bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasma renin activity and plasma prorenin assays
Authors: Sealey J E
Published: January 2020

Sensitivity and accuracy are essential features of an assay of plasma renin activity (PRA) because the normal concentration of PRA is only 1 pmol/L, and subnormal concentrations have diagnostic relevance. Conditions for blood collection need to be standardized but the conditions are not difficult for outpatients. For routine diagnostic purposes blood should be collected from ambulatory (ideally, untreated) patients on moderate sodium intake. To avoid irreversible cryoactivation of plasma prorenin (which is present in 10-fold greater concentrations than renin), samples should be processed at room temperature and stored completely frozen. Cryoactivation occurs when plasma is liquid at temperatures less than 6 degrees C. PRA is commonly measured with an enzyme kinetic assay in which angiotensin I (Ang I) is formed by the reaction of plasma renin with endogenous renin substrate (angiotensinogen). The Ang I so formed is measured by RIA; results are expressed as an hourly rate (micrograms/L formed per hour). This method, which is provided by most commercial kits, has the potential for unlimited sensitivity because the step for Ang I generation can be prolonged as long as necessary, so that enough Ang I forms to be measured accurately. Unfortunately, that sensitivity is not always exploited. Dilution of plasma during pH adjustment should be kept to a minimum. The Ang I generation step should last at least 3 h. The step should last 18 h for samples with PRA less than 1.0 micrograms/L per hour, to eliminate the errors inherent in the measurement and subtraction of immunoreactive Ang I in the untreated plasma (blank subtraction). These changes actually simplify PRA measurements because they eliminate the need for ice in the clinic and reduce by almost half the number of samples to be assayed by RIA. I also describe the method for measurement of plasma prorenin, which may be an important marker for patients with diabetes mellitus who subsequently develop vascular complications.
Abbreviated kinetic profiles in area-under-the-curve monitoring of cyclosporine therapy
Authors: Grevel J , Kahan B D
Published: January 2020

Abbreviated kinetic profiles can reduce the number of phlebotomies and drug assays, and thereby the cost of area-under-the-curve (AUC) monitoring. In the present investigation, we used two independent data sets: group 1, 101 AUC profiles from 77 stable renal-transplant patients, which included a 5-h sample in addition to the usual 0-, 2-, 4-, 6-, 10-, 14-, and 24-h samples; and group 2, 100 profiles from 50 stable renal-transplant patients before and after a change in their daily oral dose of cyclosporine. Group I demonstrated a fair correlation between cyclosporine trough concentrations and the AUC calculated from a complete set of seven concentrations (r2 = 0.820 and 0.758 for the 24- and 0-h samples, respectively). Stepwise multiple linear-regression analysis revealed that the abbreviated set of three time points (2, 6, and 14 h) explained 96% of the variance in AUC values calculated from the full set of seven samples; additional time points increased the accuracy only slightly. For group 2, we examined the difference between the observed and the predicted concentrations by linear extrapolation; the error in the observed AUC value, compared with the predicted value calculated from seven time points (-13.2% to -1.2%), was similar to the error from just three time points (-11.5% to 4.5%). Abbreviated AUC profiles involving three time points used with a model equation seem to provide a reliable alternative to full seven-point profiles.
New method for isolating and quantifying intermediate and beta-very-low-density lipoprotein cholesterol
Authors: Wikinski R L , Schreier L E , Rosental S B
Published: January 2020

We describe a new method, useful to clinical laboratories, for assessing intermediate density (IDL) or beta-very-low-density (beta-VLDL) lipoprotein cholesterol. The technique involves selective precipitation properties of the qualitative Wieland and Seidel post-electrophoretic method that immobilizes IDL and beta-VLDL in the beta-zone of an agarose slide (Clin Chem 1973;19:1139-41). In our method, we separate low-density lipoprotein (LDL) in a second electrophoretic step, in which LDL moves toward the anode, and then quantify the cholesterol of the above lipoproteins remaining in the precipitate band at the beta-zone. Replicate within-run precision (CV) of 15 aliquots of a sera pool was 10.1%. The correlation with sequential ultracentrifugation of 30 samples was r = 0.96 (P less than 0.001). Serum reference values for 30 normal individuals are 57 +/- 7.0 mg/L. Seven phenotype III hyperlipoproteinemic patients had the highest concentrations of IDL or beta-VLDL cholesterol in serum, 1620 +/- 346 mg/L.
Use of immunoglobulin heavy-chain and light-chain measurements in a multicenter trial to investigate monoclonal components: I. Detection
Authors: Jones R G , Aguzzi F , Bienvenu J , Bianchi P , Gasparro C , Bergami M R , Perinet A , Bernon H , Penn G M , Keller I
Published: January 2020

We assessed the combined use of serum protein electrophoresis (SPE) and nephelometric measurement of immunoglobulin heavy- and light-chain components for detecting serum monoclonal immunoglobulins (monoclonal components, MC) in 4788 unselected samples from 4173 patients. MC were detected in 514 samples from 390 patients. In 356 these were detected by SPE; the other 34 had a normal SPE pattern but an abnormal kappa:lambda light-chain ratio (KLR). Only 208 of the 356 (58%) samples with bands by SPE had abnormal KLRs. Samples with MC concentrations greater than 5 g/L had a higher proportion of abnormal KLRs (75%) than those with concentrations less than 5 g/L (42%). The KLR was abnormal in 13% of samples in which no MC were visible by SPE or immunofixation electrophoresis (IFE). Compared with quantitative measurements of immunoglobulin heavy and light chains, high-quality SPE remains the method of choice for the detection of MC. Quantitative methods, however, are able to detect additional MC, especially those containing free light chains, and in the absence of SPE and IFE will detect about 75% of MC present at greater than 5 g/L.
Efficient extraction of basic, neutral, and weakly acidic drugs from plasma for analysis by gas chromatography-mass spectrometry
Authors: Brooks K E , Smith N B
Published: January 2020

We describe a method for efficiently extracting basic, neutral, and weakly acidic drugs from plasma for toxicological analysis by gas chromatography-mass spectrometry (GC/MS). The 2-mL plasma sample is diluted with an equal volume of saturated NaCl containing triethylamine, 10 mmol/L, and then extracted twice with 4 mL of an equivolume solution of dichloromethane/acetone. The organic (top) phases are combined, then mixed with 1 mL of water, 200 mg of NaHCO3, and 100 microliters of acetic anhydride. This mixture is then heated at 75 degrees C until the solvents have boiled off and aqueous acetylation is complete (less than 30 min). After addition of 1 mL of water and 2 g of NaCl, the sample is extracted twice with 2 mL of dichloromethane/acetone (2/1 by vol). The combined extracts are dried and then subjected to thin-layer chromatography on a blank Toxi-Lab Toxi-A chromatogram with 1-chlorobutane as the developing solvent (about 6 min). After the lipids have migrated with the mobile phase, the drugs are eluted from the origin with acetone/triethylamine (29/1 by vol), evaporated, and reconstituted in injection solvent. With this procedure drugs are recovered relatively quickly (less than 2 h) and the GC/MS total ion chromatograms are very clean. Studies with 13 basic, neutral, and weakly acidic drugs showed that all except theophylline were extracted with recoveries of at least 75%.
High-performance liquid-chromatographic method compared with a modified radioimmunoassay of cotinine in plasma
Authors: Perkins S L , Livesey J F , Escares E A , Belcher J M , Dudley D K
Published: January 2020

Cotinine is a sensitive and specific biochemical marker of exposure to cigarette smoke. We describe a simple solid-phase extraction of cotinine from plasma before quantification by HPLC. Extraction recovery was 97.9% +/- 11.0% for plasma concentrations of 5-400 micrograms/L. Baseline separation of cotinine and caffeine was achieved within 11 min of injection onto a C18 reversed-phase column. The mobile phase was citric acid/dibasic potassium phosphate (30 mmol/L each, pH 6.0) containing 100 mL of acetonitrile per liter. Within-day and day-to-day precision (CV) were 4.7% and 8.4%, respectively. We also describe a modification of the Nicotine Metabolite RIA kit (Diagnostic Products Corp.) for quantifying cotinine in plasma. Recovery of cotinine from supplemented plasma was within 10% of the expected value with this RIA kit. Interassay precision averaged 8.1% for samples in the range 50-400 micrograms/L; intra-assay precision averaged 3.6% at 230 micrograms/L and 8.7% at 53 micrograms/L. Correlation between the two methods was RIA = 1.13 HPLC + 14.8 (n = 128, r = 0.957, P less than 0.001). Both methods are technically simple to perform.
Determination of intact parathyrin by immunoradiometric assay evaluated in normal children and in patients with various disorders of calcium metabolism
Authors: Saggese G , Baroncelli G I , Bertelloni S
Published: January 2020

We report the reference values for intact parathyrin (PTH) measured by a two-site immunoradiometric assay (IRMA) during childhood. The study has been carried out in 215 healthy children and adolescents, ages 2.0 to 18.7 years. Some patients with altered mineral homeostasis were also studied to assess the sensitivity of the method in a clinical setting. Mean intact PTH concentrations were 30.8 (SD 9.6) ng/L; the median was 28.5 ng/L. Normal reference values were 16.0-59.0 ng/L (95% confidence interval). The distribution of intact PTH values was nongaussian. We found no significant variations between males and females and no age-related variations. The IRMA used was sufficiently sensitive to detect differences in PTH concentrations between healthy children and patients with hypocalcemia or hypercalcemia.
Serum Catalase: Reversibly Formed Charge Isoform of Erythrocyte Catalase
Authors: Góth László
Published: January 2020

The different electrophoretic mobilities of erythrocyte and serum catalase (EC 1.11.1.6) were confirmed and the causes responsible for their differences were examined. The presence of a catalase-binding protein in serum that could form a complex with erythrocyte catalase was excluded by incubating serum proteins with erythrocyte catalase. No new unequivocal catalase bands representing a catalase-binding protein were detected. The erythrocyte and serum catalase proved to be charge isoforms: their molecular masses, estimated by gel permeation chromatography or polyacrylamide gel electrophoresis in a nondenaturing system, were very similar, whereas their electrophoretic mobilities were different. Assay of serum catalase by gel permeation and hydrophobic chromatography yielded a product with the same electrophoretic mobility as that of erythrocyte catalase. Different dilution of erythrocyte catalase with human sera led to a gradual decrease of its mobility, 20-fold or greater dilution yielding the same results as for serum catalase. Similarly, when serum catalase was diluted 20-fold or more with 60 mmol/L phosphate buffer, it migrated similarly to erythrocyte catalase. I detected no effect of dialyzable serum ligands, NADPH, or protection of SH groups on the electrophoretic mobility of either catalase isoform. I conclude that formation of charge isoforms of catalase is caused by a reversible, conformational modification due to matrix effect of serum.
Factors Influencing the Accuracy of the National Reference System Total Cholesterol Reference Method
Authors: Bernert John T , Akins James R , Cooper Gerald R , Poulose Abraham K , Myers Gary L , Sampson Eric J
Published: January 2020

Previous comparisons between the Reference and Definitive Methods for measuring serum cholesterol have demonstrated a small but persistent positive bias in the Reference Method, averaging about +1.6%. Here we describe the results of further investigations designed to better characterize the nature of this bias. Analysis of a well-characterized model serum sample (SRM 909) suggests that more than half of the difference in cholesterol values determined by the two methods is the result of small contributions from cholesterol precursor sterols and phytosterols, which are also measured for the Reference Method. An additional significant contribution may be from cholesterol oxidation products, particularly 7-hydroxycholesterol isomers, which are active in the Liebermann-Burchard reaction. The 7-hydroxycholesterol in SRM 909, most of which appeared to be already present in the serum rather than formed during saponification, may account for as much as 20% of the observed difference between the methods. Contributions from other possible sources, including impurities in the cholesterol standard and incomplete saponification of cholesteryl esters, are very small. Because the observed bias is both quite small and consistent among samples, the cholesterol Reference Method continues to meet all of the requirements generally expected for a dependable and effective Reference Method.
Analytical Performance of EMITTM Cyclosporine Assay Evaluated
Authors: Dasgupta Amitava , Saldana Sylvia , Desai Manju
Published: January 2020

We evaluated the EMITTM Cyclosporine Assay (Syva Co., Palo Alto, CA), using the Cobas-Mira analyzer to assess the precision, accuracy, and analytical recovery from whole-blood samples supplemented with cyclosporine. We also performed comparative analysis of whole-blood samples containing cyclosporine from liver and kidney transplant patients by using EMIT, HPLC, and RIA (IncStar Cyclo-Trac, SP assay). Before assay by EMIT or RIA, cyclosporine was extracted from whole blood with methanol. For the HPLC method, whole blood containing cyclosporine was hemolyzed with 300 mL/L acetonitrile in water; cyclosporine was extracted from the hemolysate with acetonitrile. The within-run and between-run CVs for the EMIT assay of cyclospoprine were 9.9% (means = 72.6, SD = 7.2 μg/L; n = 20) and 13.5% (means = 75.0, SD = 10.1 μg/L; n = 26) for the low control; 3.5% (means = 194.7, SD = 6.8 μg/L; n = 20) and 8.1% (means = 189.0, SD = 15.3 μg/L; n = 26) for the medium control; and 7.0% (means = 332.5, SD = 23.3 μg/L; n = 20) and 7.1% (means = 340.0, SD = 24.2 μg/L; n = 24) for the high control (Bio-Rad, whole-blood controls). Analytical recovery of cyclosporine from drug-supplemented samples averaged 99% for EMIT, 104% for HPLC, and 90% for RIA over a concentration range of 50-500 μg/L. Analysis of 196 specimens by HPLC (x) vs EMIT (y) gave the following regression statistics: y = 1.27x + 16.44; IncStar‘s RIA (x‘) vs EMIT: y = 1.12x‘ - 2.50; HPLC vs RIA: x‘ = 1.10x + 23.87.
PCR-Based Method to Create a Specific Mutation as a Reference for Oligomelting
Authors: Gudnason Vilmundur , Humphries Steve
Published: January 2020

In screening selected populations for known mutations in genes relevant to risk for hyperlipidemia, atherosclerosis, and coronary artery disease, one gene we have examined is the low-density-lipoprotein receptor (LDLR) gene in patients with familial hypercholesterolemia (FH), which has a frequency of 1:500 in most populations(1). More than 30 mutations in this gene have been described, with gross alterations accounting for about 2-6& of defects in the populations investigated (2), leaving the majority to be point mutations. Some mutations create a change of a recognition site for a restriction enzyme, allowing direct detection; the remainder can be detectedby the oligomelting technique with allele-specific oligonucleotides(ASOs) (3). One problem in using ASOs to screen for mutations is the unavailability of a DNA samplefrom a patient with the mutation to use as a positive control in the test. To overcome this problem, we have used a polymerase chain reaction (PCR)-based method to create mutations of interest, using primers containingthe mutated DNA sequence [this method is frequently used to create new restriction sites in PCR fragments via 3' mutated primers (4)].
Sensitive, Selective, and Rapid Procedure for Quantifying Cocaine in Urine
Authors: Vasiliades John
Published: January 2020

Cocaine metabolism produces two major metabolites, benzoylecgonine (BE) and ecgonine methyl ester (EME) (1-4). BE is the metabolite ordinarily used to identify cocaine misuse in urine (5). However, determination of EME and cocaine in urine can provide valuable and important forensic information as to the source of BE and the time of cocaine ingestion (4-7). Laboratories should therefore be able to provide information on EME and cocaine when requested, in addition to BE. We have already described a sensitive and selective procedure for EME analysis in urine (8). We now present a rapid and sensitive procedure for the identification and quantification of cocaine in urine.
Prognostic Value of Rate of Decline of Creatinine Kinase and MB-Isoenzyme Activity after Acute Myocardial Infarction
Authors: Clark Gregory D , Haver Virginia M
Published: January 2020

The rate of decline of total creatine kinase (CK; EC 2.7.3.2) activity during the course of an acute myocardial infarction (AMI) was shown by Weinberger at al. (1) to be of prognostic value. In their study, patients in whom CK values declined by <50% of their peak value within 48 h had an increased risk of re-infarction or dying. This prognosis was not related to sex, previous AMI, site of infarction, or coronary risk factors. We wanted to confirm these findings in our populations and determine if there was additional value/specificity in monitoring the rate of decline of the more heartspecific isoenzyme, CK-MB, within the first 24 h of patient treatment. Therefore, we examined the peak activity and rate of decline of total CK and of CK-MB in 35 patients diagnosed with an AMI.
Usefulness of Thromboxane B2 in Diagnosis of Renal Transplant Rejection
Authors: Gonzalez-Revalderia J , Sabater J , Villafruela J J , Ortuño J
Published: January 2020

Morelet et al. (1) assessed the usefulness of sequential urinary albumin measurements in 16 renal allograft recipients, eight with and eight without acute graft-rejection episodes. They concludedthat the urinary albumin/creatinine ratio is poorly specific in the diagnosis of acute renal rejection, as shown by a high proportion of false-positive results. In addition, they commented on similar findings-poor specificity and poor positive predictive value-for other urinary components, e.g., β2-microglobulin, neopterin, renal tubular enzymes, thromboxane B2 (TxB2), interleukin-2, and solubleIL-2 receptor.
Derived Quantities in Clinical Chemistry: Further Need for Improved Performance
Authors: Fuentes-Arderiu X , Ortolá J , Castiñeiras M J , Fraser C G
Published: January 2020

Westgard and Burnett (1) recently put forward a strong case for an improvement in imprecision over current requirements.
The clinical chemist
Authors: David E. Bruns
Published: January 2020

I have pretty much made up my mind to run for President. What the country wants is a candidate who cannot be injured by investigation of his past history so that the enemies of the party will be unable to rake up anything against him that nobody ever heard of before. If you know the worst about a candidate to begin with, every attempt to spring things on him will be checkmated. Now I am going to enter the field with an open record. I am going to own up in advance to all the wickedness I have done, and if any Congressional committee is disposed to prowl around my biography in the hope of discovering any dark and deadly deed that I have secreted, why—let it prowl
Immunochemiluminometric and immunoradiometric determinations of intact and total immunoreactive parathyrin: performance in the differential diagnosis of hypercalcemia and hypoparathyroidism
Authors: Endres David B , Villanueva Rocheile , Sharp Charles F , Singer FrederIck R
Published: January 2020

Parathyrin (parathyroid hormone; PTH) was measured with three immunoassays: a two-site immunochemiluminometric (ICMA) and a two-site immunoradiometric (IRMA) method for intact PTH, and a sensitive radioimmunoassay for mid-region or "total" PTH, measuring both intact hormone and inactive fragments. Single specimens from normal subjects and from individuals with primary hyperparathyroidism, hypercalcemia associated with malignancy, and hypoparathyroidism were analyzed with all three methods. All individuals with primary hyperparathyroidism showed absolutely above-normal concentrations with the mid-region RIA, 28 of 29 did with the ICMA, and 21 of 29 did with the IRMA. PTH concentrations in primary hyperparathyroidism were most increased relative to normal subjects with the mid-region assay (10.4 times), less so with the intact assays (ICMA 5.5 times; IRMA 5.3 times). Concentrations of intact PTH were suppressed below normal in nearly all patients with hypercalcemia associated with malignancy, as measured with the ICMA (26 of 30) and the IRMA (28 of 30) assays. In marked contrast, results for mid-region PTH were normal or slightly above normal, consistent with studies suggesting that the parathyroids secrete both intact hormone and inactive fragments, the former being more sensitive to suppression by hypercalcemia. In hypoparathyroidism PTH concentrations were detectable but below normal in all patients by the intact assays and in all but one patient by the mid-region assay. These low concentrations are probably due to a nonspecific serum effect that could be resolved with selection of a more appropriate standard matrix. Although all three assays are useful in the differential diagnosis of hypercalcemia, two-site intact assays are more convenient and more specific in patients with compromised renal function.
Catecholamine sulfates as internal standards in HPLC determinations of sulfoconjugated catecholamines in plasma and urine
Authors: Strobel Gɒnther , Weicker HelmuT
Published: January 2020

A method is described to measure catecholamine sulfates from human plasma and urine by isocratic reversed-phase high-performance liquid chromatography with electrochemical detection. For this measurement we use catecholamine 3-sulfate isomers as internal standards and determine the sulfoconjugates only after eliminating the catecholamines. Catecholamines that have previously been used as internal standards are shown to cause a significant overestimation (P less than 0.05) of the catecholamine sulfates--by 10% to 25% and 20% to 42% in human plasma and urine, respectively. The detection limits (signal-to-noise ratio greater than 3) in plasma and urine samples were about 80 pmol/L for each analyte. The intra-assay and interassay CVs were less than 4.0% and 10.6% in human plasma and less than 6.6% and 12.8% in human urine, respectively. The calibration curves for all catecholamine sulfates in human plasma and urine were linear (r greater than 0.96; P less than 0.001) over the respective concentration ranges of 0.1-100 nmol/L and 5-1000 nmol/L.
Simple, optimized liquid-chromatographic method for measuring total hydroxyproline in urine evaluated
Authors: Reed P , Holbrook I B , Gardner M L G , McMurray J R
Published: January 2020

We have optimized a method for measuring total hydroxyproline (HYP) in urine by HPLC after release from urinary peptides by solid-phase hydrolysis on Dowex 50W x 8 ion-exchange resin. The HYP was derivatized with 4-chlor-7-nitrobenzo-2-oxa-1,3-diazole, and excess reagent was removed with the use of a 100-mg C18 Bond-Elut cartridge. The HYP derivative was separated isocratically at 30 degrees C on a 250 x 4.6 mm reversed-phase column containing 5-microns particles of Spherisorb S5 ODS-2, with S-carboxymethylcysteine as internal standard. Total assay time was 14 min. The standard curve for the method was linear from the detection limit for HYP, 3.6 mumol/L, to 10 mmol/L. The between-batch CV was less than 5.1% and the mean analytical recovery of HYP was 95% +/- 1.4%. Comparison with a commercially available colorimetric method showed good correlation: y = 1.158x + 19.76 mumol/L (Syx = 74, n = 120), but HPLC results were 15% higher, probably from incomplete hydrolysis with the colorimetric method. This method offers a considerable improvement in assay time, specificity, sensitivity, precision, and cost compared with the colorimetric method.
Further insights into the pathophysiology of hyperapobetalipoproteinemia: role of basic proteins I, II, III
Authors: Kwiterovich Peter O , Motevalli Mahnaz , Miller Michae , Bachorik Paul S , Kafonek Stephanie D , Chatterjee Subroto , Beaty Tern , Virgil Donna
Published: January 2020

Hyperapobetalipoproteinemia (hyperapoB), a familial lipoprotein disorder characterized by an increase in small, dense, low-density lipoprotein (LDL) particles, is strongly associated with coronary artery disease. There are two metabolic defects in hyperapoB: an increased synthesis of a very-low-density lipoprotein in liver, resulting in an overproduction of LDL, and a delayed clearance of post-prandial triglyceride and free fatty acids. To date, defects in the apolipoprotein B gene do not appear to explain the hyperapoB phenotype. Defect(s) in the uptake or intracellular metabolism of free fatty acids have been found in cells from hyperapoB patients. Three basic proteins (BPs)--BP I (Mr 14,000, pI 9.10), BP II (Mr 27,500, pI 8.48), and BP III (Mr 55,000, pI 8.73)--were isolated from normal human serum. Compared with normal fibroblasts, cultured hyperapoB fibroblasts incubated with BP I, which appears to be the same protein as acylation-stimulating protein (ASP), showed 50% less stimulation of triglyceride acylation and cholesterol esterification, whereas BP II markedly stimulated cholesteryl ester formation, and BP III caused no difference in response vs normal fibroblasts. However, in cultured normal human monocyte macrophages, BP III, but not BP I or BP II, stimulated cholesteryl esterification two- to threefold. BP I, BP II, and BP III may provide new insights into normal metabolism of lipids, lipoproteins, and free fatty acids and the pathophysiology of hyperapoB.
Relation between debrisoquine oxidation phenotype and morphological, biological, and pathological variables in a large population
Authors: Vincent-Viry M , Muller J , Fournier B , Galteau M M , Siest G
Published: January 2020

Factors affecting biological variations in debrisoquine-oxidation polymorphism were investigated in a population of 3065 unrelated supposedly healthy Caucasian subjects, ages 35 to 50 years. This population, including 1526 men and 1539 women, was used to determine whether the debrisoquine-oxidation phenotype can be related with environmental factors such as alcohol intake, smoking habits or medication; with morphological variables; or with 22 blood constituents and some pathological states. A single dose of 10 mg of debrisoquine sulfate was administered to determine the oxidation phenotype. A metabolic ratio (debrisoquine/4-hydroxydebrisoquine) of 10.0 defined a poor metabolizer [frequency of 8.2% (SD 1.0%)] in this sample. Dose recoveries of debrisoquine excretion (mean and SD) were 8.9% (11.9%) and 45.1% (32.2%) in extensive and poor metabolizers, respectively. The mean (SD) amount of debrisoquine administered that was excreted in urine as 4-hydroxydebrisoquine was 17.4% (17.3%) in extensive metabolizers and 0.5% (0.9%) in poor metabolizers. The main factors differing significantly between poor and extensive metabolizers were mean cell volume, mean corpuscular hemoglobin concentration, albumin, and ponderal index. No other blood constituents (e.g., cholesterol, glucose) differed between poor and extensive metabolizers. The lack of correlation with most of the variables tested is of interest in clinical trials, because our findings indicate that no subgroups will be required, making selection of subjects easier.
Analyzing quality-control trends with moving slope charts
Authors: Smith S Jay , Myers Gary L
Published: January 2020

We have developed and evaluated a new procedure for detecting trends in quality-control measurements and applied it to laboratory data. The method requires the use of sequential or "moving" slope estimates to identify trends. Formulae are derived to estimate the regression error for the moving slope directly from the standard deviation of the analytical measurements obtained during characterization runs. Control limits for the moving slope depend only on this regression error, the span of the slope, and the desired statistical level of control. The moving slope can be plotted with control limits to determine out-of-control points. The statistical power of the moving slope is found to be much greater than that of an often-used test for trends. An example of the use of the moving slope is shown for quality-control measurements for total cholesterol obtained over several years. We conclude that the moving slope procedure has considerably more statistical power than trend rules and that it yields more useful information to the analyst.
Lipase isoforms and amylase isoenzymes: assays and application in the diagnosis of acute pancreatitis
Authors: Lott John A , Lu Carrle J
Published: January 2020

Pancreatic juice and serum from patients with acute pancreatitis contain three enzymes that have lipolytic activity: L1 and L2, which are pancreatic isoenzymes or isoforms of lipase (EC 3.1.1.3), and L3, which is probably pancreatic carboxyl ester lipase, also known as cholesterol esterase (EC 3.1.1.13). These enzymes are readily separated electrophoretically on agarose and can be developed with an overlay of Kodak Ektachem lipase slide material. The latter acts as a dry-reagent developing substrate, with the enzymes producing blue bands in the slide material. We found L1 in about one-half of normal persons, L2 in none, and L3 in all. We assayed for amylase (EC 3.2.1.1), amylase isoenzymes, lipase, and lipase isoforms in the sera of 100 patients with suspected acute pancreatitis. L2 lipase has the greatest diagnostic efficiency for the diagnosis of pancreatitis, compared with total amylase, P3 amylase, and total lipase. Lipase and L2 could replace amylase, an inefficient test, for the diagnosis of patients with suspected acute pancreatitis. In patients receiving organ transplants, a serum amylase value of greater than 300 U/L or a lipase of greater than 1000 U/L discriminated well between patients with and without complications and (or) acute rejection.
Quantification of plasma lipoproteins by proton nuclear magnetic resonance spectroscopy
Authors: Otvos J D , Jeyarajah E J , Bennett D W
Published: January 2020

A new analytical procedure for quantifying plasma lipoproteins by proton nuclear magnetic resonance (NMR) spectroscopy has been developed that potentially offers significant advantages over existing clinical methods used for assessing risk of coronary heart disease. Analysis of a single spectrum of a nonfasting plasma sample, acquired simply and rapidly at moderate magnetic field strength (250 MHz), yields a complete profile of lipoprotein concentrations: chylomicrons and very-low-, low-, and high-density lipoproteins. The method is based on curve-fitting (spectral deconvolution) of the plasma methyl lipid resonance envelope, the amplitude and shape of which depend directly on the amplitudes of the superimposed methyl resonances of the lipoprotein components. A linear least-squares curve-fitting algorithm was developed to efficiently extract the signal amplitudes (concentrations) of the lipoproteins from the plasma spectrum. These signal amplitudes correlate well with lipoprotein concentrations determined by triglyceride and cholesterol measurements.
Elimination of undesired cross-reactants by using mixtures of antibodies: experimental and theoretical evaluations of hapten radioimmunoassays
Authors: Boutten B , Ezan E , Mamas S , Dray F
Published: January 2020

This study shows that the specificity of radioimmunoassays can be improved by including a second antibody raised against an undesired cross-reactant. In a radioimmunoassay of prostaglandin E2 (PGE2) involving a monoclonal antibody, the cross-reactivity with 6-keto-prostaglandin E1 (6kPGE1) was decreased from 20% to 2% by including a high concentration of a polyclonal anti-6kPGE1. A similar increase in specificity was obtained in the assay of a larger hapten, luliberin (luteinizing hormone releasing hormone); the cross-reactivity of a luliberin analog was decreased 20-fold. Equations derived from the Law of Mass Action were used for the mathematical analysis and for the computer simulation of changes in assay affinity and specificity according to the quantity and quality of the mixed antibodies. The model gave values that agreed well with experimental data; it promises to be quite useful in designing specific radioimmunoassays.
Automated determination of creatine kinase activity in serum with use of thermostable glucokinase
Authors: Maeda Ikuhiro , Hayashi Sadao , Amino Nobuyuk , Miyai Kiyosh
Published: January 2020

We measured creatine kinase (CK, EC 2.7.3.2) activity in serum with a new reagent system utilizing thermostable glucokinase (EC 2.7.1.2). Automated determinations were performed with Toshiba's Model TBA-80S Biochemical Analyzer. Precision studies demonstrated within-run and between-run CVs of 0.4%-2.4% and 2.8%-3.1%, respectively. The response linearity was confirmed for CK activity up to 1000 U/L at 37 degrees C. CK activities correlated well (r = 0.997) with those obtained by the manual method recommended by the German Society for Clinical Chemistry (measuring at 37 degrees C) involving hexokinase (EC 2.7.1.1). However, CK activities measured by our method were consistently higher than those of the hexokinase method at reaction temperatures of 30, 37, and 40 degrees C. These data indicate that the new method with thermostable glucokinase is better than that with thermo-unstable hexokinase for determination of CK activity in serum.
Concentration of Mn-superoxide dismutase in serum in acute myocardial infarction
Authors: Usui Akihlko , Kato Kanefusa , Tsuboi Hldeyuki , Sone Takahito , Sassa Hirom , Abe Toshio
Published: January 2020

Serum concentrations of manganese-containing superoxide dismutase (Mn-SOD; EC 1.15.1.1), a mitochondrial enzyme present in high concentrations in the heart, were measured successively by enzyme immunoassay in 18 patients with acute myocardial infarction (AMI) and in eight with angina pectoris. Results were compared with creatine kinase (EC 2.7.3.2) isoenzyme MB (CK-MB) concentrations in the same specimens. The mean (and SD) serum Mn-SOD concentration in 120 healthy adults was 77.5 (18) micrograms/L. Mn-SOD concentrations exceeding 150 micrograms/L (greater than mean + 4 SD) were considered above-normal. In patients with AMI, the Mn-SOD concentration was 80 (16.8) micrograms/L on admission and increased gradually after the first hospital day. The peak, 260 (109) microgram/L, occurred on the fourth hospital day, at which time the Mn-SOD concentrations in all AMI patients were above normal. Thereafter the Mn-SOD concentration decreased slowly, but still was above normal in 14 of 18 patients on the seventh hospital day. On the other hand, in patients with angina pectoris, the Mn-SOD concentration was 78 (11) micrograms/L on admission and did not increase significantly [peak value 97.5 (42) micrograms/L on the fourth hospital day]. The serum concentration of Mn-SOD is a potentially useful marker for estimating cardiac mitochondrial damage and for diagnosing AMI, especially in the late phase.
Assessment of split-sample proficiency testing for cholesterol by use of a computer simulation model
Authors: Bennett Sterling T , Connelly Donald P , Eckfeldt John H
Published: January 2020

We developed a computer model to study the use of patients' specimens to assess compliance of cholesterol measurement performance with the 1992 goals of the Laboratory Standardization Panel of the National Cholesterol Education Program. The model uses Monte Carlo techniques to simulate cholesterol measurements that are subject to both systematic and random error. Split-sample measurements by a clinical laboratory and by a reference laboratory are compared by using linear regression to estimate clinical laboratory bias and imprecision; subsequently, according to specified decision limits, the performance of the clinical laboratory is classified as acceptable or deficient. We have quantified the influence of the bias and imprecision of the clinical laboratory, the imprecision of the reference laboratory, the number of split samples compared, and the decision limits on the accuracy of the classification of clinical laboratory performance. Unless the decision limits are carefully selected and a sufficient number of split samples are used, clinical laboratory performance will be frequently misclassified.
Improved sample preparation for accurate determination of low concentrations of lead in whole blood by graphite furnace analysis
Authors: Jacobson Beryl E , Lockitch Gillian , Quigley Gayle
Published: January 2020

The effect of low concentrations of lead on pre- and post-natal growth and development is a current concern. We describe a simple method of sample preparation for direct determination of lead in whole blood by Zeeman graphite-furnace atomic absorption spectrometry. This procedure improves analytical precision and accuracy of lead determinations at low concentrations as compared with published furnace data. At blood lead concentrations of 0.25, 1.98, and 3.76 mumol/L, within-run CVs were 3.2%, 1.8%, and 1.4% respectively; between-run CVs were 7.3%, 2.9%, and 2.2%. Accuracy, as demonstrated by analytical recovery, ranged from 99% to 102%. Our reproducibility/accuracy score in the 1989 Quebec interlaboratory comparison program was 96% compared with the target, second best of 66 participating laboratories.
Laboratory process specifications for assuring quality in the U.S. National Cholesterol Education Program
Authors: Westgard J O , Petersen P H , Wiebe D A
Published: January 2020

We have assessed the laboratory specifications necessary for ensuring that cholesterol testing processes satisfy the quality required by the U.S. National Cholesterol Education Program (NCEP). A model for setting process specifications has been developed to relate the NCEP guidelines for medical interpretation of a cholesterol test to the pre-analytical and analytical variables that can affect a test result. Using this model, we derived specifications for the imprecision (coefficient of variation, CV, or standard deviation, s) and inaccuracy (bias) that are allowable under stable operation, as well as the quality-control procedures (control rules and number of control measurements) that are necessary to detect unstable operation. The NCEP goals of an allowable CV less than or equal to 3% and an allowable bias no greater than +/- 3% are inadequate for assuring the quality of an individual or single cholesterol test when monitoring performance with many of the statistical quality-control procedures currently used in the U.S. With quality-control procedures having two control measurements per run, a CV of 3% is allowable only when bias is zero; a CV less than or equal to 2% is necessary if bias is +/- 3%. With quality-control procedures having four control measurements per run, a CV of 3% is allowable when bias is +/- 1.5%; a CV less than or equal to 2.5% is required if bias is as large as +/- 3%. For two serial tests, the NCEP 3% goals are adequate for current quality-control procedures having four control measurements per run.
Different constructs for the expression of mammalian gamma-glutamyltransferase cDNAs in Escherichia coli and in Saccharomyces cerevisiae
Authors: Angele C , Oster T , Visvikis A , Michels J M , Wellman M , Siest G
Published: January 2020

To prepare a reference material for gamma-glutamyltransferase (GGT; EC 2.3.2.2) measurements in clinical chemistry, we constructed different vectors containing either the rat kidney or the human hepatoma Hep G2 GGT cDNA downstream from an inducible promoter for expression in Escherichia coli and Saccharomyces cerevisiae. Transformed bacterial and yeast cells were tested for GGT production by use of Western blot analysis and enzymatic activity measurements. Both rat renal and Hep G2 GGT cDNAs were expressed in E. coli, producing active and nonglycosylated enzymes localized in the periplasmic space. Recombinant Hep G2 GGT was synthesized as a single-chain protein, unlike rat renal GGT, which presented two polypeptides of 62 and 30 kDa, identified as the precursor and a GGT heavy-subunit-like peptide, respectively. Rat renal GGT was produced in S. cerevisiae as two polypeptides, 55 and 30 kDa, detected by antisera against rat renal GGT. These results suggest maturation mechanisms such as glycosylation and cleavage steps, enhancing the interest of S. cerevisiae as a useful expression system for producing active mammalian proteins as reference materials.
Effect of plasma triglyceride concentrations on the accuracy of immunoturbidimetric assays of apolipoprotein B
Authors: Walmsley T A , Grant S , George P M
Published: January 2020

We have studied the influence of triglyceride-rich particles on the analytical bias of apolipoprotein B measurements by various immunoturbidimetric methods. Three commercially available methods grossly overestimate apolipoprotein B in samples with even moderately above-normal triglyceride concentrations. This effect is due to the increased relative reactivity of very-low-density lipoproteins in these reagent systems, and can be eliminated by including Tween 20 (2 g/L) in the reagent buffer. We have developed, and describe, an automated immunoturbidimetric method that allows the accurate determination of apolipoprotein B in the plasma of patients with hypertriglyceridemia.
The role of the laboratory in evaluation of kidney function
Authors: Cohen Eric P , Lemann Jacob
Published: January 2020

Evaluation of kidney function by physical examination alone is imprecise and limited. Quantitative, reproducible assessment of kidney function required laboratory measurements of substances in plasma and urine, followed by reliable interpretation. Thus, glomerular filtration, urine protein excretion, water metabolism, and electrolyte disturbances may be quantified. These data are very useful in the single and serial assessment of patients with kidney disease and in evaluation of the effects of treatment.
Rapid monitoring of benzodiazepines in clinical samples by using on-line column switching HPLC
Authors: Moore C M , Sato K , Katsumata Y
Published: January 2020

We have determined routinely administered benzodiazepines and some of their metabolites in clinical samples by using on-line column switching for sample clean-up and reversed-phase high-pressure liquid chromatography (HPLC). The plasma sample is directly injected onto a cyanopropyl (CN) pre-column, washed with buffer, and eluted onto an analytical column (C18, octadecylsilane). Analytical recovery from drug-supplemented plasma samples was greater than 85% for all the benzodiazepines tested. The response of the analytical column varied linearly with drug concentrations over the range 0.01-1 mg/L in plasma, with CVs ranging from 0.3% to 1.8% within-day and 5.5% to 15.7% between days. The detection limit (signal-to-noise ratio greater than 3) was 0.01 mg/L (1 ng on column) for all of the benzodiazepines and their metabolites. The advantages of the technique include the extreme cleanliness of the traces, no requirement for off-line sample preparation, and ease of automation.
Measurement of prostate-specific antigen and prostatic acid phosphatase concentrations in serum before and 1-42 days after transurethral resection of the prostate and orchidectomy
Authors: Price Alun , Attwood Stephen E A , Grant jhon B F , Gray Trevour A , Moore Kenneth T H
Published: January 2020

Preoperative intra-individual variation for determinations of prostate-specific antigen and prostatic acid phosphatase concentrations, 15-30% in 92 patients with benign prostatic hyperplasia, limits the diagnostic usefulness of both tumor markers. In benign prostatic hyperplasia (214 patients), concentrations of these tumor markers increased in the initial postoperative period. Prostatic acid phosphatase concentration then decreased by the third postoperative day. Prostate-specific antigen concentration remained above normal in the first postoperative week but had decreased by 42 days. In prostatic carcinoma (46 patients), the concentrations of these tumor markers did not increase postoperatively. During the first week, the concentrations of prostatic acid phosphatase began to fall, but prostate-specific antigen showed a decrease only at 42 days. After orchidectomy (11 patients), the concentrations of both markers had decreased by five days. Concentrations of prostate-specific antigen but not of prostatic acid phosphatase were significantly increased in patients with metastases at 42 days postoperatively. When the concentration of tumor marker did decrease, the magnitude of change was greater for prostatic acid phosphatase than for prostate-specific antigen. These changes were accentuated after an orchidectomy.
Enzymatic fluorometric procedure for phospholipid quantification with an automated microtiter plate fluorometer
Authors: Nanjee M Nazeem , Gebre Abraham K , Miller Norman E
Published: January 2020

We describe a new enzymatic assay for phospholipids that is rapid, sensitive, convenient, and inexpensive. The method is based on the fluorometric detection of H2O2, generated by the choline oxidase-catalyzed oxidation of choline, after liberation of choline from phospholipids by phospholipase D. Significant advantages over existing methods are that the entire reaction sequence can take place in a single vessel, a 12 x 8 well microtiter plate, and that the fluorescence intensity can be measured automatically with a Fluoroskan II (Labsystems Oy) detector. Extracting samples with organic solvents is unnecessary, although the method can be applied to extracts in isopropanol. The assay is approximately 60-fold more sensitive and has a limit of detection eightfold lower than currently available enzymatic colorimetric methods. Including solvent blanks, eight standards, and three quality-control pools, 34 samples can be pipetted and assayed in duplicate in 60 min. Results obtained by this procedure for total phospholipid choline in lipoproteins in primate plasma agreed well with those obtained for inorganic phosphorus by the Bartlett acid-digestion procedure.
Simple method for preparing a concentration gradient of serum components by freezing and thawing
Authors: Hirano Tetsuo , Yoneyama Toshiaki , Matsuzaki Hiroko , Sekine Takainitsu
Published: January 2020

We created a simple method for obtaining a series of successively more-concentrated samples from a serum without changing the ratio of its components. We froze a pooled serum and then allowed it to thaw undisturbed. The serum components formed a gradient of increasing concentration from the top of the sample to the bottom. We found that (a) in test results, each fraction of serum in the gradient showed almost the same relative concentrations of components (i.e., inorganic and organic compounds, proteins, metals, and hormones), irrespective of atomic or molecular mass; (b) the concentration gradient depended on the thawing temperature but not on the freezing temperature; (c) when we thawed the frozen sample with centrifugation, the slope of the concentration gradient increased with increasing centrifugal force; (d) when the thawed sample was fractionated into 10 fractions from the top to the bottom, the original serum concentration was always maintained between the sixth and seventh fractions from the top; and (e) the concentration gradient became steeper with repeated freezing and thawing. By using this method, one can easily prepare serum samples at gradients of concentration useful in the clinical laboratory, although the mechanism of gradient formation is still unclear.