PubMed Health⌕ Search

Biomedical subjects

P P Waymack

Publications and source records attributed to P P Waymack.

13 recordsLinked to original sources
PubMed2000

A reference method laboratory network for cholesterol: a model for standardization and improvement of clinical laboratory measurements.

BACKGROUND: Accurate and precise measurement of blood cholesterol plays a central role in the National Cholesterol Education Program's strategy to reduce the morbidity and mortality attributable to coronary heart disease. Matrix effects hamper the ability of manufacturers to adequately calibrate and validate traceability to the National Reference System for Cholesterol (NRS/CHOL). CDC created the Cholesterol Reference Method Laboratory Network (CRMLN) to improve cholesterol measurement by assisting manufacturers of in vitro diagnostic products with validation of the traceability of their assays to the NRS/CHOL. METHODS: CRMLN laboratories established the CDC cholesterol reference method (modification of the Abell-Levy-Brodie-Kendall chemical method) and are standardized using CDC frozen serum reference materials. CRMLN laboratories use common quality-control materials and participate in monthly external performance evaluations conducted by CDC. The CRMLN performance criteria require member laboratories to agree with CDC within +/-1.0% and maintain a CV < or =2.0%. RESULTS: From 1995 to 200 the CRMLN laboratories met the accuracy criterion 97% of the time and the precision criterion 99% of the time. During this time period, the CRMLN maintained an average bias to CDC of 0.01% and an average collective CV of 0.33%. CONCLUSIONS: CDC established the CRMLN as the first international reference method laboratory network. The CRMLN assists manufacturers in the validation of the calibration of their diagnostic products so that clinical laboratories can measure blood cholesterol more reliably. The CRMLN can serve as a model for other clinical analytes where traceability to a hierarchy of methods is needed and matrix effects of the field methods with processed calibrators or reference materials are present.

Calibration↗
PubMed1999

Selection, validation, standardization, and performance of a designated comparison method for HDL-cholesterol for use in the cholesterol reference method laboratory network.

BACKGROUND: Accurate and precise HDL-cholesterol (HDL-C) measurements are essential for effective application of National Cholesterol Education Program treatment guidelines. The Cholesterol Reference Method Laboratory Network (CRMLN) assists manufacturers of in vitro diagnostic products to establish traceability to the accuracy base. CRMLN sought to implement a designated comparison method (DCM) that overcomes the impracticalities of the expensive and labor-intensive reference method for HDL-C. METHODS: CRMLN evaluated candidate DCMs and selected one that uses 50-kDa dextran sulfate with magnesium ions as the precipitation reagent followed by measurement of cholesterol by the CDC reference method. After validating the method, we transferred it to all CRMLN laboratories and successfully standardized it using CDC frozen serum reference materials. CRMLN laboratories participate in monthly performance evaluations. RESULTS: CRMLN laboratories were able to meet a precision goal, as indicated by SD, of </=0.03 mmol/L (1 mg/dL) 94.4% of the time. They were able to meet a bias goal of </=0.05 mmol/L (2 mg/dL) for HDL-C <1.09 mmol/L (42 mg/dL) 97.3% of the time and a goal of </=3% for HDL-C >/=1.09 mmol/L (42 mg/dL) 95.6% of the time. CRMLN is working to further improve its performance by implementing a bias criterion of 0.03 mmol/L (1 mg/dL) for all HDL-C concentrations. CONCLUSIONS: CRMLN selected, validated, standardized, and implemented a DCM for HDL-C that is accurate, robust, transferable, and practical. The DCM is being used to assist manufacturers in calibrating their products so that ultimately, clinical laboratories using the products will more accurately measure HDL-C.

Cholesterol, HDL↗
PubMed1993

Assay instrument-dependent matrix effects in standardization of cholesterol measurements.

Human serum-based frozen reference materials have been used by the Centers for Disease Control and Prevention (CDC)-National Heart, Lung and Blood Institute Lipid Standardization Program to improve the precision and accuracy of blood cholesterol measurements. Occasionally, laboratories in the program have had problems obtaining results for patients' fresh serum samples equivalent to those obtained with frozen CDC standardization pools. This incompatibility of sample, reagent, instrument, and assay characteristics has been labeled broadly as a "matrix effect," which usually is attributed to unknown characteristics of the processed pool material. In this study we showed that a large negative bias obtained with CDC pools was attributable to use of the sample blank mode on the Cobas-Bio analyzer. However, under the same conditions, fresh patients' serum samples were analyzed accurately. The use of a blank absorbance immediately after mixing sample and reagents (the "autoblank" mode) allowed the instrument to accurately analyze both fresh serum samples and CDC standardization pools and thus allowed the documentation of traceability of the cholesterol measurements to the National Reference System for Cholesterol.

Bias↗
PubMed1992

Determination of "free" glycerol in human serum reference materials by isotope-dilution gas chromatography-mass spectrometry.

Serum free glycerol analyses are an important part of the preparation and evaluation of human serum reference materials used for the quality assurance of triglyceride assays. However, enzymatic kits for free glycerol analysis obtained from different vendors have, on occasion, provided different results for a given sample. In an effort to establish the "target" glycerol content of selected reference materials, we have established a method for the analysis of serum free glycerol by using isotope-dilution gas chromatography-mass spectrometry, incorporating [1,3-13C2]glycerol as the internal standard. The use of a simplified serum extraction and clean-up procedure resulted in (uncorrected) recoveries of glycerol averaging about 90% before derivatization, and in estimated concentrations for spiked serum pools that corresponded closely to the expected values. A comparison of enzymatic and gas chromatographic-mass spectrometric results for several reference serum pools suggest that the latter method is of value in evaluating and validating routine enzymatic methods for free glycerol analysis.

Carbon Isotopes↗
PubMed1991

Isolation and characterization of a homogeneous isoenzyme of wheat germ acid phosphatase.

An acid phosphatase (orthophosphoric monoester phosphohydrolase, acid optimum; EC 3.1.3.2) isoenzyme from wheat germ was purified 7000-fold to homogeneity. The effect of wheat germ sources and their relationship to the isoenzyme content and purification behavior of acid phosphatases was investigated. Extensive information about the purification and stabilization of the enzyme is provided. The instability of isoenzymes in the latter stages of purification appeared to be the result of surface inactivation together with a sensitivity to dilution that could be partially offset by addition of Triton X-100 during chromatographic procedures. Added sulfhydryl protecting reagents had no effect on activity or stability, which was greatest in the pH range 4-7. The purified isoenzyme was homogeneous by polyacrylamide gel electrophoresis and exhibited the highest specific activity and turnover number reported for any acid phosphatase. The molecular weights of the pure isoenzyme and of related isoenzymes from wheat germ were found to be identical (58,000). The pure isoenzyme contained a single polypeptide chain and had a negligible carbohydrate content. The amino acid composition was determined. Of the various reasons that were considered to explain isoenzyme occurrence, a genetic basis was considered most likely. The enzyme was found to exhibit substrate inhibition with some substrates below pH 6, while above pH 8 it exhibited downwardly curving Lineweaver-Burk plots of the type that are generally described as "substrate activation". The observation of a phosphotransferase activity was consistent with the formation of a covalent phosphoenzyme intermediate, while inactivation by diethyl pyrocarbonate was consistent with the presence of an active site histidine.

Acid Phosphatase↗
PubMed1991

Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.

6-Phytase↗
PubMed1988

Acute effects of a superoxide radical-generating system on DNA double-strand stability in Chinese hamster ovary cells. Determination by a modified fluorometric procedure.

The rate of oxyradical generation by a xanthine oxidase-xanthine system to acutely cause DNA strand breakage in Chinese hamster ovary cells was studied in a phosphate-buffered saline system. DNA strand breakage, measured by a fluorometric procedure, was found to increase curvilinearly as a function of oxyradical generation. Results of studying the ability of 5 mM mannitol, 10 mM dimethylthiourea, 300 micrograms superoxide dismutase/ml, or 1 mg catalase/ml to interfere with DNA damage at a high rate of oxyradical production best supported a hydrogen peroxide-promoted mechanism for DNA breakage.

Animals↗
PubMed1988

Measurement of branched-chain alpha-keto acid dehydrogenase flux rates in perfused heart and liver.

The methods described here represent a flexible set of procedures for investigating the metabolism of the branched-chain alpha-keto acids and other substances in perfused organs, notably the rat heart and liver. These procedures have been used to investigate many aspects of the metabolism of the branched-chain alpha-keto acids not discussed here, such as the effects on branched-chain alpha-keto acid metabolism by exposure to alpha-adrenergic agents, by inhibition of the monocarboxylate translocator, and by the coinfusion of other metabolites.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1980

Studies on the activation and inactivation of the branched chain alpha-keto acid dehydrogenase in the perfused rat heart.

Evidence for a reversible process resulting in stable activated and inactivated states of the mitochondrial branched chain alpha-keto acid dehydrogenase complex in isolated perfused rat heart is presented. The inactivation process is mediated by pyruvate infusion, while activation (up to 18-fold) is facilitated by branched chain alpha-keto acid substrates. The low activity state of the branched chain complex characteristic of freshly excised rat hearts could be maintained by infusion of either pyruvate or glucose. Activation of the complex in the perfused rat heart was achieved slowly by substrate-free perfusion, while rapid activation was accomplished by infusion of branched chain alpha-keto acids. The fully activated enzyme complex resulting from branched chain alpha-keto acid infusion subsequently could be inactivated maximally by infusion of pyruvate alone or intermediate degrees of inactivation could be produced by certain ratios of co-infused pyruvate and branched chain alpha-keto acid. alpha-Ketoisocaproate was an order of magnitude more effective than alpha-keto isovalerate either in preventing inactivation or in stimulating the opposing activation process when co-infused with pyruvate. The mitochondrial pyruvate transport inhibitor, alpha-cyanocinnamate, effectively prevented inactivation of the complex by infused pyruvate. Differential changes in the activation states of the branched chain alpha-keto acid dehydrogenase and pyruvate dehydrogenase complexes were evident when the two complexes were compared in apparently similar flux-inhibited (via octanoate infusion) and flux-stimulated (via dichloroacetate infusion) metabolic conditions. The differential effect of pyruvate concentration on the activity states of the two complexes was also well-defined. The results of the present study suggest distinct systems for the regulation of the activity of the two multienzyme complexes of interest. While our results argue neither for nor against an inactivation of the branched chain alpha-keto acid dehydrogenase complex by a protein kinase, the regulatory properties of such an intramitochondrial protein kinase may not be similar to the pyruvate dehydrogenase kinase. The mechanistic nature of the suggested novel regulatory system concerned with the pyruvate-mediated inactivation of the branched chain alpha-keto acid activation cannot be inferred at the present time.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗