INTRODUCTION

Vitamin B12 (cobalamin) is an important metalloenzyme which consists of a porphyrin-like macromolecule with a central cobalt atom that is bound to a nucleotide residue, the whole unit is called cobalamin. It is required as coenzymes for the metabolism of the amino acids methionine, threonine and valine, as well as for the transformation of methyl-tetrahydrofolate to tetrahydrofolate which is necessary for DNA synthesis.¹

Cobalamin is bound to two proteins, transcobalamin (TC) and haptocorrin (HC). The TC-vitamin B12 complex is called holotranscobalamin (holoTC) containing the biologically active cobalamin, which promotes the uptake of its cobalamin by all cells, via specific receptors. The much larger fraction (about 80%) of cobalamin carried by HC is considered to be metabolically inert because no cellular receptors exist, except on the liver.^2,3 Genetic absence of HC, although rare, is not a serious condition. On the other hand, genetic absence or abnormalities of TC are manifested as typical haematological, neurological and metabolic manifestations of cobalamin deficiency requiring treatment even though serum analysis may show normal cobalamin concentrations.⁴ The shorter half life for holoTC compared to holoHC makes a decrease in holoTC as one of the earliest markers of cobalamin deficiency.⁵

Measurement of total vitamin B12 suffers from some limitations, in particular, most of the cobalamin measured is that bound to HC.^6,7 A number of studies have been published to support that holoTC would be a better indicator of vitamin B12 status than total vitamin B12.^8,9 New methods based on specific anti-TC antibodies are available and confirm the usefulness of holoTC measurement for diagnosing vitamin B12 deficiency.^10,11 As expected, holoTC levels are low in patients with biochemical signs of vitamin B12 deficiency.¹²

Methods

Serum samples used in this preliminary study were randomly selected from 76 patients who were admitted or followed up at Royal Hospital, Muscat, Oman, and who had been referred to the Clinical Biochemistry Laboratory, for the assessment of vitamin B12 status, during September 2007. These patients included 48 females and 28 males with an age range of 12-69 years. A variety of clinical problems/diseases were present or investigated in these patients including; anaemia, gastrointestinal diseases, autoimmune, endocrine disorders, malignancy and chronic renal failure. Two of the female subjects were pregnant. For each patient, serum total vitamin B12 level was determined by chemiluminescent microparticle immunoassay on Architect 2000 analyzer (Abbott, USA) and holoTC (active vitamin B12) level was determined by microparticle enzyme immunoassay on Axsym analyzer (Abbott, USA).

Comparison of the data was performed to test the statistical relationship as reflected by the mean, median, standard deviation and correlation coefficient.¹⁷ Also, the data was evaluated concerning any misclassification of the results. This was performed by comparing holoTC versus total vitamin B12 values, particularly in terms of whether either or both values agree or disagree for classifying the patients as being normal (results within the reference range) or abnormal (results lower or higher than the reference range). The cut-off thresholds for the data were assessed using the kit’s quoted reference range for holoTC and for total vitamin B12. The quoted between-batch coefficient of variation

RESULTS

A comparison of holoTC and total vitamin B12 are presented in Table 1. The correlation between the two parameters was examined using linear regression analysis. A significant (P< 0.001) correlation was noted with r= 0.765 and regression equation of: y = a + bx (ie: holoTC= 1.5 + 0.14 total vitamin B12). (Fig. 1)

However, in 7 (9.2%) samples, there was a disagreement/ misclassification of results. In 1 (1.4%) case, holoTC was within the reference range but total vitamin B12 was high. The patient was a nine year old boy, with acute lymphoblastic leukaemia. The laboratory findings for the patient were; serum holoTC 74.1 pmol/L (RR 9-123), total vitamin B12 685 pmol/l (RR 140-600), folate 19.0 nmol/L (RR 4.8-30.5); and Hb 8.6 g/dL, RBC 3.9 X 1012, MCV 77 fL, MCH 25 pg, MCHC 32 g/dL, RDW 21.5 %, WBC 7.4 X 199/L, neutrophil 2.7 X 199/L, platelets 365 X 199/L.

In 6 other (7.8.%) patients, holoTC was within reference range, however, total vitamin B12 was low. Their laboratory findings were as follows:

1. Male, 14 years (with neutropenia). Serum holoTC 14.2 pmol/L, total vitamin B12 138 pmol/l, folate 24.9 nmol/L, ferritin 36 ug/L (RR 20-280); and Hb 11.7 g/dL, RBC 4.3 X 1012, MCV 84 fL, MCH 27 pg, MCHC 32 g/dL, RDW 13.2 %, neutrophils 0.8 X 109/L, platelets 434 X 199/L, blood film: moderate neutropenia.
2. Male, 36 years (with gait problem). Serum holoTC 21.1 pmol/L,total vitamiMale, 43 years (with thrombocytopenia). Serum holoTC 13 pmol/L, total vitamin B12 137 pmol/l, folate 16.2 nmol/L, ferritin 40 ug/L; and Hb 9.4 g/dL, MCV 82 fL, MCH 28 pg, MCHC 34 g/dL, RDW 14.9 %, RBC 3.4 X 1012, WBC 29.6 X 199/L, neutrophil 25.8 X 199/L, platelets 3 X 199/L, blood film: normochromic normocytic, severe thrombocytopenia, marked neutrophilia with mild hypersegmentation
3. Male, 48 years (with periodic leg movement). Serum holoTC 19.6 pmol/L, total vitamin B12 135 pmol/l, folate 26.4 nmol/L, ferritin 45 ug/L; and Hb 12.9 g%, RBC 5.5 X 1012,MCV 68 fL, MCH 22 pg, MCHC 32 g/dL, RDW 12.9 %, WBC 6.6 X 199/L, neutrophil 4.3 X 199/L, platelets 247 X 199/L

DISCUSSION

In this preliminary study, comparable results with significant correlation (r= 0.765, P< 0.001) were noted between holoTC and total vitamin B12. Using the cut-off thresholds or kits’ quoted reference range for holoTC of 9-123 pmol/L and for total vitamin B12 of 140-600 pmol/L, a parallel agreement/classification of results was noted in the majority of patients. This was evident in 90.8% of patients, who had agreement in the classification of results, both being normal or abnormal (low or high). Generally, in 80.4% of cases, both results were within the reference ranges. However, in 5.2% cases, both results were higher than the reference ranges, and also in 5.2% of cases, both results were lower than the However, in 7 (9.2%) patients (6 males and 1 female), there was disagreement or misclassification of results. Although more females (48) than males (28) were included in this comparison study, the misclassification was observed mostly among males.

Blood samples were randomly collected, and male patients who were included in the study may be more severely affected than females, otherwise there is no other explanation for this sex predominance. Also, no medication history was available for the patients. Of these seven patients, only one patient (1.4%) had holoTC of 74.1 pmol/L (within the reference range) but with high total vitamin B12 (685 pmol/l) level. The patient was a nine year old boy, with acute lymphoblastic leukaemia. The possible explanation for the raised total vitamin B12 is that, in patients with myeloproliferative disorders or liver diseases, the haptocorrin level may be high leading to high total vitamin B12, as most of the cobalamin measured is that bound to HC.^6,7,18

The other six patients (5 males, 1 female) (8.4%) had normal levels of holoTC but low total vitamin B12. Following the interpretation of the data, the range of holoTC was 13-25 pmol/L while the range for total vitamin B12 was 115-137 pmol/L. holoTC levels were in the lower reference range (cut-off 9 pmol/L) while total vitamin B12 levels were moderately decreased (cut-off 160 pmol/L). There seemed to be no clear evidence of vitamin B12 deficiency (particularly from blood film examination and lack of haematological indices of hyperchromasia, macrocytosis, neurophil hypersegmentation, thrombocytopenia), however, three patients had iron deficiency anaemia. HoloTC may appear to be a better indicator of total vitamin B12 status in these few patients, although no definite conclusion can be drawn. No gold method for comparison was used in this study, as other indices such as methylmalonic acid (MMA) and homocysteine, which are not offered on a wide scale as core investigations for vitamin B12 deficiency, were not measured in this comparitive study.^2,19

In this study, results with normal total vitamin B12 but low holoTC were not observed. This may be due to the random selection of the samples in this preliminary study, however, it would have been an area of interest for investigation.

More importantly and taking into consideration the agreement of the results in the majority of patients (90.8%) in this study, it can be recommended that both holoTC and total vitamin B12 have almost equal diagnostic efficiency in screening/diagnosing vitamin B12 deficiency. In a few patients and under certain circumstances, holoTC alone or in combination with total vitamin B12 may be required. Miller et al. in a comparison screening study using total vitamin B12 (at a cut-off 148 pmol/L), holoTC (at cut-off 25 pmol/L), MMA and homocysteine assays, their refults supported the diagnostic accuracy of both forms of vitamin B12, alone and in combination for screening of metabolic vitamin B12 deficiency.¹⁴

They suggested that holoTC is a more sensitive marker for vitamin B12 deficiency favoring its use as a first line parameter in detecting cobalamin deficiency.^20,21 Analytical studies of the different techniques that include microbiological, enzyme-labelledAgreement between serum total vitamin B12 and holoTC results were noted in the majority of patients, where a highly significant correlation was reported between the two indices. There was a disagreement in a minority of patients who had low total vitamin B12 but low-normal holoTC levels in the absence of a clear evidence of any deficiency. It can be recommended that holoTC and total vitamin B12, alone and in combination, have almost equal diagnostic efficiency in diagnosing vitamin B12 deficiency for the majority of patients. In a small number of patients, holoTC appeared to be a better reflector of vitamin B12 status. Further large-scale comparison studies based on a gold standard method for classifying vitamin B12 status are worth considering and warranted to clarify the effectiveness of holoTC in the clinical setting.