We are developing a technology based on the combined application of riboflavin (RB) and light for inactivating pathogens in blood products while retaining the biological functions of the treated cells and proteins. Virus and bacteria reduction measured by tissue culture infectivity or colony formation with UV light alone and in combination with RB yield equivalent results. The effects of RB as a sensitizing agent on DNA in white cells, bacteria and viruses in combination with UV light exposure have been evaluated. UV-mediated DNA degradation in Jurkat T cells and leukocytes in plasma as measured by the FlowTACS assay was significantly increased in the presence of RB. Agarose gel electrophoretic analysis of DNA in Escherichia coli and leukocytes in plasma demonstrated enhanced DNA degradation in the presence of RB. UV light in combination with RB prevented the reactivation of lambda phage compared with samples irradiated in the absence of RB. UV-mediated oxidative damage in calf thymus DNA was also enhanced in the presence of RB. These observations clearly demonstrate that the presence of RB and UV light selectively enhances damage to the guanine bases in DNA. These data also suggest that the type and extent of damage to DNA for virus in the presence of RB and light make it less likely to be repaired by normal repair pathways available in host cells.

OBJECTIVE

To evaluate the 1-year results of corneal collagen cross-linking (CXL) in eyes with postoperative excimer laser refractive surgery corneal ectasia.

METHODS

Thirteen eyes of 9 consecutive patients who had undergone excimer laser refractive surgery (photorefractive keratectomy [n = 3], LASIK [n = 10]) with resultant unstable corneal ectasia underwent CXL with photosensitizing riboflavin 0.1% solution and subsequent exposure to ultraviolet radiation. Study eyes underwent complete ophthalmologic examination, endothelial specular microscopy, corneal topography, and aberrometry as well as central pachymetry and Scheimpflug-based topo/tomography preoperatively and at 3-, 6-, and 12-month intervals.

RESULTS

Best spectacle-corrected visual acuity (BSCVA) improvement was statistically significant (P < .05) beyond 6 months after surgery (improvement of 0.1 logMAR at 1 year). Mean spherical equivalent refraction and mean refractive sphere reduction (improvement of 1.40 and 1.44 diopters [D], respectively) were statistically significant (P < .05) at 6 months postoperatively. At 1 year after CXL, mean endothelial cell count and keratometry (average SimK decrease of 2.02 D) as well as Klyce and Ambrósio indices did not deteriorate. Coma and spherical aberration did not change significantly. Mean pupil center pachymetry and corneal thickness at 0 and 2 mm from the thinnest corneal point decreased significantly.

CONCLUSIONS

One year after surgery, CXL appears to stabilize eyes with ectasia consequent to excimer laser refractive surgery and improve BSCVA.

OBJECTIVE

To determine the efficacy for migraine prophylaxis of a compound containing a combination of riboflavin, magnesium, and feverfew.

BACKGROUND

Previous studies of magnesium and feverfew for migraine prophylaxis have found conflicting results, and there has been only a single placebo-controlled trial of riboflavin.

METHODS

Randomized double-blind placebo-controlled trial of a compound providing a daily dose of riboflavin 400 mg, magnesium 300 mg, and feverfew 100 mg. The placebo contained 25 mg riboflavin. The study included a 1-month run-in phase and 3-month trial. The protocol allowed for 120 patients to be randomized, with a preplanned interim analysis of the data after 48 patients had completed the trial.

RESULTS

Forty-nine patients completed the 3-month trial. For the primary outcome measure, a 50% or greater reduction in migraines, there was no difference between active and "placebo" groups, achieved by 10 (42%) and 11 (44%), respectively (P=.87). Similarly, there was no significant difference in secondary outcome measures, for active versus placebo groups, respectively: 50% or greater reduction in migraine days (33% and 40%, P=.63); or change in mean number of migraines, migraine days, migraine index, or triptan doses. Compared to baseline, however, both groups showed a significant reduction in number of migraines, migraine days, and migraine index. This effect exceeds that reported for placebo agents in previous migraine trials.

CONCLUSIONS

Riboflavin 25 mg showed an effect comparable to a combination of riboflavin 400 mg, magnesium 300 mg, and feverfew 100 mg. The placebo response exceeds that reported for any other placebo in trials of migraine prophylaxis, and suggests that riboflavin 25 mg may be an active comparator. There is at present conflicting scientific evidence with regard to the efficacy of these compounds for migraine prophylaxis.

Furfural and acetic acid are two prevalent inhibitors to microorganisms during cellulosic ethanol production, but molecular mechanisms of tolerance to these inhibitors are still unclear. In this study, genome-wide transcriptional responses to furfural and acetic acid were investigated in Saccharomyces cerevisiae using microarray analysis. We found that 103 and 227 genes were differentially expressed in the response to furfural and acetic acid, respectively. Furfural downregulated genes related to transcriptional control and translational control, while it upregulated stress-responsive genes. Furthermore, furfural also interrupted the transcription of genes involved in metabolism of essential chemicals, such as etrahydrofolate, spermidine, spermine, and riboflavin monophosphate. Acetic acid downregulated genes encoding mitochondrial ribosomal proteins and genes involved in carbohydrate metabolism and regulation and upregulated genes related to amino acid metabolism. The results revealed that furfural and acetic acid had effects on multiple aspects of cellular metabolism on the transcriptional level and that mitochondria might play important roles in response to both furfural and acetic acid. This research has provided insights into molecular response to furfural and acetic acid in S. cerevisiae, and it will be helpful to construct more resistant strains for cellulosic ethanol production.

High levels of conversion of 14C-labelled pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA) were obtained in vivo in Streptomyces pristinaespiralis and in some other streptogramin A producers. This established that PIIB was an intermediate on the pathway to PIIA. In addition, in vitro studies with cell-free protein preparations demonstrated that the oxidation of PIIB to PIIA is a complex process requiring NADH, riboflavin 5'-phosphate (FMN), and molecular oxygen. Two enzymes were shown to be necessary to catalyze this reaction. Both were purified to homogeneity from S. pristinaespiralis by a coupled enzyme assay based on the formation of PIIA and by requiring addition of the complementing enzyme. One enzyme was purified about 3,000-fold by a procedure including a decisive affinity chromatography step on FMN-agarose. It was shown to be a NADH:FMN oxidoreductase (E.C. 1.6.8.1.) (hereafter called FMN reductase), providing reduced FMN (FMNH2) to the more abundant second enzyme. The latter was purified only 160-fold and was called PIIA synthase. Our data strongly suggest that this enzyme catalyzes a transient hydroxylation of PIIB by molecular oxygen immediately followed by a dehydration leading to PIIA. The native PIIA synthase consists of two different subunits with Mrs of around 50,000 and 35,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the FMN reductase seems to be a monomer with a Mr of around 28,000 and containing one molecule of tightly bound FMN. Stepwise Edman degradation of the entire polypeptides or some of their trypsin-digested fragments provided amino acid sequences for the two isolated proteins.

We tested the hypothesis that methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, folic acid deficiency and riboflavin deficiency, independently or interactively, are important determinants of genomic stability, cell death, cell proliferation and homocysteine (Hcy) concentration in 9-d human lymphocyte cultures. Lymphocytes of seven wild-type (CC) and seven mutant (TT) homozygotes were cultured under the four possible combinations of deficiency and sufficiency of riboflavin (0 and 500 nmol/L) and folic acid (20 and 100 nmol/L) at a constant L-methionine concentration of 50 micromol/L. Viable cell growth was 25% greater in TT than in CC cells (P<0.05) and 32% greater at 100 nmol/L folic acid than at 20 nmol/L folic acid (P=0.002). The comprehensive cytokinesis-block micronucleus assay was used to measure micronuclei (MNi; a marker for chromosome breakage and loss), nucleoplasmic bridges (NPB; a marker of chromosome rearrangement) and nuclear buds (NBUD, a marker of gene amplification). The MNi levels were 21% higher in TT cells than in CC cells (P<0.05) and 42% lower in the high folic acid medium than in the low folic acid medium (P<0.0001). The NBUD levels were 27% lower in TT cells than in CC cells (P<0.05) and 45% lower in the high folic acid medium than in the low folic acid medium (P<0.0001). High riboflavin concentration (500 nmol/L) increased NBUD levels by 25% (compared with 0 nmol/L riboflavin) in folate-deficient conditions (20 nmol/L folic acid medium; P<0.05), and there was an interaction between folic acid and riboflavin that affected NBUD levels (P=0.042). This preliminary investigation suggests that MTHFR C677T polymorphism and riboflavin affect genome instability; however, the effect is relatively small compared with that of folic acid.

This study was designed to identify the secular trend in the age at menarche and to investigate the possible factors that influence the age at menarche using representative Korean data from the 2005 Korean National Health and Nutrition Survey. Three thousand five hundred sixty-two women born between 1920 and 1985 were enrolled to identify secular trends in the age at menarche and 620 girls born between 1986 and 1995 were recruited to evaluate the factors influencing the age at onset of menarche. Mean age at menarche decreased from 16.90 +/- 1.25 years for women born between 1920 and 1925 to 13.79 +/- 1.37 years for those born between 1980 and 1985, indicating a downward trend of 0.68 years per decade (95% CI, 0.64-0.71) in age at menarche. Mean age at menarche of girls born between 1986 and 1995 was 13.10 +/- 0.06 years as estimated by the Kaplan-Meier method. Among girls born between 1986 and 1995, menarcheal girls had a larger waist circumference, a higher body mass index (BMI), and lower maternal menarcheal age and maternal age at birth than premenarcheal girls. The energy and nutrient intake of protein, sugar, fiber, ash, phosphate, natrium, thiamine, riboflavin, and niacin were greater for menarcheal girls than for premenarcheal girls. These data indicate a decreasing secular trend of age at menarche in a Korean population born between 1920 and 1995. Furthermore, maternal menarcheal age, BMI, maternal age at birth, and nutrition are important variables that appear to influence age at menarche in Korean girls.

ABCG2 is an ATP-binding cassette (ABC) transporter preferentially expressed by immature human hematopoietic progenitors. Due to its role in drug resistance, its expression has been correlated with a protection role against protoporhyrin IX (PPIX) accumulation in stem cells under hypoxic conditions. We show here that zinc mesoporphyrin, a validated fluorescent heme analog, is transported by ABCG2. We also show that the ABCG2 large extracellular loop ECL3 constitutes a porphyrin-binding domain, which strongly interacts with heme, hemin, PPIX, ZnPPIX, CoPPIX, and much less efficiently with pheophorbide a, but not with vitamin B12. K(d) values are in the range 0.5-3.5 μm, with heme displaying the highest affinity. Nonporphyrin substrates of ABCG2, such as mitoxantrone, doxo/daunorubicin, and riboflavin, do not bind to ECL3. Single-point mutations H583A and C603A inside ECL3 prevent the binding of hemin but hardly affect that of iron-free PPIX. The extracellular location of ECL3 downstream from the transport sites suggests that, after membrane translocation, hemin is transferred to ECL3, which is strategically positioned to release the bound porphyrin to extracellular partners. We show here that human serum albumin could be one of these possible partners as it removes hemin bound to ECL3 and interacts with ABCG2, with a K(d) of about 3 μm.

We examined the relationship between dietary intake and cognitive performance in Korean elderly people. Data for dietary intake, anthropometric measurements and cognitive function tests were collected and the relationships of the variables were analyzed. A random sample of 210 men and 239 women in Korea, aged 60 and over, was selected. Subjects were free-living elderly people who had not experienced major cognitive function impairment. Main outcome measures, 24 h dietary recall method, food behaviour variables, anthropometrics indices, health variables, and Kwon's Mini-Mental State Examination for Koreans (MMSE-K) for cognitive function test. The prevalence rate of poor cognitive function (MMSE-K score < or = 19) of Korean elderly was 22.3%: women with poor cognitive function had a higher rate (31.0%) than that in men (12.3%). Cognitive ability was related negatively with age and positively with school education level. Female subjects of poor cognitive function had significantly lower intakes of total amount of foods, cereals, vegetables, fruits, milk, spices, and also, energy, protein, fat, carbohydrate, Ca, P, Fe, vitamin A, thiamin, riboflavin, and niacin than those of the normal cognitive score >> or = 24) group (P < 0.05). Male subjects of poor cognitive function had significantly lower intakes of fruits, fiber, and vitamin C than the normal subjects (P < 0.05). The MMSE-K score of female subjects showed a significant positive correlation with total amount of foods, cereals, beans, fruits, milk, oil, spices, and energy, protein, fat, carbohydrate, Ca, Fe, P, riboflavin and niacin intakes. The consumption of adequate nutrients, by taking sufficient amounts and variety of foods, may be important in maintaining adequate cognitive function in elderly Koreans.

Mucosal-associated invariant T (MAIT) cells recognize microbial compounds presented by the MHC-related 1 (MR1) protein. Although riboflavin precursor derivatives from Gram-positive bacteria have been characterized, some level of ligand heterogeneity has been suggested through the analysis of the MAIT cell TCR repertoire in humans and differential reactivity of human MAIT cell clones according to the bacteria. In this study, using Gram-negative bacteria mutated for the riboflavin biosynthetic pathway, we show a strict correlation between the ability to synthesize the 5-amino-ribityl-uracil riboflavin precursor and to activate polyclonal and quasi-monoclonal mouse MAIT cells. To our knowledge, we show for the first time that the semipurified bacterial fraction and the synthetic ligand activate murine MAIT cells in vitro and in vivo. We describe new MR1 ligands that do not activate MAIT cells but compete with bacterial and synthetic compounds activating MAIT cells, providing the capacity to modulate MAIT cell activation. Through competition experiments, we show that the most active synthetic MAIT cell ligand displays the same functional avidity for MR1 as does the microbial compound. Altogether, these results show that most, if not all, MAIT cell ligands found in Escherichia coli are related to the riboflavin biosynthetic pathway and display very limited heterogeneity.

Ashbya gossypii is a riboflavin-overproducing filamentous fungus that is closely related to unicellular yeasts such as Saccharomyces cerevisiae. With its close ties to yeast and the ease of genetic manipulation in this fungal species, A. gossypii is well suited as a model to elucidate the regulatory networks that govern the functional differences between filamentous growth and yeast growth, especially now that the A. gossypii genome sequence has been completed. Understanding these networks could be relevant to related dimorphic yeasts such as the human fungal pathogen Candida albicans, in which a switch in morphology from the yeast to the filamentous form in response to specific environmental stimuli is important for virulence.

Vitamin B2 (riboflavin) is an essential dietary compound used for the enzymatic biosynthesis of FMN and FAD. The human genome contains 90 genes encoding for flavin-dependent proteins, six for riboflavin uptake and transformation into the active coenzymes FMN and FAD as well as two for the reduction to the dihydroflavin form. Flavoproteins utilize either FMN (16%) or FAD (84%) while five human flavoenzymes have a requirement for both FMN and FAD. The majority of flavin-dependent enzymes catalyze oxidation-reduction processes in primary metabolic pathways such as the citric acid cycle, β-oxidation and degradation of amino acids. Ten flavoproteins occur as isozymes and assume special functions in the human organism. Two thirds of flavin-dependent proteins are associated with disorders caused by allelic variants affecting protein function. Flavin-dependent proteins also play an important role in the biosynthesis of other essential cofactors and hormones such as coenzyme A, coenzyme Q, heme, pyridoxal 5'-phosphate, steroids and thyroxine. Moreover, they are important for the regulation of folate metabolites by using tetrahydrofolate as cosubstrate in choline degradation, reduction of N-5.10-methylenetetrahydrofolate to N-5-methyltetrahydrofolate and maintenance of the catalytically competent form of methionine synthase. These flavoenzymes are discussed in detail to highlight their role in health and disease.

OBJECTIVE

Malnutrition is observed frequently in patients with inflammatory bowel disease (IBD). Knowledge of the nutritional status in patients with recently diagnosed IBD is limited. The aim of this study was to establish a comprehensive picture of the nutritional status in recently diagnosed IBD patients.

METHODS

Sixty-nine IBD patients (23 Crohn's disease (CD) and 46 with ulcerative colitis (UC)) within 6 months of diagnosis and 69 age- and sex-matched population controls were included in the study.

METHODS

The nutritional status was assessed by: (1) body composition (anthropometry and dual-energy X-ray absorptiometry); (2) dietary intake (dietary history); (3) biochemical indexes of nutrition; and (4) muscle strength (isokinetic dynamometer).

RESULTS

Body weight and body mass index were significantly lower in UC patients compared with controls. The mean daily intake of carbohydrates was significantly higher in CD patients and the intakes of protein, calcium, phosphorus, and riboflavin were significantly lower in UC patients compared with controls, respectively. Serum concentrations of several nutrients (beta-carotene, magnesium, selenium and zinc) were significantly lower in UC patients compared with controls. Serum vitamin B12 concentration was significantly lower in CD patients. Muscle strength did not significantly differ between IBD patients and controls.

CONCLUSIONS

This study showed that the nutritional status of IBD patients was already affected negatively at time of diagnosis. It needs to be elucidated whether nutritional supplementation in recently diagnosed IBD patients may improve the clinical course of the disease.

How early-life colonization and subsequent exposure to the microbiota affect long-term tissue immunity remains poorly understood. Here, we show that the development of mucosal-associated invariant T (MAIT) cells relies on a specific temporal window, after which MAIT cell development is permanently impaired. This imprinting depends on early-life exposure to defined microbes that synthesize riboflavin-derived antigens. In adults, cutaneous MAIT cells are a dominant population of interleukin-17A (IL-17A)-producing lymphocytes, which display a distinct transcriptional signature and can subsequently respond to skin commensals in an IL-1-, IL-18-, and antigen-dependent manner. Consequently, local activation of cutaneous MAIT cells promotes wound healing. Together, our work uncovers a privileged interaction between defined members of the microbiota and MAIT cells, which sequentially controls both tissue-imprinting and subsequent responses to injury.

With application of molecular biology techniques, there has been rapid progress in understanding how many drugs and micronutrients (e.g., vitamins) are transferred across the choroid plexus (CP), the main transport locus of the blood-cerebrospinal fluid (CSF) barrier, and the renal tubular epithelial cells. In many cases, these molecules are transported by separate, specific carriers or receptors on the apical and/or basal side of the CP or renal epithelial cells. This commentary focuses on four micronutrient transport systems in CP (ascorbic acid, folate, inositol, and riboflavin), all of which have been recently cloned, expressed and for which knockout mice models were developed and transporter localization studies performed. Also reviewed is the recently cloned uric acid transport system in human kidney in which there exists a human "knockout" model. The implications of these transport systems for drug therapy of central nervous system and renal disorders are discussed, especially with regard to methods to circumvent the blood-brain and blood-CSF barriers to deliver drugs to the brain.

OBJECTIVE

The aims of this study were to determine the prevalence of protein-calorie malnutrition, characteristics, and clinical importance of nutrition disorders in patients with liver cirrhosis according to severity of disease.

METHODS

Nutrition assessments such as subjective global assessment, anthropometric and biochemical measurements, immunocompentency, thiamin and riboflavin status in 60 patients with cirrhosis (33 male and 27 female) were recorded between June 1999 and December 1999 at an outpatient clinic at Ramathibodi Hospital, Bangkok, Thailand. The origin of liver disease was alcohol related in 50% of patients. Child-Pugh criteria were used to establish the severity of liver disease.

RESULTS

In terms of energy malnutrition, 13.3% of patients had ideal body weights below 90% and 11.7% had body mass indexes below 18.5 kg/m(2). Protein malnutrition (low albumin) and immunoincompetence (abnormal response to skin tests) were found much more frequently (45% and 22%) than energy malnutrition. Patients with alcoholic cirrhosis had ascites (P < 0.05) and hepatic encephalopathy (P < 0.001) more frequently and less triceps skinfold thickness than those with non-alcoholic cirrhosis. Subjective global assessment and serum proteins correlated with the degree of liver-function impairment, but immunologic tests correlated inversely in cirrhosis patients. Mean values for creatinine-height index, hemoglobin, cholesterol, and complement C4 showed significant decreases in severe liver failure (Child-Pugh class C) only in patients with alcoholic cirrhosis (P < 0.05). Malnutrition was correlated with the clinical severity of liver disease.

CONCLUSIONS

The study showed that protein-energy malnutrition is a common complication of liver cirrhosis. Nutritional disorders appeared to be related to the degree of liver injury and the etiology of nutritional disorders. Nutritional disorders were more severe with alcoholic cirrhosis than with non-alcoholic cirrhosis.

When amino acids were infused at a rate of 4 g/kg/day, an infant with hypoglycemia, metabolic acidemia and chronic regurgitation showed hypersarcosinemia and excreted abnormal amounts of sarcosine, isovalerylglycine, isobutyrylglycine, alpha-methylbutyrylglycine, and beta-hydroxyisovaleric, glutaric, alpha-hydroxyglutaric, methylsuccinic, and alpha-hydroxyisobutyric acids in urine. On all other occasions, when protein intake was lower and lipid intake higher, urine organic acids were dominated by methylsuccinic, ethylmalonic, and alpha-hydroxyglutaric acids, and hypersarcosinemia was absent. Autopsy showed severe fatty changes in liver, kidneys, and skeletal muscle. A previous female sibling had died with similar autopsy findings at 4 days of age. While activity of glutaryl-CoA dehydrogenase was completely deficient in liver and almost completely so in kidney, it was normal in cultured fibroblasts in the presence of flavin adenine dinucleotide (FAD) and only marginally low in its absence. Incorporation of D-(2-14C) riboflavin into flavin mononucleotides (FMN) and FAD by kidney tissue was normal. The authors conclude that this disorder is not due to generalized deficiency of glutaryl-CoA dehydrogenase or to a defect in FAD synthesis. The amino and organic acid abnormalities noted are most consistent with a defect in the flavoprotein which transfers electrons from the FAD of sarcosine and acyl-CoA dehydrogenases into the respiratory chain, although a defect in intercompartmental transfer of C4--5 acyl CoA esters across cell membranes is not excluded. The variability of the organic aciduria, which possibly reflects changes in protein and fat intake, suggests that a previous name for this disorder, i.e., glutaric aciduria type II, is inappropriate and should be replaced, perhaps by "multiple acyl-CoA dehydrogenase deficiency."

OBJECTIVE

To compare the diet quality and weight status of girls consuming diets meeting the recommendation of the American Academy of Pediatrics for dietary fat with those of girls consuming >30% of energy from fat and to examine relationships between girls' dietary fat intake, mothers' nutrient intakes, and mothers' child-feeding practices.

METHODS

Participants were 192 white girls and their mothers, who were divided into 2 groups: >30% of energy from fat (high fat [HF]) or </=30% of energy from fat (low fat [LF]), based on girls' 3-day dietary recalls. Girls' food group and nutrient intakes, Healthy Eating Index, body mass index, and mothers' nutrient intakes and child-feeding practices were compared.

RESULTS

Girls with HF diets consumed fewer fruits, more meat, and more fats and sweets and had lower Healthy Eating Index scores than did the girls in the LF group. Mothers of girls in the HF group had higher fat intakes than did those in the LF group. Girls and mothers in the HF group had lower intakes of fiber and vitamins A, C, B6, folate, and riboflavin. Mothers in the HF group reported using more restriction and pressure to eat in feeding their daughters. Girls in the HF group showed greater increase in body mass index and skinfold thickness from age 5 to 7 years.

CONCLUSIONS

These findings provide additional support for the recommendation of the American Academy of Pediatrics to limit total dietary fat. Findings reveal that mothers' use of controlling feeding practices are not effective in fostering healthier diets among girls and that mothers' own eating may be more influential than their attempts to control the intake of their daughters. dietary fat, dietary quality, nutrient intake, overweight, feeding practices, children.

Given the high prevalence of micronutrient deficiencies and infectious diseases in infants in developing countries, an evaluation of the efficacy of different micronutrient formulations on infant morbidity is a priority. The efficacy of weekly supplementation of four different micronutrient formulations on diarrhea and acute lower respiratory infection (ALRI) morbidity was evaluated in Bangladeshi infants. In a double-blind, randomized, controlled community trial, 799 infants aged 6 mo were randomly assigned to one of the following 5 groups: 1) 20 mg elemental iron with 1 mg riboflavin, 2) 20 mg elemental zinc with 1 mg riboflavin, 3) 20 mg iron and 20 mg zinc with 1 mg riboflavin, 4) a micronutrient mix (MM) containing 20 mg iron, 20 mg zinc, 1 mg riboflavin along with other minerals and vitamins and 5) a control treatment, 1 mg riboflavin only. Health workers visited each infant weekly until age 12 mo to feed the supplement and to collect data on diarrhea and ALRI morbidity. Hemoglobin, serum ferritin and serum zinc levels of a sample of infants were measured at 6 and 12 mo. Compared with the control group, at 12 mo, serum ferritin levels were higher in the iron + zinc group, and serum zinc levels were higher in the zinc and iron + zinc groups. Simultaneous supplementation with iron + zinc was associated with lower risk of severe diarrhea, 19% lower in all infants and 30% lower in less well-nourished infants with weight-for-age Z-score below -1. Iron + zinc supplementation was also associated with 40% lower risk of severe ALRI in less well-nourished infants. MM supplementation was associated with a 15% higher risk of diarrhea in all infants and 22% higher risk in less well-nourished infants. Intermittent simultaneous supplementation with iron + zinc seems promising; it will be useful to determine whether higher doses would provide greater benefits.

Compared with other regions in the United States, the southern region has had the highest stroke mortality rate and a more prevalent and resistant hypertension. We designed this analysis of the data obtained from the National Health and Nutritional Examination Survey III (NHANES-III), which is a community-based cross-sectional survey, to describe regional variations in blood pressure and the reported consumption of nutrients, focusing on those linked to blood pressure, in the United States. We selected the following variables from the NHANES-III data for this analysis: systolic and diastolic blood pressures, protein, carbohydrates, total fatty acids, saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, cholesterol, fiber, sodium, potassium, calcium, magnesium, zinc, copper, iron, riboflavin, niacin, thiamin, alcohol and vitamins C, E, B-6 and B-12. Of the 17,752 participants in the survey who were 18 y of age or older, the south had the highest systolic and diastolic blood pressures (P < 0.005 for each) and reported the highest consumption of monounsaturated fatty acids, polyunsaturated fatty acids and cholesterol (P < 0.05 for all) and the least amount of fiber in the multivariate analysis (P < 0.005). The highest reported sodium consumption was in the south region (3.4 +/- 0.02 g), and the lowest was in the west (3.2 +/- 0.03 g; P < 0.05). The south also consumed the least potassium, calcium, phosphorous, magnesium, copper, riboflavin, niacin, iron and vitamins A, C and B-6 (P < 0.005). There was no difference among the four regions in frequency of "adding salt on the table." The region of the United States that includes the "stroke belt" has dietary patterns that may contribute to the high prevalence of hypertension and cardiovascular disease.

Measurement and quantitation of superoxide by electron paramagnetic resonance (EPR) using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) have been limited by the short half-life of the superoxide adduct DMPO-OOH (approximately 50 s at pH 7). Recently a beta-phosphorylated nitrone, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), was developed and reported to form a more stable superoxide adduct with a half-life of approximately 15 min. We evaluated the use of DEPMPO for quantitative measurement of superoxide in chemical and biochemical systems. To estimate the efficiency of trapping, EPR oximetry was used to measure oxygen consumption and the intensity of the DEPMPO-OOH signal to measure superoxide generation and adduct decay. With the superoxide-generating systems, riboflavin/light and xanthine/xanthine oxidase, DEPMPO trapped approximately 65% of the superoxide produced. The efficiency of superoxide trapping by DEPMPO was compared to the commonly used cytochrome c reduction method. When superoxide production was>> 20 microM, cytochrome c detected approximately 100% of the superoxide produced, while DEPMPO trapped 60 to 70%. However, EPR detection with DEPMPO was 40-fold more sensitive than cytochrome c. Thus, DEPMPO is an efficient spin trap which enables specific and sensitive detection and quantitation of superoxide generation.

This study describes the characterization of the riboflavin transport protein RibU in the lactic acid bacterium Lactococcus lactis subsp. cremoris NZ9000. RibU is predicted to contain five membrane-spanning segments and is a member of a novel transport protein family, not described in the Transport Classification Database. Transcriptional analysis revealed that ribU transcription is downregulated in response to riboflavin and flavin mononucleotide (FMN), presumably by means of the structurally conserved RFN (riboflavin) element located between the transcription start site and the start codon. An L. lactis strain carrying a mutated ribU gene exhibits altered transcriptional control of the riboflavin biosynthesis operon ribGBAH in response to riboflavin and FMN and does not consume riboflavin from its growth medium. Furthermore, it was shown that radiolabeled riboflavin is not taken up by the ribU mutant strain, in contrast to the wild-type strain, directly demonstrating the involvement of RibU in riboflavin uptake. FMN and the toxic riboflavin analogue roseoflavin were shown to inhibit riboflavin uptake and are likely to be RibU substrates. FMN transport by RibU is consistent with the observed transcriptional regulation of the ribGBAH operon by external FMN. The presented transport data are consistent with a uniport mechanism for riboflavin translocation and provide the first detailed molecular and functional analysis of a bacterial protein involved in riboflavin transport.

Formation of DNA-protein cross-links involving the initial formation of a guanine radical cation was investigated. For this purpose, riboflavin-mediated photosensitization of a TGT oligonucleotide in aerated aqueous solution in the presence of the KKK tripeptide was performed. We have shown that the nucleophilic addition of the epsilon-amino group of the central lysine residue of KKK to the C8 atom of either the guanine radical cation or its deprotonated form gives rise to the efficient formation of a Nepsilon-(guanin-8-yl)-lysine cross-link. Interestingly, the time course of formation of the above-mentioned cross-link was found to be not linear with the time of irradiation, and its formation rapidly reached a plateau. This is explained by secondary decomposition of the initially generated cross-link which could be further oxidized more efficiently than starting TGT oligonucleotide. One-electron oxidation of the initially generated cross-link was found to produce mainly two diastereomeric cross-links exhibiting a spiroimino-trilysine-dihydantoin structure as inferred from enzymatic digestion, CD, UV, NMR and mass spectrometry measurements. In addition, other minor cross-links, for which formation was favored at acidic pH, were assigned as lysine-guanine adducts in which the modified guanine base exhibits a guanidino-trilysine-iminohydantoin structure. A proposed mechanism for the formation of the different detected oligonucleotide-peptide cross-links is given. The high yield of formation of the detected cross-links strongly suggests that a DNA-protein cross-link involving a lysine residue linked to the C8 position of guanine could be generated in cellular systems if a lysine is located in the close vicinity of a guanine radical cation.

BACKGROUND

Plasma total homocysteine (tHcy) is a risk factor for cardiovascular disease. tHcy concentrations are partly determined by folate, cobalamin, and vitamin B(6) status, and methylenetetrahydrofolate reductase (MTHFR) and other flavoenzymes are important for the biotransformation of these vitamins. This motivates the investigation of the possible relationship between riboflavin status and tHcy.

METHODS

The study had a cross-sectional design and included 423 healthy blood donors, ages 19-69 years. We determined plasma tHcy, serum folate, serum cobalamin, serum creatinine, and MTHFR C677T genotype. In addition, we measured riboflavin and its two coenzyme forms, flavin mononucleotide and flavin adenine dinucleotide, in EDTA plasma by capillary electrophoresis and laser-induced fluorescence detection.

RESULTS

Riboflavin determined tHcy independently in a multiple linear regression model with adjustment for sex, age, folate, cobalamin, creatinine, and MTHFR genotype (P = 0.008). tHcy was 1.4 micromol/L higher in the lowest compared with the highest riboflavin quartile. The riboflavin-tHcy relationship was modified by genotype (P = 0.004) and was essentially confined to subjects with the C677T transition of the MTHFR gene.

CONCLUSIONS

Plasma riboflavin is an independent determinant of plasma tHcy. Studies on deficient populations are needed to evaluate the utility of riboflavin supplementation in hyperhomocysteinemia.