Identification of vitamin B12 deficiency in vegetarian Indians

Sadanand Naik; Namita Mahalle; Vijayshri Bhide

doi:10.1017/S0007114518000090

Identification of vitamin B12 deficiency in vegetarian Indians

Published online by Cambridge University Press: 15 February 2018

Sadanand Naik ,

Namita Mahalle and

Vijayshri Bhide

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Sadanand Naik*: Affiliation:
Pathology Laboratory, Deenanath Mangeshkar Hospital & Research Centre, Pune 411004, Maharashtra, India
Namita Mahalle: Affiliation:
Pathology Laboratory, Deenanath Mangeshkar Hospital & Research Centre, Pune 411004, Maharashtra, India
Vijayshri Bhide: Affiliation:
Pathology Laboratory, Deenanath Mangeshkar Hospital & Research Centre, Pune 411004, Maharashtra, India
*: * Corresponding author: Dr S. S. Naik, email sadu_naik@rediffmail.com

Article contents

Abstract
Methods
Results
Discussion
References

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Abstract

The prevalence of a sub-clinical vitamin B12 deficiency in the vegetarians is high. Total serum vitamin B12 concentration alone does not reliably reflect vitamin B12 status. Holotranscobalamin (holo-TC) II is a bioactive B12 fraction promoting specific uptake of B12 by cells and the circulating concentration reflects the intake of B12, whereas total homocysteine (tHcy) indicates the metabolic ability. In this study, we investigated the diagnostic value of circulating holo-TC, B12, folate and homocysteine in vegetarians who were at risk of B12 deficiency. B12-related biomarkers were measured in 119 young, healthy graduate vegetarians. None was folate deficient. As per reported definition, half were B12 deficient; 70 % of males and 50 % of females had low plasma holo-TC concentrations; and 92 % of males and half of females had hyperhomocysteinaemia. None had any clinical signs of B12 deficiency. Receiver operating characteristic curve analysis demonstrated similar AUC at the B12 concentration of 100 and 150 pmol/l when holo-TC (0·777 and 0·784) and homocysteine (0·924 and 0·928) were used as variables. Cut-off value of 100 pmol/l resulted in the highest sensitivity of 77·78 % and specificity of 71·05 % with a predictive value of 19·6 pmol/l for holo-TC and a sensitivity of 82·72 % and specificity of 89·7 % with a predictive value of 21·7 µmol/l for homocysteine. The combination of B12, holo-TC and tHcy improves the diagnostic accuracy at these cut-offs, and we suggest that for the young Indian vegetarians the cut-off for plasma B12 and holotrancobalamin is 100 pmol/l and 19·6 pmol/l, respectively, and for homocysteine it is 17·6 (females) and 27 µmol/l (males) for identifying B12 deficiency.

Keywords

Vitamin B12 Holotranscobalamin Folate Homocysteine

Information

Type: Full Papers
Information: British Journal of Nutrition , Volume 119 , Issue 6 , 28 March 2018 , pp. 629 - 635

DOI: https://doi.org/10.1017/S0007114518000090 [Opens in a new window]
Copyright: Copyright © The Authors 2018

Vitamin B₁₂ is essential for 1-C metabolism and cell division. Foods derived from animals are the main sources of vitamin B₁₂. Strict vegetarians have limited sources of vitamin B₁₂ in their diet and therefore likely to have vitamin B₁₂ deficiency⁽ Reference Antony ¹ ^– Reference Jathar, Inamdar-Deshmukh and Rege ³ ⁾. Reduced consumption of cobalamin from food or impaired intestinal absorption leads to severe deficiency when tissue stores of the vitamin are depleted. The clinical consequence of vitamin B₁₂ deficiency includes megaloblastic anaemia and progressive neurologic disease of the central and peripheral nervous system⁽ Reference Obeid, Giesel and Schorr ⁴ ⁾ and hyperhomocysteinaemia, a risk factor for CVD⁽ Reference Refsum, Smith and Ueland ⁵ ⁾. Early diagnosis of vitamin B₁₂ deficiency is useful to prevent irreversible neurological damage by cobalamin supplementation⁽ Reference Herrmann, Obeid and Schorr ⁶ ^– Reference Weir and Scott ⁸ ⁾. It has been observed that asymptomatic Indian lactovegetarians, who make up for more than half of the Indian population, had distinctly lower vitamin B₁₂ concentrations than non-vegetarians⁽ Reference Yajnik, Deshpande and Lubree ⁹ ⁾ and was confirmed by studies from different geographic regions of India. However, total plasma vitamin B₁₂ concentration may not reliably reflect vitamin B₁₂ status. To obtain more specificity and sensitivity in diagnosing vitamin B₁₂ deficiency, the concept of measuring holotranscobalamin (holo-TC) II, a transport protein, has aroused great interest. holo-TC is a biologically active vitamin B₁₂ fraction that promotes the aspecific uptake of vitamin B₁₂ by cells⁽ Reference Nexo and Hoffman-Lücke ¹⁰ ⁾.

Vitamin B₁₂ deficiency has been divided into four stages⁽ Reference Herbert ¹¹ ⁾. In stages I and II, indicated by a low plasma level of holo-TC, the plasma and cell stores become depleted. Stage III is characterised by increased plasma levels of total homocysteine (tHcy) and methylmalonic acid (MMA) in addition to lowered holo-TC. In stage IV, clinical signs become recognisable such as macroovalocytosis, elevated mean corpuscular volume (MCV) or lower Hb levels. Stage III of vitamin B₁₂ deficiency has been found in over 60 % of vegetarians⁽ Reference Herrmann and Giesel ¹² ⁾. Thus, it is important to monitor vitamin B₁₂ status in this dietary group. Measurement of plasma vitamin B₁₂, holo-TC, tHcy and MMA has been suggested for optimal monitoring of vitamin B₁₂ status in vegetarians⁽ Reference Lindenbaum, Savage and Stabler ¹³ ⁾. In this study, we investigated the diagnostic cut-off values of plasma vitamin B₁₂, holo-TC and functional marker, tHcy, of vitamin B₁₂ metabolism in Indian vegetarians.

Methods

The study was conducted at Deenanath Mangeshkar Hospital and Research Centre. Young, healthy, postgraduates and staff members of the hospital and their relatives were invited to participate in the study. Self-explained information regarding the participants was captured by an interview, which included age, vitamin supplementation, food habits and routine lifestyle. Study subjects were enrolled after the purpose and requirements of the study were explained. They were clinically examined for gross clinical signs of protein-energy under-nutrition and vitamin deficiencies (vitamin A, B-complex, C and D). Non-vegetarians and pregnant women were not included. Subjects with diabetes, cancer and those taking drugs known to influence vitamin B₁₂ absorption were also excluded. A total of 119 eligible participants (forty-six male and seventy-three female) were enrolled for the study. The study protocol was approved by the Hospital Ethical Committee and all the participants gave written informed consent (2015_APR/SN/169).

Experimental procedure

A volume of 10 ml of fasting blood sample was collected in EDTA vacutainers. Haematological parameters were measured on a five-part differential cell counter (Sysmex) and the remaining blood was centrifuged at 1500 g for 20 min. Separated plasma was stored for biochemical investigations at −20°C. Plasma holo-TC was measured using microparticle enzyme immunoassay. Microparticle enzyme intrinsic factor assay was used for the quantitative determination of plasma vitamin B₁₂. Plasma folate and tHcy were measured by the fluorescence polarisation immunoassay technique. All four biomarkers were analysed on an AxSYM immunoassay analyzer (Abbott Laboratories)⁽ Reference Naik, Bhide and Babhulkar ¹⁷ ⁾. Plasma creatinine was measured using the alkaline picrate method with a Daytona analyser (Randox)⁽ ¹⁴ ⁾.

Dietary intake data

A 24-h dietary recall questionnaire was administered to capture detailed information about all food, beverages and dietary supplements consumed in the past 24 h from midnight to midnight the previous day by an experienced nutritionist. Energy, protein, fat and vitamin B₁₂ intake was calculated using Dietsoft⁽ Reference Kanade, Rao and Kelkar ¹⁵ ^, Reference Gopalan, Rama Sastri and Balsubramanian ¹⁶ ⁾.

Definitions

Folate and vitamin B₁₂ deficiency was defined as concentrations <2 ng/ml and <148 pmol/l, respectively; hyperhomocysteinaemia as plasma tHcy concentrations <15 µmol/l⁽ Reference Naik, Bhide and Babhulkar ¹⁷ ⁾; low holo-TC concentration as <35 pmol/l; anaemia as Hb concentration <120 g/l in females and <130 g/l in males; and macrocytosis as MCV>100 fl⁽ Reference Refsum, Yajnik and Gadkari ¹⁸ ⁾.

Statistical analysis

The data are presented as medians and 25th and 75th percentiles. Between-group comparisons were calculated using Mann–Whitney U test and associations were tested using Pearson’s correlation coefficient.

Sample size justification

Prevalence of vitamin B₁₂ deficiency in vegetarian Indians is found to be 70 %⁽ Reference Yajnik, Deshpande and Lubree ⁹ ⁾. Sample size was calculated using Buderer’s method considering prevalence rate⁽ Reference Buderer ¹⁹ ⁾. We selected sensitivity and specificity to be 80 and 90 %, respectively. We chose clinically acceptable width of 95 % CI for sensitivity and specificity to be no more than 10 %, significance (a=0·05). The sample size calculated was 88 for the expected sensitivity of 80 % and 116 for the expected sensitivity of 90 %. Taking maximum of both, we arrived at the estimate of 116.

We enrolled 119 subjects. Receiver operating characteristic (ROC) curves and AUC (with 95 % CI) were used to measure the diagnostic accuracy of (cut-offs) vitamin B₁₂, holo-TC and tHcy. ROC decision plots depicting sensitivity and specificity for 100 and 150 pmol/l concentration of vitamin B₁₂ using holo-TC (<35 pmol/l) and tHcy (>15 µmol/l) variables were determined to identify the cut-off values.

Results

The participants had no clinical symptoms of vitamin B₁₂ deficiency (neurological symptoms such as paresthesia, weakness, gait abnormality, tingling of hands and feet or anaemia symptoms such as skin pallor, fatigue, shortness of breath and so on) and were not taking vitamin B₁₂ supplementation or any drugs known to influence vitamin B₁₂ absorption.

All subjects had normal plasma folate and creatinine concentrations (5·9, 6·6 ng/ml and 1·2, 0·95 mg/dl in males and females, respectively). Median plasma vitamin B₁₂, holo-TC and tHcy concentrations were 146·5 and 164 pmol/l, 22·3 and 26·5 pmol/l, and 22·6 and 14·2 µmol/l in males and females, respectively (Table 1). In all, 39 and 30 % had very low concentration (<100 pmol/l), 11 and 20 % had low concentration (100–148 pmol/l), and 50 and 52 % had normal plasma vitamin B₁₂ concentration (>148 pmol/l) in males and females, respectively. holo-TC was lower in three-fourth of the participants (22 and 24 pmol/l). A significant sex difference was found in plasma tHcy concentrations (22·6 µmol/l in males and 14·2 µmol/l in females, P=0·0001), with no sex difference in holo-TC and vitamin B₁₂ concentrations (Table 1). Group-wise distribution of vitamin B₁₂ (<100, 100–150 pmol/l) was strongly related to plasma holo-TC and inversely to tHcy concentrations in both males and females (Table 2, P<0·0001). At normal level of vitamin B₁₂ (>148 pmol/l), the median concentrations of plasma holo-TC concentrations were 27·7 and 29·8 pmol/l in vegetarian males and females, which are lower than those found in non-vegetarians (Enexo). Between plasma vitamin B₁₂ concentrations of 113 and 122 pmol/l, nineteen participants had normal holo-TC (34–52 pmol/l) with higher tHcy (34 μmol/l) concentrations, which is unexplainable with existing cut-offs (Table 3). ROC analysis demonstrated similar AUC at the vitamin B₁₂ concentration of 100 and 150 pmol/l when holo-TC (0·784 and 0·777, respectively) and homocysteine (Hcy) (0·928 and 0·924, respectively) were used as variables. Cut-off value of 100 pmol/l resulted in the highest sensitivity (77·78 %) with acceptable specificity (71·05 %) with a predictive value of 19·6 pmol/l for holo-TC and a sensitivity of 82·72 % and specificity of 89·47 % with a predictive value of 21·2 µmol/l for tHcy (Fig. 1 and 2).

Fig. 1

Plasma vitamin B₁₂ at concentrations of 150 and 100 pmol/l were used for analysis. Metabolic deficiency was defined as plasma holotranscobalamin (holo-TC)<35 pmol/l in 119 vegetarian Indians. Predictive values of plasma holo-TC were 24·58 and 19·6 µmol/l, respectively, with similar sensitivity and specificity at both 150 and 100 pmol/l of vitamin B₁₂. ROC, receiver operating characteristic.

Fig. 2

Plasma vitamin B₁₂ at 150 and 100 pmol/l were used as variables. Metabolic deficiency was defined as total homocysteine (tHcy)>15 µmol/l in 119 vegetarian Indians. Predictive values of plasma tHcy were 18·56 and 21·2 µmol/l, respectively, with better specificity (89·47 %) at 100 pmol/l of vitamin B₁₂. ROC, receiver operating characteristic.

Table 1

Baseline characteristics and biochemistry of the participants (male and female) (Medians and 25th, 75th percentiles)

holo-TC, holotranscobalamin; tHcy, total homocysteine.

* Difference between male and female.

Table 2

Sex difference in total homocysteine (tHcy) at different vitamin B₁₂ concentrations (Medians and 25th–75th percentiles)

holo-TC, holotranscobalamin.

Table 3

Plasma vitamin B₁₂ concentrations and corresponding concentrations of plasma holotranscobalamin (holo-TC) and total homocysteine (tHcy)

holo-TC, holotranscobalamin; tHcy, total homocysteine.

Discussion

Our data support the concept that the measurement of plasma holo-TC and tHcy along with vitamin B₁₂ provides a better index of cobalamin status than the measurement of vitamin B₁₂ alone⁽ Reference Lysne, Strand and Svingen ²⁰ ⁾. Plasma holo-TC and tHcy are both sensitive markers for cobalamin status⁽ Reference Solomon ²¹ ⁾. The lowest median levels of holo-TC were observed in the low vitamin B₁₂ concentration group, followed by the 100–150 pmol/l group and then by the normal group (>148 pmol/l): 13·2, 21·4 and 31·3 pmol/l, respectively (P=0·0001). At similar plasma cobalamin concentrations, men had different metabolic effects compared with women. The use of holo-TC and tHcy results enables the differentiation between storage, depletion and functional vitamin B₁₂ deficiency.

Fedosov⁽ Reference Fedosov ²² ⁾ reported that a combination of vitamin B₁₂, holo-TC, MMA and Hcy (cB₁₂) biomarkers is a reliable diagnostic tool. Fedosov et al.⁽ Reference Fedosov, Brito and Miller ²³ ⁾ derived equations that combined two, three or four biomarkers into one diagnostic indicator and provided a guidance for treatment. They suggested that adults having plasma vitamin B₁₂ levels between 116 and 119 pmol/l, holo-TC between 8·4 and 20 pmol/l, and Hcy between 19·2 and 51 μmol/l be grouped as low vitamin B₁₂ status and adults with B₁₂ between 119 and 186 pmol/l, holo-TC between 20 and 37 pmol/l, and Hcy between 13·6 and 19·2 μmol/l as transitional vitamin B₁₂ deficiency. A Brito et al.⁽ Reference Brito, Verdugo and Hertrampf ²⁴ ⁾ stated that cB₁₂ could only detect improved neurophysiological function in asymptomatic Chilean elderly with poor vitamin B₁₂ status and also suggested to identify functional indicators of sub-clinical vitamin B₁₂ deficiency.

This is the first study to investigate the diagnostic value of circulating plasma vitamin B₁₂, holo-TC and tHcy (a functional marker) concentrations associated with 1-C metabolism in Indian vegetarians. We found that at similar circulating concentrations of plasma vitamin B₁₂ and holo-TC, there is a sex difference in plasma tHcy concentrations, with men having higher tHcy concentrations than women (P<0·0001). The marked sex difference⁽ Reference Naik, Bhide and Babhulkar ¹⁷ ⁾ in plasma tHcy concentrations is confirmed in this study. The difference suggests a higher threshold for supplementation of vitamin B₁₂ to improve reproductive and cardiovascular outcomes⁽ Reference Naik, Joglekar and Bhat ²⁵ ⁾. The sex difference is not significant in participants whose plasma vitamin B₁₂ concentrations are between 105 and 148 pmol/l. The strong inverse relation between plasma vitamin B₁₂ and tHcy and direct relation with holo-TC⁽ Reference Naik, Bhide and Babhulkar ¹⁷ ⁾ concentrations are confirmed in this study. Although age and sex reference intervals of tHcy have been established⁽ Reference Naik, Joglekar and Bhat ²⁵ ^, Reference Hannibal and Blom ²⁶ ⁾, they are usually ignored in reporting tHcy levels, because of dual biochemical origin (B₁₂ and folate). Elevated Hcy in plasma has been very often used as a biomarker, but its relationship to the molecular mechanisms of disease has not been established⁽ Reference Hannibal, Lysne and Monsen ²⁷ ⁾.

In this study, we aimed to meet two criteria: (a) vitamin B₁₂ absorption capacity in Indian vegetarians and (b) optimum plasma vitamin B₁₂ concentration required for methylation of Hcy. At normal plasma vitamin B₁₂ levels (>150 pmol/l), median holo-TC concentrations were 31·3 and 35·3 pmol/l in men and women, respectively, thereby attaining normal tHcy concentrations (13·9 and 10·17 µmol/l, respectively).

A cohort of 100 known patients with CVD and sixty-three normal healthy subjects (median age 44 years) were examined for their vitamin B₁₂ status in a case–control study in Pune. Median plasma vitamin B₁₂, holo-TC and tHcy concentrations were 160 pmol/l, 24 pmol/l and 19·7 µmol/l, respectively, in normal subjects. They stated that hyperhomocysteinaemia and elevated MMA were due to vegetarianism⁽ Reference Refsum, Yajnik and Gadkari ¹⁸ ⁾. Similarly, another report from Pune, wherein subjects with low plasma vitamin B₁₂ levels were studied, showed very low plasma holo-TC concentrations (7·7 (SD 4·2) and 9·8 (SD 8·7) pmol/l in men and women, respectively), with tHcy concentrations of 29·2 (SD 19·2) and 15·3 (SD 8·3) µmol/l⁽ Reference Bhat, Thuse and Lubree ²⁸ ⁾. Low vitamin B₁₂ concentration (median 110 pmol/l) and hyperhomocysteinaemia (>15 µmol/l) have been reported to be common in Indian men, particularly in vegetarians and urban middle-class residents. Most of the participants from these studies were vegetarians. In the study by Naik et al.⁽ Reference Naik, Bhide and Babhulkar ¹⁷ ⁾, young Indian vegetarian subjects with low plasma vitamin B₁₂ status (<148 pmol/l) were found to have holo-TC and tHcy concentrations of 14·4 pmol/l and 31·9 μmol/l, respectively, and the subjects with normal status (>200 pmol/l) were found to have holo-TC and tHcy concentrations of 27·7 pmol/l and 11·9 μmol/l, respectively. Most of the Indian studies did not use plasma holo-TC measurements. A Dutch study reported 49 (8–388) pmol/l of plasma holo-TC concentrations in healthy subjects with corresponding vitamin B₁₂ concentrations of 217 (119–1210) pmol/l⁽ Reference Bor, Lydeking-Olsen and Moller ²⁹ ⁾. A study from USA reported plasma holo-TC concentration of 85 (SD 48) pmol/l for 495 (SD 119) pmol/l of vitamin B₁₂ concentrations in healthy volunteers⁽ Reference Wokes, Badenoch and Sinclair ³⁰ ⁾. In both these studies the participants were non-vegetarians with high plasma holo-TC concentrations and the referred cut-offs may not be appropriate for vegetarians.

In healthy individuals, all four biomarkers (plasma vitamin B₁₂, holo-TC, tHcy and MMA) had a strong relation to vitamin B₁₂ intake, with steady-state concentrations at a daily intake of 4–7 µg vitamin B₁₂ Reference Bor, von Castel-Roberts and Kauwell ⁽³¹⁾ . These studies suggest that all four markers may be useful for monitoring a population’s vitamin B₁₂ status over time.

Carmel⁽ Reference Carmel ³² ⁾ categorised available biomarkers as those that directly measured plasma vitamin B₁₂ and those that measured metabolites that accumulated with inadequate amounts of vitamin B₁₂. Plasma holo-TC and vitamin B₁₂ measured circulating vitamin B₁₂ concentrations. These two therefore reflected the broad vitamin B₁₂ status from high risk of severe deficiency to adequacy. Miller et al.⁽ Reference Miller, Garrod and Rockwood ³³ ⁾ have stated that holo-TC and total vitamin B₁₂ have equal diagnostic accuracy in screening for metabolic vitamin B₁₂ deficiency. Measurement of both holo-TC and total vitamin B₁₂ provided better screen for vitamin B₁₂ deficiency than either assay alone. According to Green⁽ Reference Green ³⁴ ⁾, low vitamin B₁₂ status was indicated by values lower than the reference range (for vitamin B₁₂<148 pmol/l; for holo-TC <35 pmol/l), whereas for indirect measures of metabolites (MMA or tHcy) low vitamin B₁₂ status measures were indicated by a level above the upper limit of the reference range (for MMA >260 nmol/l; for tHcy >12 µmol/l). However, Valente et al.⁽ Reference Valente, Scott and Cunningham ³⁵ ⁾ suggested a diagnostic strategy using holo-TC as the front-line test. The cut-offs for deficiency were defined as 20 pmol/l for holo-TC and 123 pmol/l for serum vitamin B₁₂ after studying employees and medical students of a local hospital at Dundee, UK.

Conclusions

holo-TC levels may prove most useful if the aim is to monitor a population with a borderline sub-optimal vitamin B₁₂ supply. In contrast, total vitamin B₁₂ may be superior if the goal is to monitor a possible surplus load of vitamin. We advocate a diagnostic cut-off level of plasma vitamin B₁₂ (105 pmol/l), holo-TC (22·6 pmol/l) and Hcy (17·6 µmol/l for females and 27·0 µmol/l for males) in vegetarian Indian population. In addition to this, we recommend that vegetarians do take a supplement of vitamin B₁₂ to ensure adequate supply of the micronutrient. This would be of particular importance for females in reproductive age, to prevent the risks associated with maternal–fetal vitamin B₁₂ deficiency.

The limitation of the study is that plasma MMA has not been measured. However, if none of the participants is folate deficient, the measurement of Hcy shall indicate vitamin B₁₂ status.

Acknowledgements

This research received no specific grant from any funding agency, commercial or not-for-profit sectors. Dr Dhanajay Kelkar, Medical Director, extended all the help. Our thanks are due to Swati, Swapnali and Sandeep and Abbott Laboratories for free kits.

S. N. designed the study. V. B. and N. M. prepared the manuscript draft. S. N. prepared the final draft.

The authors declare that there are no conflicts of interest.

References

1. Antony, AC (2003) Vegetarianism and vitamin B₁₂ deficiency. Am J Clin Nutr 78, 3–6.Google Scholar

2. Dhopeshwarkar, GA, Trivedi, JC, Kulkarni, BS, et al. (1956) The effect of vegetarianism and antibiotics upon proteins and vitamin B₁₂ in the blood. Br J Nutr 10, 105–110.Google Scholar

3. Jathar, VS, Inamdar-Deshmukh, AB, Rege, DV, et al. (1975) Vitamin B₁₂ and vegetarianism in India. Acta Hematol 53, 90–99.Google Scholar

4. Obeid, R, Giesel, J, Schorr, T, et al. (2002) The impact of vegetarianism on some hematological prameters. Eu J Hematol 69, 275–279.Google Scholar

5. Refsum, H, Smith, AD, Ueland, PM, et al. (2001) Facts and recommendations about total homocysteine determinations: an expert opinion. Br J Nutr 85, Suppl. 2, 5109–5113.Google Scholar

6. Herrmann, W, Obeid, R, Schorr, H, et al. (2003) Functional vitamin B₁₂ deficiency and determination of holotranscobalamin in population at risk. Clin Chem Lab Med 41, 1478–1488.Google Scholar

7. Bhate, V, Deshpande, S, Bhat, D, et al. (2008) Vitamin B₁₂ status of pregnant Indian women and cognitive function in their 9 yr old children. Food Nutr Bull 29, 249–254.Google Scholar

8. Weir, DC & Scott, JM (1999) Brain function in the elderly: role of vitamin B₁₂ and folate. Br Med Bull 55, 669–682.Google Scholar

9. Yajnik, CS, Deshpande, SS, Lubree, HG, et al. (2006) Vitamin B₁₂ deficiency and hyperhomocysteinemia in rural and urban Indians. J Assoc Physicians India 54, 775–782.Google Scholar

10. Nexo, E & Hoffman-Lücke, E (2011) Holotranscobalamin, a marker of vitamin B-12 status: analytical aspects and clinical utility. Am J Clin Nutr 94, 359S–365S.Google Scholar

11. Herbert, V (1994) Staging vitamin B-12 (cobalamin) status in vegetarians. Am J Clin Nutr 59, 1213S–1232SS.Google Scholar

12. Herrmann, W & Giesel, J (2002) Vegetarian lifestyle and monitoring vitamin B₁₂ status. Clin Chim Acta 326, 47–59.Google Scholar

13. Lindenbaum, J, Savage, DG, Stabler, SP, et al. (1990) Diagnosis of cobalamin deficiency: II. Relative sensitivities of serum cobalamin, methylmalonic acid and total homocysteine concentrations. Am J Hematol 34, 99–107.Google Scholar

14. Tulip Group. Creatinine kit (alkaline picrate method). http://www.tulipgroup.com/Coral_New2/html/pack_inserts/Creatinine%20Kit%20Alkaline%20Picrate%20Method.pdf Google Scholar

15. Kanade, AN, Rao, S, Kelkar, RS, et al. (2008) Maternal nutrition and birth size among urban affluent and rural women in India. Am J Clin Nutr 27, 137–145.Google Scholar PubMed

16. Gopalan, C, Rama Sastri, BV & Balsubramanian, SC (2007) Dietsoft software version 1.1.7. Developed by invincible, iDeAS, Nutritive Value of Indian Foods, revised ed. Hyderabad: NIN, ICMR. www.invincibleiDeas.com Google Scholar

17. Naik, S, Bhide, V, Babhulkar, A, et al. (2013) Daily milk intake improves vitamin B₁₂ status in young vegetarian Indians: an intervention trial. Nutr J 12, 136–144.Google Scholar

18. Refsum, H, Yajnik, CS, Gadkari, M, et al. (2001) Hyperhomocysteinemia and elevated methylmalonic acid indicate a high prevalence of cobalamin deficiency in Asian Indians. Am J Clin Nutr 74, 233–241.Google Scholar

19. Buderer, NM (1996) Statistical methodology 1. Incorporating the procedure of disease into the sample size calculation for sensitivity and specificity. Acad Emerg Med 3, 895–900.Google Scholar

20. Lysne, V, Strand, E, Svingen, GFT, et al. (2016) Peroxisome proliferator-activated receptor activation is associated with altered plasma one-carbon metabolites and B-vitamin status in rats. Nutrients 8, 26.Google Scholar

21. Solomon, LR (2015) Functional cobalamin (vitamin B₁₂) deficiency: role of advanced age and disorders associated with increased oxidative stress. Eur J Clin Nutr 69, 687–692.Google Scholar

22. Fedosov, SN (2013) Biochemical markers of vitamin B₁₂ deficiency combined in one diagnostic parameter: the age dependence and associations with cognitive function and blood haemoglobin. Clin Chim Acta 422, 47–53.Google Scholar

23. Fedosov, SN, Brito, A, Miller, JW, et al. (2015) Combined indicator of vitamin B₁₂ status: modifications for missing biomarkers and folate status and recommendations for revised cut-points. Clin Chim Lab Med 53, 1215–1225.Google Scholar

24. Brito, A, Verdugo, R, Hertrampf, E, et al. (2016) Vitamin B₁₂ treatment of asymptomatic, deficient, elderly Chileans improves conductivity in myelinated peripheral nerves but high serum folate impairs vitamin B₁₂ response assessed by combined indicator of vitamin B₁₂ status. Am J Clin Nutr 103, 250–257.Google Scholar

25. Naik, S, Joglekar, C, Bhat, D, et al. (2011) Marked gender difference in plasma total homocysteine concentrations in Indian adults with low vitamin B₁₂ . Int J Vitam Nutr Res 81, 306–316.Google Scholar

26. Hannibal, L & Blom, HJ (2016) Homocysteine and disease: causal associations or epiphenomenons? Mol Aspects Med 53, 36–42.Google Scholar

27. Hannibal, L, Lysne, V, Monsen, ALB, et al. (2016) Biomarkers and algorithms for the diagnosis of vitamin B₁₂ deficiency. Front Mol Biosci 3, 27–42.Google Scholar

28. Bhat, DS, Thuse, NV, Lubree, HG, et al. (2009) Increased plasma holotranscobalamin can be used to assess vitamin B₁₂ absorption in individuals with low plasma vitamin B₁₂ . J Nutr 23, 2119–2123.Google Scholar

29. Bor, MV, Lydeking-Olsen, E, Moller, J, et al. (2009) A daily intake of 6 ug vitamin B₁₂ appears to saturate all the vitamin B₁₂ related variables in Danish postmenopausal women. Am J Clin Nutr 89, 1078–1087.Google Scholar

30. Wokes, F, Badenoch, J & Sinclair, HM (1955) Human dietary deficiency of vitamin B₁₂ . Am J Clin Nutr 3, 375–382.CrossRef Google Scholar PubMed

31. Bor, MV, von Castel-Roberts, KM, Kauwell, GPA, et al. (2010) Daily intake of 4-7 ug dietary vitamin B₁₂ is associated with a steady concentrations of vitamin B₁₂ related biomarkers in a healthy young population. Am J Clin Nutr 91, 571–577.Google Scholar

32. Carmel, R (2011) Biomarkers of cobalamin status in the epidemiologic applications: a critical over view of context, applications and performance characteristics of cobalamin, methylmalonic acid and holotranscobalamin. Am J Clin Nutr 94, 348S–358S.Google Scholar

33. Miller, W, Garrod, MG, Rockwood, AL, et al. (2006) Measurement of both total vitamin B₁₂ and holotranscobalamin singly and in combination. Clin Chem 52, 278–285.Google Scholar

34. Green, R (2011) Indicators for assessing folate and vitamin B₁₂ status and for monitoring the efficiency of intervention strategies. Am J Clin Nutr 94, 667S–672S.CrossRef Google Scholar

35. Valente, E, Scott, JM, Cunningham, C, et al. (2011) Diagnostic accuracy of holotranscobalamin, methylmalonic acid, serum cobalamin, and other indicators of tissue vitamin B₁₂ status in the elderly. Clin Chem 57, 860–863.Google Scholar

Fig. 1 Plasma vitamin B12 at concentrations of 150 and 100 pmol/l were used for analysis. Metabolic deficiency was defined as plasma holotranscobalamin (holo-TC)<35 pmol/l in 119 vegetarian Indians. Predictive values of plasma holo-TC were 24·58 and 19·6 µmol/l, respectively, with similar sensitivity and specificity at both 150 and 100 pmol/l of vitamin B12. ROC, receiver operating characteristic.

Fig. 2 Plasma vitamin B12 at 150 and 100 pmol/l were used as variables. Metabolic deficiency was defined as total homocysteine (tHcy)>15 µmol/l in 119 vegetarian Indians. Predictive values of plasma tHcy were 18·56 and 21·2 µmol/l, respectively, with better specificity (89·47 %) at 100 pmol/l of vitamin B12. ROC, receiver operating characteristic.

Table 1 Baseline characteristics and biochemistry of the participants (male and female) (Medians and 25th, 75th percentiles)

Table 2 Sex difference in total homocysteine (tHcy) at different vitamin B12 concentrations (Medians and 25th–75th percentiles)

Table 3 Plasma vitamin B12 concentrations and corresponding concentrations of plasma holotranscobalamin (holo-TC) and total homocysteine (tHcy)

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Identification of vitamin B12 deficiency in vegetarian Indians

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