Henry N. Kirkman

Mechanisms of Protection of Catalase by NADPH KINETICS AND STOICHIOMETRY

NADPH is known to be tightly bound to mammalian catalase and to offset the ability of the substrate of catalase (H2O2) to convert the enzyme to an inactive state (compound II). In the process, the bound NADPH becomes NADP+ and is replaced by another molecule of NADPH. This protection is believed to occur through electron tunneling between NADPH on the surface of the catalase and the heme group within the enzyme. The present study provided additional support for the concept of an intermediate state of catalase, through which NADPH serves to prevent the formation (rather than increase the removal) of compound II. In contrast, the superoxide radical seemed to bypass the intermediate state since NADPH had very little ability to prevent the superoxide radical from converting catalase to compound II. Moreover, the rate of NADPH oxidation was several times the rate of compound II formation (in the absence of NADPH) under a variety of conditions. Very little NADPH oxidation occurred when NADPH was exposed to catalase, H2O2, or the superoxide radical separately. That the ratio exceeds 1 suggests that NADPH may protect catalase from oxidative damage through actions broader than merely preventing the formation of compound II.

Lactic acidemia, neurologic deterioration and carbohydrate dependence in a girl with dihydrolipoyl dehydrogenase deficiency

A girl with failure to thrive in the neonatal period was brought to the hospital at 10 weeks of age following a respiratory arrest, preceded by 12 h of vomiting and diarrhea. There was significant acidosis with a blood lactate of 8.8 mM. A high carbohydrate diet decreased her acidosis. Episodes of acidosis, often associated with infections, and accompanied by progressive neurological deterioration, have continued for 18 months. The activity of pyruvate dehydrogenase from cultured skin fibroblasts was 24% of that from normal fibroblasts. The activities of α-ketoglutarate dehydrogenase and branched-chain keto acid dehydrogenase were also deficient. The activity of the dihydrolipoyl dehydrogenase component (E3) of PDH in skin fibroblasts was 5% of that in control cell lines. Limited studies performed on liver and muscle biopsy specimens showed E3 activity in liver and muscle to be undetectable in both tissues. We conclude that the enzyme defect present in dihydrolipoyl dehydrogenase is responsible for the reduced activity of all three α-keto-acid dehydrogenase complexes and the patients symptoms. Our results provide further evidence that the E3 component of these complexes is genetically and biochemically the same protein.

Projections of a rebound in frequency of mental retardation from phenylketonuria

The present decade marks the end of a unique generation--when the public benefited from the prevention of mental retardation phenylketonuria (PKU) without having to deal fully with the problems which can be anticipated from the reproduction of successfully treated, phenylketonuric individuals. The dysgenic effect (increase in prevalence of the PKU gene) from this reproduction will have negligible influence on the frequency of mental retardation from PKU over the next few centuries. In contrast, a dysgenic effect from maternal PKU will cause a rebound in frequency of mental retardation within this decade. Failure of the PKU programs as a result of maternal PKU, could affect the public attitude toward programs for the study and prevention of mental retardation and genetic diseases. Minimizing and explaining the rebound in frequency of mental retardation will be a difficult but necessary task for workers in the field of mental retardation.

Further evidence for a racial difference in frequency of ABO hemolytic disease

The results of a Coombs test on each infant, age of onset of jaundice, and of maximum recorded serum indirect bilirubin concentration were statisically analyzed on 2,428 consecutive newborn infants who were Rh compatible with their mothers and who weighed 2.5 kg or more. The incidence of ABO isoimmune hemolytic disease was estimated by subtracting the frequency of each of these observations in ABO compatible infants from the corresponding frequency in ABO incompatible infants. This subtraction decreased the contribution of extraneous causes for jaundice and a positive Coombs reaction. As expected, these signs of hemolytic disease were found more often in ABO incompatible infants than in compatible infants. Whether detected by a positive Coombs reaction, jaundice in the first 24 hours, serum bilirubin concentrations over 10 mg/dl, or any combination thereof, the incidence of ABO disease was much higher in black neonates than in white ones. These findings indicate that early discharge of newborn infants from the hospital should not be authorized without specific assessments, especially when the infant is ABO incompatible and black.

A novel NADPH:(bound) NADP + reductase and NADH:(bound) NADP + transhydrogenase function in bovine liver catalase

Many catalases have the shared property of containing bound NADPH and being susceptible to inactivation by their own substrate, H2O2. The presence of additional (unbound) NADPH effectively prevents bovine liver and human erythrocytic catalase from becoming compound II, the reversibly inactivated state of catalase, and NADP+ is known to be generated in the process. The function of the bound NADPH, which is tightly bound in bovine liver catalase, has been unknown. The present study with bovine liver catalase and [14C]NADPH and [14C]NADH revealed that unbound NADPH or NADH are substrates for an internal reductase and transhydrogenase reaction respectively; the unbound NADPH or NADH cause tightly bound NADP+ to become NADPH without becoming tightly bound themselves. This and other results provide insight into the function of tightly bound NADPH.

Isozymes of human red cell glucose-6-phosphate dehydrogenase.

With most gel electrophoretic techniques used for genetic studies of human G‐6‐PD, the activity from hemolysates of males migrates predominantly as a single band. Under two conditions of starch gel electrophoresis, however, dual bands of red cell G‐6‐PD may be seen. These exceptions are: (a) two prominent bands of G‐6‐PD that appear after starch‐gel electrophoresis in a discontinuous‐borate buffer system, and (b) a minor band of red cell G‐6‐PD that moves about 80 percent as fast as the major band in a number of buffer and gel systems. The faster band of (a) was found to be absent or less prominent in samples that had been incubated under conditions previously shown to cause aggregation of G‐6‐PD subunits. Electrophoresis in starch gel of various concentrations revealed that the slower component of (a) is of larger molecular size than the faster component. In contrast, the minor component of (b) was found to be similar in molecular size to the major component. The isolated minor component had the electrophoretic mobility of the major band after treatment with β‐mercaptoethanol. Reappearance of a minor band was obtained by incubation of major band G‐6‐PD with another protein fraction from normal red cells, in the absence of β‐mercaptoethanol. It is proposed that bands of (a) represent simply the aggregated and disaggregated form of G‐6‐PD. The minor band of (b) appears to be a complex of a subunit of G‐6‐PD and another protein.

Genetic screening of donors for artificial insemination

A routine has been established for genetic screening of donors in an artificial insemination and frozen sperm bank program. This report is a summary and analysis of the information obtained on the first 168 donor applicants and 89 recipients who were genetically screened. The specific forms for screening, family information obtained, characteristics of the donor and recipient groups, and guidelines for acceptance or rejection of donors are discussed. The donor and recipient often failed to perceive that the disorders in the family were genetic. The simple question of whether or not there were genetic or hereditary problems in the family was ineffective, even when the donor or recipient had formal medical training.

GLUCOSE-6-PHOSPHATE DEHYDROGENASE VARIANTS AND DRUG-INDUCED HEMOLYSIS*

Deficiencies in activity of red cell glucose-6-phosphate dehydrogenase (G-6PD) are among the most prevalent hereditary enzymic defects in man. In some populations, between 10 and 60 percent of the males are affected. Of significance for the present conference, however, is the susceptibility of such G-6-PD-deficient subjects to a hemolytic anemia that is induced by certain drugs and toxic substances. The possible mechanisms of this drug-induced hemolysis are discussed in greater detail elsewhere in this symposium.1.2 This paper is a brief review of the abnormalities of human G-6-PD, with particular attention to any correlation between abnormal properties of the enzyme and clinical manifestations of the abnormality. The antimalarial drug, pamaquine, has been known since 1926 to be capable of causing an acute hemolytic anemia in some people. Subsequent observations have revealed that such susceptible people are also sensitive to primaquine and to a number of other drugs (TABLE 1) . Through the cross-transfusion experiments of Dern and coworkers, this susceptibility to primaquine-induced hemolysis was shown to be an intrinsic property of the red cells of susceptible subjects3 A clue to the nature of the defect appeared when Flanagan, Beutler, Dern and Alving found that the level of reduced glutathione (GSH) fell sharply in the red cells of sensitive subjects after several days of ingestion of the drug.4 This fall occurred just before hemolysis in vivo. Beutler was able to reproduce this abnormal fall in GSH in vitro by incubating the blood of sensitive people with a~etylphenylhydrazine.~ This test made possible the identification of sensitive subjects even before they were exposed to drugs. It allowed Childs and coworkers to demonstrate that this abnormality is a sex-linked (or X-linked) trait with intermediate dominance,6 a. conclusion also obtained subsequently by studies of the G-6-PD defect, More recently, certain other chemicals have been found to diminish the level of GSH in sensitive cells during incubation in vitro.9 Many investigators now think that the drugs causing hemolysis, or the metabolic derivatives of these drugs, can exist in both an oxidized and reduced form and serve to increase the rate of oxidation of GSH within the red cells of normal and sensitive subjects. The metabolism of normal red cells is well suited to protect itself from such oxidative stresses. Glutathione reductase (FIGURE 1 ) regenerates GSH from oxidized glutathione (GSSG) by using NADPH, which, in turn, is generated by both G-6-PD and 6-phosphogluconate dehydrogenase (6-PGD) . Carson and coworkers discovered that levels of G-6-PD are greatly reduced in sensitive cells, even when the affected subject is not exposed to drugs.1° Subsequent studies have shown that the activity of glutathione reductase is not diminished in sensitive subjects. The impairment, then, seems to be an inability to meet the increased demand for NADPH that is imposed by the enhanced

Minimizing false positive diagnoses in newborn screening for galactosemia

Heat and humidity, rather than summertime heat alone, cause extensive loss of galactose-1-phosphate uridyltransferase activity in mailed blood spots and seem to account for false positive diagnoses of galactosemia. The spots are partially protected from the effects of atmospheric humidity if they are allowed to dry and then are sealed in a plastic freezer bag before being exposed to higher humidity. Conversely, extensive loss of transferase activity occurs if the samples are sealed in the bags before the spots are dry. The fluorescence from transferase activity can be monitored with greater sensitivity fluorometrically than visually. A simultaneous fluorometric determination of phosphoglucomutase activity reveals whether decreased transferase activity represents sample deterioration or galactosemia. Mg2+ and a sulfhydryl agent, such as dithiothreitol, are needed for activity of phosphoglucomutase, an enzyme in the sequence leading to the fluorescent substance (NADPH). They must be added in certain modifications of the assay.

Altered aggregational properties in a genetic variant of human glucose 6-phosphate dehydrogenase

The Tel Hashomer variant of human G6PD migrates as two prominent components during electrophoresis in several gel systems in which red cell G6PD from other males migrates predominantly as a single band. Since human males normally have but one X-chromosome, the extra band of this variant seemed an exception to earlier biochemical and genetic observations suggesting that human red cell G6PD is determined by a locus on the X chromosome. Results of the present studies indicate that the Tel Hashomer variant is unusually susceptible to the formation of a complex which has a higher molecular weight than normal G6PD and which represents the slow electrophoretic component. The conditions of formation and disruption of this complex in crude and purified Tel Hashomer preparations suggest that it results from the formation of disulfide bridges between molecules of Tel Hashomer G6PD.