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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
PubMed2000

Nutritional rickets in African American breast-fed infants.

OBJECTIVE: To analyze the characteristics of infants and children diagnosed with nutritional rickets at two medical centers in North Carolina in the 1990s. STUDY DESIGN: The physical and radiographic findings, calcium, phosphorus, alkaline phosphatase, and 25-hydroxyvitamin D levels of infants and children diagnosed with nutritional rickets at two medical centers were reviewed. Breast-feeding data were obtained from the North Carolina Women, Infants and Children Program (WIC). RESULTS: Thirty patients with nutritional rickets were first seen between 1990 and June of 1999. Over half of the cases occurred in 1998 and the first half of 1999. All patients were African American children who were breast fed without receiving supplemental vitamin D. The average duration of breast-feeding was 12.5 months. The age at diagnosis was 5 to 25 months, with a median age of 15.5 months. Growth failure was common: length was <5th percentile in 65% of cases, and weight was <5th percentile in 43%. CONCLUSION: Factors that may have contributed to the increase in referrals of children with nutritional rickets include more African American women breast-feeding, fewer infants receiving vitamin D supplements, and mothers and children exposed to less sunlight. We recommend that all dark-skinned breast-fed infants and children receive vitamin D supplementation.

Black People↗
PubMed1999

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.

Acetaldehyde↗
PubMed1996

Predominant role of catalase in the disposal of hydrogen peroxide within human erythrocytes.

Purified enzymes were mixed to form a cell-free system that simulated the conditions for removal of hydrogen peroxide within human erythrocytes. Human glutathione peroxidase disposed of hydrogen peroxide (H2O2) at a rate that was only 17% of the rate at which human catalase simultaneously removed hydrogen peroxide. The relative rates observed were in agreement with the relative rates predicted from the kinetic constants of the two enzymes. These results confirm two earlier studies on intact erythrocytes, which refuted the notion that glutathione peroxidase is the primary enzyme for removal of hydrogen peroxide within erythrocytes. The present findings differ from the results with intact cells, however, in showing that glutathione peroxidase accounts for even less than 50% of the removal of hydrogen peroxide. A means is proposed for calculating the relative contribution of glutathione peroxidase and catalase in other cells and species. The present results raise the possibility that the major function of glutathione peroxidase may be the disposal of organic peroxides rather than the removal of hydrogen peroxide.

Catalase↗
PubMed1994

Importance of catalase in the disposal of hydrogen peroxide within human erythrocytes.

The catalase within normal, intact human erythrocytes was completely inactivated with amino triazole. The rate of 14CO2 evolution, when the cells were subsequently incubated with 14C-labeled glucose, provided a measure of the rate at which NADPH was being oxidized by the glutathione peroxidase/reductase system for the disposal of H2O2. This rate was determined in control cells and in catalase-inactivated cells while the cells were exposed to H2O2, which was generated at various constant and predetermined rates by glucose oxidase. The results indicated that catalase handles approximately half of the generated H2O2. The glutathione peroxidase/reductase mechanism accounted for the other half. These results are in agreement with our earlier findings on erythrocytes of a subject with a genetic deficiency of catalase. However, an unexpected result with the present approach was the finding that the increased dependence on the glutathione peroxidase/reductase mechanism did not occur until greater than 98% of the catalase had been inactivated. The latter observation indicates that catalase and the glutathione peroxidase/reductase system function intracellularly in a manner very different from that previously ascribed to them. An explanation of the findings requires that the two methods of H2O2 disposal function in a coordinated way, such as a sequential action in which the glutathione peroxidase/reductase system is the rate-limiting step.

Catalase↗
PubMed1993

Tetrasomy 9p: tissue-limited idic(9p) in a child with mild manifestations and a normal CVS result. Report and review.

Supernumerary isochromosomes resulting in autosomal tetrasomy are rare and have been described only for 12p, 18p, and 9p. Nineteen previous cases of tetrasomy 9p have been reported, and in 6 cases, tissue-specific mosaicism was implied with the i(9p) cell line present exclusively or predominantly in blood. We report on an infant who had apparently normal chromosomes (46,XY) on CVS. He was referred for genetic evaluation because of mild developmental delay and minor anomalies. In 75% of blood cells he had an extra isodicentric 9p chromosome (pter-->q12-->pter). The interpretation of tetrasomy 9p was confirmed by elevated GALT activity. No tetrasomy 9p cells were seen in 100 skin fibroblasts. This case demonstrates the tissue specific mosaicism in tetrasomy 9p which rendered the anomaly undetectable by CVS. It also demonstrates the mild end of the clinical spectrum associated with tetrasomy 9p.

Chromosome Aberrations↗
PubMed1993

Estimates of uridine diphosphate hexoses in erythrocytes: implications for galactosemia.

Impurities in a reagent dehydrogenase caused overestimates of erythrocytic uridine diphosphate glucose and accounted for clinically important differences in results between those of one group of investigators using enzymatic methods and those of two other groups using enzymatic methods, high-performance liquid chromatography, and nuclear magnetic resonance. These data have relevance for the current debate regarding the pathophysiologic changes in galactosemia.

Drug Contamination↗
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↗