An automated workflow approach for the analysis of flavin adenine dinucleotide by HPLC with internal standard.

Flavin adenine dinucleotide (FAD) is an active coenzyme of vitamin B2 involved in oxidation and reduction reactions. In this study we have developed a fully automated method for the analysis of FAD by fluorescence detection. FAD is extracted from whole blood samples by trichloroacetic acid, with diethyl ribityl isoalloxazine used as an internal standard. Linearity for FAD was above 0.99 (r2) up to a concentration range of 1000 nmol/L. Precision of the method (intra-day and inter-day) compared against commercial quality control material was below 8% (coefficient of variation) with recovery of FAD exceeding 90%. Accuracy of the protocol was compared against a previous cycle from an external quality assurance program (RCPAQAP) (n = 12) with satisfactory agreement. Overall, this method has increased laboratory workflow and reduced manual labour required in performing FAD analysis.

Through the eyes of a pathogen: light perception and signal transduction in Acinetobacter baumannii.

Sunlight is a ubiquitous environmental stimulus for the great majority of living organisms on Earth; therefore it is logical to expect the development of "seeing mechanisms" which lead them to successfully adapt to particular ecological niches. Although these mechanisms were recognized in photosynthetic organisms, it was not until recent years that the scientific community found out about light perception in chemotrophic ones. In this review we summarize the current knowledge about the mechanism of light sensing through the blue light receptor BlsA in Acinetobacter baumannii. We highlight its function as a global regulator that pleiotropically modulates a large number of physiological processes, many of which are linked to the ability of this opportunist pathogen to persist in adverse intrahospital environments. Moreover, we describe with some specific examples the molecular basis of how this photoregulator senses blue light and translates this physical signal by modulating gene expression of target regulons. Finally, we discuss the possible course of these investigations needed to dissect this complex regulatory network, which ultimately will help us better understand the A. baumannii physiology.

A study for the detection of kidney cancer using fluorescence emission spectra and synchronous fluorescence excitation spectra of blood and urine.

In this study, we compared different types of biomolecular markers in kidney cancer patients and in normal healthy controls, using fluorescence emission spectra and synchronous fluorescence excitation spectra. We were able to provide an accurate classification of the spectral features of kidney cancer patients relative to that of normal controls, in terms of the concentration ratios of biomolecules (viz., tryptophan, NADH, FAD, basic porphyrin, and acidic porphyrin) based on the intensity of their spectral peaks. The specificity and sensitivity of the method were 90%. The rationale of our current approach is to evolve an innovative protocol for the spectral characterization of in vitro optical analyses suitable for both small clinics and hospitals.

Quantitative fluorescence kinetic analysis of NADH and FAD in human plasma using three- and four-way calibration methods capable of providing the second-order advantage.

The metabolic coenzymes reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are the primary electron donor and acceptor respectively, participate in almost all biological metabolic pathways. This study develops a novel method for the quantitative kinetic analysis of the degradation reaction of NADH and the formation reaction of FAD in human plasma containing an uncalibrated interferent, by using three-way calibration based on multi-way fluorescence technique. In the three-way analysis, by using the calibration set in a static manner, we directly predicted the concentrations of both analytes in the mixture at any time after the start of their reactions, even in the presence of an uncalibrated spectral interferent and a varying background interferent. The satisfactory quantitative results indicate that the proposed method allows one to directly monitor the concentration of each analyte in the mixture as the function of time in real-time and nondestructively, instead of determining the concentration after the analytical separation. Thereafter, we fitted the first-order rate law to their concentration data throughout their reactions. Additionally, a four-way calibration procedure is developed as an alternative for highly collinear systems. The results of the four-way analysis confirmed the results of the three-way analysis and revealed that both the degradation reaction of NADH and the formation reaction of FAD in human plasma fit the first-order rate law. The proposed methods could be expected to provide promising tools for simultaneous kinetic analysis of multiple reactions in complex systems in real-time and nondestructively.

Fluorescence spectral diagnosis of malaria: a preliminary study.

BACKGROUND: Malaria is the most common disease transmitted by the bite by an infected female anopheles mosquito and caused by the plasmodium parasite. It is mostly prevalent in subtropical regions receiving abundant rain and supporting copious mosquito breeding. This disease is generally detected by the microscopic examination of blood films or antigen based rapid diagnostic test. Only occasionally the parasite DNA is detected using polymerase chain reaction in certain advanced, expensive laboratories. METHODS: An innovative spectral detection method based on the fluorescence spectra of a set of blood plasma biomolecules [tyrosine, tryptophan, nicotinamide adenine dinucleotide (NAD), and flavin adenine dinucleotide (FAD)] and red blood cell (RBC)-associated porphyrin is being evolved by our group. RESULTS: The research so far has exhibited sensitivity and specificity values exceeding 90% based on the spectral features of blood components of 14 malaria patients and 20 numbers of age adjusted normal controls. The fluorescent biomolecules go out of proportion when the malarial parasite breaks down the hemoglobin of blood. CONCLUSION: This technique has the potential to be used as an alternative diagnostic procedure for malaria since the instrumentation involved is portable and inexpensive. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_182.

Fluorescence spectral classification of iron deficiency anemia and thalassemia.

Thalassemia (Thal), sickle cell anemia, and iron deficiency anemia (IDA) are the most common blood disorders in many parts of the world, particularly in developing countries like India and Bangladesh. The well-established diagnostic procedure for them is the complete blood count (CBC); however, there is substantial confusion in discrimination between Thal and IDA blood samples based on such CBC. We propose a new spectral technique for reliable classification between the above two anemias. This is based on the identification and quantification of a certain set of fluorescent metabolites found in the blood samples of patients of Thal and IDA.

The relationships between plasma and red cell vitamin B2 and B6 concentrations and the systemic and local inflammatory responses in patients with colorectal cancer.

B vitamins have been implicated in cancer pathogenesis. It is therefore of interest that plasma B6 falls as part of the systemic inflammatory response (SIR), whereas red cell concentrations do not. The modified Glasgow Prognostic Score (mGPS) is a validated inflammation-based prognostic score that consists of a combination of albumin and C-reactive protein concentrations. The aim of this study was to examine the relationships between the concentrations of plasma and red cell vitamin B concentrations, the local and systemic inflammatory response in patients with colorectal cancer. Preoperative venous blood of 108 patients with colorectal cancer were analyzed for C-reactive protein, albumin, flavin adenine dinucleotide (FAD), and pyridoxal phosphate (PLP), and lymphocyte counts. Pathological slides were retrieved for assessment of inflammatory cell infiltration. Increasing mGPS was associated with lower plasma PLP concentrations (P < 0.01) but not plasma and red cell FAD and red cell PLP concentrations. Increasing tumor stage was associated with the presence of venous invasion (P < 0.01) and low-grade inflammatory cell infiltrate (P < 0.05) but not the SIR, FAD, or PLP concentrations. A low-grade inflammatory cell infiltrate was not significantly associated with any other parameter. The presence of a SIR was associated with lower concentrations of plasma PLP but not red cell PLP concentrations in patients with colorectal cancer. Neither FAD and PLP were associated with the tumor inflammatory cell infiltrate.

Relation between riboflavin, flavin mononucleotide and flavin adenine dinucleotide concentrations in plasma and red cells in patients with critical illness.

BACKGROUND: There is some evidence that the relationship between plasma and red cell vitamin B2 concentrations is perturbed in the critically ill patient. The aim of the present study was to examine the longitudinal interrelationships between riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in plasma and red cells in patients with critical illness. METHODS: Riboflavin, FMN and FAD concentrations were measured, by HPLC, in plasma and red cells in healthy subjects (n=119) and in critically ill patients (n=125) on admission and on follow-up. RESULTS: On admission, compared with the controls, critically ill patients had significantly higher plasma riboflavin and FMN concentrations (p<0.001) and lower median plasma FAD concentrations (p<0.001). In the red cell, FAD concentrations were significantly lower in critically ill patients (p<0.001). In healthy subjects, plasma riboflavin was directly associated with both plasma FMN (r(s)=0.55, p<0.001) and plasma FAD (r(s)=0.49, p<0.001). Red cell riboflavin was directly associated with red cell FMN (r(s)=0.52, p<0.001) but not red cell FAD. In the critically ill patients, plasma riboflavin was not significantly associated with either plasma FMN or FAD. Red cell riboflavin was directly associated with red cell FMN (r(s)=0.79, p<0.001) and red cell FAD (r(s)=0.72, p<0.001). Longitudinal measurements (n=60) were similar. CONCLUSIONS: The relationship between plasma riboflavin, FMN and FAD was significantly perturbed in critical illness. This effect was less pronounced in red cells. Therefore, red cell FAD concentrations are more likely to be a reliable measure of status in the critically ill patient.

Flavin adenine dinucleotide status and the effects of high-dose riboflavin treatment in short-chain acyl-CoA dehydrogenase deficiency.

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an inborn error, biochemically characterized by increased plasma butyrylcarnitine (C4-C) concentration and increased ethylmalonic acid (EMA) excretion and caused by rare mutations and/or common gene variants in the SCAD encoding gene. Although its clinical relevance is not clear, SCADD is included in most US newborn screening programs. Riboflavin, the precursor of flavin adenine dinucleotide (FAD, cofactor), might be effective for treating SCADD. We assessed the FAD status and evaluated the effects of riboflavin treatment in a prospective open-label cohort study involving 16 patients with SCADD, subdivided into mutation/mutation (mut/mut), mutation/variant (mut/var), and variant/variant (var/var) genotype groups. Blood FAD levels were normal in all patients before therapy, but significantly lower in the mut/var and var/var groups compared with the mut/mut group. Riboflavin treatment resulted in a decrease in EMA excretion in the mut/var group and in a subjective clinical improvement in four patients from this group. However, this improvement persisted after stopping treatment. These results indicate that high-dose riboflavin treatment may improve the biochemical features of SCADD, at least in patients with a mut/var genotype and low FAD levels. As our study could not demonstrate a clinically relevant effect of riboflavin, general use of riboflavin cannot be recommended.

Erythrocyte pyridoxamine phosphate oxidase activity: a potential biomarker of riboflavin status?

BACKGROUND: Riboflavin status is commonly measured by the in vitro stimulation of erythrocyte glutathione reductase with flavin adenine dinucleotide and expressed as an erythrocyte glutathione reductase activation coefficient (EGRAC). However, this assay is insensitive to poor riboflavin status in subjects with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Because G6PD deficiency is common in parts of the world where ariboflavinosis is endemic, it is important to have a measure of riboflavin status that is unaffected by differences in G6PD status. OBJECTIVE: The objective was to further develop and validate a fluorometric assay for pyridoxamine phosphate oxidase (PPO) activity as a measure of riboflavin status. DESIGN: A fluorometric assay was optimized for the flavin-dependent enzyme PPO in erythrocytes. Hemolysates from a previous riboflavin intervention study (2- and 4-mg riboflavin supplements) were used to investigate the responsiveness of the method to changes in riboflavin intake. RESULTS: PPO activity and the PPO activation coefficient (PPOAC) were used to assess riboflavin status. Both PPO activity and PPOAC responded to riboflavin supplements (P < 0.01), but only PPO showed a dose response (P < 0.001). The change from baseline to after the intervention in PPOAC and PPO enzyme activity was significantly inversely correlated (P < 0.001). Both PPO activity and PPOAC were strongly correlated with EGRAC (P < 0.001). Additionally, both PPOAC and EGRAC showed a significant inverse correlation with dietary riboflavin intake (P < 0.01); PPO activity was positively correlated with riboflavin intake (P < 0.01). CONCLUSION: PPO activity could be used as a biomarker for measuring riboflavin status, especially in populations with a high prevalence of G6PD deficiency. This trial is registered at www.isrctn.org as ISRCTN35811298.

Neutrophils with toxic granulation show high fluorescence with bis(Zn2+-dipicolylamine) complex.

Although blood neutrophils with toxic granulation provide an excellent means of evaluating acute bacterial infections, the methods are labor-intensive and their reproducibilities depend on the staining technique and the observer's judgment. We measured the flavin adenine dinucleotide (FAD) content of normal neutrophils and neutrophils with toxic granulation by flow cytometry after incubating them with bis(Zn(2+)-dipicolylamine) complex. A total of 122 blood samples (78 with neutrophils with toxic granulation, 44 with normal neutrophils without toxic granulation) were analyzed. The mean autofluorescence levels of neutrophils in the toxic granulation (+) group and toxic granulation (-) group were both 1.9 x 10(3) MESF (mean equivalent soluble fluorochrome) values. However, after incubating neutrophils with bis(Zn(2+)-dipicolylamine) complex for 15 min, the mean fluorescence intensities of neutrophils in the toxic granulation (+) and toxic granulation (-) groups were significantly enhanced by 71- and 19-fold to 138+/-78 x 10(3) MESF and 37+/-37 x 10(3) MESF, respectively (p <0.001). An MESF cutoff value of 79 x 10(3) showed a sensitivity of 93.2% and a specificity of 81.8% for neutrophils with toxic granulation. Similarly, the MESF level ratio, defined as the ratio of the MESF value after incubation with bis(Zn(2+)-dipicolylamine) complex to the autofluorescence MESF value, at a cutoff value of 41, showed a sensitivity of 92.3% and a specificity of 81.8% for the detection of neutrophils with toxic granulation. The MESF values of neutrophils after incubation with bis(Zn(2+)-dipicolylamine) complex did not correlate with the leukocyte (p >0.05) or neutrophil counts (p >0.05). In conclusion, measurement of FAD fluorescence intensity in neutrophils by flow cytometry after incubation with bis(Zn(2+)-dipicolylamine) complex is an easy, objective, and reliable method of detecting neutrophils with toxic granulation.

Multidrug transporter ABCG2/breast cancer resistance protein secretes riboflavin (vitamin B2) into milk.

The multidrug transporter breast cancer resistance protein (BCRP/ABCG2) is strongly induced in the mammary gland during pregnancy and lactation. We here demonstrate that BCRP is responsible for pumping riboflavin (vitamin B(2)) into milk, thus supplying the young with this important nutrient. In Bcrp1(-/-) mice, milk secretion of riboflavin was reduced >60-fold compared to that in wild-type mice. Yet, under laboratory conditions, Bcrp1(-/-) pups showed no riboflavin deficiency due to concomitant milk secretion of its cofactor flavin adenine dinucleotide, which was not affected. Thus, two independent secretion mechanisms supply vitamin B(2) equivalents to milk. BCRP is the first active riboflavin efflux transporter identified in mammals and the first transporter shown to concentrate a vitamin into milk. BCRP activity elsewhere in the body protects against xenotoxins by reducing their absorption and mediating their excretion. Indeed, Bcrp1 activity increased excretion of riboflavin into the intestine and decreased its systemic availability in adult mice. Surprisingly, the paradoxical dual utilization of BCRP as a xenotoxin and a riboflavin pump is evolutionarily conserved among mammals as diverse as mice and humans. This study establishes the principle that an ABC transporter can transport a vitamin into milk and raises the possibility that other vitamins and nutrients are likewise secreted into milk by ABC transporters.

Conversion of FAD to FMN and riboflavin in plasma: effects of measuring method.

The stability of flavin adenin dinucleotide (FAD) in plasma was studied under a low-intensity light and FAD was found to be converted to flavin mononucleotide (FMN) and riboflavin (RF) in both human and rat plasma. The hydrolysis rates of FAD in plasma at 4 degrees C were lower than those at 37 degrees C. In addition, the hydrolysis rates were markedly inhibited when EDTA, known as an anticoagulant, was added to plasma. These results indicated that plasma samples in pharmacokinetic studies should be pretreated with EDTA, extracted at the earliest convenience and lower temperature like 4 degrees C to keep a high stability. The pharmacokinetic study after intravenous administration of FAD at a dose of 500 nmol/kg as FAD in rats was performed with plasma samples after addition of EDTA under strict light and temperature control. A measurable amount of FAD in plasma together with rapid conversions of FAD to FMN and RF were observed in rat plasma. The AUC values (mean+/-S.D. of 4 rats) for FAD, FMN and RF were 707+/-378, 3643+/-958 and 30095+/-3544 nmol x min/l, respectively. Using excess EDTA under strict temperature and light control may be useful for assessment of vitamin B2 in the in vivo study.

The relationship between plasma and red cell B-vitamin concentrations in critically-ill patients.

BACKGROUND AND AIMS: Low vitamin B-complex status has been associated with poorer outcome in critically-ill patients. However, these findings have been based on indirect methods. Using direct methods for assessing vitamin status, we examined the effect of B-complex vitamin supplementation by measuring plasma and red blood cell B1, B2 and B6-vitamin concentrations in critically-ill patients. METHODS: Thiamine diphosphate (TDP), flavin adenine dinucleotide (FAD) and pyridoxal phosphate (PLP) concentrations were measured in plasma and red cells of normal subjects (n=49) and ITU patients (n=41). RESULTS: Compared with the normal subjects, critically-ill patients had higher C-reactive protein and lower albumin concentrations (P<0.001). Also, plasma FAD and PLP were lower (P<0.001) and red cell concentrations of both were higher (P<0.01) in critically-ill patients. Critically-ill patients were grouped according to whether (n=23) or not (n=18) they had been supplemented with B-complex vitamins. Compared with non-supplemented group, the supplemented group had significantly higher red cell TDP and PLP concentrations (P<0.01). Plasma FAD and PLP concentrations did not differ significantly between the groups. CONCLUSIONS: The results of the present study suggest that direct measurements of red cell FAD and PLP are more responsive to supplementation than plasma measurements in the critically-ill patient.

The relationship between plasma and red cell concentrations of vitamins thiamine diphosphate, flavin adenine dinucleotide and pyridoxal 5-phosphate following elective knee arthroplasty.

BACKGROUND & AIMS: Water soluble vitamins B1, B2 and B6 are essential precursors for a wide variety of coenzymes involved in intermediary metabolism and their status is usually assessed from blood samples. The aim of the study was to examine the relationship between plasma and intra-cellular B-vitamins following the systemic inflammatory response of surgery. METHODS: Patients (n = 10) who underwent an elective knee arthroplasty, had venous blood samples withdrawn pre-operatively and at 12, 24, 48, 72 and 168 h after the start of surgery for the analysis of circulating concentrations of C-reactive protein and albumin and also plasma and/ or red cell thiamine diphosphate (TDP), flavin adenine dinucleotide (FAD), pyridoxal 5-phosphate (PLP) as indicators of vitamins B1, B2, and B6 status respectively. RESULTS: Pre-operative, baseline vitamin assessments were all within population reference ranges. Over the study period of 0-168 h there was a significant increase in circulating C-reactive protein concentrations (peak 48 h, P < 0.001) and a significant fall in albumin concentrations (trough 48 h, P < 0.001). Plasma FAD and PLP concentrations fell transiently (P < 0.001) by approximately 40% reaching their nadir at approximately 48 h. CONCLUSIONS: The results of the present study indicate that plasma concentrations of FAD and PLP are transiently reduced following an inflammatory insult and therefore unlikely to be a reliable measure of status in the presence of a systemic inflammatory response. It may be that during such a response red cell concentrations provide a more reliable measure.

Normal riboflavin status in malaria patients in Gabon.

Previous publications reported commonly the occurrence of riboflavin deficiency and a positive correlation between riboflavin status and parasitemia in patients with Plasmodium falciparum malaria. In these studies, riboflavin status was determined by erythrocyte glutathione reductase activation coefficients (EGRACs). Inherited low erythrocyte glutathione reductase activity is highly prevalent in malarial regions, however. To rule out falsely diagnosed riboflavin deficiency in affected patients, we conducted an investigation using a high-performance liquid chromatography method (HPLC) instead of the EGRAC method. In 29 infants (age range, 1-5 years), 22 schoolchildren (age range, 6-12 years), and 33 adolescents and adults (age range, 13-74 years) from Lambaréné, Gabon, with acute P. falciparum malaria, plasma concentrations of riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) were measured by HPLC. Results were correlated with parasite densities. Profiles of plasma concentrations of all 3 flavin compounds were within the normal range in all patients. Concentrations of free riboflavin were not different between the 3 age groups. In adolescents and adults, FMN and FAD concentrations were higher than in infants (P = 0.002 and P = 0.001) and schoolchildren (P = 0.003 and P = 0.002). Comparing children with hyperparasitemic and uncomplicated malaria, no difference in the concentrations of either flavin compound was found. Neither the concentrations of free riboflavin nor the concentrations of one of the flavin nucleotides correlated with parasitemia within subgroups of age or of children with uncomplicated and hyperparasitemic malaria. Our data indicate that nutritional riboflavin deficiency might have been overestimated in previous malaria studies and do not support a relationship between flavin concentrations and parasitemia in P. falciparum malaria.

Riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in human plasma and erythrocytes at baseline and after low-dose riboflavin supplementation.

BACKGROUND: Vitamin B(2) exists in blood as riboflavin and its cofactors, flavin mononucleotide (FMN) and FAD. The erythrocyte glutathione reductase activation coefficient (EGRAC) has traditionally been used to assess vitamin B(2) status in humans. We investigated the relationships of EGRAC and plasma and erythrocyte concentrations of riboflavin, FMN, and FAD in elderly volunteers and their responses to riboflavin administration. METHODS: EGRAC and plasma and erythrocyte concentrations of riboflavin, FMN, and FAD were determined in 124 healthy individuals with a mean age of 69 years. The same measurements were made in a subgroup of 46 individuals with EGRAC > or =1.20 who participated in a randomized double-blind 12-week intervention study and received riboflavin (1.6 mg/day; n = 23) or placebo (n = 23). RESULTS: Median plasma concentrations were 10.5 nmol/L for riboflavin, 6.6 nmol/L for FMN, and 74 nmol/L for FAD. In erythrocytes, there were only trace amounts of riboflavin, whereas median FMN and FAD concentrations were 44 and 469 nmol/L, respectively. Erythrocyte FMN and FAD correlated with each other and with EGRAC and plasma riboflavin (P <0.05). All variables except plasma FAD responded significantly to riboflavin supplementation compared with placebo (P < or =0.04). The strongest increases were for riboflavin in plasma (83%) and for FMN in erythrocytes (87%). CONCLUSIONS: Concentrations of all B(2) vitamers except plasma FAD are potential indicators of vitamin B(2) status, and plasma riboflavin and erythrocyte FMN may be useful for the assessment of vitamin B(2) status in population studies.

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.

Reduction of hydroxamic acids to the corresponding amides catalyzed by rabbit blood.

1. The hydroxamic acids N-hydroxyphenacetin and N-hydroxy-2-acetylaminofluorene were reduced to the corresponding amides, phenacetin and 2-acetylaminofluorene respectively by rabbit blood supplemented with both NAD(P)H and FAD. These reducing activities were found in erythrocytes but not in plasma, and were sensitive to inhibition by carbon monoxide and oxygen. When blood or erythrocytes were boiled, these activities were not abolished. 2. Haemoproteins such as haemoglobin and catalase exhibited the reductase activity in the presence of both NAD(P)H and FAD under anaerobic conditions. The activity was not abolished when the haemoproteins were boiled. 3. Haematin showed a significant reducing activity in the presence of these cofactors. The activity of haematin was also observed with the photochemically reduced form of FAD. 4. The reduction system in blood was composed of NAD(P)H, FAD and haemoglobin. Reduction appears to proceed in two steps, i.e. the reduction of FAD by NADH or NADPH, followed by the non-enzymatic reduction of the hydroxamic acids to the amides by reduced FAD, catalyzed by the haem group of haemoglobin in rabbit erythrocytes.