Effect of restricted feed intake and addition of vitamins B2 and B6 and folic acid on the liver concentration of zinc and copper in rats

The aim of the present study was to investigate the influence of nutritional deficiency and dietary addition of vitamins (B₂, B₆, and folate) on hepatic concentration of zinc and copper in rats. The experiment was performed on 260 growing male Wistar rats divided into 13 groups. Animals of 11 groups were fed isocaloric diets (14.7 MJ/kg) in which the 20% of energy was derived from protein. Another two groups of rats were offered diets with 9% or 4.5% of energy originating from protein. Animals of both mentioned groups and of the control group (20% of energy from protein) were offered diets ad libitum. The other 10 groups were offered 50% and 30% of the amount consumed in the control group. Eight groups, from those 10 restricted ones, were differentiated by dietary addition of vitamins B₂ and B₆ and folate (300% addition). Restricted feed intake did not affect the liver zinc concentration but significantly increased the copper concentration. The addition of vitamin B₆ decreased the liver Zn concentration. The highest liver Cu concentration was noted in rats offered restricted diets to only 30% of intake in the control group and high in vitamin B₂ and in rats supplemented with all of studied vitamins together. It suggests that vitamin B₂ had the strongest impact on liver Cu concentration in rats fed restricted diets.     

EFFECTS OF CERTAIN LIMITING CONDITIONS ON THE SYNTHESIS OF B VITAMINS BY YEAST

In yeast crops which were grown in the presence of various inhibitors, there was considerable variation in content of the various B vitamins. A higher degree of parallelism in variation in content was found to exist between thiamine and niacin than between any other pair of vitamins; this has been interpreted as indicating that the predominant functions of these two vitamins are their established rôles in fermentation. Values for inositol indicate that it may be involved in fermentation processes, but this is not the case for other members of the B complex. Biotin appears to be unique since in no case did the biotin content of yeast grown in the presence of an inhibitor fall below that of the control yeast. There was some evidence of synthesis of biotin, or a material with biotin activity, in the presence of certain inhibitors, the most striking instance being with sulfaguanidine. An exogenous supply of biotin was essential for extensive proliferation of F. B. yeast, and yeast grown in a medium to which biotin was the only added vitamin contained the B vitamins in amounts very similar to those found in the control yeast, the most marked differences being in increased vitamin B₆ and p-aminobenzoic acid contents. In the absence of biotin, significant amounts of all of the B vitamins except biotin were synthesized, both in the presence and absence of certain other members of the B complex. The addition of thiamine, pyridoxine, inositol, and β-alanine to the culture medium caused a reduction in the amounts of vitamin B₆ and p-aminobenzoic acid synthesized. F. B. yeast was able to grow in a xylose medium only when certain of the B vitamins were present, and even then growth was limited. Evidence was obtained for some synthesis of all of the vitamins investigated except biotin and vitamin B₆. The most significant differences in vitamin content between galac yeast and the parent F. B. strain were in folic acid and vitamin B₆, the former being considerably reduced in amount, the latter being increased.     

PRODUCTION OF VITAMIN B12, THIAMINE,AND BIOTIN BY PHYTOPLANKTON1

Some ecologically important phytoplankters released vitamins into culture medium during growth. Skeletonema costatum and Stephanopyxis turris (vitamin B₁₂-requirers) produced both thiamine (vitamin B₁) and biotin when growing with either 12 or 2 ng vitamin B₁₂/liter. Gonyaulax polyedra (vitamin B₁₂-requirer) produced thiamine with 12 ng vitamin B₁₂/liter, and Coccolithus huxleyi (thiamine-requirer) produced vitamin B₁₂ and biotin with 120 ng thiamine/liter, but only biotin with 10 ng thiamine/liter. The amount of vitamin produced by an alga and rate at which it was produced varied with the phytoplankter, the concentration of the required vitamin, and incubation time. Vitamins produced during early and exponential growth were due to excretions, and those produced at stationary growth resulted from excretion and release due to cell lysis. Uptake of the required vitamin by all phytoplankters was greatest during the first few days of incubation. On continued incubation the rate of uptake/cell decreased. In the sea phytoplankters may contribute a major portion of the amount of dissolved vitamins.     

SECRETION OF VITAMINS AND AMINO ACIDS INTO THE ENVIRONMENT BY OCHROMONAS DANICA1,2

Ochromonas danica grown on a chemically defined medium under controlled conditions in the light synthesized the following vitamins: ascorbate, B₆, N⁵-methyltetrahydrofolate, tetrahydrofolate polyglutamates, oxidized folate monoglutamates, nicotinate, pantothenate, riboflavin, vitamin A, β-carotene, and vitamin E but no vitamin. B₁₂. The cells also secreted molecules into their growth medium including the vitamins ascorbate, B₆, the above folates, nicotinate, pantothenate, riboflavin, vitamin E, and the amino acids alanine, aspartic acid, leucine, and valine. The role of such secretions in nature is discussed.     

Effects of increasing amounts of extruded linseed in the diet on apparent ruminal synthesis of some B vitamins in dairy cows

Many studies have shown that metabolic efficiency of ruminants can be significantly decreased when B-vitamin supply is insufficient. Under the present state of knowledge, the amounts of B vitamins available for intestinal absorption cannot be predicted based on diet composition. Therefore, in an attempt to increase our understanding of the effects of dietary factors, on B-vitamin supply for dairy cows, the effects of increasing amounts of extruded linseed in diets based on hay (permanent grassland hay, H; Experiment 1) or corn silage (CS; Experiment 2) on apparent ruminal synthesis (ARS) of thiamin, riboflavin, niacin, vitamin B₆, folates and vitamin B₁₂ were evaluated. In each experiment, four lactating Holstein cows fitted with cannulas in the rumen and the proximal duodenum were used in a 4 × 4 Latin square design. In both experiments, the dietary treatments consisted of an increasing supply of extruded linseed representing 0%, 5%, 10% or 15% of diet DM. The forage : concentrate ratios were 50 : 50 and 60 : 40 for Experiments 1 and 2, respectively. Duodenal flow was determined using YbCl₃ as a marker. The ARS of each B vitamin was calculated as duodenal flow – daily intake. In both experiments, treatments did not affect thiamin, riboflavin, niacin and vitamin B₁₂ duodenal flow or ARS. Increasing the dietary concentration of extruded linseed decreased folate intake in Experiment 1 and vitamin B₆ intake in Experiment 2 but resulted in a greater duodenal flow of vitamin B₆ and folates regardless of the forage used in basal diet. Greater dietary linseed concentrations decreased vitamin B₆ apparent degradation in the rumen in CS-based diet only and increased folate ARS in both H- and CS-based diets. Increasing linseed concentration of isonitrogenous and isoenergetic diets increased vitamin B₆ and folate supply to dairy cows, both with H- and CS-based diets.     

Plant amino acid-derived vitamins: biosynthesis and function

Vitamins are essential organic compounds for humans, having lost the ability to de novo synthesize them. Hence, they represent dietary requirements, which are covered by plants as the main dietary source of most vitamins (through food or livestock’s feed). Most vitamins synthesized by plants present amino acids as precursors (B₁, B₂, B₃, B₅, B₇, B₉ and E) and are therefore linked to plant nitrogen metabolism. Amino acids play different roles in their biosynthesis and metabolism, either incorporated into the backbone of the vitamin or as amino, sulfur or one-carbon group donors. There is a high natural variation in vitamin contents in crops and its exploitation through breeding, metabolic engineering and agronomic practices can enhance their nutritional quality. While the underlying biochemical roles of vitamins as cosubstrates or cofactors are usually common for most eukaryotes, the impact of vitamins B and E in metabolism and physiology can be quite different on plants and animals. Here, we first aim at giving an overview of the biosynthesis of amino acid-derived vitamins in plants, with a particular focus on how this knowledge can be exploited to increase vitamin contents in crops. Second, we will focus on the functions of these vitamins in both plants and animals (and humans in particular), to unravel common and specific roles for vitamins in evolutionary distant organisms, in which these amino acid-derived vitamins play, however, an essential role.     

VITAMIN B12, THIAMINE,AND BIOTIN CONTENTS OF MARINE PHYTOPLANKTON1

An extraction procedure was developed for determining vitamin B₁₂, thiamine, and biotin contents of marine phytoplankton. Phytoplankters were collected either by centrifugation or by retention on a glass fiber filter, then heated at 100 C for I hr in 100 ml of vitamin-free seawater acidified to pH 3.5 with HCl. The extract, after debris removal, was filter-sterilized and analyzed, for vitamin B₁₂, thiamine, and biotin with standard vitamin assay procedures. The vitamin contents of haeodactylum tricornutum, Skeletonema costatum, Stephanopyxis turris, and occolithus liuxleyi were determined during growth in batch cultures. P. tricornutum (non-vitamin requirer) growing in aerated cultures contained 0.29–0.96 ng B₁₂, 5–15 ng thiamine, and 0.45–1.70 ng biotin/mg C. Under similar conditions S. costatum (B₁₂-requirer) contained about 0.06 ng B₁₂, 5–36 ng thiamine, and 0.16–2.10 ng biotin/mg C. The concentrations of vitamin were generally similar during some portion of the growth curve, eg, logarithmic growth. The vitamin B₁₂, content of S. costatum growing under nonaerated conditions decreased when medium B₁₂, was reduced. The biotin content did not change when medium B₁₂ was decreased. The thiamine content per unit weight of C. huxleyi (thiamine-requirer) growing with either 10 or 120 ng/liter thiamine decreased under both medium concentrations, indicating no net synthesis of the vitamin.     

One pathway can incorporate either adenine or dimethylbenzimidazole as an alpha-axial ligand of B12 cofactors in Salmonella enterica

Corrinoid (vitamin B₁₂-like) cofactors contain various α-axial ligands, including 5,6-dimethylbenzimidazole (DMB) or adenine. The bacterium Salmonella enterica produces the corrin ring only under anaerobic conditions, but it can form “complete” corrinoids aerobically by importing an “incomplete” corrinoid, such as cobinamide (Cbi), and adding appropriate α- and β-axial ligands. Under aerobic conditions, S. enterica performs the corrinoid-dependent degradation of ethanolamine if given vitamin B₁₂, but it can make B₁₂ from exogenous Cbi only if DMB is also provided. Mutants isolated for their ability to degrade ethanolamine without added DMB converted Cbi to pseudo-B₁₂ cofactors (having adenine as an α-axial ligand). The mutations cause an increase in the level of free adenine and install adenine (instead of DMB) as an α-ligand. When DMB is provided to these mutants, synthesis of pseudo-B₁₂ cofactors ceases and B₁₂ cofactors are produced, suggesting that DMB regulates production or incorporation of free adenine as an α-ligand. Wild-type cells make pseudo-B₁₂ cofactors during aerobic growth on propanediol plus Cbi and can use pseudo-vitamin B₁₂ for all of their corrinoid-dependent enzymes. Synthesis of coenzyme pseudo-B₁₂ cofactors requires the same enzymes (CobT, CobU, CobS, and CobC) that install DMB in the formation of coenzyme B₁₂. Models are described for the mechanism and control of α-axial ligand installation.     

Vitamin losses during frozen storage of Liza aurata (Risso, 1810), Cyprinus carpio L. 1758, Clupeonella cultriventris caspia (Nordmann, 1840), Rutilus frisii kutum (Kamenskii, 1901) and Sander lucioperca (L., 1758)

The objective of this study was to determine the fat and water‐soluble vitamins contents (A, E, D, K, B₁, B₂, B₆, B₁₂, niacin, biotin, ascorbic, folic and pantethonic acids; in mg kg^?1 fresh weight) lost during frozen storage (?24°C) in time intervals of 0, 1, 2, 3 and 4 months. Studied species included: Liza aurata (golden grey mullet), Cyprinus carpio (common carp), Clupeonella cultriventris caspia (common kilka or sprat), Rutilus frisii kutum (Caspian kutum) and Sander lucioperca (pike perch). Results showed that A, D and K vitamin losses occurred in all samples during storage, but no statistically significant differences were observed. Vitamin E losses under frozen conditions were significant in common kilka, Caspian kutum and pike perch, however, not in golden grey mullet or common carp. A decrease in vitamin C was significant in all fish species, but there were no significant decreases in other water‐soluble vitamins (B₁, B₂, B₆, B₁₂, niacin, folic acid, pantothenic acid, biotin).     

Inflammation causes tissue-specific depletion of vitamin B<Subscript>6</Subscript>

Previously we observed strong and consistent associations between vitamin B₆ status and several indicators of inflammation in patients with rheumatoid arthritis. Clinical indicators, including the disability score, the length of morning stiffness, and the degree of pain, and biochemical markers, including the erythrocyte sedimentation rate and C-reactive protein levels, were found to be inversely correlated with circulating vitamin B₆ levels. Such strong associations imply that impaired vitamin B₆ status in these patients results from inflammation. In the present study we examined whether inflammation directly alters vitamin B₆ tissue contents and its excretion in vivo. A cross-sectional case-controlled human clinical trial was performed in parallel with experiments in an animal model of inflammation. Plasma and erythrocyte and pyridoxal 5'-phosphate concentrations, urinary 4-pyridoxic acid excretion, and the activity coefficient of erythrocyte aspartate aminotransferase were compared between patients and healthy subjects. Adjuvant arthritis was induced in rats for investigating hepatic and muscle contents as well as the urinary excretion of vitamin B₆ during acute and chronic inflammation. Patients with rheumatoid arthritis had low plasma pyridoxal 5'-phosphate compared with healthy control subjects, but normal erythrocyte pyridoxal 5'-phosphate and urinary 4-pyridoxic acid excretion. Adjuvant arthritis in rats did not affect 4-pyridoxic acid excretion or muscle storage of pyridoxal 5'-phosphate, but it resulted in significantly lower pyridoxal 5'-phosphate levels in circulation and in liver during inflammation. Inflammation induced a tissue-specific depletion of vitamin B₆. The low plasma pyridoxal 5'-phosphate levels seen in inflammation are unlikely to be due to insufficient intake or excessive vitamin B₆ excretion. Possible causes of decreased levels of vitamin B₆ are discussed.     

Postpartum depressive symptoms: the B-vitamin link

Objective This study examined longitudinal relationships between maternal red-cell folate status and dietary intakes of vitamins B₆, B₁₂ and folate before and during pregnancy and subsequent postpartum depressive symptoms.Study design and setting Within a cohort study of women aged 20–34 years (the Southampton Women''s Survey) dietary data were obtained before pregnancy and at 11 and 34 weeks'' gestation. Red-cell folate was measured before pregnancy and at 11 weeks'' gestation. We derived relative risks of postpartum depressive symptoms using an Edinburgh Postnatal Depression Scale (EPDS) score of ≥ 13 administered from 6 months to 1 year postpartum.Results No significant differences were found between those with postpartum depressive symptoms (n = 905) and those without (n = 1951) in relation to red-cell folate concentration or dietary intake of folate, vitamin B₁₂ and vitamin B₆, before or during pregnancy. A prior history of mental illness (relative risk (RR) 1.83; 95% confidence interval (CI) 1.53–2.19) was associated with postpartum depressive symptoms, and women who breastfed until 6 months were less likely to experience postpartum depressive symptoms (RR 0.68; 95% CI 0.55–0.84).Conclusion This study suggests that folate status and dietary folate, B₆ and B₁₂ intakes before and during pregnancy are not associated with postpartum depressive symptoms. A history of mental illness, however, was a strong risk factor.     

Dependence of Betaine Stimulation of Vitamin B12 Overproduction on Protein Synthesis

The betaine-stimulated differential synthesis of vitamin B₁₂, i.e., the increase in B₁₂ per increase in dry cell weight, by Pseudomonas denitrificans was inhibited by rifampin and chloramphenicol but not by benzylpenicillin and carbenicillin at concentrations of antibiotic that inhibit growth. The level of the first enzyme of corrin (and porphyrin) biosynthesis, δ-aminolevulinic acid synthetase, was decreased to a much greater degree by rifampin and chloramphenicol than by the penicillins. These data support the concept that betaine stimulation of B₁₂ synthesis is a result of its stimulation of synthesis of δ-aminolevulinic acid synthetase, a labile and presumably rate-limiting enzyme of corrin formation requiring continuous induction. In further support of this hypothesis, it was found that chloramphenicol immediately interfered with both vitamin B₁₂ and δ-aminolevulinic acid synthetase formation, no matter when it was added to the system.     

Paternal B Vitamin Intake Is a Determinant of Growth,Hepatic Lipid Metabolism and Intestinal Tumor Volume in Female Apc1638N Mouse Offspring

Background

The importance of maternal nutrition to offspring health and risk of disease is well established. Emerging evidence suggests paternal diet may affect offspring health as well.

Objective

In the current study we sought to determine whether modulating pre-conception paternal B vitamin intake alters intestinal tumor formation in offspring. Additionally, we sought to identify potential mechanisms for the observed weight differential among offspring by profiling hepatic gene expression and lipid content.

Methods

Male Apc^1638N mice (prone to intestinal tumor formation) were fed diets containing replete (control, CTRL), mildly deficient (DEF), or supplemental (SUPP) quantities of vitamins B₂, B₆, B₁₂, and folate for 8 weeks before mating with control-fed wild type females. Wild type offspring were euthanized at weaning and hepatic gene expression profiled. Apc^1638N offspring were fed a replete diet and euthanized at 28 weeks of age to assess tumor burden.

Results

No differences in intestinal tumor incidence or burden were found between male Apc^1638N offspring of different paternal diet groups. Although in female Apc^1638N offspring there were no differences in tumor incidence or multiplicity, a stepwise increase in tumor volume with increasing paternal B vitamin intake was observed. Interestingly, female offspring of SUPP and DEF fathers had a significantly lower body weight than those of CTRL fed fathers. Moreover, hepatic trigylcerides and cholesterol were elevated 3-fold in adult female offspring of SUPP fathers. Weanling offspring of the same fathers displayed altered expression of several key lipid-metabolism genes. Hundreds of differentially methylated regions were identified in the paternal sperm in response to DEF and SUPP diets. Aside from a few genes including Igf2, there was a striking lack of overlap between these genes differentially methylated in sperm and differentially expressed in offspring.

Conclusions

In this animal model, modulation of paternal B vitamin intake prior to mating alters offspring weight gain, lipid metabolism and tumor growth in a sex-specific fashion. These results highlight the need to better define how paternal nutrition affects the health of offspring.     

THE EFFECT OF VITAMIN B12 ON THE ANEMIA AND COMBINED SYSTEM DISEASE OF ADDISONIAN PERNICIOUS ANEMIA

The effect of the parenteral administration of vitamin B₁₂ has been observed in eight patients with Addisonian pernicious anemia.Vitamin B₁₂ in initial doses of 50 micrograms or 25 micrograms induced satisfactory reticulocyte response and a return of erythrocyte count to within normal range in 60 days.In only two of the patients were secondary reticulocyte responses induced on a second injection of vitamin B₁₂.Concurrently with the hemopoietic response, the bone marrow changed from megaloblastic hyperplasia to normoblastic distribution.The paresthesias associated with combined system disease as well as disturbances in position sense and locomotor function may be entirely relieved or greatly diminished following injections of vitamin B₁₂.Maintenance injections of vitamin B₁₂ may be from 30 to 50 micrograms at intervals of one month, the amount depending upon the individual case.Vitamin B₁₂ may be used without untoward symptoms in patients previously sensitive to liver extract.     

B vitamins, methionine and alcohol intake and risk of colon cancer in relation to BRAF mutation and CpG island methylator phenotype (CIMP)

Background

One-carbon metabolism appears to play an important role in DNA methylation reaction. Evidence suggests that a low intake of B vitamins or high alcohol consumption increases colorectal cancer risk. How one-carbon nutrients affect the CpG island methylator phenotype (CIMP) or BRAF mutation status in colon cancer remains uncertain.

Methods

Utilizing incident colon cancers in a large prospective cohort of women (the Nurses'' Health Study), we determined BRAF status (N = 386) and CIMP status (N = 375) by 8 CIMP-specific markers [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1], and 8 other CpG islands (CHFR, HIC1, IGFBP3, MGMT, MINT-1, MINT-31, p14, and WRN). We examined the relationship between intake of one-carbon nutrients and alcohol and colon cancer risk, by BRAF mutation or CIMP status.

Results

Higher folate intake was associated with a trend towards low risk of CIMP-low/0 tumors [total folate intake ≥400 µg/day vs. <200 µg/day; the multivariate relative risk = 0.73; 95% CI = 0.53–1.02], whereas total folate intake had no influence on CIMP-high tumor risks (P_{heterogeneity} = 0.73). Neither vitamin B₆, methionine or alcohol intake appeared to differentially influence risks for CIMP-high and CIMP-low/0 tumors. Using the 16-marker CIMP panel did not substantially alter our results. B vitamins, methionine or alcohol intake did not affect colon cancer risk differentially by BRAF status.

Conclusions

This molecular pathological epidemiology study suggests that low level intake of folate may be associated with an increased risk of CIMP-low/0 colon tumors, but not that of CIMP-high tumors. However, the difference between CIMP-high and CIMP-low/0 cancer risks was not statistically significant, and additional studies are necessary to confirm these observations.     

Enrichment of some B-vitamins in plants with application of organic fertilizers

A review of the literature showed that plants grown with organic fertilizers often contain higher concentrations of vitamins B₁ (thiamin) and B₁₂ (cyanocobalamin) as compared with plants grown with inorganic fertilizers. Since plant roots were recently shown to be able to absorb B₁ and B₁₂, it was thus suspected that organic fertilizers (such as manure of diverse sources or sewage sludges which often contain relatively high concentrations of several vitamins) introduce additional vitamins into the soil which in turn leads to increased vitamins in the plants. This possibility was studied by measuring the B₁₂ content in the seeds of soybean and barley and in the leaves of spinach plants grown in soils amended with pure B₁₂ or cow dung (which is naturally rich in B₁₂). The addition of pure B₁₂ or cow dung did not alter the B₁₂ content in the soybean seeds but significantly increased that in the barley kernels and in the spinach leaves. For example, the addition of cow dung at the rate of 10 g kg^–1 increased the B₁₂ content in barley kernels by more than threefold (from 2.6 to 9.1 ng g^–1 DW) and in spinach leaves by close to twofold (from 6.9 to 17.8 ng g^–1 DW). Long-term addition of organic fertilizers to the soil also significantly increased the soil content of this vitamin. Since plants cannot synthesize B₁₂ and thus plant foods are normally fully devoid of (or have very low concentrations of) this vitamin, the finding that plants grown with organic fertilizers may contain relatively higher concentrations of this vitamin may have nutritional consequences in that the consumption of these plants by humans would inadvertently increase their intake of this vitamin. This may be of special benefit to people living by choice or by necessity on strict vegetarian diets who are known to be in danger of B₁₂ deficiency.     

INFLUENCE OF TEMPERATURE AND SOLAR RADIATION ON PERSISTENCE OF VITAMIN B12, THIAMINE,AND BIOTIN IN SEAWATER1

Ecologically important concentrations of vitamin B₁₂ and thiamine in charcoal-treated, filter-sterilized seawater stored in the dark at 5, 18, 28, and 37 C generally did not change over a 9-week period, although there was some breakdown of B₁₂ at 37 C. Biotin activity under similar conditions generally increased, indicating its decomposition to more active products. Solutions kept at–20 C had unchanged vitamin activity. B₁₂ and biotin in seawater exposed to sunlight were rapidly destroyed. The course of thiamine destruction in sunlight indicated a breakdown to a stable, biologically active product(s)).     

Vitamin analysis of five planktonic microalgae and one macroalga

Vitamin analysis was carried out on five microalgae used in aquaculture:Tetraselmis suecica, Isochrysis galbana, Pavlova lutheri, Skeletonema costatum andChaetoceros calcitrans and one macroalga,Sargassum muticum, which is invasive on the Atlantic shores of France. Both liposoluble (provitamin A, E, K) and hydrosoluble (B₁, B₂, B₆, B₁₂, C, PP) vitamins were quantified. For most of them, greater amounts were obtained in the algal products than in the usual sources. On a dry weight basis,Tetraselmis suecica contained 4280 μg g^?1 provitamin A and 6323 μg g^?1 vitamin E,Pavlova lutheri 1162 μg g^?1 vitamin B₁₂ and 837 μg g^?1 vitamin C,Isochrysis galbana 2690 μg g^?1 vitamin PP and 183 μg g^?1 vitamin B₆, andSkeletonema costatum 710 μg g^?1 vitamin B₁.     

Effect of Vegetarianism and Smoking on Vitamin B12, Thiocyanate,and Folate Levels in the Blood of Normal Subjects

Vitamin B₁₂, thiocyanate, and folate levels in the blood were estimated in 69 apparently normal subjects, of whom 26 were non-vegetarian non-smokers, 19 non-vegetarian smokers, 15 vegetarian non-smokers, and nine vegetarian smokers. The serum total (cyanide-extracted) B₁₂ level (value A) ranged from 105 to 728 pg/ml, with a mean of 292 pg/ml. The highest values were found in non-vegetarian non-smokers and the lowest in vegetarian smokers. There was no significant difference in value A between smokers as a group and non-smokers as a group. On the other hand, in vegetarians value A was very significantly lower than in non-vegetarians regardless of their smoking habits.It is suggested that A may represent both the protein-bound and free forms of vitamin B₁₂ in the blood, and B mainly the free B₁₂, which may be the physiologically active form. The plasma thiocyanate level varied from 1·0 to 15 μmol/100 ml, being, as expected, much higher in smokers (mean 8·20 μmol/100 ml) than in non-smokers (mean 2·02 μmol/100 ml). There was a rough correlation between falling B₁₂ levels and rising thiocyanate levels. The serum folate level ranged from 2·75 to 15·75 ng/ml, and was slightly but significantly higher in vegetarians (mean 6·60 ng/ml) than in non-vegetarians (mean 4·79 ng/ml), reflecting the greater content of folate in a vegetarian diet.     

Relationships between biotin and vitamin B12. Effects of biotin and vitamin B12 on folic acid metabolism

1. The effects of dietary biotin compared with vitamin B₁₂ on the total content and on the distribution of the various folate derivatives in the liver of rats given a biotin-free diet have been studied. The effect of both vitamins on the conversion in vitro of folic acid into citrovorum factor in the same experimental conditions was also examined. 2. In biotin-treated rats as well as in vitamin B₁₂-treated rats the total content of folic acid-active substances measured microbiologically by Pediococcus cerevisiae, Streptococcus faecalis and Lactobacillus casei is significantly higher than that in biotin-deficient rats. The liver distribution of various folate derivatives in the three groups of animals is also markedly modified. 3. The amount of citrovorum factor formed in systems with liver homogenate of rats receiving biotin or vitamin B₁₂ is higher than that with liver homogenates of deficient rats. 4. The results obtained demonstrate the influence of biotin in the metabolism of folic acid, and the similar actions at this level of both biotin and vitamin B₁₂. These results are discussed in relation to the participation of the two vitamins in the metabolism of C₁ units, as a biochemical interpretation of the relationships between vitamin B₁₂ and biotin.