Pierre Boivin

Blood polymorphonuclear dysfunction in patients with alcoholic cirrhosis

Abstract. Polymorphonuclear leucocyte function was investigated in twenty patients with alcoholic cirrhosis and three patients with cryptogenic cirrhosis. Bacterial ingestion, oxygen‐dependent bactericidal capacity, and chemotactic response were measured. Serum dependent abnormalities were common; they included deficiencies of ingestion and of all subsequent oxygen‐dependent metabolic events (three patients), all oxygen‐dependent metabolic events (one patient), cytochrome c reduction and iodination deficiencies (six patients), isolated cytochrome c reduction deficiency (ten patients), and chemotactic deficiencies (fourteen out of eighteen patients). Serum‐independent abnormalities were much less common; they included increased ingestion rate (four patients), decreased stimulated reduction of nitroblue tetrazolium (three patients), and decreased myeloperoxidase content (eight patients). Polymorphonuclear leucocyte abnormalities are frequent in cirrhosis and may account in part for increased susceptibility to infection in that disease.

Acquired Erythroenzymopathies in Blood Disorders: Study of 200 Cases

Enzyme abnormalities are frequently found in the red cells of patients with various acquired blood disorders. In leukaemias, preleukaemic states and bone marrow insufficiencies with or without sideroblastosis, changes in enzyme activity are usually characterized by the coexistence of deficiency of some enzymes and an increased activity of others.

Molecular mechanism of glucose-6-phosphate dehydrogenase deficiency.

SummaryWith an electro-immunodiffusion technique the authors immunologically titrated 10 deficient glucose-6-phosphate dehydrogenase variants in the leukocytes, platelets, and red blood cells.By comparison between the immunological reactivity and the enzymatic activity, the relative importance of the molecular instability, the decrease in molecular specific activity, and the post-translational modifications of the muted protein could be appreciated.The Gd(-) A, Gd(-) West Bengale, Gd(-) Seattle, and Gd(-) Worcester-like variants had a normal specific activity and were only unstable.The Gd(-) Mali, Gd(-) Fort de France, Gd(-) Ankara, Gd(-) Mediterranee, Gd(-) Matam, and Gd(-) Benevento-like variants were also unstable and they had a diminished molecular specific activity, which entirely or partly explained the leukocyte deficiency.

Evidence for structural differences between human glucose-6-phosphate dehydrogenase purified from leukocytes and erythrocytes.

Summary The C-terminal end of pure glucose-6-phosphate dehydrogenase from human leukocytes and red cells has been determined by subjecting these enzymes to partial proteolysis by carboxypeptidase A and B. The C-termini of the leukocyte enzyme were Lysine-Leucine, while Glycine was found as the C-terminal residue of erythrocyte glucose-6-phosphate dehydrogenase. From the results reported in this paper and from data previously reported we may conclude that aging of glucose-6-phosphate dehydrogenase in the red cells is associated with partial proteolysis of the C-terminal end of the enzyme molecules.

Quantitative Iodination of Human Blood Polymorphonuclear Leukocytes

Abstract. It was shown by Pincus and Klebanoff that a correlation existed between leukocytic iodination measured in vivo and microbicidal leukocytic activity. We have analysed the results of this test in relation to time and in the presence of variable quantities of polymorphonuclear leukocytes (PMN). The values observed per time and PMN unit proved to be equivalent in the presence of 2. 5 × 105 PMN or 5. 0 × 105 PMN per 0.5 ml of incubation medium, measured after 10, 20 and 30 minutes or in the presence of 1. 0 × 106 PMN, measured after 10 minutes. That is to say iodination is proportional to leukocyte concentration and incubation time. Increase of either the quantity of cells or the incubation time, beyond the area we defined, reduce iodination per cell and per unit of time. Concerning the patients with an insufficient iodination, we have studied 2 parameters in the presence of 5. 0 × 105 PMN:1) initial iodination measured after 10 and 20 minutes and 2) stability of iodination measured after 60 minutes. These two parameters were equally affected in two cases with myelofibrosis, 3 patients with acquired refractory anaemia, one with chronic lymphoid leukaemia, one with erythroleukaemia, one with hairy cell leukaemia, one with systemic mastocytosis and almost complete myeloperoxidase deficiency, one with sickle cell disease, two with liver diseases and two with chronic myeloid leukaemia. The iodination at the 60th minute was more affected than at the 10th minute with a patient with myelofibrosis and 4 other patients with acquired refractory anaemias. The significance of these differences is not well understood; however the meaning of the decrease in the iodination of whatever type is that a PMN anomaly exists directly related to the myeloperoxidase H2O2 halogenation system, or to one of the stages of engulfment and/or metabolic events preceeding it and leading to the production of H2O2. This test, with the alterations we introduced, is suggested as a test for detection of functional PMN abnormalities.

Studies on the nature of different molecular forms of glucose-6-phosphate dehydrogenase purified from human leukocytes.

Several molecular forms of human glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) corresponding to different stages of post-synthetic modifications have been purified from human leukocytes. The various enzyme forms were different in their specific activity, their kinetic properties and their isoelectrofusing pattern. The molecular weight of the subunits of the different forms was not modified. The changes in the electrofocusing pattern were not due to modifications of the N-terminal ends, the oxidation of thiol groups or the non-covalent fixation of an acid molecule upon the enzyme. Carboxypeptidase B cleaved a C-terminal lysine from the different enzyme forms and shifted the isoelectric point of the different enzyme active bands towards the acid pH. The different enzyme forms studied here seemed to result from the action upon native glucose-6-phosphate dehydrogenase of modifying factors especially abundant in some leukemic granulocytes. The modifying factors did not seem to be consumed during the modification of glucose-6-phosphate dehydrogenase. Moreover, the storage for one year of unmodified enzyme resulted in changes in its electrofocusing pattern similar to those quickly induced by the modifying factors. Consequently it appears that the modifying factors are catalysts of the modification of special residues of glucose-6-phosphate dehydrogenase. The hypothesis that this modification involves the deamination of asparagine or glutamine residues is put forward.

Glucose-phosphate isomerase deficiency due to a new variant (GP I barcelona) and to a silent gene: Biochemical, immunological and genetic studies

A 12-year-old girl of Spanish origin was found to be double heterozygote for a deficient GP I variant (GP I Barcelona) and for a silent GP I gene. The mother was heterozygote for GP I Barcelona and the father was heterozygote for the silent gene. GP I Barcelona was a fast variant (116%) with an increased isoelectric point (9.55), lability to heat and to urea, and shift of the pH curve towards the acidic pH. The other kinetic characteristics were normal. The ratio of enzymatic activity to immunological reactivity was normal in erythrocytes and white blood cells of the father and the mother but decreased to 75% of normal in blood cells of the daughter. The genetic and molecular mechanisms of GP I deficiency of this patient are discussed.

A spanish family with erythrocyte pyruvate kinase deficiency: Contribution of various immunologic methods in the study of the mutant enzyme

Erythrocyte PK deficiency was detected in a 38-year-old man from Catalonia, in Spain. His father and his three children were proven to be heterozygous for the same mutant PK variant. This variant was characterized by low immunologic specific activity, normal (or slightly increased) stability to heat and to urea; normal isoelectric point, increased K0.5 for phosphoenolpyruvate, increased inhibition by ATP and normal activation by 0.35 mM fructose 1,6-diphosphate. The mutant PK variant was antigenically identical with wild enzyme as tested against anti wild erythrocyte PK serum by double immunodiffusion and micro complement fixation. The utility and the significance of the immunologic methods to be used in the study of mutant PK variants are discussed.

Subcellular superoxide dismutase activity in phagocytozing human blood polymorphonuclear leucocytes.

Both cyanide-sensitive and -insensitive superoxide dismutase (SOD) (Superoxide oxidoreductase EC 1.15.1.1 )have been identified in human polymorphonuclear leucocytes (PMN) [1,2] and located both in the cytosol and in the granule fraction of the cells [3]. This enzyme catalyzes the dismutation of superoxid e anion (OF)into hydrogen peroxide (H202) and is thus thought to be protective against the highly reactive O~ in PMN as welt as in other tissues of aerobic organism [2-5] . However, the functional activity of PMN depends on the production of O~ [6] and an inhibitory effect of SOD on the leukocytic bactericidal activity has been reported [7]. On the other hand, SOD shows a strong inhibitory effect on the O~-mediated chain reaction involved in the aerobic oxidation of NAD(P)H, likely responsible for the cyanide-insensitive burst of oxygen associated with phagocytosis [8-10] . These observations suggested that the function of SOD in PMN, could be more complicated than initially proposed, and needed to be further investigated. In this way, the present communication is concerned with the determination of the effect of phagocytosis on SOD activity in subcellular fractions isolated from human PMN.

Studies on the mechanism of NADPH oxidation by the granule fraction isolated from human resting polymorphonuclear blood cells.

Various factor affecting NADPH-oxidation by resting human leucocyte granules (LG) at acid pH, have been investigated. It was found that: 1) oxidation of NADPH by LG was increasingly inhibited by increased cyanide concentrations in the medium and was abolished by 4 mM cyanide. 2) with or without cyanide in the incubation medium, LG omitted, Mn++ in the presence of NADPH induced superoxide anion (O- WITH 2) production, as evidenced by oxygen consumption and H2O2 production, which were abolished (in the absence of cyanide) by cytochrome C (a potent O- with 2 scavenger). 3) Both NADPH oxidation in the presence of 2 mM cyanide (cyanide-resistant) and in its absence (cyanide-sensitive) by LG occurred only in the presence of Mn++, and both were inhibited by superoxide dismutase. 4) Cyanide-resistant NADPH oxidation by LG generated H2O2, was inhibited by H2O2 and was not modified by active catalase. The ratio of cyanide-resistant NADPH oxidation/O2 uptake was 1 up to 1.25 mM NADPH, and increased above this concentration. 5) Cyanide-sensitive NADPH oxidation was inhibited by catalase and increased upon addition of H2O2. The ratio of cyanide-sensitive NADPH oxidation/O2 uptake was 2. It was concluded that after initiation by O - with 2, produced independently of LG, two sequential types of LG dependent NADPH oxidations occur. First, an O - with 2-dependent protein mediated NADPH oxidation (cyanide-resistant) which generates H2O2 and O - with 2 occurs. Second, NADPH peroxidation (cyanide-sensitive) which utilizes H2O2 takes place.