Emerging roles for riboflavin in functional rescue of mitochondrial ß-oxidation flavoenzymes.

Riboflavin, commonly known as vitamin B2, is the precursor of flavin cofactors. It is present in our typical diet, and inside the cells it is metabolized to FMN and FAD. As a result of their rather unique and flexible chemical properties these flavins are among the most important redox cofactors present in a large series of different enzymes. A problem in riboflavin metabolism or a low intake of this vitamin will have consequences on the level of FAD and FMN in the cell, resulting in disorders associated with riboflavin deficiency. In a few number of cases, riboflavin deficiency is associated with impaired oxidative folding, cell damage and impaired heme biosynthesis. More relevant are several studies referring reduced activity of enzymes such as dehydrogenases involved in oxidative reactions, respiratory complexes and enzymes from the fatty acid ß-oxidation pathway. The role of this vitamin in mitochondrial metabolism, and in particular in fatty acid oxidation, will be discussed in this review. The basic aspects concerning riboflavin and flavin metabolism and deficiency will be addressed, as well as an overview of the role of the different flavoenzymes and flavin chemistry in fatty acid ß-oxidation, merging clinical, cellular and biochemical perspectives. A number of recent studies shedding new light on the cellular processes and biological effects of riboflavin supplementation in metabolic disease will also be overviewed. Overall, a deeper understanding of these emerging roles of riboflavin intake is essential to design better therapies.

Vitamins, minerals and supplements: part two.

Vitamins and minerals are organic food substances found only in plants and animals and are essential to the normal functioning of the body. Although only required in small amounts, as previously discussed in the past decade there has been an increased use of vitamin, mineral, herbal and nutritional supplements in the general population. While deficiencies in such nutrients can be harmful to health, conflicting claims have been made about the health benefits of such supplementation. In the second of an occasional series on vitamins, minerals, and supplements, JUNE THOMPSON gives an overview of the role that water-soluble vitamins play in the health of the individual, including their functions, and the potential impact of any deficiency of these.

Water-soluble vitamins: research update.

For more than 50 years, the Food and Nutrition Board of the National Academy of Sciences has been reviewing nutrition research and defining nutrient requirements for healthy people, referred to as the recommended dietary allowances (RDA). As new nutrition research is published, the importance of vitamins as vital nutrients is underscored, and new physiologic roles and applications to human health are examined and considered with regard to updating the RDA. Each year a substantial amount of research is published on vitamins. This article examines and summarizes noteworthy research published on individual water-soluble vitamins (excluding vitamin C) in the past 12 months, provides relevant background information on these vitamins, and offers critical reviews as appropriate.

Riboflavin, overproduced during sporulation of Ashbya gossypii, protects its hyaline spores against ultraviolet light.

Riboflavin (vitamin B2), essential in tiny amounts as a precursor for oxidoreductase coenzymes, is a yellow pigment. Although it causes cytotoxicity via photoinduced damage of macromolecules, several microorganisms are striking overproducers. A question, unanswered for decades, is whether riboflavin overproducers can benefit from this property. Here, we report an ultraviolet (UV) protective effect of riboflavin. The spores of Ashbya gossypii, a riboflavin-overproducing fungus, are more sensitive to UV than those of Aspergillus nidulans. The addition of riboflavin to suspensions improves the UV resistance of both spore types. Interestingly, we show that regulation of sporulation and riboflavin overproduction in A. gossypii are linked. In batch culture, both were elevated when growth ceased. At constant growth rates, obtained in a chemostat culture, neither was elevated. Supplementation of cultures by cAMP, a known stress signal, negatively affected sporulation as well as riboflavin overproduction, establishing a second, independent argument for the linkage.

Effect of physical activity on thiamine, riboflavin, and vitamin B-6 requirements.

Because exercise stresses metabolic pathways that depend on thiamine, riboflavin, and vitamin B-6, the requirements for these vitamins may be increased in athletes and active individuals. Theoretically, exercise could increase the need for these micronutrients in several ways: through decreased absorption of the nutrients; by increased turnover, metabolism, or loss of the nutrients; through biochemical adaptation as a result of training that increases nutrient needs; by an increase in mitochondrial enzymes that require the nutrients; or through an increased need for the nutrients for tissue maintenance and repair. Biochemical evidence of deficiencies in some of these vitamins in active individuals has been reported, but studies examining these issues are limited and equivocal. On the basis of metabolic studies, the riboflavin status of young and older women who exercise moderately (2.5-5 h/wk) appears to be poorer in periods of exercise, dieting, and dieting plus exercise than during control periods. Exercise also increases the loss of vitamin B-6 as 4-pyridoxic acid. These losses are small and concomitant decreases in blood vitamin B-6 measures have not been documented. There are no metabolic studies that have compared thiamine status in active and sedentary persons. Exercise appears to decrease nutrient status even further in active individuals with preexisting marginal vitamin intakes or marginal body stores. Thus, active individuals who restrict their energy intake or make poor dietary choices are at greatest risk for poor thiamine, riboflavin, and vitamin B-6 status.

Thiol homeostasis and supplements in physical exercise.

Thiols are a class of organic sulfur derivatives (mercaptans) characterized by the presence of sulfhydryl residues. In biological systems, thiols have numerous functions, including a central role in coordinating the antioxidant defense network. Physical exercise may induce oxidative stress. In humans, a consistent marker of exercise-induced oxidative stress is blood glutathione oxidation. Physical training programs have specific effects on tissue glutathione metabolism that depend on the work program and the type of tissue. Experimental studies show that glutathione metabolism in several tissues sensitively responds to an exhaustive bout of exercise. Study of glutathione-deficient animals clearly indicates the central importance of having adequate tissue glutathione to protect against exercise-induced oxidative stress. Among the various thiol supplements studied, N-acetyl-L-cysteine and alpha-lipoic acid hold the most promise. These agents may have antioxidant effects at the biochemical level but are also known to influence redox-sensitive cell signaling.

Vitamin B status in patients with chronic fatigue syndrome.

Some patients with chronic fatigue syndrome say they benefit from taking vitamin supplements. We assessed functional status for the B vitamins pyridoxine, riboflavin and thiamine in 12 vitamin-untreated CFS patients and in 18 healthy controls matched for age and sex. Vitamin-dependent activities--aspartate aminotransferase (AST) for pyridoxine, glutathione reductase (GTR) for riboflavin, transketolase (TK) for thiamine--were measured in erythrocyte haemolysates before and after in-vitro addition of the relevant vitamin. For all three enzymes basal activity (U/g Hb) was lower in CFS patients than in controls: AST 2.84 (SD 0.62) vs 4.61 (1.43), P < 0.001; GTR 6.13 (1.89) vs 7.42 (1.25), P < 0.04; TK 0.50 (0.13) vs 0.60 (0.07), P < 0.04. This was also true of activated values: AST 4.91 (0.54) vs 7.89 (2.11), P < 0.001; GTR 8.29 (1.60) vs 10.0 (1.80), P < 0.001; TK 0.56 (0.19) vs 0.66 (0.08), P < 0.07. The activation ratios, however, did not differ between the groups. These data provide preliminary evidence of reduced functional B vitamin status, particularly of pyridoxine, in CFS patients.

Estado nutricional con respecto a vitamina B2 en un grupo de adultos, antes y después de la suplementación / Ribofavin nutritional status of a group of adults, before and after supplementation

Se evaluó el estado nutricional con respecto a vitamina B2 (riboflavina), en 61 mujeres y 15 varones, clínicamente sanos,universitarios de la Universidad Nacional de Buenos Aires, que no tomaban suplementos vitamínicos. Se calculó la ingesta de vitamina B2 mediante una encuesta dietética de 7 días. Se determinó, en hemolizado de glóbulos rojos, el grado de estimulación de la glutation reductasa eritrocitaria o coeficiente de actividad, que aumenta en la inadecuación nutricional a valores superiores a 1,30. En los individuos con valores de inadecuación se estudió el efecto de administración de 5 mg/día de riboflavina, durante una semana. Las ingestas de vitamina B2 (mg/día) fueron, en mujeres y varones, respectivamente: 1,19 ñ 0,44 y 1,65 ñ 0,74, representando 91,5 y 97,0 por ciento de las ingestas recomendadas, siendo inferiores a éstas en 75,4 por ciento de mujeres y en 53,3 por ciento de varones. Desde el punto de vista bioquímico el porcentaje de individuos con inadecuación fue 26,2 por ciento en las mujeres y 26,7 por ciento en los varones. En estos casos el índice se normalizó luego de la administración de la riboflavina (1,51 ñ 0,14 vs. 1,13 ñ 0,14; p < 0,0001). Estos datos indican deficiencia específica de vitamina B2, de menor incidencia que la reflejada por la ingesta diaria, que se relacionó con el bajo consumo de productos lácteos

Estado nutricional con respecto a vitamina B2 en un grupo de adultos, antes y después de la suplementación / Ribofavin nutritional status of a group of adults, before and after supplementation

Se evaluó el estado nutricional con respecto a vitamina B2 (riboflavina), en 61 mujeres y 15 varones, clínicamente sanos,universitarios de la Universidad Nacional de Buenos Aires, que no tomaban suplementos vitamínicos. Se calculó la ingesta de vitamina B2 mediante una encuesta dietética de 7 días. Se determinó, en hemolizado de glóbulos rojos, el grado de estimulación de la glutation reductasa eritrocitaria o coeficiente de actividad, que aumenta en la inadecuación nutricional a valores superiores a 1,30. En los individuos con valores de inadecuación se estudió el efecto de administración de 5 mg/día de riboflavina, durante una semana. Las ingestas de vitamina B2 (mg/día) fueron, en mujeres y varones, respectivamente: 1,19 ñ 0,44 y 1,65 ñ 0,74, representando 91,5 y 97,0 por ciento de las ingestas recomendadas, siendo inferiores a éstas en 75,4 por ciento de mujeres y en 53,3 por ciento de varones. Desde el punto de vista bioquímico el porcentaje de individuos con inadecuación fue 26,2 por ciento en las mujeres y 26,7 por ciento en los varones. En estos casos el índice se normalizó luego de la administración de la riboflavina (1,51 ñ 0,14 vs. 1,13 ñ 0,14; p < 0,0001). Estos datos indican deficiencia específica de vitamina B2, de menor incidencia que la reflejada por la ingesta diaria, que se relacionó con el bajo consumo de productos lácteos (AU)

Vitamin B2-based blue-light photoreceptors in the retinohypothalamic tract as the photoactive pigments for setting the circadian clock in mammals.

In mammals the retina contains photoactive molecules responsible for both vision and circadian photoresponse systems. Opsins, which are located in rods and cones, are the pigments for vision but it is not known whether they play a role in circadian regulation. A subset of retinal ganglion cells with direct projections to the suprachiasmatic nucleus (SCN) are at the origin of the retinohypothalamic tract that transmits the light signal to the master circadian clock in the SCN. However, the ganglion cells are not known to contain rhodopsin or other opsins that may function as photoreceptors. We have found that the two blue-light photoreceptors, cryptochromes 1 and 2 (CRY1 and CRY2), recently discovered in mammals are specifically expressed in the ganglion cell and inner nuclear layers of the mouse retina. In addition, CRY1 is expressed at high level in the SCN and oscillates in this tissue in a circadian manner. These data, in conjunction with the established role of CRY2 in photoperiodism in plants, lead us to propose that mammals have a vitamin A-based photopigment (opsin) for vision and a vitamin B2-based pigment (cryptochrome) for entrainment of the circadian clock.

Antioxidative vitamins in prematurely and maturely born infants.

Postnatally a rapid change occurs from a relatively hypoxic to a relatively hyperoxic environment, especially during artificial ventilation with all risks of ROS-formation. Among the non enzymatic antioxidative strategies the vitamins E, C, A and B2 are of major importance. Vitamin E is considered the most important radical scavenging vitamin of the lipid soluble compartment. Hereby vitamin E itself is converted into a radical which is handed over to vitamin C and glutathione into the water soluble compartment. The vitamin E content of the fetus increases with the fetal fat mass mainly during the last trimester of pregnancy. Placenta is only slightly permeable to lipid soluble vitamins. Vitamin E deficiency may rapidly develop typically at about 6-8 weeks of age. Vitamin E is able to prolong significantly the onset of retinopathic changes during oxygen therapy and may prevent intraventricular hemorrhage. Vitamin C is together with glutathione a major representative of the non enzymatic antioxidative system in the water soluble compartment. The best determinant of the vitamin C status is its concentration in leukocytes. Vitamin C reduces iron to the divalent state which supports the hydroxyl radical formation (Haber-Weiss reaction). This should be considered mainly in cases of intraventricular hemorrhage. Vitamin B2 acts mainly as cofactor of glutathione reductase which keeps glutathione in the reduced state. It can therefore be considered an indirect antioxidative vitamin. Vitamin B2 is destroyed by light. Phototherapy has been recognized as a cause of riboflavin deficiency. Vitamin A comprises all retinols with properties like trans-retinol. Retinol storage in the fetal liver increases during late pregnancy. In both, premature and mature newborns, the serum concentrations amount to only about 50% of those of their mothers. Vitamin A has a paramount importance for fetal lung development, because the individual surfactant proteins are selectively regulated by retinoic acid.

In vivo effect of dietary factors on the molecular action of aflatoxin B1: role of riboflavin on the catalytic activity of liver fractions.

Weanling rats were kept on a synthetic riboflavin-free diet for 4 weeks, and subsequently on the same diet but supplemented with riboflavin for 2 weeks. The ability of liver microsomes to catalyze reactions of aflatoxin B1 (AFB1) leading to its activation and DNA adduct formation was measured after each period of experimental feeding. A decrease in both activities was evident during riboflavin deficiency, and this could be restored after normal supply of the vitamin. The decrease was attributed to a fall in the endogenous flavin content, specifically the coenzyme flavin adenine dinucleotide which forms an integral part of the microsomal monooxygenase that catalyzes the activation reactions. The vitamin and its coenzymes, however, inhibit the microsomal enzyme activity when added in excess in the in vitro system. It is envisaged that riboflavin may play a role in regulating the carcinogenic activity of AFB.

Partial characterization of the microsomal and solubilized hypothalamic progesterone 5 alpha-reductase.

Microsomal progesterone 5 alpha-reductase activity from female rat hypothalamus has been solubilized and partially characterized in terms of kinetic and physical properties. The solubilization of progesterone 5 alpha-reductase has been accomplished through the use of a digitonin/KCL-extraction. Both the microsomal and solubilized enzyme activities exhibit similar kinetic and physical characteristics. These include their apparent Km for progesterone (Microsomal Km = 113 +/- 11 nM; solubilized Km = 144 +/- 20 nM) and their affinity (approximately 7 nM) for the 5 alpha-dihydroprogesterone analog, 4-aza-4-methyl-5 alpha-pregnane-3,20-dione, which is a potent inhibitor of progesterone 5 alpha-reduction. Both activities are inhibited by divalent cations (Zn2+ and Cu2+) and the sulfhydryl-blocking agent p-chloromercuribenzoic acid. Studies aimed at optimizing isolation and assay conditions for the hypothalamic progesterone 5 alpha-reductase indicate that the microsomal activity is enhanced in the presence of monovalent cations (particularly K+ and Li+) and the metal chelator EDTA, but is unaffected by the sulfhydryl reducing agent dithiothreitol. The activity of the solubilized enzyme is also enhanced by EDTA but slightly stimulated by dithiothreitol. Analysis of hypothalamic progesterone 5 alpha-reduction for possible flavin involvement (as a hydride carrier between NADPH and the steroid) indicates that the enzyme activity is decreased by high levels of flavins, flavin analogs and riboflavin deficiency.

Role of placenta in maternal-fetal vitamin transfer in humans.

Vitamins B12, B6, biotin, folate, thiamine, riboflavin, pantothenate, and nicotinate were determined in maternal and fetal blood and placental tissue of normovitaminemic and hypovitaminemic mothers who disclaimed supplemental vitamin intake during pregnancy. No biotin or pantothenate deficits were observed in the gravidas. Hypovitaminemic mothers transferred less B12, folate, and B6 to the fetus and placenta than normovitaminemic mothers. Vitamins given by mouth increased maternal fetal, and placental levels of folate, but B6 increased only in maternal blood and the placenta; biotin and pantothenate increased only in fetal blood. Except for riboflavin, nicotinate, and pantothenate, the intramuscular administration of vitamins increased the levels of other vitamins in maternal and fetal blood and placental tissue. Results suggest that the placenta stores vitamins and the tissue vitamin receptors must be saturated before adequate transfer of vitamins to the fetus occurs.

New concepts in phototherapy: photoisomerization of bilirubin IX alpha and potential toxic effects of light.

New information is summarized, indicating that configurational photoisomerization of bilirubin at the 5 and 15 carbon bridges is the major mechanism of bilirubin photocatabolism in vivo, and that singlet oxygen photooxidation plays only a minor role. The literature is reviewed concerning potentially damaging photodynamic reactions that are observed in vitro with vitamins, proteins, lipids, and nucleic acids, and their possible relationships to the limited number of toxic side-effects that have been detected with clinical phototherapy of neonatal jaundice. Secondary toxic effects, mediated by bilirubin photoderivatives or by retina-neuroendocrine pathways are also considered. Areas requiring further investigations are delineated.

Riboflavin requirement for the cultivation of axenic Entamoeba histolytica.

Riboflavin was found to be essential for the cultivation of axenic Entamoeba histolytica. This is the first demonstration of a flavin requirement by the organism. Panmede, the principal source of flavins in the axenic medium, was treated with activated carbon to remove flavins. Medium made with this flavin-deficient Panmede, and supplemented with ribonucleic acid failed to support the multiplication of amebae in serial subculture, but did so when riboflavin was added. The concentration of riboflavin required to achieve maximal growth was about 1.3 microgram per ml medium. Studies on riboflavin uptake revealed that amebae lack a high-affinity transport system for this vitamin. The rate of riboflavin uptake was equivalent to the rate of pinocytotic uptake of fluid as previously determined.