Measurement of pancreatic lipase activity in serum by a kinetic colorimetric assay using a new chromogenic substrate.

We evaluate a new assay reagent for lipase determination, based on the use of 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester (DGGR) as substrate. DGGR is cleaved by lipase, resulting in an unstable dicarbonic acid ester which is spontaneously hydrolysed to yield glutaric acid and methylresorufin, a bluish-purple chromophore with peak absorption at 580 nm. The rate of methylresorufin formation is directly proportional to the lipase activity in the sample. Bile salts, colipase and calcium chloride are included to provide optimal reactivity and specificity. Analysis of total imprecision gave a coefficient of variation of between 5.7% and 9.6%. Anticoagulants, common interfering substances and carboxylesterase had no effect on the assay, but interference by increased concentrations of serum triglycerides was noted. Good correlations were obtained with turbidimetry and a coupled enzymatic method. The estimated reference interval was 6-38 U/L. The unique characteristics of the chromogenic substrate qualify the present method as an innovative approach to serum lipase analysis.

Multicenter evaluation of five assays for myoglobin determination.

BACKGROUND: Lacking assay standardization, different myoglobin methods may produce results that differ significantly. METHODS: A multicenter study was carried out to compare the analytical performance of five commercially available assays for myoglobin measurement. Linearity, imprecision, interferences, and method comparison were studied according to NCCLS guidelines, whereas reference values were determined following IFCC recommendations. RESULTS: The BNA and Opus showed relatively high imprecision (all but one total CV >7.4%). Other assays showed lower CVs, but they varied among laboratories, particularly at a normal myoglobin concentration (Access, 6.0-11%; Hitachi, 3.8-5.8%; Stratus, 3.4-6.5%). Results were lower in anticoagulated samples on the Access, in heparin and citrate samples on the Stratus, and in citrate samples on the BNA and Opus, and increased in heparin and EDTA samples on the Hitachi. Use of separator gel produced results significantly lower (P <0.001) on the Hitachi and higher (P = 0.016) on the Opus. Bilirubin, turbidity, and hemoglobin had no effect on evaluated methods, but rheumatoid factor affected the Access. In method comparisons, high correlation coefficients (>/=0.98) were obtained. The Stratus gave higher results; however, the Access and BNA gave the lowest. The following upper reference limits (microgram/L) for men and women, respectively, were obtained: Access, 70 and 52; BNA, 51 and 49; Hitachi, 67 and 58; Opus, 80 and 50; and Stratus, 86 and 63. CONCLUSION: The possibility of high imprecision and marked disagreement among commercial myoglobin assays should be carefully considered in clinical practice.

Rapid and specific immunoassay for cardiac troponin I in the diagnosis of myocardial damage.

We evaluated a new immunometric assay for the quantitation of cardiac troponin I with the Behring Opus analyzer. All assay steps are performed automatically by the analyzer, the complete procedure requiring 20 min for the first test result. Total precision (coefficient of variation) varied between 5.6% and 13.0%. Comparison with the Pasteur immunoenzymometric assay showed good correlation (r = 0.959), but the Opus assay gave approximately tenfold higher values. Cardiac troponin I was undetectable (< 0.5 microgram/l) in sera from healthy subjects (n = 46), patients with severe skeletal muscle damage (n = 8), and all but 1 patient with chronic renal failure (n = 39). In patients with myocardial infarction (n = 21), investigated for 4 days after onset by frequent sampling, cardiac troponin I peaked at 20.8 +/- 8.1 h after onset with a mean concentration of 164.8 +/- 361.3 micrograms/l, remaining elevated in 89% of patients on the 4th day. In patients with unstable angina (n = 15), cardiac troponin I measurement was valuable in predicting the type of lesion morphology on coronary angiography and the short-term outcome.

Determination of glycated apolipoprotein B in serum by a combination of affinity chromatography and immunonephelometry.

We have developed and optimized a procedure for the quantitation of non-enzymatically glycated apolipoprotein B (apo B). Glycated and non-glycated apo B were separated from serum using m-aminophenylboronate affinity chromatography, determined by immunophelometry and the percentage of glycated apo B was calculated. The measuring range of the assay was 2.9-185 mg/dL apo B. The within- and between-run coefficients of variation were < 7.4% and 14.6%, respectively, and recovery was > 98%. Free glucose in serum did not affect the results at concentrations below 25 mmol/L. In 45 non-diabetic subjects the mean concentration of glycated apo B was 4.3% (SD 1%). In type 1 (n = 17) and Type 2 (n = 60) diabetic patients the mean glycated apo B concentrations were 5.3% (SD 0.7%) and 5.9% (SD 1.1%), respectively, significantly higher than in controls (P < 0.001).

An immunoinhibition assay for determination of creatine kinase isoforms in serum.

We report a preliminary evaluation of an immunoinhibition assay for creatine kinase isoform quantification. The procedure employs the monoclonal antibody CKM-G01, which inhibits the native M subunit of creatine kinase. The antibody does not inhibit the M subunit modified by removal of lysine by plasma carboxypeptidase N. Residual activity after treatment with the antibody is therefore due to serum delysinated isoforms. The ratio inhibited/residual activity correlated directly with the ratio tissue/serum isoforms. Analysis of the total imprecision of isoform ratio measurement gave a coefficient of variation between 5.9 and 21.1%. Reference intervals for the ratio were 0.14-0.79 in females and 0.19-0.95 in men (p = 0.0046). Analytical and clinical comparison with alternative isoform procedures gave good results, showing that this assay can be used as alternative to the widely accepted electrophoretic method for measurement of the creatine kinase isoform ratio.

Clinical and analytical evaluation of a continuous enzymatic method for measuring pancreatic lipase activity.

We report the evaluation of a new commercial kit for the determination of pancreatic lipase activity. The kit is based on the use of a 1,2-diglyceride as substrate and a specific monoglyceride lipase. The detection step is the continuous colorimetric measurement of hydrogen peroxide produced from glycerol by glycerol kinase, glycerol-3-phosphate oxidase, and peroxidase reactions. The procedure appears to be precise (between-day CV < 9%) and the results show good correlation with those obtained by alternative procedures (vs turbidimetry, r = 0.965; vs ultraviolet absorbance-enzymatic method, r = 0.995; vs Ektachem, r = 0.976; vs immunometry, r = 0.970). However, the method is susceptible to interference by increased concentrations (> 4.5 mmol/L) of serum triglycerides. We estimated the reference interval for healthy adults to be 8-44 U/L. When we evaluated clinical efficacy by using receiver-operating characteristic curves and the overlap index, no significant differences were found between the commercial kit and a common turbidimetric assay for diagnosing patients with acute pancreatitis; both methods performed satisfactorily.

Pre-analytical and biological variability of prostatic acid phosphatase and prostate-specific antigen in serum from patients with prostatic pathology.

We determined the pre-analytical and biological variation of prostatic acid phosphatase and prostate-specific antigen in the same patient samples. Prostatic acid phosphatase and prostate-specific antigen were both stable when stored for at least 3 weeks with acidification (acetate buffer) or without acidification, except for prostate-specific antigen in samples stored unacidified at 4 degrees C. A significant elevation of prostate-specific antigen was noted in four patients with benign prostatic hyperplasia between 1/2 and 6 hours after prostatic massage. No significant effect was shown of changes in the glomerular filtration rate on prostate-specific antigen concentration, in spite of its low molecular mass. The estimate of within-subject biological variation showed a coefficient of variation of 33.8% for prostatic acid phosphatase and 14% for prostate-specific antigen. Desirable analytical imprecisions based on these findings were about 17% for prostatic acid phosphatase and 7% for prostate-specific antigen, these goals being achieved in practice for marker values higher than or equal to the upper reference limit.

Diagnostic value of four assays for lipase determination in serum: a comparative reevaluation.

We assessed the diagnostic value of four commercially available methods for determining pancreatic lipase (LPS) in serum (the turbidimetric procedure from Boehringer, two enzymatic approaches from Kodak and Poli, and an immunochemical assay) in a population of 46 hospitalized patients with acute abdominal pain. In 31 cases (67.4%), the final diagnosis was acute pancreatitis. When evaluated by means of receiver-operating characteristic (ROC) curves, no significant differences were found among the procedures. Concerning clinical efficiency, all the assays had values equal to or greater than 90%. Using the calculation of the overlap index (OI) as a statistical approach to quantify the clinical utility of various LPS assays, the test having the greatest potential for differentiating between patients with and without acute pancreatitis was the turbidimetric assay (OI = 0.14).

Diagnostic value of prostatic acid phosphatase and prostate-specific antigen in patients with prostatic cancer.

To compare the clinical usefulness of the measurement of prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in serum of patients with prostatic carcinoma, we studied 128 patients with prostatic pathology, sixty (46.9%) of whom had prostatic cancer. Receiver-operating characteristics (ROC) curves were constructed and the underlying areas were calculated and compared to study clinical efficiency of the two markers regardless of the cutoff level selected. The area for PSA (0.90 +/- 0.30) was significantly higher (p less than 0.001) than that of PAP (0.71 +/- 0.05) showing that PSA was a better discriminator of the patients with or without prostatic cancer. The maximal clinical efficiency of the two tests at selected cutoff levels (0.8 U/L for PAP and 10 micrograms/L for PSA) was 0.787 and 0.883, respectively, confirming the superiority of PSA. However, the associated determination of the two markers improved the clinical specificity with no false-positive cases.

Evaluation of a new commercial assay kit for quantification of lactate dehydrogenase isoenzyme 1 in serum.

A new method for measuring lactate dehydrogenase (EC 1.1.1.27) isoenzyme 1 using sodium perchlorate as a chaotropic chemical selective inhibitor of all lactate dehydrogenase isoenzymes containing M-subunit was evaluated. Results with this method were precise (between-day coefficient of variation less than 9%), highly linear (coefficient of correlation = 0.9998) and correlated well with lactate dehydrogenase isoenzyme 1 as determined by the conventional immunological method (coefficient of correlation = 0.976). The reference interval for 307 healthy subjects was estimated to be 23-46 U/l (95% central range, determined non-parametrically). Being simple, convenient and amenable to automation, the method provides a substantial saving in labour and reagent costs when compared with alternative analytical approaches.

An immunochemical procedure for determination of creatine kinase 3(1) (serum-specific) isoform in human serum evaluated.

Changes in the proportions of individual isoforms of creatine kinase (CK) in serum promptly reflect both myocardial infarction and coronary reperfusion. A new commercial kit has been introduced for measuring CK-3(1) isoform in serum (ISOFOR-MM, International Immunoassay Labs.). This is an immunochemical assay containing CK-3(1) specific monoclonal antibody, bound to magnetizable particles, used to immunoextract this isoform. The CK activity of the sample is measured before and after immunoextraction and the difference in the two values gives the measure of CK-3(1). Extraction of CK-3(1) was complete at less than or equal to 1200 U/L. Analysis of between-day imprecision gave CV between 2.9-7.9%. The method was not susceptible to interference by CK-3(2) and CK-3(3) isoforms, CK-2 isoenzyme, or mitochondrial CK. Reference interval for CK-3(1) (expressed as percent of total CK-3) was 42-69%. Correlation between percent CK-3(1) by isoform electrophoresis (x) and evaluated procedure (y) was y = 0.83x + 7.6, with r = 0.957 (n = 40). The ISOFOR-MM performed well enough in this evaluation to replace electrophoresis or isoelectric focusing for measurement of CK-3(1) isoform.

A two-tube immunochemical method for determination of CK-MB isoenzyme in serum evaluated.

A new commercial kit (Impres-MB; International Immunoassay Labs.) recently was introduced for measuring the MB isoenzyme of creatine kinase (CK-MB) based on the use of monoclonal antibodies. After antibodies to CK-MM isoenzyme are added to precipitate the CK-MM, antibodies to CK-M monomer are added to precipitate the M-subunit isoenzymes of CK. Subtracting the enzymatic activity of the second supernate from the residual activity in the first yields the activity of CK-MB. Results are not affected by CK-BB, mitochondrial CK, or adenylate kinase. However, the anti-CK-MM antibodies precipitated only about 98% of serum CK-MM and may have partly precipitated CK-MB isoenzyme (average analytical recovery of CK-MB, 86.6%). Comparison between Impres-MB (y) and electrophoresis (x) yielded the following linear-regression equation: y = 0.79x + 3 (r = 0.982, n = 97). Data for CK-MB temporal kinetics, obtained from patients with myocardial infarction, correlated significantly in both methods; however, peak activity values of CK-MB were significantly different, confirming that the difference between the new method and the electrophoretic method average 20%.