Pigmentation in Megaloblastic Anaemia Associated with Pregnancy and Lactation

Generalized skin pigmentation in five African women with megaloblastic anaemia in the postnatal period was associated with low serum folate levels, as distinct from vitamin B₁₂ deficiency. It is suggested that the occurrence of pigmentation in both folate and vitamin B₁₂ deficiency may reflect a common abnormality of metabolism.     

Serum Folate and Vitamine B12 Levels in Acute and Chronic Renal Disease. Effect of Peritoneal Dialysis

Serum folate and vitamin B₁₂ levels have been measured in 32 patients with renal failure. The initial mean serum folate level was raised above normal in seven patients with acute renal failure whereas the mean level in eight patients severely ill from chronic renal failure was significantly lower than normal. Serum folate levels fell during peritoneal dialysis and rose between dialyses in all these patients and also in one patient who was dialysed for acute pancreatitis.The mean serum B₁₂ level was raised in patients with both acute and chronic renal failure, but there was no consistent change in serum B₁₂ level during dialysis.Hypersegmented polymorphs were present in the peripheral blood film of most of the patients with acute or chronic renal failure. Their presence bore no relation to the clinical state, blood urea, serum folate, or serum B₁₂ level of the patients.     

Vitamin B12 Status in Pregnancy among Immigrants to Britain

Haemoglobin, serum vitamin B₁₂, and serum and red cell folate levels have been measured in 322 pregnant immigrant women in London at their first booking and in a proportion at 34 weeks of gestation and postnatally. The Indian, East-African Indian, and Pakistani and Bangladeshi patients showed significantly lower initial mean serum vitamin B₁₂ levels than the European group, the levels being lower in Hindu and Sikh patients than in Moslems. The patients of West Indian, Indian, and East-African Indian origin showed significantly lower initial mean haemoglobin levels than the immigrants from European countries. Though there was no overall correlation between haemoglobin and serum vitamin B₁₂ level the incidence of hypersegmented polymorphs and macrocytosis in the peripheral blood was highest in the Indian and East-African Indian patients, and both these features were particularly frequent in patients with subnormal serum vitamin B₁₂ levels. Only one patient, however, had overt megaloblastic anaemia due to vitamin B₁₂ deficiency. The Indian patients whose red cell folate levels were less than 200 ng/ml also had a lower mean serum vitamin B₁₂ level than those with red cell folate levels greater than 200 ng/ml. The Indian patients had smaller babies than the Europeans but this was not related to the differences in vitamin B₁₂ status between the two groups. However, out of 39 babies of the Indian group 5 (13%) showed subnormal serum vitamin B₁₂ levels in the first 10 days of life, the lowest level being 120 pg/ml.Though there was an overall statistically significant fall in serum vitamin B₁₂ between first booking and 34 weeks of pregnancy there was no significant fall in serum vitamin B₁₂ in those who initially had subnormal levels. Thus many Indian women are vitamin B₁₂ deficient in pregnancy, and this is associated with morphological blood abnormalities in many cases, but megaloblastic anaemia due to this deficiency is relatively infrequent.     

Impact of Helicobacter pylori on the development of vitamin B12 deficiency in the absence of gastric atrophy

Background. Cobalamin (vitamin B₁₂) deficiency is associated with Helicobacter pylori infection. This study examined how serum vitamin B₁₂ levels relate to gastric mucosa H. pylori density and histology, and to hematological findings in patients with minimal or no gastric atrophy. A second aim was to confirm that H. pylori eradication therapy increases serum B₁₂. Materials and Methods. Biopsies of the gastric mucosa from a population of dyspeptic patients were graded for level of chronic inflammation, neutrophil activity, atrophy, and H. pylori density. A total of 145 H. pylori‐infected patients with minimal or no atrophy were included in the study. Serum cobalamin level, hemoglobin level, and mean corpuscular volume were measured in the 145 patients before eradication therapy, and in 65 of the subjects after treatment. The hematologic findings before and after eradication therapy and correlations between serum vitamin B₁₂ level and histologic parameters, hematologic findings, and patient age were statistically analyzed. Results. There was no significant correlation between serum cobalamin level and patient age. Before treatment all the histopathological scores were inversely correlated with serum vitamin B₁₂ level (p < .01) on univariate analysis. Only H. pylori density was significantly associated with B₁₂ level on multivariate analysis. Serum hemoglobin and cobalamin levels were significantly increased after treatment, regardless of H. pylori eradication status (p < .001). Conclusion. The findings provide strong evidence that H. pylori infection is associated with cobalamin deficiency, and show that this is true even in patients with nonulcer dyspepsia and minimal or no gastric atrophy.     

Mouse transcobalamin II metabolism: The effects of antibiotics on the clearance of vitamin B12 from the serum transcobalamin II-vitamin B12 complex and the reappearance of the free serum transcobalamin II in the mouse

The mechanism of the clearance of vitamin B₁₂ from the serum transcobalamin II-vitamin B₁₂ (Tc-II-B₁₂) complex and the reappearance of free Tc-II in mouse have been studied. When a saturating dose of vitamin B₁₂ is given parenterally to normal mice, a portion of the Tc-II-bound vitamin B₁₂ is rapidly cleared and free Tc-II promptly reappears until it reaches a constant level in 6–8 h. The remaining vitamin B₁₂ is cleared slowly from the rest of the Tc-II-B₁₂ complex. In cycloheximide or puromycin-treated mice, free Tc-II fails to reappear and the bound Tc-IIdecreases. Treatment with actinomycin D has no effect on the reappearance of free Tc-II. The probable mechanism of this inhibition is discussed. The results suggest that mouse serum Tc-II has a stable messenger RNA template and a fast turnover. The free Tc-II which reappears in the serum after Tc-II has been saturated with vitamin B₁₂, appears to be newly synthesized.     

Assessment of Deoxyuridine Suppression Test in Diagnosis of Vitamin B12 or Folate Deficiency

Deoxyuridine (dU) suppression tests have been performed on virtually all marrow samples aspirated at this hospital over the past 12 months. Of the 110 samples studied 26 gave abnormal results, and these 26 samples came from patients deficient in either vitamin B₁₂ or folate. The dU suppression test was found to be of particular value in the diagnosis of vitamin B₁₂ or folate deficiency in non-anaemic patients with macrocytosis and equivocal changes in marrow morphology and in patients in whom the serum vitamin B₁₂ or red cell folate levels were within the normal range.     

IL-12 serum levels in patients with type 2 diabetes treated with sulphonylureas

Interleukin-12 (IL-12) has been identified as a pro-inflammatory cytokine which is thought to contribute to the development of atherosclerosis. However, to date, the various associations between factors related to the course of type 2 diabetes, like metabolic compensation, beta cell secretory dysfunction, insulin resistance and IL-12 serum levels, remain unclear. Our study involved 41 patients with type 2 diabetes, 19 patients with coronary artery disease (CAD), and 19 healthy controls. We measured serum levels of fasting glucose, HbA₁c, 1,5-anhydro-d-glucitol, and lipids. In addition, serum levels of C-peptide, insulin, proinsulin and IL-12 were assayed. HOMA_IR score was calculated. The serum concentrations of IL-12 were higher in diabetics than in either patients with CAD or healthy controls, and were correlated with BMI, C-peptide, insulin, HOMA_IR, proinsulin and HDL serum levels. Multiple regression analysis revealed that the IL-12 serum level in type 2 diabetics primarily is dependent upon fasting proinsulin concentration. Our results demonstrate that elevated IL-12 serum levels in type 2 diabetics treated with sulphonylureas are induced especially by peripheral insulin resistance and beta cells dysfunction, as expressed by fasting serum proinsulin levels. This finding gives us hope that treatment to decrease peripheral insulin resistance and to avoid excessive proinsulin secretion might be successful in the prevention of IL-12-induced atherosclerosis.     

Effect of pH on vitamin B12 binding by transcobalamins

An investigation has been made of the effect of varying pH at constant ionic strength on vitamin B₁₂ binding by human serum and by two transcbalamin fractions separated from serum by gel filtration. It was found that the methodology used had a considerable influence on the results obtained. Genuine effects of pH were largely confined to reduced vitamin B₁₂ binding at very acid and very alkaline pH. However, due to an adsorption artefact involving transcobalamin II, certain methods appeared to demonstrate a marked decrease in vitamin B₁₂ binding between pH 4.5 and pH 10.3, especially in the range of pH 5.3–7.5.     

Vitamin-B12 Status of Patients on Long-term Metformin Therapy

Vitamin-B₁₂ malabsorption has been found in 21 (30%) of 71 diabetic patients taking long-term metformin therapy in addition to dietary management. The patients with evidence of B₁₂ malabsorption had significantly lower haemoglobin levels (and significantly higher serum folic acid levels) than those with normal B₁₂ absorption. Steatorrhoea was found in only one patient. Stopping metformin therapy resulted in reversion of B₁₂ absorption to normal in most patients examined. Four patients with B₁₂ malabsorption were found to have pathologically low serum B₁₂ levels. The causes and implications of these findings are discussed and it is concluded that all patients on long-term metformin therapy should have annual serum B₁₂ estimations.     

Vitamin B12 deficiency-induced increase of osteoclastic bone resorption caused by abnormal renal resorption of inorganic phosphorus via Napi2a

Vitamin B₁₂ deficiency is a risk factor for bone disorders via mechanisms not fully understood. In this study, an increase in serum inorganic phosphorus (Pi) concentrations was associated with a vitamin B₁₂ deficiency. Napi2a, a renal cotransporter for Pi reabsorption, accumulated on plasma membranes in a vitamin B₁₂ deficiency suggests that vitamin B₁₂ plays an important role in Pi homeostasis.     

Pharmacological characterization of the biosynthesis of prostanoids and hydroxyeicosatetraenoic acids in human whole blood and platelets by targeted chiral lipidomics analysis

Platelet 12-lipoxygenase(p-12-LOX) is highly expressed in human platelets, and the development of p-12-LOX inhibitors has the potential to be a novel antithrombotic tool by inhibiting thrombosis without prolonging hemostasis. A chiral liquid chromatography-mass spectrometry(LC-MS/MS) method was used to assess the impact of three commercially available LOX inhibitors[esculetin(6,7-dihydroxycoumarin), ML-355(N-2-benzothiazolyl-4-[[(2-hydroxy-3-methoxyphenyl)methyl]amino]-benzenesulfonamide), CDC(cinnamyl-3,4-dihydroxy-α-cyanocinnamate) and acetylsalicylic acid(ASA; a cyclooxygenase-1 inhibitor) on the generation of prostanoids and HETEs(hydroxyeicosatetraenoic acids) in human whole blood allowed to clot for 1 h at 37 °C(serum), platelet-rich plasma(PRP) stimulated with collagen or TRAP-6(a peptide activating thrombin receptor) and washed platelets. In serum, ML-355 did not affect eicosanoid generation, while CDC caused an incomplete reduction of 12S-HETE levels; esculetin inhibited both 12S-HETE and thromboxane(TX)B₂ production; ASA selectively affected TXB₂ production. In washed platelets stimulated with thrombin, esculetin, and CDC inhibited both 12S-HETE and TXB₂ while ML-355 was almost ineffective. In PRP, ML-355, CDC, and esculetin did not affect platelet aggregation associated with incomplete effects on eicosanoid biosynthesis. ASA alone or in combination with ticagrelor(a P2Y₁₂ blocker) affected platelet aggregation associated with profound inhibition of TXB₂ generation. P2Y₁₂ receptor signaling contributed to platelet 12S-HETE biosynthesis in response to primary agonists. In conclusion, ML-355, esculetin, and CDC were not selective inhibitors of p-12-LOX in different cellular systems. They did not affect platelet aggregation induced in PRP by collagen or TRAP-6. The characterization of 12-LOX inhibitors on eicosanoids generated in human whole blood is useful for information on their enzyme selectivity, off-target effects, and the possible influence of plasma components on their pharmacological effects.     

Bone turnover in rats treated with 1,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3 or 24,25-dihydroxyvitamin D3

Female rats were given 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), 0.25 g per 100 g body weight (bw), 25-hydroxyvitamin D₃ (25(OH)D₃), 1.7 g/100 g bw or 24,25-dihydroxyvitamin D₃ (24,25(OH)₂D₃) 1.7 g/100 g bw, subcutaneously three times a week for 12 weeks. Traditional variables pertaining to calcium homeostasis and growth, i.e. blood and urine calcium (Ca) and phosphate (P), serum levels of vitamin D₃ metabolites parathyroid hormone, (PTH), calcitonin (CT), prolactin (PRL) and growth hormone (GH) were measured every four weeks. This data pool was correlated with bone matrix turnover parameters, i.e. serum levels of alkaline phosphatase (ALP) and urinary hydroxyproline (u-HYP) excretion. After 12 weeks of treatment, 1,25(OH)₂D₃ significantly enhanced serum total and ionized Ca, urine Ca and urine P, and also diminished urine cAMP due to reduced renal function (creatinine clearance). However, 25(OH)D₃ administration had no such impact. 24,25(OH)₂D₃ opposed the effect of 1,25(OH)₂D₃ after 12 weeks by significantly augmenting serum P and diminishing serum levels of total Ca and ionized Ca. Cross sectional group analyses showed that criculating levels of ALP were directly related with serum 1,25(OH)₂D₃ and inversely related to serum 24,25(OH)₂D₃ and CT. Total u-HYP and per cent non-dialysable HYP (ndHYP) were reciprocally and positively correlated with serum PRL, respectively. However, no such relations were observed with serum GH.It appears that rats with elevated circulating levels of 1,25(OH)₂D₃ exhibit increased bone resorption, while augmented 24,25(OH)₂D₃ is associated with the opposite. Apparently, high bone turnover (i.e. reduced total urinary HYP and enhanced ndHYP) is associated with high serum PRL.     

Clinical Trial of the Effect of Vitamin B12 in Elderly Subjects with Low Serum B12 Levels

A clinical trial of the effect of vitamin B₁₂ therapy was conducted in 39 elderly subjects who had been found, in a community screening survey, to have low levels of serum B₁₂ without a macrocytic anaemia or neuropathy. The study produced no evidence which suggests that in such subjects B₁₂ is superior to placebo in effecting an improvement in psychiatric state or general well-being. There was a clear tendency for all the subjects to show an improvement during the trial, but this probably represents the therapeutic effect of involvement in a research exercise of this kind.     

Free and Total Vitamin B12 in Cerebrospinal Fluid

Free and total vitamin B₁₂ levels in serum and cerebrospinal fluid (CSF) were bioassayed, since there were no available data on the relationship between free and total vitamin B₁₂ in CSF or between free vitamin in serum and CSF vitamin B₁₂. The subjects were 43 neurological patients. Serum levels were normal in 40 of 43 patients. Values for free and total vitamin B₁₂ in CSF were the same in 42 of 43 patients. Mean CSF vitamin B₁₂ was 21 μμg./ml. In 17 cases CSF vitamin B₁₂ equalled free vitamin B₁₂ level in serum, in 16 cases CSF vitamin B₁₂ was lower than the free level in serum, and in 10 cases CSF vitamin B₁₂ was higher than the free vitamin level in serum. There was no apparent diagnostic correlation. The findings suggest that vitamin B₁₂ is not bound in CSF and that there is some selective control of passage of vitamin B₁₂ across the blood-CSF barrier.     

Muscular differentiation of chicken myotubes in a simple defined synthetic culture medium and in serum supplemented media: Expression of the molecular forms of acetylcholinesterase

We attempted to cultivate muscle cells from chick embryos in a serum-free, defined medium similar to that proposed by Bottenstein and Sato (1979) for the growth and differentiation of a murine neuronal cell-line. (1) We found that muscle cells from the legs of 11-day old chick embryos can be cultivated in a medium containing the different components indicated by Bottenstein and Sato, with 2 g/l bovine serum albumin, without serum or chick embryo extract. Myoblasts attached to the gelatin-coated dishes without any addition of attachment factors. They differentiated into myotubes in a similar manner as in classical serum supplemented media. (2) The level of cellular AchE activity was comparable in cultures grown in the presence of fetal calf serum (FCS), of horse serum (HS) and in the defined medium. The percentage of A₁₂ form was however higher in the defined medium (25–30%) than in FCS supplemented medium (about 5–6%). In HS supplemented medium the A₁₂ form was not detectable, partly because horse serum contains immunoglobulins which bind chicken AChE. The addition of defined medium components to FCS medium cultures did not lead to an increase of A₁₂. In contrast, the addition of a small amount (1%) of fetal calf serum to DM cultures reduced the level of A₁₂ in a drastic manner. FCS components therefore seem to repress the biosynthesis of A₁₂ AChE, or increase its degradation. (3) We estimated intracellular and extracellular compartments of AChE. The ratio of endocellular to ectocellular AChE decreased with the age of the cultures. The G₁ form was intracellular at all stages analyzed, but the other molecular forms were located in both cellular compartment, in different proportion: A₁₂ and G₄ seemed to be located preferentially in the external compartment, whereas G₂ was preferentially intracellular. (4) Muscle cultures grown in the defined medium and in the presence of serum secreted globular forms of AChE in a similar manner.     

Radiolabeled biomolecules with 186Re: Potential for radioimmunotherapy

Rhenium-186 has been theoretically determined to be among the best therapy radiolabels due to its unique half-life, paniculate and γ emissions and chelation properties. Traditionally, rhenium chelates have been synthesized by the tin reduction method at low pH which frequently produces denaturation of acid labile proteins. A comparative study has been carried out to assess three techniques of reducing ¹⁸⁶ReO⁻₄ in order to label biologically active macromolecules (i.e. human serum albumin (HSA), anti-human serum albumin antibody (HSA-Ab) and a monoclonal antibody to T-cells [T-101]). These experiments showed that stannous and dithionite reduction methods provide for an overall labeling yield of between 5 and 18% with an associated immunoreactivity of 12–40%. The hypophosphorous acid (H₃PO₂) reduction method, however, yielded no usable radiolabeled product. The preparation of ¹⁸⁶Re-DTPA-HSA produced an 18.2% radiochemical yield (7.4 × 10⁶ Bq/mg) and 40% retention of binding affinity. Using the stannous or dithionite reduction methods for the radiolabeling of HSA-Ab and T-101 resulted in a relatively low yield (9%), but the labeled product retained binding affinity of 12–25% with Protein A.     

Vitamin B12 Excretion in Patients with Various Skin Diseases

The excretion in the urine of ⁵⁸Co after an oral dose of ⁵⁸Co vitamin B₁₂ given together with intrinsic factor has been found to be reduced in a number of patients with psoriasis, eczema, and other less common dermatoses. There is a correlation between the abnormality and the extent of the rash. A reduced glomerular filtration rate was found in a few of the patients in whom it was measured, and this must have been responsible, at least in part, for the reduced excretion of vitamin B₁₂ in these patients, but abnormal vitamin B₁₂ excretion also occurred in the absence of impaired renal function. Our evidence is insufficient to show whether malabsorption or increased tissue utilization of vitamin B₁₂ was the explanation in other cases. Certainly a number of patients had steatorrhoea, and in these it is most likely that malabsorption was the major factor. In patients without steatorrhoea a lone malabsorption of vitamin B₁₂ cannot be excluded. A decreased serum concentration of vitamin B₁₂ was found in only one of the patients.     

Common variant in FUT2 gene is associated with levels of vitamin B12 in Indian population

Vitamin B₁₂ is an essential micronutrient synthesized by microorganisms. Mammals including humans have evolved ways for transport and absorption of this vitamin. Deficiency of vitamin B₁₂ (either due to low intake or polymorphism in genes involved in absorption and intracellular transport of this vitamin) has been associated with various complex diseases. Genome-wide association studies have recently identified several common single nucleotide polymorphisms (SNPs) in fucosyl transferase 2 gene (FUT2) to be associated with levels of vitamin B₁₂—the strongest association was with a non-synonymous SNP rs602662 in this gene. In the present study, we attempted to replicate the association of this SNP (rs602662) in an Indian population since a significant proportion has been reported to have low levels of vitamin B₁₂ in this population. A total of 1146 individuals were genotyped for this SNP using a single base extension method and association with levels of vitamin B₁₂ was assessed in these individuals. Regression analysis was performed to analyze the association considering various confounding factors like for age, sex, diet, hypertension, diabetes mellitus and coronary artery disease status. We found that the SNP rs602662 was significantly associated with the levels of vitamin B₁₂ (p value < 0.0001). We also found that individuals adhering to a vegetarian diet with GG (homozygous major genotype) have significantly lower levels of vitamin B₁₂ in these individuals. Thus, our study reveals that vegetarian diet along with polymorphism in the FUT2 gene may contribute significantly to the high prevalence of vitamin B₁₂ deficiency in India.     

Vitamin B12 in neurology and ageing; Clinical and genetic aspects

The classic neurological and psychiatric features associated with vitamin B₁₂ deficiency have been well described and are the subject of many excellent review articles. The advent of sensitive diagnostic tests, including homocysteine and methylmalonic acid assays, has revealed a surprisingly high prevalence of a more subtle ‘subclinical’ form of B₁₂ deficiency, particularly within the elderly. This is often associated with cognitive impairment and dementia, including Alzheimer's disease. Metabolic evidence of B₁₂ deficiency is also reported in association with other neurodegenerative disorders including vascular dementia, Parkinson's disease and multiple sclerosis. These conditions are all associated with chronic neuro-inflammation and oxidative stress. It is possible that these clinical associations reflect compromised vitamin B₁₂ metabolism due to such stress. Physicians are also increasingly aware of considerable inter-individual variation in the clinical response to B₁₂ replacement therapy. Further research is needed to determine to what extent this is attributable to genetic determinants of vitamin B₁₂ absorption, distribution and cellular uptake.     

Correlation of Serum and Urine Vitamin B12

The relation between the serum vitamin B₁₂ level and the daily loss of vitamin B₁₂ in urine was examined in patients with normal serum vitamin B₁₂ levels and in patients suffering from vitamin B₁₂ deficiency. A linear correlation was found between the two measurements, suggesting that the serum vitamin B₁₂ level is a governing factor in the urinary loss of vitamin B₁₂. The contribution by this loss to the total loss of vitamin B₁₂ from the body is small under normal circumstances but becomes quantitatively more important with the depletion of body stores.