bluuebunny/crossref_metadata_2025 huggingface dataset:
Optimized method for measuring aspartate aminotransferase activity with the CentrifiChem Analyzer, with automatic preincubation of serum.
Authors: Ertingshausen G , Amsellem-Winzelberg L , Richert J F , Davids R
Published: January 2020

We propose a routine method for the mechanized measurement of aspartate aminotransferase with the CentrifiChem Analyzer, which is based on the recommendations of both the International Federation of Clinical Chemistry and the Société Française de Biologie Clinique. A modification of the CentrifiChem pipettor permits simultaneous pipetting of two reagents, thus achieving automatic preincubation of the serum in the transfer disk. Owing to the fixed reagent volumes dispensed by the pipettor, preincubation conditions had to be modified, but the recommendations for the final reagent concentrations in the assay cuvet were observed. The totally automated method correlates very well with one involving manual pipetting to reproduce the detailed step-by-step recommendations of the International Federation of Clinical Chemistry. Intra-assay precision ranged from 3.4 to 4.9% and interassay precision from 1.7 to 7.5%. We assayed 135 sera and obtained a correlation coefficient of 0.996 (a = 1.025, b = 0.08).
Analysis for nickel in plasma and urine by electrothermal atomic absorption spectrometry, with sample preparation by protein precipitation.
Authors: Andersen I , Torjussen W , Zachariasen H
Published: January 2020

We describe and evaluate a method for determining nickel in plasma and urine by atomic absorption spectrometry. Proteins are precipitated with trichloroacetic acid and sulfuric acid; ammonium pyrrolidinedithlocarbamate is used as the chelating agent for nickel, and methyl isobutyl ketone as extraction solvent. The results were compared with results obtained by the acid-digestion technique for removing proteins and other organic substances. Analyses for both plasma and urine were better by the present procedure. The mean and standard deviation for nickel in plasma from 15 healthy individuals was 2.13 +/- 0.58 microgram/liter by this method. For nickel in urine from 15 healthy men the mean and standard deviation was 4.45 +/- 1.9 microgram/liter. The coefficient of variation for plasma was 11.9%, and for urine 12.2% in 10 analyses of the same plasma and urine with the protein-precipitation procedure, as compared with 26.0 and 38.2%, respectively, by the acid-digestion technique.
An aqueous primary standard for use with the Technicon SMA 6/60.
Authors: Balkcom R M
Published: January 2020

An aqueous primary standard containing sodium, potassium, chloride, carbon dioxide, glucose, and creatinine was prepared for use with the Technicon SMA 6/60 continuous-flow analyzer. This standard, compared with a protein-base primary standard, showed no significant difference in slope, gamma-intercept, and linearity for these methods. Precision with use of the aqueous primary standard was acceptable, comparable to that for a commercial reference serum. The standard was stable for at least 30 days.
Dual-column cation-exchange chromatographic method for beta-aminoisobutyric acid and beta-alanine in biological samples.
Authors: Kuo K C , Cole T F , Gehrke C W , Waalkes T P , Borek E
Published: January 2020

A rapid, automated chromatographic method has been developed for the quantitation of the nucleic acid catabolites beta-aminoisobutyric acid and beta-alanine in urine, serum, and other physiological fluids. The analyses were performed on a modified Beckman 121M amino acid analyzer with dual ion-exchange columns and the use of a single sodium citrate buffer (pH 4.38, 0.20 mol/liter). By carefully matching the elution pattern for the two ion-exchange columns and alternating use of these columns, analyses are completed every 40 min. The chromatography, regeneration, and equilibration of the two columns are precisely programmed, thus the detector sees only the elution of beta-aminoisobutyric acid and beta-alanine alternately from each column. Long-term precision and analytical recovery for the two metabolites in urine were 1.9 and 102%, and 3.3 and 101%, respectively. Their normal physiological values were determined in human serum and urine. Their excretion in the urine was also studied as a function of collection time, to validate a more convenient, less costly method of sampling. This study shows that randomly collected samples are acceptable when the concentration of the two metabolites are expressed in terms of creatinine excretion. In addition, the distribution of the free and conjugated forms of the two metabolites in urine and serum was studied. A preparative method was also developed for the quantitative isolation of beta-amino-isobutyric acid from urine samples. The alternating dual-column technique may be applied to any ion-exchange chromatographic method where many analyses must be performed. This method is currently used in our laboratories for measuring these beta-amino acids in urine and serum of patients with various types of cancers.
Chromatographic separation and continuously referenced, on-line monitoring of creatine kinase isoenzymes by use of an immobilized-enzyme microreactor.
Authors: Denton M S , Bostick W D , Dinsmore S R , Mrochek J E
Published: January 2020

We describe a new concept in continuously referenced monitoring of the isoenzyme activities of creatine kinase (EC 2.7.3.2) after liquid-chromatographic separation. After separation on a diethylaminoethyl-Sephacel column, the three isoenzymes of creatine kinase undergo a series of coupled enzyme reactions, ultimately resulting in the formation of ultraviolet-detectable NADPH. A major advantage of this detection system is the immobilized-enzyme microreactor (2 X 17 mm), which may be removed and stored refrigerated when not in use. A split-stream configuation allows self-blanking of endogenous ultraviolet-absorbing constituents in authentic sera samples, which would otherwise make definitive diagnosis and quantitation difficult or impossible. This system is applicable to the automated analysis of creatine kinase isoenzymes in the clinical laboratory.
Radioimmunoassay for urinary albumin.
Authors: Woo J , Floyd M , Cannon D C , Kahan B
Published: January 2020

We describe a rapid, sensitive, and precise radioimmunoassay for urinary albumin (Ualb). Aliquots of diluted urine were incubated at room temperature for 1 h with 125I-labeled albumin and a rabbit antiserum monospecific for human albumin. Phase separation was effected by the double-antibody technique. The dose-response curve as linear in the range of 15.6-10000 ng, equivalent to 4 to 3000 mg/liter of urine. The limit of sensitivity was 16 ng of albumin. The coefficient of assay variation was 4.8%, both at 44 mg/liter and at 1304 mg/liter. A displacement curve obtained with a serially diluted urine sample of high albumin concentration was completely superimposable with the curve for which human albumin was used as a standard. In 26 normal individuals the range for Ualb was 2.2--12.6 mg/24h, and for albumin clearance (Calb, 1.8 x 10(-5)-19.6 x 10(-5) ml/min. After renal homografts in 25 patients, Ualb ranged from 16.9 to 9928 mg/24 h, and Calb from 2.7 x 10(-4) to 1.7 x 10(-1) ml/min. Both increased Ualb and Calb correlated well with the severity of renal homograft rejection.
Simple radioimmunoassay of cortisol in diluted samples of human plasma.
Authors: Connolly T M , Vecsei P
Published: January 2020

We describe a simple radioimmunoassay of plasma cortisol, which can be performed in 3 h and which requires no purification, heating, or refrigeration steps. The plasma proteins are inhibited through direct competition between them and the antiserum at room temperature, at which the antiserum's affinity exceeds that of the binding proteins. Plasma, diluted with water, is incubated for 3 h at room temperature with [3H]cortisol and the antiserum. We compared results with those by the usual extraction method. The correlation between methods on evaluating normal samples with one antiserum. We compared results with those by the usual extraction method. The correlation between methods on evaluating normal samples with one antiserum was r = 0.954 (P less than 0.001), and the slope was 0.661. With three other antisera it was r = 0.922 (P less than 0.001) and slope 0.644. Plasmas with abnormal protein concentrations (i.e., from preganant women, and after corticotropin administration), tested to examine the validity of the method for routine use, and to define the role of the protein carriers, showed r = 0.859 (P less than 0.001) and slope 0.726 for the four antisera used. Additional samples, assayed with diluted standards plus stripped plasma, showed a correlation with the usual extraction method of r = 0.945 (P less than 0.001) and slope 1.026.
Measurement of free calcium ion in capillary blood and serum.
Authors: Fogh-Andersen N , Christiansen T F , Komarmy L , Siggaard-Andersen O
Published: January 2020

We describe a new calcium ion-selective electrode for measurement of the substance concentration of free calcium ion [Ca2+] in the plasma phase of whole blood and in serum at 37 degrees C. A sample volume of 50 microliter suffices to obtain simultaneous values of pH and [Ca2+]. We found the within-series analytical standard deviation for serum to be 0.013 mmol/litre (CV, 1.1%) and day-to-day precision to be 0.022 mmol/litre (CV, 1.7%). The reference interval for [Ca2+] (at pH 7.40) in serum was found to be 1.184 +/- 0.054 mmol/litre (2 SD) from measurements on sera from 121 healthy blood donors. Measurements on capillary blood from 29 healthy volunteers gave a mean (+/- 2 SD) value for [Ca2+] (at pH 7.40) of 1.22 +/- 0.072 mmol/litre.
Plasma calcium, copper, magnesium, and zinc concentrations in patients with the alcohol withdrawal syndrome.
Authors: Bogden J D , Troiano R A
Published: January 2020

We determined zinc, copper, magnesium, and calcium concentrations by atomic absorption spectrophotometry in the plasma of 30 patients hospitalized for treatment of seizures during a period of alcohol withdrawal. Those patients who developed delirium tremens or a prolonged hallucinatory state had significantly higher plasma copper concentrations (P = 0.026), significantly lower zinc concentrations (P = 0.004), and significantly higher copper/zinc ratios (P = 0.001) than the patients who recovered uneventfully. Zinc deficiency may be one of the factors that contribute to the neurologic complications of alcoholism. A determination of the plasma copper/zinc ratio early in the course of alcohol withdrawal could be of value in indicating which patients have the most substantial underlying disease or metabolic imbalance and therefore may be at greatest risk of developing delirium tremens or prolonged hallucinosis.
Blood intermediary metabolite and insulin concentrations after an overnight fast: reference ranges for adults, and interrelations.
Authors: Foster K J , Alberti K G , Hinks L , Lloyd B , Postle A , Smythe P , Turnell D C , Walton R
Published: January 2020

Concentrations of several metabolites in deproteinized blood and plasma were determined in 100 subjects after an overnight fast, to establish reference ranges. Blood glycerol, nonesterified fatty acids, and ketone body concentrations and the 3-hydroxybutyrate/acetoacetate ratio increased with age while blood alanine concentrations delined. Serum insulin and blood alanine concentrations were also related to weight. No marked sex-related differences were found, although blood glycerol concentration was slightly higher in women. Distribution of all metabolite concentrations was positively skewed, except for glucose, but skewness could be corrected by logarithmic transformation. Skew was particularly marked for blood ketone body concentrations. Concentrations of all metabolites in plasma exceeded those in whole blood, but this was significant for only lactate and pyruvate when concentrations in plasma were compared with calculated concentrations in erythrocyte water.
Use of the vertical rotor to facilitate estrogen and progesterone receptor analysis.
Authors: Hofman L F , Moline C , McGrath G , Barron E J
Published: January 2020

Sucrose density-gradient analysis is one method of identifying specific estrogen and progesterone receptors in mammary tumor tissue. Use of the vertical rotor makes this practical for routine applications in the clinical laboratory by increasing the number of samples that can be run at one time and shortening the centrifuge time from 18 to less than 2 h. The separations and reproducibility compare favorably with those obtained with a swinging-bucket rotor.
Performance comparison of the Corning thin-film agarose and the Hyland thick-film agar methods for immunoelectrophoresis.
Authors: Gerson B , LaBrie J , Copeland B E
Published: January 2020

Many clinical laboratories do immunoelectrophoresis for qualitative assessment of proteins in biological fluids. Commercial kits are available that supply some or all of the necessary components, but the nature of these components varies. Seeking a reliable method for most easily preserving the original immunoelectrophoresis pattern as a permanent record, we compared a thick-film agar method to a thin-film agarose method in immunoelectrophoresis of a total of 70 serum and urine samples. For each sample, either method resulted in the same interpretation. The thin-film agarose method not only yields a stained, permanent record in about the same time that the thick film agar is ready for interpretation but not preservation, but also requires less antiserum, a smaller sample, and may not require purchase of additional hardware.
A further comparison of radioassay results for serum and plasma.
Authors: Kubasik N P , Sine H E
Published: January 2020

We evaluated results for serum vs. plasma for 12 selected radioassay procedures. Blood samples were collected with heparin, oxalate-fluoride, or ethylenediaminetetraacetate as anticoagulant and compared with the corresponding serum samples. In combination with a previous report [Kubasik, N.P., and Sine, H.E., Clin. Chem. 24 137 (1978)] we have now tested a total of 25 different analytes, using 31 manufacturers' kits. Differences were again demonstrated between serum and plasma that may be of sufficient magnitude to alter clinical interpretation of the results. Assays also demonstrated differences based on the procedure used.
Suggested isosbestic wavelength calibration in clinical analyses.
Authors: Hoxter G
Published: January 2020

I recommend the use of isosbestic points for conveniently checking the wavelength scale of spectrophotometers in the ultraviolet and visible regions. Colorimetric pH indicators, hemoglobin derivatives, and other radiation-absorbing substances that are convertible into stable isomers of different absorption spectra provide a means for calibrating many different wavelengths by comparing the absorptivities of these isomers in equimolar solutions. The method requires no special precautions and results are independent of substance concentration and temperature between 4 and 45 degrees C. Isosbestic calibration may be important for (e.g.) coenzyme-dependent dehydrogenase activity determinations and in quality assurance programs.
Radioimmunoassay for chlorpromazine in plasma.
Authors: Midha K K , Loo J C , Hubbard J W , Rowe M L , McGilveray I J
Published: January 2020

A radioimmunoassay for chlorpromazine in plasma is described. The antiserum was obtained by immunizing rabbits with a conjugate of bovine serum albumin and N-(2-carboxyethyl)desmethylchlorpromazine. It is specific for chlorpromazine and its minor active metabolite, N-desmethylchlorpromazine. Other known active or inactive chlorpromazine metabolites and other psychotropic drugs tested do not cross react with the antiserum. Less than 34 pg of the drug can be detected in 200 muL of plasma. As many as 100 samples can be processed in a day by one technician. Concentrations of chlorpromazine can be measured in 200-muL samples of plasma collected as late as 48 h after a single oral 25-mg dose.
Automated enzymatic assay for plasma ammonia.
Authors: Humphries B A , Melnychuk M , Donegan E J , Snee R D
Published: January 2020

An enzymatic method for determining plasma ammonia with the Du Pont Automatic Clinical Analyzer (aca) is described. The assay requires a sample volume of 500 muL for a kinetic ammonia measurement. The reaction is initiated with glutamate dehydrogenase and the rate of depletion of NADPH is monitored with two measurements, 17 s apart, at 340 nm. Reaction conditions have been optimized for maximum sensitivity through both one-factor-at-a-time and multiple variable response surface optimization techniques. Linearity to 1000 mumol of ammonia per liter of plasma has been achieved. No significant interferences were observed from anticoagulants or endogenous blood components, including pyruvate and oxalacetate. Use of the coenzyme NADPH (instead of NADH) in this aca procedure eliminates the lengthy pre-incubation otherwise required for endogenous dehydrogenase reactions.
Relation of creatine kinase isoenzyme MB to postoperative electrocardiographic diagnosis in patients undergoing coronary-artery bypass surgery.
Authors: Roe C R , Wagner G S , Young W G , Curtis S E , Cobb F R , Irvin R G
Published: January 2020

We compared (a) the frequency of detection of isoenzyme MB of creatine kinase (CK; EC 2.7.3.2) in serum of patients undergoing coronary-artery bypass surgery, (b) the interval during uhich its activity was supranormal in serum, and (c) an index of the amount of CK released into blood ("CK-MB area") with postoperative electrocardiographic changes in 80 patients. The frequency of detection of CK-MB is a function of frequency of sampling during the early postoperative period. Because the duration of appearance and the calculated CK-MB area increased as the electrocardiogram became more specific for infarction (p less than 0.01), a twice-daily sampling schedule proved clinically relevant. Only 5.4% of patients had electrocardiographic evidence of infarction when CK-MB was absent by the second postoperative morning. When CK-MB was still detected at that time, 69.6% of patients had persistent new Q waves, consistent with infarction. In three patients who died postoperatively, significant myocardial necrosis was demonstrated. All three had had persistently increased values for CK-MB, related to electrocardiographic changes of infarction in one patient and ischemic changes in two. Evidently CK-MB is a more sensitive indicator of myocardial necrosis than the electrocardiogram and CK-MB area should be a useful criterion in evaluating methods of intra-operative myocardial protection.
Increase in dialyzable calcium associated with therapy with lithium.
Authors: Toffaletti J , McComb R B , Bowers G N
Published: January 2020

We measured total and dialyzable calcium concentrations in consecutive sera submitted for routine lithium analysis. Of 98 samples from 61 different individuals, six (6.1%) total calcium results and 32 (33%) dialyzable calcium results were above the respective reference intervals. By comparison, when both total and dialyzable calcium were measured on 50 different apparently healthy volunteers, no results were outside either reference interval (2.20--2.58 mmol/L for total and 1.30--1.47 mmol/L for dialyzable calcium). These increases were not due to age or sex differences between the patients and controls. From the dialyzable calcium data, there appears to be an even higher incidence of mild hypercalcemia in patients receiving oral lithium salts than is indicated by the total calcium concentration alone.
Comparison of theophylline concentrations in venous blood and capillary blood in asthmatic children.
Authors: Umstead G S , Rosen J P
Published: January 2020

Twenty children, five to 16 years old, diagnosed as having asthma, were studied to compare the serum concentrations of theophylline obtained by standard venipuncture with capillary concentrations obtained by finger lancet. The mean theophylline concentration in venous samples was 9.27 mg/L and in capillary samples was 9.26 mg/L. Comparison by the paired t-test showed no statistical difference between kinds of samples. When surveyed as to their preference, nine patients preferred the venous method, five the capillary method, and six had no preference.
A commercial digoxin radioassay modified for use as an emergency ("stat") procedure.
Authors: Chen I W , Sperling M , Maxon H R
Published: January 2020

A recent "stat" procedure recommended by the manufacturer for use with a commercial digoxin radioassay involves doubling the amount of antibody used in the regular procedure, to compensate for the decrease in binding that results from use of a shorter (10 min) incubation. This has two obvious disadvantages: increased material cost and decreased sensitivity because of the increase in the binding capacity of the assay. In our modified procedure, we kept the binding capacity constant by decreasing the volume of the incubation mixture without increasing the amount of antibody. Total binding and the rate of binding on 10-min incubation are 50.4 and less than 1.3% per minute, respectively. Results by our modified stat procedure compare well with those by the manufacturer's suggested regular procedure in terms of sensitivity, accuracy, and precision, and are more sensitive and economical than those by the manufacturer's suggested stat procedure.
Muscle protein analysis. I. High-resolution two-dimensional electrophoresis of skeletal muscle proteins for analysis of small biopsy samples.
Authors: Giometti C S , Anderson N G , Anderson N L
Published: January 2020

We have been developing a clinically useful method for high-resolution two-dimensional electrophoretic analysis of small (5--10 mg) human muscle biopsy samples with sufficient resolution to resolve the major contractile proteins and enzymes. Using rabbit psoas muscle as a model, we describe methods for sample preparation and two-dimensional electrophoresis. Basic proteins, which appear as streaks when conventional isoelectric focusing is used in the first dimension, are resolved through a modification of the nonequilibrium pH gradient electrophoresis method [Cell 12, 1133 (1977)]. In the two-dimensional patterns obtained from rabbit muscle, we identify the components of 10 enzymes and of myosin, actin, tropomyosin, and troponin. These patterns indicate charge heterogeneity in a large fraction of the proteins. Comparison of rabbit and normal human muscle patterns shows many similarities, but much additional work is required to confirm identifications. We conclude that analysis of small biopsy samples is feasible, but that all aspects of human sample acquisition, storage (when necessary), and preparation require thorough study before the method becomes routine in human muscle research and, ultimately, in the diagnosis of some muscle diseases.
Determination of manganese in whole blood and serum.
Authors: Pleban P A , Pearson K H
Published: January 2020

We describe methods for determination of manganese in whole blood and serum with Zeeman-effect flameless atomic absorption spectroscopy. These analyses are performed on a twofold or fourfold dilution of the specimen in Triton X-100, 1 g/L. No predigestion or extraction procedures are required. The method of standard additions was used for quantitation. Within-run coefficients of variation for whole-blood manganese were 7.0 and 5.5% for 2.29 and 5.67 micrograms/L, respectively. For determination of serum manganese, coefficients of variation were 10.3 and 5.3% for 0.97 and 3.01 micrograms/L, respectively. Manganese detection limits for the assays were 3.0 pg. Whole-blood manganese concentrations, determined for 60 subjects, yielded a mean (+/- SD) of 9.03 (+/- 2.25) micrograms/L. Mean serum manganese concentration, determined for 20 subjects, was 1.82 (+/- 0.64) microgram/L. No correlation was found between blood manganese concentrations and age, sex, or smoking status.
Response-surface co-optimization of reaction conditions in clinical chemical methods.
Authors: Rautela G S , Snee R D , Miller W K
Published: January 2020

We describe the theory and applications of the response-surface approach to simultaneous optimization (co-optimization of multiple interdependent variables. The co-optimization experiments are designed according to a selected response-surface model. Computer-assisted analysis includes fitting the data to the model, testing the resulting fit for statistical validity, and plotting contour maps of the model for simple interpretation. Co-optimizations of the reaction parameters of three methods--creatine kinase, lipase, and aspartate aminotransfrease--are discussed to illustrate the application of the approach. In contrast to the commonly used optimization strategies in which each factor is varied in turn while the others are kept constant, the response-surface approach allows study of several responses (reaction rates, sensitivities) and effects (linear, curvature, interaction) at the same time. It also allows determination of accurate optima, which is necessary for the formulation of analytically reliable clinical methods.
The performance of delta check methods.
Authors: Sheiner L B , Wheeler L A , Moore J K
Published: January 2020

The percentage of mislabeled specimens detected (true-positive rate) and the percentage of correctly labeled specimens misidentified (false-positive rate) were computed for three previously proposed delta check methods and two linear discriminant functions. The true-positive rate was computed from a set of pairs of specimens, each having one member replaced by a member from another pair chosen at random. The relationship between true-positive and false-positive rates was similar among the delta check methods tested, indicating equal performance for all of them over the range of false-positive rate of interest. At a practical false-positive operating rate of about 5%, delta check methods detect only about 50% of mislabeled specimens; even if the actual mislabeling rate is moderate (e.g., 1%), only abot 10% of specimens flagged a by a delta check will actually have been mislabeled.
Is antiglycolysis required for routine glucose analysis?
Authors: Sazama K , Robertson E A , Chesler R A
Published: January 2020

We obtained -68 pairs of simultaneously drawn serum and fluoride-oxalate plasma samples from patients and analyzed them by a continuous-flow (AutoAnalyzer II) glucose oxidase method. Glucose concentrations ranged from 370 to 3530 mg/L. Glucose concentrations for samples obtained in the fluoride-oxalate preservative averaged 42 +/- 35 mg/L (mean +/- SD) higher than serum. The magnitude of this difference was independent of glucose concentration. Linear-regression analysis of 270 pairs for which the time from collection to separation was recorded indicated that the difference between serum and plasma increased by 0.32 mg/L per minute of delay over a time span of 15 to 295 min. These differences are smaller than those described in standard textbooks. We conclude that, with the specimen-handling process used in our hospital, serum glucose determinations are clinically acceptable.
Analysis for underivatized theophylline by gas-chromatography on a silicone stationary phase, SP-2510-DA.
Authors: Schwertner H A
Published: January 2020

I describe a flame-ionization gas-chromatographic method for the quantitative analysis of serum theophylline without the need for derivatization. The analysis is performed isothermally on a new silicone stationary phase, 2% SP 2510-DA (Supelco). With this liquid phase, gas-chromatographic properties and sensitivities are comparable to methods requiring derivatization. A salt-solvent pair of ammonium sulfate and methylene chloride/hexane/acetic acid (80:20:0.1 by vol) is used for extraction. Ammonium sulfate greatly reduces co-extractable interferences from icteric, lipemic, and hemolyzed serum. The density of methylene chloride is adjusted with hexane so that the solvent is less dense than the salt-saturated serum and can be removed by decanting. Acetic acid, used to adjust the pH of the serum samples, is combined with the extraction solvent rather than being added individually to the serum samples. Theophylline values obtained by this method agree closely with those determined by enzyme immunoassay (correlation coefficient, 0.988).
Radioimmunoassay of conjugated cholic acid, chenodeoxycholic acid, and deoxycholic acid from human serum, with use of 125I-labeled ligands.
Authors: Mäentausta O , Jänne O
Published: January 2020

We describe a method for radioimmunoassay of conjugated cholic acid, chenodeoxycholic acid, and deoxycholic acid in serum. In the method, 125I-labeled bile acid conjugates are used as the tracers along with antibodies raised against individual bile acid-bovine serum albumin conjugates. Antibody-bound and free bile acids were separated by polyethylene glycol precipitation (final concentration, 125 g/L). Before radioimmunoassay, 0.1-mL serum samples were precipitated with nine volumes of ethanol, and portions from the supernate were used in the assays. The lowest measurable amounts of the bile acids, expressed as pmol/tube, were: cholic acid conjugates, 2; chenodeoxycholic acid conjugates, 0.5; and deoxycholic acid conjugates. 2. Analytical recovery of bile acids added to bile acid-free serum ranged from 85 to 110%; intra-assay and inter-assay CVs ranged from 3.2 to 5.3% and from 5.3 to 12.2%, respectively. Concentrations (mean +/- SD) of the bile acid conjugates in serum from apparently healthy women and men (in mumol/L) were: cholic acid conjugates, 0.43 +/- 0.17 (n = 126); chenodeoxycholic acid conjugates, 0.47 +/- 0.23 (n = 111); and deoxycholic acid conjugates, 0.33 +/- 0.11 (n = 96). The values for primary bile acids were greatly increased in patients with various hepatobiliary diseases.
Lactate-to-pyruvate or pyruvate-to-lactate assay for lactate dehydrogenase: a re-examination.
Authors: Howell B F , McCune S , Schaffer R
Published: January 2020

The pyruvate-to-lactate assay for determining lactate dehydrogenase (EC 1.1.1.27) can now yield linearity equal to or better than that obtained by the lactate-to-pyruvate assay. In addition, there are significant advantages to the pyruvate-to-lactate reaction: (a) a greater change in absorbance per unit time, which allows more accurate spectrophotometric readout; (b) lower reactant concentrations are required, which substantially reduces the cost per assay; (c) solid reagents are used to prepare the assay solution; and (d) reagent solutions are more stable. However, impurities present in commercial NADH preparations may substantially affect measured lactated dehydrogenase activities; therefore, a Standard Reference Material for NADH is being developed for issuance by the National Bureau of Standards.
Stability of dilute solutions of gentamicin and tobramycin.
Authors: Josephson L , Houle P , Haggerty M
Published: January 2020

Dilution of sera containing gentamicin or tobramycin in glass containers results in substantial adsorption of the antibiotic to the surface of the container, which can be prevented if acidic or basic solutions are used to dilute such sera. We describe protocols for radioimmunoassays of gentamicin and tobramycin that include solutions and containers that obviate this problem.
Activities of enzymes in plasma should be measured at 37 degrees C.
Authors: Duggan P F
Published: January 2020

There are considerable differences of opinion as to the most desirable temperature at which activities of enzymes in plasma should be measured. Debate centers around 25 degrees C because of its widespread use in physico-chemical measurements, 30 degrees C because of its supposed technical convenience, and 37 degrees because of its physiological significance. There is, however, widespread agreement that the activities of intracellular enzymes, and of enzymes having a natural function in plasma, be measured at 37 degrees C. Perhaps because estimations of the activities of those enzymes in plasma that have leaked from damaged tissues have been subject to almost continuous modifications, temperature has simply become just another variable to take into account. In this article I describe the relevance of a temperature near 37 degrees C to the activities of all enzymes in man, intracellular and extracellular. I conclude that 37 degrees C is to be favored as the temperature for assaying all parameters in human biochemistry affected by temperature. These include not only enzyme activities but also pH, blood-gas equilibria, binding data, and, in fact, all kinetic activities in the body.
Fluoroimmunoassay: present status and key problems.
Authors: Soini E , Hemmilä I
Published: January 2020

Fluorescence immunoassay of biological fluids (for example, blood samples) is discussed. We attempt to chart present methods of assay as well as new possibilities. Different fluorescent probes, their detection limit, and methods for reduction of background are discussed; methods for separating the free and bound fraction are also reviewed. Special consideration is given to the possibilities of enhancing sensitivity by developing both instruments and chemical methods, and in particular to the possibilities inherent in time-resolved fluorometric applications and to the use of metal chelates in this application.
Influence of a between-run component of variation, choice of control limits, and shape of error distribution on the performance characteristics of rules for internal quality control.
Authors: Westgard J O , Falk H , Groth T
Published: January 2020

A computer-stimulation study has been performed to determine how the performance characteristics of quality-control rules are affected by the presence of a between-run component of variation, the choice of control limits (calculated from within-run vs. total standard deviations), and the shape of the error distribution. When a between-run standard deviation (Sb) exists and control limits are calculated from the total standard deviation (St, which includes Sb as well as the within-run standard deviation, Sw), there is generally a loss in ability to detect analytical disturbances or errors. With control limits calculated from Sw, there is generally an increase in the level of false rejections. The presence of non-gaussian error distribution appears to have considerably less effect. It can be recommended that random error be controlled by use of a chi-square or range-control rule, with control limits calculated from Sw. Optimal control of systematic errors is difficult when Sb exists. An effort should be made to reduce Sb, and this will lead to increased ability to detect analytical errors. When Sb is tolerated or accepted as part of the baseline state of operation for the analytical method, then further increases in the number of control observations will be necessary to achieve a given probability for error detection.
Liquid-chromatographic determination of acetaminophen in serum.
Authors: Fletterick C G , Grove T H , Hohnadel D C
Published: January 2020

We describe a sensitive and precise "high-pressure" liquid-chromatographic method for determining acetaminophen in serum. The 2-acetaminophenol and 3-acetaminophenol structural isomers of acetaminophen are used as internal standards. The method, which involves solvent extraction and adsorption chromatography on silica, provides excellent sensitivity, accuracy, and selectivity. The standard curve is linear over the range of acetaminophen concentrations of 0.5 to 300 mg/L, which makes the method useful for both pharmacokinetic studies and overdose monitoring. Analytical recovery is 97% for acetaminophen concentrations ranging from 5 to 300 mg/L. Many commonly used drugs were tested and found not to interfere. The procedure has been successfully adapted as a microscale method requiring only 50 microL of sample. The microscale method is particularly useful for pediatric and neonatal patients for whom sample size is a major concern.
Cytosine and orotic acid in urine of immunodeficient children.
Authors: Mills G C , Schmalstieg F C , Newkirk K E , Goldblum R M
Published: January 2020

We describe procedures for determining cytosine and orotic acid in urine. We determine cytosine by cation-exchange analysis with either HCl or pH 5.2 buffer as eluent. Orotic acid is first separated by an anion-exchange separative procedure; after lyophilization, the product is subjected to "high-pressure" liquid chromatography for further separation and detection. We analyzed urine from normal subjects and from immunodeficient children. Three children with severe combined immunodeficiency had increased levels of cytosine in urine (23-160 mmol/mol creatinine); one child with severe combined immunodeficiency and two children with other immunodeficiencies had normal urinary levels (less than 2 mmol/mol creatinine). Orotic acid excretion in urine was normal (1-5 mmol/mol creatinine) in all of th immunodeficient children. We discuss the possible significance of the increased cytosine excretion in the three children with severe combined immunodeficiency.
Importance of blood-collection tubes in plasma lidocaine determinations.
Authors: Stargel W W , Roe C R , Routledge P A , Shand D G
Published: January 2020

In 25 clinical samples serum lidocaine concentrations fell from a mean of 6.5 +/- 2.1 mg/L (mean +/- SD) to 4.9 +/- 1.8 mg/L (p less than 0.001) when the blood sample was allowed to make contact with the stopper of the Vacutainer collection tube. In vitro experiments showed that this effect of the stopper occurred only with whole blood and was dependent on sample concentration. The plasma binding of lidocaine decreased from a normal value of 56% +/- 2.2 (mean +/- SD) to 28% +/- 2.2 (p less than 0.001) when exposed to the Vacutainer stopper. We conclude that a chemical leached from such stoppers displaces lidocaine from its plasma-binding sites and that the drug is then redistributed into the erythrocytes, producing spuriously low lidocaine concentrations in plasma or serum. Such artifacts are important in therapeutic drug monitoring and can lead to erroneous clinical decisions.
Relative stabilities of purified human mitochondrial and cytoplasmic isoenzymes of aspartate aminotransferase in lyophilized materials.
Authors: Sampson E J , McKneally S S , Whitner V S , Burtis C A , Bayse D D
Published: January 2020

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.
A simple, direct radioimmunoassay for plasma cortisol, featuring a 125I radioligand and a solid-phase separation technique.
Authors: Riad-Fahmy D , Read G F , Gaskell S J , Dyas J , Hindawi R
Published: January 2020

A simple, direct radioimmunoassay for cortisol in human serum and plasma is described. An antiserum, raised in sheep to a cortisol-3-(O-carboxymethyl)oxime/bovine serum albumin conjugate, is coupled to microcellulose. No extraction is required because plasma samples and standards are incubated with the antiserum and an 125I radioligand in a low-pH buffer, which denatures cortisol-binding globulins. The assay satisfies accepted validation criteria. In addition, results from the radioimmunoassay compare well with those obtained by a gas chromatographic-mass spectrometric technique (r = 0.968; FRIA = 0.97 FGCMS + 2.0 nmol/L). The latter procedure features the very high intrinsic specificity obtained by selected ion monitoring at high mass-spectrometric resolution (M/deltaM = 8500) with a Varian MAT-731 instrument. The simplicity of the radioimmunoassay procedure, with use of reagents prepared "in house," makes this a very practical and economical assay for use in the medium or large endocrine laboratory.
Outliers in clinical chemistry quality-control schemes.
Authors: Healy M J
Published: January 2020

Results submitted to large-scale quality-control schemes are commonly judged against the mean and standard deviation (SD) of the results from other laboratories. It is desirable to ignore outlying values in estimating this mean and standard deviation, and results more than 2.5 or 3 SD from the mean are commonly rejected. I show that an outlier can so inflate the estimated SD that its presence is not detected by this method. Alternative estimators that are less influenced by outliers are described, and their application to quality-control data is discussed.
Elemental composition of platelets. Part III. Determination of Ag, Au, Cd, Co, Cr, Cs, Mo, Rb, Sb, and Se in normal human platelets by neutron activation analysis.
Authors: Kasperek K , Iyengar G V , Kiem J , Borberg H , Feinendegen L E
Published: January 2020

The elements Ag, Au, Cd, Co, Cr, Cs, Mo, Rb, Sb, and Se were determined in platelets from seven normal donors. The results, in ng/g wet weight, for plasma-free platelets follow: "Pure" platelets: Ag = 29 +/- (18), au = 0.22 +/- (0.22), Cd = 6.2 +/- 3.4, Cs = 54.8 +/- 19.2, Cr = 6.1 +/- 2.5, Co = 7.5 +/- (5.0), Mo = 3.4 +/- 1.3, Rb = 10400 +/- 3000, Sb = 18 +/- (26), and Se = 782 +/- 127. "Impure" platelets: Au = 0.23 +/- (0.28), Cd = 6.4 +/- 2.6, Cs = 35.2 +/- 13.8, Cr = 8.2 +/- 2.9, Co = 2.9 +/- (3.0), Mo = 3.2 +/- 0.8, Rb = 8700 +/- 1700, Sb = 13.2 +/- (8.7), and Se = 679 +/- 57. To our knowledge, none of these 10 trace elements has been determined in platelets before. The selenium concentration in platelets exceeds that in other tissues (e.g., liver). We suggest that glutathione peroxidase or other unknown selenoenzymes are particularly important in platelet metabolism. Platelets are crucial for triggering thrombosis, and so may be involved as links between selenium deficiency and the concomitant increased death rate from cardiovascular disease.
Limitations of immunochemical measurement of ceruloplasmin.
Authors: Buffone G J , Brett E M , Lewis S A , Iosefsohn M , Hicks J M
Published: January 2020

We describe a quantitative immunochemical method for the nephelometric measurement of ceruloplasmin. Results are compared with those from a radial immunodiffusion technique. We examine factors affecting the results, such as antiserum source and calibrators, and list reference limits for a pediatric population.
Evaluation of automated enzyme immunoassays for five anticonvulsants and theophylline adapted to a centrifugal analyzer.
Authors: Urquhart N , Godolphin W , Campbell D J
Published: January 2020

We report a clinical evaluation of the enzyme immunoassay (EMIT) performed with the GEMSAEC centrifugal analyzer as compared to gas-liquid and liquid chromatography for anticonvulsant drugs and theophylline, respectively. A good correlation was obtained for all drugs, although some difficulties were experienced with one lot of reagent for ethosuximide. The analyzer has an economic advantage if many samples are being analyzed for few drugs in each sample.
An evaluation of the detection capacity of a computer-assisted real-time delta check system.
Authors: Sher P P
Published: January 2020

We developed of computer programs to evaluate the clinical reliability of test results by comparing each new result with previous results for the same patient, and to signal discrepancies in real time. These "delta check" discrepancies are noted, and they must be reviewed by the laboratory staff before results can appear on a patient's record. During a month, I reviewed 1403 such delta check messages and detected 55 (3.9%) that could not be explained on the basis of the patient's clinical condition. Of these, 23 represented true laboratory errors, which were corrected. The recognition of discrepancies before they appear on patients' reports has facilitated the operation of the clinical chemistry laboratory. Mislabeled and otherwise mishandled specimens are discovered before erroneous results appear on a patient's record.
Application of Bayes's theorem to results of quantitative clinical chemical determinations.
Authors: van der Helm H J , Hische E A
Published: January 2020

The diagnostic implication of a certain test result with regard to a certain condition can be expressed as a single number, L, the likelihood ratio of this result. This ratio allows Bayes's theorem to be written in a convenient form. We show that the practice of calculating predictive values for the results of quantitative tests by use of discrimination limits leads to incorrect predictive values. Including L values in laboratory reports seems a more logical approach to optimum interpretation of laboratory results than the use of discrimination values.
Creatine kinase B-subunit activity in serum after immunoinhibition of M-subunit activity.
Authors: Gerhardt W , Waldenström J
Published: January 2020

Creatine kinase (EC 2.7.3.2) B-subunit activity in serum may be routinely measured as residual activity after specific immunoinhibition of the M-subunit. We assessed the inhibition kinetics, specificity, completeness of inhibition, and inhibitory capacity of three different anti-M preparations, with use of isolated human BB, MM, and MB isoenzymes. The Scandinavian-recommended reaction system was used. We suggest a set of tentative quality requirements for anti-M for use in diagnosing acute myocardial infarction. The need to measure and subtract sample residual adenylate kinase activity was demosntrated. We describe a routine photometric method for determining B-subunit activity in serum. With the Scandinavian CK method the upper reference value for total creatine kinase in serum was found to be 150 U/L for women, 270 U/L for men. By bioluminescence, we found the upper reference value for B-subunit activity to be 6 U/L for both sexes. We discuss three different modes for applying B-subunit determinations to the diagnosis of acute myocardial infarction.
Urinary excretion of conjugated homovanillic acid, 3,4-dihydroxyphenylacetic acid, p-hydroxyphenylacetic acid, and vanillic acid by persons on their usual diet and patients with neuroblastoma.
Authors: Muskiet F A , Groen A
Published: January 2020

We report quantitative data on beta-glucuronidase- and sulfatase-hydrolyzable conjugates of homovanillic acid, 3,4-dihydroxyphenylacetic acid, p-hydroxyphenylacetic acid, and vanillic acid in the urine of 20 apparently normal and healthy control persons and of three patients with neuroblastoma. We used organic solvent extraction and capillary gas chromatography. There was considerable person-to-person variation in the conjugation percentages calculated. Mean conjugated percentages of the four compounds for 16 normal healthy persons 2.5--40 years of age were, respectively, 12%, 33%, 14%, and 35%. For newborns and patients with neuroblastoma, these percentages were somewhat different. Increased amounts of vanillic acid were found in the urine of the patients with neuroblastoma, but results of a small metabolic study in rats suggest that this increase most probably is of dietary origin.