Simultaneous quantitation of folates, flavins and B6 metabolites in human plasma by LC-MS/MS assay: Applications in colorectal cancer.

An analytical method using electrospray ionization and high- performance liquid chromatography/tandem mass spectrometry (LC/ESI-MS/MS) was developed to quantify the vitamin B metabolites found in the folate one-carbon metabolism, using 50 µL of human plasma. Analytes in plasma were extracted using protein precipitation after being stabilized in 1% ascorbic acid. The analytes were separated using a Kinetex 2.6 µm Pentafluorophenyl (2.1 × 30 mm) column utilizing a gradient mobile phase system of 0.1% formic acid in water and 100% acetonitrile in a 13.2 min run. The MS detector run using a positive multiple reaction monitoring with parameters optimized for each analyte's ion pair. The assay was selective and linear for all analytes at defined dynamic ranges. The recoveries were generally above 80% except for the folate metabolites whose recoveries dipped possibly due to the drying process. The inter-day precision (%coefficient of variation) and accuracy (%calculated concentration of the nominal concentrations) for six replicates of all quality control samples were ≤14% and within 12.2%, respectively. The lower limit of quantification ranged from 0.2 to 3.9 nM. No significant instability was observed after repeated freezing and thawing or in processed samples. The LC-MS/MS assay was found applicable for sensitive, accurate and precise quantitation of vitamin B metabolites in plasma of healthy volunteers and colorectal cancer patients.

Rapid analysis of trace levels of flavins by pressurized capillary electrochromatography-laser induced fluorescence detection with sulfonated N-octadecyl methacrylate monolith.

In this paper, pressurized capillary electrochromatography (pCEC) with laser induced fluorescence detection (LIF) was demonstrated as a viable approach for the separation and determination of trace flavins in human plasma, where flavins tend to be degraded ex vivo. Using a sulfonated N-octadecyl methacrylate monolithic column in isocratic pCEC separation, symmetrical peak shapes and rapid separation could be obtained in a weakly acidic mobile phase. Baseline separation of riboflavin, flavin mononucleotide and flavin adenine dinucleotide could be achieved within 4.5 min in a mobile phase containing 60% (v/v) acetonitrile and 40% (v/v) of 20 mmol L(-1) phosphate buffer (pH 4.0), with -22 kV of applied voltage and 290 psi of supplementary pressure and 0.02 mL min(-1) of flow rate. Based on a 473 nm laser diode double pumped solid state source, flavins could be determined by LIF with the detection limit (LOD) as low as 0.5 nmol L(-1) (S/N=3). The concentration ranges were 0.005-2 micromol L(-1) for RF and FMN, and 0.02-40 micromol L(-1) for FAD. Owing to the weakly acidic condition selected in this experiment, the high fluorescence quantum yields and good stability of flavins contributed to a preferable analysis. Combined with a simple clean-up procedure, this method has been proved to be effective for the rapid and selective analysis of trace levels of flavins in human plasma without sample preconcentration.

Quantification of the bioavailability of riboflavin from foods by use of stable-isotope labels and kinetic modeling.

BACKGROUND: Discrepancies have been reported between estimates of the prevalence of riboflavin deficiency based on intakes of riboflavin and estimates based on measures of riboflavin status. One reason for this may be an overestimate of the bioavailability of riboflavin from foods, about which relatively little is known. OBJECTIVE: We aimed to quantify the bioavailability of riboflavin from milk and spinach by using stable-isotope labels and a urinary monitoring technique and by a plasma appearance method based on kinetic modeling. DESIGN: Twenty healthy women aged 18-65 y were recruited for a randomized crossover study performed with extrinsically labeled (13C) milk and intrinsically labeled (15N) spinach as sources of riboflavin. An intravenous bolus of labeled riboflavin was administered with each test meal to assess the apparent volume of distribution of riboflavin in plasma. RESULTS: No significant differences were noted in riboflavin absorption from the spinach meal and from the milk meal according to either the urinary monitoring technique (60 +/- 8.0% and 67 +/- 5.4%, respectively; P = 0.549) or the plasma appearance method (20 +/- 2.8% and 23 +/- 5.3%, respectively; P = 0.670). CONCLUSIONS: A large fraction of newly absorbed riboflavin is removed by the liver on "first pass." The plasma appearance method therefore underestimates riboflavin bioavailability and should not be used to estimate riboflavin bioavailability from foodstuffs. Urinary monitoring suggests that riboflavin from spinach is as bioavailable as is riboflavin from milk.

Proliferation of peripheral blood mononuclear cells increases riboflavin influx.

Previously we demonstrated that proliferation of peripheral blood mononuclear cells (PBMC) causes a five-fold increase in cellular uptake of biotin; this increase is mediated by an increased number of biotin transporters on the PBMC surface. In the present study, we investigated the specificity of this phenomenon by determining whether the cellular uptake of riboflavin also increases in proliferating PBMC and whether the increase is also mediated by an increased number of transporters per cell. We characterized [3H]riboflavin uptake in both quiescent and proliferating PBMC. In quiescent PBMC, [3H]riboflavin uptake exhibited saturation kinetics and was reduced by addition of unlabeled riboflavin (P < 0.05) or lumichrome (P < 0.01). These observations are consistent with transporter-mediated uptake. [3H]Riboflavin uptake was reduced at 4 degrees C compared with 37 degrees C (P < 0.01) and by 2, 4-dinitrophenol (P < 0.05) but not by ouabain or incubation in sodium-free medium. These data provide evidence for an energy-dependent but sodium-independent transporter. Proliferating PBMC accumulated approximately four times more [3H]riboflavin than quiescent PBMC (P < 0.05). Because both transporter affinity and transporter number per cell (as judged by maximal transport rate) were similar in quiescent and proliferating PBMC, we hypothesize that the increased riboflavin uptake by proliferating PBMC reflects only increased cellular volume. To test this hypothesis, PBMC volume was reduced using hyperosmolar medium; [3H]riboflavin uptake decreased to about 50% of isotonic controls (P < 0.01). Thus we conclude that proliferating PBMC increase cellular content of riboflavin and biotin by two different mechanisms.

Riboflavin and riboflavin-derived cofactors in adolescent girls with anorexia nervosa.

BACKGROUND: Thyroid hormones, riboflavin, riboflavin cofactors, and organic acids were assessed in girls with anorexia nervosa. OBJECTIVE: The objective was to examine the effect of malnutrition and low thyroid hormone concentrations on erythrocyte and plasma riboflavin metabolism and their relation with urinary organic acid excretion. DESIGN: Seventeen adolescent girls with anorexia nervosa [body mass index (BMI; in kg/m2): 14.8 +/- 2.2] and 17 age-matched, healthy girls (control subjects; BMI: 20.5 +/- 2.2) took part in the feeding study. Erythrocyte and plasma riboflavin as well as riboflavin cofactors (flavin mononucleotide and flavin adenine dinucleotide) were assessed by HPLC, whereas urinary organic acids were assessed by gas chromatography-mass spectrometry. RESULTS: Anorectic patients who began a feeding program had higher erythrocyte riboflavin (3.5 +/- 2.2 compared with <0.1 nmol/mol hemoglobin; P < 0.001), lower plasma flavin adenine dinucleotide (57.8 +/- 18.5 compared with 78.5 +/- 54.3 nmol/L; P < 0.05), and higher urinary ethylmalonic acid (7.12 +/- 4.39 compared with 1.3 +/- 2.8 micromol/mmol creatinine; P < 0.001) and isovalerylglycine (7.65 +/- 4.78 compared with 3.8 +/- 0.9 micromol/mmol creatinine; P < 0.05) concentrations than did control subjects. Triiodothyronine concentrations were low and negatively correlated with plasma riboflavin concentrations (r = -0.69, P < 0.01). Not all patients showed improvements in these biochemical indexes after 30 d of refeeding. CONCLUSIONS: The low triiodothyronine concentrations observed in anorexia nervosa could alter the extent of riboflavin conversion into cofactors, thus leading to high erythrocyte riboflavin concentrations, low plasma flavin adenine dinucleotide concentrations, and high rates of ethylmalonic acid and isovalerylglycine excretion.

Riboflavin deficiency caused by treatment with adriamycin.

The present study was undertaken to determine whether administration of adriamycin causes the depletion of riboflavin content. Rats received intraperitoneal injections of adriamycin (4 mg per kg body weight) for 6 consecutive days. Urinary riboflavin excretion began to increase after 2 days of treatment with adriamycin. Erythrocyte FAD levels decreased gradually and plasma lipid peroxide contents increased markedly at the 6th day. The activity coefficient of erythrocyte glutathione reductase showed a significant increase before the decrease of flavin content and the elevation of lipid peroxide level. Therefore, the value of this coefficient obtained from erythrocyte appears to be a reliable index of riboflavin deficiency, particularly during the early stage.

In vivo kinetics of intestinal absorption of riboflavin in rats.

To investigate absorption kinetics of riboflavin under in vivo conditions, with blood and lymph circulation intact, the small intestine of anesthetized rats was perfused with [14C]riboflavin in a concentration range between 0.31 and 10.00 mumol/L. Apart from the uptake of riboflavin from the perfusate, passage of the vitamin into the portal (vena portae) and peripheral (vena femoralis) blood was determined. The absorption proved to be a dual process: at low substrate concentrations (less than 2 mumol/L) a saturable component predominated; at higher concentrations simple diffusion was found to be the prevailing uptake mechanism. The apparent transport constant of the saturable component was calculated to be 0.38 mumol/L. [14C]flavin concentrations in the portal and peripheral blood were estimated as a function of the riboflavin concentration of the perfusion media. The dual character of the absorption was reflected by the portal blood flavin levels. Due to the high retaining and equalizing capacity of the liver, the [14C]flavin level of the peripheral blood was relatively low and obeyed saturation kinetics. Constants of elimination, determined by pharmacokinetic calculations, were different for the two blood compartments but independent of the concentration of riboflavin in the perfusion media.

Generation of adriamycin radical by interaction of adriamycin with serum.

ESR signal of the adriamycin (AD) free radical was obtained when adriamycin interacted with serum under anaerobic conditions, but it was not observed by the interaction of AD with erythrocytes or the lysate. The intensities of the ESR signals were dependent upon the concentrations of adriamycin and serum, and cytochrome c completely diminished the ESR signal. Addition of reduced pyridine nucleotides stimulated the formation of AD radical. Between pH 5.0 and 8.0, more of AD radical was formed at higher pH. Allopurinol and arsenite, which are flavin enzyme inhibitors, partially inhibited the AD radical formation by serum. From these results, it seems that several flavin enzymes in serum are involved in the AD radical formation.

Two cytosolic components of the neutrophil NADPH oxidase, P47-phox and P67-phox, are not flavoproteins.

Two cytosolic proteins, p47-phox and p67-phox, have been shown to be essential components of the NADPH-dependent oxidase of human neutrophils, although the specific role of each of these proteins in the multicomponent electron transport complex is undetermined. The superoxide-generating activity of this oxidase can be reproduced in a cell-free system, combining cytosol and membranes from unstimulated neutrophils in the presence of fatty acid and NADPH. In the present studies, cytosol was treated with myristic acid, arachidonic acid, or sodium dodecyl sulfate in the absence of membranes and the resultant precipitate collected by centrifugation and analyzed. Both p47-phox and p67-phox precipitated in the presence of fatty acid. However, neither FAD nor FMN was localized in the precipitates, even though substantial amounts of p47-phox and p67-phox precipitated. These results suggest that neither p47-phox nor p67-phox is a flavoprotein and that neither, therefore, is the oxidase component which accepts electrons from NADPH.

Cytochrome b, flavins, and ubiquinone-50 in enucleated human neutrophils (polymorphonuclear leukocyte cytoplasts).

Neutrophilic granulocytes contain an oxidase system in their plasma membrane that can be activated to generate superoxide radicals and hydrogen peroxide. Cytochrome b, flavoprotein, and ubiquinone-50 have been proposed as components of this oxidase system. These components have been quantitated, but the results are obscured by different isolation procedures for plasma membranes from resting and activated neutrophils. This problem has now been avoided by the use of enucleated neutrophils (polymorphonuclear leukocyte cytoplasts), which are almost completely devoid of intracellular structures but contain an intact, activatable oxidase system (Roos, D., Voetman, A.A., and Meerhof, L.J. (1983) J. Cell Biol. 97, 368-377). Membranes of resting and phorbol myristate acetate-stimulated cytoplasts contain equal amounts of cytochrome b (4 pmol/milliunit of alkaline phosphatase) and also equal amounts of noncovalently bound FAD (2 pmol/milliunit of alkaline phosphatase). These findings refute the hypothesis that incorporation of cytochrome b and/or a flavoprotein into the plasma membrane constitutes the mechanism of activation of the oxidase system. Ubiquinone-50 is present neither in intact neutrophils nor in cytoplasts, excluding a role for this compound in the generation of bactericidal oxygen species by neutrophils.

Purification and properties of soluble NADH-cytochrome b5 reductase of rabbit erythrocytes.

Soluble NADH-cytochrome b5 reductase was purified from rabbit erythrocytes to homogeneity by simple procedures developed in this study including fractionation with ammonium sulfate, gel filtration on a Sephadex G-75 column, and affinity chromatography on a 5'-AMP-Sepharose 4B column. The enzyme was purified about 12,000-fold from hemolysate in terms of NADH-cytochrome b5 reductase activity with a high yield of 40%. The purified enzyme has absorption maxima at 273, 390, and 462 nm, and shoulders at 370, 435, and 488 nm. The ratio of the absorbance at 273 nm to that at 462 nm of the purified enzyme was 5.6-5.8. The prosthetic group of the enzyme was found to be FAD, and the flavin content in the enzyme was 1 mol/mol of the enzyme. The molecular weight of the purified enzyme was estimated to be 33,000 and 32,000 by gel filtration on a Sephadex G-75 column, and by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, respectively. The NADH-cytochrome b5 reductase activity decreased strikingly as the buffer or salt concentration in the assay mixture was increased, and the optimal pH for the reduction of cytochrome b5 with NADH was determined to be 6.6 in Tris-maleate buffer of constant ionic strength. The maximum velocity of NADH-cytochrome b5 reductase activity of the purified enzyme was very high, 1,280 mumol/min/mg of protein in 10 mM phosphate buffer (pH 6.6). The Michaelis constants for NADH and cytochrome b5 were determined to be 2.5 and 4 microM, respectively. The reduction of cytochrome b5 with NADH by the enzyme was suggested to follow the ordered-type reaction mechanism based on the modes of product inhibition. From these results, and also from the estimated enzyme content in the erythrocytes (16-20 mg protein per liter of packed erythrocytes), the possible contribution of the enzyme to functions other than methemoglobin reduction in rabbit erythrocytes is discussed.

Pregnancy suppression by active immunization against gestation-specific riboflavin carrier protein.

A riboflavin carrier protein isolated from chickens cross-reacts with a gestation-specific rodent carrier for riboflavin. Active immunization of female rats of proved fertility with the purified chicken carrier protein completely yet reversibly suppressed early pregnancy without impairing implantation per se. Concurrently there were no discernible adverse effects on maternal health in terms of weight gain, vitamin status, and fertility.

Congenital methaemoglobinaemia due to NADH methaemoglobin reductase deficiency: successful treatment with oral riboflavin.

A Japanese family with congenital methaemoglobinaemia is described. The family pedigree was compatible with autosomal recessive type of inheritance. The increased methaemoglobin concentration was ascribed to the red cell NADH diaphorase deficiency associated with the almost complete lack of one of the two peaks of the diaphorase activity as separated by DEAE Sephadex column chromatography. The NADH diaphorase and NADH methaemoglobin reductase deficiency was limited to the red cells. The methaemoglobin content in the blood of the propositus was 17.8% and isoelectric focusing analysis on a polyacrylamide gel plate showed that the haemoglobin consisted of 65.2% oxyhaemoglobin (alpha 2+ beta 2+)2, 29.6% half-oxidized forms, 20.9% (alpha 3+ beta 2+)2 and 8.7% (alpha 2+ beta 3+)2, and 3% full-oxidized methaemoglobin (alpha 3+ beta 3+)2. Oral administration of riboflavin 120 mg/d resulted in a gradual but significant decrease in the level of the met-form haemoglobins in parallel with a gradual increase in the red cell flavin content. Riboflavin is considered to be effective by activating the NADPH diaphorase (NADPH flavin reductase) system and appears to be useful for the treatment of congenital methaemoglobinaemia.

Enzymatic reduction of hemoglobins M Milwaukee-1 and M Saskatoon by NADH-cytochrome b5 reductase and NADPH-flavin reductase purified from human erythrocytes.

Enzymatic reduction of the hemoglobin (Hb) M group was studied. Hb M Milwaukee-1 and Hb M Saskatoon were reduced by NADH-cytochrome b5 reductase highly purified from human erythrocytes. Hb M Saskatoon was also reduced by another enzyme in red cells, NADPH-flavin reductase. The reduction rates of Hb M Saskatoon by both enzymes were almost the same as those of MetHb A. The reduction of Hb M Milwaukee-1 by NADH-cytochrome b5 reductase progressed much more slowly than that of Hb M Saskatoon and MetHb A. It took 1/2 h and 10 h for the 50% reduction of Hb M Saskatoon and Hb M Milwaukee-1, respectively. These two methemoglobin reductases from erythrocytes did not reduce other hemoglobins M such as Hb M Iwate, Hb M Boston, or Hb M Hyde Park. A possible role of these abnormal hemoglobins as oxygen carriers and the reason for cyanosis in the patients of Hb M Saskatoon and Hb M Milwaukee-1 are discussed.