Methods are described for detection of lactate dehydrogenase (LDH) inhibitors in preparations of ... more Methods are described for detection of lactate dehydrogenase (LDH) inhibitors in preparations of reduced nicotinamide adenine dinucleotide. They are (a) comparison of values by kinetic methods with those measured for highly purified NADH and (b) examination of Lineweaver-Burk plots. Chromatographic inhomogeneities are correlated with deviant values for the kinetic constants of NADH preparations. Lineweaver-Burk plots that curve upward at the high concentrations or have a larger or smaller than normal slope may indicate the presence of inhibitor. As determined in bicarbonate buffer (0.11 mol/liter, pH 7.9) by use of 0.600 mmol/liter pyruvate and NADH freshly separated from impurities by chromatography on diethyl-aminoethyl-cellulose, the Km (apparent) of NADH at 25 degrees C has the value 8.11 +/- 0.71 mumol/liter (SD, n = 28) with LDH-1 (pig heart, 2.48 +/- 0.05 U per milliliter of reaction mixture, or 41.3 +/- 0.8 nmol/liter per second). Under similar conditions, the Km (apparent) of NADH has the value of 8.57 +/- 1.58 mol/liter (SD, n = 21) with LDH-5 (pig muscle, 1.77 +/- 0.03 U/ml of reaction mixture), or 29.4 +/- 0.6 nmol/liter per second). At infinite substrate concentrations with the same pH, buffer, and temperature, the Km (apparent) for NADH was 26.0 +/- 0.63 mumol/liter with LDH-1 and 23.2 +/- 4.6 mumol/liter with LDH-5.
The American Journal of Clinical Nutrition, Dec 1, 1991
Plasma supplemented with ascorbic acid was prepared; the stability of these samples was character... more Plasma supplemented with ascorbic acid was prepared; the stability of these samples was characterized and the accuracy of the supplementation was established. Studies on the accuracy, precision, and sources of methodological bias in the measurement of ascorbic acid were summarized. Measurements of the ratio of ascorbic acid to dehydroascorbic acid in clinical samples was evaluated and was shown to be relatively constant in plasma taken from blood stored at 12 degrees C for 6 h. These results imply that whole blood has the capacity to maintain a constant ascorbic-dehydroascorbic acid ratio and suggest that this ratio may be of physiological significance.
We describe two independent HPLC procedures for the rapid, accurate analysis for ascorbic acid in... more We describe two independent HPLC procedures for the rapid, accurate analysis for ascorbic acid in human plasma. No sample extraction or phase separation is required. We also describe a procedure for preparing a human plasma reference material for use in clinical laboratory analysis for ascorbic acid. The ascorbic acid in plasma can be determined in 15 min, with as little as 50 microL of sample. Analytical recoveries are near 100% with direct injection of deproteinized plasma. Extensive stability data under several conditions, with dithiothreitol as a preservative (antioxidant), indicate that ascorbic acid remains stable in stored plasma for as long as 57 weeks. CVs for round-robin analysis of 11 normal human blood samples by two independent methods were between 0.1% and 5.3%. These clinical samples appear to be stable for at least 50 days under the described conditions of stabilization and sample treatment. Finally, because ascorbic acid prepared by the described procedures is stable at room temperature for at least 18 h, these methods can be readily adapted to clinical laboratory automation at room temperature.
The chemical shifts of the isoleucine and histidine protons of angiotensin I were assigned and th... more The chemical shifts of the isoleucine and histidine protons of angiotensin I were assigned and the chemical shifts of the protons of the other amino acids in the peptide were confirmed at a field strength of 400 MHz. These chemical shift assignments were used to determine the amino acid composition of angiotensin I. These data were then compared to the amino acid composition which was determined by chromatographic analysis of the peptide hydrolysate. The results obtained by the chromatographic method were similar to those obtained by the NMR method. The standard deviations of the results were similar, indicating that these methods are equally precise. The major advantages of the NMR method are that it permits the recovery of the peptide after completion of the analysis and improves the quantitation of amino acids which are either partially destroyed by the hydrolysis procedure or require special derivatization methods for detection and quantitation.
The presence of a new lactate dehydrogenase inhibitor on the trailing edge of the NADH peak from ... more The presence of a new lactate dehydrogenase inhibitor on the trailing edge of the NADH peak from chromatography on diethylaminoethyl-celluose [Loshon et al., Clin. Chem., this issue] was verified. It was resolved from the NADH by high-performance liquid chromatography on muBondapak C18. When the new inhibitor was present in a reaction mixture to the extent that, of the initial 260-nm absorbance, about 5% was contributed by the inhibitor, the rate of NADH oxidation by lactate dehydrogenase decreased by 65%. The inhibitor absorbs at 260 and 340 nm, and is different from the Strandjord-Clayson inhibitor [J. Lab. Clin. Med. 67, 144 (1966)] by both types of chromatography. Because this new inhibitor has ultraviolet properties similar to those of NADH and chromatographs with the NADH on DEAE-cellulose, the high-performance liquid chromatographic method must be used to ensure its absence in preparations of NADH used for clinical assay.
We developed an analytical reverse-phase high-performance liquid chromatographic procedure for ra... more We developed an analytical reverse-phase high-performance liquid chromatographic procedure for rapid assessment of the purity of NADH. The method completely separates adenosine monophosphate and adenosine diphosphoribose from NADH. By use of this analytical technique we found that preparative chromatography on DEAE-cellulose gives NADH that is free from adenine nucleotides as well as other impurities that commonly are present in NADH. The absorbance ratio at 260 and 340 nm of the purified NADH in 1.8 mmol/liter ammonium carbonate is 2.261 +/- 0.002 (+/- 1 SD).
The Journal of Chemical Thermodynamics, Feb 1, 1993
ABSTRACT Molar enthalpies reaction for the hydrolysis of d-arginine(aq) to {d-ornithine(aq) + ure... more ABSTRACT Molar enthalpies reaction for the hydrolysis of d-arginine(aq) to {d-ornithine(aq) + urea(aq)} and for the hydrolysis of d -arginine(aq) to {d-citrulline(aq) + ammonia(aq)} have been measured by microcalorimetry. These reactions are catalyzed, respectively, by arginase and by arginine deiminase. The effects of variations in pH, temperature, and ionic strength on the molar enthalpies of reaction were studied. The results have been analyzed with a model which accounts for the complex equilibria in solution. The results obtained at T = 198.15 K for the reference reactions are: ΔrHom = -(21.4±0.5) kJ·mol-1 for d-arginine+(aq) + H2O(l) = d-ornithine+(aq) + urea(aq) and ΔrHom = -(31.9±0.8) kJ·mol-1 for d-arginine+(aq) + H2O(l) = d-citrulline(aq) + NH+4(aq). These results are discussed in terms of thermodynamic-cycle calculations and in terms of the metabolic urea cycle.
Methods are described for detection of lactate dehydrogenase (LDH) inhibitors in preparations of ... more Methods are described for detection of lactate dehydrogenase (LDH) inhibitors in preparations of reduced nicotinamide adenine dinucleotide. They are (a) comparison of values by kinetic methods with those measured for highly purified NADH and (b) examination of Lineweaver-Burk plots. Chromatographic inhomogeneities are correlated with deviant values for the kinetic constants of NADH preparations. Lineweaver-Burk plots that curve upward at the high concentrations or have a larger or smaller than normal slope may indicate the presence of inhibitor. As determined in bicarbonate buffer (0.11 mol/liter, pH 7.9) by use of 0.600 mmol/liter pyruvate and NADH freshly separated from impurities by chromatography on diethyl-aminoethyl-cellulose, the Km (apparent) of NADH at 25 degrees C has the value 8.11 +/- 0.71 mumol/liter (SD, n = 28) with LDH-1 (pig heart, 2.48 +/- 0.05 U per milliliter of reaction mixture, or 41.3 +/- 0.8 nmol/liter per second). Under similar conditions, the Km (apparent) of NADH has the value of 8.57 +/- 1.58 mol/liter (SD, n = 21) with LDH-5 (pig muscle, 1.77 +/- 0.03 U/ml of reaction mixture), or 29.4 +/- 0.6 nmol/liter per second). At infinite substrate concentrations with the same pH, buffer, and temperature, the Km (apparent) for NADH was 26.0 +/- 0.63 mumol/liter with LDH-1 and 23.2 +/- 4.6 mumol/liter with LDH-5.
The American Journal of Clinical Nutrition, Dec 1, 1991
Plasma supplemented with ascorbic acid was prepared; the stability of these samples was character... more Plasma supplemented with ascorbic acid was prepared; the stability of these samples was characterized and the accuracy of the supplementation was established. Studies on the accuracy, precision, and sources of methodological bias in the measurement of ascorbic acid were summarized. Measurements of the ratio of ascorbic acid to dehydroascorbic acid in clinical samples was evaluated and was shown to be relatively constant in plasma taken from blood stored at 12 degrees C for 6 h. These results imply that whole blood has the capacity to maintain a constant ascorbic-dehydroascorbic acid ratio and suggest that this ratio may be of physiological significance.
We describe two independent HPLC procedures for the rapid, accurate analysis for ascorbic acid in... more We describe two independent HPLC procedures for the rapid, accurate analysis for ascorbic acid in human plasma. No sample extraction or phase separation is required. We also describe a procedure for preparing a human plasma reference material for use in clinical laboratory analysis for ascorbic acid. The ascorbic acid in plasma can be determined in 15 min, with as little as 50 microL of sample. Analytical recoveries are near 100% with direct injection of deproteinized plasma. Extensive stability data under several conditions, with dithiothreitol as a preservative (antioxidant), indicate that ascorbic acid remains stable in stored plasma for as long as 57 weeks. CVs for round-robin analysis of 11 normal human blood samples by two independent methods were between 0.1% and 5.3%. These clinical samples appear to be stable for at least 50 days under the described conditions of stabilization and sample treatment. Finally, because ascorbic acid prepared by the described procedures is stable at room temperature for at least 18 h, these methods can be readily adapted to clinical laboratory automation at room temperature.
The chemical shifts of the isoleucine and histidine protons of angiotensin I were assigned and th... more The chemical shifts of the isoleucine and histidine protons of angiotensin I were assigned and the chemical shifts of the protons of the other amino acids in the peptide were confirmed at a field strength of 400 MHz. These chemical shift assignments were used to determine the amino acid composition of angiotensin I. These data were then compared to the amino acid composition which was determined by chromatographic analysis of the peptide hydrolysate. The results obtained by the chromatographic method were similar to those obtained by the NMR method. The standard deviations of the results were similar, indicating that these methods are equally precise. The major advantages of the NMR method are that it permits the recovery of the peptide after completion of the analysis and improves the quantitation of amino acids which are either partially destroyed by the hydrolysis procedure or require special derivatization methods for detection and quantitation.
The presence of a new lactate dehydrogenase inhibitor on the trailing edge of the NADH peak from ... more The presence of a new lactate dehydrogenase inhibitor on the trailing edge of the NADH peak from chromatography on diethylaminoethyl-celluose [Loshon et al., Clin. Chem., this issue] was verified. It was resolved from the NADH by high-performance liquid chromatography on muBondapak C18. When the new inhibitor was present in a reaction mixture to the extent that, of the initial 260-nm absorbance, about 5% was contributed by the inhibitor, the rate of NADH oxidation by lactate dehydrogenase decreased by 65%. The inhibitor absorbs at 260 and 340 nm, and is different from the Strandjord-Clayson inhibitor [J. Lab. Clin. Med. 67, 144 (1966)] by both types of chromatography. Because this new inhibitor has ultraviolet properties similar to those of NADH and chromatographs with the NADH on DEAE-cellulose, the high-performance liquid chromatographic method must be used to ensure its absence in preparations of NADH used for clinical assay.
We developed an analytical reverse-phase high-performance liquid chromatographic procedure for ra... more We developed an analytical reverse-phase high-performance liquid chromatographic procedure for rapid assessment of the purity of NADH. The method completely separates adenosine monophosphate and adenosine diphosphoribose from NADH. By use of this analytical technique we found that preparative chromatography on DEAE-cellulose gives NADH that is free from adenine nucleotides as well as other impurities that commonly are present in NADH. The absorbance ratio at 260 and 340 nm of the purified NADH in 1.8 mmol/liter ammonium carbonate is 2.261 +/- 0.002 (+/- 1 SD).
The Journal of Chemical Thermodynamics, Feb 1, 1993
ABSTRACT Molar enthalpies reaction for the hydrolysis of d-arginine(aq) to {d-ornithine(aq) + ure... more ABSTRACT Molar enthalpies reaction for the hydrolysis of d-arginine(aq) to {d-ornithine(aq) + urea(aq)} and for the hydrolysis of d -arginine(aq) to {d-citrulline(aq) + ammonia(aq)} have been measured by microcalorimetry. These reactions are catalyzed, respectively, by arginase and by arginine deiminase. The effects of variations in pH, temperature, and ionic strength on the molar enthalpies of reaction were studied. The results have been analyzed with a model which accounts for the complex equilibria in solution. The results obtained at T = 198.15 K for the reference reactions are: ΔrHom = -(21.4±0.5) kJ·mol-1 for d-arginine+(aq) + H2O(l) = d-ornithine+(aq) + urea(aq) and ΔrHom = -(31.9±0.8) kJ·mol-1 for d-arginine+(aq) + H2O(l) = d-citrulline(aq) + NH+4(aq). These results are discussed in terms of thermodynamic-cycle calculations and in terms of the metabolic urea cycle.
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Papers by Sam A Margolis