Effects of Inadequate Folate Intake on the Onset and Progression of Hypertensive Vascular Injury.

We investigated the effects of inadequate folate intake on the onset and progression of hypertensive organ injury. In the present study, 5-wk-old male stroke-prone spontaneously hypertensive rats (SHRSP) were fed with a normal-folate (control; 160-170 µg of folate/100 g diet) or low-folate (8-10 µg of folate/100 g diet) diet until they reached 25 wk of age. After the animals reached 10 wk of age, the bodyweight of the rats in the low-folate group was lower than that of the rats in the control group. Regarding blood pressure, both groups had severe hypertension of ≥230 mmHg at 12 wk of age that was not significantly different between the groups. At 16 wk of age, the low-folate group had a low number of blood cell types. The folate levels in the serum, liver, and kidneys of these rats were significantly lower (p<0.01) and the serum homocysteine level in the low-folate group was significantly higher than in the controls. The low-folate group had a significantly lower testicular weight than the control group (p<0.05) and arterial hypertrophy, spermatogenesis arrest, and interstitial connective tissue hyperplasia were observed. However, there was no clear difference in lesions in other organs. These results indicated that under low folate status, SHRSP causes hematopoietic disorders and exacerbates hypertensive vascular injury at various degrees by organ type.

Growth Response of Lactobacillus rhamnosus Chloramphenicol-Resistant Strain ATCC 27773 to Pteroyl-Mono- and -Di-Glutamates.

This study investigated the effect of various concentrations of pteroyl-mono-γ-glutamate (PteGlu1) and pteroyl-di-γ-glutamate (PteGlu2) on the growth of Lactobacillus rhamnosus strains ATCC 7469 (wild-type strain) and ATCC 27773 (chloramphenicol-resistant strain) used for folate microbiological assays. At concentrations of 0.025-0.20 nmol/L, the growth of the chloramphenicol-resistant strain was stimulated to a greater extent by PteGlu1 than by PteGlu2, but the wild-type strain did not show such phenomena. L. rhamnosus ATCC 27773 bioassays were used to determine the total folate content of various foods treated with a chicken pancreas folate conjugase. This showed a significantly lower value when PteGlu1 was used as a calibrator than with PteGlu2. These results indicated that PteGlu2 should be the standard folate compound when chicken pancreas folate conjugase is used in preparing samples for L. rhamnosus ATCC 27773 bioassay.

Nutritional role of folate.

Folate functions as a coenzyme to transfer one-carbon units that are necessary for deoxythymidylate synthesis, purine synthesis, and various methylation reactions. Ingested folate becomes a functional molecule through intestinal absorption, circulation, transport to cells, and various modifications to its structure. Associations between nutritional folate status and chronic diseases such as cardiovascular disease, cancer, and cognitive dysfunction have been reported. It has also been reported that maternal folate nutritional status is related to the risk of neural tube defects (NTDs) in the offspring. It has also been recommended that folate be consumed in the diet to promote the maintenance of good health. To reduce the risk of NTDs, supplementation with folic acid (a synthetic form of folate) during the periconceptional period has also been recommended. This paper describes the basic features and nutritional role of folate.

Effects of Biotin Deficiency on Biotinylated Proteins and Biotin-Related Genes in the Rat Brain.

Biotin is a water-soluble vitamin that functions as a cofactor for biotin-dependent carboxylases. The biochemical and physiological roles of biotin in brain regions have not yet been investigated sufficiently in vivo. Thus, in order to clarify the function of biotin in the brain, we herein examined biotin contents, biotinylated protein expression (e.g. holocarboxylases), and biotin-related gene expression in the brain of biotin-deficient rats. Three-week-old male Wistar rats were divided into a control group, biotin-deficient group, and pair-fed group. Rats were fed experimental diets from 3 wk old for 8 wk, and the cortex, hippocampus, striatum, hypothalamus, and cerebellum were then collected. In the biotin-deficient group, the maintenance of total biotin and holocarboxylases, increases in the bound form of biotin and biotinidase activity, and the expression of an unknown biotinylated protein were observed in the cortex. In other regions, total and free biotin contents decreased, holocarboxylase expression was maintained, and bound biotin and biotinidase activity remained unchanged. Biotin-related gene (pyruvate carboxylase, sodium-dependent multivitamin transporter, holocarboxylase synthetase, and biotinidase) expression in the cortex and hippocampus also remained unchanged among the dietary groups. These results suggest that biotin may be related to cortex functions by binding protein, and the effects of a biotin deficiency and the importance of biotin differ among the different brain regions.

Consumption of a low-carbohydrate and high-fat diet (the ketogenic diet) exaggerates biotin deficiency in mice.

OBJECTIVE: Biotin is a water-soluble vitamin that acts as a cofactor for several carboxylases. The ketogenic diet, a low-carbohydrate, high-fat diet, is used to treat drug-resistant epilepsy and promote weight loss. In Japan, the infant version of the ketogenic diet is known as the "ketone formula." However, as the special infant formulas used in Japan, including the ketone formula, do not contain sufficient amounts of biotin, biotin deficiency can develop in infants who consume the ketone formula. Therefore, the aim of this study was to evaluate the effects of the ketogenic diet on biotin status in mice. METHODS: Male mice (N = 32) were divided into the following groups: control diet group, biotin-deficient (BD) diet group, ketogenic control diet group, and ketogenic biotin-deficient (KBD) diet group. Eight mice were used in each group. RESULTS: At 9 wk, the typical symptoms of biotin deficiency such as hair loss and dermatitis had only developed in the KBD diet group. The total protein expression level of biotin-dependent carboxylases and the total tissue biotin content were significantly decreased in the KBD and BD diet groups. However, these changes were more severe in the KBD diet group. CONCLUSION: These findings demonstrated that the ketogenic diet increases biotin bioavailability and consumption, and hence, promotes energy production by gluconeogenesis and branched-chain amino acid metabolism, which results in exaggerated biotin deficiency in biotin-deficient mice. Therefore, biotin supplementation is important for mice that consume the ketogenic diet. It is suggested that individuals that consume the ketogenic diet have an increased biotin requirement.

Biochemical alterations in the palatal processes in fetuses of biotin-deficient mice.

To clarify the role of biotin in palatal formation, we investigated the effects of biotin deficiency on the development of palatal processes in mouse fetuses at midgestation. We also investigated protein expressions in the palatal processes. Pregnant mice were given either a biotin-deficient diet or a biotin-supplemented (control) diet from day 0 of gestation (dg 0). Some dams in the biotin-deficient group were changed to a biotin-supplemented diet on dg 12, 13 or 14. On dg 15, the palatal processes were dissected from these fetuses and their peptides were characterized using two-dimensional electrophoresis and liquid chromatography/tandem mass spectrometry (LC-MS/MS) system. Regarding Trasler's stage for the growth of the palatal processes in mouse fetuses on dg 15, the average stage of palatal development was 5.83 +/- 0.39 in the biotin-supplemented group, 5.39 +/- 0.66 in the dg 13-supplemented group, and 4.64 +/- 0.90 in the biotin-deficient group. The development of the palatal processes significantly increased in relation to the earlier day of biotin supplementation. In a protein analysis of palatal processes by isoelectro focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 19-kDa spot was confirmed around position at pI 6-7 in the biotin-supplemented group, but this protein was not present in either the biotin-deficient group or the dg 13-supplemented group. From the MS/MS database of peptides, adenosine diphosphate (ADP)-ribosylation factor 2 (arf2) and alpha-crystallin were detected in the mesenchyme of the palatal processes. It is suggested that the expression of these proteins may be downregulated by biotin deficiency, inducing the inhibited development of palatal processes.

Dietary intake of seven B vitamins based on a total diet study in Japan.

The present study estimated the dietary intake of seven B vitamins using a total diet study (TDS) in Japan. The daily intake of vitamins estimated by TDS was calculated based on the mean contents of vitamins in 18 food groups, and the amount of food intake in the Nation Health and Nutrition Survey in Japan, 2006. The estimated daily intake of these vitamins for all ages was 22.8 mg NE/d for niacin, 7.4 µg/d for vitamin B(12), 146 µg/d for folic acid, 4.52 mg/d for pantothenic acid, 1.06 mg/d for riboflavin, and 1.44 mg/d for pyridoxine. The estimated daily intake of the vitamins of niacin, vitamin B(12) and pyridoxine exceeded the dietary reference values for adults aged 18-29 y. The estimated daily intake of these vitamins by TDS was higher than the daily intake reported in the National Health and Nutrition Survey in Japan, 2006. There was a strongly positive correlation between the intake levels estimated by TDS and those reported in the National Health and Nutrition Survey. This suggests that TDS is an effective dietary survey for estimating the dietary intake of water-soluble vitamins. Therefore, when being determined by TDS, the estimated daily intake of biotin was 51.0 µg/d for all ages.

Effects of biotin deficiency on embryonic development in mice.

OBJECTIVE: The purpose of this investigation was to determine the effects of biotin deficiency on maternal metabolism and embryonic development in pregnant mouse dams. METHODS: The pregnant mice were randomly assigned to one of three dietary groups and given a biotin-deficient diet, biotin-supplemented diet, or biotin-control diet during gestation. On days of gestation (dgs) 0, 4, 8, 12, and 16, organic acids including 3-hydroxyisovaleric acid in urine were discovered by high-performance liquid chromatography, and the biotin level in the serum and urine was determined by a bioassay. On dg 18, fetuses were examined for morphologic development. RESULTS: In the biotin-deficient group, biotin excretion in urine decreased on dg 4 and was subsequently below the lower limit, whereas the urinary concentration of 3-hydroxyisovaleric acid increased after dg 12. In contrast, the biotin concentration in urine significantly increased on dgs 4, 8 and 12 in the biotin-supplemented group, but decreased on dg 16 in the biotin-supplemented and biotin-control groups. The urinary excretion of pyruvic acid in the biotin-deficient group was significantly higher than that in the biotin-supplemented group throughout the entire gestation. These concentrations in urine significantly increased on dg 16 compared with dg 0. The inhibition of embryonic development and external malformations such as cleft palate (100%), micrognathia (100%), and micromelia (91.4%) were also detected in biotin-deficient fetuses. CONCLUSION: These findings indicated that, as the requirement of biotin increases during gestation and/or embryonic development, a large amount of biotin is necessary for maintaining normal reproductive performance during the late stage of gestation.

Biotin deficiency affects the proliferation of human embryonic palatal mesenchymal cells in culture.

It has recently been demonstrated that pregnancy in women may cause mild biotin deficiency without any clinical signs. However, the teratogenicity of biotin deficiency in humans has not been well investigated. On the other hand, our previous studies have shown that maternal biotin deficiency induces many kinds of malformations, such as cleft palate, micrognathia, and micromelia, in all animal fetuses. However the mechanism for cleft palate induction under biotin-deficient conditions is unknown. Therefore, to investigate the possible mechanisms for cleft palate induction in embryos, we investigated the effects of biotin deficiency on human embryonic palatal mesenchymal (HEPM) cells in culture in this study. HEPM cells were cultured in biotin-deficient and biotin-physiological (control) media for 5 wk. The proliferative availabilities of HEPM cells in the biotin-deficient state were significantly lower after wk 2 of culture (41.3% of the control). Biotin concentrations in biotin-deficient cells were drastically lower after wk 1 of culture, whereas those in the control cells remained at almost the same level. Biotinidase activities were also lower in biotin-deficient cells. Holocarboxylases in biotin-deficient cells were fewer after the first week of culture and were almost undetectable after wk 2. The amount of biotinylated histones in the nuclei of biotin-deficient cells was lower than in the control cells. This suppressed proliferation of mesenchymal cells may delay or inhibit the growth of palatal processes in embryos and thus it may partially contribute to the mechanisms for cleft palate induction.

Vitamin B12 deficiency results in the abnormal regulation of serine dehydratase and tyrosine aminotransferase activities correlated with impairment of the adenylyl cyclase system in rat liver.

The aim of the present study was to elucidate the mechanism of the vitamin B(12) deficiency-induced changes of the serine dehydratase (SDH) and tyrosine aminotransferase (TAT) activities in the rat liver. When rats were maintained on a vitamin B(12)-deficient diet, the activities of these two enzymes in the liver were significantly reduced compared with those in the B12-sufficient control rats (SDH 2.8 (sd 0.56) v. 17.5 (sd 6.22) nmol/mg protein per min (n 5); P < 0.05) (TAT 25.2 (sd 5.22) v. 41.3 (sd 8.11) nmol/mg protein per min (n 5); P < 0.05). In the B(12)-deficient rats, the level of SDH induction in response to the administration of glucagon and dexamethasone was significantly lower than in the B(12)-sufficient controls. Dexamethasone induced a significant increase in TAT activity in the primary culture of the hepatocytes prepared from the deficient rats, as well as in the cells from the control rats. However, a further increase in TAT activity was not observed in the hepatocytes from the deficient rats, in contrast to the cells from the controls, when glucagon was added simultaneously with dexamethasone. The glucagon-stimulated production of cAMP was significantly reduced in the hepatocytes from the deficient rats relative to the cells from the control rats. Furthermore, the glucagon-stimulated adenylyl cyclase activity in the liver was significantly lower in the deficient rats than in the controls. These results suggest that vitamin B(12) deficiency results in decreases in SDH and TAT activities correlated with the impairment of the glucagon signal transduction through the activation of the adenylyl cyclase system in the liver.

Maternal vitamin B12 deficiency affects spermatogenesis at the embryonic and immature stages in rats.

To evaluate the role of cobalamin (Cbl) on spermatogenesis, the effect of dietary vitamin B(12) deficiency on early spermatogenesis was histologically investigated in male fetuses and newborns in the first filial generation (F(1) males) of rats. There was no difference in the number of gonocytes and supporting cells of Sertoli in the gonad in male fetuses on day 16 of gestation and in the testes in F(1) males at 0 days of age between vitamin B(12)-deficient (VB12-D) and vitamin B(12)-supplemented (VB12-S) groups. However, at 21 days of age, a decreased number of spermatogonia and no spermatocytes were observed in the VB12-D group. Numerous TUNEL positive cells were located among spermatocytes of the spermatogenic epithelium. The ultrastructural features examined using transmission electron microscopy were considered to be indicative of apoptosis. The incidence of seminiferous tubules having apoptotic cells was 51.5% in the VB12-D group. At 60 days of age, aplasia of the spermatids and spermatozoa was detected in the VB12-D group. In the connective tissue between the seminiferous tubules, many interstitial Leydig cells and blood vessels were observed in the VB12-D group, as compared with the VB12-S group. These changes produced by vitamin B(12) deficiency can be reversed by providing a VB12-S diet after weaning at 21 days of age. From these findings, such a vitamin B(12) deficiency during gestation and lactation could affect the germ cells and especially damage spermatocytes in F(1) male rats, which indicates that Cbl may be an essential constituent in the meiosis of spermatogenesis.

Abnormal increase in the expression level of proliferating cell nuclear antigen (PCNA) in the liver and hepatic injury in rats with dietary cobalamin deficiency.

Dietary cobalamin (Cbl; vitamin B12) deficiency resulted in severe growth retardation in rats, and body weight in the Cbl-deficient rats at 20 wk of age was significantly lower compared with the age-matched Cbl-sufficient control rats. In contrast, liver weight, when normalized to body weight, was greater in the Cbl-deficient rats than in the controls (p<0.05). The expression level of proliferating cell nuclear antigen (PCNA), a marker for cell proliferation, in the liver was significantly enhanced in the deficient rats, suggesting that cell proliferation is abnormally activated in the liver under Cbl-deficient conditions. In addition, plasma alanine aminotransferase (ALT) activity, a marker for hepatic injury, was also significantly elevated in the deficient rats. When L-carnitine, which is used clinically for the treatment of Cbl-deficient patients with methylmalonic aciduria, was administered to the Cbl-deficient rats by intraperitoneal injection twice per day for 2 wk (each 0.5 mmol), the amount of methylmalonic acid excreted into the urine was significantly reduced, and the plasma ALT activity was lowered to a normal level. However, the PCNA expression in the liver was barely influenced by the treatment with carnitine. In contrast, when the deficient rats were fed an L-methionine-supplemented diet (4 g of L-methionine per kg of the diet) for 2 wk, the increased expression of PCNA was normalized.

Measurement of 3-hydroxyisovaleric acid in urine of biotin-deficient infants and mice by HPLC.

We developed an assay for measuring urinary 3-hydroxyisovaleric acid (3-HIA) using HPLC after derivatization with 2-nitrophenylhydrazine hydrochloride (2-NPH . HCl). The derivatized 3-HIA was extracted into n-hexane and separated isocratically on a C8 reversed-phase column for fatty acids (YMC-Pack FA). We used this method to measure 3-HIA in urine extracts from mice fed a biotin-deficient diet for >4 wk and in an infant who was fed a special Japanese formula and was suspected of being biotin deficient. Urinary 3-HIA could be assayed within the range of 0.42-8.5 mmol/L with high accuracy by this method, as an indicator of biotin deficiency. Therefore, the HPLC method for 3-HIA described here may be a useful tool clinically as well as in the research laboratory.

The effects of dietary vitamin B12 deficiency on sperm maturation in developing and growing male rats.

To evaluate the role of vitamin B12 on spermatogenesis, the effects of dietary vitamin B12 deficiency on sperm maturation in developing rat fetuses and young growing rats were examined. The vitamin B12-deficient diet was given to all the animals for three different periods: whole period (gestation to mature), gestation period (gestation to weaning), or immature period (3-12 weeks postnatal). Sperm examination revealed that the sperm count was markedly lower in male progeny (F1) that were vitamin B12-deficient during the whole period. In addition, a significantly higher number of abnormal sperm, such as tailless and amorphous sperm, was observed. In male rats that were vitamin B12-deficient during the immature period, the incidence of abnormal sperms was 14.4% and 4.8% for tailless and short tail, respectively. The motion rates, such as path velocity and straight line velocity, were decreased to 20-40% of the control value in rats that were vitamin B12-deficient both during the whole and gestation periods. However, no effects of vitamin B12 deficiency on sperm motility were observed during the immature and mature periods. From these findings, we suggest that dietary vitamin B12 deficiency during pregnancy may induce irreversible damage in the germ cells of embryos and affect the maturation of spermatozoa.

Hypoxia-induced megaloblastosis in vitamin B12-deficient rats.

In rats, in contrast with human subjects who develop megaloblastic anaemia due to vitamin B12 deficiency, haematological abnormalities with anaemia were not observed under normoxic conditions even though plasma vitamin B12 concentration was reduced to <15 % of a normal concentration by depleting dietary vitamin B12. To elucidate whether erythropoiesis was affected by vitamin B12 deficiency in rats, these vitamin B12-deficient rats were exposed to hypoxia (10.5 % O2) to stimulate erythropoiesis. In the vitamin B12-sufficient control rats, erythrocyte count was significantly (P<0.05) increased 1 week after starting the hypoxic exposure. However, the hypoxia-induced erythropoiesis was affected by vitamin B12 deficiency, and no significant increase in the erythrocyte count was observed even after 6-week exposure to hypoxia in the vitamin B12-deficient rats. In the vitamin B12-deficient rats in hypoxia, erythrocytes became abnormally enlarged, and haemoglobin concentration in peripheral blood was increased in proportion to the increase of mean corpuscular volume. However, the level of the increase in the haemoglobin concentration was significantly (P<0.05) lower in the vitamin B12-deficient rats compared with that in the -sufficient controls. In addition, in the vitamin B12-deficient rats, in contrast to the -sufficient rats, serum erythropoietin concentration was not normalized even after 6-week exposure to hypoxia. These results indicate that a megaloblastic anaemia-like symptom is induced when the vitamin B12-deficient rats are exposed to hypoxia.

Characterization and bioavailability of vitamin B12-compounds from edible algae.

Substantial amounts of vitamin B12 were found in some edible algae (green and purple lavers) and algal health food (chlorella and spirulina tablets) using the Lactobacillus delbrueckii subsp. lactis ATCC7830 microbiological assay method. Corrinoid-compounds were purified and characterized from these algae to clarify the chemical properties and bioavailability of the algal vitamin B12. True vitamin B12 is the predominate cobamide of green and purple lavers and chlorella tablets. Feeding the purple laver to vitamin B12-deficient rats significantly improved the vitamin B12 status. The results suggest that algal vitamin B12 is a bioavailable source for mammals. Pseudovitamin B12 (an inactive corrinoid) predominated in the spirulina tablets, which are not suitable for use as a vitamin B12 source, especially for vegetarians. algal health food, bioavailability, cobalamin, edible algae, vitamin B12