In search of causal pathways in diabetes: a study using proteomics and genotyping data from a cross-sectional study.

AIMS/HYPOTHESIS: The pathogenesis of type 2 diabetes is not fully understood. We investigated whether circulating levels of preselected proteins were associated with the outcome 'diabetes' and whether these associations were causal. METHODS: In 2467 individuals of the population-based, cross-sectional EpiHealth study (45-75 years, 50% women), 249 plasma proteins were analysed by the proximity extension assay technique. DNA was genotyped using the Illumina HumanCoreExome-12 v1.0 BeadChip. Diabetes was defined as taking glucose-lowering treatment or having a fasting plasma glucose of ≥7.0 mmol/l. The associations between proteins and diabetes were assessed using logistic regression. To investigate causal relationships between proteins and diabetes, a bidirectional two-sample Mendelian randomisation was performed based on large, genome-wide association studies belonging to the DIAGRAM and MAGIC consortia, and a genome-wide association study in the EpiHealth study. RESULTS: Twenty-six proteins were positively associated with diabetes, including cathepsin D, retinal dehydrogenase 1, α-L-iduronidase, hydroxyacid oxidase 1 and galectin-4 (top five findings). Three proteins, lipoprotein lipase, IGF-binding protein 2 and paraoxonase 3 (PON-3), were inversely associated with diabetes. Fourteen of the proteins are novel discoveries. The Mendelian randomisation study did not disclose any significant causal effects between the proteins and diabetes in either direction that were consistent with the relationships found between the protein levels and diabetes. CONCLUSIONS/INTERPRETATION: The 29 proteins associated with diabetes are involved in several physiological pathways, but given the power of the study no causal link was identified for those proteins tested in Mendelian randomisation. Therefore, the identified proteins are likely to be biomarkers for type 2 diabetes, rather than representing causal pathways.

[Assessment of the concentrations of carbonylated proteins and carbonyl reductase enzyme in mexican women with breast cancer: A pilot study]. / Determinación de proteínas carboniladas y enzima carbonil reductasa en mujeres mexicanas con cáncer de mama: estudio piloto.

Oxidative stress could promote the development of cancer and implicate carbonylated proteins in the carcinogenic process. The goal of this study was to assess the concentrations of carbonylated proteins and carbonyl reductase enzyme in women with breast cancer and determine whether these markers were possible indicators of tissue damage caused by the disease. A total of 120 healthy women and 123 women with a diagnosis of breast cancer were included. The concentration of carbonylated proteins in plasma and the concentration of carbonyl reductase enzyme in leukocytes were determined using the ELISA assay. There was a 3.76-fold increase in the amount of carbonylated proteins in the plasma from the patient group compared with healthy control group (5±3.27 vs. 1.33±2.31 nmol carbonyls/mg protein; p<0.05). Additionally, a 60% increase in the carbonyl reductase enzyme was observed in the patient group compared with the healthy control group (3.27±0.124 vs. 2.04±0.11 ng/mg protein; p<0.05). A positive correlation (r=0.95; p<0.001) was found between both measurements. These results suggest the presence of tissue damage produced by cancer; therefore, these parameters could be used to indicate tissue damage in cancer patients.

[Aldehyde reductase activity and blood aldo-keto reductase spectrum in adolescents with neuroendocrine obesity].

Investigation of aldehyde-reductase activity and blood aldo-keto reductase spectrum has been performed in 13-15 and 16-18-years old adolescents with obesity to clear up the mechanisms of neuroendocrine obesity at the age of puberty. It has been established that basal aldehyde reductase activity and blood aldo-keto reductase spectrum of healthy adolescents in early puberty do not differ from those of healthy adolescents in late puberty. A decreased aldehyde reductase activity and some alterations in blood aldo-keto reductase spectrum have been observed in late puberty in adolescents with neuroendocrine obesity. In adolescents with obesity there have been registered some changes in blood aldo-keto reductase spectrum which are not accompanied by any alterations in its aldehyde reductase activity. The results obtained suggest that certain prerequisites are formed in late puberty to complicate the course of neuroendocrine obesity.

Feasibility of fetal-derived hypermethylated RASSF1A sequence quantification in maternal plasma--next step toward reliable non-invasive prenatal diagnostics.

We determined the feasibility of universal fetal marker detection in maternal circulation. Using real-time PCR, we compared the levels of fetal (SRY and hypermethylated RASSF1A) and total (GLO gene and total RASSF1A) extracellular DNA and fractions of extracellular fetal DNA (SRY/GLO vs. hypermethylated RASSF1A/total RASSF1A) in maternal circulation. Sensitivity and specificity reached 100% as the fetal-specific hypermethylated RASSF1A sequence was detected in all 151 examined plasma samples derived from 70 normal pregnancies with a singleton male (n=51) or female (n=19) fetus sampled throughout gestation and absent in non-pregnant individuals (n=29). A strong positive correlation was observed between fetal-derived hypermethylated RASSF1A and SRY (ρ=0.66, P<0.001), total RASSF1A and GLO (ρ=0.65,P<0.001), SRY/GLO vs. hypermethylated RASSF1A/total RASSF1A ratio (ρ=0.62, P<0.001) in maternal plasma. The results indicate that a universal fetal marker could be useful not only for the confirmation of the presence of fetal cell-free DNA in maternal plasma but could enable quantification of cell-free fetal DNA in pregnancy associated disorders, independently of the sex of the fetus.

[The state of hemostatic system and platelet metabolism in patients with complicated hypertensive disease].

The study designed to estimate the state of the homeostatic system, levels of NAD and NAD-dependent dehydrogenases included 90 patients with complicated hypertensive disease and coronary heart disease following ischemic stroke. Hypercoagulation and enhanced platelet aggregation occurred throughout the entire follow-up period. Acute stage of ischemic stroke was associated with marked changes in bioenergetic and plastic processes.

Bone mass does not correlate with the serum fibroblast growth factor 23 in hemodialysis patients.

Circulating fibroblast growth factor 23 (FGF23) increases renal phosphate excretion, decreases bone mineralization and is markedly increased in hemodialysis patients. Bone cells express fibroblast growth receptor 1, suggesting that FGF23 could alter bone mineralization by means of a direct effect on the skeleton and/or secondarily due to hypophosphatemia. To distinguish between these possibilities we measured serum concentrations of FGF23, parathyroid hormone, phosphate, calcium, and markers of bone remodeling, and assessed bone mineral density in 99 hemodialysis patients. FGF23 concentrations were increased in all hemodialysis patients, even in those without hyperphosphatemia, and positively correlated with serum phosphate but not with parathyroid hormone. Hemodialysis did not decrease the serum FGF23 concentration. We found no significant correlation between serum FGF23 levels and bone mineral density. Further analysis by gender or T-score did not modify these results. Serum markers of bone remodeling significantly correlated with parathyroid hormone but not with FGF23 levels. The increase in serum FGF23 concentration in hemodialysis patients cannot be solely ascribed to hyperphosphatemia. Our study suggests that the effects of FGF23 on bone mineralization are mainly due to hypophosphatemia and not a direct effect on bone.

Glyoxylate reductase activity in blood mononuclear cells and the diagnosis of primary hyperoxaluria type 2.

BACKGROUND: Primary hyperoxaluria type 2 (PH2) is a rare monogenic disorder characterized by an elevated urinary excretion of oxalate. Increased oxalate excretion in PH2 patients can cause nephrolithiasis and nephrocalcinosis, and can, in some cases, result in renal failure and systemic oxalate deposition. The disease is due to a deficiency of glyoxylate reductase/hydroxypyruvate reductase (GRHPR) activity. A definitive diagnosis of PH2 is currently made by the analysis of GR activity in a liver biopsy. GRHPR is expressed in virtually every tissue in the body, suggesting that utilization of more readily available cells could be used to determine GRHPR deficiency. In this study, we have evaluated the potential of determining GR and d-glycerate dehydrogenase (DGDH) activity in blood mononuclear cells (BMC) as a diagnostic indicator of PH2. METHODS: Blood samples were obtained from 10 male and 10 female normal subjects, median age 31, range 21-63, at the Wake Forest University Medical Center and from primary hyperoxaluria patients at the Mayo Clinic. The BMC were isolated and GR and DGDH activities measured in cell lysates. RESULTS: An assay of 20 normal individuals indicated that BMC contained a DGDH and GR activity of 0.97+/-0.20 (range 0.62-1.45), and 10.6+/-3.3 (range 8.3-16.6) nmol/min/mg protein, respectively. The intra-assay coefficient of variation for DGDH and GR activity was 8.2 and 11.5%, respectively. The BMC lysates from normal adult subjects and patients with PH1 showed similar GR and DGDH activities. This was confirmed by the presence of immunoreactive GRHPR protein by western blot analysis. In contrast, PH2 BMC lysates did not exhibit DGDH or GR activity, and showed no immunoreactive GRHPR by western blot analysis. CONCLUSION: These results suggest that the assay of DGDH or GR activity in BMC could be used as a minimally invasive diagnostic test for PH2.

Krebs cycle anions in metabolic acidosis.

For many years it has been apparent from estimates of the anion gap and the strong ion gap that anions of unknown identity can be generated in sepsis and shock states. Evidence is emerging that at least some of these are intermediates of the citric acid cycle. The exact source of this disturbance remains unclear, because a great many metabolic blocks and bottlenecks can disturb the anaplerotic and cataplerotic pathways that enter and leave the cycle. These mechanisms require clarification with the use of tools such as gas chromatography-mass spectrometry.

Evaluation of oxidative stress after repeated intravenous iron supplementation.

Parenteral iron has been recommended for the treatment of iron deficiency in the majority of maintenance hemodialyzed (HD) patients. However, iron supplementation and consequent over saturation of transferrin and high iron levels, may aggravate oxidative stress already present in these patients. This study aimed to further clarify the role of repeated intravenous iron therapy as a supplementary cause of oxidative stress in HD patients. Markers of free radical activities (carbonyl reactive derivatives, CRD, thiol groups, SH, malondialdehyde, MDA) and antioxidant enzyme activities (superoxide dismutase, SOD and glutathione peroxidase, GPX) were determined in plasma and red blood cells (RBC) of 19 hemodialysis patients given a total iron dose of 625 mg (ferrogluconat, Ferrlecit, 62.5 mg). Blood samples were taken before the first and after the last dose of iron. Twenty apparently normal subjects served as healthy controls. Before iron treatment, HD patients exhibited increased concentrations of MDA and CRD in plasma and red blood cells, accompanied with impaired antioxidant capacity. All patients responded to iron therapy with a significant increase in their serum ferritin, serum iron, hemoglobin, and red blood cells levels. However, iron treatment resulted in enhanced oxidative stress in plasma of HD patients, since significant increase in plasma MDA and CRD concentrations, together with a decrease in nonprotein SH groups levels were detected. Supplementation with iron did not significantly influence plasma SOD and GPX activities, nor did any of the red blood cell parameters tested. Our data show that, despite improvement in hematological parameters, an increase in iron stores due to supplementation could also contribute to increased free radical production in HD patients.

Functional tetrahydrobiopterin synthesis in human platelets.

BACKGROUND: Previous studies have provided evidence for the importance of platelet-derived nitric oxide (NO) for the regulation of hemostasis. Tetrahydrobiopterin (BH4) is an essential cofactor and regulator of NO synthase activity in the vasculature; however, it is as yet unknown whether platelets dispose over a functional BH4 synthesis. METHODS AND RESULTS: We quantified mRNA expression of genes involved in BH4 synthesis, measured enzymatic activities, and determined intraplatelet levels of pteridines in platelets from healthy volunteers and from patients treated for prolonged periods of time with glucocorticoids. Freshly isolated platelets from healthy volunteers show functional BH4 synthesis, as evidenced by the presence of mRNA species and enzymatic activity of GTP cyclohydrolase I (GTPCH), 6-pyruvoyl tetrahydropterin synthase, and sepiapterin reductase. Biopterin was the major intraplatelet pteridine, whereas no neopterin was found. mRNA expression and enzymatic activity of GTPCH were undetectably low in platelets that had been stored for 5 days, and no pteridines were found in these platelets. Freshly isolated platelets from patients treated with glucocorticoids had decreased mRNA expression and activity of GTPCH compared with platelets from healthy volunteers. CONCLUSIONS: Human platelets dispose over a functional de novo BH4 synthesis. Furthermore, our results indicate the potential of external factors, eg, prolonged storage or glucocorticoid therapy, to significantly affect BH4 synthesis within platelets. Together, these findings offer new insights into the biology and pathobiology of platelet function in humans.

2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency in a 23-year-old man.

2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.178) deficiency is a recently described defect of isoleucine catabolism. The disorder is characterized by normal early development followed by a progressive loss of mental and motor skills. Deterioration may be rapid or may follow a slower decline with a possible stabilization of the disorder on a low-protein diet and appropriate medication. We report a 23-year-old man with 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency with a very mild clinical course. He had apparently normal early development and remained relatively well until the age of 6 years, when he contracted measles. Following this illness, his motor skills and school progress deteriorated. At 15 years he had significant dysarthria, and generalized rigidity with some dystonic and unusual posturing. He was then treated with a low-protein high-carbohydrate diet with a good response in terms of balance and gait. At 18 years he was given benzhexol (Artane), increased slowly from 2 mg to 6 mg daily, resulting in improvement in tremor and dystonia. At 23 years he can dress himself and works in sheltered employment but remains severely dysarthric.

Mutations in the sepiapterin reductase gene cause a novel tetrahydrobiopterin-dependent monoamine-neurotransmitter deficiency without hyperphenylalaninemia.

Classic tetrahydrobiopterin (BH(4)) deficiencies are characterized by hyperphenylalaninemia and deficiency of monoamine neurotransmitters. In this article, we report two patients with progressive psychomotor retardation, dystonia, severe dopamine and serotonin deficiencies (low levels of 5-hydroxyindoleacetic and homovanillic acids), and abnormal pterin pattern (high levels of biopterin and dihydrobiopterin) in cerebrospinal fluid. Furthermore, they presented with normal urinary pterins and without hyperphenylalaninemia. Investigation of skin fibroblasts revealed inactive sepiapterin reductase (SR), the enzyme catalyzing the final two-step reaction in the biosynthesis of BH(4). Mutations in the SPR gene were detected in both patients and their family members. One patient was homozygous for a TC-->CT dinucleotide exchange, predicting a truncated SR (Q119X). The other patient was a compound heterozygote for a genomic 5-bp deletion (1397-1401delAGAAC) resulting in abolished SPR-gene expression and an A-->G transition leading to an R150G amino acid substitution and to inactive SR as confirmed by recombinant expression. The absence of hyperphenylalaninemia and the presence of normal urinary pterin metabolites and of normal SR-like activity in red blood cells may be explained by alternative pathways for the final two-step reaction of BH(4) biosynthesis in peripheral and neuronal tissues. We propose that, for the biosynthesis of BH(4) in peripheral tissues, SR activity may be substituted by aldose reductase (AR), carbonyl reductase (CR), and dihydrofolate reductase, whereas, in the brain, only AR and CR are fully present. Thus, autosomal recessive SR deficiency leads to BH(4) and to neurotransmitter deficiencies without hyperphenylalaninemia and may not be detected by neonatal screening for phenylketonuria.

[Changes in the L-serine and L-threonine dehydrogenase activities in the blood serum of those who worked in the cleanup of the aftermath of the accident at the Chernobyl Atomic Electric Power Station who became ill with chronic acalculous cholecystitis]. / Izmeneniia aktivnosti L-serin- i L-treonindegidrogenaz v syvorote krovi likvidatorov posledstvii avarii na ChAES, bol'nykh khronicheskim beskamennym kholetsistitom.

Activity was studied of blood serum plasmic enzymes L-serine and L-threonine dehydrogenazes (SDG and ThDG) in 92 liquidators of aftermath of the Chernobyl atomic power plant breakdown, presenting with chronic non-calculous cholecystitis during the stage of moderately severe exacerbation with no clinical and laboratory and sonographic signs of affection of the liver. A quarter of the examinees demonstrated an increased activity of the enzymes under study, which fact is regarded by the authors as a preclinical sign of reactive hepatitis. Recommendations are given as to the outpatient registration and prophylactic management and therapy of those persons having taken part in the elimination of the effects of the Chernobyl accident, presenting with biliary pathologies.

Biological inactivation of 5-oxo-6,8,11,14-eicosatetraenoic acid by human platelets.

Neutrophil-derived 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent activator of neutrophils and eosinophils. In the present study we examined the biosynthesis and metabolism of this substance by platelets. Although platelets contain an abundant amount of 5-hydroxyeicosanoid dehydrogenase, the enzyme responsible for the formation of 5-oxo-ETE, they synthesize only very small amounts of this substance from exogenous 5-hydroxyeicosatetraenoic acid (5-HETE) unless endogenous NADPH is converted to NADP+ by addition of phenazine methosulfate. Similarly, relatively small amounts of 5-oxo-ETE were formed by A23187-stimulated mixtures of platelets and neutrophils, which instead formed substantial amounts of two 12-hydroxy metabolites of this substance, 5-oxo-12-HETE and 8-trans-5-oxo-12-HETE, which were identified by comparison with authentic chemically synthesized compounds. These metabolites were also formed from 5-oxo-ETE by platelets stimulated with thrombin or A23187. In contrast, unstimulated platelets converted 5-oxo-ETE principally to 5-HETE. Neither 5-oxo-12-HETE nor 8-trans-5-oxo-12-HETE had appreciable effects on neutrophil calcium levels or platelet aggregation at concentrations as high as 10 micromol/L, but both blocked 5-oxo-ETE-induced calcium mobilization in neutrophils with IC50 values of 0.5 and 2.5 micromol/L, respectively. We conclude that platelets can biologically inactivate 5-oxo-ETE. Unstimulated platelets convert 5-oxo-ETE to 5-HETE, with a 99% loss of biological potency, whereas stimulated platelets convert this substance to 12-hydroxy metabolites, which possess antagonist properties.

Identification of mutations causing 6-pyruvoyl-tetrahydropterin synthase deficiency in four Italian families.

6-Pyruvoyl-tetrahydrobiopterin synthase (PTPS) is involved in tetrahydrobiopterin (BH4) biosynthesis, the cofactor for various enzymes including the hepatic phenylalanine hydroxylase. Inherited PTPS deficiency leads to BH4 depletion, causes hyperphenylalaninemia, and requires cofactor replacement therapy for treatment. We previously isolated the human PTPS cDNA and recently characterized its corresponding gene, PTS. Here we developed PCR-based mutation analysis with newly designed primers to detect genomic alterations and describe five mutations, four of which are novel, in the PTS gene of four Italian families with affected individuals. The mutant alleles found included three missense mutations (T67M, K129E, D136V), a previously described triplet deletion (delta V57), and a single c-3-->g transversion in the 3'-acceptor splice site of intron 1, leading to cryptic splice site usage that resulted in a 12 bp deletion (mutant allele delta (K29-S32)). Except for K129E, all mutant alleles were inactive and/or unstable proteins, as shown by recombinant expression and Western blot analysis of patients' fibroblasts. The PTPS-deficient patient with the homozygous K129E allele had transient hyperphenylalaninemia, did not depend on BH4 replacement therapy, and showed normal PTPS immunoreactivity, but no enzyme activity in primary fibroblasts and red blood cells. In contrast to its inactivity in these cells, the K129E mutant was 2-3 fold more active than wild-type PTPS when transfected into COS-1 or the human hepatoma cell line Hep G2. K129E appears thus as a mutant PTPS whose activity depends on the cell type.

Interindividual variation in the enzymatic 15-keto-reduction of 13,14-dihydro-15-keto-prostaglandin E1 in human liver and in human erythrocytes.

OBJECTIVE: The therapeutic response to PGE1 is highly variable, and a contribution by variable formation of its active tertiary metabolite PGE0 is in question. Hence, the objective of this study was to assess the person-to-person variation of the reduction of the inactive intermediate metabolite 15-KD PGE1 by human liver and human erythrocytes in forming the active metabolite PGE0. METHODS: Source of enzyme was lysed erythrocytes from 29 donors, and a bank of 37 donor livers including specimens from 15 children. Tritium-labelled 13,14-dihydro-15-keto-prostaglandin E1 (15-KD PGE1) was used at low nanomolar concentrations and found to be converted almost exclusively to the more polar compound 13,14-dihydro-prostaglandin E1 (PGE0) by an NADPH-dependent carbonyl reductase. The identity of the product PGE0 was established by comparison of its chromatographic and mass spectral characteristics with authentic PGE0. RESULTS: Lysed erythrocytes had readily measurable enzymatic activity; differences between the preparations from 29 subjects were very small with only a twofold range of variation. In contrast to lysed erythrocytes, intact erythrocytes did not catalyse the reaction so that the erythrocyte activity should be medically immaterial. 15-KD PGE1 15-ketoreductase activity of liver cytosol averaged 61.1 fmol.min-1.mg-1 protein in preparations from 37 human livers. Individual activities varied over an almost tenfold range, with indications of a non-normal distribution. Kinetic studies of selected specimens showed substantially different Vmax values but indistinguishable kM values, suggesting that the individual variation in 15-KD PGE1 15-ketoreduction is the result of differences in enzyme concentration rather than of structural enzyme variations. The activity in 15 livers from children was significantly lower than in those from adults. Inhibition data suggest that both the liver and the erythrocyte enzymes belong to the class of carbonyl reductases. CONCLUSIONS: The variations in hepatic enzyme activity may be expected to affect the transformation of 15-KD PGE1 to the active metabolite PGE0 in vivo. The clinical significance remains to be explored.

Relative contribution of human erythrocyte aldehyde dehydrogenase to the systemic detoxification of the oxazaphosphorines.

Detoxification of cyclophosphamide is effected, in part, by hepatic class 1 aldehyde dehydrogenase (ALDH-1)-catalyzed oxidation of aldophosphamide, a pivotal aldehyde intermediate, to the nontoxic metabolite, carboxyphosphamide. This enzyme is found in erythrocytes as well. Detoxification of aldophosphamide may also be effected by enzymes, viz. certain aldo-keto reductases, that catalyze the reduction of aldophosphamide to alcophosphamide. Such enzymes are also found in erythrocytes. Not known at the onset of this investigation was whether the contribution of erythrocyte ALDH-1 and/or aldo-keto reductases to the overall systemic detoxification of circulating aldophosphamide is significant. Thus, NAD-linked oxidation and NADPH-linked reduction of aldophosphamide catalyzed by relevant erythrocyte enzymes were quantified. ALDH-1-catalyzed oxidation of aldophosphamide (160 microM) to carboxyphosphamide occurred at a mean (+/- SD) rate of 5.0 +/- 1.4 atmol/min/rbc (red blood cell). Aldo-keto reductase-catalyzed reduction of aldophosphamide (160 microM) to alcophosphamide occurred at a much slower rate, viz. 0.3 +/- 0.2 atmol/min/rbc. Thus, at a pharmacologically relevant concentration of aldophosphamide, viz. 1 microM, estimated aggregate erythrocyte ALDH-1-catalyzed aldophosphamide oxidation, viz. 2.0 micromol/min, was only about 3% of estimated aggregate hepatic enzyme-catalyzed aldophosphamide oxidation, viz. 72 micromol/min; however, this rate is greater than the estimated flow-limited rate of aldophosphamide delivery to the liver by the blood, viz. 1.5 micromol/min. These observations/considerations suggest an important in vivo role for erythrocyte ALDH-1 in systemic aldophosphamide detoxification. Erythrocyte ALDH-1-effected oxidation of other aldehydes to their corresponding acids, e.g. retinaldehyde to retinoic acid, may also be of pharmacological and/or physiological significance since a wide variety of aldehydes are known to be substrates for ALDH-1.

Glucose-6-phosphate dehydrogenase deficiency severely restricts the biotransformation of daunorubicin in human erythrocytes.

Recognition and analysis of distinct mechanisms by which primaquine and other hemolytic drugs activate the hexose monophosphate shunt (HMS) have suggested a hitherto unsuspected pharmacogenetic interaction between daunorubicin metabolism and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Because this deficiency is very common, and because anthracyclines are indispensable antitumor antibiotics that are biotransformed mainly by carbonyl reductase, we have compared the reductase-mediated conversion of daunorubicin to daunorubicinol and the conversion of doxorubicin to doxorubicinol in G6PD-deficient and nondeficient erythrocytes. We found that even without G6PD deficiency, the HMS dehydrogenases selectively limited daunorubicin metabolism, as contrasted with that of doxorubicin. The milder GdA- variety of G6PD deficiency restricted the biotransformation of daunorubicin at therapeutic levels, in hemolysates and intact erythrocytes, within 15 minutes, for at least 24 hours. The bioconversion defect was even more severe in Gd Mediterranean G6PD deficiency. Primaquine aldehyde competed with daunorubicin as a substrate for carbonyl reductase. These studies show that HMS dehydrogenase activity controls carbonyl reductase-dependent biotransformation. New issues arise concerning possible effects of G6PD deficiency on the oncolytic and toxic properties of anthracyclines that are effective substrates for carbonyl reductase and also on non-xenobiotic reactions catalyzed by this enzyme.

The presence of 2-keto-3-deoxygluconic acid and oxoaldehyde dehydrogenase activity in human erythrocytes.

2-Keto-3-deoxygluconic acid (3-DGA) is produced from 3-deoxyglucosone (3-DG:a highly reactive glycation intermediate) through oxidation by the enzyme oxoaldehyde dehydrogenase (OAD) in animals. We developed a specific assay method for 3-DGA using high-performance liquid chromatography [Fujii, E. et al. (1994) J. Chromatogr. B 660, 265-270] and measured it in the hemolysate and plasma of diabetic patients and healthy subjects. Both human erythrocytes and plasma contained considerable amounts of 3-DGA. However, human erythrocyte contained about 30-50 times higher 3-DGA than human plasma did and also had the same ability to convert 3-DG to 3-DGA as OAD had. Erythrocyte 3-DGA levels of diabetic patients were 990 +/- 370 nmol/gHb (n = 57, Mean +/- SD) and were significantly higher compared with healthy subjects (527 +/- 194 nmol/gHb, n = 7, p < 0.01). In all diabetic patients and healthy subjects (n = 64), there was only one patient who had a very low level of erythrocyte 3-DGA and lacked the ability to convert 3-DG to 3-DGA. When erythrocytes were incubated at 37 degrees C for 8 hours in phosphate buffer containing 0.35 mM 3-DG, 3-DG was easily taken into the erythrocytes and was converted to 3-DGA. Our results suggest the contribution of OAD not only to the prevention of glycation of hemoglobin but also to that of blood vessels by scavenging plasma 3-DG into erythrocytes.