Vitamin B12 is a water soluble micronutrient, essential for the normal functioning of nervous and haematological system of our body.1 It is produced naturally by microorganisms, and the main dietary sources are of animal origin.2 Vitamin B12 has the most complex and largest chemical structure among all vitamins; adenosylcobalamin and methylcobalamin are biologically active forms. Hydroxycobalamin and cyanocobalamin are two other forms that may be metabolized into either methylcobalamin or adenosylcobalamin and play a functional roles in the human cells. Vitamin B12 is used in the transfer of methyl group in a methionine synthase requiring reaction, which converts homocysteine to methionine. This reaction activates folate, which is needed for DNA synthesis of red blood cells (RBC’s), which helps in the maturation of nucleus.1 Vitamin B12 is essential for fatty acid and amino acid metabolisms and it also plays a significant role in the synthesis of neurotransmitters, phospholipids and methylation of myelin.3 Diagnostic criteria for vitamin B12 deficiency include a serum cobalamin level < 200 picogram/mililiter (148 picomol/liter) in the presence of signs and symptoms and/or haematological indices of vitamin B12 deficiency.4 Common causes of vitamin B12 deficiency are low intake, food bound cobalamin malabsorption (FBCM), autoimmune and in various conditions causing malabsorption. Pregnancy and drugs like metformin, proton pump inhibitors, oral contraceptives, hormone replacement therapy also leads to vitamin B12 deficiency.1 Vitamin B12 is stored in the liver in a sufficient amounts, development of symptoms of deficiency usually takes five or more years. Deficiency of vitamin B12 is characterised by haematological, neurological and neuropsychiatric features, ranging from milder manifestations like fatigue, paraesthesia, to severe manifestations like pancytopenia and subacute combined degeneration of spinal cord. Usually symptoms develop early before a decrease in serum vitamin B12 below the reference value. The patient may have shortness of breath, pallor, dizziness, tinnitus, fatigability, lethargy and palpitation. Since the underlying pathologic process consists of demyelination of peripheral nerves, the spinal cord and the cerebrum, the signs and symptoms include numbness, paraesthesia, weakness, ataxia, poor finger coordination, diminished reflexes and loss of vision. Participants may come up with severe mental problems like depression, memory loss, irritability, behavioural changes, panic attacks and insomnia. Untreated vitamin B12 deficiency can cause pulmonary embolism, deep vein thrombosis, suppression of immune system and bone marrow failure.5 The prevalence of vitamin B12 Deficiency is 47% in north Indian population.6 Data documenting vitamin B12 status in India in general population is limited. Documentation of more data from different parts of country may spark well informed debate on replacement strategies and also on food fortification. With this in mindset, we had conducted a cross-sectional study on population of rural part of north western Rajasthan, India, to know the prevalence of vitamin B12 deficiency among them. Aim: to estimate the prevalence of vitamin B12 deficiency in this population.

Present study was conducted on rural population of Bikaner district in Rajasthan. Research population was divided into different strata geographically and then sample population taken by grid sampling from 6 villages (i.e. Bholasar, Bikkampur, Diatra, Kakda, Panchu and Uttamdesar) selected randomly from 3 tehsils selected randomly. It was a cross sectional study conducted within 6 months between December 2023 and May 2024 and samples collected by stratified random sampling. Calculated sample size was 421 but we had tested some extra samples in view of inadequacy or spillage of blood samples during the processing and we got results of 427 samples, which we had included in our study. We excluded person with age <14 years, person not willing to participate in research study and subject residing in selected rural area of north western Rajasthan since less than 6 months. Research population was divided into different strata geographically in Bikaner district and then sample population taken randomly from different villages selected randomly (Villages – Bholasar, Bikkampur, Diatra, Kakda, Panchu, Uttamdesar) from each strata. Each selected villages is divided into group of houses to decrease sampling bias and sample taken from each group via grid sampling. We went to a total of 603 people and asked them to participate in the study among them 548 given consent for participation in the study and only 481 participants had given consent for blood sample collection, among them 457 samples were sent to laboratory and rest were found clotted and broken vials out of them 427 samples were tested in laboratory and rest were declared inadequate for testing, so we had included data of these 427 samples in our study. All participants was interviewed with a pre-designed questionnaire including age, sex, occupation, socioeconomic status (by modified Kuppuswamy scale)7, dietary habits like ingestion of animal milk or milk products, ingestion of meat, clinical symptoms related to vitamin B12 deficiency, any concomitant illness history etc. Weight and height of the participants were taken by electronic weighing machine and stadiometer scale to measure BMI based on WHO criteria. The questionnaires were completed with the assistance of a trained person, in the local language. 3 ml venous blood samples were drawn from peripheral vein with aseptic precaution from every subject after obtaining an informed verbal and written consent and transferred from collection site to testing site in cold storage box for biochemical assessment of serum vitamin B12 level (reference range 120-914 pg/ml) by using UniCelDxl 800, Access Immunoassay System S/N 607846, Version 5.3.1 in Immunoassay laboratory, Department of Biochemistry, S.P. Medical College, Bikaner.

In our study 187 male (43.79%) and 240 female (56.21%) participants had participated with a male female ratio of 3:4. In this study most of the participants were in age group of 21–40 years (42.86%). Among male participants 36.36% were belongs to 21-40 years age group and among female participants 47.92% in age group 21-40 years. Table-1- Participant distribution according to various parameters Parameter Male (n=187) Female (n=240) Total (n=427) No. % No. % No. % Age group (in years) <20 46 24.60 45 18.75 91 21.31 21 – 40 68 36.36 115 47.92 183 42.86 41 – 60 50 26.74 55 22.92 105 24.6 >60 23 12.30 25 10.41 48 11.24 Socioeconomic class Upper Class 1 0.53 1 0.41 2 0.46 Upper Middle Class 40 21.39 33 13.75 73 17.1 Lower Middle Class 7 3.74 4 1.67 11 2.58 Upper Lower Class 139 74.33 202 84.17 341 79.86 Lower Class 0 - 0 - 0 - Dietary habit Vegetarian 124 66.31 226 94.16 350 81.97 Non Vegetarian 63 33.69 14 5.83 77 18.03 Addiction habits Smoking 55 29.41 2 0.83 57 13.35 Smokeless Tobacco 36 19.25 8 3.33 44 10.30 Alcohol 33 17.64 2 0.83 35 8.20 Mean age of the study participants was 36.86+ 17.42 years with an age range of 14 to 86 years. We observed in this study that most of the participants were in upper lower class (79.86%) with 74.33% male and 84.17% female from this class. In our study most of the participants were vegetarian (81.97%). Our study shows that 13.35% of the study participants were smoker and 10.30% were smokeless tobacco chewer and 8.20% were alcoholic. Table-2 - Distribution of severity of vitamin B12 deficiency and its relation to different age group and gender Severity of vitamin B12 deficiency (pg/ml) Male (n=187) Female (n=240) Total (n= 427) No. % No. % No. % Border line deficiency (200–299) 31 16.58 49 20.42 80 18.73 Mild deficiency (150 –199) 4 2.14 6 2.5 10 2.34 Moderate deficiency (100–149) 11 5.88 14 5.83 25 5.85 Severe deficiency (<100) 6 3.21 16 6.67 22 5.15 Total Deficient 52 27.81 85 35.42 137 32.08 B12 Deficiency distribution in different age group Age (years) Male (n=52) Female (n=85) Total (n=137) <20 10 19.23 13 9.48 23 16.79 21 – 40 14 10.22 44 32.12 58 42.34 41 – 60 19 13.87 17 12.41 36 26.28 >60 9 6.56 11 8.03 20 14.60 We observed that out of 427 participants, 137 participants (32.08%) had vitamin B12 level deficiency, severity levels and demographic variations associated with deficiency were also studied. Among elderly out of total 48 elderly (>60Years) participated in study 20 (41.67%) were B12 deficient. Most of the non-deficient was from age group of 21–40 years in both male and female population i.e. 18.62 and 24.48 % respectively. Table-3– Correlation between vitamin B12 deficiency and non-deficiency participants on the basis of socioeconomic status, BMI and dietary habits Socioeconomic Class B12 deficiency (n=137) Non deficiency (n=290) p value No. % No. % Upper Class 0 – 2 0.69 – Upper Middle Class 27 19.70 46 15.86 0.32 Lower Middle Class 9 6.56 2 0.69 0.0003 Upper Lower Class 101 73.72 240 82.76 0.02 Lower Class 0 – 0 – – BMI (Kilogram/Meter2) <18 8 5.84 12 4.14 0.43 18–24.9 115 83.94 258 88.97 0.14 25 – 29.9 13 9.49 19 6.55 0.23 >30 1 0.72 1 0.34 Type of vegetarian food consumption Milk and milk products 112 81.75 237 81.72 0.99 Fruits and vegetables 111 81.02 238 82.07 0.79 We also observed that among vitamin B12 deficient participants most of were belonging from upper lower class (73.72%). We have found a highly significant result in lower middle class with p value 0.0003 and upper lower class (p<0.02) when compared between vitamin B12 deficient and non-deficient population. There was no significant correlation of BMI in vitamin B12 level deficient and non-deficient population.

After getting laboratory results we analyzed all our data and compared the results of our study with previously conducted studies, with a special emphasis on comparison with other studies conducted in India. In our study female participants were more as compared to male, possible reason behind this may be because, most of the male population in rural area go out from home for work on daily basis, in our study we had visited house to house to take samples, so less male population were participated. In this study we had taken a cut-off of 299pg/ml to include most of the possible deficiency population. Yao, Y et al also made similar observations on 100 consecutive, unselected geriatric out patients of a primary care setting in Ulster County, New York, they found that 16% of the participants had serum cobalamin levels of 200 pg/ml or below, and 21% had levels between 201 and 299 pg/ml, which was 37% total but they had taken only geriatric outpatients but our study had data on whole population with age range of 14 to 86 years when compared to severity levels of vitamin B12 in this study.8 Meena S et al also reported that prevalence of vitamin B12 deficiency was 19.52%, which was also consistent with our observation difference was due to different cut off for deficiency.9 In 2019 Singh G et al also found overall prevalence of vitamin B12 deficiency in urban northern Indian population was 45%, which was higher as compared to our study.10 According to gender distribution, deficiency was more among females in our study as compared to males because female participants were more and also we can correlate with dietary habits of females in rural area, most of the females were vegetarian by diet contributing to their deficiency status. Our results were in contrast to study done by Meena S et al in which they reported that prevalence of vitamin B12 deficiency was more in males (18.33%) as compared to females (15.83%) but data was statistically not significant.9 Sharma P et al in 2018 also found that vitamin B12 deficiency was higher in males i.e. 29.6% as compared to females i.e. 22.2%, and deficiency was much higher in urban males than urban females as compared to rural males and females with a significant difference and p value of 0.05.11 Younger and elderly both are more deficient as compared to other age categories in our study. According to Wong C. W. et al elderly people are particularly at risk of vitamin B12 deficiency due to the high prevalence of atrophic gastritis-associated food-cobalamin mal-absorption, and the increasing prevalence of pernicious anaemia with advancing age.12 Meena S et al observed in their study that vitamin B12 deficiency was more in young adult (20-40 years) i.e. 29.16% than older person (1.66%). They also showed that in population age <20 years, deficiency was 3.33%, our study also showed similar results with most common age group having vitamin B12 deficiency was 21-40 years.9 We had found a significant correlation of vitamin B12 deficiency with lower middle class and upper lower class population. This is possibly due to poor dietary practices and poor access to health care in middle and lower socioeconomic groups. Similar observation was made by Patel S.V. et al in 2022 that vitamin B12 deficiency was more common in lower socioeconomic class 84.4% and 15.6% in middle class, our results was also consistent with this study.13 Yajnik CS et al also showed that among urban middle class, 81% had low vitamin B12 concentration. We observed in this study that most of the participants were having normal BMI and those with overweight and obesity had no significant correlation with the B12 deficiency status of the population.14 In rural area most of the population is working in farming and not having sedentary lifestyle attributing to their normal BMI status. Study conducted by Chakraborty S et al in 2018 found that among vitamin B12 deficient participants normal weight were 28.1%, overweight 39.8% and obese 51.2%, with a P value < 0.001.15 This study indicates a significant risk of vitamin B12 deficiency in vegetarians as compared to non vegetarians. There is lots of literature available on dietary association of vitamin B12 deficiency status, here in rural area most of the population is vegetarian attributing to their B12 deficiency status. Pawlak R et al also showed that prevalence of vitamin B12 deficiency among vegetarians was ranging from about 11 to 90%.16 Meena S et al also showed that prevalence of vitamin B12 deficiency in vegetarian and non-vegetarian were 28.33% and 5.83% respectively, vegetarian dietary habit was found to be a substantial risk factor for vitamin B12 deficiency. They also found high rates of vitamin B12 deficiency, even among participants labeled non vegetarian.9 Singh G et al also found that prevalence of vitamin B12 deficiency was higher in vegetarians (35.5%) as compared non vegetarians (9.5%). All these studies were consistent with our study that vegetarians had more vitamin B12 deficiency than non-vegetarians.10 In this study vitamin B12 deficiency had no significant correlation with smoking, tobacco and alcohol consumptions. Various studies showed some connection between metabolism of vitamin B12 and that of cyanide, smoking releases cyanide and other toxins, which results in vitamin B12 deficiency among smokers. Vora JH, Oza HN et al in their study reported that in tobacco consuming patients vitamin B12 levels in serum were low and they concluded that association between tobacco use and vitamin B12 deficiency was found statistically significant.17 Patel S.V. et al showed that vitamin B12 deficiency was more common in alcoholics (19.4%) and smokers (12.5%) as compared to non-alcoholics (15.6%) and non-smokers (6.9%), which was consistent with our observation in this study.13 Limitations: In this study we had not excluded people who might have already taken supplements containing vitamin B12. Same limitation was faced by other researchers worldwide. Current data collected from rural population may underestimate the prevalence of vitamin B12 deficiency as it was a cross-sectional collection of data from general population after visiting to them at their home, so those people who had participated may be more health conscious than other general population. We might had got overestimation of vitamin B12 deficiency, due to no further enzymatic testing in borderline deficiency people due to some financial constrains in our study. Further studies with enzyme estimation (e.g. MMA) for diagnosis of vitamin B12 deficiency are needed.

This is one of the largest studies on vitamin B12 status in rural population of India. Study data were taken from general population of rural part of north western Rajasthan; which was likely to be representative of north Indian population at large. We had found 32.08% prevalence of vitamin B12 deficiency in our study. This study also provides critical data on clinical features and factors associated with vitamin B12 deficiency will aid in the development of effective prevention, and management techniques tailored to the Indian population. Vegetarian diet, lower socioeconomic status, smoking, tobacco and alcohol were most common risk factors associated with vitamin B12 deficiency. Preventive methods can be adopted to reduce the burned of vitamin B12 deficiency by food fortification for general population. Furthermore, health administrators might use the information provided in our study for planning of health programs and resource distribution. More research is required to conduct such studies in different part of the world as prevalence varies in different geographical areas.

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