T.F. Draisey

Glutathione metabolic enzyme activities in diabetic platelets as a function of glycemic control

Type 1 diabetic subjects categorized on the basis of the glycated haemoglobin content of their blood (low less than 7%; medium, greater than 7% and less than 11%; high, greater than 11%) were analyzed for total intraplatelet GSH as well as for the steady-state kinetic parameters (apparent KM and apparent Vmax) of some glutathione metabolic enzymes including glutathione reductase, glutathione peroxidase, gamma-glutamyltrans-peptidase and glutathione-S-transferase. This study indicates that intraplatelet GSH content of subjects with low glycated-haemoglobin is approximately 2-fold higher than those with medium glycated-haemoglobin. There was no further decrease in intraplatelet-GSH in subjects with high glycated-haemoglobin. The kinetic parameters of the platelet-enzymes studied (glutathione reductase, gamma-glutamyltranspeptidase and glutathione-S-transferase) were essentially independent of the glycation state of the subject. However, the apparent KM of glutathione peroxidase was approximately 4-fold higher in the subjects with high glycated-haemoglobin, in comparison to low subjects. This decrease in affinity could possibly result from the susceptibility of this enzyme to non-enzymatic glucosylation as purified samples of glutathione peroxidase incubated in vitro with glucose showed similar increases in apparent KM. These results are discussed in terms of the potential contribution of glutathione peroxidase impairment, to the hyperaggregability of the diabetic platelet.

A procedure for the direct determination of micromolar quantities of lecithin employing enzymes as reagents

Abstract A procedure has been proposed for the determination of micromolar concentrations of lecithin. This procedure, which utilizes enzymes as reagents, is relatively quick, simple, and inexpensive, and involves no extractions. Clinical studies of the applicability of this procedure for the determination of lecithin concentrations in amniotic fluid have, as yet, not been concluded. The synthesis of sodium 2-hydroxy-3, 5-dichlorobenzenesulfonate was described. This material in purified form has proven to be very convenient for routine use in a peroxidase-catalyzed coupling to 4-aminoantipyrine.

The determination of lecithin and total choline-containing phospholipids in amniotic fluid employing enzymes as reagents

Abstract Two assays which employ enzymes as reagents have been described for the determination of lecithin and TCCP in amniotic fluid. Both of these assays are relatively quick, simple, inexpensive, precise, involve no extractions, and are amenable to automation. Hemoglobin, blood cell membranes, and bilirubin, common interferences in L S ratio determinations, have been studied. Their effects on these two assays appear to be negligible. It can be inferred from preliminary studies that the described assays correlate well with each other, and with the clinical status of the fetus. The values obtained for lecithin and TCCP cmncentrations are in the same range as previous reports employing different procedures.

Colorimetric determination of non-enzymatically glycated albumin

A colorimetric method was developed for the determination of nonenzymatically glycated albumin and adapted to a Flexigem centrifugal analyzer. Albumin was separated from serum or plasma using Sepharose-blue dextran affinity chromatography. The stable ketoamine linkage in glycated albumin reduced a tetrazolium salt to its colored formazan. Glycated human serum albumin was used as the standard and optimum conditions for the assay were established. Recovery of glycated albumin was quantitative. The coefficients of variation for within-run and day-to-day precision were 4.6% and 8.5%, respectively. The labile aldimine fraction, lipemia, icterus, hemolysis and type of anticoagulant used did not affect the results. The non-diabetic reference interval for this method was 7.9-11.6% glycated albumin, and normal and diabetic populations can be clearly discriminated (p less than 0.005). Values obtained with this method correlated well with a thiobarbituric acid assay (r = 0.974) but less so with those for glycated hemoglobin (r = 0.35).

Fluorometric determination of cholesterol using an oxidase-peroxidase-resorufin system

Abstract A fluorometric method for the enzymatic determination of cholesterol in serum was developed. It involves the use of resorufin and peroxidase for the measurement of hydrogen peroxide generated when cholesterol oxidase acts upon cholesterol. The ratio of serum to reagent was 1:1999 which permitted the determination of cholesterol concentrations of between 0.8 and 10 μmol/liter in the final solution. The within-run and between-run CVs for two control sera containing 3.19 and 5.66% mmol/liter cholesterol were 1.9 and 2.6%, and 0.88 and 1.22%, respectively. Interferences from triglycerides up to 10.25 mmol/liter and hemoglobin up to 5.6 g/liter were insignificant, whereas the bilirubin interference is significant starting from 115 μmol/liter. Recoveries for between 2 and 6 mmol/liter standard additions to a pooled serum containing 5.87 mmol/liter were between 94.5 and 104.3%. Serum samples were analyzed using a modified Trinder method and the proposed method. The regression equation was Y = 1.02X − 0.13 (r = 0.98, N = 56). The proposed method for the determination of cholesterol is simple, reliable, and can be easily automated.

Determination of non-enzymatically glycated apolipoprotein A1

A procedure for the quantitation of non-enzymatically glycated apolipoprotein A1 (GApoA1) was developed and optimized. Glycated total protein was separated from plasma using m-aminophenyl-boronate affinity chromatography. Apolipoprotein A1 present in the glycated and non-glycated fractions of each sample was determined by rate nephelometry, and the percent glycated apo A1 calculated. The measuring range of the assay was 0.5-8.0% GApoA1. The within- and between-run CVs were less than 5.2 and 7.9%, respectively, and recoveries were greater than 92%. Free glucose did not affect the results. In a group of female non-insulin diabetic subjects the mean GApoA1 was 3.8 +/- 1.6% (mean +/- SD). In non-diabetic subjects the mean level of GApoA1 was 2.1 +/- 0.8% (mean +/- SD).

An improved fluorometric coupled enzymatic method for the determination of pyrophosphate in plasma and platelets

Abstract A fluorometric coupled enzymatic method for the determination of pyrophosphate is described. The kinetic method can determine pyrophosphate in amounts as low as 0.5 pmol. Intra- and interassay CVs were both less than 1% and recoveries were found to be quantitative. No significant interference was observed from EDTA, heparin, magnesium, or calcium. Correlation with an established method was significant (r = 0.87). Clinical studies of the applicability of the procedure were demonstrated by the measurement of pyrophosphate in plasma and platelets. Mean pyrophosphate levels in plasma and platelets of “normal” subjects were found to be 3.27 ± 0.2 μM and 2.40 ± 0.10 nmol/108 platelets or 17.59 ± 1.44 nmol/mg platelet protein, respectively. Pyrophosphate levels in plasma and platelets of patients on hemodialysis were determined and found to be significantly lower than the “normal” population. This finding may in part explain the occurrence of metastatic calcification seen in this patient population.

A fluorometric coupled enzymatic method for the determination of γ-aminobutyric acid transaminase in platelets

Abstract A fluorometric, coupled enzymatic method for the determination of γ-aminobutyric acid transaminase in platelets is described. The limit of linearity of the method is 0.08 pmol GABA/min/mg platelet protein. Within- and between-run C.V.s were both less than 4.6% and recoveries were found to be quantitative. Correlation with an established radiochemical procedure was significant ( r = 0.999). Mean (±SEM) GABA-T levels in platelets of “normal” subjects ( n = 35) were 23.3 ± 14.8 pmol GABA/min/mg platelet protein.

Assessment of the EMIT™ technique as a screening test for opiates and methadone for a methadone maintenance clinic and its calibration by bayesian statistics

1. The performance of the semi-quantitative enzyme multiplied immunoassay technique (EMIT, Syva Corp.) for opiates and methadone has been examined as a screening procedure of urine samples from a methadone maintenance programme. The predictive value model that is based upon Bayesian statistics was used to determine screening levels for the EMIT assays. 2. With a predictive value of a negative result of 100%, the EMIT opiate assay can be used to show the absence of the indicated drugs, while positive results can be confirmed by a non-immunological technique. A screening level of 1.0 micrograms/ml for the opiate assay, conforms to this model. 3. The EMIT methadone assay was shown to have a predictive value of a negative result of 28% with respect to thin-layer chromatographic (TLC) results. This discrepancy between EMIT and TLC can not be explained by sensitivity alone. The manufacturers recommended 0.5 micrograms/ml cutoff has been used therefore for the methadone assay.

LOW-DENSITY LIPOPROTEIN BINDING ASSAY USING THE CALF ADRENOCORTICAL LOW-DENSITY LIPOPROTEIN RECEPTOR

The low-density lipoprotein (LDL) receptor was purified to a semipure solubilized form from calf adrenocortical tissue. This receptor was found to be a suitable substitute for the human LDL receptor for studying human LDL binding. The apparent dissociation constant of the receptor from calf adrenocortical cells, using human LDL as the ligand, was found to be 8.8 +/- 1.0 micrograms 125I-LDL/mL, similar to that reported for the human LDL receptor (4-10 micrograms LDL/mL). The calf adrenocortical LDL receptor demonstrated specificity toward human lipoprotein fractions that was identical with that of the human LDL receptor. A competitive binding assay was optimized using the semipurified solubilized calf adrenocortical receptor. This facilitated the study of nonenzymatically glycosylated human LDL by a binding assay that is much simpler and faster than previous studies, which used intact cultured cells. The present assay requires only a 1-h incubation of LDL with the receptor and a simple filtration procedure to remove unbound LDL. Using the present assay, it was shown that nonenzymatic glycosylation of LDL on the order of what is seen in diabetics, that is, modification of 2-5% of lysine residues, caused a decreased ability of the LDL to bind to the receptor.