Determination of vitamin B12 using the enzyme glycerol dehydrase

Glycerol dehydrase is an enzyme that catalyzes dehydration of glycerol into β‐propionaldehyde. It requires 5′‐deoxyadenosylcobalamin, one of the forms of vitamin B₁₂, as a coenzyme. The enzyme is inactivated in vitro by all forms of vitamin B₁₂ stoichiometrically. The objective of this study was to determine vitamin B₁₂ content by utilizing the inactivation of the enzyme by vitamin B₁₂. After various examinations, an excellent standard curve was obtained up to 1?pmol vitamin B₁₂ using 14?mU of the enzyme per tube. Glycerol dehydrase does not respond to vitamin B₁₂ if it is bound to haptocorrin, a vitamin B₁₂‐binding protein. This necessitates a procedure for extraction of vitamin B₁₂ from samples before assay. The enzyme was less inactivated by 5′‐deoxyadenosylcobalamin than any other form of vitamin B₁₂. However, this did not matter because all forms of vitamin B₁₂ were converted into cyanocobalamin during the extraction procedure cited above, which was performed in a buffer containing potassium cyanide.     

Methoxistasis: Integrating the Roles of Homocysteine and Folic Acid in Cardiovascular Pathobiology

Over the last four decades, abnormalities in the methionine-homocysteine cycle and associated folate metabolism have garnered great interest due to the reported link between hyperhomocysteinemia and human pathology, especially atherothrombotic cardiovascular disease. However, clinical trials of B-vitamin supplementation including high doses of folic acid have not demonstrated any benefit in preventing or treating cardiovascular disease. In addition to the fact that these clinical trials may have been shorter in duration than appropriate for modulating chronic disease states, it is likely that reduction of the blood homocysteine level may be an oversimplified approach to a complex biologic perturbation. The methionine-homocysteine cycle and folate metabolism regulate redox and methylation reactions and are, in turn, regulated by redox and methylation status. Under normal conditions, a normal redox-methylation balance, or “methoxistasis”, exists, coordinated by the methionine-homocysteine cycle. An abnormal homocysteine level seen in pathologic states may reflect a disturbance of methoxistasis. We propose that future research should be targeted at estimating the deviation from methoxistasis and how best to restore it. This approach could lead to significant advances in preventing and treating cardiovascular diseases, including heart failure.     

Serum vitamin B12 and homocysteine levels in pregnant women with neural tube defect

Objective.?The aim of the study is to investigate the importance of serum vitamin B12 levels in pregnant women with foetal neural tube defect (NTD).

Study design.?This study consists of 31 pregnant women having fetuses with NTD. The pregnant women in the study group were selected among cases with normal folate levels. Serum vitamin B12 levels were investigated. Additionally, complete blood count, serum iron level, iron binding capacity, ferritin, folate, free T₃, free T₄, thyroid stimulating hormone and plasma homocysteine levels were measured. Control group consisted of 32 pregnant women who did not have a history of NTD in previous pregnancies and did not have fetuses with NTD in present pregnancy. NTD was diagnosed between14th and 20th gestational age. The mean gestational age of members of control group was the same as those of NTD group.

Results.?There was no statistically significant difference between pregnants with NTD and control group according to number of cases with vitamin B12 deficiency.

Conclusion.?It seems that vitamin B12 deficiency does not play a causative role in the development of foetal NTD. Monitoring maternal homocystein levels might be important in understanding the aetiologies of foetal NTD.     

在线微生物浊度法测定多维元素片中微量维生素B_(12)的含量

目的:建立在线微生物浊度法测定多维元素片中微量维生素B12的含量。方法:以莱士曼氏乳酸杆菌为实验菌,以缺乏维生素B12的培养基为检测培养基,采用浊度法在线检测。结果:维生素B12浓度在0.02～0.10 ng.mL-1范围内呈现良好线性关系(r=0.9985),平均回收率为101.8%。结论:该方法快速、简便、灵敏,在线检测结果准确可靠,适用于多维元素片中微量维生素B12的含量测定。     

Cobalamin disorder Cbl‐C presenting with late‐onset thrombotic microangiopathy

Two siblings, a boy age 12 and his sister age 4 years, presented with proteinuria and hematuria, hypertension, and chronic hemolytic anemia. At age 13 years, the boy developed an episode of severe hypertensive encephalopathy and transient renal failure. Both children are attending normal school, have no neurologic symptoms, and only minimal pigmentary retinal abnormalities. Renal biopsy showed a chronic thrombotic microangiopathic nephropathy. Both patients had hyperhomocysteinemia and mild methylmalonic aciduria. Fibroblasts showed decreased cobalamin uptake, reduced methyl‐ and adenosyl‐cobalamin formation, and deficient incorporation of formate and propionate, compatible with the Cbl‐C complementation group, but milder than that found in cells from most patients. Both patients and their father carry a balanced reciprocal translocation. Parenteral hydroxycobalamin treatment reduced the homocysteine levels, and methylmalonic acid disappeared. Increasing the dosage of hydroxycobalamin from 1 to 2.5, then 5 mg daily together with betaine, further reduced homocysteine levels (boy from 118 to 23 μM and girl from 59 to 14 μM). With this treatment, hemolysis has stopped, hematuria has disappeared, proteinuria has almost normalized, and creatinine clearance has been stable. Investigations for chronic thrombotic microangiopathy should include testing for this unusual but treatable disorder, regardless of age of presentation. © 2002 Wiley‐Liss, Inc.     

Subtle and atypical cobalamin deficiency states

Evidence for cobalamin (vitamin B₁₂) deficiency usually involves some combination of low serum cobalamin levels, clinical abnormalities (classically, megaloblastic anemia and neurologic defects), metabolic abnormalities, and response to therapy. However, cobalamin deficiency may often display few of the expected clinical findings. Identification of the underlying cause is also important in the diagnosis of deficiency, and its value may be particularly great when the expression of deficiency is subtle. The cause of cobalamin deficiency is usually malabsorptive, but may sometimes be limited to malabsorption of food cobalamin while free cobalamin is absorbed normally. Nongastroenterologic entities may sometimes also be found. All of these considerations allow the proposal of four patterns of cobalamin deficiency. The first type is classical deficiency; typical megaloblastic anemia with or without neurologic dysfunction occurs because of classical cobalamin malabsorption such as lack of intrinsic factor (pernicious anemia). The second type consists of classical cobalamin malabsorption in which the cobalamin deficiency is expressed subtly rather than in classical fashion. There is no megaloblastic anemia and sometimes the only evidence of deficiency may be metabolic. In the third type, cobalamin deficiency is expressed classically but is attributable to a subtle or atypical cause, such as food-cobalamin malabsorption. In the fourth type, deficiency is both expressed subtly and arises from subtle or atypical causes. Such presentations require further investigation but are a challenging expansion of our understanding and recognition of cobalamin deficiency.     

Low serum cobalamin levels occur frequently in the acquired immune deficiency syndrome and related disorders

Low serum cobalamin levels in 10 patients with AIDS or AIDS-related complex led us to also prospectively survey 40 homosexual men in our AIDS clinic. 8 of the latter (20%) had low cobalamin values. We found no evidence of megaloblastic changes in the blood or bone marrow. Assessment disclosed malabsorption of cobalamin in only 1 of 6 cases tested for it. 6 of the patients were treated with cobalamin and had no hematologic response. It appears that low serum cobalamin levels in AIDS and related disorders do not usually represent overt cobalamin deficiency. While malabsorption is occasionally responsible for the low cobalamin level, in most cases the cause is unknown and may reflect a serum abnormality similar to that in multiple myeloma. AIDS and related disorders should be considered in the differential diagnosis of unexplained low cobalamin levels.     

Cobalamin forms and analogues in plasma and myeloid cells during chronic myelogenous leukaemia related to clinical condition

Plasma and buffy coat specimens of CML patients with untreated disease, in chronic and accelerated phase, and in overt blastic crises were analysed for the cobalamin patterns using non-polar extraction, thin-layer chromato-graphy and bioautography. Splenic tissue specimens from four splenectomized patients in accelerated phase were analysed similarly. Cellular extracts were separated into two compartments by adsorption to haptocorrin antibodies. Plasma concentrations of all cobalamin forms were increased in CML. The proportion of methylcobalamin was significantly lower than in a reference population, and a low plasma proportion of methylcobalamin was associated with a poor prognosis. Buffy coat cells and splenic tissue had a higher proportion of 5'-deoxyadenosylcobalamin and a lower proportion of methylcobalamin than plasma; still, there was relatively more methylcobalamin than in normal tissue. The haptocorrin-bound compartment differed from the non-haptocorrin compartment in untreated or chronic phase patients by binding less methylcobalamin. An estimate of cobalamin analogues in plasma was achieved by comparing two different isotope dilution assays employing a cobalamin-specific binder, intrinsic factor, and a nonspecific binder, hog non-intrinsic factor. Values for total cobalamin and analogues were increased to the same degree in CML plasma.     

Erythropoietin levels in cobalamin deficiency: comparison of anemic and non-anemic, subtly deficient patients.

The major stimulus for erythropoietin secretion is the circulating hemoglobin level, but other poorly understood factors appear to influence the erythropoietin level as well. Therefore, the effect of cobalamin deficiency was studied in patients who were not anemic, even though many of them had macrocytosis or metabolic deficiency of the bone marrow cells. All 15 cobalamin-deficient patients without anemia had normal erythropoietin levels, including the 4 patients with macrocytosis and 3 in whom metabolic cobalamin deficiency of the marrow cells was documented with the deoxyuridine suppression test. Moreover, among 21 cobalamin-deficient patients with anemia, the 4 least anemic patients also had normal erythropoietin levels. The other 17 anemic patients had elevated erythropoietin levels. Erythropoietin levels correlated with the severity of the anemia (r = 0.423, p less than 0.05). However, wide individual variations were observed; 4 patients with hemoglobin levels of 37-43 g/l had erythropoietin levels ranging from 69 to 3300 units/l, for example. These observations support the hypothesis that the hemoglobin level is the major, but not the sole factor that determines erythropoietin levels. As long as it does not produce anemia, however, cobalamin deficiency does not raise erythropoietin levels even when it induces metabolic deficiency of the bone marrow and macrocytosis.