Subjects: One hundred and seventy five healthy men and women (>60 yr) were recruited between April and December 2009 from various Senior Citizen Associations in Bangalore (84%) and outpatient department at St. John's Medical College Hospital (14%), Bangalore. The subjects were recruited based on convenience sampling technique. Sample size was estimated based on expected prevalence of vitamin B12 deficiency of 30 per cent with a relative precision of 25 per cent. This was estimated to be 150. The purpose of the study was explained and a written consent was obtained from each of the participants. Demographic, socio-economic and lifestyle information was obtained using a questionnaire. Education was recorded as completed years of formal education and monthly income was categorized.
Detailed history, physical examination and neurological assessment were carried out by a qualified physician. Medical history that included gastrointestinal (GI) symptoms, neurologic and psychiatric symptoms, underlying medical and surgical conditions, was obtained using a structured questionnaire. Subjects with underlying heart disease, renal failure, stroke and major surgery within the last year were excluded. Subjects with previous neurologic or psychiatric symptoms were also excluded. This ensured inclusion of only healthy elderly subjects in this study. Subjects with well controlled diabetes and hypertension were included. The Institutional Ethical Review Board of St. John's Medical College approved the research protocol.
: Habitual dietary intake for the preceding six months was assessed using a food frequency questionnaire12
that was interviewer administered by a trained nutritionist. This questionnaire was adapted from the one developed for the urban middle class residing in south India. It has a food list of 108 items, derived from a food database developed over a period of many years from studies at the Division of Nutrition, St John's Medical College, and has four frequency categories (daily, weekly, monthly and yearly)12
. Nutrient composition of the food item was calculated using standard food conversion tables for the ingredients12
. Wherever available, Indian data were used. However, for some nutrients, for which Indian data were not available, USDA data in the public domain were used13
. A replica sheet of the questionnaire was made in Microsoft Excel and the information was entered. The program computed nutrient scores by multiplying the relative frequency of consumption of each food item by its nutrient content of the standard portion size. Nutrient information was obtained on total caloric intake, daily folate and vitamin B12
intake and many other macro- and micro-nutrients. Information on dietary supplements being consumed was separately recorded to arrive at the total daily intake of vitamin B12
: The cognitive measures consisted of a series of neuropsychological tests applicable for use in elderly population. The cognitive battery included neuropsychological tests specially adapted from the Consortium to Establish a Registry for Alzheimer's disease (CERAD)14
and the Indo-U.S. Cross National Dementia Epidemiology Study15
. This battery consisted of 10 sub-tests, which measured various domains of cognitive abilities and was administered by a trained clinical psychologist. The cognitive domains that were assessed included verbal reproduction and language function, language capacity and impairment of expressive language or speech, mathematical abilities, attention and concentration, visual perception and motor execution and various aspects of memory such as immediate memory, delayed recall and delayed recognition. In addition, subjects were also assessed on an Indian adaptation of "Mini Mental Status Examination" (MMSE) - a widely used screening instrument for the detection of cognitive deficits16
. The Indian adaptation of the “Mini Mental Status Examination” is known as the “Hindi Mental Status Examination” (HMSE)17
. The Kannada version of this instrument was used to suit the local population; this was done using the translation-back translation procedure18
. Depression, which can confound assessment of cognitive abilities in elderly, was measured on “Geriatric Depression Scale”19
. Finally, the functional status of the subjects was assessed using “everyday ability scale”20
adapted for Indian population.
Biochemical measurements: Approx. 4 ml of blood was collected from each subject and routine haematological work up, including haemoglobin, total leucocyte count, platelet count, haematocrit, red cell indices, blood smear evaluation, neutrophil lobe count, blood sugars and serum creatinine was done on all the subjects using standard haematology and biochemical techniques. Whole blood was treated with ascorbic acid and stored for red cell folate analysis. The plasma was separated and stored at -80°C until analysis for vitamin B12, Hcy and MMA. Red cell folate and vitamin B12 was measured by the electrochemiluminescence method (Elecsys 2010, Roche Diagnostics Mannheim, USA). The intra- assay coefficient of variation (CV) for the trilevel level controls for red cell folate and vitamin B12 were 2.6, 2.4, 1.7; 4.6, 4.2, 2.8; and inter-assay were 6.4,5.1,5.8; 6.8, 5.3, 3.2; respectively.
The combined measurement of Hcy and MMA was performed by gas chromatography-mass spectrometry (GC-MS) method. An aliquote of 500 μl of plasma was lyophilized in a freeze dryer (Labconco, Kansas, MO, USA) to concentrate the analytes. The lyophilized mass was reconstituted with 200 μl of MiliQ water. The plasma was treated with D,L-dithioerythritol containing deuterated homocysteine (d8
-Hcy), to cleave disulphide bonds in the deuterated homocysteine, protein and cysteine bound homocysteine and dimeric form of homocysteine. Plasma was deproteinized by ethanol containing deuterated methyl malonic acid (d3
-MMA). The supernatant containing Hcy, MMA and their deuterated standards were derivatized with methycholorformate and extracted into toluene. The N(S)-methoxycarbonyl ethyl ester derivatives in the extract were injected onto the GC-MS (Varian 3800, Palo Alto, CA, USA) and separated on a CP sil 24-CB low-bleed/MS capillary column (15m X 0.25 mm (i.d); film thickness, 0.25 μm) from Varian, USA. The molecules were analyzed in the selective ion monitoring mode of the MS. The ion pairs of m/z 174/177 for MMA/d3
-MMA and 233/237 for Hcy/d4
-Hcy were quantified. The concentrations of Hcy and MMA were computed from calibration curves drawn using area ratios of analyte and deuterated analyte, against known concentration of the analyte. The intra- and inter-assay CV for Hcy and MMA were 4.6, 6.2 and 9.3, 10.1 respectively21
Statistical analysis: Data recorded on a pre-designed proforma were entered on an excel spreadsheet and entries double checked for any errors. Normality was checked using Kolmogorov-Smirnov (KS) test. Vitamin B12 intake and plasma levels of MMA and Vitamin B12 were not normally distributed, and therefore, non-parametric statistical tests were used for vitamin B12 and its metabolites. Categorical data were presented using number and percentage and continuous data as median and quartiles. Since plasma vitamin B12 levels were not normally distributed, Spearman's correlation was performed between vitamin B12 and the metabolites to assess the extent of their correlation. The data were divided into two groups based on supplement intakes and were compared using Mann-Whitney U test. Categorical variables were compared using Chi-square test. The cognitive parameters were compared between the study groups using Mann-Whitney U test. P<0.05 was considered significant. Data were analyzed using SPSS version 11.5.