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Biomedical subjects

H N Kirkman

Publications and source records attributed to H N Kirkman.

At least 19 recordsLinked to original sources
PubMed1992

Erythrocytic uridine diphosphate galactose in galactosaemia.

An earlier claim of a deficiency of uridine diphosphate galactose in erythrocytes of galactosaemia patients was not confirmed. Enzymic techniques similar to those of the earlier investigators were used to determine not only the concentration of uridine diphosphate galactose but also the ratio of this concentration to the sum of the uridine sugar diphosphates (uridine diphosphate galactose and uridine diphosphate glucose). The values in erythrocytes of galactosaemic subjects were similar to those of non-galactosaemic children on a galactose-restricted diet and to those of normal adults. These results cast doubt on the claim of a major deficiency of uridine diphosphate galactose in galactosaemia and on the need for treating galactosaemic children with uridine.

Adult↗
PubMed1992

31P NMR analysis of red blood cell UDPGlucose and UDPGalactose: comparison with HPLC and enzymatic methods.

The levels of uridine diphosphogalactose (UDPGal) and uridine diphosphoglucose (UDPGlu) in trichloroacetic acid extracts of human red blood cells (RBC) were measured by 31P NMR spectroscopy. Individual determinations were compared to results obtained by enzymatic and high-pressure liquid chromatographic (HPLC) methods. The characteristic doublet of the P beta resonance signals of both UDPGal and UDPGlu were detected in proton-decoupled spectra of extracts. Quantitative analyses were obtained by employing a standard, methylene diphosphonate, in an external capillary tube during data acquisition for periods of 14 to 24 h using an "inverse-gated" pulse sequence. The ratio of the integrated area of each of the uridine sugar nucleotide doublets to the area of the external reference peak was linear with concentrations between 0.03 and 0.50 mM. There was no difference between the mean value obtained by 31P NMR of 6.6 +/- 1.4 mumol UDPGlu/100 g Hgb or 2.1 +/- 0.6 mumol UDPGal/100 gHgb and the corresponding levels determined enzymatically or by HPLC in identical RBC extracts. When analyzed as paired data, only UDPGlu by NMR was found to be lower than the value obtained by HPLC. As a quantitative analytical tool, NMR spectrometry validated both the enzymatic and HPLC methods used for measurement of uridine sugar nucleotides in our laboratories.

Adult↗
PubMed1992

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.

Dithiothreitol↗
PubMed1992

A molecular deletion of distal chromosome 4p in two families with a satellited chromosome 4 lacking the Wolf-Hirschhorn syndrome phenotype.

We report two families with a satellited chromosome 4 short arm (4ps). Satellites and stalks normally occur on the short arms of acrocentric chromosomes; however, the literature cites several reports of satellited nonacrocentric chromosomes, which presumably result from a translocation with an acrocentric chromosome. This is the first report of 4ps chromosomes. Our families are remarkable in that both unaffected and affected individuals carry the 4ps chromosome. The phenotypes observed in affected individuals, although dissimilar, were sufficient to encourage a search for a deletion of chromosome 4p. By Southern blot analysis and fluorescence in situ hybridization, a deletion of material mapping approximately 150 kb from chromosome 4pter was discovered. This deletion is notable because it does not result in the Wolf-Hirschhorn syndrome and can result in an apparently normal phenotype. We speculate that homology between subterminal repeat sequences on 4p and sequences on the acrocentric short arms may explain the origin of the rearrangement and that position effect may play a role in the expression of the abnormal phenotype.

Abnormalities, Multiple↗
PubMed1989

Catalase and glutathione peroxidase are equally active in detoxification of hydrogen peroxide in human erythrocytes.

Genetic deficiencies of glucose-6-phosphate dehydrogenase (G6PD) and NADPH predispose affected erythrocytes to destruction from peroxides. Conversely, genetic deficiencies of catalase do not predispose affected erythrocytes to peroxide-induced destruction. These observations have served to strengthen the assumption that the NADPH/glutathione/glutathione peroxidase pathway is the principal means for disposal of H2O2 in human erythrocytes. Recently, however, mammalian catalase was found to have tightly bound NADPH and to require NADPH for the prevention and reversal of inactivation by its toxic substrate (H2O2). Since both catalase and the glutathione pathway are dependent on NADPH for function, this finding raises the possibility that both mechanisms destroy H2O2 in human erythrocytes. A comparison of normal and acatalasemic erythrocytes in the present study indicated that catalase accounts for more than half of the destruction of H2O2 when H2O2 is generated at a rate comparable to that which leads to hemolysis in G6PD- deficient erythrocytes.

Carbon Dioxide↗
PubMed1988

Equilibrium of 5,6-hydration of NADH and mechanism of ATP-dependent dehydration.

At equilibrium, water addition to the 5,6 double bond of NADH was observed to favor the hydrate by a factor of approximately 100. Hydration generates two epimers of NADHX (beta-6-hydroxy-1,4,5,6-tetrahydronicotinamide adenine dinucleotide). Only the 6S epimer of the hydrate was found to serve as a true substrate for an ATP-dependent dehydratase from yeast that regenerates NADH. Yet enzymatic conversion of both epimers of the hydrate to NADH was found to proceed essentially to completion in the presence of ATP and dehydratase. This is explained by the observed ability of the epimers to undergo rapid spontaneous equilibration, so that it is unnecessary to postulate a lack of stereospecificity in the dehydratase.

Adenosine Diphosphate Ribose↗
PubMed1987

The function of catalase-bound NADPH.

Catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) is of historical interest for having been the subject of some of the earliest investigations of enzymes. A feature of catalase that has been poorly understood for several decades, however, is the mechanism by which catalase remains active in the presence of its own substrate, hydrogen peroxide. We reported recently that catalase contains tightly bound NADPH. The present study with bovine and human catalase revealed that NADPH both prevents and reverses the accumulation of compound II, an inactive form of catalase that is generated slowly when catalase is exposed to hydrogen peroxide. Since the effect of NADPH occurs even at NADPH concentrations below 0.1 microM, the protective mechanism is likely to operate in vivo. This discovery of the role of catalase-bound NADPH brings a unity to the concept of two different mechanisms for disposing of hydrogen peroxide (catalase and the glutathione reductase/peroxidase pathway) by revealing that both mechanisms are dependent on NADPH.

Animals↗
PubMed1986

Regulation of glucose-6-phosphate dehydrogenase in human erythrocytes.

Glucose-6-phosphate dehydrogenase catalyzes the initial and committed step of the pathway that is the principal source of NADPH in many cells. The intracellular rate of the enzyme in human erythrocytes was estimated from the rate at which the cells generated 14CO2 from 14C-labeled glucose in the presence of different amounts of methylene blue. This investigation differed from earlier studies in that: (a) accumulations of 6-phosphogluconate were considered in calculations of rate and (b) the cells were suspended in Krebs-Ringer bicarbonate buffer, which is the buffer system for erythrocytes in vivo. As with earlier studies, however, the intracellular enzyme was under unexplained inhibition or restraint relative to kinetic properties of the purified enzyme. Also, the intracellular enzyme exhibited sigmoid kinetics. In contrast, the isolated enzyme has been found to exhibit classical kinetics. In the course of dilution/ultrafiltration of the hemolysate a possible cause for the reduced activity of the enzyme was found: most of the NADP was bound to soluble macromolecules of the erythrocyte. The amount of NADP available to the enzyme was much less than the amount indicated by measurements of total (bound and unbound) NADP.

Carbon Dioxide↗
PubMed1986

NADP-binding proteins causing reduced availability and sigmoid release of NADP+ in human erythrocytes.

Glucose-6-phosphate dehydrogenase catalyzes the initial and rate-limiting step of the pathway that is the principal source of NADPH in many cells. Earlier studies of cells from several species indicated that the intracellular enzyme is under severe and unexplained restraint or inhibition. Moreover, the intracellular enzyme of human erythrocytes exhibits sigmoid kinetics, whereas the purified enzyme exhibits only classical kinetics. We here report that most of the NADP in the human erythrocyte is bound by soluble proteins. In addition, the fraction of unbound NADP that is in the oxidized form, [NADP+]/[NADP], varies in a sigmoid manner relative to the fraction of bound NADP that is in the oxidized form. These features of intracellular binding of NADP: 1) account for the previously unexplained inhibition and sigmoid kinetics of glucose-6-phosphate dehydrogenase within human erythrocytes and 2) represent a system in which activity of a rate-limiting enzyme is largely determined by the binding and release of substrate and product by intracellular proteins other than the enzyme itself.

Bicarbonates↗
PubMed1978

Prenatal diagnosis by linkage: hemophilia A and polymorphic glucose-6-phosphate deydrogenase.

Close linkage between the loci for G6PD and hemophilia A allows prenatal diagnosis of hemophilia in the fetuses of certain women who are heterozygous for two electrophoretic types of G6PD. A pregnant woman, whose mother was an obligate heterozygote for hemophilia, had factor VIII levels and a G6PD phenotype that failed to indicate clearly whether or not she was heterozygous for hemophilia. The G6PD phenotype of her male fetus revealed that the fetus was unlikely to have hemophilia.

Female↗
PubMed1977

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.

ABO Blood-Group System↗