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1 Department of Clinical Biochemistry, AKH, and
2
Department of Clinical Biochemistry, AAS, Aarhus University Hospital, DK 8000 Aarhus C, Denmark.
3 Institute of Medicine and
4
Department of Pharmacology, University of Bergen, N-5021 Bergen, Norway.
5 The Protein Chemistry Laboratory, Aarhus University, DK-5000 Aarhus C, Denmark.
6 Locus for Homocysteine and Related Vitamins, University of Bergen, N-5021 Bergen, Norway.
aAddress correspondence to this author at: Department of Clinical Biochemistry, AKH, Aarhus University Hospital, Norrebrogade 44, DK 8000 Aarhus C, Denmark. Fax 45-8949-3060; e-mail E.Nexo{at}dadlnet.dk.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Patients (n = 88; age range, 38–80 years) undergoing coronary angiography (part of the homocysteine-lowering Western Norway B-Vitamin Intervention Trial) were allocated to daily oral treatment with (a) vitamin B12 (0.4 mg), folic acid (0.8 mg), and vitamin B6 (40 mg); (b) vitamin B12 and folic acid; (c) vitamin B6; or (d) placebo. EDTA blood was obtained before treatment and 3, 14, 28, and 84 days thereafter.
Results: The intraindividual variation for patients not treated with B12 was 
10% for plasma total cobalamin, total TC, apo-TC, and apo-HC, and <20% for holo-TC and TC saturation. In B12-treated patients, the maximum change in concentrations was observed already after 3 days for total TC (-16%), holo-TC (+54%), and TC saturation (+82%). At this time holo-HC (+20%) and plasma total cobalamin (+28%) showed an initial burst, but had increased further at 84 days. All changes were highly significant compared with the control group (P <0.0001).
Conclusions: Oral vitamin B12 treatment produces maximal effects on total TC, holo-TC, and TC saturation within 3 days, whereas maximal increases in holo-HC and plasma total cobalamin occur later. The results support the view that holo-TC is an early marker of changes in cobalamin homeostasis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TC is essential for the transport of cobalamin from the intestine and into most cells of the body; patients lacking this protein eventually develop cobalamin deficiency (2). TC carries one-third of the circulating cobalamin (holo-TC), but the major portion of this protein is present in its unsaturated form (apo-TC) (1)(3). Recently, improved methods for the measurement of holo-TC have been developed (4)(5). Whether measurement of holo-TC can help in the elucidation of cobalamin metabolism is a current topic of discussion (6).
HC carries most of the circulating cobalamin (holo-HC), and typically, only a minor fraction of this protein is present in its unsaturated state (apo-HC) (1)(3). The function of HC is still being debated (7).
In the present work we studied the effect of oral vitamin B12 treatment on plasma total cobalamin and its binding proteins in patients not suffering from vitamin B12 deficiency.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recruited patients were randomized into four groups in a 2 x 2 factorial block design to daily oral treatment with vitamin B12 (0.4 mg), folic acid (0.8 mg), and vitamin B6 (40 mg; group A); vitamin B12 (0.4 mg) and folic acid (0.8 mg; group B); vitamin B6 (40 mg; group C); or placebo (group D). For the first 2 weeks the folic acid groups (A and B) received an additional capsules with a loading dose of folic acid (5 mg/day); the other two groups (C and D) received additional placebo capsules. Packages of study capsules were prepared and given serial numbers in random order by Alpharma ApS (Copenhagen, Denmark).
Patients were grouped according to treatment with vitamin B12 (A and B; treatment group) or without vitamin B12 (C and D; control group). Two patients in the control group were excluded because of regular treatment with B12 injections or multivitamins containing vitamin B12.
EDTA blood was collected before and 3, 14, 28, and 84 days after the start of vitamin treatment. Eleven patients (6 treated and 5 controls) had only four, 1 control had only three, and 1 control had only two samples collected. The blood samples were immediately placed on ice and centrifuged within 30 min; plasma was stored at -80 °C until additional processing.
biochemical analyses
Plasma total cobalamin was determined by a commercial method (Bayer Corporation) on a Centaur instrument (analytical imprecision <10%).
Plasma total TC and plasma holo-TC were measured by ELISA as described recently (analytical imprecision, 5% for total TC and 8% for holo-TC) (4)(8).
Plasma apo-HC and apo-TC were measured after separation of the cobalamin-saturated proteins with silica gel (analytical imprecision <10%) (9).
statistical analysis
Biochemical markers for the cobalamin-treated group (A plus B) and the control group (C and D) were compared by use of an independent-samples t-test. We also used independent-sample t-tests to compare changes in the biochemical markers between the treatment group and control group. The Levene test was used to test for equal variances. Linear regression analysis was used for analyzing the association between the biochemical markers and age. The intraindividual variation was estimated from the SD of the biochemical markers. The analyses were carried out according to the intention-to-treat principle. Data were log-transformed as appropriate to assure gaussian distribution. P values <5% were regarded as statistically significant. Data were analyzed using SPSS 10.0 (SPSS Inc.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After intervention, no differences were observed regarding changes in total cobalamin and cobalamin-binding proteins between the two groups not receiving vitamin B12 (C and D) or between the two groups receiving vitamin B12 (A and B; data not shown). Thus, intervention with vitamin B6 had no influence on the markers studied.
The intraindividual variation was calculated from the serial measurement of the analytes in the control group (C and D). The intraindividual variation was 10% for plasma total cobalamin and its binding proteins except for plasma holo-TC and TC saturation, which showed somewhat higher values (Table 1
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The changes in plasma total cobalamin and cobalamin-binding proteins were followed at timed intervals after treatment with oral vitamin B12 or placebo (Fig. 1
). No major changes in these variables were observed for the control group, whereas the treatment group showed highly significant changes in all variables when compared with the baseline values and with values obtained for the control group (P <0.0001 for all days studied). The most interesting patterns were observed for holo-TC, TC saturation, and total TC. After 3 days, both holo-TC and TC saturation reached their highest values (means, +54% and +82%, respectively), whereas total TC reached its lowest value (mean, -16%). At this point the TC saturation showed a broad range of values, reflecting a combination of opposite changes in holo-TC and total TC, but after 84 days the interindividual variation was low and only one participant had plasma TC saturation <0.1.
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The concentrations of holo-HC and plasma total cobalamin increased to means of +20% and +28%, respectively, after 3 days, but continued to increase and reached mean values of +44% and +45%, respectively, after 84 days. At this point, no participant had a plasma holo-HC <175 pmol/L or a plasma total cobalamin <325 pmol/L.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The dose of vitamin B12 used in the present study is more than 100 times the daily requirement, but it is low compared with the dose recommended for oral treatment of patients suffering from vitamin B12 deficiency attributable to a lack of intrinsic factor (10).
Absorption of an increased amount of cobalamin was evident after 3 days of treatment. The most remarkable changes were observed for the TC-associated markers. Holo-TC increased and total TC decreased, whereas their ratio (TC saturation) increased from a median of 0.14 to almost twice this value. The variations in TC saturation were considerable, but TC saturation did not exceed 0.6 in any of the patients, indicating that the carrier capacity of TC was in excess of the amount of cobalamin absorbed. This is in contrast to patients receiving parental vitamin B12. In these patients [e.g., as part of the Schilling test (11)], TC is completely saturated with cobalamin and the excess free vitamin is excreted into the urine.
The reason for the significant decrease in total TC that we observed is unknown. One possibility is that the plasma clearance of holo-TC is faster than the clearance of apo-TC. Another possibility is down-regulation of TC synthesis by excess cobalamin.
The pattern observed for plasma total cobalamin and especially holo-HC was different from the changes in the TC-derived markers. Holo-HC showed an increase after 3 days and continued to increase moderately thereafter throughout the 84 days of the study. The corresponding reduction in apo-HC was considerably smaller than the increment observed for holo-HC, suggesting a net increase in the amount of circulating HC. We did not analyze these changes in detail because it would involve a very indirect calculation of total HC as plasma total cobalamin minus holo-TC plus apo-HC.
In summary, our study suggests that holo-TC and TC saturation reflect sudden changes in vitamin B12 homeostasis, whereas holo-HC and total plasma cobalamin seem to reflect the accumulation of vitamin B12.
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Acknowledgments |
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Footnotes |
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References |
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The following articles in journals at HighWire Press have cited this article:
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) before intervention and 3, 14, 28, and 84 days thereafter.