Within-run CV Research Articles

A new endogenous immunoenzyme assay for prostatic acid phosphatase evaluated.

I evaluated the performance of the "ORTHO PAP-IA," an endogenous immunoenzyme assay for prostatic acid phosphatase. The procedure is based on double-antibody precipitation of the antigen, followed by quantification of its enzymic activity. The antibodies are in large excess, to speed the reactions and minimize sensitivity to variations in assay conditions. Enzymic activity is measured via an extremely sensitive colorimetric reaction, the analytical sensitivity of which exceeds that of radioimmunoassay. Absorbance is linearly related to activity concentration up to an absorbance of 4.0, and only a single calibration standard is required. Within-run CV was less than 2%, between-run CV about 4%. Neither individual blanks nor assay in duplicate is required. All reagents, including the enzyme standard, are stable solutions. Results correlated well with those by a standard radioimmunoassay (r = 0.993, n = 38).

Einfache und rasche Bestimmung von Thiopental im Serum mit HPLC

An HPLC method is described for the determination of thiopental in serum. It offers the advantage of a simple extraction procedure and a low detection limit. Including sample preparation, the analysis takes 20 minutes. The within-run and between-run CV's were 1.5% and 3.8% respectively.

Zeeman effect electrothermal atomic absorption spectrophotometry with matrix modification for the determination of arsenic in urine.

Arsenic was determined in urine by electrothermal atomic absorption spectrophotometry using Zeeman Effect background correction and after matrix modification with 5% nickel nitrate. Mean recovery of urine samples spiked with 100 micrograms . L-1 dimethylarsinic acid was 97.9 micrograms . L-1. Within-run CV was 2.4%, between-run CV was 7.3% and the detection limit was determined to be 10.0 micrograms . L-1. The method can be used for the rapid screening of urine samples for elevated arsenic levels.

Quantification of urinary oxalate with oxalate oxidase from beet stems.

We describe an automated (ABA-100) enzymic method for determination of urinary oxalate by use of oxalate oxidase (EC 1.2.3.4) isolated from beet stems. The H2O2 produced by the oxidation of oxalate by oxalate oxidase is measured by coupling with oxidation and conjugation of 3-methyl-3-benzothiazolinone hydrazone with N,N-dimethylaniline with catalysis by horseradish peroxidase. The resulting indamine dye is measured spectrophotometrically by the difference in absorption at 500 and 600 nm. Interfering substances are removed by oxidation with acidic ferric chloride and by cation-exchange chromatography. The assay is sensitive to 5 mg of urinary oxalate per liter, the standard curve is linear to 70 mg/L, and the procedure requires less than 3 h for completion. The within-run CV was less than 1.6%, the between-day CV less than 5.6%. The oxalate oxidase method results in a mean and reference interval for oxalate excretion that are comparable with those by isotope dilution, gas-chromatographic, colorimetric, and other enzymic procedures.

Evaluation of a modified colorimetric assay for the determination of acetaminophen in serum.

An evaluation of a new colorimetric assay (SIGMA-430) for the rapid determination of acetaminophen in serum or plasma was performed. Acetaminophen was reacted with nitrous acid to form a 2-nitro-5-acetamidophenol derivative, which was measured colorimetrically in an alkaline medium. The assay was linear over a concentration range of 50-500 mg/L. Within-run and between-run CV's with a 300 mg/L sample were 1.51% (n = 12) and 3.00% (n = 20), respectively. Mean analytical recovery of acetaminophen added to plasma at 50-600 mg/L was 102.0%. Seventeen commonly prescribed weakly acidic or neutral drugs were also analyzed by the method with no significant interferences. A comparison of 21 consecutive serum samples with the colorimetric assay and an HPLC method yielded a correlation coefficient of 0.989. Colorimetric values were statistically higher.

Improved turbidimetric assay for lysozyme in urine.

I describe an improved turbidimetric assay for lysozyme in urine. This method is simple, reproducible, linear over a 100-fold concentration range, sensitive to concentrations as low as 10 micrograms/L, and only commercially available products are required. Evaluation of urinary lysozyme concentration with this assay demonstrated a within-run and between-run CV of 9.9% and 4.8%, respectively, for normal urine, minimal chemical interference, and a mean analytical recovery of 104.8%. There were differences in the rate of clearing and in the linearity of the assay with use of three commercial preparations of Micrococcus lysodeikticus, and significant differences in results when different lysozyme standards were used. Reference values were established by use of data on 169 healthy subjects, ages six months to 61 years. Clinical efficacy of the assay was demonstrated in children with renal disorders, especially those associated with chronic renal failure or tubular dysfunction.

Dipalmitoylphosphatidylcholine in amniotic fluid quantified by fast-atom-bombardment mass spectrometry.

Dipalmitoylphosphatidylcholine (DPC) is quantified by taking advantage of stable-isotope-labeled d9-DPC as internal standard. Use of a mass spectrometer to measure the ratio of d9/d0 makes this procedure a quantitative one. d9-DPC was synthesized by refluxing dipalmitoylethanolamine with d3-methyl iodide in methanol in the presence of sodium bicarbonate for 26 h. The yield of d9-phosphatidylcholine (d9-lecithin) was 89% after column-chromatographic purification. Fast atom bombardment was used to desorb the preformed phosphatidylcholine ions in a mass spectrometer of Nier-Johnson geometry. In our assessment of accuracy and precision of this technique, we found a correlation coefficient of 0.9994 between signal and sample concentration. The method was less precise when the total d0- plus d9-DPC was less than 0.2 micrograms or when the ratio of d0- to d9-lecithin exceeded 100. The within-run CV was about 1.0%. The amount of DPC in amniotic fluid samples assessed by mass spectrometry was compared with results for total phosphatidylcholine quantified by thin-layer chromatography. The fate of DPC in various laboratory manipulations was also studied.

"Sandwich" enzyme immunoassay for serum ferritin with polypropylene test tubes as the solid phase.

A "sandwich" enzyme immunoassay for ferritin in human serum is described, in which horseradish peroxidase-labeled antibody and a very sensitive chromogen, 2,2'-azino-di(3-ethyl-benzthiazolin-6-sulfonate), are used. The solid phase is polypropylene test tubes treated with glutaraldehyde; immunologic reactions are developed during constant rotation of the tubes. The assay requires 20 microL of serum per assay and takes less than 3 h. The standard curve is linear between 0 and 400 micrograms of serum ferritin per liter. Analytical recoveries of various amounts of standard added to a serum sample ranged from 92 to 98.6%. The sensitivity is 2 micrograms/L, which translates to 0.4 ng per assay tube. Within-run CV ranges from 1.6 to 5.4% and between-run from 4.2 to 7.8% according to the ferritin concentration. Results by competitive radioimmunoassay correlated well (r = 0.98). No "hook effect" was observed for concentrations up to 13 mg/L. The reference interval is 40-240 micrograms/L for men, 8-100 micrograms/L for women.

Determination of glycosylated hemoglobin by affinity chromatography: comparison with colorimetric and ion-exchange methods, and effects of common interferences.

An affinity-chromatographic method for determination of glycosylated hemoglobin (Anal. Lett. 14: 649-661, 1981) is compared with the thiobarbituric acid colorimetric (I) (Clin. Chem. 27: 669-672, 1981) and the ion-exchange liquid-chromatographic (II) (Diabetes 29: 623-628, 1980) methods. A correlation of 0.98 was obtained for the affinity method vs II and 0.97 for affinity vs I (n = 51). The within-run CV was 1.9% for specimens from non-diabetic individuals and 1.0% for those from diabetics. The respective between-run CVs were 3.4% and 2.4%. Failure to remove "labile" glucose adducts by 5-h incubation of erythrocytes in isotonic saline (37 degrees C) contributed an average error of 13.1% for II, 5.4% for I, and 1.6% for the affinity method. Affinity chromatography gave a decrease of 0.1-0.2% glycosylated hemoglobin for each 1.0 degree C temperature increase between 18 and 27 degrees C. Varying the pH of the wash buffer used in the affinity procedure from 7.75 to 8.25 (pH 8.0 optimum) produced at net change of 0.5% in glycosylated hemoglobin with one diabetic specimen. Using the affinity method, we determined the reference interval for glycosylated hemoglobin in 124 apparently healthy individuals to be 5.3 to 7.5% (mean 6.36%, SD 0.55%). Rechromatography by II and isoelectric focusing analysis of the fractions obtained by the affinity separation revealed a substantial population of glycosylated hemoglobins not measured by II. The affinity method offers a rapid, simple, precise, and accurate alternative to methods currently in use and gives substantial freedom from many common interferences.

Liquid-chromatographic determination of pentobarbital in plasma with use of a resin column and an alkaline mobile phase.

We describe a liquid-chromatographic method involving a new, nonsilica column (XAD-2, Hamilton Co.) for pentobarbital in plasma. Plasma is extracted with chloroform after addition of the internal standard, 5-ethyl-5-p-tolyl-barbituric acid. Acidic drugs are back-extracted into alkali, then chromatographed on the resin-base reversed-phase column. The use of alkaline mobile phase allows enhanced sensitivity and detection of barbiturates at 240 nm. The within-run CV for 10 samples was 1.9%, the between-run CV 1.8%. Ten commonly used barbiturates are separated isocratically in less than 15 min. Other commonly prescribed acidic drugs do not interfere with determination of pentobarbital.

Simplified fluorometry of total metanephrines in urine.

We describe a simplified fluorometric method for quantitatively determining metanephrines in urine. The method is based on the elution of metanephrines from an ion-exchange column followed by production of a fluorescent derivative that has excitation and emission maxima of 405 nm and 505 nm, respectively. Metanephrine concentrations as great as 7.0 mg/L were within the linear range of the assay. Analytical recovery of metanephrines was 82-87%. Cross contamination by catecholamines was insignificant. The upper range of normal was 520 micrograms/24 h. Within-run and between-run CV's were 5% and about 9%, respectively. Twenty-four-hour urine specimens from five patients with pheochromocytoma showed above-normal amounts of the metanephrines by this method.

Standardization of serum cholesterol assays by use of serum calibrators and direct addition of Liebermann-Burchard reagent.

Serum cholesterol concentrations of subjects in epidemiological studies were measured after direct addition of Liebermann-Burchard reagent; results were calibrated with human serum pools assayed according to Abell et al. (J. Biol. Chem. 195:357-366, 1952). Accuracy and precision were monitored for six years by analysis of internal-control pools and blind external-control pools. For various internal-control pools, the imprecision (CV) of the long-term averages of run means ranged from 0.5 to 0.9%. The within-run CV for internal control and patients' sera was about 1%. For blind control sera with different concentrations (provided by the Centers for Disease Control, Atlanta, GA, over the same period), the average difference per three-month period between the values found and the target values was usually between -0.5% and +0.7% for medium-concentration pools and between -2% and +2% for low- and high-concentration pools (extreme values: -2.4% and +2.5%). The CV per three-month period ranged from 0.6 to 2.7%. Sera from subjects on diets of high or low linoleic acid content were analyzed to study the effect of the fatty acid portion of serum cholesterol esters; the differences between values obtained with the comparison method and the direct method was insignificant on both diets. We conclude that the use of serum calibrators eliminates the bias inherent in the direct method.

Direct determination of the linoleate/oleate ratio in serum cholesterol esters by liquid chromatography.

We describe a convenient method for the direct determination of the serum cholesterol linoleate/cholesterol oleate (L/O) ratio by reversed-phase "high-performance" liquid chromatography. After removal of phospholipids by silicic acid chromatography, a serum extract is analyzed on a 5-micrometers particle size Ultrasphere-ODS column, eluted isocratically with acetonitrile/isopropanol (30/70, by vol). Detection is at 200 nm. Cholesterol palmitoleate interferes with the measurement when the analysis is based on peak area, but not when peak height is used. The overall precision of L/O measurements by this method was very similar to that observed with a gas-liquid chromatographic procedure, in which the cholesterol esters are first isolated and transesterified to the methyl esters. In both cases, the within-run CV for six replicate analyses was less than 2%. Analysis of 53 human serum samples by both methods yielded very similar L/O ratios. A plot of the data (our method = y) vs the usual gas-liquid chromatographic procedure gave a correlation coefficient of 0.988 and a regression equation of y = 1.03x + 0.013. Furthermore, direct analysis of serum cholesterol ester L/O ratios by our liquid-chromatographic method is simpler, quicker, and more readily adaptable to automation.

Automated analysis for plasma epinephrine and norepinephrine by liquid chromatography, including a sample cleanup procedure.

We describe a fully automated method of analysis for plasma epinephrine and norepinephrine by use of a two-column system of "high-performance" liquid chromatography. Catecholamines in deproteinized plasma are purified on the first (preparation) column, then transferred automatically to the second (analytical) column in which epinephrine and norepinephrine are resolved. These compounds are then determined fluorometrically with a continuous-flow reaction system by the trihydroxyindole method. The minimum sensitivity of the method is 0.02 and 0.04 pmol for epinephrine and norepinephrine, respectively. One assay can be completed in 30 min, and greater than 1 mL of plasma is required for the procedure. The within-run CV in the chromatographic determination of pooled plasma was greater than 3%, and the analytical overall recovery of the two compounds was 95%. The concentrations in plasma of medical students at rest were 0.32 (SE 0.06) nmol/L for epinephrine and 0.98 (SE 0.08) nmol/L for norepinephrine.

Colorimetry of angiotensin-I converting enzyme activity in serum.

This simple, accurate, and reproducible colorimetric method for determining the activity of angiotensin-I converting enzyme is based upon colorimetry of the quinoneimine dye produced from the substrate p-hydroxyhippuryl-L-histidyl-L-leucine by action of this enzyme through the following series of reactions. The enzyme acts on the substrate to yield p-hydroxyhippuric acid and L-histidyl-L-leucine. The former is then hydrolyzed in the presence of hippuricase to produce p-hydroxybenzoic acid and glycine. Finally, oxidative coupling of p-hydroxybenzoic acid with 4-aminoantipyrine is catalyzed by peroxidase in the presence of hydrogen peroxide, producing a quinoneimine dye, the concentration of which is measured at its absorbance maximum at 505 nm to evaluate the activity of ACE. The Km value for the above-mentioned substrate is 0.32 mmol/L, the optimum pH is 8.3. Results by the present method and Cushman and Cheung's method (Biochem. Pharmacol. 20: 1637, 1971) correlate closely (r = 0.986). The within-run CV is 2.93%.

Automated determination of copper in undiluted serum by atomic absorption spectroscopy.

An injection method has been adapted for the determination of copper concentration in untreated, undiluted serum by flame atomic absorption spectroscopy. Serum, 50 or 100 microliter, is automatically injected by a commercial microprobe system into a plastic cone connected to the capillary tube of the burner, at a rate of 240 samples per hour. The required sample volume is considerably decreased, and sensitivity is increased 20- to 40-fold. After 500 measurements we observed no memory effects, carryover, or clogging of the burner. We discuss common difficulties with calibration standards due to viscosity and other physicochemical interferences, and suggest the use of pooled human serum as a secondary standard. Within-run CV was 1.8%, the day-to-day CV 2.2%. Comparison with a dilution method gave a correlation coefficient exceeding 0.98.

Homogeneous substrate-labeled fluorescent immunoassay for theophylline in serum.

A substrate-labeled fluorescent immunoassay for theophylline in serum is described. 8-(3-Aminopropyl)-theophylline is covalently attached to a fluorogenic enzyme substrate, 7-beta-galactosylcoumarin-3-carboxylic acid. Hydrolysis of this theophylline-labeled substrate by beta-galactosidase yields a fluorescent product. When antibody to theophylline interacts with this substrate, the resulting complex is inactive as an enzyme substrate. For measuring theophylline, competitive protein-binding reactions are set up, with the theophylline in the sample competing with the substrate for the antibody-binding sites. The substrate not bound to antibody is hydrolyzed by beta-galactosidase, producing fluorescence that is proportional to the theophylline concentration. Results for theophylline determined by this method in clinical samples of serum correlated well (r > 0.96) with results obtained by gas-chromatographic or enzyme immunoassay procedures. The within-run CV for three control samples ranged from 1.1 to 2.8%, the between-run CB from 2.3 to 4.5%.

Automated creatine kinase-MB estimation by immuno-inhibition: a clinical evaluation.

We detected the presence of creatine kinase (EC 2.7.3.2) MB isoenzyme (CK-MB) in serum by using an immunoinhibition technique to inactivate the creatine kinase isoenzyme of muscle origin (CK-MM). The GEMSAEC centrifugal analyzer is ideally suited for this procedure because it rapidly mixes reagents and has a throughput of 60 samples/h. The within-run CV was acceptable when the CK-MB value was 7.5 U/L or more. CK-MM was totally inactivated up to 5000 U/L, a value well above that usually seen in myocardial-infarct patients. We assessed the predictability of the assay for detecting myocardial infarction among 120 coronary-care patients when 2% of total CK activity was considered diagnostic and compared the results with those obtained with an electrophoresis technique. The positive predictive value of the immunoinhibition assay was 97.8%, as compared to 92% with the CK electrophoresis technique, when lactate dehydrogenase (EC 1.1.1.27) isoenzyme results were included in both cases. The optimal negative predictive value was 95.1% with the immunoinhibition assay vs 98.2% with electrophoresis. We conclude that the immunoinhibition technique for estimating CK-MB can be automated to assess myocardial status rapidly, precisely, inexpensively, and sensitively.

Automated creatine kinase-MB estimation by immuno-inhibition: a clinical evaluation.

Abstract We detected the presence of creatine kinase (EC 2.7.3.2) MB isoenzyme (CK-MB) in serum by using an immunoinhibition technique to inactivate the creatine kinase isoenzyme of muscle origin (CK-MM). The GEMSAEC centrifugal analyzer is ideally suited for this procedure because it rapidly mixes reagents and has a throughput of 60 samples/h. The within-run CV was acceptable when the CK-MB value was 7.5 U/L or more. CK-MM was totally inactivated up to 5000 U/L, a value well above that usually seen in myocardial-infarct patients. We assessed the predictability of the assay for detecting myocardial infarction among 120 coronary-care patients when 2% of total CK activity was considered diagnostic and compared the results with those obtained with an electrophoresis technique. The positive predictive value of the immunoinhibition assay was 97.8%, as compared to 92% with the CK electrophoresis technique, when lactate dehydrogenase (EC 1.1.1.27) isoenzyme results were included in both cases. The optimal negative predictive value was 95.1% with the immunoinhibition assay vs 98.2% with electrophoresis. We conclude that the immunoinhibition technique for estimating CK-MB can be automated to assess myocardial status rapidly, precisely, inexpensively, and sensitively.

Continuous-flow fluorometry of low galactose concentrations in blood or plasma.

Clearance of 0-100 mg/L concentrations of galactose from the blood depends on nutrient hepatic blood flow. We can measure such concentrations, which was not previously possible, by a continuous-flow method involving the use of galactose oxidase and peroxidase, the latter being coupled to a fluorogenic substrate, p-hydroxyphenylacetic acid. Interfering substances in the peroxidase reaction are removed by zinc/alkali precipitation. Sensitivity is maximized by using saturating concentrations of the enzymes and substrate. In prepared plasma test samples with galactose concentrations of 10, 40, 70, and 100 mg/L, the within-run CV's ranged from 2.1 to 8.6%, and day-to-day CV's from 2.2 to 17.2%, the largest CV's being for the 10 mg/L concentration. Normal subjects are shown to clear galactose more efficiently than subjects with moderate cirrhosis.