Search PubMed⌕ Search

Biomedical subjects

E J Sampson

Publications and source records attributed to E J Sampson.

At least 55 records · Page 3Linked to original sources
PubMed1983

Creatine kinase response surfaces explored by use of factorial experiments and simplex maximization.

We conducted a five-component, five-level response-surface experiment to optimize the pH and the concentrations of magnesium, creatine phosphate, adenosine diphosphate, and buffer in an assay for creatine kinase. Under optimal conditions, creatine kinase activity was about 5% greater than that obtained with a previously reported assay (Clin Chem 23: 1569, 1977). We also applied a simplex maximization algorithm to the response-surface equation to locate areas of maximum sensitivity. Reaction conditions for two such areas were found, each yielding approximately 11% more activity than with the previously reported method.

Adenosine Diphosphate↗
PubMed1982

The development and evaluation of a homogeneous immunoassay for the isoenzymes of aspartate aminotransferase.

Purified isoenzymes of aspartate aminotransferase (AST) from human liver (mitochondrial) and erythrocytes (cytoplasmic) were used to elicit antisera in rabbits. Each antiserum was characterized for titer and specificity. Complexes formed upon addition of each isoenzyme to its specific antiserum were demonstrated to be catalytically inactive. Results obtained when either filtration or centrifugation was used for separating the complexes (heterogeneous assay) were comparable to those obtained when the complexes were not separated (homogeneous assay) from the mixture before assay. A quality control system was designed to monitor specificity in addition to the usual parameters. The precision for the inhibition of cytoplasmic (CV 4.8%) and mitochondrial (CV 3.5%) isoenzymes was within that of the enzymatic assay. Several parametric conditions of the enzyme-antibody reactions were examined, and the assay was adapted for semi-automation. The homogeneous assay was evaluated with a series of pseudo specimens containing known mixtures of pure isoenzymes to determine the extent of recovery (99.8%) for a particular isoenzyme in the presence of varying concentrations of the other isoenzyme. In addition, sera from patients having elevated AST concentrations were examined for isoenzyme contributions to total AST activity. A mean recovery of 96% was obtained for these specimens.

Antibody Specificity↗
PubMed1982

Stability of creatine kinase-3 in lyophilized materials.

We examined the stability of creatine kinase (EC 2.7.3.2) isoenzyme-3 (CK-3) in lyophilized bovine albumin matrices in the presence and absence of various sulfhydryl compounds and ADP. We initially purified CK-3 from human myocardium and skeletal muscle by the batch-chromatographic technique and by gradient elution column chromatography to specific activities of 293 and 93 kU/g, respectively. To assess stability, we subjected the lyophilized materials to storage studies at 4, 25, 37, 42, 56, and 65 degrees C and compared first-order rate constants for the decay of creatine kinase activity at 42 degrees C. Our most stable matrix contained, per liter, 2 mmol of ADP and 10 mmol of N-acetylcysteine, and had an extrapolated first-order half-life (Arrhenius plot) at -20 degrees C of approximately 60000 years.

Creatine Kinase↗
PubMed1982

Multivariate and univariate optimization studies of liquid-chromatographic separation of steroid mixtures.

We developed a high-performance liquid-chromatographic separation of five steroids (estriol, estradiol, cortisol, progesterone, and testosterone), eluting with a water-acetonitrile gradient from a reversed-phase (C18) column. By applying a simplex search algorithm to maximize a chromatographic-response function, we sought to optimize the original conditions of the chromatographic analysis, which did not separate two pairs of overlapping peaks. Our chromatographic-response function incorporated both peak separation and total time of analysis. Three factors were varied simultaneously to maximize this function: flow rate, column temperature, and gradient shape. From the simplex optimization, we selected a flow rate of 1.50 mL/min, a temperature of 52 degrees C, and a linear gradient for our analysis. Subsequent univariate studies of the initial mobile phase composition showed that acetonitrile/water (20/80 by vol) gave an adequate separation.

Analysis of Variance↗
PubMed1981

Characterization and intermethod relationships of materials containing purified human pancreatic and salivary amylase.

We describe the preparation and characterization of materials containing human pancreatic and salivary alpha-amylase (EC 3.2.1.1) and examine their relationship to endogenous amylase in human serum. Amylase was purified from human pancreas and saliva by solvent- and salt-fractionation and column chromatography to specific activities of 63 and 279 kU/g, respectively. Four liquid pools, differing only in activity, were prepared from each source of amylase, each in a matrix containing, per liter: 30 g of human albumin, 50 mmol of sodium chloride, 1 mmol of calcium chloride, and 50 mmol of Tris hydrochloride buffer, pH 7.4. Characterization of the pools showed that the amylase activity in the materials was stable for at least six months at 25 degrees C; among-vial variability of amylase activity was less than or equal to 0.5% (2 CV); and the pools were free from eight possible contaminating enzymes. Plots of salivary vs pancreatic amylase activity measure in our materials with eight commercially available methods showed least-squares slopes ranging from 0.51 to 1.0. The intermethod "commutability" of the materials (i.e., how closely they mimic endogenous serum amylase) was examined in relationship to approximately 100 human sera.

Amylases↗
PubMed1980

A coupled-enzyme equilibrium method for measuring urea in serum: optimization and evaluation of the AACC study group on urea candidate reference method.

We describe a coupled-enzyme equilibrium method for measuring urea in serum, which is performed on supernates prepared by treating each specimen with Ba(OH)2 and ZnSO4 (Somogyi reagent). Analytical recovery of [14C]urea added to a variety of matrices was essentially complete (mean, 100.6%) for the supernates after precipitation. Nine variables were univariately examined in arriving at the reaction conditions for the method: glutamate dehydrogenase, urease, 2-oxoglutarate, ADP, Tris . HCI, NADH, EDTA, pH, and temperature. The reagent is stable for at least 48 days at--20 degrees C and for 23 days at 4 degrees C. Mean analytical recovery of urea (14 mmol/L) added to seven different specimens (three different matrices) was 100.8%. The analytical linear range of the method extends to 30 mmol of urea per liter. Of 22 potential interferents, only bilirubin at 1 mmol/L (580 mg/L), hemoglobin at 10 g/L, and hydroxyurea at 6 mmol/L showed more than 2% interference. We discuss precision and effects of specimen dilution, and compare results for 100 human serum specimens with those measured for the same specimens with four other urea methods. We examined the effects of measuring a blank, consisting of sample and reagent without urease, with each specimen.

Animals↗
PubMed1979

Relative stabilities of purified human mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase in lyophilized materials.

Eight different pools of purified human mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase were prepared, to examine the effects of the following matrix variables: the matrix support material (bovine serum albumin and polyvinylpyrrolidone), endogenous pyridoxal concentration, and azide as an antimicrobial preservation. Storage temperatures of 25 and 37 degrees C were used as a rapid and convenient means of accelerating the degradation process. Activity of the enzyme was measured with and without pyridoxal in the reaction solution. We found that the mitochondrial isoenzyme was consistently more labile than the cytoplasmic isoenzyme under identical storage conditions. Both isoenzymes were more stable in matrixes containing bovine serum albumin than in those containing polyvinylpyrrolidone. No apparent difference in the stability of either isoenzyme was observed at matrix pyridoxal concentrations of 15 micromol/L and 150 micromol/L. Only the mitochondrial isoenzyme in matrixes containing bovine serum albumin and 15 micromol of pyridoxal per liter had increased activity (about 9%) when pyridoxal was added to the enzymatic reagent. The amount of activity in reconstituted specimens did not apparently change after 72 h at 4 degrees C.

Aspartate Aminotransferases↗
PubMed1979

Column chromatography and immunoassay compared for measuring the isoenzymes of aspartate aminotransferase in serum.

We compare a column-chromatographic method and a homogeneous immunoassay method for separately measuring the mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase. Analytical recovery for the two methods averaged 102% (SD, 2%) and 103% (SD, 4%), respectively, for 11 pools prepared by adding the purified isoenzymes to serum and 102% (SD 8.9%) and 89% (SD, 8.1%) for 26 unaltered specimens of human serum. In comparing the results of the immunoassay method (y) to the chromatographic method (x), our measurements agreed closely for the mitochondrial (y = 0.947 X + 7, r = 0.9991, standard error of estimate = 2.9 U/L) and cytoplasmic (y = 0.92x-6, r = 0.9995, standard error of estimate = 2.1 U/L) isoenzymes in pools prepared from the purified isoenzymes. Similar measurements of the 26 human serum specimens yielded the following results for least-squares evaluation; cytoplasmic isoenzyme y = 1.03x-11, r = 0.994, and standard error of estimate = 6.0 U/L; mitochondrial isoenzyme y = 0.75x+0, r = 0.927, and standard error of estimate = 3.9 U/L.

Aspartate Aminotransferases↗
PubMed1979

Chemical inhibition used in a kinetic urease/glutamate dehydrogenase method for urea in serum.

We describe a fixed-time-interval, kinetic inhibition method, with use of a competitive inhibitor (l) of the urease/glutamate dehydrogenase reaction to increase the "apparent" Michaelis constant by a factor of (1 + [l]lKl). This allows greater flexibility in selecting an appropriate sample dilution for kinetic determinations of urea in serum (i.e., [S]lKm ratio). Nine compounds were screened as potential inhibitors for this study. Adding 5 mmol of hydroxyurea per liter increases the "apparent" Michaelis constant for the coupled enzyme reaction by 10-fold. We used a sample dilution of 21-fold vs. dilutions of 141- to 350-fold for previously reported kinetic methods. Mean analytical recovery with this method was 100.2%. Reaction rate vs. urea concentration was linear, and complete recovery extended to 30 mmol of urea per liter. Of 22 potential interferents, only fluoride (250 mmol/L) and bilirubin (1 mmol/L, or 580 mg/L) caused greater than 5% interference. We discuss precision and effects of specimen dilution, and compare results for 100 specimens with those by a manual Berthelot-indophenol method, a manual diacetyl monoxime method, and a diacetyl monoxime method adapted to continuous-flow analysis.

Autoanalysis↗
PubMed1978

Column-chromatographic separation of isoenzymes of aspartate aminotransferase.

We describe a column-chromatographic method for separating the mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase in human serum. Bed height of the ion exchanger, pH, and salt concentrations in the eluting buffers are shown to be variables affecting the separation of the isoenzymes. Under the optimized conditions selected for this study, a 30% increase in volume was observed in one fraction, associated with changing the salt concentration of the eluting buffer and attributed to a contraction of the DEAE-Sephadex A-50. Elution profiles (enzyme activity vs. fraction number) were examined with highly purified mitochondrial and cytoplasmic isoenzymes of human origin in bovine serum albumin and human serum. Recovery of the enzyme in the eluted fractions averaged 102% (SD, 2.0%) for specimens prepared from the purified isoenzymes and 104% (SD, 10.7%) for 38 human serum specimens. The separation technique showed linearity to catalytic concentrations in excess of 200 U/liter (reaction temperature 30 degrees C) for each isoenzyme. Additional information is presented regarding among-day precision and the effect of specimen dilution.

Aspartate Aminotransferases↗
PubMed1978

An interlaboratory study of measurement of aspartate aminotransferase activity with use of purified enzyme materials.

The Center for Disease Control (CDC), the New York State Department of Health (NYSDH), the College of American Pathologists, and 23 manufacturers of diagnostic products participated in an interlaboratory study of aspartate aminotransferase (EC 2.6.1.1) methodologies. Six different lyophilized materials were prepared and characterized and then distributed to 293 laboratories for aspartate aminotransferase measurements. The specimens included one human serum; four catalytic concentrations of the cytoplasmic isoenzyme, two purified from human erythrocytes, and two from porcine heart; and one matrix bovine serum albumin (30 g/liter) blank. The purified isoenzymes were prepared in the matrix. We present data on Michaelis parameters (Km and Vmax), Arrhenius plots, activation with pyridoxal 5-phosphate, vial-to-vial variability, and stability on reconstitution. The 281 responses showed that most of the laboratories used NADH-detection methods (91.1%), monitored at 340 nm (79.4%), and reported results in U/liter (89.4%). The percentage of laboratories reporting use of reaction temperatures of 30 and 37 degrees C was evenly divided, i.e., 42.7 and 42%, respectively. Analytical values reported by participating laboratories were categorized by reporting temperature, instrument, and method. Results were most consistent for a selected group of laboratories that supplemented optimized reaction solutions with pyridoxal 5-phosphate.

Aspartate Aminotransferases↗
PubMed1977

Temperature dependence of the absorbance of alkaline solutions of 4-nitrophenyl phosphate--a potential source of error in the measurement of alkaline phosphatase activity.

The absorbance of an alkaline solution of 4-nitrophenyl phosphate is a function of temperature. Quantitative evaluation of this phenomenon indicates that it (a) depends on the concentration of the compound and is independent of source, buffer concentration, and pH above 9.0; (b) is reversible; (c) is not a result of alkaline hydrolysis or 4-nitrophenol contamination; and (d) correlates with a temperature-induced shift of its absorbance spectrum. The phenomenon may represent a potential analytical problem in methods for alkaline phosphatase in which this compound is the substrate. If thermal equilibrium is not reached and maintained during an alkaline phosphatase assay, the thermochromic response will be included in the measured rate. The magnitude of this error depends on the thermal response and control characteristics of each particular instrument and the reaction conditions under which such an analysis is performed.

Alkaline Phosphatase↗
PubMed1976

Comparison of serum gastrin radioimmunoassay kits.

1. Two commercially available kit procedures for gastrin measurement were investigated. 2. While the precision of both kits appeared comparable, discrepancies were observed in both accuracy and parallel studies and in the comparison of kit standards and patients' samples.

Evaluation Studies as Topic↗
PubMed1976

A comparative study of micro-ammonia determinations in plasma using two different methods.

Two commercially available kits for determining blood ammonia were compared, an enzymatic method requiring a sample volume of 100mul and a nonenzymatic method requiring 1 ml of sample. Calibration curves for both kits were linear to 270 mumol/l, however correlation studies on plasma samples revealed a lack of agreement between methods (r equal to 0.825, Y equal to 0.76X +51). Improved correlation occurs when protein interference is eliminated from the enzymatic procedure. Finally, the nonenzymatic method is modified to accommodate 100 mul of sample and compared to the original method.

Ammonia↗