REASSESSING THE ROLE OF HOMOCYSTEINE AND HOLOTRANSCOBALAMIN LEVELS IN DIAGNOSING VITAMIN B12 DEFICIENCY ANEMIA

Background: Vitamin B12 deficiency can lead to irreversible neurological damage, megaloblastic anemia, osteoporosis, cerebrovascular, and cardiovascular diseases, and thus, early diagnosis is essential.
Objective: The objective of the study was to assess homocysteine (Hcy) and holotranscobalamine (HoloTC) levels among patients with Vitamin B12 deficiency and to see if Hcy and HoloTC level assay can help us in diagnosis of Vitamin B12 deficiency.
Methods: We carried out a cross-sectional observational study on 60 patients of Sr. B12 deficient male and female patients between the ages of 18 and 65 years in IPD and OPD patients at multispecialty hospital. Data were collected from predefined pro forma and were asked about their diet, socioeconomic status, and history. Then, these patients further undergone anthropometric measurements and investigated for Hcy and HoloTC level. The statistical analysis was done using Statistical Package for the Social Sciences (SSPS) software (version 11).
Results: About 60% of cases were vegetarian and 40% of cases were non-vegetarian. Mean age of study participants was 43.67 years, mean of mean cellular volume was 90.7 fl, mean of B12 was 138 pmol/L, mean of HoloTC was 60.84 pmol/L, and mean of Hcy was 34.17 umol/L. Out of 60 patients, 10 patients had anemia, 21 patients had neurological manifestation, and 29 patients had gastrointestinal (GI) manifestation. In male group, out of 32, 11 patients had HoloTC <8.9, 19 patients had value between 8.9 and 128, and two patients had HoloTC more than 128. In female group, out of 28, seven patients had HoloTC <8.9, 14 patients had value between 8.9 and 128, and seven patients had HoloTC >128. In group of 32 male patients, none of male patients showed Hcy value <5.9, four patients showed Hcy between 5.9 and 16, and 28 patients showed Hcy value >16. Out of 28 female patients, none of female patients showed Hcy <3.36, nine patients showed Hcy between 3.36 and 20.4, and 19 patients showed Hcy >20.4. p <0.001 is highly statistically significant.
Conclusion: In our study, we found that 31.33% of cases also showed decreased HoloTC along with B12 deficiency, but this correlation was statistically insignificant. We also found that 78.33% of cases showed increased Hcy along with serum B12 deficiency, which was statistically significant, so we concluded that there is a strong association between serum B12 and Hcy. We found that all patients with elevated Hcy also had low HoloTC except in two cases, but this correlation was not found to be statistically significant.


INTRODUCTION
There are many patients with Vitamin B12 deficiency where it is difficult to pinpoint the etiology of Vitamin B12 deficiency. Most of the times, the etiology of B12 deficiency is multifactorial and the diagnosis is challenging in many patients. The diagnosis depends on many investigations and at times even after an exhaustive series of investigations, we fail to detect the exact cause of Vitamin B12 deficiency. Vitamin B12 is essential for the synthesis of S-adenosyl methionine and is involved in the metabolism of proteins, phospholipids, and neurotransmitters. Its deficiency leads to several neurological manifestations and affects all age groups [1]. Its deficiency can take 3-6 years to develop after absorption of dietary B12 had ceased and the initial clinical manifestations are subtle and nonspecific or are attributed to the normal aging process. This problem was also underestimated in the past because of the belief that deficiency is unlikely except in strict vegetarian and pernicious anemia patients are known to be cobalamin deficient and that it usually takes about 20 years for stores of the vitamin to become depleted. Since a deficiency in this vitamin can lead to irreversible neurological damage [2] (subacute combined degeneration of the spinal cord and cognitive impairment in the elderly), megaloblastic anemia, osteoporosis [3], cerebrovascular, and cardiovascular diseases [4], early diagnosis is essential. In recent years, new and sensitive diagnostic markers to determine a person's cobalamin status have become available.
Even though the human body can store Vitamin B12 to last for up to 5 years, its deficiency is not very uncommon. The diagnosis is frequently made based on a low serum Vitamin B12 level or megaloblastic bone marrow or both [5]. Vitamin B12, apart from causing neuropsychiatric symptoms, leads to hyperhomocysteinemia and methylmalonic acidemia which can have serious health implications. Low serum Vitamin B12 levels have low sensitivity and specificity in terms of tissue deficiency [6]. Homocysteine (Hcy) and methylmalonic acid estimations are adjunct and aid in diagnosis of B12 deficiency but still serum Vitamin B12 measurement is the extensively applied standard method by practical purposes. To obtain a more sensitive marker of Vitamin B12 status, a new test involving measurement of the levels of holotranscobalamine (HoloTC)transcobalamin-Vitamin B12 complex has been introduced [7]. HoloTC promotes global cellular uptake of cobalamin by specific receptors; therefore, it may be more sensitive than serum Vitamin B12 levels in indicating Vitamin B12 status [8].
The racial, religious, ethnic, and socioeconomic heterogeneity of the people in India greatly influences their dietary habits. This study was carried out to assess Hcy and HoloTC levels among patients with Vitamin B12 deficiency and to see if Hcy and HoloTC level assay can help us in diagnosis of Vitamin B12 deficiency.

METHODS
We carried out a cross-sectional observational study among inpatients and outpatients of tertiary care center for a period of 6 months. Age group of the study population was between 18 and 65 years. We selected a convenient sample of 95 patients with B12 deficiency, randomly from laboratory of patients with B12 less than 200 pg/dl and after retrogradely seeing patients, we excluded patients with renal, hepatic, and heart diseases due to possibility of falsely elevated Hcy level. Out of 95, a total of 60 patients were included in the study. Data were collected from predefined pro forma and were asked about their diet, socioeconomic status, and history. Then, these patients further undergone anthropometric measurements and investigated for Hcy and HoloTC level. B12 was estimated by the electrochemiluminescence immunoassay "ECLIA." Holotranscobalamin assay was a precision test for the AxSYM microparticle enzyme immunoassay (MEIA) technology. Informed consent was taken, and identity of the patients was kept confidential. The statistical analysis was done using Statistical Package for the Social Sciences (SSPS) software (version 11). Chi-square was used to test the significance between groups and Pearson's correlation coefficient was used, to test the correlation between different variables with p<0.05 which was accepted as statistically significant. The test results were tabulated and graphically represented using Microsoft Word 2007.

RESULTS
This study was conducted on 60 patients of Sr. B12 deficient male and female patients between the ages of 18 and 65 years in IPD and OPD patients at multispecialty hospital. About 60% of cases were vegetarian and 40% of cases were non-vegetarian. Mean age of the study participants was 43.67 years, mean of mean cellular volume (MCV) was 90.7 fl, mean of B12 was 138 pmol/L, mean of HoloTC was 60.84 pmol/L, and mean of Hcy was 34.17 umol/L ( Table 1). Out of 60 patients, 10 patients had anemia, 21 patients had neurological manifestation, and 29 patients had gastrointestinal (GI) manifestation (Figs. 1 and 2). It showed that out of 32 male patients, 28 patients had normal MCV and four patients had MCV more than 100. Out of 28 female patients, 25 patients showed normal MCV and two patients showed MCV more than 100. In male group, out of 32, 11 patients had HoloTC <8.9, 19 patients had value between 8.9 and 128, and two patients had HoloTC more than 128. In female group, out of 28, seven patients had HoloTC <8.9, 14 patients had value between 8.9 and 128, and seven patients had HoloTC >128. In group of 32 male patients, none of male patients showed Hcy value <5.9, four patients showed Hcy between 5.9 and 16, and 28 patients showed Hcy value >16 ( Table 2). Out of 28 female patients, none of female patients showed Hcy <3.36, nine patients showed Hcy between 3.36 and 20.4, and 19 patients showed Hcy >20.4. p<0.001 is highly statistically significant. Table 3 shows relation of MCV, HoloTC, and Hcy with clinical manifestations among male and female patients. Table 4 shows distribution of different laboratory values among different age groups of study participants.
In our study, we found that 31.33% of cases also showed decreased HoloTC along with B12 deficiency, but this correlation was statistically insignificant. We also found that 78.33% of cases showed increased Hcy along with serum B12 deficiency, which was statistically significant, so we concluded that there is a strong association between serum B12 and Hcy. We found that all patients with elevated Hcy also had low HoloTC except in two cases, but this correlation was not found to be statistically significant.

DISCUSSION
Deficiency of Vitamin B12 is very common because of inadequate dietary intake and/or malabsorption. The deficiency state has a very wide presentation and can cause or exacerbate neuropsychiatric and other vague symptoms. It has been observed that Vitamin B12 deficiency is far more prevalent than expected and majority of the cases remain undiagnosed. In early stage, Vitamin B12 deficiency might present with subtle and slight cognitive impairments. Hence, early recognition becomes crucial for preventing irreversible damage.

Variables Patients
Age (  Dietary Vitamin B12 deficiency is a severe problem in the Indian subcontinent [9] as seen in this study. The mean Vitamin B12 level was observed to be 138 pmol/L which itself was on a lower side. The findings from several studies performed in different clinical settings have confirmed that serum Vitamin B12 is a relatively poor marker with low sensitivity and specificity in predicting Vitamin B12 status and that HoloTC is a useful diagnostic indicator for this purpose [7]. In addition, a few studies performed in different countries and ethnic groups have shown differences in the prevalence of Vitamin B12 Vitamin B12 acts as cofactor, in Hcy metabolism [10], so elevated serum Hcy levels have been linked to Vitamin B12 deficiency [11]. Hcy levels have been considered to be more sensitive than serum Vitamin B12 levels for determining Vitamin B12 status [11]. This study was conducted to know the role of Hcy and HoloTC in serum B12 deficiency. Vegetarian diet has always been suspected to contribute toward the development of B12 deficiency. Although dietary data history recovered was partial in the present survey, it could be assessed that vegetarian dietetic practice offered considerable risk for developing B12 deficiency, rate being 60%. This is in consistence with several studies [12].
In the present study, Vitamin B12 measurement was used as the first-line test and the definition of Vitamin B12 deficiency was based on low level of serum Vitamin B12 although the measurements of metabolites such as methylmalonic acid and Hcy have been shown to be more sensitive in the diagnosis of Vitamin B12 deficiency [13].
Hcy is known as a sensitive functional marker of inadequate cellular Vitamin B12 concentration. Deficiency of this vitamin has important health consequences, in addition to role in Hcy metabolism [14]. In our study, we observed raised Hcy level in 78.33% of subjects along with Vitamin B12 deficiency (p<0.001). A weak direct correlation was found in between serum Vitamin B12 and HoloTC (panel A: r=0.09723, p=0.45) (Fig. 3). As shown in Fig. 4 Out of that 13.33% in between 18 and 30 years, 18.33% in between 31 and 40 years, 20% in between 41 and 50 years and 26.66% in between 51 and 65 years age group. This is consistent with -Fenech et al. [15] reported that 75% of the cases of high Hcy concentration were associated with low B12 concentration. Campbell et al. [16] reported that high serum Hcy (≥13 μmol/L) was found in 50.9% of deficient plasma B12 (<200 pg/mL). We also observed decrease in HoloTC level along with Vitamin B12 deficiency in 31.66% of patients, (p>0.05) out of that, 5% of patients in 18-30 years age, 8.33% in 31-40 years, and in 41-50 years age and 10% in 51-65 years age group. This is consistent with -Al Aisari et al. [17] reported that low serum transcobalamin level was found in 40% of plasma deficient patient.    30 years, 50% in between 31 and 40 years, 20% in between 41-and 50 years age group, and 20% in between 51-and 65 years age group. It showed that the prevalence of anemia with Vitamin B12 deficiency is highest in reproductive age group. In these groups, 90% of patients show raised Hcy level in blood but only 30% of patients show decreased HoloTC level in blood. This is consistent with Craig et al. [18] who did not find such a correlation between vitamin serum B12 deficiency and MCV. Stott et al. [19] found that only 23% of patients with serum B12 deficiency had raised MCV. Luong and Nguyen [20] told that he did not found any patient with macrocytosis in group of patients with anemia and Vitamin B12 deficiency.

Limitations
First, ours was a small convenient sample. Hence, study findings cannot be extrapolated. Macrocytic anemia may be masked in vegetarians by excess folate intake or by concomitant iron deficiency. We did not check Sr. folate and Sr. iron level. Our study had possible ethnic differences and selection bias. Our study did not show significant correlation in between Vitamin B12 deficiency and serum HoloTC, the serum HoloTC levels can be affected by several factors, for example, food intake, amount of absorbed Vitamin B12, and subclinical renal and hepatic dysfunction. We have not considered the percentage of total Vitamin B12 bound to transcobalamin in the assessment of Vitamin B12 status. Although this percentage typically ranges from 20% to 30%, it can vary from 10% to 70% and this variation may influence the results.

CONCLUSION
Thus, we conclude that the vegetarians investigated in this study had more Vitamin B12 deficiency than non-vegetarians, which were related to the degree of animal product restriction. Hyperhomocysteinemia was linked to Vitamin B12 deficiency. According to our data, the assessment of HoloTC II, accompanied by that of metabolic markers such as Hcy, may offer sensitive and reliable tools for early diagnosis and hence proper intervention can be carried out in persons who are prone to Vitamin B12 deficiency. This study also suggests that Hcy levels can be early markers for tissue Vitamin B12 deficiency, even before hematologic manifestations occur.

ACKNOWLEDGMENTS
I acknowledge and thank to all my coauthors and study participants.

AUTHORS' CONTRIBUTIONS
All authors have contributed to the preparation of manuscript.

CONFLICTS OF INTEREST
Nil.