 70 muscle strength has been shown to decline by around 30% with even higher declines experienced after age 80. This decline is thought to be largely the consequence of a decreased size and number of muscle ﬁbers. Physical inactivity also contributes to the loss of muscle strength, particularly in antigravity or postural muscles required for good upright posture. Posture refers to the biomechanical alignment of the individual body parts as well as the orientation of the body to the environment. A good posture is crucial for good balance. When one is standing quietly in space, one’s goal is to align each body part vertically and thereby to spend the least amount of internal energy needed to maintain an upright and stable position relative to gravity. To counteract the energy of gravity, a number of muscles are active during quiet standing. Anticipatory postural control refers to those actions that can be planned beforehand, whereas reactive postural control referes to those situations that cannot be planned in advance of actions being required. Reactive postural control is needed when we have to respond quickly to an event we did not expect. Mobility is the ability to transfer oneself independently and safely from one place to another. Adequate levels of mobility are required for many different types of activities we perform in our daily lives. These may include transfers, e.g. rising from a chair,

24 climbing or descending stairs, walking or running and other types of recreational activities (e.g. gardening, sport, dancing). As already mentioned in chapter 1, impaired mobility is another major concern for the elderly and to keep their independence and to perform ADL sufficient strength in hands, arms and legs must retained. The programmes to improve mobility are commonly built around measuring and addressing specific impairments, such as balance capacity and the reduced strength of arms and legs. Data from our research group have found how strength values and MMSE were positively associated with hand grip strength, and arm and leg strength (p < .01) (in review in Ageing and Mental Health). Structural and functional changes that occur within the Central Nervous System (CNS) with advancing age appear to have the most profound and observable effect on motor function as a whole. And at a behavioural level, these cumulative changes as a consequence of the ageing nervous system [that can be aggravated as a consequence of vitamin B12 deficits] appear to manifest them- selves in a reduced ability to perform a variety of complex movements that require speed and accuracy, balance, strength, or coordination and are necessary to maintain independence and to be able to perform utilitarian or basic ADLs. Handgrip strength is recorded by clinicians as a quick and viable measure of hand impairment and function that provides a useful evaluation of the hand status [69]. Poor hand strength as measured by handgrip is a predictor of disability in older people. The handgrip strength test was highly predictive of functional limitations and disability 25 years later among 45 to 68y old men concluding that good muscle strength in midlife may protect people from old age disability by providing a greater safety margin above the disability threshold [205][180]. When multiple systems become impaired, the quality of the interaction between the impaired systems declines and results in observable motor dysfunction. Adverse changes occurring in the visual system make it more difficult to use visual information for balance, information from the somatosensory and vestibular systems could be used to compensate for the impaired visual system in most situations this would not be the case when these systems are also impaired as in the case of a general nervous system deterioration.

2.8 Laboratory Diagnosis of Vitamin B Deﬁciency

Most people with suspected vitamin B12 deﬁciency do not present anaemia as the clas- sical sign, but do show vague non speciﬁc symptoms such as neurologic or intestinal complaints. That´s why several laboratory tests should be considered.

25 Table 3: Overview of different cut-offs used to deﬁne vitamin B12 and folate deﬁciency in selected Studies, sorted by vitamin B12

n Age B12 bc HoloTC bc MMA bc tHcy bc sFolate bc RBC folate bc No. References City (Country) a (all; m, f) (y) (pmol/L) (pmol/L) (µmol/L) (µmol/L) (nmol/L) (nmol/L) 1 Joosten et al. 1993 [154] (BE, DE, NL) 350; 135, 215 61 - 97 d 103 0.247 13.9 5.4 2 Naurath et al. 1995 [187] 13 centres (BE) 285; 105, 180 65 - 96 d 103 0.247 13.9 5.4 3 Bjorkegren and Svardsudd 2001 [25] Skutskär (SE) 224; 94, 130 78 (77.2 - 78.9) e 115 0.37 15 3.7 275 4 Ray et al. 2000 [206] Toronto (CA) 711; 403, 308 58 f 120 15 215 5 Goringe et al. 2006 [112] Cardiff (GB) 49 < 75 125 38 0.47 15 6 Bor et al. 2006 [32] Copenhagen (DK) 98; 0, 98 41 - 75 d 130 50 0.28 11.9 7 Herrmann et al. 2000 [131] Saarland (DE) 232; 79, 153 69 (19 - 102) g 132 0.271 15 6.4 8 Lloyd-Wright et al. 2003 [166] Oxford (GB) 195; 195, 0 44 (18 - 78) g 135 50 0.28 12 6.8 London (GB) 9 Carmel et al. 1999 [41] Los Angeles (US) 591; 345, 246 > 60 140 0.37 17.1 (m) 5.7 16.8 (f) 10 Flicker et al. 2006 [98] Perth (AU) 299 ≥ 75 140 15 11 Figlin et al. 2003 [96] Tel-Hashomer (IS) 1271 147 0.24 15 11

26 12 Andres et al. 2006 [10] Strasbourg (FR) 201; 57, 144 67 ± 6 h 148 13 6.8 13 von Castel-Dunwoody et al. 2005 [257] Florida (US) 359; 0, 359 20 - 30 d 148 35 14 Lewerin et al. 2003 [163] Gothenburg (SE) 209 76 (70 - 93) i 148 0.34 16 8.6 15 Miller et al. 2006 [183] Sacramento (US) 1789; 751, 1038 > 60 148 35 0.35 13 6.8 363

16 Morris et al. 2002 [185] (US) 1145 ≥ 65 148 0.37

17 Sola et al. 2006 [111] Granada (ES) 218; 82, 136 79.2 (60 - 105) g 148 45 0.3 15 6 397 18 Solomon 2005 [236] New Heaven (US) 304 28 - 82 d 148 0.376 13.6 19 Fenech et al. 1997 [91] Adelaide (AU) 64; 64, 0 50 - 70 d 150 10 6.8 317 20 Loikas et al. 2003 [168] Lieto (FI) 333 ≥ 65 150 37 0.45 19 21 Nilsson et al. 2004 [191] Lund (SE) 209; 91, 118 73 ± 11 h 150 40 0.41 19.9 7 22 Refsum et al. 2001 [208] Puna (IND) 205; 169, 35 27 - 55 d 150 35 0.26 15 5 23 Riggs et al. 1996 [212] Boston (US) 70; 70, 0 54 - 81 d 150 14 6.8 24 Smulders et al. 2006 [234] Amsterdam (NL) 1; 1, 0 51 150 0.35 18 6.5 25 Gamble et al. 2005 [105] Araihazar (BD) 1650; 677, 973 20 - 65 d 151 11.4 (m) 9 10.4 (f) 26 Herrmann et al. 2003 [130] Saarland (DE) 545; 251, 294 57 (18 - 92) g 156 35 0.271 12 7 27 Hirsch et al. 2002 [137] Santiago (CL) 108; 43, 65 74.4 ± 3.7 h 165 14 6.8 Table 4: (Continuation) Overview of different cut-offs used to deﬁne vitamin B12 and folate deﬁciency in selected Studies, sorted by vitamin B12

n Age B12 bc HoloTC bc MMA bc tHcy bc sFolate bc RBC folate bc No. References City (Country)a (all; m, f) (y) (pmol/L) (pmol/L) (µmol/L) (µmol/L) (nmol/L) (nmol/L) 28 Holleland et al. 1999 [138] Oslo (NO) 224 18 - 90 d 170 0.376 15 5

29 Chen et al. 2005 [45] (US) and (ES) 62 56 f 180 30 0.279 14.9 (m) 14.5 (f) 30 Stott et al. 2005 [240] Leiden (NL) 185; 88, 97 ≥ 65 184 124 Glasgow (GB) 31 Flood et al. 2006 [99] Sydney (AU) 2963 ≥ 50 185 15 6.8 32 Obeid et al. 2004 [194] Homburg (DE) 228; 72, 156 > 65 196 29 0.28 14.1 11.1

33 Clarke et al. 2003 [57] Oxford City (GB) 1502; 618, 884 ≥ 65 200 0.35 15 34 Clarke et al. 2004 [56] (GB) 3511 ≥ 65 200 20 35 Hvas and Nexo 2005 [144] Aarhus (DK) 937; 349, 588 72 (19 - 102) i 200 40 0.28 11.9 350

36 Hvas and Nexo 2003 [143] Aarhus (DK) 143; 51, 92 72 (24 - 90) i 200 50 0.29 15 350 27 37 Lindgren et al. 1999 [165] Gothenburg (SE) 101; 35, 66 52 (18 - 80) i 200 35 0.4 13 38 Nexo et al. 2002 [189] Haukeland (NO) 90; 69, 21 38 - 80 d 200 40 0.27 39 Refsum and Smith 2003 [207] Oxford (GB) 116; 43, 73 73 f 200 40 0.3 14 15 40 Koehler et al. 1996 [157] Albuquerque (US) 100; 50, 50 68 - 96 d 221 0.271 16.2 5 41 Pennypacker et al. 1992 [199] Denver (US) 152 65 - 99 d 221 0.376 21.6 42 Rajan et al. 2002 [203] Seattle (US) 315; 203, 112 65 - 100 d 221 0.271 13.9 7.3 43 Johnson et al. 2003 [151] Georgia (US) 103; 21, 82 76 ± 8 h (60 - 95) d 258 0.271 13.9 6.8 295 44 Lindenbaum et al. 1994 [164] New York (US) 548; 200, 348 77.5 (67 - 96) i 258 0.376 21.3 11.3

45 Robertson et al. 2005 [219] London (GB) 421; 215, 206 66 (37 - 90) g 258 0.271 14 46 Stabler et al. 1999 [237] Baltimore (US) 762; 0, 762 ≥ 65 258 0.271 13.9 11.4 47 Wolters et al. 2003 [267] Hanover (DE) 178; 0, 178 60 - 70 d 258 0.271 12 7 320 48 van Asselt et al. 1998a [252] Nijmegen (NL) 105; 46, 59 76 (74 - 80) i 260 0.32 19.9

49 Eussen et al. 2005[85] Wageningen (NL) 486 ≥ 70 300 0.26 a. ISO 3166-1 country code elements. b. Abbreviations: m, male; f, female; B12, vitamin B12; HoloTC, holotranscobalamin; sFolate, serum folate; RBC, red blood cell; MMA, methylmalonic acid; tHcy, total homocysteine. c. SI conversion factors: To convert vitamin B12 and HoloTC to ng/L, multiply with 1.3554; folate to ng/mL, multiply with 2.266; MMA to mg/L, divide by 8.475; d. range.; e. mean (95 % CI); f. mean; g. mean (range); h. mean ± SD; i. median (range). 2.8.1 Indicators of Haematologic Status

Hemoglobin, hematocrit, red blood cell count, and mean corpuscular volume [122] are all suitable measurements. However, the 120-day red blood cell survival time response time explains the slow response time of these indices. Therefore, affirming solely with these indices the diagnosis for vitamin B12 deficiency is not sufficient to identify a subclinical vitamin B12 deficiency. Hypersegmented neutrophils occur before macrocytosis [246]; however, the sensitivity of this finding has recently been questioned [40].

2.8.2 Serum or Plasma Cobalamin Levels

Concentrations of serum or plasma cobalamin are suitable initial diagnostic tests for vitamin B12 deﬁciency because they are widely available, cheap and because of the familiarity with the test due to its time on the market. Serum and plasma cobalamin determine both the fraction available for the cells that is linked to transcobalamin (20- 30%) and the fraction that is linked to haptocorrin (70-80%). Currently and commonly used assays for measuring serum or plasma cobalamin concentrations are run on automatic analyzers and based on the competition between labelled vitamin B12 and cobalamin of the sample for binding to IF. The design of the assay and consequently the interval of reference vary depending on the manufacturer. Therefore, it is advisable to use the reference interval from the manufacturer. Decreased values considered as values well below the lower level of the interval of reference most often indicate vitamin B12 deﬁciency except for pregnant women and patients with decreased haptorrin levels.

2.8.3 Holotranscobalamin (HoloTC, Active B12)

Holotranscobalamin refers to the fraction of cobalamin linked to transcobalamin and thereby the part of the circulating cobalamin that is responsible for receptor-mediated uptake of vitamin B12 into cells. It is synthesized by the liver and binds only a small fraction of plasma vitamin B12 (7%–20%) to form the transcobalamin-vitamin B12 complex. Two different types for determining holoTC are commonly used, one that measures holoTC by a transcobalamin specific ELISA after removal of unsaturated transcobalamin [189] and one that directly measures transcobalamin saturated with cobalamin [34]. Only the latter method is available on an automatic platform. From a theoretical point of view holoTC (active B12) should be the first marker to show alterations in vitamin B12 deficiency at early stages. So far methods for measurement of holoTC are available only on one automated analyzer consequently the clinical experience from the use of holoTC measurements in routine clinical use is scarce.

28 2.8.4 Serum Methylmalonic Acid

Methylmalonic acid (MMA) values rise when vitamin B12 is subadequate and tend to rise in the elderly [153]. The metabolite is currently considered as the most speciﬁc marker of vitamin B12 deﬁciency [117]. The metabolite is measured via chromato- graphic methods such as GC-MS, but till now, there is no method available for analzing it on an automatic system. Consequently, analysis of MMA is not widely accessible and is costly. The interpretation of MMA in serum has to take into consideration that MMA is correlated to kidney function and thereby to serum creatinine. The upper limit for the reference interval has been indicated as values between 0.28 and 0.55 umol/I. The uncertainty concerning the exact value does not relate to methodological issues but rather to the reference populations included for the establishment of the interval. Borderline elevations in serum methylmalonic acid levels will not respond to cobalamin therapy in the presence of renal failure [184], although Lindenbaum et al. [164] reported that moderate renal dysfunction in the absence of renal failure did not affect methylmalonic acid values as strongly as did inadequate vitamin B12 status.

2.8.5 Serum Homocysteine Concentration

Serum homocysteine levels show a strong inverse association with folate plasma levels, but there is also an inverse association (albeit weaker) with vitamin B12 and B6 plasma levels. Inadequate plasma concentrations of one or more of the above three B vitamins appear to account for 67% of cases of high homocysteine levels (more than 14 pmol/liter) in an elderly population. Because hyperhomocysteinemia is also observed in renal insufficiency or hypovolemia, serum creatinine is useful for interpretation. Because elevated serum homocysteine concentrations are not specific for vitamin B12 deficiency, it is of limited usefulness for evaluation of vitamin B12 status [117].

2.9 Oral Supplements as Alternatives to Conventional Vitamin B12 Treatment

It is indifferent for the intracellular metabolism in which form cobalamin is offered to the cells (cyano-, hydroxy-, or mehthyl cobalamin, because the ligands are split off during the liberation from the lysosomes and the reduction process). Preformed methylcobalamin as well has to be remethylated after liberation from the lysosomes and reduced to Cbl2+ [50] [158]. Therefore, metyhlcobalamin does not have the edge over hydroxy- or cyanocobalamin. Due to the small resorption of cyanocobalamin of between 1 and 3%, parenteral administration is usually preferred. However, an oral therapy can be worthwile for some deﬁned patient groups with hypersensitivity for intramuscular administration and with elevated bleeding tendency (haemophilia, oral anticoagulants) that showed good compliance.

29 Adverse reactions

Pure oral as well as parenteral vitamin B12 is also in high dosage highly compatible and practicallly without adverse reactions. Although, cobalamin as a drug has been on the market for more than 50 years and as is regularly prescribed in high amounts, adverse reactions are described only in a few single cases. The mayoritiy of adverse reaction notations (mainly allergies) are attributed to parenteral induced sensibilisation, which probably is not caused by the molecule itself, but most likely is caused by associated material (trace elements of the biosynthesis, culture media, streptomycin, liver extract) or rather by additives (preservatives). After parenteral administration single cases of acne, as well as anaphylactic reactions were also described which are most likely also attributable to galenic dependent substances

30 HYPOTHESES AND OBJECTIVES

3 HYPOTHESESAND OBJECTIVES

The hypotheses and objectives were classiﬁed according to the three Studies of the Thesis.

3.1 Hypotheses of Study I

The main experimental hypothesis of this study was that a period of one year would be sufficient to show a worsening of the vitamin, haematological and lipid profile as well as of the functional and cognitive status of institutionalized elderly. A secondary experimental hypothesis was that the known association among several parameters found in younger subjects would be confirmed in this population group. To test these hypotheses, null hypotheses were established for the statistical analyses.

3.2 Objectives of Study I

In this context, in order to contrast these hypotheses, the following objectives were specified: 1. To analyze the evolution of vitamin B profile, selected haematological parameters, lipid profile, selected anthropometric measurements, functional and cognitive status over one year in institutionalized elderly 2. To quantify the prevalence of health-related risk factors over one year in institutionalized elderly 3. To study the relationship between cobalamin status and cognitive and functional condition in institutionalized elderly 4. To study the diagnostic efficacy of holo-transcobalamin compared to other biomarkers regarding vitamin B12 status

3.3 Hypotheses of Study II

The experimental hypothesis was that an intervention with an oral supplement (providing 500 µg cyanocabalamin) for 28 days would be effective to improve vitamin B status in institutionalized elderly. The statistical hypothesis was formulated as a null hypothesis that there were not any differences between pre and post supplementation values.

33 3.4 Objectives of Study II

In order to test this hypothesis the following objectives were speciﬁed: 5. To analyze the effect of 500 µg oral cyanocobalamin during 28 days on vitamin B12 status and related parameters in institutionalized elderly 6. To investigate the effect of the intervention on holo-transcobalamin as a marker of vitamin B12 status

3.5 Hypotheses of Study III

The main experimental hypothesis was that the medication intake affects values of vitamin B status and homocysteine levels. The null hypothesis was that there were not statistical differences in the percentage of people with adequate or low (in the case of homocysteine elevated) values and the percentage of people taking or not-taking certain groups of medication.

3.6 Objectives of Study III

To contrast this hypothesis the following objectives were established: 7. To analyze if drug intake affects vitamin B12, folate and homocysteine status in institutionalized elderly 8. To assess the number and type of medications which are taken routinely by Spanish institutionalized elderly

34 METHODOLOGY

4 METHODOLOGY

For the present thesis three different studies were performed in four old people’s homes in the Region of Madrid. The first one (I) was a longitudinal descriptive study, the second one (II) was an intervention study with an oral supplement of 500 µg cyanocoabal- amin, and the last one (III) was a descriptive study about drug use and its association with vitamin B status. Selection of the old people’s home Selection of the old people’s homes was not random. With the support of the “Aso- ciación de Familiares de Alzheimer (AFAL, Spain)”, we got in contact with twelve old people’s homes, both private and public, in the Region of Madrid. In order to reduce the influence of confounders, the minimal number of residences was aimed for, to se- lect the desired number of subjects. After visiting all of them, explaining the aims of the project and asking for their willingness to participate, finally four were selected to be included in the studies. Inclusion criteria for the old people’s homes were: geo- graphical distribution, urban/rural area, private or public ownership, and willingness of the medical staff to support the study in situ. From the four residences finally selected, one was of private ownership and the other three of public ownership, all four covering different metropolitan areas of Madrid. Sample size For both the intervention and the longitudinal study, the sample size was calculated to be 60, on the basis of the parameter with most variance, which for these studies was holotranscobalamin. In the case of the longitudinal study, at least 20 additional subjects were recruited bearing in mind the possible experimental mortality, in order to guarantee the final number. Participants From the selected residences, volunteers of both sexes were recruited to participate in one of the studies. Inclusion criteria were to be older than 60 years, not clinically diagnosed with dementia and having signed the informed written consent. Exclusion criteria were vitamin B12 and folate supplement intake, neuropathy and Hunter’s glossitis. The concomitant intake of medication related to affect Cbl absorption (such as metformin, H2-antagonists, and proton pump inhibitors) was not considered as an exclusion criterion if the medication had been started at least three months before the study and was taken continuously by the study participant for the whole duration of the intervention period.

37 4.1 Study Designs

4.1.1 Study I (Longitudinal)

The study design was a prospective longitudinal study (Figure 5). The study population was visited four times, the ﬁrst one to establish reference baseline data and three controls at four months intervals over a one year period. The information gathered during data collection was identical at each examination point.

Figure 5: Study design for longitudinal study and distribution of drop outs in the course of follow-up

4.1.2 Study II (Intervention)

The study design was an intervention study with pre- post comparisons (Figure 6). The study population was examined before and after the treatment period of 28 days.

Figure 6: Study design for intervention study

4.1.3 Study III (Polypharmacy)

The study design was a descriptive cross-sectional study with a posteriori comparisons.

38 4.2 Ethical Aspects

All three studies were performed in the framework of a broader study dealing with the early diagnosis and treatment of vitamin B12 deﬁciency in Spanish elderly. The study proposal was sent to the Human Research Review Committee of the University of Granada for its approval and was checked for respecting people’s rights and dig- nity. The Board reviewed the proposals to ensure that ethical issues would be handled correctly and that the study procedures met all the legal requirements necessary to con- duct research and approved the proposal. The study was performed in accordance with the Helsinki Declaration of 1964 and later amendments, the last one revised in 2000 in Edinburgh, with the Convention on Human Rights and Biomedicine of Oviedo in 1997. A written informed consent was obtained from all study participants, family representatives or guardian if participants were unable to provide consent.

4.3 Materials and Equipment

4.3.1 Equipment

The materials and equipment used to collect the data and analysis are shown in Table 5. Medication proﬁle forms (for chronic as well as acute diseases) were extracted from the local data management systems.

4.3.2 Procedures

Phlebotomy and Blood Distribution

4.3.2.1 Blood analyses Blood was collected following established venipuncture pro- tocol and procedure from the participants after an overnight fast in situ at the old people’s homes. Blood specimens were collected into Vacutainer Tubes containing EDTA as anticoagulants or gel for serum. The EDTA tubes were used for whole blood count and red blood cell folate (RBC folate) and the gel tubes were immediately placed on ice. After blood clotting, they were centrifuged at 3000 g for 15 min, aliquoted and frozen at -86° C until analysis in the biochemical laboratory of the Faculty for Sport Sciences, UPM, Madrid (Registered Lab number 242, from Madrid Community laboratory network [Red de Laboratorios de la Comunidad de Madrid]).

39 Table 5: Equipment used for analyses Type of analyses Equipement / Materials Manufacturer / Battery For the blood analyses: Phlebotomy Vacutainer® System Becton, Dickinson and Company, Franklin 20 Lakes, NJ Vitamin B12 status Autoanalyzer AxSYM Abbott Park, Illinois, USA) Biochemical proﬁle Hitachi 912 Roche Diagnostics, Mannheim, Germany Complete Blood Cound Beckman Coulter MaxM / HMX IZASA, Madrid, Spain For the anthropometric measurements: Weight Weighing scale Seca 709 Seca Hamburg, Germany Knee Height Anthropometer GPM, Switzerland

40 Skinfold thickness Skinfold caliper Holtain Ltd., Crymych, UK Girths Anthropometric tape For cognitive status cognitive parameters MMSE questionnaire™ Official translation to Spanish of PAR Inc. Lutz, Florida, USA Wristwatch Pencil or ballpen Some blank paper Piece of paper with CLOSE YOUR EYES written in large letters For the functional tests: Hand grip strength Digital hand grip dynamometer Takei TKK 5101 Tokyo, Japan Upper body strength Dumbbells (2kg for women and 4kg for men), Stopwatch Rikli Jones Lower body strength Standardized chair (Height = 43.18 cm), Stopwatch Rikli Jones Static balance Stopwatch Eurofit Vitamin B12 Status Serum cobalamin: Total sCbl concentrations were determined by a Microparticle Enzyme Immunoassay (MEIA; Abbott AxSYM, Abbott Park, USA, total CV ≤ 11%) [64] [266]. Serum holoTC: Serum holoTC was analysed by a two-step sandwich MEIA (Abbott AxSYM, total CV ≤ 10%) [248] [197][34]. Serum folate (sFolate): Serum and RBC folate were measured by an ion-capture immunoassay (ICIA; Abbott AxSYM, Abbott Park, USA, total CV ≤ 19 %) [233]. RBC folate was determined in whole blood from EDTA-coated tubes. Serum tHcy: Serum tHcy was determined by a fluorescence polarization immunoassay (FPIA; Abbott AxSYM, Abbott Park, USA, total CV ≤ 6%) [200]. The samples for sCbl, holoTC, sFolate, RBC folate, and tHcy of each subject were measured within one run to avoid between-run variation. For project II, the samples for sCbl, holoTC, sFolate, RBC folate, and tHcy of each participant were measured within one run to avoid between-run variation.

Haematological Parameters Complete blood count (CBC) was performed within four hours after extraction and was obtained by an automated haematology analyser (Beckman Coulter MaxM, IZASA, Madrid, Spain). The CVs for the analyses were the following: haemoglobin (Hb, CV ≤ 1.5%), haematocrit (HCT), mean corpuscular volume (MCV, CV ≤ 2.0%). The analyses were conducted by a local clinical laboratory (CLIMESA, Gabinete Medico Conde-Duque, Madrid, Spain).

Biochemical Status Basic clinical chemistry variables including lipid proﬁle and creatinine, were obtained by standard spectrophotometric assays on a Hitachi 912 (Roche Diagnostics, Mannheim, Germany) and were conducted by a local clinical laboratory (CLIMESA, Gabinete Medico Conde-Duque, Madrid Spain).

4.3.2.2 Anthropometric Measurements Weight: The participant stood in light cloth- ing in the center of a scale platform. Body weight was recorded to the nearest tenth of a kilogram. Height Estimated from Knee Height To estimate height, knee-heel height was measured to the nearest 1 mm using a GPM spreading anthropometer (Siber-Hegner GPM, Switzerland, range 0-2100mm) at the left side in a sitting position, lower legs relaxed, with the knee ﬂexed at a 90° angle. Standing height was calculated by the equations proposed by Chumlea and Guo [53].

41 Table 6: Analysed variables and methods used Variable Index measured Sample/Method B12 vitamin B12 Serum HoloTC Serum Folate Folate Serum RBC Folate Serum tHcy tHcy Serum Lipid proﬁle Triglycerides Serum total cholesterol Serum LDL-cholesterol Serum HDL-cholesterol Serum Body composition Height Estimation from Knee Height SF-thickness Anthropometry (SF caliper) Functional tests Hand grip strength Dynamometry Strength of arm ﬂexors Arm curl tests Leg strength 30-Second Chair Stand Balance Stork Stand Cognitive status MMSE Folstein Questionnaire

Girths Girths were measured using a anthropometric tape of inextensible material and were measured in the following anatomical locations: waist (obvious narrowing between the rib and the iliac crest), hip (horizontal level of greatest gluteal protuberance). Skinfolds Skinfold measurements were measured with a Holtain skinfold caliper (Crosswell, Crymych, UK, pressure 10 g/mm²). The laboratory manual of the International So- ciety for Advancement of Kinanthropometry (ISAK) served as a compendium for examination but the left side has been chosen following the recommendations by the International Biological Programs [176] [134]. All examiners were previously trained. Anatomical landmarks were marked for reference. For each location three measurements were taken and their means were used as representative values. The skinfold measurements were asssessed in the following anatomical locations: - Biceps (vertical fold, anterior aspect of the arm over the belly of the biceps muscle, 1 cm above the level used to mark the triceps site, arm held freely to the side of the body) - Triceps (vertical fold, posterior midline of the upper arm halfway between the acromion (shoulder) and olecranon (elbow), arm held freely to the side of the body) In the above mentioned anatomical locations, the skinfolds were taken avoiding including muscle tissue. The hand pressure was maintained constant when measuring, and the reading was taken after two or three seconds after having situated the caliper. For greater precision, the whole measurement was repeated three times [60].

42 4.3.2.3 Cognitive Status Cognitive functioning was assessed using the official translation of the Mini Mental State Examination™ (MMSE) [101] [Psychological Assess- ment Resources (PAR), Inc., Lutz, FL]. MMSE questionnaire was administered in a personal interview by an expert in a quite room. The data and final score were recorded on the working sheet (see Appendix C). After having got the patient’s confidence with some introductory ice-breaking questions, each question was read as it was exactly written. If the patient had not understood acoustically, the question was repeated up to a maximum of three times without any additional explaination. During the interview, any hints for the patient were forbidden as well as any indication that they answered incorrectly or to correct the patient if they had answered incorrectly. The seven categories, representing different cognitive domains were scored as follows: 1. Orientation to time (5 points) 2. Orientation to place (5 points) 3. Registration of three words (3 points) Names of three unrelated objects were presented clearly and slowly, about one second for each name. After all three names were said, the patient was asked to repeat them. Scoring: The first repetition determines scoring (0-3) but the words can be repeated until the patient can repeat all three, up to six trials. If all three are not eventually learned, recall cannot be meaningfully tested but should nevertheless be attempted. 4. Attention and calculation (5 points) 5. Recall of three words (3 points) 6. Language (8 points: name items (2 points); repeat (1 point); understanding (3 points); reading (1 point); and writing (1 point). 7. Visual construction (1 point)

The patient had to draw two five-sided figures intersected by a four-sided figure.

For total scoring, the points obtained for each item were summed and recorded on the last page of the questionnaire. If the patient did not respond to the items, 0 was scored. The maximum possible score was 30.

4.3.2.4 Functional Tests Strength was evaluated by the following tests. Handgrip strength: It was assessed with a handgrip dynamometer (Takei TKK 5101, Tokyo, Japan, range 5-100 kg, precision .1 kg). Due to the disability of some of the participants to maintain the upright position without any help, the handgrip strength was performed in a sitting position performing the test with the elbow in extension and

43 in a vertical position. Two trials were recorded on both hands with at least one minute break between them and the average value was included in the statistical analysis. Body strength Body strength was assessed by two items from the Senior Fitness Test (SFT) of Rikli and Jones [215]: the upper limb (arm curl) and the lower limb (30-Second Chair Stand). Upper-body strength [215] The strength of the upper extremities was assessed by the “arm curl” test. The participant sat in a an upright position on a chair of standard size (43.18 cm seat height) with feet flat on the floor holding a dumbbell in his/her hand down at the side, perpendicular to the floor, in a handshake grip. Instead of the 5 pounds (2.27 kg) of load for women and 8 pounds (3.63 kg) of load for men suggested in the battery, the load were adjusted to “kg”, the International System (SI) unit routinely used in Spain, resulting in an adaption to the load of 2 kg for women and 4 kg for men. The participants were in- structed to perform the biceps curls with his/her dominant arm and had 30 seconds for performing the greatest possible number of repetitions. The palm had to rotate up during the curl-up phase and then should return to a handshake position at extension. The upper arm had to remain still throughout the test. The participants got the instructions and made an attempt to see if the execution was correct. Participants were encouraged verbally to improve their motivation. The test was repeated twice with the dominant arm, leaving a break of about 3 min for recuperation between each performance. The average of both trials was calculated and included in the statistical analyses. Lower body strength [215] The strength of the lower extremities was assessed by the “30-Second Chair Stand”. Participants who were able to perform the test sat on a straight back chair (43.18 cm seat height) with a solid seat and feet flat on the floor and arms across chest; then they were asked to rise to a full stand and to sit down again to a fully seated position. The participants got the instructions and performed one complete extension and flexion in order to check if the execution was correct. The participants repeated the action at his/her own tempo for 30 s. The final test score were the number of times that the participant got to a full stand from the seated position with arms folded within 30 s. This test was only performed once. Static balance (Stork Stand) Static balance was evaluated by the stork stand test on the foot of the dominant leg. The participant stood on the dominant leg with opened eyes, and placed the other foot on the inside of the supporting knee and placing the hands on the hips. Upon a given signal, the participant tried to maintain balance as long as possible without placing the

44 non supporting foot on the floor or touching it. The final score was the greatest number of seconds during which the participant kept balance on three trials. Only the highest score was recorded. Participants who failed at the first attempt were retested.

4.4 Delimitations of the Studies

Common for all the three Studies: The samples chosen were limited to Spanish elderly volunteers aged over 60 residing in old people’s homes in the Madrid Autonomous Community. Owing to the impossibility to control the medication taken by the participants and its specificity drug intake was recorded. The diet given in the old people’s homes was kept as usual. Serum vitamin B12, serum folate and RBC folate, holoTC, the metabolite serum homocysteine, full blood count and biochemical parameters were analyzed with the same equipment which is considered of high precision. Functional evaluation was assessed by hand grip strength and additionally by the “Arm Curl Test” of the Rikli Jones battery and the “30-Second Chair Stand” of the SFT battery and static balance was assessed by the Stork Stand test of the Eurofit battery. All tests were analyzed with the same equipment which is considered of high precision. Staff was trained beforehand to perform the tests. Specific to the Studies: In study I, the analyzed parameters were examined at regular four months intervals for determining the evolution and avoiding possible variations due to seasons as a possible time varying covariate. The data used for statistical analyses were the data of those participants for whom data of the key outcomes were complete for all four examination points. No significant differences were found between the means of the studied key outcomes of the persons whose data were complete for all four examination points and the participants lost during follow-up. In study II, the treatment of the intervention was set at 500 µg cyanocobalamin given as an oral daily supplement. The analysis of the effectiveness of the intervention was assessed by means of a factorial pre post test in which the participants were their own control. In study III, the medication intake was recorded from the local data management system. The medication was classified according to the Anatomical Therapeutic Chemical (ATC) Classification System. The statistic analysis of the possible effects of the medication intake on the status of vitamin B was based on the known and unknown effect of the medication type.

45 4.5 Justiﬁcation of the Chosen Tests

Height Estimated from Knee Height Accurate and valid measurements of standing height are particularly difficult to ob- tain from elderly people because of the difficulty for most of them to stand erect and of bowing of the legs [54] [253] [26]. Suffering chronic diseases, such as osteoporo- sis, arthritis and Parkinson-like disorders, which influence the neuromuscular system, aggravate this problem. Body height decreases in the course of senescence [16] [72] [140] [161] [68] due to pathologic changes in the spine including increased kyphosis (curvature of the spine), collapsed disks and vertebral bodies (compression of vertrebal disc space) [52]. Body Mass Index (BMI) Body mass index is a commonly used measurement to estimate obesity. In 1977, the WHO proposed a classification system for overweight and obesity and this classification system has been utilized all around the world since the year 2000. Overweight is defined as values between 25.0 and 29.9 and obesity for values over 30. Overweight and obesity are associated with an overall death rate whose relationship is curvilinear. A major risk increases when BMI exceeds 30 although BMI values between 25 and 30 are associated with an increase risk for many diseases among them heart disease and hypertension [265]. Despite these assumptions, several authors think that BMI is not an accurate index of body composition. However, it has been and it still is an indicator frequently used to control and evaluate body weight and body composition in Health Sciences. Skinfolds According to Lohman et al. [167] the subscapular skinfold thickness is a measure of subcutaneous adipose tissue and skin thickness on the posterior aspect of the torso. It is an important measure of nutritional status and, in combination with other skinfold measurements, it is a useful predictor of total body fat, blood pressure and blood lipids. It has been recommended in combination with the tricipital skinfold by the International Biological Programme (IBP) and the WHO as a relatively new tool. Changes in the proportions and densities of the Fat Free Body (FFB) Mass component due to ageing limit the usefullness of the two component model for estimating relative body fatness. Relative mineral content of the FFB decreases approximately 1% per year between 50 and 70 years of age, and because of these changes the density of the FFB of the elderly, particularly elderly women, changes and the prediction equations based on the models using series of Brozeck’s equations tend to overestimate the relative body fat [135]. Because of the lack of skinfold cross-validaded equations

46 to estimate body density for elderly people the WHO has recommended to use two skinfolds, subscapular and tricipital [264]. Waist-to-Hip ratio The Waist-to-Hip ratio (W-H ratio) is the circumference of the waist divided by the circumference of the hips and has been used as a simple method for determining body fat distribution [36]. Waist Waist circumference can be chosen singly as an indicator of health risk because ab- dominal obesity is the primary concern. Furthermore, the proportion of mortality attributable to large waist circumference among never smoking men was found to be three-fold the proportion attributable to high BMI [256]. The Expert Panel on the Iden- tification, Evaluation and Treatment of Overweight and Obesity in Adults provided a classification of disease risk based upon both BMI and waist circumference [86]. Fur- thermore, a newer risk stratification scheme for adults based on waist circumference has been proposed [35]. Assessments for risk stratification to evaluate chronic disease risk are recommended to include a minimum of either waist circumference or BMI but preferably both. Functional Tests The chosen functional tests were easy-to-perform tests that are considered not be in- fluenced by learning; however, the time period between the examination dates were considered to be sufficient for wiping out possible effects of learning. Handgrip Strength There are different protocols for examining hand grip strength [196] [94]. The main differences are participant’s position at the moment of test performance (lying, sitting or standing) and the arm position, especially the one of the elbow (flexed or extended) when performing the test. There is evidence that better results are obtained if the test is performed in a standing position [175][245][147]. However, as some of the participants were uncapable of staying in an upright position the test was performed in a sitting position. Regarding elbow position, there is divergence of opinion. Some authors showed that better results were obtained when the elbow was flexed [71]; others, when the elbow was in extension [19] [82] [198], although also some authors showed that the results were similar [190] [93]. Lower Body Strength The test, “30- Second Chair Stand” was used to measure lower body strength. Rikli and Jones demonstrated that this test is a valid and reliable test for measuring proximal lower limb (LL) strength in older adults [213] [66] [214] [152].

47 Static Balance Regarding the population studied the chosen static balance test, stork stand test, was used as a factor in general motor ability tests [196] [150]. The Mini-Mental State Examination (MMSE) MMSE by Folstein [101] is a screening tool for quantitatively estimating the severity of cognitive impairment and for documenting cognitive changes. It is widely used because of its easy and fast administration (5-10 minutes). In addition, this questionnaire is recommended by the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) to document the clinical diagnosis of probable Alzheimer’s Dis- ease.

4.6 Limitations of the Studies

In addition to limitations arising from the above delimitations the following ones can be pointed out: Common for all the three Studies: The results can be applied to elderly people with similar characteristics to the studied population. The results can be applied to the context of institutions where the evaluations were performed. Specific for the Studies: Regarding study I, there were 36 dropouts due to the special characteristics of the study group such as death, change of residence or dropped during following-up, but their values were similar to the ones who finished the whole evaluation period and fulfilling the calculated sample size of 60 elders with complete main outcome data. Regarding study II, the used dosis was sufficient for demonstrating significant differences after a 28 days supplementation. Nevertheless, further investigation is needed for determining the effectiveness of supplements or rather treatments of various doses and/or different durations. The time lapse of one month between the two examination dates was unlikely to be the reason to explain the observed differences due to matura- tion and or history. Regarding study III, the complexity and diversity of the medications taken by the participants and the possible interactions among them and on the vitamin B status makes it difficult to make definitive conclusions of the effects.

48 4.7 Statistical Analysis

For the statistical analysis, continuous variables were checked for Gaussian normal distribution with the Kolmogorov-Smirnov test and whenever a normal distribution could not be achieved with the raw data, logarithmic transformation was applied for significance testing. Statistical analyses were performed using SPSS for Windows (ver.19.0, SPSS Inc). All tests were performed two-tailed and a P value ≤ .05 was considered statistically significant. Cutoff values for each biomarker were based on a literature report [183] and were as follows: vitamin B12 < 148 pmol/L, holoTC < 35 pmol/L, tHcy > 13 µmol/L, serum folate < 15.8 nmol/L (< 7 ng/mL) and RBC folate < 362.4 nmol/L (< 160 ng/mL). For study I, statistical analyses were carried out with data from those participants who had complete data (n=60). Differences of means of each analysed parameter over time (four examination dates) were evaluated by using a general lineal mixed model for repeated measures with time as repeated factor and gender as fixed factor. Correlation between variables was evaluated by using partial Pearsons’ Correlations coefficients with time as control. For study II, geometric and arithmetic means with standard deviations of pre- and postsupplementation concentrations were computed for tables. Besides, the trimmed mean was also calculated to check the degree of variation. Differences between means or rather medians were analysed with a t-test for paired samples and Wilcoxon matched- pairs signed-ranks test in the case of sCbl. Since there is still no consensus on cut-off values to define Cbl deficiency, neither for serum cobalamin nor for holoTC values, and given that the distribution of cobalamin concentration values was not normal, participants were grouped in clusters (k-means clustering) according to the initial cobalamin status in order to achieve maximum homogeneity within the groups. GLM repeated measures analysis with “sCbl clusters” as a fixed factor and post-hoc Bonferroni were used in order to assess the interaction of the initial sCbl status on the effect of the supplementation. Results were reported as arithmetic means. For study III, the reference central intervals (95%), established by the manufacturers of the inmunoassays were used to divide the participants in two groups, (adequate or deficient level of the according parameter). The values were as follows: HoloTC (19.1-119.3 pmoL/L), Hcy (4.72-14.05 µmoL/L), serum folate (7.2-15.4 ng/mL) and Cbl (208-963.5 pg/mL). The medications was classified into 39 groups following the Anatomical Therapeutic Chemical (ATC) Classification System [63] and a dichotomous variable (medication intake / no medication intake) was created for the comparisons. In order to examine the

49 possible inﬂuence of the variables of medication intake on biochemical variables con- tingence tables of the chi square test applying the exact test of Fisher for dichotomous variables were used.

50 RESULTS

5 RESULTS

5.1 Study I (Longitudinal)

Regarding disposition of the study sample, 98 elders were eligible for participation after screening. Thirty-eight were lost to follow-up, 36 because they moved out of study area or died and were therefore excluded for the final analysis due to missing key outcome information. Another two were excluded because they started folate or rather cobalamin supplementation during the observation time period so that the final analytic sample consisted of sixty elders fulfilling the established sample size. The mean (SD) age of the study group was 84 (7) years, and 68% were women. The mean weight at baseline (month 1) was 70.0 kg (11.0) for men and 59.3 kg (12.9) for women, respectively. Men presented a BMI of 27.9 kg/m2 (4.1) and women of 28.3 kg/m2 (5.2) (Table 7).

Table 7: Characteristics of the study population Characteristics Mean±SD Number 60 Age (y)a 83.7 (7.4) Gender (n) 19 men/41 women Weight (kg)a 62.9±13.2 Men 70.0±11.0 Women 59.3±12.9 BMI (kg/m²)a,b 28.2±4.8 Men 27.9±4.1 Women 28.3±5.2 aMean (SD) bHeight was estimated from knee height.

Tables 8 - 12 show the averages (means or medians) of the analyzed parameters at baseline and at the other three examination dates (5th ,9th,13th month). The results are classified according to the following catagories: vitamin B status, selected haematological variables, lipid profile, anthropometric parameters, functional tests and MMSE. Parameters related to vitamin B status Descriptive data for selected vitamin parameters over one year period are shown in Table8 . Median serum cobalamin, serum folate as well as serum RBC folate and serum creatinine values were within the normal range on all four examination dates. Median values for holoTC were in the normal range except at month 9 (median: 42.8 pmol/L). In contrast, median serum homocysteine values were elevated on all four examination dates, independently of whether 13 or 15 µmol/L (= the most commonly found cut-offs revising the literature) were used as decision criteria. Despite median values for serum cobalamin of the overall study population being within the normal range, 10% of the study population showed deficient levels (de-

53 fined as < 200 pg/mL) which remained stable over the year. When holoTC is taken as criterion for deficient vitamin B status (defined as < 45 pmol/L), these prevalences were higher ranging between 45 and 52%. Whereas the prevalence for deficient RBC folate levels showed quite big oscilations (4%, 7%, 25%, 14%), the prevalence taking serum folate as the criterion was higher, but more stable (43%, 40%, 38%, 37%). Prevalence for elevated Hcy levels was very high, when taking 15 µmol/L prevalence was found between 60 and 70% and when taking 13 µmol/L even around 82% to 90%. Seventeen per cent of men and between 17 and 27% of women showed renal alterations (creatinine values < 1.1 for women and < 1.4 for men mg/dL). Regarding the pattern of these parameters, two parameters showed significant changes over the studied time period: serum folate levels increased from 6.2 to 7.0 ng/mL (F=2.86, df=3, p<.05) whereas serum cobalamin values decreased continuously from 388.3 to 319.1 pg/mL (F=3.43, df = 2.35, p<.05). Selected haematological variables The evolution of the haematological parameters are summarized in Table 9. Mean haematocrit and haemoglobin values as well as median MCV values of the study group were within their corresponding normal ranges. Statistical analysis revealed statistical differences for the intrasubject effect time as well as for the between gender effect for the parameter haematocrit (Ftime=4.21, df = 2.51, p<.01 and Fgender= 20.25, df = 1, p<.001) and also for the parameter haemoglobin (Ftime= 4.09, df = 2.53, p<.01 and Fgender= 22.96, df = 1, p<.001), whereas it was only significant for MCV after time (F=19.8, df=3, p≤.001). Lipid profile The evolution of the parameters related to the lipid profile are shown in Table 10. Overall, TG values increased significantly from 92 to 106 mg/dL (F = 9.53, df = 3, p<.001) and HDL-cholesterol decreased significantly from 49.8 to 47.1 mg/dL (F = 3.21, df =3, p<.05). The values for total cholesterol and LDL cholesterol showed a slight increase although not significant, but showed significant differences between gender (Fgender = 7.30, df = 1, p<.001 and Fgender = 4.77, df = 1, p<.05, respectively). Anthropometric measurements The evolution of the anthropometric measurements are summarized in Table 11. Mean values for BMI and median values of skinfold thickness of the triceps and waist-to- hip ratio did not show significant changes over the studied year. Mean values for subscapular skinfold thickness oscillated significantly over the year (F = 3.29, df = 2.48, p<.05) and the mean values of waist girths increased from 84.8 to 87.3 cm (F =

54 Table 8: Evolution of parameters related to vitamin B status over a one year period Month 1 Month 5 Month 9 Month 13

N Average Dispersion Average Dispersion Average Dispersion Average Dispersion F df Serum cobalamin 60 388.3 (283.0-480.3) 373.8 (300.0-475.1) 341.4 (272.3-442.1) 319.1 (276.2-486.5) 3.43* 2.35 (pg/mL)a,c,d % abnormalb 10 5 10 10 HoloTC (pmol/L)a,c,d 60 46.0 (30.7-57.9) 46.2 (34.9-66.2) 42.8 (34.2-57.0) 47.7 (34.4-60.7) 1.64 2.49e % abnormalb 48 45 52 47 Serum folate 60 6.2 (4.8-7.4) 6.7 (5.2-9.6) 6.8 (5.3-9.5) 7.0 (5.2-8.4) 2.86* 3 (ng/mL)a,c,d % abnormal 43 40 38 37 RBC folate (ng/mL)a,c,d 57 276.8 (224.5-341.6) 277.1 (227.8-308.6) 248.9 (173.7-337.0) 271.3 (192.5-359.3) b 55 % abnormal 4 7 25 14 tHcy (µmol/L)a,c,d 60 17.1 (13.9-21.6) 16.9 (14.7-23.1) 17.2 (14.1-27.0) 17.0 (14.0-24.4) 0.90 2.40e % abnormalb 63(>15) 70(>15) 60(>15) 68(>15) 87(>13) 88(>13) 90 (>13) 82(>13) Creatinine (mg/dL)a,c,d 59 0.8 (0.7-1-1) 0.8 (0.7-1.1) 0.8 (0.6-1.0) 0.8 (0.7-1.2) 3.12* 2.66e Men 18 1.0 (0.78-1.30) 0.95 (0.80-1.30) 0.85 (0.78-1.03) 1.0 (0.80-1.15) 3.06 1 % abnormalb 17 17 17 17 Women 41 0.8 (0.60-1.10) 0.7 (0.70-1.00) 0.8 (0.60-1.00) 0.7 (0.70-1.20) % abnormalb 17 20 20 27 aMedian Q2 (interquartile range; Q1-Q3) bCut-off values were the following: serum cobalamin < 200 pg/mL; HoloTC < 45 pmol/L; serum folate < 6 ng/mL, RBC folate < 175 ng/mL; tHcy > 15 /13 µmol/L; creatinine < 1.1 for women and < 1.4 mg/dL for men. cGLM for repeated measurements with epsilon correction by Greenhouse-Geisser whenever Mauchy tests results violated spherity. ddata lg 10 transformed for mean comparisons edf corrected (epsilon) according to Greenhouse-Geisser *p≤0.05 **p≤0.01 ***p≤0.001 Signiﬁcant results are marked with bold typeface. Table 9: Evolution of related haematologic parameters over a one year period Month 1 Month 5 Month 9 Month 13

N Average Dispersion Average Dispersion Average Dispersion Average Dispersion F/Chi-square df Haematocrit (%)a,d 57 38.3 5.1 39.7 4.8 39.8 5.2 39.0 4.5 4.21** 2.51f Men 18 41.9 5.2 43.5 3.3 43.2 4.6 41.6 4.1 20.25### 1 % abnormalc 38.9 16.7 22.2 27.8 Women 39 36.7 4.1 40.0 4.4 38.3 4.7 37.8 4.3 % abnormalc 35.9 25.6 25.6 25.6 Haemoglobin (g/dL)a,d 57 12.7 1.6 13.1 1.5 13.00 1.6 12.7 1.6 4.09** 2.53f Men 18 13.7 1.6 14.3 1.2 14.1 1.5 13.8 1.3 22.96### 1 c 56 % abnormal 38.9 22.2 22.2 27.8 Women 39 12.2 1.3 12.6 1.4 12.4 1.4 12.2 1.5 % abnormalc 35.9 20.5 28.2 38.5 MCV (fL)b,d,e 57 90.1 (87.8-93.8) 89.8 (88.1-93.6) 90.8 (88.9-93,7) 91.2 (88.7-95.1) 19.79*** 3 % abnormalc 5.3 5.3 5.3 3.5 aMean (SD) bMedian Q2 (interquartile range; Q1-Q3) cCut-off values were the following: MCV < 80fL; for men Hgb < 13.5 , for women Hgb < 11.5 g/dL dGLM for repeated measurements with epsilon correction by Greenhouse-Geisser whenever Mauchy tests results violated spherity. edata lg 10 transformed for mean comparisons fdf corrected (epsilon) according to Greenhouse-Geisser gChi-square of non-parametric test for k groups Friedman *: Intrasubject effect (time): *p≤0.05 **p≤0.01 ***p≤0.001 #: Between gender effect: #p≤0.05 ##p≤0.01 ###p≤0.001 Table 10: Evolution of lipid proﬁle over a one year period Month 1 Month 5 Month 9 Month 13

N Average Dispersion Average Dispersion Average Dispersion Average Dispersion F df TG (mg/dL)b 59 92 (67-121) 98 (75-129) 110 (87-139) 106 (83-132) 9.53*** 3 % abnormalf 5(B);5(H) 5(B);5(H) 10(B);10(H) 14(B);8(H) Men 18 95 (62.5-109.3) 101 (74.3-126.8) 105 (78.0-129.8) 101 (89.3-119.3) 1.87 1 % abnormalf 6(B);0(H) 0(B);0(H) 0(B);0(H) 11(B);0(H) Women 41 92 (70.0-129.0) 96 (76.5-133.5) 113 (78.4-163.5) 112 (81.5-150.5) % abnormalf 5(B);7(H) 7(B);7(H) 15(B);15(H) 15(B);12 (H) Total chol (mg/dL)b 59 189.00 (168-207) 195.00 (179-215) 198.00 (175-218) 196.00 (169-213) 1.20 3 % abnormalf 24(B);5(H) 31(B);15(H) 37(B);8(H) 29 (B);12(H) Men 18 171.5 (156.8-197.5) 177.9 (117.9-200.5) 174.4 (139.8-206.8) 184.3 (166.0-197.5) 7.30## 1 % abnormalf 11(B);0(H) 22(B);6(H) 33(B);0 (H) 6(B);11(H) Women 41 193.0 (174.0-220.5) 201.0 (181.5-236.5) 201.0 (180.0-225.0) 200.0 (174.0-214.0) % abnormalf 29(B);7(H) 34(B);20(H) 39(B);12(H) 39(B);12(H) LDL-chol (mg/dL)a 59 119.2 29.4 124.9 30.1 124.3 34.3 123.3 31.1 1.06 3

57 % abnormal 25(B);8(H) 29(B);15(H) 37(B); 8 (H) 29(B);10(H) Men 18 107.9 23.7 112.1 26.9 109.0 29.8 117.4 25.9 4.77# 1 % abnormalf 17(B);0(H) 17(B);6(H) 28(B);0(H) 17(B);11(H) Women 41 124.2 30.5 130.6 30.0 131.0 34.3 125.8 33.1 % abnormalf 29(B);12(H) 34(B);20(H) 41(B);12(H) 34(B);10(H) HDL-chol (mg/dL)a 59 49.8 11.9 49.6 11.7 46.4 11.4 47.1 13.0 3.21* 3 % abnormalf 19 24 29 34 Men 18 47.4 13.4 45.8 10.5 44.7 13.9 45.8 16.2 1.15 1 % abnormalf 17 33 44 44 Women 41 50.8 11.1 51.3 11.9 47.2 10.3 47.7 11.5 % abnormalf 20 20 22 29 aMean (SD) bMedian (innerquartile P25-P75) cCut-off values were the following: Abbreviations: TG Triglycerides; (B) borderline; (H) high; chol cholesterol ddata lg 10 transformed for mean comparisons eGLM for repeated measurements with epsilon correction by Greenhouse-Geisser whenever Mauchy tests results violated spherity. fTG 150-199 mg/dL borderline and > 200 mg/dL high; total cholesterol 200-239 mg/dL borderline and > 240 mg/dL high; LDL 130-159 mg/dL borderline high and 160-189 mg/dL high; HDL < 40 mg/dL high risk *: Intrasubject effect (time): *p≤0.05 **p≤0.01 ***p≤0.001 #: Between gender effect: #p≤0.05##p≤0.01###p≤0.001 9.42, df = 2.21, p<.001). As expected, the latter values were significantly different for men and women (Fgender = 29.22, df = 1, p<.001). Functional tests Table 12 summarizes the evolution of the functional parameters over a one year period. There were no changes over time in grip strength of both hands and in arm strength. However, as expected, the values of right and left hand grip strength and the strength of the dominant arm were significantly different for genders (Fgender = 54.41, F gender = 70.01, F gender = 17.47, respectively, all df = 1, all p <.001). MMSE The evolution of the MMSE scoring is shown in Table 13. Mean MMSE scoring values did not change significantly over time, but gender differences were found women having lower values (Fgender = 11.30, df = 1, p<.001). Correlations Table 14 illustrates the partial (controlled for 4 time measures) Pearsons’ Correlation coefficients and significance levels for the analyzed parameters. Serum vitamin B12 was correlated positively with HoloTC and RBC folate (p≤.001) and MMSE scores (p≤.01) and negatively with tHcy (all p≤.001). HoloTC was correlated positively with RBC folate (p≤.01) and negatively with tHcy (p≤.05). Serum folate was correlated positively with RBC folate and negatively with tHcy (both p≤.001) and RBC folate was correlated negatively with tHcy (p≤.001). Age correlated negatively with serum vitamin B12 and MMSE (both p≤.001) and HoloTC (p≤ .05) and positively with tHcy (p≤.001). Creatinine values were correlated positively with age and HoloTC (both p≤.01) and tHcy (both p≤.001) and negatively with serum folate (p≤.001). As expected, the right hand grip strength correlated positively with left hand grip strength and arm strength (both p≤.001); and with the MMSE scoring (p≤.001) and negatively with age (p<.01). The left hand grip strength correlated negatively with age (p≤.001) and positively with creatinine (p≤0.05), arm strength and MMSE scoring (both p≤.001). Arm strength correlated negatively with age (p≤.05) and positively with MMSE p≤.001). Correlation between body composition and lipid profile BMI was positively correlated to waist circumference, triceps and subscapular skinfold (p≤.001). Waist circumference was positively correlated with Waist-to-Hip ratio, triceps and subscapular skinfolds (all p≤.001) and negatively correlated with HDL- cholesterol (p≤.01). Waist-to-Hip ratio was negatively correlated to triceps skinfold

58 Table 11: Evolution of anthropometric parameters over a one year period Month 1 Month 5 Month 9 Month 13 N Average Dispersion Average Dispersion Average Dispersion Average Dispersion F df BMIa(kg/m²) 36 27.4 4.4 27.8 4.3 27.7 4.6 27.6 5.0 0.82 3 Men 13 27.7 3.4 28.1 3.4 28.6 3.4 28.5 3.8 0.38 1 Women 23 27.2 5.0 27.6 4.7 27.3 5.1 27.1 5.6 SF triceps (mm)b 47 12.9 (9.3-18.0) 14.5 (11.2-21.0) 14.4 (10.9-20.3) 13.5 (11.1-18.2) 2.78 2.25f Men 15 11.9 (7.5-15.5) 12.1 (9.1-16.1) 12.6 (9.2-14.1) 11.8 (9.8-13.5) 3.11 1 Women 32 13.7 (11.1-20.5) 16.0 (12.5-22.8) 16.5 (11.3-23.3) 16.2 (11.3-21.0) SF subscapular (mm)a 41 16.5 7.8 18.6 8.6 17.8 8.8 16.4 7.8 3.29* 2.48f Men 14 18.9 7.2 20.1 8.2 18.9 7.6 18.3 6.9 1.06 1 Women 27 15.3 7.9 17.9 8.9 17.2 9.4 15.4 8.1

59 Waist (cm)a 37 84.8 10.6 85.6 10.5 87.7 10.4 87.3 11.6 9.42*** 2.21f Men 15 93.1 7.1 94.3 7.9 95.3 8.0 96.6 9.6 29.22### 1 Women 22 79.2 8.9 79.7 7.5 82.5 8.5 80.9 8.0 W-H ratiob 34 0.86 (0.80-0.95) 0.87 (0.78-0.97) 0.89 (0.81-0.97) 0.87 (0.80-0.96) 2.06 3 Men 15 0.95 (0.91-0.98) 0.98 (0.95-0.99) 0.97 (0.94-1.01) 0.97 (0.93-1.01) 70.26### 1 Women 19 0.80 (0.77-0.84) 0.80 (0.77-0.83) 0.82 (0.77-0.86) 0.81 (0.79-0.86) aMean (SD) bMedian Q2 (interquartile range; Q1-Q3) cCut-off values were the following: ddata lg 10 transformed for mean comparisons eGLM for repeated measurements fdf corrected (epsilon) according to Greenhouse-Geisser *: Intrasubject effect (time): *p≤0.05 **p≤0.01 ***p≤0.001 #: Between gender effect: #p≤0.05##p≤0.01###p≤0.001 Abbreviations: SF: skinfold; W-H ratio: Waist-to-Hip ratio Table 12: Evolution of functional tests over a one year period Month 1 Month 5 Month 9 Month 13 N Average Dispersion Average Dispersion Average Dispersion Average Dispersion F df Functional tests Right hand grip strength (kg)b 44 11.59 (7.6-20.4) 11.75 (7.9-22.3) 10.5 (8.0-21.7) 11.8 (8.1-19.4) 0.14 2.20f Men 15 25.55 (18.7-29.2) 24.6 (21.8-26.2) 24.8 (19.9-29.8) 24.3 (19.1-30.3) 54.41### 1 Women 29 8.9 (7.4-12.9) 9.1 (7.0-12.9) 9.2 (7.2-12.2) 9.5 (7.0-12.3) Left hand grip strength (kg)b 45 10.65 (7.1-18.2) 10.00 (7.6-15.2) 10.2 (6.5-13.3) 10.5 (6.6-14.3) 2.18 2.41f Men 15 23.4 (16.8-29.1) 20.9 (12.6-24.6) 21.1 (11.5-24.9) 22.25 (13.6-24.8) 70.01### 1 Women 30 8.38 (6.5-11.2) 8.88 (6.4-10.7) 7.38 (6.0-11.0) 7.9 (4.9-10.8) Arm strength (Rept. Nr.)a 46 9.77 6.0 10.65 6.4 10.77 6.5 10.33 6.1 1.46 3 Men (Load 4 kg) 16 13.13 5.8 14.84 4.4 15.56 5.4 14.28 5.00 17.47### 1 60 Women (Load 2 kg) 30 7.98 5.4 8.42 6.2 8.22 5.6 8.22 4.6 Leg strength (Rept. Nr.)b 39 5.00 (0-9) 5.00 (0-11) 4.00 (0-11) 0.00 (0-10) Men 11 7.00 (0-11) 10.00 (0-14) 11.00 (0-11) 2.00 (0-12) Women 28 5.00 (0-8) 5.00 (0-10) 4.00 (0-10) 0.00 (0-9) Balance (sec)b 25 1.87 (0-3.25) 1.66 (0-2.71) 1.40 (0.43-2.00) 1.10 (0.42-1.88) Men 10 2.13 (0-6.16) 2.18 (0.65-3.69) 1.97 (1.50-3.06) 1.56 (1.05-5.87) Women 15 1.00 (0-2.55) 1.00 (0.2.10) 0.83 (0-1.44) 0.72 (0-1.59) aArithmetic mean ±SD bMedian Q2 (interquartile range; Q1-Q3) cArm volume load = Set x Nr repetitions x Load ddata lg 10 transformed for mean comparisons eGLM for repeated measurements fdf corrected (epsilon) according to Greenhouse-Geisser #: Between gender effect: #p≤0.05 ##p≤0.01 ###p≤0.001 Table 13: Evolution of MMSE scores over a one year period Month 1 Month 5 Month 9 Month 13 N Average Dispersion Average Dispersion Average Dispersion Average Dispersion F df 61 MMSE (Score)a 46 18 7.3 18 7.8 18 7.8 18 8.0 Men 17 23 5.5 22 7.1 22 6.7 22 7.8 0.98 3 Women 29 16 7.0 16 7.2 15 7.2 15 7.7 11.30## 1 aArithmetic mean ±SD #: Between gender effect: #p≤0.05##p≤0.01###p≤0.001 Table 14: Partial Pearsons Correlations (with time as control) coefﬁcients and signﬁcance level for selected blood parameters

Age B12a HoloTCa sFolatea RBC folatea tHcya Creatininea Right handa Left handa Arm MMSE Age 1 B12a -.235*** 1 HoloTCa -.136* .646*** 1 sFolatea .039 .105 -.047 1 62 RBC Folatea -.064 .221*** .172** .455*** 1 tHcya .349*** -.287*** .152* -.436*** -.317*** 1 Creatininea .184** .073 .199** - .231*** -.080 .543*** 1 Right handa -.212** -.036 .037 -.037 .019 .016 .104 1 Left handa -.237*** .060 .103 -.137 .007 -.059 .168* .895*** 1 Arm -.137* .079 .117 .012 .128 -.039 .077 .676*** .617*** 1 MMSE -.318*** .206** .047 .038 .072 -.085 -.045 .488*** .476*** .561*** 1 *p≤0.05**p≤0.01***p≤0.001 adata lg 10 transformed Table 15: Partial Pearsons Correlations (with time as control) coefficients and signficance level between anthropometric parameters and lipid profile

BMI Waist W-H ratioa Tricepsa SF subscapular LDL (mg/dL) HDL (mg/dL) Cholesterola TGa BMI 1 Waist .561*** 1 W-H ratioa .053 .598*** 1 63 Tricepsa .523*** .259*** -.352*** 1 SF subscapular .442*** .500*** .007 .465*** 1 LDL (mg/dL) .005 .020 -.155* .168* .049 1 HDL (mg/dL) -.052 -.194** -.254*** .116 .043 .033 1.000 Cholesterola -.024 -.069 -.278*** .227** .072 .927*** .276*** 1 TGa .046 -.044 -.188** .168** .078 .339*** -.341*** .400*** 1 *p≤0.05**p≤0.01***p≤0.001 adata lg 10 transformed (p≤.001) and negatively correlated with LDL-cholesterol (p≤.05), and TG (p≤.01) and HDL and total cholesterol (both p≤.001). Triceps skinfold thickness was positively correlated with subscapular skinfold (p≤.001), total cholesterol and TG (both p≤.01) and LDL- cholesterol (p≤.05). LDL-cholesterol was positively correlated with total cholesterol and TG values (both p≤.001) and HDL-cholesterol was positively correlated with total cholesterol and negatively with TG values (both p≤.001). Total cholesterol was positively correlated with TG values (p≤.001).

5.2 Study II (Intervention)

The characteristics of the whole study population are summarised in Table 16. The study group comprised 64 participants, 40 (63%) women and 24 (37%) men, with a mean age of 82 (range 63-93 y). At baseline, 13 (20%) participants of the study population showed serum cobalamin values lower than 200 pmol/l and 23 (36%) serum holo transcobalamin levels lower than 40 pmol/l. Anaemia, defined as haemoglobin concentrations < 13 g/dl in men and < 12 g/dl in women was present in 28% of the study group. Only one participant had MCV levels above 100 fl. Folate deficiency, defined as serum folate ≤ 6 ng/ml was present in 12 participants (19%). For each analysed parameter the trimmed mean at 5% was calculated and no significant differences were found between their means and trimmed means. Creatinine values were within reference range.

Table 16: Characteristics of the study population Characteristics N Mean±SD Age (y) 64 82.1±6.7 Gender (Male/Female(n)) 64 24/40 Weight (kg) 64 63.5±13.9 Knee Height (cm) 64 47.0±3.4 Height (cm) 64 153.0±7.7 BMI 64 27.2±5.8 Creatinine (mg/dl) 64 0.92±0.32 Haemoglobin (g/dl) 64 13.32±1.65 Haematocrit (%) 64 41.36±5.12 MCV (ﬂ) 64 89.78±5.58 Erythrocytes (1012/l) 64 4.61±0.54

Mean serum cobalamin levels improved from 308.4 to 558.3 pmol/l (P < .001, Table 17); the individual absolute changes for serum cobalamin, HoloTC and tHcy are shown in Figure 7 and 8. After supplementation, from the above mentioned 13 subjects with inadequate serum cobalamin levels 11 (85%) reached normal levels (deﬁned as > 200 pmol/l) and from the 23 with inadequate holoTC levels, 18 (78%) reached normal values (deﬁned as > 40 pmol/l). The quick cluster analysis delineated four clusters according to initial sCbl values. The “low” sCbl cluster consisted of eight subjects who had serum cobalamin values of

64 Figure 7: Individual changes of serum cobalamin and HoloTC values after a 28 day supplementation with 500 µg oral cyanocabalamin

65 Table 17: Wilcoxon test (z) for Pre and Post mean values for sCbl and its absolute and relative change 66 Variables N Pre Post Wilcoxon (z) Change (%) sCbl (pmol/l) 64 308.4 (132.9) 558.3(262.2) -6.95*** 249.9(172.8) 88.67(58.6) *** p < 0.001 Values are presented as mean (SD) Table 18: t-test for Pre and Post mean values (geometric and arithmetic) for holoTC, serum and RBC folate, tHcy, MCV, HCT, Hb and erythrocytes and their absolute and relative changes Variables N Pre GM Pre Post GM Post t Change (%) HoloTC (pmol/l) 64 45.77 53.8(32.5) 84.49 95.7(47.2) -14.11*** 41.8(28.0) 97.3(85.5) sFolate (nmol/l) 64 19.38 20.97(8.6) 19.21 21.02(9.1) 0.31 0.04(4.8) 1.5(21.3) 67 RBC Folate (nmol/l) 64 779.7 859.2(417.7) 863.8 945.3(434) -2.55* 86.1(334.8) 15.8(37.4) tHcy (µmol/l) 64 - 15.1(4.5) - 13.1(3.5) 8.55*** -2.0(1.9) -11.9(10.9) MCV (ﬂ) 64 - 89.69(5.57) - 89.35(5.64) 1.74 -0.34(1.56) -0.37(1.71) HCT (%) 64 - 41.4(5.15) - 41.03(4.96) 1.38 -0.37(2.14) -0.72(5.13) Hb (g/dl) 64 13.2 13.33(1.66) 13.5 13.56(1.59) -2.69** 0.23(0.70) 1.91(5.42) Erythrocytes (1012/l) 64 - 4.62(0.54) - 4.60(0.53) 0.76 -0.02(0.24) -0.36(5.24) * p < .05 ** p < .01 *** p < .001 Figure 8: Individual changes of serum tHcy values after a 28 day supplementation with 500 µg oral cyanocabalamin

126.8 (25.3) pmol/l, the “medium” cluster consisted of 17 with mean serum cobalamin levels of 220.3 (26.6) pmol/l. The “high” cluster comprised 30 and the “very high” nine participants with mean serum cobalamin concentrations of 332.6 (38.2) and 555.5 (107.3) pmol/l, respectively. The “low” sCbl cluster had serum holo transcobalamin values of 21.4 (15.2) pmol/l, the “medium” cluster of 46.0 (16.7), and the “high” and “very high” of 54.4 (21.6) and 95.8 (52.3) pmol/l, respectively. The “low” sCbl cluster had serum tHcy values of 18.5 (6.6) µmol/l, the “medium” cluster of 15.1 (3.7), and the “high” and “very high” of 13.8 (3.8) and 16.2 (4.9) µmol/l, respectively. There was a significant increase from pre- to posttreatment sCbl values (308.4 to 558.3 pmol/l, z = -6.95, df = 63, P < .001, Table 17). Similarly, holoTC concentrations rose significantly (53.8 to 95.7 pmol/l, t = -14.11, df = 63, P < .001, Table 18) as a result of the treatment. Cobalamin supplementation lowered significantly mean tHcy concentrations (15.1 to 13.1 µmol/l, t = 8.55, df = 63, P < .001, Table 18). RBC folate increased significantly from pre to postsupplementation (859.2 to 945.3 nmol/l, t = - 2.55, df = 55, P < .05, Table 18). The haemoglobin values increased significantly (13.3 to 13.6 g/dl, t = -2.69, df = 63, P < .01). No significant changes were found for sFolate, MCV, HCT and erythrocyte values (Table 18). The comparison of the pre and post sCbl, holoTC and tHcy values within each cluster showed significant changes (all P < .01); sCbl and holoTC concentrations increased whereas tHcy values decreased in each cluster (Figures 9,10,11). Regarding tHcy, the

68 Table 19: Summary table One-Way ANOVA with repeated measures and one ﬁxed factor (serum cobalamin cluster) for mean serum cobalamin (sCbl), holo transcobalamin (holoTC) and total homocysteine (tHcy) concentrations Source Sum of Square df Mean Square F sCbl 1763,621.618 1 1763,621.618 138.45*** sCbl * Cluster sCbl 176,145.652 3 58,715.217 4.61** Error (sCbl) 764,275.388 60 12,737.923 HoloTC 43,688.135 1 43,688.135 108.83*** HoloTC * Cluster sCbl 549.343 3 183.114 0.46 Error (HoloTC) 24,084.607 60 401.410 tHcy 122.437 1 122.437 71.81*** tHcy * Cluster sCbl 10.047 3 3.349 1.96 Error (tHcy) 102.300 60 1.705 ** p < .01 *** p < .001

“low” and “very high” sCbl clusters showed pre as well as posttreatment tHcy levels with a high variation and the “very high” sCbl cluster had higher initial tHcy concentrations than the “high” and “medium” group but without statistical significance. When applying the GLM model for repeated measurements with the initial sCbl as a fixed factor in order to compare the effect of the supplement according to the pretreatment sCbl status, only the increase of sCbl depended on the initial status (F = 4.61, P < .01). The effect of the supplement on holoTC and tHcy was not significantly affected by the initial sCbl status (F = 0.46 and F = 2.0, respectively, both P > .05, Table 19).

Figure 9: Change from pre to posttreatment mean serum cobalamin (sCbl) concentrations by clusters of initial sCbl concentrations

69 Figure 10: Change from pre to posttreatment mean holo transcobalamin (holoTC) concentrations by clusters of initial serum cobalamin (sCbl) concentrations

Figure 11: Change from pre to posttreatment mean total homocysteine (tHcy) concentrations by clusters of initial serum cobalamin (sCbl) concentrations

70 5.3 Study III (Polypharmacy)

Study participants were the participants from the first two projects at baseline, con- sisting of 167 elderly people from four old people’s homes of the Madrid Area. The population studied took a total of 858 medications of 292 different brands, which is equivalent to an average of 5.13 medications per resident. More than half of the residents (59%) took medications belonging to five or more different ATC groups. The most frequent groups were the psycholeptics (ATC group N05), taken by 83 residents (53%), antiacids (A02), taken by 82 residents (53%), antithrombotics (B01) 47% and psychoanaleptics (N06) 39%. Table 20 shows the complete results of the groups of medications taken. Table 21 shows the values of the biochemical variables both for the total population and for the population in which some of these variables were outside the reference interval. A deficit of vitamin B12 was found in 10.7%, of serum folate in 52.1%, and of holo-TC in 8% of the residents. There was mild hyperhomocysteinemia in 65.9% (average Hcy = 19.31 µmol/L). The influence of the variables of medication on the biochemical variables was significant for vitamin B12 and the anti anaemia medications (group ATC B03, p<0.05). The percentage of subjects with a vitamin B12 deficit was greater for the group which did not take anti anaemia medications. Hcy increased significantly with the administration of medications for obstructive pulmonary disorders (R03, p<0.05), the holo-TC deficit was lesser when psychoanaleptic medications were taken (N06, p<0.05) and the percentage of elderly people who had a deficit of serum folate was lower if they took urological preparations (G04, p<0.05) or systemic corticosteroids (H02, p<0.05). The pattern of the percentages was the same for Hcy, vitamin B12 and holo-TC, while serum folate showed an inverse pattern between the taking or not of medications (Figure 12).

71 Table 20: Number and percentage of elderly people who take one of the ATC group of medication ATC code* Definition n % ATC code* Definition n % N05 Psycholeptics 89 53 C07 Betablocking agents 11 7 A02 Drugs for acid related disorders 88 53 M05 Drugs for treatment of bone diseases 10 6 B01 Antithrombotic agents 78 47 C02 Antihypertensives 9 5 N06 Psychoanaleptics 65 39 H03 Thyroid therapy 9 5 C09 Agents acting on the renin-angiotensin system 62 37 C05 Vasoprotectives 7 4 C03 Diurectics 56 34 H02 Corticosteroids for systemic use 7 4 N02 Analgesics 53 32 C04 Peripheral vasodilators 6 4 A06 Laxatives 43 26 M04 Antigout preparations 6 4 C01 Cardiac Therapy 36 22 L02 Endocrine therapy 5 3 72 A12 Mineral supplements 25 15 A03 Drugs for functional gastrointestinal disorders 4 2 C08 Calcium channel blockers 24 14 J01 Antibacterials for systemic use 3 2 R03 Drugs for obstructive airway diseases 23 14 A05 Bile and liver therapy 2 1 B03 Antianemic preparations 18 11 N07 Other nervous system drugs 2 1 A10 Drugs used in diabetes 17 10 R05 Cough and cold preparations 2 1 C10 Lipid modifying agents 17 10 A11 Vitamins 1 1 G04 Urologicals 17 10 D03 Prepartions for treatment of wounds and ulcers 1 1 N03 Antiepileptics 17 10 D06 Antibiotics and chemotherapeutics for dermatological use 1 1 S01 Ophthalmologicals 17 10 G02 Other gynecologicals 1 1 N04 Antiparkinson’s drugs 13 8 R06 Antihistamines for systematic use 1 1 M01 Anti-inflammatory and antirheumatic products 12 7 *Anatomical Therapeutic Chemical (ATC) Classification System Table 21: Concentrations for the biochemical parameters by population Global population Population with concentrations outside of the reference interval Median p25 p75 Condition* n % Median p25 p75 73 tHcy (µmol/L) 15.91 13.2 20.29 >14.05 110 65.9 19.31 16.03 21.98 Serum folate (ng/mL) 6.9 5.3 9.4 <7.20 87 52.1 5.3 4.45 6.3 HoloTC (pmol/L) 46 29.53 62.26 <19.1 13 7.8 11.76 7.45 17.43 B12 (pg/mL) 390.35 288.92 499.92 <208 18 10.7 158.2 139.4 178.8 *Minimum or maximum concentration from which the variable was defined as being outside of the normality interval Figure 12: Percentages of participants outside and inside of the reference interval of tHcy, B12, HoloTC and folate separated by taking or not-taking medications

74 DISCUSSION

6 DISCUSSION

6.1 Study I (Longitudinal)

The lack of data related to the evolution of risk factors for cardiosvascular diseases and cognitive impairment which especially affect institutionalized people of advanced age makes it necessary to evaluate these parameters in this population for determining their evolution. To the best of our knowledge, this is the first study observing vitamin B status over a one-year period in Spanish insitutionalized elderly. The present discussion is framed in this context. After the data analysis, it could be observed, that the majority of the parameters showed a stable evolution over the studied year. Regarding vitamin B status it has to be stressed that serum cobalamin values decreased significantly and continuously over the studied period of one year. The percentage of people showing deficient serum vitamin B12 levels was the same on three of the four examination dates (10%) and even lower in one control (5%, at month 9). The percentage of deficiency found was similar to the ones found by other investigators which ranged from around 5% [154][193][171] to 19.71% [260] using a similar cutoff value as in the present thesis. When holoTC is chosen as the criterion for vitamin B deficiency (defined as < 45pmol/L), the percentages found were higher; nearly half of the participants studied showed deficient levels (46% in the beginning and 48% in the end of the study) which were higher than the ones found by other investigators for independently-living elderly (25%-34% depending on the age groups [58]). The values of holoTC found in this study were lower than those found by Hin et. al. [136] and even more than half as low as the ones found by Kivelto et al. [156]. In this context, it has to be stressed that both cut-off points are under discussion. Different cut-off points have been suggested ranging from 103 to 300 pmol/L for serum vitamin B12 (see Table 3), leading to divergence about the number of prevalences found in the literature. Regarding folate status, a notable percentage (37-43%) of participants showed low serum folate concentrations (defined as < 6 ng/mL), but only a small percentage showed folate deficiency when RBC folate was chosen as the criterion, but the latter showed a greater variation (between 4% at month 1 and 25% at month 9). Keeping in mind that RBC folate reflects folate stores, it can be stated that folate status showed a tendency to worsen over the study period. Both B12 and folate deficiencies are associated with the appearance of macrocytic- megaloblastic anaemia and immunological irregularities [22][129]. In the present study, anemia (HCT < 40% in men and HCT < 35% in women) was present in 22 and 16 participants from the first to the fourth examination time point, respectively. The

77 anemia was typically normocytic (MCV between 80 –100 fL) throughout the study except for one person. Mean Hgb concentrations were at the lower end of the reference range (men: 13.5-18.0 g/dl; women: 11.5-16.0 g/dL), and did not change during the study and were slightly lower to the ones found by other investigators [156] [136]. No significant changes were observed for median serum tHcy concentrations throughout the study duration but remained with a median value of around 17 µmol/L above the commonly two used cutoff points of 13 and 15 µmol/L. These levels were lower than the ones found by Kivipelto et al. [156] for community-living elderly although other investigators [136] found lower tHcy levels in free-living elderly. Total Hcy concentrations were significantly inversely correlated with serum and RBC folate, respectively as reported in others studies [154][164] [156] as well as with HoloTC and serum vitamin B12 [156]. In this study, prevalence of moderate hyperhomocysteinemia defined by serum tHcy of 14 –30 µmol/L did not increase over time. Of the B vitamins, folate intake generally seems to have the greatest influence on Hcy concentrations, although vitamin B12 may be more important for elderly people who suffer from cobalamin malabsorption [232]. The hypothesis that a negative cobalamin balance, occurring prior to a fall in serum cobalamin, may have resulted in a different pattern of tHcy values over the observation period could not be confirmed including in the GLM for Hcy groups with low and normal initial holoTC values (cut off point 45 pmol/L) as a fixed factor (data not shown). Other risk factors for cardiovascular diseases Among the risk factors for cardiovascular diseases are an unfavourable lipid profile, overweight and high percentage of body fat and its distribution. Lipid profile TG levels of the overall group increased whereas HDL-cholesterol decreased, both significantly; but the changes occurred within the corresponding normal reference values. However, subdividing the group by gender, the values for women showed an increment whereas the values for men were stable. Total cholesterol and LDL-cholesterol remained stable over time, but gender differences were found with women having higher values. Whereas in the case of total cholesterol these slight variations occurred within the normal range, LDL-cholesterol levels surpassed the lower threshold for high borderline values according to the ACM’s Guidelines for Exercise Testing and Prescription [8] at month 5 and 9 . Body composition Among middle aged adults (50-65y), overweight and obesity are important public health problems in various countries. For elderly people, 65y+ the health risk of overweight is unclear; in fact, data indicate that moderate overweight at advanced ages is

78 related to lower mortality [12] [209]. Among those 80y+, thinness and loss of lean body mass may be a more significant problem than overweight. A U-shaped relationship between BMI and mortality has been reported for Finnish men [217]. Lowest mortality occurred at a somewhat higher BMI among men over 75 years of age than among younger men, but the curve showed excess mortality at the tails of the distribution. Among thin men mortality from cardiovascular causes increased with BMI in the younger cohorts but not in those aged 55-90. A U-shaped relationship between BMI and mortality was also apparent in young women, but was more uncertain for older women, who showed little variation and mortality with BMI. Overweight did not reduce life expectancy in Finnish women aged 65-79 years [218]. Indeed, a modest degree of overweight seemed to be protective against death. The most favourable BMI was 27-31, a considerably higher figure than was observed in Norway. A follow-up study in Finland of 95 men and 431 women over 85 years of age showed that a low BMI was a more important predictor of risk of death than a high BMI [132]. High- est 5-year mortality was reported in the group with BMI < 20.0 and the lowest in the group with BMI greater than 30. It was concluded that overweight ceases to be a risk factor for death in this age group. Both overweight and thinness appear to carry risk for mortality, but in the elderly thinness carries a greater risk than overweight. Weight change, especially involuntary weight loss, also poses considerable risk, which may interfere with the interpretation of the data from large studies. In our study, the median values of BMI for men remained stable (between 28 and 29); same was observed for women (BMI values between 27 and 28), which are considered to be normal for this age group according to the criteria used by the WHO Expert Committee [264]. Com- paring the results to the ones found by Camiña Fernández et al. [38], the values for the women correspond to the category of good to average whereas the values for men were a little higher than the average. As BMI was stable during the studied year, the risk for cardiovascular diseases and stroke seem to be not affected by this parameter. Skinfold thickness (triceps and subscapular) Skinfolds are considered a good measure of subcutaneous fat in different group of subjects but older subjects of the same gender and body density have proportionally less subcutaneous fat than their younger counterparts [160]. They also have a higher proportion of internal fat and the proportion of fat located internally decreases as overall fat increases. The sum of several skinfolds has been proposed to estimate body fat but biological variation in fat distribution is affected by age, gender and degree of fatness [134]. Skinfold thickness of the triceps remained stable (between 12 and 13 mm for men and between 14 and 17 mm for women, respectively). Subscapular skinfold thickness varied between 18 and 22 mm for men and 15 and 18 mm for women, respectively. The subscapular skinfold is considered a useful predictor of lipid profile; however, in

79 our study, triceps skinfold but not the subscapular skinfold correlated positively with the values of total cholesterol and TG but not with HDL or LDL-cholesterol. The values registered in this study can be used for comparisons among groups with similar characteristics to the ones of the present study. Waist and Waist-to-Hip ratio Health risk increases with W-H ratio levels, and standards for risk range with age and gender. Health risk is very high for young men when W-H ratio exceeds 0.95 and for young women when WHR exceeds 0.86, whereas for people 60 to 69 years old, the cut-offs for the same risk classiﬁcation are greater than 1.03 for men and greater than 0.90 for women [100] [135]. In our study, the median as well as the whole interquartile section were in the normal range and were similar to the ones found in the Exernet Study by Ara et al. [13] (0.96 for men and 0.89 for women, respectively). Waist circumferences increased 4 cm for both genders (from 93 cm to 97 cm for men and from 79 cm to 83 cm for women over the year). For both genders those values were smaller than the ones found in the Exernet study whose P50 values were 98.8 cm for men and 94.3 cm for women, respectively [13], although the change of waist circumferences in men over the studied year entered in the category of increased mortality (WC between 94 and 102 cm) revealed by the results of the Rotterdam Study, a population-based cohort study with a mean follow-up of 5.4 y [256]. Functional tests and MMSE Presenting a deteriorated vitamin status can lead to functional implications of clinical importance. Since physical impairment is one reason for long-term institutionalization, impaired strength may be expected for elderly people residing in old people’s homes. Poor hand strength as measured by hand-grip is a predictor of disability in older people [109]. In the present study, median right and left hand grip were stable for men and women with higher values in men compared to women over the entire duration of the study. The range of the right and left hand grip strength of our male participants (21 and 23 kg for left hand and between 24 and 26 kg for right hand strength, respectively) was similar to the average category of hand strength reported by Camiña Fernández et al. [38] (20.7-30.6 kg) for elderly active people aged over 80 participating in a programme of physical activity. The range of the right and left hand grip strength of our female participants (between 9 and 10 kg for right hand and between 7 and 9 kg for the left hand grip strength) was smaller than the lowest category of hand strength reported by Camiña Fernández et al. [38] (<12.4 kg). Arm strength remained stable over the year. The mean value for men which ranged between 13 and 16 Nr. of repetitions were the same as the ones of the age group 80+ from the Exernet study group [13] whereas the women in this study only performed

80 half of the repetitions of the women of the age group 80+ of the Exernet study group [13] (8 compared to 15 Nr of repetitions, respectively). The values for both gender were notably lower than the ones found in the study performed by Vallejo et al. [251] in which Spanish male participants performed 25.0 ± 4.8 (3.63kg load) and the female participants 20.1 ± 3.6 (2.27kg load) Nr. of repetitions, respectively. Regarding leg strength, men as well as women experienced a notably decrease from month 9 to month 13. The values of the present study were lower compared to the ones found within the Exernet study [13] (14 (=P50) for both gender for the age group 80+) and also to the ones found by Vallejo et al. [251] (men 18.27 ± 4.94; women 14.86 ± 1.98 for an elderly group of 33 people, 22 women and 11 men with a mean age of 67.37 ± 4.79y). The latter group studied was younger and both mentioned elderly groups studied were active; however, the great differences are still indicating very poor status of leg strength which might be due to lack of use because of a sedentary life and the unnecessary use of wheel chairs. It has to be stressed that several participants were not able to perform the test and that the results of the subjects who performed were extremely low which might indicate that this test is not appropriate when evaluating this population group. In terms of balance, male participants performed the balance test with a median average of 2 sec, whereas the female participants were able to stand on one foot for 1 sec. Those results were lower than the values found for the group aged over 80 of active Spanish elderly people who participated in the EXERNET [13] study (P50 for men 6.2 sec and for women 5.6 sec, respectively). As mentioned before for leg strength a high percentage of participants was not able to perform the test which again might indicate that this test is not appropriate as a measurement for this study group. Mean MMSE scores showed a stable pattern over the year of examination with higher scoring for men compared to women (between 22 and 23). In the present study, a positive correlation was found between the MMSE scores and serum vitamin B12 but not for holoTC concentrations, nor with serum folate or RBC folate, nor between MMSE scores and serum tHcy concentrations. This was in agreement with some studies [235][23] [224] that found no association between folate status and cognitive state although on the other hand, Riggs et al. [212] and Gottfries et al. [113] showed that serum Hcy concentrations can be considered an early and sensitive marker for cognitive impairment. Lack of association between serum tHcy concentrations and MMSE scores in the present study may be related to the length of the study duration and to the small number of study population.

81 6.2 Study II (Intervention)

The main results of the study were that Hcy values decreased significantly whereas serum cobalamin and holoTC values increased significantly after only 4 weeks of daily 500 µg cyanocobalamin supplementation. The observed effect improves the results of a former investigation of a dose-finding trial conducted by Eussen et al. [85] who concluded that when comparing a daily oral dosage of 500 µg vs 1000 µg cyanocobalamin administered for eight weeks that 500 µg was the lowest oral dose associated with a maximum increase of holoTC levels in mild vitamin B12 deficient subjects, deficiency defined as serum vitamin B12 levels of 100-300 pmol/l. In the study by Eussen et al. [85] they did not analyse a period of 4 weeks as was done in our study. The increase in the subjects included in the “low” cluster in our study was slightly higher than the one presented by the group in the dose-finding trial by Eussen et al. [85] which received the same amount of oral cyanocobalamin (186 vs 182 pmol/l) but during double of time. In addition, the increase of the “medium” cluster even reached the same effect as the group receiving 1000 µg/d of Eussen´s et al. dose-finding trial [85] for eight weeks and the milk trial in the study performed by Dhonukshe-Rutten [73], who administered a milk drink enriched with 1000 µg crystalline Cbl or the same amount containing cap- sules for 12 weeks (249 vs 248 vs 250 pmol/l) to mildly cobalamin deficient elderly defined as serum vitamin B12 levels of 100-300 pmol/l. Highly significant increases were also observed for holoTC values, both for the whole study group and for each cluster. The increase of the “low” and “medium” clusters were smaller (36 vs 45 vs 49 pmol/l) than the one presented by the 500 µg oral Cbl receiving group in the dose-finding trial [85]. Regarding tHcy, mean values of the “medium” cluster decreased less than in the 500 µg oral Cbl administered subgroup in the dose-finding trial [85] (-1.5 vs -1.9 µmol/l) and the capsule trial (-1.8 µmol/l) of the above named study performed by Dhonukshe- Rutten [73]. In contrast, the “low” cluster showed a higher response of -3.4 µmol/l. But it has to be mentioned that both, pre and posttreatment values, presented a great variation. The “very high” sCbl cluster had higher pretreatment tHcy concentrations than the ones for the “high” and even “medium” group. It has to be mentioned that in our study, high tHcy values were mostly independent of Cbl status. In relationship to folate, folate deficiency according to serum folate levels was present in 12 subjects (18.5%) of the whole study population. Only two subjects had tHcy values < 13 µmol/L. This seems to indicate that other factors beyond Cbl and folate status such as lifestyle factors [67] are affecting tHcy concentrations in this population group. Fur- thermore, that cluster consisted of few people and showed a big variance and pretreatment value did not reach statistical significance compared to the pretreatment values

82 of the other clusters. The oral vitamin B12 supplementation did not significantly change mean sFolate. RBC folate levels increased (P < .05) in the whole study population. These findings are supported by a double-blind, placebo-controlled trial [84] where oral vitamin B12 was supplemented alone or together with folic acid. In the latter study, mildly vitamin B12 deficient subjects (n = 195) were randomly assigned to receive 1000 µg vitamin B12, 1000 µg vitamin B12 in combination with 400 µg folic acid, or a placebo. After 12 weeks of supplementation, mean RBC folate values increased significantly in the vitamin B12 group and in the vitamin B12 plus folic acid group. In the above-mentioned study performed by Dhonukshe-Rutten et al. [73] after 12 weeks both intervention sets also presented an increase of RBC folate, but the increase reached statistical significance only in the milk trial. After having performed the quick cluster analysis, in our study the significant increase of RBC folate disappeared (data not shown). To the best of our knowledge, only two studies included haematological parameters in the outcome. The results of the present study are consistent with previous trials that reported no significant changes in haematological parameters within one month of Cbl administration such as the study conducted by Seal et al. [227] who compared the effectiveness of 10 and 50 µg oral cyanocobalamin or placebo administered daily for four weeks in 31 older patients with deficient or borderline serum vitamin B12 levels between 100 and 150 pmol/l. Hvas et al. [145] found no significant changes in blood Hb and MCV between the treatment group (n = 70) and the placebo group (n = 70) in a Danish clinical trial with participants older than 18 y. It has to be mentioned that these studies administered much lower doses than was the case in our study. The results support the fact that a daily dose of 500 µg of cyanocobalamin during 28 days improves vitamin B12 status in institutionalized elderly. No control group was included because foods provided at the nursing homes followed a regular pattern so dietary factors were unlikely to be important. Additionally, as has been mentioned above, dietary vitamin B12 intake as a determinant of blood concentrations decreases in importance during the ageing process because of increasing malabsorption problems[130][11].

6.3 Study III (Polypharmacy)

The ﬁrst contribution of this study is a description of the type of medications which the institutionalized population of old people in the Region of Madrid take every day, data which are currently very scarce. The second is the high percentage of elderly with concentrations which are considered pathological of mainly Hcy (hyperhomocysteinemia) and serum folate (deﬁcit). These percentages are very similar to those found by other

83 authors in populations of institutionalized elderly [111][183] except in the case of holo- TC where the percentage of elderly with a deficit was lower than that described in other studies. The reason is the use of different cut off points for the deficit of holo-TC. As has been stated already, for this study the cut offs recommended by the immunoassay manufacturer (19.10 pmoL/L) were used while other authors used higher cut off points, between 30 and 40 pmol/L depending on the measurement procedure used [183]. A folate deficit can be corrected with a folate rich diet or even with folate supplements. However, hyperhomocysteinemia, even when mild, is more difficult to resolve, as it is not always due to a lack of vitamin B12 or folate [15]. Thus the importance of car- rying out frequent follow ups of homocystein levels on this type of population, as the relation is known between hyperhomocysteinemia and the increased risk of a cardiovascular event and other problems mentioned above. A significant influence on Hcy levels was only found for the medications for pulmonary obstruction. Several authors have already described slight increases of Hcy in obstructive pulmonary pathologies, in particular COPD [228]. It is also known that bronchodilators derived from theo- phylline can increase Hcy levels [97]. Thus the increased level of Hcy in patients who took this group of medications could be due to the reasons mentioned above. The anti anaemia medications, formed by compounds of vitamin B12, folates, and iron or a combination of them, revealed a significant influence on the normalization of vitamin B12 levels. The levels of folate tended to normalize under the influence of the psychoanaleptic medications, mainly the antidepressants, taken by some residents. Here the reason for the influence is not clear. Some studies show that the risk of suffering from dementia or depression increases when the levels of vitamin B12 and/or folate are low [178]. In fact it is thought that supplementation with vitamin B12 and folate can potentiate the action of the psychoanaleptics [49]. It is in fact unknown whether or not there is a direction relation between levels of holo-TC and depressive or cognitive pathologies. Lastly the mechanisms or reasons why urological preparations or systemic corticoids could increase levels of serum folate are still unexplained. To carry out a statistical study to show the influence of a determined group of medications on a biochemical parameter in this type of population is complex. Most of the subjects take medications from more than five different groups, which cause an interrelation of all the medications, making it difficult to study individual relations between a medication and the biochemical variable. As an example, although there is a relation between levels of Hcy and vitamin B12, medications which had a significant influence on one of the variables had no influence on the other. Another additional problem is the impossibility of working with a control group of elderly people who take no medications.

84 CONCLUSIONS

7 CONCLUSIONS

The following conclusions address the topics presented in the hypotheses as well as the objectives of this thesis. 1. Over one year, in relationship to vitamin B status, serum cobalamin decreased, serum folate and mean corpuscular volumen increased significantly and total homocysteine concentrations were stable. Regarding blood lipid profile, triglycerides increased and HDL-cholesterol decreased significantly. Regarding selected anthropometric measurements, waist circumference increased significantly. No significant changes were observed for the rest of the parameters. 2. Prevalence of hyperhomocysteinemia was high in the studied elderly, ranging from 60% to 90 % over the year depending on the cut-off used for the classification. LDL- cholesterol values were high, especially among women, and showed a tendency to increase over the year. Results of the balance test showed a deficiency and a tendency to decrease; this indicates that the population studied is at high a risk for falls. Lower extremity muscular function was deficient and showed a tendency to decrease. A highly significant relationship was observed between the skinfold of the triceps and blood lipid profile. 3. Low cobalamin concentrations correlated significantly with low MMSE scores in the elderly studied. No correlations were observed between vitamin B12 status and functional parameters. 4. Regarding vitamin B12 status, holotranscobalamin seems to be more sensitive for diagnosis; 5-10% of the elderly were deficient using serum cobalamin as a criterion, and 45-52% were deficient when using serum holotranscobalamin as a criterion. 5. 500 µg of cyanocobalamin administered orally during 28 days improved significantly vitamin B12 status and decreased significantly total homocysteine concentrations in institutionalized elderly. No effect of the intervention was observed on functional and cognitive parameters. 6. The relative change (%) of improvement of vitamin B12 status was higher when using serum holotranscobalamin as a criterion than when using serum cobalamin. 7. Antiaenemic drug intake normalized cobalamin, urologic drugs and corticosteroids serum folate, and psychoanaleptics holotranscobalamin levels. Drugs against pulmonary obstruction increased total homocysteine concentration significantly. 8. The daily mean drug intake was 5.13. Fifty-nine percent of the elderly consumed medication belonging to 5 or more ATC different groups. The most prevalent were psycholeptic (53%), antiacid (53%) and antithrombotic (47%) drugs.

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115

8 Appendices

8.1 Case Report Form Longitudinal Study

8.2 Case Report Form Intervention Study

8.3 MMSE approved form (version date September 6, 2002)

117

CASE REPORT FORM

PROJECT: - La deficiencia de vitamina B12 como factor de riesgo de enfermedades neurodegenerativas (cognitivas y funcionales) en las personas mayores - Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people

Date Code

Name

Date of birth Age Sex

Entry date to the residence Profession

I. STUDY START: Yes No The subject has signed the informed consent form

Date and signature of the investigator:

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 1/14

II. SELECTION OF SUBJECTS a) Inclusion criteria Yes No 01 Male and female subjects aged 60 and more 02 Subclinical vitamin B12 deficiency b) Exclusion criteria Yes No 01 Neuropathy 02 Hunter’s glossitis 03 Addison’s/Megaloblastic anaemia 04 Vitamin B12 intake (supplements) 05 Folate intake (supplements)

Date and signature of the investigator:

III. NUTRITIONAL ASSESSMENT The subject has completed the MMSE Yes No A B C D

Comments:

Date and signature of the investigator (A):

Date and signature of the investigator (B):

Date and signature of the investigator (C):

Date and signature of the investigator (D):

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 2/14

IV. PSYCHOLOGICAL ASSESSMENT The subject has participated this part of the study Yes No A B C D

The subject has completed the MMSE Yes No A B C D

Incidences:

Date and signature of the investigator (A):

Date and signature of the investigator (B):

Date and signature of the investigator (C):

Date and signature of the investigator (D):

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 3/14

V. PHYSICAL EXAMINATION The subject has participated this part of the study Yes No A B C D

A B C D 01 Fuerza y presión manual 02 Fuerza piernas 03 Fuerza brazos 04 Equilibrio (flamenco) 05 Equilibrio dinámico 06 Transferencia de fichas 07 Guardar cerillas

Incidences:

Date and signature of the investigator (A):

Date and signature of the investigator (B):

Date and signature of the investigator (C):

Date and signature of the investigator (D):

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 4/14

VI. ANTHROPOMETRIC DATA The subject has participated this part of the study Yes No A B C D

A B C D 01 Weight 02 Height 03 Skin fold thickness a) Bicipital b) Tricipital c) Subscapular d) Thigh e) Calf 04 Circumferences a) Arm b) Biceps c) Thigh d) Calf e) Waist f) Hip 05 Diameter a) Wrist b) Elbow joint c) Knee joint

Incidences:

Date and signature of the investigator (A):

Date and signature of the investigator (B):

Date and signature of the investigator (C):

Date and signature of the investigator (D):

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 5/14

VII. ANALYTIC (SCREENING) Blood samples have been collected Yes No A B C D

Date Hour Sample (dd/mm/yyyy) (hh/mm) A B C D

Has the subject been fasting at the time of blood sampling? Yes No A B C D

If no, please indicate taken foods and/or drinks A

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 6/14

VIII. ALIQUOTS Aliquots A B C D 01 3 mL Vacutainer with EDTA (haematology) 02 5 mL Vacutainer with heparin (vitamin B6) 03 8 mL Vacutainer with gel (serum) 04 8 mL Vacutainer with gel (serum)

A B C D 01 Genotype thermolabile MTHF reductase ------02 Plasma folate 03 Erythrocyte folate 04 Homocysteine 05 Methylmalonic acid 06 Vitamin B12 07 Holotranscobalamin II 08 Plasma vitamin B2 09 Plasma vitamin B6 10 Hematology 11 Biochemistry 12 Coagulation 13 14 Serothek

Comments:

Date and signature of the investigator (A):

Date and signature of the investigator (B):

Date and signature of the investigator (C):

Date and signature of the investigator (D):

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 7/14

IX. CLINICAL DATA Yes No 01 Cardiovascular disease 02 Thrombotic incidences 03 Diabetes 04 Alzheimer 05 Hypertension 06 Cancer 07 Others:

Comments:

X. INTESTINAL HABIT Yes No 01 Diarrhoea 02 Constipation

Comments:

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 8/14

XI. (A) CURRENT TREATMENT (Medication or supplements) Has the subject taken any treatment for more than 7 days during Yes No the last 30 days?

Start End On- If yes, please specify when what was taken: date date going 01

Comments:

Date and signature of the investigator:

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 9/14

XII. (B) CURRENT TREATMENT (Medication or supplements) Has the subject taken any treatment for more than 7 days during Yes No the last 30 days?

Start End On- If yes, please specify when what was taken: date date going 01

Comments:

Date and signature of the investigator:

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 10/14

XIII. (C) CURRENT TREATMENT (Medication or supplements) Has the subject taken any treatment for more than 7 days during Yes No the last 30 days?

Start End On- If yes, please specify when what was taken: date date going 01

Comments:

Date and signature of the investigator:

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 11/14

XIV. (D) CURRENT TREATMENT (Medication or supplements) Has the subject taken any treatment for more than 7 days during Yes No the last 30 days?

Start End On- If yes, please specify when what was taken: date date going 01

Comments:

Date and signature of the investigator:

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 12/14

XV. SERIOUS ADVERSE EVENT REPORT Investigator aware of serious adverse event Date:

01 Death * 02 Hospitalisation ** 03 Life threatening 04 Persistent or significant disability/incapacity 05 Important medical event 06 Other:

* If death: Date of death: Probable cause of death: Autopsy performed? Yes No

** If hospitalised: Date of hospitalisation: Date of discharge::

Description of the adverse event 01 Symptoms

02 Course

03 Diagnostic

04 Investigations

05 Results

06 Treatment

07 Other comments

Date and signature of the investigator:

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 13/14

XVI. STUDY TERMINATION Has the subject completed all exams and questionnaires of the Yes No study?

If no, please specify:

Date of subject’s withdrawal Main reason for premature discontinuation: 01 Expressed personal request 02 Exclusion criteria 03 Adverse event 04 Others: please specify:

Comments:

Date and signature of the investigator:

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 14/14

CASE REPORT FORM

Date Code

Name

Date of birth Age Sex

Entry date to the residence Profession

STUDY START: Subject’s informed consent Yes No The subject has signed the informed consent form

Date and signature of the investigator:

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 1/10

SELECTION OF SUBJECTS Inclusion criteria Yes No 01 Male and female subjects aged 60 and more 02 Subclinical vitamin B12 deficiency

Exclusion criteria Yes No 01 Neuropathy 02 Hunter’s glossitis 03 Addison’s/Megaloblastic anaemia 04 Vitamin B12 intake (supplements) 05 Folate intake (supplements)

Date and signature of the investigator:

NUTRITIONAL ASSESSMENT The subject has completed the following questionnaires: Yes No 01 Mini Nutritional Assessment (MNA) 02 Questionnaire B12

Comments:

Date and signature of the investigator :

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 2/10

PSYCHOLOGICAL ASSESSMENT A B The subject has participated this part of the study Yes No Yes No

The subject has completed the following questionnaire A B Yes No Yes No Mini Mental State Examination (MMSE)

Incidences:

Date and signature of the investigator (A) :

Date and signature of the investigator (B) :

PHYSICAL EXAMINATION A B The subject has participated this part of the study: Yes No Yes No

A B Yes No Yes No 01 Test of Rikli and Jones 02 Test of Groningen for adults aged over 55 years 03 Dinamometria 04 Equilibrium 05 Barthel Index [3]

Incidences:

Date and signature of the investigator (A) :

Date and signature of the investigator (B) :

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 3/10

ANTHROPOMETRIC DATA A B The subject has participated this part of the study: Yes No Yes No

A B Yes No Yes No 01 Weight 02 Height 03 Skin fold thickness a) Bicipital b) Tricipital c) Subscapular d) Thigh e) Calf 04 Circumferences a) Arm b) Biceps c) Thigh d) Calf e) Waist f) Hip 05 Diameter a) Wrist b) Elbow joint c) Knee joint

Incidences:

Date and signature of the investigator (A):

Date and signature of the investigator (B):

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 4/10

ANALYTIC (SCREENING) A B Blood samples have been collected Yes No Yes No

A B Sample Date (dd/mm/yyyy) Hour (hh/mm)

A B Has the subject been fasting at the time of blood Yes No Yes No sampling?

If no, please indicate taken foods and/or drinks A

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 5/10

Aliquots A B Yes No Yes No 01 3 mL Vacutainer with EDTA (haematology) 02 5 mL Vacutainer with heparin (vitamin B6) 03 8 mL Vacutainer with gel (serum) 04 8 mL Vacutainer with gel (serum)

A B Yes No Yes No 01 Genotype thermolabile MTHF reductase ------02 Plasma folate 03 Erythrocyte folate 04 Homocysteine 05 Methylmalonic acid 06 Vitamin B12 07 Holotranscobalamin II 08 Plasma vitamin B2 09 Plasma vitamin B6 10 Hematology 11 Biochemistry 12 Coagulation 13 14 Serothek

Comments:

Date and signature of the investigator (A):

Date and signature of the investigator (B):

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 6/10

CLINICAL DATA Yes No 01 Cardiovascular disease 02 Thrombotic incidences 03 Diabetes 04 Alzheimer 05 Hypertension 06 Cancer 07 Others:

Comments:

INTESTINAL HABIT Yes No 01 Diarrhoea 02 Constipation

Comments:

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 7/10

CURRENT TREATMENT (Medication or supplements) Has the subject taken any treatment for more than 7 days during Yes No the last 30 days?

If yes, please specify when what was taken: Start End On- date date going 01

Comments:

Date and signature of the investigator :

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 8/10

SERIOUS ADVERSE EVENT REPORT Investigator aware of serious adverse event Date:

01 Death * 02 Hospitalisation ** 03 Life threatening 04 Persistent or significant disability/incapacity 05 Important medical event 06 Other:

* If death: Date of death: Probable cause of death: Autopsy performed? Yes No

** If hospitalised: Date of hospitalisation: Date of discharge::

Description of the adverse event 01 Symptoms

02 Course

03 Diagnostic

04 Investigations

05 Results

06 Treatment

07 Other comments

Date and signature of the investigator :

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 9/10

STUDY TERMINATION Has the subject completed all exams and questionnaires of the Yes No study?

If no, please specify:

Date of subject’s withdrawal Main reason for premature discontinuation: 01 Expressed personal request 02 Exclusion criteria 03 Adverse event 04 Others: please specify:

Comments:

Date and signature of the investigator:

Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 10/10

DOCUMENTO FUENTE – N.º DE PROTOCOLO Iniciales del paciente: Randomización N.º Fecha: Visita: ______Página 1 de 4

Mini-Mental State Examination (MMSE)

Instrucciones: las palabras en negrita deben leerse en voz alta y clara a la persona a la que se evalúa. Entre paréntesis aparecen las distintas alternativas para las preguntas. La prueba debe realizarse en privado y en la primera lengua del examinando. Rodee el 0 si la respuesta es incorrecta, o 1 si es correcta. Comience haciendo las siguientes preguntas: ¿Tiene algún problema de memoria? ¿Puedo hacerle unas preguntas sobre su memoria?

ORIENTACIÓN EN EL TIEMPO RESPUESTA PUNTUACIÓN (marque una) ¿ En qué año estamos? ______0 1 estación ______0 1 mes del año ______0 1 día de la semana ______0 1 fecha ______0 1

ORIENTACIÓN EN EL ESPACIO*

¿Dónde estamos ahora? ¿En qué ... estamos?

estado (provincia) ______0 1 país (o ciudad/pueblo) ______0 1 ciudad/pueblo ______0 1 (o zona de la ciudad/barrio) edificio (nombre o tipo) ______0 1 piso del edificio ______0 1 ( nº habitación o dirección)

*Se pueden utilizar y anotar otras palabras de lugar que encajen en la serie y sean más precisas.

MEMORIA INMEDIATA

Escuche atentamente. Voy a decir tres palabras. Repítalas cuando yo termine. ¿Está listo? Estas son: MANZANA [pausa], CÉNTIMO [pausa], MESA [pausa]. Ahora repítame las palabras. [Repítalo 5 veces, pero puntúe sólo la primera serie]

MANZANA ______0 1 CÉNTIMO ______0 1 MESA ______0 1

Ahora memorice estas palabras. Le pediré que las repita dentro de unos minutos.

PAR Psychological Assessment Resources, Inc./ 16204 North Florida Avenue/Lutz FL 33549/Toll-Free 1.800.331.TEST MMSE Copyright © 1975, 1998, 2001 por Minimental, LLC. Todos los derechos reservados. Publicado en 2001 por Psychological Assessment Resources, Inc. No deberá copiarse ni reproducirse en modo alguno, ni parcial ni completamente, sin el permiso expreso por escrito de Psycological Assessment Resources, Inc.

MMSE Version date: September 6, 2002 DOCUMENTO FUENTE – N.º DE PROTOCOLO Iniciales del paciente: Randomización N.º Fecha: Visita: ______Página 2 de 4

ATENCIÓN Y CÁLCULO [Serie 7s]*

Ahora tiene que restar 100 menos 7. Luego siga restando 7 del resultado que obtenga hasta que le diga que puede parar.

¿Cuánto es 100 menos 7? [93] ______0 1 Si es necesario, diga : Continúe [86] ______0 1 Si es necesario, diga : Continúe [79] ______0 1 Si es necesario, diga : Continúe [72] ______0 1 Si es necesario, diga : Continúe [65] ______0 1

Sólo se deben hacer otras preguntas (MUNDO al revés) si la persona a la que se está evaluando se niega a realizar el ejercicio de la Serie 7. Realice y puntúe esta pregunta sólo si la persona que está siendo evaluada se niega a realizar el ejercicio de la Serie 7s. Deletree MUNDO hacia delante y luego hacia atrás. Si al deletrear hacia delante se ha equivocado corríjale, pero puntúe sólo cuando deletree hacia atrás. ______(O=1) (D=1) (N=1) (U=1) (M=1) (0 a 5)

RECUERDO DIFERIDO

¿Qué tres palabras le pedí que recordara? [No dé ninguna pista]

MANZANA ______0 1 CENTIMO ______0 1 MESA ______0 1

NOMBRAR

¿Qué es esto? (Señale un lápiz o bolígrafo) ______0 1 ¿Qué es esto? (Señale un reloj) ______0 1

REPETICIÓN

Ahora voy a pedirle que repita lo de que yo diga. ¿Está preparado? “EL FLAN TIENE FRUTILLAS Y FRAMBUESAS”. Ahora dígalo. [Repítalo hasta 5 veces, pero puntúe sólo la primera.]

“EL FLAN TIENE FRUTILLAS Y FRAMBUESAS” ______0 1

MMSE Version date: September 6, 2002 COMPRENSIÓN

Escuche con atención porque voy a pedirle que haga algo. Coja este papel con su mano derecha [pausa], dóblelo por la mitad [pausa], y póngalo en el suelo (o mesa).

COGER CON LA MANO DERECHA ______0 1 DOBLAR POR LA MITAD ______0 1 PONER EN EL SUELO (o MESA) ______0 1

LECTURA

Por favor lea esto y haga lo que dice. [Muéstrele el impreso de estímulo]

CIERRE LOS OJOS 0 1

ESCRITURA

Por favor, escriba una frase. [Si no responde, diga: Escriba sobre el tiempo.] 0 1

Coloque el trozo de papel en blanco (sin doblar) frente a la persona que realiza el examen y dele un bolígrafo o un lápiz. Anote 1 punto si la frase es comprensible y contiene sujeto y predicado. Ignore los errores de gramática u ortografía.

DIBUJO

Por favor, copie este dibujo. [ Muestre los pentágonos del impreso de estímulo.] 0 1

Anote 1 punto si el dibujo consta de dos figuras con cinco lados que forman una intersección para formar una figura de cuatro lados.

Anote este valore en la página de puntuación de las evaluaciones del CRD. Puntuación total = ______

(Sume todos los puntos) (máx. 30)

Nombre del evaluador: ______

Firma del evaluador: ______

Fecha: ______

Hora de comienzo de la evaluación: ______(00:00 – 23:59)

MMSE Version date: September 6, 2002 MMSE Version date: September 6, 2002 CIERRE LOS OJOS

AGRADECIMIENTOS

Escribir una tesis doctoral no es una tarea sencilla, aunque resulte mucho más fácil cuando se cuenta con un equipo que te apoya en tu trabajo y personas que realizan parte de las diversas gestiones necesarias. Durante esta etapa son muchas las personas que me han rodeado. No solamente personas sin más, sino sobre todo, un equipo humano muy especial, al que no puedo dejar de mencionar. Desde los últimos años de estudiante de la carrera de nutrición una mujer me ha guiado, la Profesora María Marcela González Gross y a ella le debo en gran parte el constante empuje para seguir con mi andadura en la investigación. He aprendido mucho de ella y han sido muy gratiﬁcantes todos estos años. En realidad, no existen palabras para agradecer todo el apoyo y conﬁanza que ella ha puesto en mi durante todo este tiempo. He contado con su apoyo, cuento y estoy segura que contaré con él. Quiero expresar mi agradecimiento a mi co-director Dr. Agustín Meléndez Ortega que siempre ha estado a mi lado, por su motivación, por sus comentarios y críticas constructivas que poco a poco me han permitido completar el trabajo, por sus consejos, especialmente en los momentos menos agradables y por la amistad que como persona me ha demostrado a lo largo de la realización de esta tesis. Aunque no lo parezca, sí me han quedado bien claras sus historias y moralejas y ya poseo un buen “saco de historias” que contar. Quiero agradecerle a los Prof. Klaus Pietrzik y Prof. Manuel Castillo su contribución a mi formación y a los trabajos presentados en diversas ocasiones. Gracias también a los doctores Gabriel Rodríguez Romo, Pedro Jesús Jiménez Martín y Enrique López Adán, por sus comentarios, correcciones y consejos durante la pre- lectura de la tesis que me han permitido detectar errores y conseguir una mayor claridad en su redacción. Quiero agradecer también a Ma Jesús Morala del Campo y Blanca Clavijo Juaneda de la Asociación de Familiares de Alzheimer (AFAL) el apoyo facilitado para conseguir la participación de las residencias de ancianos seleccionadas y al personal tanto sani- tario como auxiliar de éstas, la cooperación prestada para la realización de las pruebas y la recogida de las muestras. Merecen especial mención los médicos Javier Caballero, José Luis Tobaruela, Patricia Morán y Juan Carlos Pastor de dichas residencias por su total apoyo y colaboración. No puedo olvidar la sincera cooperación de las personas mayores residentes que participaron de forma desinteresada e ilusionada en estos estudios, que me dieron un extraordinario ejemplo de dignidad. A Gonzalo Palacios por su dedicación y colaboración y por su apoyo profesional y personal sincero.

149 A David, Víctor, Ana, María, Rocío, y Jara que participaron de forma activa en la recogida de datos y especialmente a Raquel por su más estrecha colaboración. A Monika, Christina, Eva, Rebecca e Ilaria por toda su ayuda, interés y dedicación en todos estos proyectos. Quiero expresar mi más sincero agradecimiento a Pilar Irureta-Goyena Sánchez y al personal de la Biblioteca de la Facultad por su ayuda y gestiones para conseguir algu- nas referencias difíciles de encontrar; igualmente, quiero agradecerle al personal del Rectorado su amable trato y la información que me han proporcionado. A Laura Barrios del Consejo Superior de Investigaciones Cientíﬁcas (CSIC) por su asesoramiento en el tratamiento estadístico de los datos y a Gádor Faura y especialmente a Paloma Navarro por las gestiones administrativas necesarias para la realización de esta tesis. Al mismo tiempo, quiero agradecer a la Universidad Politécnica de Madrid la oportu- nidad que me ofrecieron para realizar el doctorado concediéndome la beca predoctoral y la posibilidad de la realización de mis estancias en el extranjero. Cómo olvidar a Rosa Torres, siempre dispuesta a colaborar y con la capacidad de poder tolerar bien los momentos tensos de la elaboración de la tesis debidos a cuestiones todavía sin resolver o partes del trabajo sin completar o a Ms Diane Schoﬁeld que ha revisado las diferentes copias de esta tesis y los artículos, comunicaciones y posters originales de estos estudios. I would like to thank all the people I got the chance to know during my internships in Lisbon, Lille and Coleraine. You are all so different and also all so very important for me. Meinem Vater Carl-Heinz für sein großes Verständnis. Meiner Mutter Christina für ihre einzigartige Sichtweise der Dinge und meinem Bruder Benedikt für seine allumfassende Liebe. Mögen sie beide in Frieden ruhen. Me gustaría agradecer también a todos mis profesores de idiomas su gran dedicación a la enseñanza y por haber podido mantenerme motivada, “CHAPEAU”! A Ricardo Sola por su asesoramiento en los temas hematológicos y a Reme por apor- tarme su experiencia en los temas del laboratorio. Quiero agradecer especialmente a Rosa Carazo, Rosa Madre y Pilar Carazo no sólo su maravillosa hospitalidad, sino su empatía, su capacidad de tranquilizarme y ponerme la cabeza donde debería estar en tiempos de emociones alteradas, sea mirando cactuses de verdad y/o en documentales o sea llevándome para pedir una tabla en Rossini; cabe resaltar que con el último van incluidas lecciones de Español a un Nivel mucho más alto o elevado del Nivel Europeo C2 por parte del dueño.

150 A Kati Schulz y Eva Olsson por ser tan buenas personas y por la amistad que en este tiempo me han dispensado. A Patri, por haber sido mi apoyo afectivo durante la última fase de la tesis y por pro- porcionarme esos fantásticos momentos que hacen que las cosas sean más fáciles. Por último, quiero agradecer a las empresas Axis Shield LTD, Abbott Diagnostics S.A., Whitehall-Much GmbH su apoyo y destacar especialmente la colaboración con el Dr. Thomas Schettler y el Dr. Edward Valente. Espero que los muy individuales lazos establecidos con cada una de estas personas no se rompan jamás ahora que voy a comenzar un nuevo camino.

151

CURRICULUM VITAE

EDUCATION

2008-2012: PhD student Universidad Politécnica de Madrid (UPM), Madrid, Spain 01.01.2008-31.12.2011: Grant for the realization of the PhD programme funded by Universidad Politécnica de Madrid (UPM) 01.05.2006-31.12.2007: Grant related to laboratory research funded by the I+D Contract Programme signed by the UPM and the Madrid Community 01.01.-31.04.2006: Grant within the vitamin B12 project funded by Universidad Politécnica de Madrid (UPM) 30.09.-31.12.2005: Research contract within the HELENA project. University of Bonn, Germany. 1999-2005: German Diploma (= 5 years) in Nutrition Sciences, University of Bonn, Germany (Rheinische Friedrich-Wilhelms –Universität)

PARTICIPATION IN RESEARCH PROJECTS

Physical activity, fitness, ageing and quality of life: functional, cognitive, social and biomedical determinants. The FELIX study (Project code: c1538b684423560a069997e544772e65) 300.000 € (passed first round, without funding). 2012-2014. Marcela González-Gross

Estudio Longitudinal EXERNET: Genética y su relación con el deterioro de la composición corporal y la condición física en personas mayores de 65 años no institucionalizadas (147/2011). Ministerio de Sanidad, Política Social e Igualdad-Instituto de Mayores y Servicios Sociales (IMSERSO) 2012-2013. Marcela González-Gross/coordinador: Ignacio Ara Royo (Universidad de Castilla-La Mancha)

Determinantes de riesgo de primeros eventos cardiovasculares. Un estudio coordinado de casos y controles anidado de la cohorte PREDIMED. Antioxidantes y estrés oxidativo (PI11/01791) Instituto de Salud Carlos III. 2012-2014. Marcela González-Gross/coordinador: Pep Tur (Universidad de las Islas Baleares)

Estudio de las alteraciones neurodegenerativas en la población geriátrica debidas a la deficiencia de vitamina B12 [Research Project of the neurodegenerative changes in the geriatric population due to vitamin B12 deficiency] (GRS 309/A/08). Regional Administration for Health of the Public Health Service of Castilla y León (SACYL, Sanidad de Castilla y León). 2008-2009. Principal researchers: Belén Cantón Álvarez, Carmen de la Fuente Hontañón, Carmina Vázquez Pedrazuela

La composición del ternasco de Aragón, cualidades dietéticas y su influencia en la salud humana [Composition of the lamb meat of Aragón (GI), dietetic properties and its influence on human health]. (PI08/0022 – PET2007-07-C08). Association for lamb meat (Ternasco) of Aragón. Hospital “Obispo Polanco”, Teruel. Superior School for Gastronomy of Aragón, National Institute for Agrarian and Alimentary Investigation and Technology (INIA, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria). 2008-2009. Principal researcher: Luis Alberto Moreno Aznar. Personal researcher: Grupo GENUD (Growth, Exercise, Nutrition and Development), University school for Health Sciences, University of Zaragoza, Spain

Estudio Multi-céntrico para la Evaluación de los Niveles de Condición Física y su relación con Estilos de Vida Saludables en la población mayor española no institucionalizada [Multicenter study for the assessment of physical fitness of Spanish free-living elderly and its relation to a healthy life style]. Spanish Ministry for Labour and Social Affairs. 2008-2009. Marcela González-Gross / coordinator: Ignacio Ara Royo (University of Zaragoza)

EXERNET - Research Network in Physical Exercise and Health for Special Populations. (DEP2005- 00046/ACTI). Strategic Action on Sports and Physical Activity, National Plan for I+D+i 2004-2007. Spanish Ministry of Education and Science. 2006-2008. Responsible researcher: Jose Antonio Casajús Mallen (University of Zaragoza) Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA). 6th Framework Programme of the European Community (Contract FOOD-CT-2005-007034). 2005-2008. Marcela González-Gross/ coordinator: Luis Moreno (University of Zaragoza)

Vitamin B12 deficiency as a risk factor for neurodegenerative disease. Longitudinal study for the evaluation of changes in the vitamin B12 status in Spanish elderly (P061100509). Axis-Shield Diagnostics Ltd. 2006- Feb 2009. Responsible researcher: Marcela González-Gross

Vitamin B12 deficiency as a risk factor for neurodegenerative disease. Effect of a cobalamin supplement during one month. (P051100649) Whitehall Much GmbH. 2006-2007. Responisble researcher: Marcela González-Gross

Vitamin B12 deficiency as a risk factor for neurodegenerative disease. Masking of the deficiency by folic acid (pteroil-monoglutamic acid) and effect of the nutritional intervention with 5-methyl- tetrahydrofolate. EPROVA Merck GMBH. 2004-2005. Responsible researchers: Klaus Pietrzik/Manuel J. Castillo/ Marcela González-Gross

EDUCATION AND TRAINING

Accreditation course for anthropometric assessment by ISAK, Level I (16.-19.10.2007). Organized by Universidad Complutense de Madrid (UCM) in collaboration with the International Society for the Advancement of Kinanthropometry (ISAK)

Training course for the user of the AxSYM System (24.-25.05.2006). Organized by Abbott Diagnostics, Madrid, Spain

HELENA Cross-sectional Study Training Workshop. (15.-20.01.2006). Organized by E.U. Ciencias de la Salud. University of Zaragoza, Spain

Summer school “Fundamentals of Nutritional Epidemiology”. (19.-30.07.2004). Organized by German Institute of Human Nutrition (DIfE). Potsdam, Germany

PUBLICATIONS

Journal Publications

• Albers U, Palacios G, Pedrero-Chamizo R, Meléndez A, Pietrzik K, González-Gross M. Polymedication in institutionalized elderly and its effect on vitamin B12, folate, and homocysteine levels. Nutr Hosp. 2012;27(1): 296-300. • Albers U, Pedrero R, Meléndez A, Pietrzik K, Castillo MJ, González-Gross M. Effectiveness of a 28 days oral cyanocobalamin supplementation on vitamin B status in Spanish institutionalized elderly. (accepted in Int J Vit Nutr Res). • Spinneker A, Egert S, González-Gross M, Breidenassel C, Albers U, Stoffel-Wagner B, Huybrechts I, Manios Y, Venneria E, Molnar D, Widhalm K, Moreno L, Stehle P. Lipid, lipoprotein and apolipoprotein profiles in European adolescents and its associations with gender, biological maturity and body-fat-The HELENA Study. Eur J Clin Nutr. 2012 Jan 18. doi: 10.1038/ejcn.2011.222. [Epub ahead of print]. • Gomez-Cabello A, Pedrero-Chamizo R, Olivares PR, Luzardo L, Juez-Bengoechea A, Mata E, Albers U, Aznar S, Villa G, Espino L, Gusi N, Gonzalez-Gross M, Casajus JA, Ara I. Prevalence of overweight and obesity in non-institutionalized people aged 65 or over from Spain: The Elderly EXERNET Multi-center Study. Obesity Reviews 2011;12(8):583-92. • Ortega FB, Chillón P, Ruiz JR, Delgado M, Albers U, Alvarez-Granda JL, Marcos A, Moreno LA, Castillo MJ. Sleep patterns in Spanish adolescents: associations with TV watching and leisure- time physical activity. Eur J Appl Physiol 2010;110(3):563-73. • González-Gross M, Breidenassel C, Gómez S, Ferrari M, Beghin L, Spinneker A, Díaz L, Maiani G, Demailly A, Al-Tahan J, Albers U, Wärnberg J, Stoffel-Wagner B, Libersa C, Pietrzik K, Marcos A, Stehle P, on behalf of the HELENA study group: Use of fresh blood samples to analyse multiple nutritional biomarkers in a European Multicenter Nutritional study. Experience from the HELENA cross sectional pilot study. Int J Obes 2008; 32(suppl 5): S66-S75. • González-Gross M, Sola R, Albers U, Barrios L, Alder M, Castillo MJ, Pietrzik K: B-Vitamins and Homocysteine in Spanish institutionalized elderly. Int J Vit Nutr Res 2007;77:22-33. • González-Gross M, Benser J, Breidenassel C, Albers U, Huybrechts I, Valtueña J, Spinneker A, Segoviano M, Widhalm K, Molnar D, Moreno LA, Stehle P, Pietrzik K on behalf of the HELENA Study group. Characterizing blood folate, vitamin B12 and vitamin B6 levels in adolescents in Europe: The HELENA Study. (submitted). • Maroto B, Valtueña J, Albers U, Benito PJ, González-Gross M. Acute physical exercise, both of maximal and submaximal intensity, increase Homocysteine concentrations in young trained male subjects. (submitted). • Pedrero-Chamizo R, Albers U, Tobaruela JL, Meléndez A, Castillo MJ, González-Gross M. Relation between physical strength and cognitive function in Spanish institutionalized elderly". (submitted).

Book chapters

• Meléndez Ortega A, Albers U, Pedrero-Chamizo R, López Torres O. Aspectos sociológicos del envejecimiento en Ejercicio Físico y Salud en Poblaciones Especiales in: Casajus JA, Vicente- Rodríguez G. Ejercicio físico y salud en poblaciones especiales. EXERNET. Editorial CSD. Colección ICD. ISBN 9788479492168. 2011, pp . 101-121. • Meléndez A, Albers U: Respuesta fisiológica al ejercicio en edad infanto-juvenil. in: Redondo Figuero C, González-Gross M, Moreno LA, García-Fuentes M, eds. Actividad física, deporte, ejercicio y salud en niños y adolescentes. Editorial Everest. ISBN 9788444102597. 2010, pp 53-66.

Communications presented at congresses

• Albers U, Pedrero R, Moran P, Cañada D, Meléndez A, González-Gross M. One year follow-up of biochemical and cobalamin status among Spanish institutionalized elderly. 11th European Nutrition Conference (FENS). Madrid, Spain. 26-29.10.2011. Ann Nutr Metab 2011, 58(suppl 3): 48. • González-Gross M, Pedrero-Chamizo R, Albers U. Interrelación entre la condición física, el estado nutricional vitamínico y el estado cognitivo y su influencia sobre la calidad de vida en mayores institucionalizados y de vida independiente. 4° Congreso internacional de Actividad Física Deportiva para Mayores. Málaga, Spain. 4-6.03.2011. Proceedings ISBN 978-84-777858980. pp. 71-86. • Vicente-Rodríguez, G; Rey-López, JP; Mesana, MI; Poortvliet, E; Albers, U, Sjöström, M and Moreno, LA, on behalf of the HELENA study group. Bicipital and tricipital are the best predictive skinfolds for Bod Pod body density using t-distribution modeling. The HELENA Study. Child Health in Europe, The IDEFICS Study. Zaragoza, Spain. 8-9.11.2010. • Albers U, Palacios G, Pedrero-Chamizo R, Meléndez A, Pietrzik K, González-Gross M. Plurimedication among Spanish institutionalized elderly, a fact that should be considered when examining their nutritional status. II World Congress of Public Health Nutrition and I Latin American Congress of Community Nutrition. Porto, Portugal. 23-25.09.2010. Public Health Nutr 2010, 13(9A): 248. • Pedrero-Chamizo R, Albers U, Valtueña J, Casajús JA, Castillo MJ, González -Gross M. Strength training is essential in elderly women. 5th World Conference on Women and Sport. Sydney, Australia. 20-23.05.2010. Abstract Book ISBN 978-0-9806738-0-7. p. 209. • Pedrero-Chamizo R, Albers U, Valtueña J, Moreno S, Pietrzik K, Castillo MJ, González-Gross M. Relation Between Physical Strength and Cognitive Function in Spanish Institutionalized Elderly. 3rd International Congress on Physical Activity and Public Health (ICPAPH). Toronto, Canada. 5-8.05.2010 • Chamizo R, Albers U, Morencos E, Pastor JC, Meléndez A, Castillo MJ, González–Gross M. Valoración del estado nutricional en mayores institucionalizados a través de diferentes marcadores. II Congreso de la FESNAD. Barcelona (Spain). 3-5.03.2010. • Albers U, Pedrero-Chamizo R, Meléndez A, Castillo MJ, Pietrzik K, González-Gross M: How effective can a 500 µg oral supplement of vitamin B12 for just 28 days be for institutionalized elderly? XIXth IAGG World congress of gerontology and geriatrics. Paris, France. 5-9.07.2009 JNHA 2009, 13 (suppl 1): S641. • Pedrero-Chamizo R, Albers U, Valtueña J, Cañada D, Jathe R, Meléndez A, Castillo MJ, González- Gross M. Evolución de los niveles de fuerza en población mayor institucionalizada de la Comunidad de Madrid. III Jornadas Nacionales de Medicina del Deporte. Zaragoza, Spain. 29-30.05.2009. • Al-Tahan J, Albers U, Valtueña J, Spinneker A, Breidenassel C, Azzini E, Diaz LE, Marcos A, Gottrand F, Kunert J, Manios Y, Pietrzik K, Stehle P, González-Gross M, on behalf of the HELENA study group. Folate and vitamin B12 status in European adolescents: associations with gender, age, and maturity. Health behaviour and health status in European adolescents: The HELENA project. Pre congress of the 17th European Congress on Obesity. Amsterdam, Netherlands. 6.05.2009. • Pedrero-Chamizo R, Albers U, Valtueña J, Jiménez-Pavón D, Meléndez A, González-Gross M. Valores de referencia en fuerza de prensión manual en población mayor institucionalizada y de vida independiente. III Congreso Internacional de Actividad Físico Deportiva para Mayores. Málaga, Spain. 12-14.03.2009. • Pedrero-Chamizo R, Albers U, Piera M, Jiménez-Pavón D, Meléndez A, Castillo MJ, González- Gross M: Gender, age and cognitive score effects on strength in Spanish institutionalized elderly. 13th Annual Congress of the European College of Sport Sciences. Estoril, Portugal. 9-12.07.2008. • Pedrero-Chamizo R, Albers U, Valtueña J, Pietrzik K, Meléndez A, Castillo MJ, González-Gross M. Vitamin B12 deficiency. Effects on physical strength and cognitive health in Spanish institutionalized elderly. 6th international Conference on Nutrition and Fitness. Athens, Greece. 15-17.05.2008. • Albers U, Pedrero R, Díaz V, Tobaruela JL, Pietrzik K, Castillo MJ, Meléndez A, González-Gross M. Gender differences in vitamin B12 status and functional fitness parameters in Spanish institutionalized elderly. 6th World Congress on the Aging Male. Tampa, Florida, United States, 21- 24.02.2008. • Pedrero R, Albers U, Jiménez-Pavón D, Cupeiro R, Meléndez A, Castillo MJ, Gutiérrez A, González-Gross M. Valoración funcional en personas mayores institucionalizadas. XII Congreso Nacional de la Federación Española de Medicina del Deporte. Sevilla, Spain. 24-27.10.2007. • Albers U, Meléndez A, Pietrzik K, Tobaruela JL, Castillo MJ, González-Gross M. Effectiveness of oral Vitamin B12 supplementation in Spanish institutionalized elderly. 7th International Conference VITAMINS - Nutrition and Diagnostics. Prague, Czech Republic. 19-21.09.2007. • Albers U, Díaz V, Peinado A, Alvarez M, Sola R, Pietrzik K, Caballero J, Meléndez A, Castillo MJ, González-Gross M. Physical strength and Vitamin B12 in relation to cognitive score in Spanish institutionalized elderly. 10th European Nutrition Conference. Paris, France. 10-13.07.2007. Ann Nutr Metab 2007; 51 (suppl 1): 119-120. • Spinneker A, Albers U, González-Gross M, Sola R, Castillo MJ, Pietrzik K. Difficulty in proving the masking of cobalamin by folic acid supplementation – PGA vs 5-MTHF. 10th European Nutrition Conference. Paris, France. 10-13.07.2007. Ann Nutr Metab 2007; 51 (suppl 1): 216. • Moran P, Albers U, Pedrero R, Sola R, Castillo MJ, González-Gross M. La deficiencia de vitamina B12 como factor de riesgo de enfermedades neurodegenerativas (cognitivas y funcionales) en las personas mayores. 49 Congreso de la Sociedad Española de Geriatría y Gerontología. Palma de Mallorca, Spain. 6-9.06.2007. • Albers U, González-Gross M, Sola R, Castillo MJ, Pietrzik K. Einfluss von 5-MTHF- und PGA- Supplementen auf den hämatologischen Status von spanischen Heimbewohnern. 43rd Congress of the German Nutrition Society (Deutsche Gesellschaft für Ernährung e. V. / DGE). Stuttgart, Germany. 9-10.03.2006.

ORGANIZATION OF CONGRESSES

• Congress PRONAF. Madrid, Spain. 15-17.12.2011. • International Symposium HEALTH(A)WARE 2009. Physical Activity and Health Education in European Schools. Madrid, Spain. 23.04-24.04.2009. ATTENDANCE AT CONGRESSES, SYMPOSIA AND COURSES

• Bial forum of Neurology 2009, organized by Bial. Lisbon, Portugal. 21.11.2009. • Jornada “Doença de Alzheimer e outras formas de demência: Que políticas?” organized by Alzhei- mer Portugal. Lisbon, Portugal. 29-30.10.2009. • International Symposium HEALTH(A)WARE 2009. Physical Activity and Health Education in European Schools. Madrid, Spain. 23.04-24.04.2009. • II National Symposium about Women in Sports “Promotion of women in sports: programmes and activities” organized by the Spanish National Sports Council. Madrid, Spain. 14-15.02.2009. • Annual meeting of the committees of the forum Gain Health (Gana Salud) organized by the forum Gain Health in collaboration with the Community of Madrid. Madrid, Spain. 28-30.01.2009. • International symposium of exercise and health in special populations. Huesca. Spain. 23-24.11.2008. • HELENA Symposium organized by the European Consortium HELENA. Granada, Spain. 20-22.04.2008. • I National Symposium about Women in Sports “Promotion of women in sports: programmes and activities” organized by the Spanish National Sports Council. Madrid, Spain. 14-15.02.2008. • V Conference of the University Complutense of Madrid (UCM) for Human Nutrition and Dietetics: “Health and Fast Food”. Madrid, Spain. 5.11.2007. • III International University Conference for Health Science and Sports organized by the Comité Olímpico Español (COE), Real Madrid and Atlético de Madrid, Madrid, Spain. 18-20.10.2007. • International Symposium about Physical Activity and Health organized by the General Directorate for the Promotion of Sports, The Sports and Cultural Council of the Madrid Community, Madrid, Spain. 27-29.11.2006. • International symposium: ”Dementia: a growing epidemic” organized by the Foundation Ramón Aceres, Madrid, Spain. 15-16.11.2006. • Regional conference about the recent progresses in Alzheimer disease organized by the Association of the family members of Alzheimer patients (Asociación de Familiares de Enfermos de Alzheimer y otras Demencias de la Comarca de Medina del Campo, AFAMEC). Medina del Campo, Spain. 28.09.2006. • Dietary Supplements and Health; organized by the Novartis Foundation in collaboration with the Royal Society of Medicine and The Physiological Society, London, UK. 12.05.2006. • II Conference of nutrition, food technology and food safety; organized by the University San Pablo CEU, Madrid, Spain. 4-5.05.2006. • 41st Annual Congress of the German Nutrition Society (Deutsche Gesellschaft für Ernährung e. V. / DGE), Freising-Weihenstephan, Germany. 11.-12.03.2004. • Conference: Nutrition science as a multi disciplinary focusing. German Nutrition Society (Deutsche Gesellschaft für Ernährung e. V. / DGE). Bonn, Germany. 9-10.10.2003.

WORK EXPERIENCE IN LABORATORY

2005-ongoing: Biochemical laboratory of the Facultad de Ciencias de la Actividad Física y del Deporte- INEF. Universidad Politécnica de Madrid. Technics used: Axsym System (Abbott Diagnostics), automatic blood cell counter Celltac E (Nihon Kohden), ELISA for adiponectin and leptin with ELISA reader Multiscan (Thermo Fisher).

08.11-15.11.2007: Universidad de Cantabria, Santander, Spain. Technics used: determination of the methylenetetrahydrofolate gene (MTHFR) C677T polymorphism including DNA extraction, amplification by Frost, enzymatic digestion with Hinf I (GE Healthcare), electrophoresis.

01.11.2003-01.02.2004: Facultad de Medicina de la Universidad Granada, Dpto. Fisiología Médica in collaboration with the Hospital Clínico Universitario San Cecilio (Granada). Technics used: ImX (Abbott Diagnostics), blood smears coloured with Giemsa, DNA Extraction via QIAGEN kits. 04.02.-10.04.2002: Microbiologic laboratory of the Universitá degli studi de Perugia, Italy, Dipartimento de scienze degli alimenti; Sezione di microbiologia agro-alimentare. Technics used: determination of bioactive substances in various milk types, identification of lactic acid produced by bacteria (LAB), test of antibacterial activity, DNA purification of the “LAB”, determination of proteins via “Lowry method”.

05.03.-12.04.2001: Nordmilch eG Beesten, Germany. Technics used: chemical and microbiologic analyses of milk products, component analyses with infrared instruments, fat content determination via “Röse-Gottlieb”, purity grade and contents of milk and milk powder, determination of the dried extract of milk with sand at 102°C, analyses of industrial wastewater, Microbiology: method by Koch (agar plates), counting of yeast, enterobacteria and fungi.

MENTORING OF FINAL PROJECTS

• Title: ”Cognitive function and B vitamins in institutionalizedelderly“ Student: Ilaria Mattoni, Rheinische Friedrich-Wilhelms-Universität Bonn Supervisors: Marcela González-Gross and Prof. Klaus Pietrzik). Year: 2010. • Title: “Вody composition and health-related aspects in institutionalized elderly” Student: Rebecca Jathe , Rheinische Friedrich-Wilhelms-Universität Bonn Supervisors: Marcela González-Gross and Prof. Klaus Pietrzik. November 2010. • Title: “Vergleichende Betrachtung von Homocystein, Lipiden und weiteren biochemischen Risikoparametern unter Alters- und Gesundheitsaspekten bei spanischen Altenheimbewohnern” Student: Eva Kiesswetter, Rheinische Friedrich-Wilhelms-Universität Bonn Supervisors: Marcela González-Gross and Prof. Klaus Pietrzik. February 2009. • Title: “Experimental evaluation of changes in the vitamin B12 status in Spanish elderly and theoretical implications from Transcobalamin 776C>G polymorphisms” Student: Christina Behrendt, Rheinische Friedrich-Wilhelms-Universität Bonn Supervisors: Marcela González-Gross and Prof. Klaus Pietrzik. June 2008. • Title: “Influence of oral vitamin B12 supplementation on selected haematological and biochemical parameters in Spanish institutionalized elderly” Student: Monika Alder, Rheinische Friedrich-Wilhelms-Universität Bonn Supervisors: Marcela González-Gross and Prof. Klaus Pietrzik. Year: March 2007.

TEACHING EXPERIENCE

01.01.2010-31.12.2011, participating in lectures of

• Dietetics (Dietética, Cod. 2209, Licenciatura en Ciencia y Tecnología de los Alimentos 2º ciclo, 6 credit points) • Nutrition and Ergogenic Aids in Sport (Nutrición y Ayudas Ergogénicas en el Deporte, Cod. 0645, Licenciatura de Ciencias de la Actividad Física y del Deporte, 4 credit points)

LANGUAGES

Mother tongue: German Other language(s): Spanish: Diploma of Spanish as a Foreign Language (Diploma de Español como Lengua Extranjera, DELE), Superior level (C2*) by Cervantes Institute English: Certificate in Advanced English, CAE (C1*) by University of Cambridge * Common European Framework of Reference (CEF) level