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R J Bondar

Publications and source records attributed to R J Bondar.

8 recordsLinked to original sources
PubMed1980

Improved separation of creatine kinase isoenzymes by use of DEAE-Sepharose Cl-6B.

We describe here a simple, rapid chromatographic procedure for quantitatively separating serum creatine kinase isoenzymes (EC 2.7.3.2), with diethylaminoethyl (DEAE)-Sepharose CL-6B as the anion-exchanger. We established the column bed height and the elution parameters by use of a simplex procedure. DEAE-Sepharose CL-6B equilibrated in tris(hydroxymethyl)aminomethane (50 mmol/L, pH 7.5, and containing 30 mmol of NaCl per liter) is packed into a miniature polystyrene column with bed dimensions of 0.7 x 1.5 cm. The DEAE-Sepharose column system was evaluated and compared with a DEAE-Sephadex A-50 column system. The results indicate that the Sepharose column yields MM, MB, and BB isoenzymes uniquely, without cross contamination. Coefficients of variation (CV's) for 10 replicate column assays were 2.8, 5.9, and 15.2% for 569 U of MM per liter, 82.3 U of MB per liter, and 9.0 U of BB per liter, respectively. The serum sample was enriched with baboon heart extract. Day-to-day reproducibility for a serum control assayed on 10 days yielded CV's of 4.6, 9.9, and 40.3% for 391, 45.3 and 1.9 U of isoenzymes MM, MB, and BB per liter, respectively. A reference interval for each isoenzymes was derived from data on 81 men and 63 women.

Animals↗
PubMed1979

Separation of five isoenzymes of serum lactate dehydrogenase by discontinuous gradient elution from a miniature ion-exchange column.

We describe a simple, fast chromatographic technique for quantitatively separating the five isoenzymes of lactate dehydrogenase (LD; EC 1.1.1.27) in serum. A 250-microL serum sample is applied to a 6.0 x 0.7 cm column of QAE-Sephadex A-50 and eluted stepwise with five different buffers. The isoenzyme fractions, assayed by the method of Wroblewski and LaDue [Proc. Soc. Exp. Biol, Med. 90, 210 (1955)], are stable at room temperature for 24 h. For all five isoenzymes the average within-day coefficient of variation is 4.3%; the day-to-day CV for 20 days is 6.3%. Of some common potentially interfering substances tested, only sodium fluoride (238 mmol/L) was found to do so, by slowing the elution from the column and making the fractions turbid. The expected range in international (IUB) united and percent for each isoenzyme was determined from data on 73 men and 70 women. From these data we calculate, by a percentile estimate of a nongaussian distribution, a normal range of LD-1/LD-2 ratio of 0.55--0.87 for men and 0.52--0.91 for women. The LD isoenzyme patterns in both normal and above-normal samples of 147 sera, as evaluated by the present column method and by electrophoresis, correlated well.

Brain↗
PubMed1975

Kinetic enzymatic method for determining serum creatinine.

Creatinine amidohydrolase is used to measure serum creatinine in a totally enzymatic procedure. Creatine, produced by hydrolysis, is acted upon by creatine kinase, and then by pyruvate kinase and lactate dehydrogenase, to result in a change in absorbance at 340 nm. The amount of creatinine present is related to the rate of change in A340 and is determined from a standard curve. Absorbance and concentration are linearly related to 100 mg/liter and only 250 mul of serum is required. At 1.0 g/liter, heparin, oxalate, citrate, ethylenediaminetetraacetate, ascorbate, or glucose had no significant effect on the accurate determination of creatinine; higher concentrations (30 g/liter) had inhibitory effects on the test. Analytical recovery of creatinine added to either normal or abnormal sera averaged 102%. When results of this procedure and of the standard direct Jaffé test were compared, the latter were significantly higher. Unlike the Jaffé method, the present method of determining creatinine is rapid (about 10 min per test), subject to few or no interfering substances, and requires no serum deproteinization.

Amidohydrolases↗