Erythrocyte and leucocyte enzymes in a case of paroxysmal nocturnal haemoglobinuria.

In a patient with paroxysmal nocturnal haemoglobinuria (PNH) enzymatic activities of erythrocytes and leucocytes were studied. Studies of autohaemolysis were also performed. The following erythrocytary enzymes were measured: Glucose-6-phosphate dehydrogenase (G-6-PD), pyruvate kinase (PK), glutathione reductase (GR), and acetylcholinesterase (AcChE). The following enzymes were measured in leucocytes: Adenosine deaminase, purine nucleoside phosphorylase, adenine phosphoribosyltransferase, hypoxanthine phosphoribosyltransferase and adenosine kinase. Normal activity of G-6-PD, GR and PK in erythrocytes was found. In leucocytes and lymphocytes activity of purine nucleoside phosphorylase was reduced. Auto-haemolysis in vitro was increased, which could not be compensated by addition of glucose or ATP.     

Enzymes of purine metabolism in B-chronic lymphocytic leukemia

In order to study if enzymes of purine metabolism could be used as cell markers in B-chronic lymphocytic leukemia (B-CLL), the activities of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and 5′-nucleotidase (5′N) were repeatedly measured in blood mononuclear cells from B-CLL patients and were compared to those obtained in normal controls. Enzyme activities in patients were also compared to other biological parameters indicative of B-CLL to activities of ADA and PNP in erythrocytes. Results show that B-leukemic cells display abnormal enzyme patterns: subnormal ADA activity is characteristic; 5′N activity is depressed in 60% of the cases but increased in 15%. An inverse relationship between PNP activity and corresponding lymphocytosis is observed in leukemic but not in normal cells. The enzymatic anomalies seem to be linked to the presence of an unusual peripheral lymphocytic population, induced by the leukemic process. Indeed, ADA and PNP are not abnormal in erythrocytes. In untreated nonevolutive patients, the enzyme profile tends to remain stable throughout the course of the illness; normalization of enzyme patterns in treated patients occurs only when therapy induces improvement in T and B cell distribution.     

肝脏中糖代谢关键酶作为糖尿病药物靶点的研究进展

葡萄糖激酶、葡萄糖-6-磷酸酶、磷酸烯醇式丙酮酸羧激酶、果糖-1,6-二磷酸酶和糖原磷酸化酶是肝脏中精代谢的关键酶.糖尿病的一个特征就是葡萄糖激酶活性下降和葡萄糖-6-磷酸酶、磷酸烯醇式丙酮酸羧激酶、果糖-1,6-二磷酸酶和糖原磷酸化酶活性的升高.最近的研究都致力于通过激活或抑制这儿种酶来降低血糖并使胰岛素正常分泌.因此这5种酶将有可能是治疗糖尿病的全新靶点.     

Deformability and enzymes activities of red blood cells in hemodialysed and peritoneal dialysed patients with chronic renal disease

These studies were focused on the influence of two treatment methods of patients with chronic renal disease on RBC deformability. In peritoneal dialysed-patients (PD) erythrocytes exhibited a decrease in their deformability as compared to control subjects whereas this parameter in RBC deriving from hemodialysed patients (HD) was not altered. The alleviating effect of plasma components on deformability of erythrocytes was confirmed after the isolation of a pure faction of these cells as the parameter became worse. The activity of studied enzymes: acetylcholinesterase (Ache), dehydrogenase glucose-6-phosphate (G-6-PD) and glutathione reductase (GR) maintained their physiological values in both dialysis groups.     

Studies on the Mechanism of the Erythrocyte Enzyme Abnormalities Induced by Chemotherapy

With the aim of determining the possible mechanisms of the red cell enzyme deficiencies induced by chemotherapy, deficient red cell glucose-6-phosphate dehydrogenase (G-6-PD), pyruvate kinase (PK) and phosphofructokinase (PFK) from 17 patients were purified and characterized. In all cases G-6-PD showed normal kinetics, electrophoretic mobility and thermostability suggesting that a decreased enzyme synthesis was possible for the deficient enzyme activity. In each case studied, at least one of the PK properties was modified, either in affinity for phosphoenol pyruvate, thermal stability or electrophoretic mobility, indicating a primary or secondary molecular abnormality. In some patients PFK had significantly increased affinity for citrate inhibitor; however, neither the quantity nor quality of the M subunits seemed to be altered. Thus it appears that chemotherapy can induce qualitative as well as quantitative red cell enzyme abnormalities by different mechanisms. These are similar to those observed in spontaneous leukaemia and preleukaemic states. Such a similarity poses the question of whether or not the red cell enzyme abnormalities induced by chemotherapy could be considered as the first sign of secondary leukaemia due to treatment by oncostatic drugs.     

骨髓增生异常综合征红细胞酶谱测定临床应用研究

本文比较了２３例ＭＤＳ和９例ＡＡ患者４种红细胞酶的活性，除醛缩酶外，丙酮酸激酶嘌呤核苷磷酸化酶、腺苷脱氨酸在ＡＡ患者均接近正常，和ＭＤＳ差异显著，尤其是ＰＫ和ＰＮＰ差异显著。据此联合检测红细胞４种酶活性，建立了ＭＤＳ和ＡＡ的差别函数式：ＰＭＤＳ＝－５．０４１０７＋０．２５６７４ＰＫ＋１．６７６５１ＡＬＤ＋０．０３４８１ＰＮＰ＋０．２５０２４ＡＤＡ，ＰＡＡ＝－７．９００３８＋０．３８４６ＰＫ＋１．４     

Inhibitory effect of a fava bean component on the in vitro development of Plasmodium falciparum in normal and glucose-6-phosphate dehydrogenase deficient erythrocytes

We examined the hypothesis that G-6-PD deficiency associated with fava bean ingestion confers resistance to malaria by studying the in vitro interactions between malaria parasites (Plasmodium falciparum), human erythrocytes with varying degrees of G-6-PD deficiency, and isouramil (IU), a fava bean extract that is known to cause oxidant stress and hemolysis of G-6-PD-deficient erythrocytes. Untreated G-6-PD-deficient and normal erythrocytes supported the in vitro growth of P. falciparum equally well. However, after pretreatment with IU, G-6-PD-deficient erythrocytes did not support parasite growth in vitro, whereas growth remained high in normal erythrocytes. Parasite growth was proportional to the G-6-PD activity of the IU-treated erythrocytes. In contrast, when parasitized erythrocytes were exposed to IU, parasites even in normal erythrocytes were destroyed. Ring forms were much less sensitive than late trophozoites and schizonts. The results suggest that there are two modes by which IU affects the development of P. falciparum and demonstrate in vitro that G-6-PD deficiency confers resistance against malaria under conditions of fava-bean-associated oxidant stress.     

Purine metabolism in leukocytes and erythrocytes in Graves' or Hashimoto's disease

INTRODUCTION: Adenosine deaminase (ADA), purine nucleoside phosphorylase (PNPase), S-adenosylhomocysteine hydrolase (SAHH), 5'-nucleotidase (5N), and deoxycytidine kinase (dCK) are involved in purine salvage metabolism. Changes of the activities of the above enzymes have been observed in blood cells in patients with immunological disorders. MATERIALS AND METHODS: The activities of ADA, PNPase, SAHH, 5'N, and dCK in lysates of leukocytes and erythrocytes, obtained from patients with Graves' or Hashimoto's disease, were measured, using chromatographic analysis. Serum concentrations of antithyroglobulin (Tg Ab) and antithyroperoxidase (TPO Ab) antibodies were measured by an immunoenzymatic method. RESULTS: (1) ADA activity in leukocytes, obtained from patients with Hashimoto's disease, was significantly higher than in control leukocytes, as well as in leukocytes from patients with Graves' disease; (2) dCK activities in leukocytes from patients with both Graves' and Hashimoto's diseases were approximately four and five times higher, respectively, than in leukocytes of control subjects; (3) a positive correlation was observed between dCK activity in leukocytes and serum Tg Ab concentration in patients with Graves' disease. In conclusion, the increased ADA and dCK activities in leukocytes from patients with Graves' and Hashimoto's diseases may be regarded as indicators of autoimmunological thyroid diseases.     

Erythrocyte Superoxide Dismutase,Catalase and Glutathione Peroxidase in Glucose-6-Phosphate Dehydrogenase Deficiency

Glucose-6-phosphate dehydrogenase (G-6-PD) deficient erythrocytes are particularly sensitive to oxidant stress. In order to evaluate if these cells are protected against oxidant damage, we assayed the antioxidant enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px) in erythrocytes of G-6-PD deficient (hemizygous and heterozygous) subjects. Normal levels of antioxidant enzymes were found in all subjects examined both with positive and negative histories of haemolytic crisis after fava bean or drug ingestion. In contrast, high levels of catalase and GSH-Px were found in a small group of G-6-PD deficient subjects (hemizygous and heterozygous) with β-thalassaemia trait, probably by reason of the chronically enhanced oxidant stress which is present in β-thalassaemia.     

A new glucose 6-phosphate dehydrogenase variant (G-6-PD Verona) in a patient with myelodysplastic syndrome

A 67-year-old woman investigated because of 'myelodysplastic syndrome' was found to have a 4-fold increase in G-6-PD activity in her erythrocytes. The enzyme was partially purified and characterized. On grounds of: (a) reduced electrophoretic mobility, (b) abnormal cathodic band(s) in isoelectrofocusing, (c) increased Michaelis constant for glucose 6-phosphate, (d) abnormal thermostability, and (e) abnormal interaction with the ligand NADPH, we conclude that this is a new structural variant which we designate G-6-PD Verona. G-6-PD Verona was the sole apparent source of G-6-PD activity in the patient's erythrocytes; by contrast, the patient's fibroblasts had only normal G-6-PH (type B). The patient's haematological course terminated into acute myeloid leukaemia. We believe G-6-PD Verona was the result of a somatic mutation in an X-chromosome which took place in a haemopoietic cell clone which subsequently underwent neoplastic transformation.     

Reduced purine 5'-nucleotidase activity in lymphocytes of patients with systemic lupus erythematosus: results of a pilot study

OBJECTIVE: To investigate purine metabolism in patients with systemic lupus erythematosus (SLE) for possible abnormalities that might be related to their overall impaired immune function. METHODS: This pilot study included 17 patients with SLE (2 men, 15 women). Enzyme activities of the purine enzymes 5'-nucleotidase (5'NT), purine nucleoside phosphorylase (PNP), and hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) were measured in peripheral blood mononuclear cells (PBMC) and also in fractions of T cells (differentiation antigen CD3+) (n = 12) and B cells (CD19+) (n = 9). The activity of the thiopurine enzyme thiopurine-methyltransferase (TPMT) was measured in red cell lysate. Routine blood tests and indices of disease activity were measured as well. Results were compared with those of healthy volunteers. RESULTS: Compared with their controls, the female SLE patients had a more than 50% reduced activity of 5'NT in the T cell fraction (p = 0.001) and in PBMC (p < 0.000). 5'NT activity was also lower in B cells, but this was not statistically significant. Enzyme activities did not correlate with indices of disease activity, disease duration or the B cell/T cell ratio and no influence of medication was found. CONCLUSION: Reduced lymphocyte 5'NT activity is a novel finding in SLE. These results indicate that purine metabolism in SLE may be disturbed. Consequences of a low 5'NT activity may be an intracellular accumulation of (deoxy)purine nucleotides and a reduction of adenosine production. It is hypothesised that these factors may play a part in the overall impaired immune function and in the chronicity of inflammation in SLE.     

Lymphospecific toxicity in adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency: Possible role of nucleoside kinase(s)

Inherited deficiencies of the enzymes adenosine deaminase (adenosine aminohydrolase; EC 3.5.4.4) and purine nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase; EC 2.4.2.1) preferentially interfere with lymphocyte development while sparing most other organ systems. Previous experiments have shown that through the action of specific kinases, nucleosides can be "trapped" intracellularly in the form of 5'-phosphates. We therefore measured the ability of newborn human tissues to phosphorylate adenosine and deoxyadenosine, the substrate of adenosine deaminase, and also inosine, deoxyinosine, guanosine, and deoxyguanosine, the substrates of purine nucleoside phosphorylase. Substantial activities of adenosine kinase were found in all tissues studied, while guanosine and inosine kinases were detected in none. However, the ability to phosphorylate deoxyadenosine, deoxyinosine, and deoxyguanosine was largely confined to lymphocytes. Adenosine deaminase, but not purine nucleoside phosphorylase, showed a similar lymphoid predominance. Other experiments showed that deoxyadenosine, deoxyinosine, and deoxyguanosine were toxic to human lymphoid cells. The toxicity of deoxyadenosine was reversed by the addition of deoxycytidine, but not uridine, to the culture medium. Based upon these and other experiments, we propose that in adenosine deaminase and purine nucleoside phosphorylase deficiency, toxic deoxyribonucleosides produced by many tissues are selectively trapped in lymphocytes by phosphorylating enzyme(s).     

Three new variants of glucose-6-phosphate dehydrogenase associated with chronic nonspherocytic hemolytic anemia: G-6-PD Lincoln Park, G-6-PD Arlington Heights, and G-6-PD West Town

Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency was identified in three children who were evaluated because of chronic nonspherocytic hemolytic anemia. One child is of German extraction, another Puerto Rican, and the third Mexican. In each of the patients the hemolytic process was well compensated, but each had one or more episodes of anemia following exposure to an oxidant drug or with infections. The electrophoretic, functional, and kinetic properties of the mutant enzymes, derived both from the patients' erythrocytes and from cultured fibroblasts, allowed each to be distinguished from G-6-PD variants previously described.     

The Reduction of Methemoglobin in Erythrocytes of a Patient with Congenital Methemoglobinemia, Subjects with Erythrocyte Glucose-6-Phosphate Dehydrogenase Deficiency, and Normal Individuals

The pathways for the reduction of methemoglobin to hemoglobin that aredependent upon the generation of reduced pyridine nucleotides were studiedin normal human erythrocytes, in erythrocytes deficient in G-6-PD activityand in erythrocytes of a subject with congenital methemoglobinemia. Reduction of methemoglobin, produced by treatment with nitrite, occurred atequivalent rates in normal and G-6-PD deficient erythrocytes, but failed tooccur in the erythrocytes of the patient with congenital methemoglobinemiaupon incubation with glucose or inosine. The DPNH-utilizing diaphorase-likesystem was normal in hemolysates of G-6-PD deficient erythrocytes, but wasmarkedly deficient in hemolysates of erythrocytes of the subject with congenital methemoglobinemia. The marked acceleration of methemoglobinreduction that occurred upon the addition of methylene blue to normal erythrocytes and to the erythrocytes of the woman with congenital methemoglobinemia did not occur with G-6-PD deficient erythrocytes. The TPNH-utilizing methemoglobin reductase system was normal in hemolysates oferythrocytes of the patient with congenital methemoglobinemia, but was reduced to about 50 per cent of normal activity in hemolysates of G-6-PD deficient erythrocytes.

The reduction of methemoglobin to hemoglobin in intact normal and G-6-PD deficient human erythrocytes probably proceeds by way of a DPNH-utilizing, diaphorase-like system that is deficient in the erythrocytes of onetype of congenital methemoglobinemia. The TPNH-utilizing methemoglobinreductase appears to be a reserve system that requires an artificial electroncarrier, such as methylene blue, to become fully effective in reducing methemoglobin to hemoglobin. The TPNH-methemoglobin reductase system isimpaired in G-6-PD deficient erythrocytes not only because of a deficientsource of TPNH, but, perhaps, also because of a defect in this methemoglobinreductase system itself.

Accepted on December 11, 1962     

Membrane deformability of erythrocytes with glucose-6-phosphate dehydrogenase Hamburg.

Deformability of erythrocytes of a patient with chronic nonspherocytic hemolytic anemia caused by a G-6-PD variant (G-6-PD Hamburg) in red cells was studied. The flow rate of erythrocytes from this patient through 5 mum polycarbonate sieves was higher than that of red cells from healthy adults. Even under oxidative stress in vitro, the deformability of erythrocytes of the patient was only slightly decreased. The residual hemoglobin content of red cell membranes (ghosts) from this patient was lower than that of ghosts from healthy adults and of ghosts prepared from comparable reticulocyte-rich blood withoug G-6-PD deficiency. In contrast to the low enzymatic activity of G-6-PD Hamburg in the hemolysate, a high activity of this enzyme could be demonstrated in the ghosts from the patient. In view of the flexibility of this patient's erythrocytes splenectomy is not to be recommended.     

Ischemic nucleotide breakdown increases during cardiac development due to drop in adenosine anabolism/catabolism ratio

Our earlier work on reperfusion showed that adult rat hearts released almost twice as much purine nucleosides and oxypurines as newborn hearts did [Am J Physiol 254 (1988) H1091]. A change in the ratio anabolism/catabolism of adenosine could be responsible for this effect. We therefore measured the activity of adenosine kinase, adenosine deaminase, nucleoside phosphorylase and xanthine oxidoreductase in homogenates of hearts and myocytes from neonatal and adult rats. In hearts the activity of adenosine deaminase and nucleoside phosphorylase (10-20 U/g protein) changed relatively little. However, adenosine kinase activity decreased from 1.3 to 0.6 U/g (P less than 0.025), and xanthine oxidoreductase activity increased from 0.02 to 0.85 U/g (P less than 0.005). Thus the ratio in activity of these rate-limiting enzymes for anabolism and catabolism dropped from 68 to 0.68 during cardiac development. In contrast, the ratio in myocytes remained unchanged (about 23). The large difference in adenosine anabolism/catabolism ratio, observed in heart homogenates, could explain why ATP breakdown due to hypoxia is lower in neonatal than in adult heart. Because this change is absent in myocytes, we speculate that mainly endothelial activities of adenosine kinase and xanthine oxidoreductase are responsible for this shift in purine metabolism during development.     

Glycolysis in Plasmodium falciparum results in modulation of host enzyme activities

BACKGROUND & OBJECTIVES: Plasmodium falciparum, the causative agent of the most serious form of malaria, infects about 5-10% of the world human population per year. It is well established that the erythrocytic stages of the malaria parasite rely mainly on glycolysis for their energy supply. In the present study, the glucose utilisation of erythrocyte population with parasitaemia levels similar to that of malaria patients was measured. The results allowed us to assess the effect of the parasites on the glucose utilisation of the vast majority of uninfected erythrocytes. METHODS: Using [2-13C]glucose and nuclear magnetic resonance (NMR) technique, the glucose utilisation in normal red blood cell (RBC) and P. falciparum infected red blood cell (IRBC) populations was measured. The IRBC population consisted of > 96% RBC and < 4% of parasite infected red blood cells (PRBC). The glycolytic enzymes were assayed to assess the effect of infected red cells on the enzymatic activities of uninfected ones. RESULTS: The rate of glucose utilisation by IRBC was considerably higher than that of RBC. Upon addition of 25% v/v conditioned culture medium (CM) of IRBC, RBCs exhibited a significant decrease in glucose utilisation. The CM could directly inhibit the activities of RBC glycolytic enzymes-phosphofructokinase (PFK) and pyruvate kinase (PK), without interfering with the activity of the pentose phosphate pathway enzyme-glucose-6-phosphate dehydrogenase (G-6-PD). INTERPRETATION & CONCLUSION: The present study showed that the clinical level of P. falciparum infected RBCs (< 4% parasitaemia) significantly enhance the glycolytic flux as well as down-regulate the glucose utilisation rate in the majority of uninfected RBC population. The mechanism of inhibition seems to be direct inhibition of the regulatory glycolytic enzymes-PFK and PK.     

Enzymes of the purine interconversion system in chronic lymphatic leukemia: Decreased purine nucleoside phosphorylase and adenosine deaminase activity

Summary Activities of adenosine deaminase (ADA), adenosine kinase (AK), adenine phosphoribosyltransferase (APRT), hypoxanthine guanine phosphoribosyltransferase (HGPRT), and purine nucleoside phosphorylase (PNP), all enzymes of the purine interconversion system, were determined in lymphocytes of 25 patients with chronic lymphatic leukemia (CLL) and in 23 controls. A statistically significant decrease of PNP activities and a reduction of ADA activities at borderline levels were found in the patients, whereas for the other enzymes assayed no deviation from normal values was observed.This work was supported in part by the Austrian Research Fund (Project No. 3796).     

Decreased adenosine deaminase (ADA) and 5′nucleotidase (5NT) activity in peripheral blood T cells in hodgkin disease

The purine metabolic enzymes adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and 5′nucleotidase (5NT) play an important role in normal lymphocyte differentiation. Abnormal levels of one or all of these enzymes have been associated with immunodeficiency diseases and lymphoproliferative disorders. ADA, PNP, and 5NT activity was measured in peripheral blood T cells from 24 patients with Hodgkin disease (HD) (12 in complete remission and 12 with active disease) to determine whether an association existed between enzyme abnormalities and the decreased cellular immune function previously described in this disorder. HD patients had a significantly decreased absolute lymphocyte count (1,618 ± 1107/mm³; mean ± SD) compared to controls (2,320 ± 980; p <.001). ADA, PNP, and NT activity was assessed in lymphocyte extracts by measuring the conversion of radiolabeled substrates to products over time. ADA activity expressed as mean ± SEM nanomoles/10⁶ lymphocytes/hr was significantly decreased in T cells from HD patients (84.6 ± 7.5) compared to controls (128 ± 12.3; p < 0.025). Likewise, 5NT was significantly decreased in HD patients (12.7 ± 1.3) compared to controls (24.0 ± 3.6; p <.005). There was not a significant difference in PNP activity between both groups. Low 5NT activity was present irrespective of whether patients had active disease (12.1 ± 1.5) or were in unmaintained complete remission (14.5 ± 2.4). These findings suggest that biochemical abnormalities may be responsible for or related to the persistent abnormalities in T-cell function noted throughout the clinical course of HD.     

Changes in glucose-6-phosphate dehydrogenase, copper, zinc-superoxide dismutase and catalase activities, glutathione and its metabolizing enzymes, and lipid peroxidation in rat erythrocytes with age

We have studied the activities of enzymes (glucose-6-phosphate dehydrogenase (G-6-PD), copper, zinc-superoxide dismutase (Cu,Zn-SOD), catalase (CAT), selenium-dependent glutathione peroxidase (Se-GSH-Px) and glutathione-S-transferase (GST)), and the levels of reduced glutathione (GSH), oxidized glutathione (GSSG) and thiobarbituric acid-reactive substances (TBARS) in rat erythrocytes and estimated the ratio of GSH/GSSG and the redox index. Male Wistar rats at ages of 1, 6 and 12 months were used. The activities of G-6-PD and Cu,Zn-SOD, the levels of GSSG and TBARS were increased, while the activity of Se-GSH-Px and the level of GSH were decreased with age. GSH/GSSG ratio was significantly decreased with age. We found a positive correlation between age and G-6-PD (r=0.476, p<0.01), Cu,Zn-SOD (r=0.291, p<0.01), CAT (r=0.254, p<0.01) and GST activities (r=0.250, p<0.05), and GSSG (r=0.708, p<0.05) and TBARS levels (r=0.802, p<0.01), whereas the correlation between age and Se-GSH-Px activity (r=-0.376, p<0.05), GSH level (r=-0.603, p<0.01) and GSH/GSSG ratio (r=-0.685, p<0.05) were negative. We found age-related differences in erythrocyte antioxidant enzyme activities, GSH, GSSG, total GSH and TBARS levels, GSH/GSSG ratio and the redox index.