Red Blood Cell Enzyme Research Articles

Insulin Resistance, Its Consequences, and Coronary Heart Disease

Approximately 25 years ago, our research group1 2 used quantitative methods to show that resistance to insulin-mediated glucose disposal was present in patients with either impaired glucose tolerance or NIDDM. These initial observations have been confirmed on multiple occasions since then, and there is now a large body of evidence that shows that this defect is present in the vast majority of glucose-intolerant individuals.3 4 5 6 7 8 In addition, several reports9 10 11 demonstrated that first-degree relatives of patients with NIDDM are also relatively insulin resistant compared with well-matched volunteers without a family history of NIDDM. Finally, prospective studies12 13 14 15 16 have identified insulin resistance and/or compensatory hyperinsulinemia as the initial defect in the development of NIDDM. Thus, the central role of insulin resistance in the pathogenesis and clinical course of patients with NIDDM has been well established. Of greater relevance to the IRAS study of “Insulin Sensitivity and Atherosclerosis” published in this issue of Circulation 17 is recent awareness that the ability of insulin to mediate glucose disposal can differ by as much as 10-fold in subjects with normal glucose tolerance.18 19 Because the degree of insulin resistance in a substantial portion of these persons approaches that seen in patients with impaired glucose tolerance or NIDDM, it is assumed that they are able to secrete enough insulin to overcome the insulin resistance and maintain normal glucose tolerance. Unfortunately, the philanthropic response on the part of the pancreatic β-cell is not without cost, and in 1988,20 it was emphasized that these individuals were at increased risk to have higher plasma TG and lower HDL cholesterol concentrations and higher blood pressure. More importantly, it was suggested that the cluster of abnormalities associated with insulin resistance, designated as Syndrome X, significantly increased …

Three cases of hereditary nonspherocytic hemolytic anemia associated with red blood cell glutathione deficiency

Three unrelated Japanese patients with chronic nonspherocytic hemolytic anemia wer found to have marked deficiency of red blood cell (RBC) reduced glutathoine (GSH) (4.4%, 13.1%, and 6.9% of normal, respectively). A panel of RBC enzyme assays showed that one patient had decreased glutathione synthetase activity and the other two were moderately deficient in gamma-glutamylcystine synthetase. Some family members of each patient showed mild deficiency of the respective enzymes. RBCs of these patients also showed a decreased level of glutathione-S-transferase as in previously described GSH-deficient cases. Hemolytic anemia was their only manifestation, and neither 5- oxoprolinemia nor 5-oxoprolinuria, which are usually associated with to generalized type of glutathione synthetase deficiency, was noted in our patients.

Three Cases of Hereditary Nonspherocytic Hemolytic Anemia Associated With Red Blood Cell Glutathione Deficiency

Three unrelated Japanese patients with chronic nonspherocytic hemolytic anemia wer found to have marked deficiency of red blood cell (RBC) reduced glutathoine (GSH) (4.4%, 13.1%, and 6.9% of normal, respectively). A panel of RBC enzyme assays showed that one patient had decreased glutathione synthetase activity and the other two were moderately deficient in gamma-glutamylcystine synthetase. Some family members of each patient showed mild deficiency of the respective enzymes. RBCs of these patients also showed a decreased level of glutathione-S-transferase as in previously described GSH-deficient cases. Hemolytic anemia was their only manifestation, and neither 5-oxoprolinemia nor 5-oxoprolinuria, which are usually associated with to generalized type of glutathione synthetase deficiency, was noted in our patients.

Whey from cultured skim milk decreases serum cholesterol and increases antioxidant enzymes in liver and red blood cells in rats

The study was carried out to examine the effect of whey from bovine skim milk fermented with bifidobacteria and lactic acid bacteria on plasma cholesterol and antioxidant enzymes in rats. Rats were maintained for 6 wks on a purified diet (reference diet) and on the purified diet supplemented milk whey or whey from cultured skim milk with Bifidobacterium longum (B. longum), Lactobacillus acidophilus (L. acidophilus) or Streptococcus salivarius subsp. thermophilus (S. thermophilus). Diets containing the product with S. thermophilus resulted in the lowest concentration of plasma cholesterol. The activity of superoxide dismutase (SOD) in red blood cells (RBC) and the activity of catalase in liver were elevated on cultured product-diets compared with the reference diet; in addition, the activity of glutathione peroxidase (GSHPx) in RBC was higher on the L. acidophilus diet compared with the reference diet. Although there were no significant differences in the concentrations of thiobarbituric acid-reactive substances and α-tocopherol in plasma, plasma d<1.063 g/ml lipoprotein fractions prepared from rats fed on the B. longum- and L. acidophilus-diets were resistant to the oxidative stress induced by a transition metal ion when compared with those from rats fed on the reference diet. The non-fermented whey diet was not as effective in lowering plasma cholesterol and in increasing antioxidant enzymes as were the fermentation product diets. These results therefore suggest that wheys from cultured milk may exert spesific effects on hypercholesterolemia and oxidative stress.

Modulation of hypoxic pulmonary vasoconstriction by antioxidant enzymes in red blood cells.

To determine whether antioxidant mechanisms within red blood cells (RBCs) significantly contribute to preserving hypoxic pulmonary vasoconstriction (HPV) in both the absence and the presence of oxidative stress, we investigated HPV changes in isolated rabbit lungs perfused with solutions containing RBCs treated with various inhibitors of superoxide dismutase (SOD), anion channels, catalase (CAT), or glutathione peroxidase (GSH-Px). Perfusion was maintained at a constant flow rate of 70 ml/min, and lung temperature at 37 to 38 degrees C. Hematocrit was adjusted to 7%. In the absence of overt oxidative stress, HPV was significantly enhanced in the perfusate containing control RBCs (untreated RBCs) as compared with that in Krebs-Henseleit buffer. Inhibition of SOD, CAT, and GSH-Px within RBCs, as well as anion channels located on the RBC membrane, had little influence on HPV. Neither exogenous SOD nor CAT altered HPV. In the presence of high levels of reactive oxygen species (ROS), generated by addition of xanthine (100 microM) and xanthine oxidase (10 mU/ml) to the reservoir, HPV was considerably suppressed in the perfusate containing only buffer but was restored in the perfusate with control RBCs. Inhibition of CAT or GSH-Px in RBCs preserved the HPV, whereas inhibition of SOD or anion channels failed to preserve HPV obtained during exposure to high ROS levels. Addition of exogenous SOD, but not CAT, to the perfusate containing RBCs in which endogenous SOD had been inhibited restored HPV under high ROS conditions. In conclusion, (1) although RBCs augment HPV in the absence of ROS, this finding is not attributable to the antioxidants in RBCs. (2) RBCs restore HPV upon exposure to high ROS. This finding may well be explained by antioxidant mechanisms operating within RBCs, especially those of endogenous SOD.

The ontogeny of the glucose-6-phosphatase enzyme in human embryonic and fetal red blood cells.

We have shown for the first time that the microsomal glucose-6-phosphatase enzyme protein is present in human embryonic and fetal red blood cells and the ontogeny of its expression has been determined. In the earliest embryos, red cells are predominantly of the primitive megaloblastic type. Circulating red cells in the primitive megaloblastic series are predominantly nucleated and glucose-6-phosphatase immunopositive. Non-nucleated, immunoreactive megaloblastic cells are in a minority. In fetuses > 12 weeks gestation, the erythrocytes are of the definitive normoblastic series and in the transitional period of switch-over in late embryonic-early fetal life, up to 30% of glucose-6-phosphatase immunopositive cells are definitive normoblastic in type, with a variable contribution from nucleated and non-nucleated cells. Thereafter, the number of immunopositive cells in the definitive normoblastic series decreases such that after 12 weeks gestation it is less than 5%. The fact that a predominantly hepatic protein in adults (glucose-6-phosphatase) is present in embryonic and fetal red blood cells, particularly nucleated red cells, raises the possibility of diagnosis of disorders of liver protein expression in nucleated fetal red cells isolated from the first trimester maternal circulation.

Frequencies of glucose 6-phosphate dehydrogenase pyruvate kinase and hexokinase deficiencies in the Basrah population of Iraq

Introduction: The frequencies of enzyme deficiencies that cause hemolytic anemia among Iraqi people in southern Iraq have been studied. These hereditary red blood cell enzymes include glucose 6-phosphate dehydrogenase (G6PD), pyruvate kinase (PK) and hexokinase (HK). The most common enzyme deficiency is G6PD deficiency, an X-linked recessive trait. PK deficiency is associated with chronic hemolytic anemia, but HK deficiency is a rare autosomal recessive enzymopathy. Methods: A total of 1497 clinically normal neonates and 1629 apparently healthy adults from Iraqi Arab original inhabitants of Basrah were studied. Blood samples obtained from the umbilical cord of the newborns or by venipuncture from adults were collected in ACD anticoagulant and stored at 4 °C. Erythrocyte G6PD activity was measured qualitatively by the fluorescent spot test or methemoglobin reduction test and confirmed quantitatively by spectrophotometric analysis. PK and HK activities were estimated only quantitatively by spectrophotometry. Results: Only seven partially deficient infants out of 506 infants and 343 adults were detected with PK deficiency. On this basis, the incidence would be 0.82 with an estimated gene frequency of about 0.004. Partial HK deficiency was found in only one female infant out of 220 infants and 246 adults in both sexes. No case of complete deficiency of these enzymes was identified. G6PD deficiency was identified in 131 out of 1040 male infants and adults. All the affected males seemed to have severe deficiency (hemizygotes). On this basis, we estimated the gene frequency to be about 12.6%. In the female population, 31 out of 771 female infants and adults had severe enzyme deficiency, a frequency of 4.0% (possible homozygosity) while 67 females showed intermediate or moderate enzyme activity (heterozygosity), a prevalence of 8.7%. Discussion: Among the Iraqi Arab populations that are original inhabitants of Basrah, erythrocytes G6PD deficiency is by far the most common enzyme deficiency that causes hemolytic anemia. The gene frequencies for male infants and adults were 11.7% and 13.1%, respectively. The observed female incidences, for both infants and adults, were far from those expected. Factors that might be the causes of such discrepancies were discussed.

No evidence for inhibition of human glutathione reductase by valproic acid

The human red blood cell enzyme glutathione reductase (GR) was reported to be inhibited by the anticonvulsant drug valproic acid (VPA) [Cotariu et al., Biochem Pharmacol 43: 425–429, 1992]. When attempting to reproduce and extend these experiments, we could not detect any significant effect of VPA on glutathione reductase in haemolysates from 20 healthy children and 10 children under VPA therapy, no matter which concentration of the drug (0.9 or 1.8 mM in a haemolysate diluted by a factor of 50 or 1.8 mM directly in the assay), which incubation time (0–60 min) and which assay system were chosen. An influence of VPA on FAD-free apoglutathione reductase was also excluded. GR-activities of 10 children under VPA therapy (1.08 ± 0.14 U/mL blood or 7.57 ± 0.94 U/g Hb) were almost identical with the activities of age- and sex-matched controls (1.04 ± 0.17 U/mL or 7.79 ± 1.32 U/g Hb). No correlation between erythrocyte GR activity and serum levels of VPA was observed. Finally, incubation of crystalline human GR with VPA did not lead to enzyme inhibition; rather, in most experiments the enzyme was stabilized by incubation with VPA. Possible explanations for the discrepancies between the results of Cotariu et al. and our data are discussed.

Lipid peroxidation products and antioxidant enzymes in red blood cells during normal and diabetic pregnancy

Oxygen free radicals produced during normal aerobic metabolism have been implicated in several pathophysiological mammalian processes. The importance of free radical-mediated fatty acid oxidation has received much attention. The generation of active oxygen species may lead to lipid peroxidation and formation of reactive products, which may be involved in severe damage of cell molecules and structures. Free radical metabolism in pregnancy and in diabetes mellitus is still unclear. To add new insights to the question, changes in lipid peroxidation products and activities of three antioxidant enzymes: catalase (CAT), glutathione peroxidase (GPX) and superoxide dismutase (SOD) in maternal red blood cells haemolysates were evaluated in pregnant women with insulin-dependent diabetes mellitus (IDDMPW) and in healthy pregnant women (HPW). Healthy non-pregnant women were the control group for IDDM-PW and HPW, respectively. Pregnancy provoked an increase of lipoperoxidation products and an high SOD activity since early pregnancy, while CAT and GPX activities did not change during gestation. IDDM-PW showed higher content of lipoperoxidation breakdown products and lower SOD activity at each trimester, if compared with HPW; moreover, a slight increase of CAT and SOD acvitity is reported during late diabetic pregnancy. IDDM-PW were in very good metabolic control at time of sampling. The variations reported suggest an easier membrane lipoperoxidability and, consequently, an easier membrane damage during diabetic gestation.

Cobalamin Deficiency in the Elderly

Cobalamin Deficiency in the Elderly

Antioxidant enzymes in RBCs as a biological index of age related macular degeneration.

The present study was undertaken to assess the levels of antioxidant enzymes in red blood cells of subjects with age-related macular degeneration and age-matched controls. The results obtained show a significant decrease in activities of superoxide dismutase (p < 0.001) and glutathione peroxidase (p < 0.001) as compared to the controls. A good correlation (r = -0.99) was also observed between age and decreased activity of antioxidant enzymes in controls, and also correlated well with age-related macular degeneration. In conclusion, oxidative stress as assessed by antioxidant enzymes is more pronounced in subjects with age-related macular degeneration as compared to age-matched controls.

Oxidative Injury to Erythrocytes, Cell Rigidity and Splenic Hemolysis in Hemodialyzed Patients before and during Erythropoietin Treatment

The oxidative injury to erythrocytes, red blood cell (RBC) rigidity and splenic hemolysis was assayed in 17 chronically hemodialyzed patients before and during recombinant erythropoietin (EPO) treatment. When a stable hematocrit between 30 and 35% had been established for at least 4 months, a statistically significant increase in RBC volume, hemoglobin concentration, hematocrit, reticulocyte count, and several RBC enzymes (2,3-diphosphoglycerate, glucose 6-phosphate dehydrogenase, pyruvate kinase, hexokinase) was noted. This indicated significant RBC rejuvenation under the influence of EPO. However, no significant improvement in the RBC oxidative sensitivity, RBC deformability, splenic RBC volume, slow mixing splenic RBC volume, and the intrasplenic RBC transit time could be disclosed. These data confirm the existence of an extra-erythrocytic factor in uremic plasma, which is partly responsible for a reduced RBC life span in hemodialysis patients despite EPO treatment.

Identification, purification and reconstitutions of thiamin metabolizing enzymes in human red blood cells

Thiamin and its mono- (TMP), di- (TDP) and triphosphate (TTP) were assayed in adult human whole blood using high-performance liquid chromatography (HPLA). TDP and TTP were detected in red blood cells (RBC), but not in plasma. After incubation with 20 μM thiamin and 5 mM glucose for 2 h, the TDP and TTP contents of RBC increased fron 111 to 222 and 0.6 to 2.2 nmol/1 of packed RBC, respectively, suggesting enzymatic convertion of thiamin to TDP and then to TTP. Thiamin pyrophosphokinase (TPK, EC 2.7.6.2) had not been isolated from human materials, nor had cytosolic kinase (AK1, EC 2.7.4.3) in human RBC been demonstrated to catalyze the phosphorylation of TDP to TTP, although AK1 from pig and chicken skeletal muscles possess TTP-synthesizing activity. TPK and AK1 in a human RBC lysate were therefore purified by a series of the conventional techniques. The specific activity of the purified TPK, which was obtained as a single protein, was 720 nmol TDP formed/mg protein per h at 37°C. A partially purified AK1 preparation catalyzed the formation of TTP from TDP (specific activity, 170 nmol/mg protein per h at 37°C) in addition to its proper reaction to form ATP from ADP. After Incubation of the purified TPK and AK1 with 20 μM thiamin in the presence of ATP, ADP and Mg 2+ at 37°C for 48 h, the amounts of TDP and TTP synthesized were 465 and 54.0 pmol/250 μ1 reaction mixture, respectively. Neither TDP nor TTP was formed when TPK was omitted from the reaction mixture and an omission of AK1 resulted in the formation of TDP alone. These results indicate that thiamin is converted to TDP by TPK and, subsequently, to TTP by AK1 in human RBC.

Decreased Pyrimidine Nucleoside Monophosphate Kinase Activity in Sickle Erythrocytes

We have previously shown that physiologic concentrations of hemin cause marked inhibition of several red blood cell (RBC) enzymes. Because endogenous heme content is elevated in sickle RBCs, we have examined the activity of hemin-sensitive enzymes in these RBCs. One of the hemin-sensitive enzymes, pyrimidine nucleoside monophosphate kinase (PNMK), was shown to have decreased activity in sickle RBCs relative to RBCs of equivalent cell age. The other hemin-sensitive enzymes, including adenylate kinase (AK), pyrimidine 5'-nucleotidase (P5N), 6-phosphogluconate dehydrogenase (6PGD), and aldolase, had activities that were appropriate for cell age. We have also examined the affinity of the hemin-sensitive enzymes to hemin. Using two different methods, PNMK was shown to have the highest binding affinity to hemin. The exquisite sensitivity of PNMK to inhibition by hemin, coupled with the enzyme's high affinity to hemin, may account for the decrease in PNMK activity and the lack of significant decrease in the other hemin-sensitive enzymes in sickle RBCs. These results suggest that the increased endogenous heme content in sickle RBCs may be responsible for the decrease in PNMK activity. Whether the increased endogenous heme content of sickle RBCs can cause hemolysis indirectly by inhibiting RBC enzymes remains to be determined.

Decreased pyrimidine nucleoside monophosphate kinase activity in sickle erythrocytes

We have previously shown that physiologic concentrations of hemin cause marked inhibition of several red blood cell (RBC) enzymes. Because endogenous heme content is elevated in sickle RBCs, we have examined the activity of hemin-sensitive enzymes in these RBCs. One of the hemin- sensitive enzymes, pyrimidine nucleoside monophosphate kinase (PNMK), was shown to have decreased activity in sickle RBCs relative to RBCs of equivalent cell age. The other hemin-sensitive enzymes, including adenylate kinase (AK), pyrimidine 5′-nucleotidase (P5N), 6- phosphogluconate dehydrogenase (6PGD), and aldolase, had activities that were appropriate for cell age. We have also examined the affinity of the hemin-sensitive enzymes to hemin. Using two different methods, PNMK was shown to have the highest binding affinity to hemin. The exquisite sensitivity of PNMK to inhibition by hemin, coupled with the enzyme's high affinity to hemin, may account for the decrease in PNMK activity and the lack of significant decrease in the other hemin- sensitive enzymes in sickle RBCs. These results suggest that the increased endogenous heme content in sickle RBCs may be responsible for the decrease in PNMK activity. Whether the increased endogenous heme content of sickle RBCs can cause hemolysis indirectly by inhibiting RBC enzymes remains to be determined.

Low thyroxine levels in female psychiatric inpatients with riboflavin deficiency: implications for folate-dependent methylation.

Intermediates in the folate-dependent methylation pathways may play a role in the etiology and treatment of such mental disorders as major depression. These pathways include a step dependent on a riboflavin (B2)-derived coenzyme, flavin adenine dinucleotide (FAD), which is reportedly sensitive to thyroid status and to phenothiazine and tricyclic drug exposure. In a sample of 52 male and female acute psychiatric inpatients, 17% (n = 9) showed B2 deficiency (i.e., insufficient FAD activity) on a functional red blood cell enzyme assay, but only one B2-deficient individual showed deficiency in another B-complex vitamin (folate). All patients with B2 deficiency were women, who were also significantly younger than the rest of the sample. The B2-deficient women had significantly lower thyroxine levels, even when controlling for sex and covarying for age. B2-deficient patients exhibited a nonsignificant trend toward more unipolar depression (44% vs 14%), but not toward bipolar or schizophrenic disorders. As in a previous study, drug exposure did not show a relationship to riboflavin deficiency in this sample. The findings suggest that B2 (FAD) activity may serve as a sensitive marker of thyroxine status in certain female psychiatric inpatients and that B2 deficiency may play an etiological role in defects of the methylation pathways in a subset of mentally ill individuals.

Preparative purification of pig red blood cell hexokinase type III using a new efficient chromatographic support.

In this paper we report the purification of pig erythrocyte hexokinase type III, at preparative level, using 52 liters of starting material (hemolysate). This was possible using a new efficient anion exchanger support, the Toyopearl DEAE 650 M which allows completely to change the strategy of removing hemoglobin from hemolysates, permitting to handle large amounts of starting material and reducing work would have required months using conventional anion exchanger supports, to only 2-3 days. Furthermore, we have tested the binding of other red blood cell enzymes to the Toyopearl DEAE 650 M, showing a wider potential use of this chromatographic support for their purification at a preparative level.

Inhibition of human red blood cell glutathione reductase by valproic acid

Glutathione reductase (GR) one of the enzymes of the glutathione redox cycle, plays a salient role in maintaining appropriate cellular levels of reduced glutathione. The enzyme in human red blood cells is inhibited in vitro by the anticonvulsant drug valproic acid (VPA). The inhibition is dose-dependent, reversible, uncompetitive and does not depend on the redox state of the enzyme. VPA also inhibits red blood cell GR activity in children being treated with the drug. The level of serum VPA correlates significantly with the suppression of GR activity.

Interstrain differences in red cell enzyme activities in mice and rats

1. 1. Interstrain differences in red blood cell enzyme activities were studied in mice (BALB/c, C57BL/6, C3H/He, DBA/2 and ddY) and rats (Donryu, F344/N, SD, Wistar and Wistar/ST), and were also compared with hamster, guinea-pig and rabbit. 2. 2. The enzyme activities measured were; glutathione S-transferase (GST), glucose-6-phosphate dehydrogenase (G-6-PD), 6-phosphogluconate dehydrogenase (6-PGD), NADPH-diaphorase (ND), hexokinase (Hx), glutamate oxaloacetate transaminase (GOT), lactate dehydrogenase (LDH) and acetylcholinesterase (AChE). 3. 3. There were marked variations in the activities of some red cell enzymes (e.g. GST, Hx, ND), while others (e.g. G-6-PD, 6-PGD) were much less variable both within different strains and species.

Red blood cell enzymes in the diagnosis of genetic disease

Erythrocytes are uniform cells which contain only those proteins that are synthesized during the reticulocyte stage. The relationship of red cell enzymes to gene dosage and gene expression enables the use of red cell enzyme assays to determine the presence or absence of gene defects causing enzyme deficiencies leading to various metabolic diseases; in addition, the mode of inheritance of these defects can frequently be ascertained by analyzing red cell enzymes. However, indirect evidence favoring other enzyme deficiency states can sometimes be obtained from a study of red cell enzyme activities, because apparent enzyme deficiencies may result from the accumulation of inhibitory metabolites formed due to an enzyme deficiency in other tissues. The polymorphic expression of many red cell enzymes lends itself to biochemical analysis which can produce highly accurate and specific diagnostic information.