Comparison of Serum Holotranscobalamin with Serum Vitamin B12 in Population Prone to Megaloblastic Anemia and their Correlation with Nerve Conduction Study

Abstract

Early detection of megaloblastic anemia and associated neurological complications is crucial for management. This study was conducted to compare serum holotranscobalamin level with serum vitamin B12 level as early biomarker in people prone to megaloblastic anemia and to evaluate co-relation between these biomarkers and nerve conduction study in study patients. 83 adult patients (Hb > 12 gm/dl) prone to megaloblastic anemia were studied for basic haematological investigations, random blood sugar, thyroid function test, liver function test, kidney function test, serum vitamin B12, serum holotranscobalamin and serum folic acid levels. 45 patients among them underwent nerve conduction studies. All study patients were classified in 6 groups on the basis of risk factors for megaloblastic anemia. 29 patients (34.9%) were on antiepileptic drugs, 26 (31.3%) were chronic alcoholic, 10 patients (12%) each, had malabsorption and ileal tuberculosis, 6 (7.22%) had chronic pancreatitis and 2 (2.4%) had ileal resection. 30 patients (36.14%) had low serum holotranscobalamin, including 7 patients (8.43%) with low serum vitamin B12 level also, unmasking vitamin B12 deficiency in 23 patients (27.7%). 7 patients (8.43%) had mean corpuscular volume (MCV) > 100fL and 8 patients (9.63%) had vitamin B12 deficiency related changes on peripheral smear. Serum vitamin B12 and holotranscobalamin levels were significantly low in patients with peripheral smear changes, with p value 0.039 and 0.041 respectively, while no such association seen with MCV. Subclinical peripheral neuropathy was detected in 18 (40%) out of 45 patients on nerve conduction study. Serum holotranscobalamin levels were significantly lower (p = 0.031) than serum vitamin B12 levels (p = 0.2) in patients with neuropathic changes. Rest investigations and serum folic acid levels were normal in all patients. Holotranscobalamin levels can be considered early and reliable marker for vitamin B12 deficiency and deficiency associated peripheral neuropathy, even in patients who are prone to megaloblastic anemia, and not yet anemic or symptomatic for neuropathy.

Introduction

Megaloblastic anemia refers to a group of disorders distinguished by special morphological appearance of developing red blood cells and ineffective hematopoiesis in the bone marrow. It is commonly caused by vitamin B12/folate deficiency or other genetic or acquired defects affecting metabolism of these two vitamins or any defect in DNA synthesis (not related to vitamin B12 or folate) [1].

Megaloblastic anemia is a very common condition in Indian scenario with prevalence ranging from 2 to 40%, as testified in various Indian studies [2]. Vitamin B12 deficiency is found to be one of the most common causes of megaloblastic anemia, but, overall prevalence of vitamin B12 deficiency is highly variable due to heterogenous nature of disease, variable clinical presentation and disputed normal serum range [3].

Vitamin B12 is an essential water-soluble vitamin required for cellular division and metabolism, neuronal integrity and DNA synthesis. Meat, fish, egg is the primary source of vitamin B12 for humans, with recommended dietary allowance of 1–3 mcg/day. The only source of vitamin B12 in strict vegetarians is green leafy vegetables, with contaminating harmless bacteria [4]. Vitamin B12 is majorly absorbed by intrinsic factor mediated process in ileum and less than 1% of oral dose is absorbed through gastrointestinal tract mucosa, independent of intrinsic factor. In systemic circulation, vitamin B12 is bound to proteins, transcobalamin II (TC2) and haptocorrin. Transcobalamin II bound vitamin B12 is called holotranscobalamin (holoTC). Holotranscobalamin represents 5–20% of total transcobalamin II and carries 6–20% of total vitamin B12 in circulation. Majorly, holotranscobalamin transports vitamin B12 to all cells. Vitamin B12 homeostasis is maintained with help of enterohepatic circulation and reabsorption of holotranscobalamin in proximal renal tubular cells. There are two active forms of vitamin B12 at the cellular level – methylcobalamin and adenosylcobalamin. Deficiency of these leads to ineffective DNA synthesis, cellular proliferation and synthesis of fatty acids and myelin sheath [4–6].

Globally, the most common cause of vitamin B12 deficiency is pernicious anemia. Other causes include dietary deficiency, chronic alcohol intake, gastrointestinal disease, chronic liver disease, hypothyroidism, human immunodeficiency virus infection, drugs (metformin, proton pump inhibitors, anti-epileptic drugs, cytotoxic drugs, nitrous oxide inhalation) and abnormal or deficient enzymes/binding proteins involved in vitamin B12 metabolism (like transcobalamin II deficiency) [1, 4, 5, 7]. Dietary deficiency is seen in vegans and strict vegetarians (their practice of thoroughly washing vegetables removes harmless contaminating bacteria, which produce vitamin B12) with 4.4 times higher risk than non-vegetarians [8]. Gastrointestinal causes include chronic gastritis, atrophic gastritis, post gastrectomy, malabsorption syndromes, chronic pancreatitis, ileocecal tuberculosis, post ileal resection, etc.

Megaloblastic anemia and vitamin B12 deficiency can present with wide variety of symptoms with involvement of multiple organ systems. Dyserythropoiesis, demyelination and impaired cellular proliferation is responsible for most of clinical manifestations. Common clinical features include fatiguability, glossitis, angular cheilosis, jaundice, knuckle pigmentation, hair loss and diarrhoea [4, 5, 9]. Deficiency can also cause serious neuropsychiatric and cardiovascular complications. Cardiovascular complications include arterial/venous thromboembolism and atherosclerotic cardiovascular diseases. Hyperhomocysteinemia in vitamin B12 deficiency is associated with cardiovascular complications [10].

Psychiatric complications include dementia, mania, delusion, depression, irritability, psychosis (megaloblastic madness), hallucinations. Neurological complications include myelopathy (being more common), neuropathy and myeloneuropathy. Common neurological disorders include subacute combined degeneration, sensorineural deafness, dysosmia, dysgeusia, cerebral/cerebellar degeneration, spinocerebellar ataxia, optic neuritis/atrophy, peripheral neuropathy, erectile dysfunction, urinary/bowel incontinence and postural hypotension [1, 4, 11–13]. Methionine deficiency, methylmalonyl CoenzymeA excess and elevated level of cytokines, tumour necrosis factor, epidermal growth factor impair myelin synthesis and damages neural tissues, which is predominantly responsible for neurological features [14, 15]. Also, hyperhomocysteinemia is independently associated with increased risk of Alzheimer`s disease [12]. These complications are responsible for increased mortality and morbidity in patients. Further, neurological involvement is a matter of grave concern, because deficient patients receiving inadequate treatment possess high risk of developing irreversible neurological sequelae, including peripheral neuropathy and subacute combined degeneration of spinal cord.

Diagnosis of vitamin B12 deficiency in suspected patients is based on haematological and biochemical investigations [4, 5, 16–18]. Haematological investigations include complete blood count, red blood cell indices and peripheral smear. Biochemical markers available for a long period of time for diagnosis are serum vitamin B12 level, serum homocysteine level and serum methylmalonic acid level. These markers have various limitations, due to which serum holotranscobalamin was investigated as new biomarker for diagnosis. Holotranscobalamin, being carrier of metabolically active fraction of vitamin B12, is considered to be better than former markers in terms of early diagnosis of vitamin B12 deficiency in patients of megaloblastic anemia [5].

Megaloblastic anemia is very common in Indian population, with wide variety of clinical features. The thing making it more dreadful and problematic is that it may sometime present only with neurological features, without anemia. Also, if untreated, neurological changes may become irreversible. This makes it necessary to detect the illness at an early stage, when it is still treatable. Further, there is paucity of data on subclinical neuropathy in megaloblastic anemia. Thus, present study is aimed to use serum holotranscobalamin as an early biomarker to detect vitamin B12 deficiency as well as deficiency related subclinical peripheral neuropathy in people who are prone to developing megaloblastic anemia, to start treatment earlier and prevent irreversible neurological changes.

Materials and Methods

The study was conducted in Department of Medicine and Department of Biochemistry, Maulana Azad Medical College and associated Lok Nayak Hospital, and Department of Neurology, Govind Ballabh Pant Institute of Postgraduate Medical Education and Research, after being approved by the ethical committee of the institute. It was a prospective observational study. Based on previous literature research for prevalence of vitamin B12 deficiency in asymptomatic population, and keeping a level of significance at 95%, power of study at 80% and allowable error of 5% as acceptable, the sample size was calculated as 377. However, due to budgetary constraints and availability of testing kits for serum holotranscobalamin estimation, we kept sample of convenience to be 83 in study. 83 adult people were enrolled in study, who were prone to develop vitamin B12 deficiency and megaloblastic anemia on basis of their clinical condition. Patients with anemia, diabetes, chronic liver/kidney disease and thyroid disorder were excluded. Anemia was defined by haemoglobin less than 13gm/dl in males and 12 gm/dl in females [19]. Patients were divided in 6 groups based on risk factors for vitamin B12 deficiency. 29(34.93%) were on anti-epileptic drugs (taking drugs for more than 1 year), 26(31.32%) were alcoholics (who consumed alcohol for more than 5 years), 10(12.04%) each in group of malabsorption and ileal tuberculosis, 6(7.22%) had chronic pancreatitis and 2(2.4%) had ileal resection.

A well-informed consent was taken from every patient, followed by detailed history and physical examination. All patients were asymptomatic for anemia and neurological symptoms. 5 ml blood sample was collected from patients by venepuncture using aseptic precautions. All patients were subjected to following investigations—Complete blood count with red blood cell (RBC) indices, peripheral smear, liver function test (by spectrophotometry), kidney function test (by spectrophotometry), random Blood glucose (by spectrophotometry), thyroid function test (by electrochemiluminescence immuno assay), serum folic acid level (by electrochemiluminescence immuno assay), serum vitamin B12 level (by electrochemiluminescence immuno assay) and serum holotranscobalamin level (by Enzyme Linked ImmunoSorbent Assay {ELISA}). Normal range for serum folic acid level is 3.1–17.5 ng/ml and for serum vitamin B12 is > 250 pg/ml. (as per our laboratory standards). Holotranscobalamin level estimation was done by Direct ELISA method with wells precoated with purified human holotranscobalamin antibody. Detection range of serum holotranscobalamin with used ELISA kit was 7.65–500 pg/ml. Normal range of serum holotranscobalamin is kept 54–270 pg/ml, as per literature [6]. Nerve conduction study was performed in 45 patients after obtaining informed consent. For motor conduction, median, ulnar, common peroneal and tibial nerves were tested for action potential latency, amplitude of potential, F wave latency and conduction velocity. For sensory conduction, median, ulnar and sural nerves were tested for action potential latency, amplitude of action potential and conduction velocity.

For Statistical analysis, data was entered in MS-excel and analysed using statistical package for social sciences (SPSS) version 17.0 for windows. Qualitative data was expressed in proportion/percentage and difference between proportions was expressed in chi square test or fisher exact test. Quantitative data was expressed in mean ± SD or median ± interquartile range and differences were analysed using students t test. Statistical analysis was done to assess serum holotranscobalamin levels, to compare serum holotranscobalamin levels with serum vitamin B12 levels and to assess association of these biomarkers with presence of subclinical peripheral neuropathy in these patients. p value of < 0.05 was kept as significant.

Each patient was given a consent form to fill. Patients were explained the purpose of study and their right to quit at any time without having to give reason. Patient information dealt with confidentiality. Any abnormality detected during screening was appropriately managed.

Results

Age of patients was in the range of 20–51 years, with mean age 30.43 ± 8.33 years. Maximum number of patients, 50 (60%) were between 20 and 30 years, followed by 23(28%) between 31 and 40 years and 10 (12%) between 41 and 51 years. 51 patients (61%) were male and 32(39%) were female. Table 1 shows complete blood count and RBC indices profile of study patients. 1 patient (1.2%) had leucopenia, 19(22.89%) had thrombocytopenia, 7(8.43%) had macrocytosis with Mean Corpuscular Volume (MCV) > 100 fL and 4(4.81%) had elevated Mean Corpuscular Hemoglobin (MCH). All patients had normal Mean Corpuscular Hemoglobin Concentration (MCHC) and serum folic acid.

Table 1.

Complete blood count, RBC indices and serum folic acid level of study patients

Investigation	Mean of study group	Normal range [19]
Hemoglobin	13.49 ± 0.81 gm/dl	12–15.5 gm/dl
Total leucocyte count	7060.96 ± 1630.31 cells/cumm	4300–10,800 cells/cumm
Platelet count	1.77 ± 0.47 lakh cells/cumm	1.5–4.5 lakh cells/cumm
MCV	89.78 ± 6.03 fL	82–98 fL
MCH	30.4 ± 1.98 pg	27–33 pg
MCHC	33.46 ± 1.13 gm/dl	31–35 gm/dl
Serum folic acid	10.72 ± 5.41 ng/ml	3.1–17.5 ng/ml

8 patients (9.63%) had findings on peripheral smear suggestive of megaloblastic changes. Megaloblastic changes were defined by presence of macro-ovalocytes, hypersegmented neutrophils, leucopenia, thrombocytopenia or pancytopenia, giant platelets [1]. Baseline biochemical investigations including liver function, kidney function, thyroid function and blood sugar are depicted in Table 2. Normal range of these investigations are as per our laboratory standards. These investigations were normal in all patients.

Table 2.

Baseline biochemical profile of study patients

Investigation	Mean of study group	Normal range
Blood urea	28.37 ± 9.42 mg/dl	20–40 mg/dl
Serum creatinine	0.86 ± 0.31 mg/dl	< 1.5 mg/dl
Serum bilirubin	0.67 ± 0.29 mg/dl	< 1.2 mg/dl
Alanine transaminase	30.55 ± 9.56 IU/L	< 35 IU/L
Aspartate transaminase	30.73 ± 8.9 IU/L	< 40 IU/L
Free T3	5.09 ± 0.87 pmol/l	3.1–6.8 pmol/l
Free T4	17.29 ± 2.34 pmol/l	12–22 pmol/l
Thyroid stimulating hormone	2.90 ± 1.15 mIU/L	0.5–4.7 mIU/L
Random blood sugar	125.80 ± 21.96 mg/dl	< 200 mg/dl

Vitamin B12 and Holotranscobalamin

In the study, mean serum vitamin B12 level was 578.36 ± 214.67 pg/ml. 7 patients (8.4%) had low serum vitamin B12 levels (< 250 pg/ml). Among these, maximum 4 out of 7 patients (4.81%) were between 20 and 30 years, and regarding presence of risk factors, maximum 4 patients (4.81%) were chronic alcoholics. Mean serum holotranscobalamin level was 131.95 ± 80.96 pg/ml. 30 patients (36.14%) had low holotranscobalamin levels (< 54 pg/ml), comprising 18(21.66%) males and 12(14.48%) females. Among these, maximum 21 patients (25.3%) were in age group 20–30 years, and maximum 11 patients (13.25%) were on antiepileptic drugs. Distribution of patients with deficiency of two biomarkers with respect to age group and risk factors is depicted in Table 3.

Table 3.

Age and risk factor based distribution of patients with low serum vitamin B12 and serum holotranscobalamin level

Age (years)	Number of patients		Risk factors	Number of patients
	Low serum vitamin B12 (n = 7)	Low serum holoTC (n = 30)		Low serum vitamin B12 (n = 7)	Low serum holoTC (n = 30)
20–30	4 (4.81%)	21 (25.3%)	Anti-epileptic drugs	0	11 (13.25%)
31–40	2 (2.4%)	8 (9.63%)	Alcohol	4 (4.81%)	7 (8.43%)
41–51	1 (1.2%)	1 (1.2%)	Malabsorption	2 (2.4%)	7 (8.43%)
			Chronic Pancreatitis	1 (1.20%)	3 (3.61%)
			Ileal TB	0	2 (2.4%)

Percentage out of total study patients

7 patients with low serum vitamin B12 level were holotranscobalamin deficient also. This gave us 23 patients (27.71%), who had low serum holotranscobalamin levels only, despite having normal serum vitamin B12 levels, thus unmasking early vitamin B12 deficiency in 27.7% of study patients, as depicted in Fig. 1.

Fig. 1.

Prevalence of vitamin B12 deficiency

Biomarkers and Mean Corpuscular Volume

7 patients (8.43%) had MCV > 100 fl. Among these, 5 patients (6.02%) had low serum holotranscobalamin levels, including 3(3.61%) with low serum vitamin B12 level also. In 7 patients with macrocytosis, mean serum vitamin B12 level was 451 ± 298.18 pg/ml and mean serum holotranscobalamin level was 88.26 ± 60.13 pg/ml. In remaining 76 patients with normal MCV, mean serum vitamin B12 level was 539.53 ± 198.65 pg/ml and mean serum holotranscobalamin level was 116.13 ± 78.08 pg/ml. Students t-test was used to compare mean serum level of biomarkers, between these 2 groups of patients, as depicted in Table 4. P value was 0.1 and 0.15(> 0.05) for serum vitamin B12 and serum holotranscobalamin, respectively. Thus, mean serum level of both biomarkers was lower in patients with MCV > 100 fl, compared to that in remaining patients. But the difference in mean level of biomarkers was statistically insignificant between two patient groups.

Table 4.

Mean serum vitamin B12 and serum holotranscobalamin level in patients with MCV > 100 fl and patients with MCV < 100 fl

Mean serum level	Patients with MCV > 100 fl	Patients with MCV < 100 fl	P value
Mean serum vitamin B12 level	451 ± 298.18 pg/ml	539.53 ± 198.65 pg/ml	0.10
Mean serum holotranscobalamin level	88.26 ± 60.13 pg/ml	116.13 ± 78.08 pg/ml	0.15

Biomarkers and Peripheral Smear

8 patients (9.63%) had findings on peripheral smear suggestive of megaloblastic anemia. Among them 5 patients (6.02%) had low serum holotranscobalamin level, including 3(3.61%) with low serum vitamin B12 level also. In these 8 patients, mean serum vitamin B12 level was 386 ± 231 pg/ml and mean serum holotranscobalamin level was 58.43 ± 56.39 pg/ml. In remaining 75 patients, mean serum vitamin B12 level was 570.28 ± 205.56 pg/ml and mean serum holotranscobalamin level was 145.59 ± 78.5 pg/ml. Mean values are depicted in Table 5. Students t-test was used to compare mean level of two biomarkers between these two groups. P value was 0.039 and 0.041(< 0.05) for serum vitamin B12 and holotranscobalamin, respectively, thus suggesting that mean value of both biomarkers was significantly lower in patients with positive findings on peripheral smear.

Table 5.

Mean serum vitamin B12 and serum holotranscobalamin level in patients with peripheral smear findings suggestive of megaloblastic anemia and remaining other patients

Mean serum level	Patients with peripheral smear suggestive of megaloblastic anemia	Patients with peripheral smear not suggestive of megaloblastic anemia	P value
Mean serum vitamin B12 level	386 ± 231 pg/ml	570.28 ± 205.56 pg/ml	0.039
Mean serum holotranscobalamin level	58.43 ± 56.39 pg/ml	145.59 ± 78.5 pg/ml	0.041

Nerve Conduction Studies (NCS)

45 patients underwent NCS. None of the patient had clinical signs or symptoms of peripheral neuropathy. Variable conduction abnormalities were detected in 18 out of 45 patients (40%), with, some patients having combination of abnormalities. There was involvement of ulnar nerve in 10 patients (22.22%), median nerve in 4 patients (8.88%), isolated common peroneal nerve and tibial nerve in 1 patient (2.22%), each, ulnar and sural nerve both in 1 patient (2.22%), ulnar and median nerve both in 1 patient (2.22%). As per abnormalities detected, there was prolonged latency in 9 patients (20%), decreased sensory nerve action potential (SNAP) in 7 patients (15.55%), decreased conduction velocity in 4 patients (8.88%), decreased compound muscle action potential (CMAP) in 2 patients (4.44%) and f wave latency chronodispersion in 1 patient (2.22%). In terms of risk factors, 7 patients (15.55%) were on antiepileptic drugs, 5(11.11%) had malabsorption, 3(6.67%) were alcoholics, 2(4.44%) had chronic pancreatitis and 1(2.22%) had ileal TB.

Among 18 patients with subclinical peripheral neuropathy, 16 (88.89% of 18) had low serum holotranscobalamin levels, but only 4 (22.22% of 18) among these 16 had low serum vitamin B12 levels also. This gave us 12 patients (66.67% of 18) with neuropathy and isolated low holotranscobalamin levels, with normal serum vitamin B12 levels. In these 18 patients, mean serum vitamin B12 level was 490.72 ± 223.74 pg/ml and mean serum holotranscobalamin level was 55.68 ± 34.56 pg/ml. In remaining 27 patients with normal NCS, mean serum vitamin B12 level was 523.92 ± 214.65 pg/ml and mean serum holotranscobalamin level was 105.06 ± 77.48 pg/ml. Mean value of biomarkers in these two patient groups is tabulated in Table 6.

Table 6.

Mean serum vitamin B12 and serum holotranscobalamin level in patients with subclinical neuropathy and patients with normal conduction studies

Mean serum level	Patients with subclinical peripheral neuropathy	Patients with normal NCS	P value
Mean serum Vitamin B12 level	490.72 ± 223.74 pg/ml	523.92 ± 214.65 pg/ml	0.2
Mean serum holotranscobalamin level	55.68 ± 34.56 pg/ml	105.06 ± 77.48 pg/ml	0.031

Students t-test was used to compare mean serum level of both biomarkers between these two groups of patients. P value was 0.2 (> 0.05) and 0.031(< 0.05) for serum vitamin B12 and for holotranscobalamin, thus suggesting that, holotranscobalamin levels were significantly low in majority of patients with neuropathic changes, while no such significant difference was seen for serum vitamin B12 levels.

Discussion

The vitamin B12 status of people who are prone to develop vitamin B12 deficiency and megaloblastic anemia, is an important scientific issue. Also, very commonly studied hematological changes in vitamin B12 deficiency, like anemia or macroovalocytosis, usually appear late in disease process. So, it is extremely necessary to have diagnostic biomarkers for early vitamin B12 deficiency, to prevent irreversible neurological changes with simple vitamin supplementation. Over time, total vitamin B12, homocysteine, methylmalonic acid and holotranscobalamin have been most studied biomarkers. This study was conducted to compare holotranscobalamin with vitamin B12 level as a diagnostic marker to detect vitamin B12 adequacy in megaloblastic prone population and to study association between these biomarkers and nerve conduction abnormalities. In this study, age of patients ranges from 20–51 years with mean age 30.43 ± 8.33 years, and maximum number of patients (60%) in age group 20–30 years. Patients were categorised in 6 groups on the basis of risk factors, with maximum patients, 29(34.93%) were on anti-epileptic drugs, 26(31.32%) were alcoholics, 10(12.04%) patients each had malabsorption and ileal tuberculosis, 6(7.22%) had chronic pancreatitis and 2(2.4%) had prior ileal resection. Baseline biochemical investigations (liver function, kidney function, thyroid function, blood sugar) and folic acid level were normal in all patients.

Total Vitamin B12 and Holotranscobalamin

In current study, mean serum vitamin B12 level was 578.36 ± 214.67 pg/ml. Only 7/83 patients (8.4%) had low total vitamin B12 level. Out of 7, maximum patients (57%) were in 20–30 years of age. In terms of risk factors, maximum patients (57%) were alcoholic. Prevalence of low total vitamin B12 level in this study is comparable to study by Bailey et al. [20]. However, much higher prevalence, ranging from 30 to 50%, was reported in various other studies, conducted in different regions of India [2, 21–25].

Mean serum holotranscobalamin level was 131.95 ± 80.96 pg/ml. 30/83 patients (36.14%) were holotranscobalamin deficient. Among these 30, maximum (70%) were in age of 20–30 years. Regarding risk factors, maximum (36.7%) were on anti-epileptic drugs, followed by patients with malabsorption and chronic alcohol users. However, previous studies have reported higher prevalence of holotranscobalamin deficiency compared to current study [22, 24–26].

Holotranscobalamin deficient group comprised all 7 patients with low serum vitamin B12 level. Thus, 23 patients (27.71%) in the current study had isolated low serum holotranscobalamin levels, despite of normal serum vitamin B12 levels. Holotranscobalamin estimation helped to unmask vitamin B12 deficiency in these 27.71% patients, who would have been misdiagnosed, if only serum vitamin B12 estimation was used. Similar difference in estimation of vitamin B12 status was also reported in a study on Indian population by Refsum et al. [22]. Superiority of holotranscobalamin over total vitamin B12 in estimating vitamin B12 deficiency, seen in current study is in line with similar results, as reported in various studies and review articles [6, 25, 27–31]. Following explanations and findings have been presented over time to prove superiority of holotranscobalamin – it is carrier of metabolically active fraction of vitamin B12 available for tissues; it has a half-life of 6 min, so recent and small changes in the vitamin B12 pool are better reflected by changes in its serum levels [24]; serum level is minimally affected by age, sex and pregnancy [6]; no co-relation seen between the severity of anemia and serum vitamin B12 levels [2]; false high levels of serum vitamin B12 seen in alcoholic liver disease, occult malignancies and renal disease [32]. This study provides evidence for these explanations and support holotranscobalamin over total vitamin B12, as a better marker for detecting early vitamin B12 deficiency in patients prone to megaloblastic anemia.

8 patients (9.63%) had findings on peripheral smear suggestive of megaloblastic anemia. Serum level of both biomarkers were significantly lower in these patients (p value < 0.05), compared to remaining patients, with, 62% of these patients being actually holotranscobalamin deficient. But no such association was seen between serum level of biomarkers and MCV > 100 fl.

Subclinical Peripheral Neuropathy

45 patients underwent NCS, among which only 18(40%) had conduction abnormalities suggesting subclinical peripheral neuropathy without any overt neurological signs and symptoms. Conduction abnormalities detected in study patients were very subtle, due to which, neuropathy could not be labelled as typical axonal or demyelinating type. However, as per literature, combined axonopathy and demyelination is common, with axon damage in excess of myelin damage. Sensory fibres and large fibres are more affected by axonopathy [33]. Ulnar nerve was most commonly affected in 12/18 (66%) patients. There is paucity of data on subclinical neuropathy in vitamin B12 deficiency. Still, it was reported that 25% patients with diagnosed megaloblastic anemia may have subclinical neuropathy and a prevalence of 5% was reported by Kaur et al. in patients of megaloblastic anemia [2]. For clinical neuropathy, variable prevalence ranging from 25 to 63% have been reported in symptomatic vitamin B12 deficient patients [12, 33–35].

12 out of 18 patients (66.67%) had low serum holotranscobalamin level only, despite of normal serum vitamin B12 level and only 4 out of 18 (22.22%) had deficiency of both biomarkers. 18 Patients with subclinical neuropathy had statistically significant lower holotranscobalamin level, compared to remaining patients, with majority (89%) of these patients being actually holotranscobalamin deficient. Total vitamin B12 levels were not significantly different in these two groups. Thus, suggesting low serum levels of holotranscobalamin were significantly associated with subclinical neuropathic changes, while low serum levels of vitamin B12 were not. It has been previously reported also, that neurological features in vitamin B12 deficiency have poor association with hemoglobin or vitamin B12 level [2]. Further, 20% patients with neurological symptoms, actually, don`t even have anemia [12]. This study shows that holotranscobalamin is a better marker to predict early neurological involvement in vitamin B12 deficiency.

Conclusion

Role of vitamins in maintenance of good health has received remarkable attention in recent times. Vitamin B12 deficiency is now not just a laboratory finding but a very well-known clinically relevant issue which needs to be properly investigated. Vitamin B12 deficiency is very common entity, has wide variety of hematological and neurological features, neuropsychiatric symptoms appearing in early stage without anemia, total vitamin B12 level being an unreliable marker and most importantly it is easily preventable with oral supplements. All these reasons, make it necessary to diagnose deficiency as earlier as possible, to prevent irreversible neurological changes. With this study, we can conclude that holotranscobalamin is a better and reliable marker to detect vitamin B12 deficiency, as well as, to predict peripheral neuropathy associated with deficiency in early stage of disease.

Limitations

1. Small sample size.

2. Lack of control group.

3. Nerve conduction study could not be done for all patients.

Author contribution

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by AV, SA, SK. The first draft of the manuscript was written by AV and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

No funding was received for conducting this study.

Declarations

Conflict of interest

The authors have no conflicts of interests to declare that are relevant to the content of this article.

Ethical approval

Approval was obtained from the ethics committee of Maulana Azad Medical College. The procedures used in this study adhere to the tenets of the Declaration of Helsinki.

Consent to participate

Informed consent was obtained from all individual participants included in the study.