Red Blood Cell Enzyme Research Articles

Activity of Red Blood Cell Enzymes and Oxygen Affinity of Hemoglobin in Athletes during Physical Exercise

Activity of Red Blood Cell Enzymes and Oxygen Affinity of Hemoglobin in Athletes during Physical Exercise

Population structure of the rural communities on the island of Krk (Croatia): A comparison of genetic, cultural, and geographic data

The population structure of seven rural communities from the island of Krk (Croatia) was studied on the basis of its genetic constitution and genetic structure, and different sets of biological (genetic), biocultural (migration), cultural (linguistic), and geographic data. Genetic constitution was estimated from the allelic frequencies of nine tetranucleotide STR loci, three HLA class I loci, five HLA class II loci, and five red blood cell enzyme and serum protein loci. Genetic structure was based on coefficients of genetic diversity GST and genetic kinship RST. Population structure was assessed by the structure of mutual relationships among biological, biocultural, cultural, and geographic matrices through Mantel's test, or by relationships among biological, biocultural, cultural measures of distance and/or similarity, and geographic distance with regression analysis of the “isolation by distance” model. One group of genetic markers (red blood enzymes, serum proteins, and HLA polymorphisms) reflects heterogeneity of the rural communities (a reflection of important historical processes and migration patterns), and a second group of genetic markers (STR polymorphisms) reflects the currently observed genetic homogeneity of the rural population. The positive correlations between the matrices of genetic (HLA) and linguistic distances indicate their possible co-evolution under conditions of genetic isolation (low gene flow). Furthermore, the negative correlations between the matrices of genetic (STR) distances and the migration kinship of the second and the third cohort indicate temporal coincidence between genetic constitution and structure assessed from STRs and migration patterns in the period between 1892 and 1940. Finally, the positive correlations between the matrices of genetic (STR) and geographic distances indicate isolation by distance. Am. J. Hum. Biol. 12:509–525, 2000. © 2000 Wiley-Liss, Inc.

Congenital infiltrating lipomatosis of the face: Case report

vated methemoglobin levels and the occurrence of Heinz bodies are frequently, but not always, perceptible indicators of oxidative damage, often having disappeared by the time hemolysis is obvious. Often, circulating red blood cells with apparent pieces missing from their perimeter appear on blood smears.5 Hemoglobinemia and hemoglobinuria are usually of moderate degree during the explosive episode, but typically hemolysis is limited in duration, because only relatively old cells are vulnerable. The more nearly normal G-6-PD activity found in younger red blood cells, which become more abundant in the circulation in response to hemolysis, protects them from excessive oxidative damage. Consequently, the red blood cell count may return to normal even though administration of the offending drug is continued, and diagnosis of a G-6-PD deficiency by assay of red blood cell enzyme activity may be difficult in this equilibrium phase when reticulocytosis is marked.6 Confirmation of the clinical diagnosis often must await the return of the red blood cell age distribution to normal, which is what was found in second test sample in this case. At least 100 million persons in the world have red cell G-6-PD deficiency,3 so that efforts to screen populations at high risk using 1 of many simple screening tests for this deficiency should be encouraged. Screening, performed when the patient is well, can serve to forewarn both patient and physician of increased susceptibility to such things as oxidant drugs, fava beans, and infections.

Pyridoxalated hemoglobin polyoxyethylene: a nitric oxide scavenger with antioxidant activity for the treatment of nitric oxide-induced shock

Hemoglobins modified for therapeutic use as either hemoglobin-based oxygen carriers or scavengers of nitric oxide are currently being evaluated in clinical trials. One such product, pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), is a human-derived and chemically modified hemoglobin that has yielded promising results in Phase II clinical trials, and is entering a pivotal Phase III clinical trial for the treatment of shock associated with systemic inflammatory response syndrome (SIRS). Shock associated with SIRS is a NO-induced shock. PHP, a new mechanism-based therapy, has been demonstrated in clinical trials to have the expected hemodynamic activity of raising blood pressure and reducing catecholamine use, consistent with its mechanism of action as a NO scavenger. PHP is conjugated with polyoxyethylene, which results in a surface-decorated molecule with enhanced circulation time and stability as well as in attachment of soluble red blood cell enzymes, including catalase and superoxide dismutase. PHP thus contains an antioxidant profile similar to the intact red blood cell and is therefore resistant to both initial oxidative modification by oxidants such as hydrogen peroxide and subsequent ferrylhemoglobin formation. These studies suggest both that the redox activity of modified hemoglobins can be attenuated and that modified hemoglobins containing endogenous antioxidants, such as PHP, may have reduced pro-oxidant potential. These antioxidant properties, in addition to the NO-scavenging properties, may allow the use of PHP in other indications in which excess NO, superoxide, or hydrogen peroxide is involved, including ischemia-reperfusion injury and hemorrhagic shock.

Transfusion Approach to Neonatal Anemia

After completing this article, readers should be able to: 1. Describe the basis for guidelines for red blood cell (RBC) transfusions to infants. 2. Delineate the guidelines for storing and administering RBCs to preterm infants as a means of reducing donor exposure without causing hyperkalemia or acidosis. 3. Describe an alternative to selecting blood from cytomegalovirus antibody-negative donors to prevent transfusion-transmitted infection. 4. Delineate the procedure used to prevent graft-versus-host disease in preterm infants receiving RBC transfusions. Neonates are given RBC transfusions for several reasons, including hemolytic disease of the newborn due to maternal alloimmunization, congenital abnormalities of RBC enzymes and membranes, and anemia of prematurity due to multiple interacting factors, the most important of which are phlebotomy blood loss and low erythropoietin levels. Although great efforts have been made during the past few years to optimize the transfusion of RBCs to these tiny patients, several questions have not been answered definitively. Accordingly, transfusion practices vary among physicians caring for neonates. This lack of precise approach stems from incomplete knowledge of the cellular and molecular biology of hematopoiesis during the perinatal period as well as an incomplete understanding of the infant’s response to severe anemia. Generally, RBC transfusions are administered in small volumes (15 mL/kg per dose) to maintain a hematocrit (HCT) level that is believed to be most desirable in terms of oxygen delivery and tissue oxygenation for each neonate’s clinical status (Table 1⇓ ). It is widely recognized that this clinical approach is imprecise, and more physiologic indications for RBC transfusions, such as circulating RBC mass, available oxygen, and measurements of oxygen delivery and tissue extraction, have been suggested. However, the means by which these techniques can best be applied to daily clinical practice remain to be defined. Because clearly established indications for neonatal RBC transfusions based on controlled scientific studies …

Chronic non-spherocytic haemolytic disorders associated with glucose-6-phosphate dehydrogenase variants

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect, being present in over 400 million people world wide. In a small number of cases, G6PD deficiency can lead to mild-to-severe chronic haemolysis, which is further exacerbated by oxidative stress. Such G6PD variants have been described all over the world and are responsible for chronic non-spherocytic haemolytic anaemia (CNSHA). To date 61 G6PD molecular variants associated with CNSHA have been identified, only some of which can cause the severe reduction in stability of the red blood cell enzyme. The distribution of the different mutations shows a predominance of small mutational events, and many have been found repeatedly in different parts of the world. By revisiting the 61 class I variants described so far, we can observe that a low inhibition constant (Ki) for NADPH, a higher Kmfor substrates and a reduced thermostability are common.

Population structure of the rural communities on the island of Krk (Croatia): A comparison of genetic, cultural, and geographic data.

The population structure of seven rural communities from the island of Krk (Croatia) was studied on the basis of its genetic constitution and genetic structure, and different sets of biological (genetic), biocultural (migration), cultural (linguistic), and geographic data. Genetic constitution was estimated from the allelic frequencies of nine tetranucleotide STR loci, three HLA class I loci, five HLA class II loci, and five red blood cell enzyme and serum protein loci. Genetic structure was based on coefficients of genetic diversity G(ST) and genetic kinship R(ST). Population structure was assessed by the structure of mutual relationships among biological, biocultural, cultural, and geographic matrices through Mantel's test, or by relationships among biological, biocultural, cultural measures of distance and/or similarity, and geographic distance with regression analysis of the "isolation by distance" model. One group of genetic markers (red blood enzymes, serum proteins, and HLA polymorphisms) reflects heterogeneity of the rural communities (a reflection of important historical processes and migration patterns), and a second group of genetic markers (STR polymorphisms) reflects the currently observed genetic homogeneity of the rural population. The positive correlations between the matrices of genetic (HLA) and linguistic distances indicate their possible co-evolution under conditions of genetic isolation (low gene flow). Furthermore, the negative correlations between the matrices of genetic (STR) distances and the migration kinship of the second and the third cohort indicate temporal coincidence between genetic constitution and structure assessed from STRs and migration patterns in the period between 1892 and 1940. Finally, the positive correlations between the matrices of genetic (STR) and geographic distances indicate isolation by distance. Am. J. Hum. Biol. 12:509-525, 2000. Copyright 2000 Wiley-Liss, Inc.

Hemolytic anemia: Part 1.

1. Kwesi Sackey, MD* 1. 2. *Associate Professor, The University of Texas Medical Branch, Galveston, TX. After completing this article, readers should be able to: 1. Explain the role of immune reactions, red blood cell membrane defects, red blood cell enzyme defects, or hemoglobin abnormalities in the development of hemolytic anemia. 2. List the characteristic findings of hemolytic anemia. 3. Describe the significance of a low reticulocyte count in a patient who has chronic hemolysis. 4. Describe the potential complications of red blood cell transfusions. Hemolytic anemia arises from a shortened survival of red blood cells (RBCs) due to an inherent abnormality of the cell, environmental factors, or both. It can be characterized by varying degrees of anemia, jaundice, an enlarged spleen, or combinations of these conditions. If the hemolysis is massive, the urine may become dark due to hemoglobinuria. Findings on examination of the peripheral blood typically include changes in the morphology of the RBCs and an increase in reticulocyte count. A low reticulocyte count usually indicates poor production of RBCs. The major categories of hemolytic anemia are: 1) immune-mediated (alloimmune or autoimmune), 2) membrane defects (spherocytosis, elliptocytosis), 3) enzyme defects (glucose-6-phosphate dehydrogenase[ G6PD] deficiency, pyruvate kinase deficiency), and 4) hemoglobin defects (sickle cell disease, thalassemia). ### DEFINITION AND EPIDEMIOLOGY This hemolysis arises from a reaction between an individual’s RBCs and “naturally occurring antibodies” that usually are not present in that individual. The primary alloimmune types are Rh (anti-D) hemolytic disease in the newborn and ABO hemolytic disease. ABO hemolytic disease is several times more common than Rh hemolytic disease. ### PATHOGENESIS AND PATHOPHYSIOLOGY In Rh disease, the mother does not have the Rh antigen on her RBCs (ie, Rh-negative) and, therefore, develops antibodies to the Rh antigen in response to Rh-positive RBCs that leak into her circulatory system during late pregnancy or delivery. The antibodies (immunoglobulin G [IgG]) leak back from the mother into the fetal circulation during subsequent pregnancies, leading to hemolysis only in subsequent …

Rat brain thioltransferase: regional distribution, immunological characterization, and localization by fluorescent in situ hybridization.

Thioltransferase (TTase) is a member of the family of thiol-disulfide oxidoreductases that are involved in the maintenance of sulfhydryl homeostasis in cells by catalyzing thiol-disulfide interchange reactions. One of the major consequences of oxidative stress in brain is the formation of protein-glutathione mixed disulfides (through oxidation of protein thiols), which can be reversed by TTase during the recovery of brain from oxidative stress. We therefore examined the presence of TTase in brain regions from rat. In the rat, TTase activity in the whole brain was comparable with the corresponding activity in liver, but significantly higher in hippocampus. The enzyme activity was significantly lower in striatum and cerebellum compared with activity in whole brain. Rat brain TTase shared immunological similarity with the human red blood cell enzyme, but not with the pig liver enzyme. The constitutive expression of the mRNA to TTase was demonstrable by northern blotting. Localization of the TTase mRNA in rat brain by fluorescent in situ hybridization showed the presence of high amounts of mRNA in the olfactory bulb, cortex, and hippocampus and its predominant localization in the neurons. TTase mRNA was also present in Purkinje cells in the cerebellum, in giant reticular neurons in the midbrain, and in the striatal and thalamic neurons. This study demonstrates the constitutive presence of a functional TTase system in brain and delineates the regional and cellular localization of the enzyme in rat brain.

Differential effects of dietary flavonoids on drug metabolizing and antioxidant enzymes in female rat

1. Gavage administration of the natural flavonoids tangeretin, chrysin, apigenin, naringenin, genistein and quercetin for 2 consecutive weeks to the female rat resulted in differential effects on selected phase 1 and 2 enzymes in liver, colon and heart as well as antioxidant enzymes in red blood cells (RBC). 2. Glutathione transferase (GST) activity assayed by use of the substrate 1-chloro-2,4- dinitrobenzene was significantly induced by apigenin, genistein and tangeretin in the heart but not in colon or liver. 3. In RBC chrysin, quercetin and genistein significantly decreased the activity of glutathione reductase (GR), catalase (CAT) and glutathione peroxidase (GPx), whereas superoxide dismutase (SOD) was only significantly decreased by genistein. 4. The oxidative status of the animal, measured as plasma malondialdehyde, revealed that chrysin, quercetin, genistein, and beta-naphthoflavone (BNF) significantly protected against, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP)-induced oxidative stress. Hepatic PhIP-DNA adduct formation was not affected by any of the administered flavonoids, whereas PhIP-DNA adduct formation in colon was slightly, but significantly, inhibited by quercetin, genistein, tangeretin and BNF. 5. The observed effects of chrysin, quercetin and genistein on antioxidant enzymes, concurrently with a protection against oxidative stress, suggest a feedback mechanism on the antioxidant enzymes triggered by the flavonoid antioxidants. 6. Despite the use of high flavonoid doses, which by far exceed the human exposure levels, the effect on drug metabolizing and antioxidant enzymes was still very minor. The role of singly administered flavonoids in the protection against cancer and heart disease is thus expected to be limited.

Expression, Purification, and Characterization of a Recombinant Erythroid-Specific Hexokinase Isozyme

ABSTRACT: Hexokinase type I (HK I; ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), the predominant glucose-phosphorylating enzyme in red blood cells, exists in human erythrocytes in multiple molecular forms that differ in isoelectric point and are separable by ion-exchange chromatography. The major forms, designated HK Ia, Ib and Ic, have similar kinetic properties but are characterized by different age-dependent decay and different intracellular distribution in reticulocytes. HK Ib, which elutes between HK I and HK II in the DEAE ion-exchange chromatography, appears to be unique to RBCs and different from any other hexokinase isozyme previously described. Indeed, Murakami and Piomelli recently reported the presence of a specific HK isozyme (named HKr) expressed in K562 cells and in human reticulocytes and, moreover, the resolution of the human HK I gene structure provided the direct evidence of an erythroid-specific exon 1. To further investigate the microheterogeneity of HK I in human RBCs we established a prokaryotic expression system for the HKr isozyme, using the pET plasmid, inducible with IPTG. The recombinant HKr, expressed in bacterial cells as a catalytically active enzyme, was purified to homogeneity by a combination of DEAE ionexchange chromatography followed by hydrophobic interaction chromatography and dye-ligand affinity chromatography. The kinetic and chromatographic properties of the homogeneous recombinant HKr suggest that this erythroid-specific HK isozyme in fact corresponds to the HK isoform previously described in human RBCs and referred to as HK Ib.

Sulphydryl groups and their relation to the antioxidant enzymes of chelonian red blood cells.

Thiol groups of hemoglobin and blood glutathione are higher in Geochelone carbonaria than in Geochelone denticulata. Exposure of stripped hemolysate of both tortoises to terc-butyl hydroperoxide, resulted in a higher ferroheme oxidation of G. denticulata hemoglobin. In this example glutathione reductase and glutathione peroxidase, were not active due to the absence of GSH and NADPH, suggesting that the thiol groups of G. carbonaria hemoglobin act as antioxidant, similar to GSH. In the total hemolysate, however, where the antioxidant enzymes are active, both species showed similar levels of hemoglobin oxidation, suggesting that the protective effect of thiol groups of hemoglobin are less effective for heme protection. The activity of glutathione reductase and glutathione peroxidase was higher in erythrocytes of G. denticulata and the activity of catalase and superoxide dismutase was higher in erythrocytes of G. carbonaria.

Erythrocyte superoxide dismutase activity and plasma malondialdehyde levels in children with Henoch Schönlein purpura

Reactive oxygen molecules (ROM) have been suggested to contribute to many pathological conditions including vasculitides and renal diseases. In the present study we measured the activity of superoxide dismutase (SOD) as an antioxidant enzyme in red blood cells and the level of malondialdehyde (MDA), which is a product and an indicator of lipid peroxidation, in the plasma of 16 children (7M, 9F) with Henoch Schönlein purpura (HSP) at the onset of the disease (SOD 1 and MDA 1) and at the remission period (SOD 2 and MDA 2). The results were compared with the results of 17 healthy children studied as a control group. There was no significant difference for SOD activities between the patients in each period and the control group (p > 0:05). There was a statistically significant difference between MDA 1 and MDA 2 levels (p < 0:01), each of which were also significantly different from the MDA levels of control group (p < 0:001 and p < 0:01, respectively). The effect of ROMs on different clinical conditions of HSP was also examined and lipid peroxidation was found to be increased more in patients with renal involvement. It is concluded that oxidant stress especially lipid peroxidation plays an important role in the pathogenesis of HSP and in development of renal injury.

Activity of Free Radical Scavenging Enzymes in Red Cells and Plasma of Patients Undergoing Extracorporeal Low‐Density Lipoprotein Apheresis

There is evidence that reactive oxygen species (ROSs) are generated in extracorporeal circuits. Free radical scavenging enzymes (FRSEs) such as glutathione reductase (GSSG-R), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) protect against the damaging effect of ROSs. The influence of extracorporeal treatment on FRSE activity was investigated in the plasma and red blood cells (RBCs) of 21 patients undergoing regular low-density lipoprotein (LDL) apheresis. The FRSEs GSSG-R, GSH-Px, and SOD were measured. Determinations were made before and after a single treatment. Because all apheresis patients suffered from coronary heart disease (CHD), 201 CHD patients and 90 individuals without CHD, neither group undergoing apheresis, served as controls. In apheresis patients, GSH-Px (33.9+/-8.2 U/g Hb) and GSSG-R (7.6+/-0.9 U/g Hb) activities were increased whereas SOD activity (5.4+/-1.5 U/g Hb) was decreased in RBCs before a single treatment compared to controls. Plasma FRSEs of apheresis patients were not different from those of controls. There was no effect of a single treatment on FRSEs in RBCs. However, a significant decrease in plasma GSH-Px activity (209.9+/-24.9 U/ml) due to the extracorporeal treatment was observed. These data show that long-term extracorporeal therapy with LDL apheresis modulates the activity of antioxidant enzymes in RBCs whereas a single treatment was without major effect on FRSE activity in RBCs and plasma, except for plasma GSH-Px.

Erythrocyte superoxide dismutase activity and plasma malondialdehyde levels in children with Henoch Schönlein purpura.

Reactive oxygen molecules (ROM) have been suggested to contribute to many pathological conditions including vasculitides and renal diseases. In the present study we measured the activity of superoxide dismutase (SOD) as an antioxidant enzyme in red blood cells and the level of malondialdehyde (MDA), which is a product and an indicator of lipid peroxidation, in the plasma of 16 children (7M, 9F) with Henoch Schönlein purpura (HSP) at the onset of the disease (SOD 1 and MDA 1) and at the remission period (SOD 2 and MDA 2). The results were compared with the results of 17 healthy children studied as a control group. There was no significant difference for SOD activities between the patients in each period and the control group (p > 0.05). There was a statistically significant difference between MDA 1 and MDA 2 levels (p < 0.01), each of which were also significantly different from the MDA levels of control group (p < 0.001 and p < 0.01, respectively). The effect of ROMs on different clinical conditions of HSP was also examined and lipid peroxidation was found to be increased more in patients with renal involvement. It is concluded that oxidant stress especially lipid peroxidation plays an important role in the pathogenesis of HSP and in development of renal injury.

Red Blood Cell Enzymes and their Clinical Application

Red blood cell enzyme activities are measured mainly to diagnose hereditary nonspherocytic hemolytic anemia associated with enzyme anomalies. At least 15 enzyme anomalies associated with hereditary hemolytic anemia have been reported. Some nonhematologic disease can also be diagnosed by the measurement of red blood cell enzyme activities in the case in which enzymes of red blood cells and the other organs are under the same genetic control. Progress in molecular biology has provided a new perspective. Techniques such as the polymerase chain reaction and single-strand conformation polymorphism analysis have greatly facilitated the molecular analysis of erythroenzymopathies. These studies have clarified the correlation between the functional and structural abnormalities of the variant enzymes. In general, the mutations that induce an alteration of substrate binding site and/or enzyme instability might result in markedly altered enzyme properties and severe clinical symptoms.

An Erythroid-Specific Exon Is Present in the Human Hexokinase Gene

To the Editor: Human hexokinase type I (EC 2.7.1.1) is the predominant glucose-phosphorylating enzyme in red blood cells. By a number of methods[1][1] it has been proved, and is now widely accepted, that this enzyme is largely heterogeneous and present in multiple molecular forms. Hexokinase

Bio-Analytical Studies of Bile Acids.

Bile acids are synthesized from cholesterol in the liver excreted into duodenum via the bile duct as their glycine and taurine conjugates, and converted in part into secondary bile acids by intestinal bacteria. It is well-known that further hydroxylation at C-1, -2, -4, -6 and -19, and conjugation at C-3 with glucuronic acid or sulfuric acid take place in the liver. In this paper bio-analytical studies on bile acids by hyphenated mass spectrometry related to drug metabolism will be discussed. In the first, to clarify an extrahepatic reduction system, 18O labeled 3-oxo bile acids were synthesized and after incubation with human blood, biotransformed products were separated and characterized by GC-MS. The 3-oxo group was reduced into the 3α- and 3β-hydroxyl function catalyzed by the enzyme (s) in human red blood cells. This result strongly implies that biologically active compounds such as drugs with the oxo group are also transformed into hydroxylated metabolites in human blood. On the biosynthesis of bile acids, the final step involving the oxidative cleavage of a side chain of 5β-cholestanoic acid, which has a chiral center at C-25, to form primary bile acids takes place in the peroxisomes. From a stereochemical point of view, the dehydrogenation mechanism of the biotransformation of 5β-cholestanoic acid into (24E)-5β-cholest-24-enoic acid was then studies with GC-MS. Substrates labeled with 2H at C 24 and C-25 stereoselectively were incubated with a rat liver light mitochondrial fraction and the stereospecific elimination of a pro-R hydrogen at C-24 in both (25R) and (25S)-cholestanoic acids indicating syn-elimination for the former, whereas anti-elimination for the letter was observed. When CoA thioesters of (25R)-and (25S)-5β-cholanoic acids were incubated individually with a rat liver peroxisomal fraction, rapid epimerization to form an equal mixture of stereoisomers from either direction was occurred. Moreover only the 25S antipode was easily transformed into dehydrogenated 5β-cholestanoic acid by peroxisomal acyl-CoA oxidase. The conjugation of carboxylic acids with n-glucuronic acid to form acyl glucuronides plays a significant role in the metabolism and disposition of drugs. The formation of 24-acyl glucuronides of bile acids in the rat liver are also discussed.

The level of antioxidant enzymes in red blood cells of patients with chronic obstructive pulmonary disease

The level of antioxidant enzymes in red blood cells of patients with chronic obstructive pulmonary disease

Isoelectric subtyping of the PGM1 in the Icelandic population

Phenotypes for the red blood cell enzyme phosphoglucomutase (PGM1) were determined by isoelectric focusing for a population of 2,501 Icelandic individuals. All ten phenotypes were observed, and the frequencies of four alleles at the PGM1 locus were as follows: PGM1 1+=0.6875; PGM1 1−=0.1124; PGM1 2+=0.1419, and PGM1 2−=0.0582. These results have been compared with those found in other northern European populations.