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

BackgroundThere 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.MethodsRiboflavin, 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.ResultsOn 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.ConclusionsThe 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.     

Mechanisms underlying the differential effects of ethanol on the bioavailability of riboflavin and flavin adenine dinucleotide.

Chronic alcoholism is associated with a high prevalence of riboflavin deficiency. Experiments were designed in an animal model to determine whether ethanol alters selectively the absorption of riboflavin and flavin adenine dinucleotide (FAD), the predominant dietary form of the vitamin. Rats received by gavage a liver homogenate to which either [14C]riboflavin or [14C]FAD was added with either ethanol or isocaloric sucrose solutions. Ethanol markedly diminished the bioavailability of [14C]FAD to a greater degree than that of [14C]riboflavin. Corroboration of an ethanol-impaired intraluminal hydrolysis of FAD was provided by using everted jejunal segments and measuring mucosal uptake of [14C]riboflavin together with nonradiolabeled FAD. In subsequent studies with mucosal cell extracts, ethanol markedly inhibited activities of FAD pyrophosphatase and flavin mononucleotide (FMN) phosphatase. These findings suggest that dietary sources of riboflavin (FMN and FAD) are not absorbed as well in the presence of ethanol than are vitamin preparations containing riboflavin, which is utilized more readily.     

Reduced nicotinamide adenine dinucleotide and the reduction of oxidized glutathione in human erythrocytes

The ability of reduced nicotinamide adenine dinucleotide (NADH), generated through the activity of lactic acid dehydrogenase, to support the reduction of endogenous oxidized glutathione in intact human erythrocytes and in hemolysates was investigated. Rapid initial oxidation of endogenous reduced glutathione was effected with methyl phenylazoformate. Freshly obtained normal erythrocytes and erythrocytes deficient in glucose-6-phosphate dehydrogenase activity were unable to regenerate reduced glutathione upon incubation with lactate. Only normal erythrocytes were capable of reducing oxidized glutathione after preincubation with glucose, inosine, or a medium which promoted the synthesis of increased amounts of intracellular NAD. This regeneration of reduced glutathione could be explained by the generation of reduced nicotinamide adenine dinucleotide phosphate through the metabolism of accumulated phosphorylated intermediates of glycolysis. Hemolysates prepared from both normal erythrocytes and from erythrocytes deficient in glucose-6-phosphate dehydrogenase activity were able to reduce oxidized glutathione in the presence of added lactate and NAD. The results obtained indicated either an inability of the intact erythrocyte to utilize the NAD at the concentrations attained or an altered behavior of the system for the regeneration of reduced glutathione after lysis of the cell.     

Effects of riboflavin-2',3',4',5'-tetrabutyrate and flavin adenine dinucleotide on the platelet aggregation induced by hydrogen peroxide

Both riboflavin-2',3',4',5'-tetrabutyrate and flavin adenine dinucleotide (FAD), especially the latter, could inhibit H2O2-induced platelet aggregation. FAD enhanced the glutathione reductase activity of platelets. FAD might exert its inhibitory effect on the H2O2-induced platelet aggregation via the glutathione reductase and peroxidase system.     

The heart production in energy-depleted human erythrocytes induced by glucose, inosine and adenine.

The heat production (HP) of glucose deprived human red blood cells was measured, using glucose, adenine and inosine as substrates. Inosine induced a significantly higher HP than glucose and adenine induced no significant HP. At low pH the HP of glucose decreased more than that of inosine, corresponding to an equally lowered lactate production. The results indicate that it should be possible to use the system developed to study the functional state both of the complete glycolytic system and the lower part of it in intact red blood cells during various clinical conditions.     

Screening for adenine and hypoxanthine phosphoribosyltransferase deficiencies in human erythrocytes by high-performance liquid chromatography

A screening method using high-performance liquid chromatography (HPLC) for the simultaneous detection of deficiencies of adenine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT) activities in human erythrocytes is described. Both enzyme reactions of APRT and HPRT in lysates treated with a charcoal-dextran were simultaneously carried out in the same reaction tube and the enzyme activities were determined by measuring the increases in absorbance at 260 nm of adenosine and inosine converted from adenosine-5'-monophosphate and inosine-5'-monophosphate with alkaline phosphatase. Adenosine and inosine were separated from adenine and hypoxanthine by a reversed-phase column.

The method could detect 1% of normal APRT activity and 0.3% of normal HPRT activity. The within-run coefficients of variation for APRT and HPRT activities were 3.2 and 3.4%, respectively.     

Purine metabolism of erythrocytes preserved in adenine, adenine- inosine, and adenine-guanosine supplemented media

Four different media for erythrocyte preservation have been compared by studying 16 variables mainly describing the purine metabolism and glycolysis of the erythrocyte. The concentrations of the additives in the erythrocyte suspensions were as follows: adenine, 0.25 mmol/l; adenine, 0.75 mmol/l; adenine, 0.25 and inosine, 0.50 mmol/l; and adenine, 0.25 and guanosine, 0.50 mmol/l. Evaluated from the concentrations of glycerate 2,3-bisphosphate and purine nucleoside triphosphates, the medium with adenine-guanosine was superior to the others. In this medium with adenine-guanosine was superior to the others. In this medium the guanosine was rapidly split to guanine, which was slowly used for nucleotide synthesis or deaminated to xanthine. Differences between ATP and GTP in their reactivity with the two enzymes phosphofructose kinase seems to explain the beneficial effect of guanosine on preservation of erythrocytes.     

Experiences of fatigue and depression before and after low-dose 1-thyroxine supplementation in essentially euthyroid individuals

Hypothyroidism is a relative state, long associated with fatigue and depression. Individuals may experience thyroid-related symptoms such as fatigue and depression before thyroid indices become abnormal. However, because of clinicians' diverse interpretations of appropriate circumstances for its use, low-dose, 1-thyroxine supplementation often is overlooked as a therapeutic agent for symptom treatment. The purpose of this exploratory, hermeneutic study was to describe euthyroid individuals' experiences of fatigue and depression before and after low-dose 1-thyroxine supplementation. For women participants, the collective influence of fatigue and depression prior to treatment interfered significantly with their day-to-day lives, despite their euthyroid status. For men, the influence of symptoms was far less substantial than for women. In general, participants responded favorably, both physically and emotionally, to low-dose 1-thyroxine supplementation. Furthermore, no participant experienced 1-thyroxine induced hyperthyroidism or untoward side effects attributable to 1-thyroxine. Further study of effects of 1-thyroxine on symptoms is needed. © 1997 John Wiley & Sons, Inc. Res Nurs Health 20: 389–398, 1997     

Coenzyme Q10 in plasma and erythrocytes: comparison of antioxidant levels in healthy probands after oral supplementation and in patients suffering from sickle cell anemia

BACKGROUND: The membrane-associated antioxidant coenzyme Q10 (CoQ10) or ubiquinone-10 is frequently measured in serum or plasma. However, little is known about the total contents or redox status of CoQ10 in blood cells. METHODS: We have developed a method for determination of CoQ10 in erythrocytes. Total CoQ10 in erythrocytes was compared to the amounts of ubiquinone-10 and ubihydroquinone-10 in plasma using high-pressure liquid chromatography (HPLC) with electrochemical detection and internal standardisation (ubiquinone-9, ubihydroquinone-9). RESULTS: Investigations in 10 healthy probands showed that oral intake of CoQ10 (3 mg/kg/day) led to a short-term (after 5 h, 1.57+/-0.55 pmol/microl plasma) and long-term (after 14 days, 4.00+/-1.88 pmol/microl plasma, p<0.05 vs. -1 h, 1.11+/-0.24 pmol/microl plasma) increase in plasma concentrations while decreasing the redox status of CoQ10 (after 14 days, 5.37+/-1.31% in plasma, p<0.05 vs. -1 h, 6.74+/-0.86% in plasma). However, in these healthy probands, CoQ10 content in red blood cells remained unchanged despite excessive supplementation. In addition, plasma and erythrocyte concentrations of CoQ10 were measured in five patients suffering from sickle cell anemia, a genetic anemia characterised by an overall accelerated production of reactive oxygen species. While these patients showed normal or decreased plasma levels of CoQ10 with a shifting of the redox state in favour of the oxidised part (10.8-27.2% in plasma), the erythrocyte concentrations of CoQ10 were dramatically elevated (280-1,093 pmol/10(9) ERY vs. 22.20+/-6.17 pmol/10(9) ERY). CONCLUSIONS: We conclude that normal red blood cells may regulate their CoQ10 content independently from environmental supplementation, but dramatic changes may be expected under pathological conditions.     

Determination of cyanide in whole blood, erythrocytes, and plasma

We describe a method for determination of cyanide in whole blood, erythrocytes, and plasma after stabilization of cyanide by addition of silver ions. The cyanide is then transferred from the acidified sample, by aeration, into sodium hydroxide and quantified by the K?nig reaction, with sodium hypochlorite as the chlorinating agent. A rapid loss of measurable cyanide found when cyanide was added to plasma in the absence of silver ions was attributed to a reaction with serum albumin. Cyanide added to whole blood was bound to a saturable component in erythrocytes, which we identified as methemoglobin.     

Activation of human neutrophil nicotinamide adenine dinucleotide phosphate, reduced (triphosphopyridine nucleotide, reduced) oxidase by arachidonic acid in a cell-free system.

Sonicates from unstimulated human neutrophils produce no measurable superoxide since the superoxide-generating enzyme, NADPH oxidase, is inactive in these preparations. Previous attempts to activate the oxidase in disrupted cells with conventional neutrophil stimuli have been unsuccessful. This report describes a cell-free system in which arachidonic acid (82 microM) was able to activate superoxide generation that was dependent upon the presence of NADPH and the sonicate. For activation to occur, both the particulate and supernatant fractions of the sonicate must be present. Calcium ions, which are required for activation of intact neutrophils by arachidonate, were not necessary in the cell-free system. In quantitative terms, the superoxide-generating activity in the cell-free system could account for at least 20-50% of the superoxide rate observed in intact neutrophils stimulated with arachidonate. Sonicates from patients with chronic granulomatous disease (CGD) could not be activated by arachidonic acid in the cell-free system. In three patients representing both genetic forms of CGD, the defect appeared to reside in the particulate fraction. The soluble cofactor was normal in all three patients and could be used to activate normal neutrophil pellets in the presence of arachidonic acid. Thus, at least a portion of the activation mechanism in the neutrophil, that residing in the soluble phase, appeared to be normal in patients with CGD.     

Temporal intrapersonal physiological variability of cholinesterase activity in human plasma and erythrocytes.

Erythrocyte and plasma cholinesterase activities were measured biweekly in one group of 22 subjects for a year and daily for three weeks in another group of nine men. The average range [i.e., (range/mean) X 100] of activity of erythrocyte cholinesterase in men during a year was 8% and during three weeks was 5%. For plasma, the corresponding values were 25% and 12%. The average ranges for erythrocyte and plasma cholinesterase activity in women during a year were 12% and 24%. Erythrocyte cholinesterase activity varies less than do hematocrit, hemoglobin, or erythrocyte count.     

Simultaneous analysis of coenzyme Q10 in plasma, erythrocytes and platelets: comparison of the antioxidant level in blood cells and their environment in healthy children and after oral supplementation in adults

BACKGROUND: Coenzyme Q10 (CoQ10) originates from food intake as well as from endogenous synthesis. While plasma concentrations may be influenced by dietary uptake, little is known whether concentrations in plasma reflect or influence intracellular concentrations. METHODS: For clinical routine investigation of intracellular CoQ10 contents, blood erythrocytes and platelets were isolated by Ficoll separating solution and CoQ10 analysed using HPLC. The intracellular concentrations were compared to environmental plasma concentrations of 50 clinically healthy infants and additionally after exogenous pharmaceutical supplementation of CoQ10 (3 mg/kg/day) to 12 adult probands for 14 days. RESULTS: In healthy children, no correlation between plasma concentration and content in blood cells was found. A negative correlation exists between the year of life of the infants and CoQ10 concentrations in plasma correlated to cholesterol content. Probands supplemented with CoQ10 showed a distinct response in plasma concentrations after 14 days. While excessive environmental supplementation was without influence on erythrocyte concentrations, a positive correlation exists between plasma content and concentrations in platelets as mitochondria containing cell lines. CONCLUSIONS: Under physiologically normal conditions, blood cells or organs may regulate their CoQ10 content independently from environmental supply. Effects may be expected in situations of deficiency or excessive supply. Erythrocyte concentration of CoQ10 keeps independent from environmental supply. Thus incorporation into outer cell membranes may be limited. However, an excessive environmental supply may influence inner compartments like mitochondrial membranes.     

Glutathione and γ-glutamylcysteine in whole blood,plasma and erythrocytes

A method for the simultaneous determination of glutathione and γ-glutamyl-cysteine in capillary blood samples is described.The whole blood levels of glutathione and γ-glutamylcysteine are 1.09 ± 0.20 mmol/l and 25 ± 8 μmol/l (M ± S.D.), respectively. The plasma concentration is approximately 4 μmol/l for both compounds. It is important to treat the blood samples with a reducing agent before protein precipitation to release glutathione and γ-glutamylcysteine from disulfide linkages since, otherwise, 30–40% of the glutathione and 80% of the γ-glutamylcysteine is lost with the protein precipitate. Whole blood is preferable to washed erythrocytes for the analysis since the washing procedure involves losses of, especially, γ-glutamyl-cysteine.     

PTCA术后红细胞滤过指数与过氧化脂质、超氧化物歧化酶的关系

目的　探讨经皮腔内冠状动脉成形术 (PTCA)术后红细胞滤过指数 (IF)与血浆过氧化脂质 (LPO)、超氧化物歧化酶 (SOD)的关系。方法　将 5 0例行PTCA术的冠心病患者分别于术前、术后即时、术后 1周抽血 ,检测其IF、血浆LPO及SOD浓度。另以 4 1例健康体检者为对照组。结果　红细胞IF于PTCA术前、术后即时及术后 1周分别为 0 38± 0 0 3、0 5 1±0 17及 0 2 6± 0 0 8,对照组为 0 2 4± 0 0 5 ,术后即时的IF较术前、术后 1周及对照组均显著增高 (P <0 0 1)。LPO及SOD的检测显示 ,冠心病患者术后即时LPO的增高及SOD的减少与手术其它时段及对照组相比 ,差异均有显著意义 (P <0 0 1,P <0 0 5 ) ,相关分析表明 :LPO与IF呈正相关 (rMDA -IF =0 75 8) ,与SOD呈负相关 (rMDA -SOD =- 0 812 )。结论　PTCA术后早期红细胞变形能力下降 ,血浆LPO浓度增高 ,SOD浓度下降 ,尤以术后即时变化明显     

Aspartate aminotransferase, alanine aminotransferase, and glutathione transferase in plasma during and after sedation by low-dose isoflurane or midazolam.

To assess the effect of prolonged administration of midazolam or isoflurane on hepatocellular integrity, we measured the concentrations of glutathione transferase (EC 2.5.1.18) B1 subunit and the activities of alanine aminotransferase (ALT; EC 2.6.1.2) and aspartate aminotransferase (AST; EC 2.6.1.1) in 40 patients who required long-term sedation with low-dose midazolam or isoflurane. Blood samples were collected before and 24 h after the start of the sedation and 0, 24, 72, 120, and 172 h after the last dose. ALT and AST activities did not change appreciably, but the glutathione transferase B1 concentration decreased significantly (P less than 0.03) at all times studied. The patients who received isoflurane and those who received midazolam showed no significant differences in any of the enzyme tests. We conclude that long-term sedation with midazolam or isoflurane is unlikely to affect hepatocellular integrity.     

Free amino acids in plasma and erythrocytes during and after cardiopulmonary by-pass in open-heart surgery.

The concentrations in plasma and erythrocytes of free amino acids before, during and after cardiopulmonary by-pass have been determined. During by-pass and the early postoperative period the total alpha-amino nitrogen decreased, mainly because of changes in glucogenic amino acids; ketogenic amino acids, on the other hand, increased. The phenylalanine/tyrosine ratio was markedly increased in all cases.