bluuebunny/crossref_metadata_2025 huggingface dataset:
Production and certification of an enzyme reference material for gamma-glutamyltransferase (CRM 319). Part 1: Preparation and characterization.
Authors: Schiele F , Muller J , Colinet E , Siest G
Published: January 2020

We have produced a batch of lyophilized gamma-glutamyltransferase as enzyme reference material. The "light" enzyme form was purified from pig kidney to a relatively high specific activity (120 kU/g) and was essentially free of contaminating enzymes. The partly purified gamma-glutamyltransferase, lyophilized in a matrix containing bovine serum albumin (Fraction V, 60 g/L), yielded a batch of 4000 ampules and was stored at -20 degrees C. The vial-to-vial variability with respect to the catalytic concentration of the final product (CV 0.6%) and its stability (predicted loss of activity at -20 degrees C was less than 0.01% per year) were considered sufficient to allow the use of this preparation for a certification procedure. The behavior of the reference material in comparison with human serum samples was evaluated three ways: (a) by kinetic characteristics, (b) by the ratio of activities for duplicate determinations by different methods, and (c) by use of the reference material to convert values obtained by various methodologies to those by the IFCC proposed method. The material appeared to be commutable for the two methods studied. The difference in the ratios obtained for patients' samples and reference material was less than +/- 5%, and the recalculated values for patients' samples as determined with the reference material differed from values determined by the IFCC method by no more than 4.8%.
Production and certification of an enzyme reference material for gamma-glutamyltransferase (CRM 319). Part 2: Certification campaign.
Authors: Schiele F , Muller J , Colinet E , Siest G
Published: January 2020

We describe the process of certification for a gamma-glutamyltransferase reference material (CRM no. 319). Fifteen laboratories participated to this interlaboratory evaluation. All steps of the measurements were controlled in an effort to locate potential sources of variations. In particular, the exclusion of some data was strictly documented or justified by the non-observance of the IFCC method and (or) discrepancies in instrumentation, reconstitution of the lyophilized samples, or measurement technique. Inaccuracy in the reconstitution of the lyophilized material was +/- 0.68%, and the molar absorptivity of the 5-amino-2-nitrobenzoate reported by each laboratory was within +/- 2% limits of the value reported by the IFCC. Calculated from the sets of accepted results, the total CV among samples was 2.6% and the overall CV was 3.2%. Within-day and between-day CVs were 1.1% and 1.4%, respectively. The greatest variation for a single component was the between-laboratory variability (CV 3.1%); the within-laboratory CV, including the day effect, was 1.8%. Finally, the certified value for the catalytic concentration of this enzyme in the reconstituted lyophilized reference material was 86.8 U/L with an uncertainty of +/- 2.1 U/L (0.95 confidence interval). The uncertainty appeared to be compatible with the end-use of this reference material.
Biotin radioligand assay with an 125I-labeled biotin derivative, avidin, and avidin double-antibody reagents.
Authors: Livaniou E , Evangelatos G P , Ithakissios D S
Published: January 2020

We describe a new radioligand assay for determining biotin in biological fluids by using a mixture of N-[beta-(4-OH-3-125I-phenyl)ethyl]- and N-[beta-(4-OH-3,5-di-125I-phenyl)ethyl]biotinamides as radiotracer, avidin as a binding protein, and an avidin double-antibody as a separation reagent. The radiotracer is synthesized by coupling (at pH 8.5, 20-22 degrees C, 90 min) N-hydroxysuccinimidobiotin to radioiodinated tyramine. The assay curve is linear and the assay itself is sensitive (less than 10 ng/L), reproducible (intra- and interassay CVs 4.1% and 7.0%, respectively), and allows the simultaneous handling of more than 100 samples in less than 4 h. Serum samples from apparently normal subjects contained 100-840 ng of biotin per liter (mean 340 ng/L). Pregnant women had low concentrations of biotin (100-300 ng/L) in their serum. Patients undergoing chronic hemodialysis treatment showed high concentrations (0.5-3.0 micrograms/L), which may be ascribable to the inability of avidin, which was used as the assay binding protein, to distinguish biotin from biotinyl derivatives with an intact ureido ring.
An examination of heart proteins by two-dimensional electrophoresis.
Authors: Gianazza E , Osio L , Grazioli G , Astrua-Testori S , Righetti P G , Accinni R , Renoldi I , Repossini A
Published: January 2020

We examined specimens from explanted human hearts by two-dimensional electrophoresis. The protocol selected includes: (a) solubilization of the sample in a urea-detergent mix; (b) charge fractionation in the presence of urea and nonionic detergent on a pH 4-10 immobilized pH gradient; (c) size fractionation on a polyacrylamide concentration gradient in the presence of sodium dodecyl sulfate; and (d) staining with silver nitrate. The method is sensitive enough for analysis of biopsies in the 1-3 mg range (wet tissue). We saw, for explanted hearts, variations in the protein pattern with the site of sample dissection. Results are presented for 11 explanted human hearts: one control organ and 10 pathological samples. The recorded pathologies included dilatative cardiomyopathy (six cases), valvulopathy (one case), ischemic cardiopathy (two cases), and graft rejection (one case). The patterns for whole extracts as well as for cytoplasmic proteins and myofibril components are compared. Extensive individual variability was observed both between control and pathological cases and among the abnormal samples.
Selecting the optimum specimen for assessing slight albuminuria, and a strategy for clinical investigation: novel uses of data on biological variation.
Authors: Howey J E , Browning M C , Fraser C G
Published: January 2020

To investigate the optimum specimen for quantifying low, but abnormal, concentrations of albumin in urine, we assessed the analytical and biological components of variation in first morning, random untimed, and 24-h urine specimens from 11 apparently healthy individuals. The results were expressed in terms of albumin concentration, albumin/creatinine ratio, and albumin excretion rate. Analytical methods generally can meet the analytical goal of CV less than or equal to 18%. For reasons detailed herein, we prefer measurement of the albumin concentration in the first morning specimen. Expressing results as an albumin/creatinine ratio has little advantage. Albumin concentrations in first morning urines from 16 diabetic subjects showed larger intra-individual variation than for nondiabetic subjects but clearly fell into two groups: those with consistently normal albumin concentrations in urine and those with abnormal concentrations in some specimens. The intrinsic biological variation of the latter group means that the ideal 100% nosological specificity cannot be achieved with any cutoff point without inclusion of a large proportion of the former. Qualitative testing with a latex-agglutination technique also demonstrates this problem. Use of data on biological variation allows development of an appropriate clinical strategy to investigate diabetic patients.
Free cortisol and creatinine in urine of healthy children.
Authors: Bertrand P V , Rudd B T , Weller P H , Day A J
Published: January 2020

Cortisol and creatinine were measured in two consecutive overnight urine collections from 103 healthy school children, ages seven to 18.5 years. Mean cortisol and creatinine concentrations were respectively 194 nmol/L and 12.7 mmol/L. The data were statistically analyzed to simultaneously assess any effects of sex, age, weight, day of collection, and urine volume. Mean urine volume for boys exceeded that for girls, increased with body weight, and was greater on the second day of collection than on the first. Cortisol concentration was independent of sex, age, and weight, but decreased with urine volume. Boys excreted more cortisol than did girls, and the amount increased with urine volume. Creatinine concentration increased with weight, decreased with urine volume. Total creatinine increased with weight, was greater for boys than girls, and increased with urine volume. The cortisol/creatinine ratio was valueless as an index of adrenocortical status.
Determination of unconjugated 3-methoxy-4-hydroxyphenylglycol by liquid chromatography for monitoring inhibition of monoamine oxidase activity in plasma.
Authors: Gupta R N , Steiner M , Lew M
Published: January 2020

We describe a simple method for extracting 3-methoxy-4-hydroxyphenylglycol (MHPG) from plasma. The sample is applied to a 1-mL "Extrelut" column (EM Industries) and eluted 15 min later with ethyl acetate. After mixing the eluate with pentane and back-extraction into water, we inject 25 or 50 microL of the aqueous phase onto a Beckman 15 cm x 4.6 mm (i.d.) column packed with 5-micron ODS particles (Beckman). Peaks are detected with a coulometric detector. The resulting chromatogram is clean, with few extraneous peaks. The extraction recovery of MHPG is 55-60%, which allows for detection of as little as 0.2 microgram of the analyte per liter. The decrease in the concentration of MHPG in plasma of patients treated with phenelzine agrees with the decrease in monoamine oxidase activity in platelets of these patients.
Analytical and physiological characteristics of prostate-specific antigen and prostatic acid phosphatase in serum compared.
Authors: Schifman R B , Ahmann F R , Elvick A , Ahmann M , Coulis K , Brawer M K
Published: January 2020

We did a comparative analysis of the physiological and analytical properties of prostate-specific antigen (PSA), acid phosphatase (ACP; EC 3.1.32) activity, and acid phosphatase antigen (PAP) in serum. The PSA assay is sensitive to 0.2 microgram/L and demonstrates good linearity (y = 1.01x + 0.74). The CV was 3.9% at 40 micrograms/L, 8.0% at 3.1 micrograms/L. PSA and PAP are less stable at 4 degrees C than at -20 degrees C. Serum PAP and ACP concentrations showed large intra-individual fluctuations (average CVs of 22% and 24%, respectively), which were not observed with PSA measurements (average CV 6.2%). We saw significant correlation with the magnitude of physiological change when analytes were compared for serially collected split samples [y(PSA) = 0.14x(PAP) + 0.00, r = 0.767], which indicates that a common factor is influencing this variation. The excellent analytical performance, tissue specificity, and small degree of intra-individual variance are characteristics that favor the measurement of PSA in serum for monitoring patients with prostatic cancer.
Improved measurement of urinary total protein (including light-chain proteins) with a Coomassie brilliant blue G-250-sodium dodecyl sulfate reagent.
Authors: Wimsatt D K , Lott J A
Published: January 2020

The Coomassie Brilliant Blue G-250 method for urinary proteins underestimates urinary immunoglobulin light chains when albumin or pooled serum is used as the protein standard. The specific color yields of these and other proteins can be brought closer together by adding sodium dodecyl sulfate to the reagent; however, there is some loss of sensitivity. We found such a reagent to be satisfactory for assaying urinary proteins on studying 43 patients with light-chain proteinuria, 19 of whom had multiple myeloma and six possible multiple myeloma.
Diagnostic performance of sensitive measurements of serum thyrotropin during severe nonthyroidal illness: their role in the diagnosis of hyperthyroidism.
Authors: Bayer M F , Macoviak J A , McDougall I R
Published: January 2020

Serum thyrotropin (TSH) concentrations were measured serially in 14 heart-transplant recipients (group 1) and 21 patients undergoing coronary artery bypass surgery (group 2), all without thyroid disease, and randomly in 158 patients hospitalized for various other nonthyroidal illnesses, including 144 judged euthyroid (group 3), six with increased FT4 and (or) T3 (group 4), and eight classified hypothyroid by conventional tests. The serial measurements indicated profound fluctuations. In group 1, TSH was subnormal in 21% of studies and increased in 10%. In group 2, corresponding abnormalities were found in 7% and 13%, respectively. Transiently low or high TSH tended to be associated with normal free thyroxin (FT4), prolonged subnormal TSH (greater than 1 week) with subnormal FT4. By contrast, subnormal TSH plus elevated FT4, or high TSH plus low FT4, were not encountered, making it unlikely that they occur by chance in severely ill patients who are not also hyper- or hypothyroid. In group 3, a suppressed TSH (plus borderline high FT4, T3/FT3) identified four cases of subclinical hyperthyroidism; however, another 11% of patients had subnormal and 10% had above-normal TSH, paired with normal FT4 and no evidence of thyroid disease. In group 4, suppressed TSH confirmed hyperthyroidism in five of six patients, and all in group 5 had increased TSH. We conclude that, in the hospital setting, sensitive TSH measurement can help to detect or confirm mild hyperthyroidism, but the positive predictive value of TSH alone may be as low as 35%.
Flow-cytometric determination of high-density-lipoprotein binding sites on human leukocytes.
Authors: Schmitz G , Wulf G , Brüning T , Assmann G
Published: January 2020

In this method, leukocytes were isolated from 6 mL of EDTA-blood by density-gradient centrifugation and subsequently incubated with rhodamine isothiocyanate (RITC)-conjugated high-density lipoproteins (HDL). The receptor-bound conjugate particles were determined by fluorescent flow cytometry and compared with 125I-labeled HDL binding data for the same cells. Human granulocytes express the highest number of HDL binding sites (9.4 X 10(4)/cell), followed by monocytes (7.3 X 10(4)/cell) and lymphocytes (4.0 X 10(4)/cell). Compared with conventional analysis of binding of 125I-labeled HDL in tissue-culture dishes, the present determination revealed significantly lower values for nonspecific binding. In competition studies, the conjugate competes for the same binding sites as 125I-labeled HDL. With the use of tetranitromethane-treated HDL3, which fails to compete for the HDL receptor sites while nonspecific binding is not affected, we could clearly distinguish between 37 degrees C surface binding and specific 37 degrees C uptake of RITC-HDL3, confirming that the HDL receptor leads bound HDL particles into an intracellular pathway rather than acting as a "docking" type of receptor. Patients with familial dysbetalipoproteinemia showed a significantly higher number of HDL binding sites in the granulocyte population but normal in lymphocytes and monocytes, indicating increased uptake of cholesterol-containing lipoproteins. In patients with familial hypercholesterolemia, HDL binding was increased in all three cell types, indicating increased cholesterol uptake and increased cholesterol synthesis. The present method allows rapid determination of HDL binding sites in leukocytes from patients with various forms of hyper- and dyslipoproteinemias.
Concentrations of superoxide dismutase and superoxide anion in blood of patients with respiratory infections and compromised immune systems.
Authors: Umeki S , Sumi M , Niki Y , Soejima R
Published: January 2020

To investigate the involvement of oxygen free radicals and their scavenger systems in the defenses of compromised hosts against pulmonary infections, we determined superoxide anion (SOA) and superoxide dismutase (SOD; EC 1.15.1.1) concentrations in the blood of compromised hosts and noncompromised hosts, with or without pneumonia. In the compromised hosts without pneumonia (compromised controls), SOD concentrations were lower than in noncompromised hosts (healthy controls). However SOA values in compromised controls did not differ statistically from that in healthy controls. Similar changes were observed in noncompromised hosts with pneumonia. In compromised hosts with pneumonia, SOD concentrations were further decreased by pulmonary infections. By contrast, SOA values were increased in pneumonia. There were, however, no differences in the values for ceruloplasmin among all the groups. The values for alpha 2-macroglobulin and alpha1-antitrypsin were within normal limits in compromised controls but were greater in compromised hosts with pneumonia. These results suggest that a decreased activity concentration of SOD in compromised controls may be partly responsible for the depression of the host's immune defenses.
An international collaborative study on standardization of apolipoproteins A-I and B. Part I. Evaluation of a lyophilized candidate reference and calibration material.
Authors: Smith S J , Cooper G R , Henderson L O , Hannon W H
Published: January 2020

We evaluated a lyophilized serum preparation for use as a candidate Reference Material for apolipoproteins (apo) A-I and B. An international collaborative study was conducted with 28 participating laboratories, selected on the basis of participation and demonstrated expertise in a 1983 survey of apolipoproteins A-I and B. The analytical suitability of the material was established by confirming linearity of its dose-response curves over a desired concentration range and demonstrating that its response curves paralleled those for fresh sera. Differences in dilution-adjusted mass units ascribable to the five analytical methods used by the various laboratories constituted only 1% of the total variation for apo A-I, but 32% for apo B. The dominant source of error, however, for both apo A-I and B was the variability among laboratories, rather than variability among methods and antisera. The assigned consensus mass-concentration units based on study data are 1.124 g/L for apo A-I, 0.589 g/L for apo B. For these estimates the coefficients of variation were 13% and 27%, respectively. These findings on the proposed Reference Material meet the requirements suggested by the World Health Organization's Expert Committee on Biological Standards for a candidate WHO Reference Preparation.
Automated solid-phase extraction and liquid chromatography for assay of cyclosporine in whole blood.
Authors: Kabra P M , Wall J H , Dimson P
Published: January 2020

In this rapid, precise, accurate, cost-effective, automated liquid-chromatographic procedure for determining cyclosporine in whole blood, the cyclosporine is extracted from 0.5 mL of whole blood together with 300 micrograms of cyclosporin D per liter, added as internal standard, by using an Advanced Automated Sample Processing unit. The on-line solid-phase extraction is performed on an octasilane sorbent cartridge, which is interfaced with a RP-8 guard column and an octyl analytical column, packed with 5-microns packing material. Both columns are eluted with a mobile phase containing acetonitrile/methanol/water (53/20/27 by vol) at a flow rate of 1.5 mL/min and column temperature of 70 degrees C. Absolute recovery of cyclosporine exceeded 85% and the standard curve was linear to 5000 micrograms/L. Within-run and day-to-day CVs were less than 8%. Correlation between automated and manual Bond-Elut extraction methods was excellent (r = 0.987). None of 18 drugs and four steroids tested interfered.
Liquid-chromatographic determination of inosine, xanthine, and hypoxanthine in uremic patients receiving hemodialysis treatment.
Authors: Severini G , Aliberti L M
Published: January 2020

We used a liquid-chromatographic method to measure xanthine, hypoxanthine, and inosine in plasma of uremic patients before and after hemodialysis. The concentrations of all three were higher than in normal subjects. After dialysis treatment, the values were significantly lower than before dialysis, but still above normal.
Reference intervals for plasma lipoperoxides: age-, sex-, and specimen-related variations.
Authors: Knight J A , Smith S E , Kinder V E , Anstall H B
Published: January 2020

We measured lipoperoxides, as malondialdehyde (MDA), by liquid chromatography in plasma from 230 male and 148 female adult blood donors, to establish reliable reference values and to compare possible sex-, age-, and specimen-related differences. Our studies show that mean have higher MDA concentrations in plasma than do women (P less than 0.05), older men have higher values than younger men (P less than 0.05), and older women have higher values than young women (P less than 0.001). These age-related results support earlier studies in experimental animals that lipid peroxidation increases with increasing age. In addition, plasma from liquid EDTA-anti-coagulated blood has significantly lower MDA concentrations than does serum or plasma from blood treated with lithium heparin, sodium citrate, or CPDA-1 (P less than 0.001).
Use of a competitive inhibitor in a kinetic enzymatic method for measuring ethanol in serum.
Authors: Young E , Rafter-Tadgell B
Published: January 2020

We describe a kinetic enzymatic method for ethanol in serum, based on the use of pyrazole, a competitive inhibitor for alcohol dehydrogenase (EC 1.1.1.1). The method is rapid and is easily automated for the Cobas Bio centrifugal analyzer. No specimen pretreatment is necessary and the total reaction time is 120 s. The standard curve is linear up to 140 mmol/L. The within-run CV was between 1.9% and 3.5%; the between-run CV ranged from 2.6% to 4.1%. Mean analytical recovery of ethanol added to serum was 100.2%. We compared results of the kinetic enzymatic method with those from a gas-liquid chromatographic (GLC) method and another commercial alcohol dehydrogenase method (TDx REA Ethanol; Abbott Diagnostics). Linear regression analysis gave the following equations: kinetic = 0.991GLC - 0.354 mmol/L (r = 0.992, n = 110) and kinetic = 0.998TDx - 1.741 mmol/L (r = 0.993, n = 70). No interference from methanol or isopropanol was seen.
Medical utility frequency.
Authors: Castañeda-Méndez K
Published: January 2020

Judging the medical acceptability and comparing the medical utility of laboratory methods requires standards for reproducibility with regard to medical performance. A completely general and flexible performance standard for reproducibility consists of the reproducibility criterion, the allowable error, and the concentration value. Neither precision, accuracy, nor total error correctly addresses this standard. The medical utility frequency of a method does do so, however, its use making method evaluation and comparison simple, completely flexible, and exact.
Simple spectrophotometric determination of urinary albumin by dye-binding with use of bromphenol blue.
Authors: Schosinsky K H , Vargas M , Luz Esquivel A , Chavarria M A
Published: January 2020

This procedure for routine quantification of albumin in urine is based on the dye-binding properties of albumin with bromphenol blue. The absorbance of 100 microL of urine mixed with 3 mL of color reagent is measured against blank reagent at 610 nm after 30 s. Results vary linearly with albumin concentration up to 6 g/L. The reaction is pH independent in the physiological range. It is not subject to substantial interference by uric acid, creatinine, calcium, sodium chloride, or bilirubin. The presence of globulins produces a small positive error. Within-run precision (CV) was 4.8, 1.5, and 0.9%, and day-to-day precision was 11.2, 2.0, and 1.9%, for samples containing albumin at about 0.1, 1.0, and 6.0 g/L, respectively. Results by a radial-immunodiffusion method (x) correlated well with those by the proposed method (y): r = 0.986; y = 0.98x + 0.096; n = 64. The method can also be used to detect globulins, such as Bence Jones protein, by measuring the ratio of the absorbance at 30 min to that at 30 s.
Noninvasive evaluation of mineral content of bone without use of ionizing radiation.
Authors: Brown C E , Allaway J R , Brown K L , Battocletti J H
Published: January 2020

The mineral content of bone can be quantified by recording a 31P NMR spectrum while the bone is stationary. The quantity of mineral in the bone is determined from the spectrum with a reference standard by comparison of relative peak areas. The phosphate of bone mineral is readily distinguished from inorganic phosphate and phosphorylated metabolites dissolved in cytosol and from the head groups of phospholipids in membranes. The technical feasibility of constructing a cost-effective instrument for analysis of bone mineral content in the extremities is demonstrated. The possible utility of such a noninvasive assay for detecting osteoporosis and for monitoring the progress of treatment is discussed.
Kinetic immunochemical method for the simultaneous quantification of creatine kinase isoenzymes.
Authors: Weiser W E , Pardue H L
Published: January 2020

We describe the development and evaluation of a kinetic immunochemical method for the simultaneous quantification of isoenzymes. Specifically, we use the inhibition of the M subunit of creatine kinase (CK; EC 2.7.3.2) by antibodies to quantify isoenzymes CK-MM and CK-MB in serum matrices. Nonlinear least-squares data-processing is used to compute the enzyme activities from the time-dependent response of absorbance vs time. Variables affecting the method, namely temperature, substrate concentration, and antibody concentration as well as their interactions, are evaluated by using response-surface methodology. For several concentrations of CK-MM and CK-MB in the range of diagnostic significance, least-squares fits of computed (y) vs expected (x) values yielded equations of y = 0.98x + (2.0 X 10(-5)) s-1 for CK-MM and y = 1.04x - (4.6 X 10(-5)) s-1 for CK-MB for rates between 0 and 3.5 X 10(-3) s-1 (CK-MM) and 0.5 X 10(-3) s-1 (CK-MB). The equations for comparison of kinetic results (y) with results by a kit method (x) were y = 0.97x + (6.5 X 10(-5)) s-1 for CK-MM and y = 1.02x + (4.3 X 10(-5)) s-1 for CK-MB. Pooled day-to-day relative standard deviations (CVs) for the kinetic method were 4.1% and 2.8% for CK-MM and CK-MB, respectively.
Interference of sex-hormone binding globulin in the "Coat-A-Count" testosterone no-extraction radioimmunoassay.
Authors: Slaats E H , Kennedy J C , Kruijswijk H
Published: January 2020

We evaluated a "no-extraction" assay system for serum testosterone [Diagnostic Products "Coat-A-Count" testosterone (direct)] with respect to the influence of sex-hormone binding globulin (SHBG). We conclude that the SHBG concentration strongly affects this estimation. The quantity of testosterone measured in the presence of 90 nmol of SHBG per liter (the upper reference limit of SHBG for women) was only about 40% of that measured at the lower limit of the SHBG reference interval (30 nmol/L). For SHBG concentrations less than 30 nmol/L one can expect the testosterone concentrations measured to be relatively too high.
Salivary amylase and pancreatic enzymes in Sjögren's syndrome.
Authors: Pal B , Griffiths I D , Katrak A , Junglee D , Dandona P
Published: January 2020

Concentrations of immunoreactive trypsin (IRT) and pancreatic and salivary amylase activities were measured in 22 patients with primary Sjögren's syndrome (SS) and in 13 patients with secondary SS. Nineteen of the 22 patients with primary SS had above-normal IRT, and six had above-normal pancreatic isoamylase activity. Six of the 13 patients with secondary SS had above-normal IRT; none had above-normal isoamylase activities. Serum IRT and pancreatic isoamylase were correlated significantly (r = 0.7; p less than 0.0001). Above-normal values for IRT and pancreatic isoamylase were more frequent in patients who had SS for longer than 10 years, but were not related to the presence of salivary gland autoantibodies or to salivary isoamylase activity. We conclude that the concentration and activity of pancreatic enzymes are frequently abnormal in SS; that the abnormality is greater and more frequent in patients with primary SS; and that it increases with the duration of the disease.
Enzyme immunoassay method for comprehensive drug screening in micro-samples of urine.
Authors: Black D L , Goldberger B A , Caplan Y H
Published: January 2020

We adapted the reagents from 11 different enzyme-multiplied immunoassay technique (EMIT; Syva Co., Palo Alto, CA 94304) drug-detection kits for use in a centrifugal analyzer. The antibody reagents were mixed into a single dilute solution, and the enzyme-labeled drug derivatives were combined similarly (Mixed EMIT reagents), for use in testing urine samples for the presence of multiple drugs. The assay, a rapid comprehensive drug-screening technique, requires 100 microL of sample and is capable of testing seven samples, in duplicate, simultaneously, in less than 10 min. Clinical evaluation (n = 325) by comparison with thin-layer chromatography (TLC) had the following results: 230 samples were negative by TLC and Mixed EMIT, 77 samples were positive by TLC and Mixed EMIT, 16 samples were negative by TLC and positive by Mixed EMIT, and two samples were positive by TLC and negative by Mixed EMIT.
Comparison of Flavobacterium and Sphingobacterium species by enzyme profiles, with use of pattern recognition of two-dimensional fluorescence data.
Authors: Pau C P , Patonay G , Moss C W , Hollis D , Carlone G M , Plikaytis B D , Warner I M
Published: January 2020

Enzyme profiles of eight Flavobacterium species and one Sphingobacterium species were compared after using a two-dimensional fluorescence technique. Enzyme contents and corresponding activities were rapidly determined for whole-cell preparations after incubation with a mixture of preselected fluorogenic substrates. A two-dimensional fluorescence spectrum of the resulting product mixture, measured with a video fluorometer, provided a characteristic "fingerprint" for each organism. Comparison of fluorescent spectra was facilitated by a Fourier-transform-based pattern-recognition algorithm and by a clustering technique involving the Pearson product-moment correlation coefficient. F. multivorum, F. thalpophilum, and S. mizutae formed one cluster; F. indologenes, F. spiritivorum, F. odoratum, and F. balustinum formed a second. F. meningosepticum was intermediate between the first and second cluster, whereas F. breve was different from all other strains examined, based on their spectral dissimilarity indices and correlation coefficients.
A method for serum C1q based on its hydroxyproline content.
Authors: Rosano C L , Braun C B , Hurwitz C
Published: January 2020

The radial immunodiffusion assay overestimates the C1q in serum. Here we describe a convenient, accurate procedure for measuring C1q in 250 microL of dialyzed serum. This method is based on our previous findings that all C1q in serum precipitates with the euglobulin fraction and that all other serum proteins containing hydroxyproline are excluded from this fraction. Because C1q is 4.3% hydroxyproline, the concentration of C1q in serum can therefore be calculated from the hydroxyproline content of the euglobulin fraction. The procedure, all done in the same tube, consists of precipitating the euglobulin fraction, digesting it with HClO4, and converting hydroxyproline to the corresponding pyrrole, which is extracted with toluene and measured by absorbance at 560 nm.
Transferrin binding of Al3+ and Fe3+.
Authors: Martin R B , Savory J , Brown S , Bertholf R L , Wills M R
Published: January 2020

An understanding of Al3+-induced diseases requires identification of the blood carrier of Al3+ to the tissues where Al3+ exerts a toxic action. Quantitative studies demonstrate that the protein transferrin (iron-free) is the strongest Al3+ binder in blood plasma. Under plasma conditions of pH 7.4 and [HCO3-]27 mmol/L, the successive stability constant values for Al3+ binding to transferrin are log K1 = 12.9 and log K2 = 12.3. When the concentration of total Al3+ in plasma is 1 mumol/L, the free Al3+ concentration permitted by transferrin is 10(-14.6) mol/L, less than that allowed by insoluble Al(OH)3, by Al(OH)2H2PO4, or by complexing with citrate. Thus transferrin is the ultimate carrier of Al3+ in the blood. We also used intensity changes produced by metal ion binding to determine the stability constants for Fe3+ binding to transferrin: log K1 = 22.7 and log K2 = 22.1. These constants agree closely with a revision of the reported values obtained by equilibrium dialysis. By comparison with Fe3+ binding, the Al3+ stability constants are weaker than expected; this suggests that the significantly smaller Al3+ ions cannot coordinate to all the transferrin donor atoms available to Fe3+.
Recognizing hyperthyroidism in the elderly.
Authors: Nyström E , Lindstedt G , Lundberg P A , Lundholm K , Smith U
Published: January 2020

Hyperthyroidism in the elderly frequently presents with atypical symptoms such as apathy, anorexia, diarrhea, atrial fibrillation, and (or) cardiac decompensation. The diagnosis may therefore be delayed or missed, as illustrated by three cases. Laboratory thyroid evaluation is justified by a wide variety of indications in elderly persons who seek medical advice or care.
Characterization and assay of tartrate-resistant acid phosphatase activity in serum: potential use to assess bone resorption.
Authors: Lau K H , Onishi T , Wergedal J E , Singer F R , Baylink D J
Published: January 2020

We improved the spectrophotometric assay of tartrate-resistant acid phosphatase (TrACP; EC 3.1.3.2) activity in serum. During development of the assay we found that human serum contains a dialyzable, mixed-type noncompetitive inhibitor(s) of TrACP activity, the effects of which on the assay were substantially lessened by diluting the serum sample with water before assay and increasing the substrate concentration. Hemolysis releases into serum a significant amount of TrACP activity from erythrocytes, which can be inactivated by incubating the serum at 37 degrees C for 1 h before assay. Our improved assay was reproducible (CV = 5%), and measured within 10% of the amount of added bovine skeletal TrACP activity. Preliminary application of the assay revealed that the amount of serum TrACP activity in patients with skeletal diseases differed from normal values and changed in the same direction as the expected change in bone turnover, suggesting that TrACP activity in serum could be useful clinically as a marker of bone metabolism, possibly of bone resorption.
Enzymic ethanol assay: a new colorimetric method based on measurement of hydrogen peroxide.
Authors: Prencipe L , Iaccheri E , Manzati C
Published: January 2020

We describe a new colorimetric method for measuring ethanol in plasma by use of a peroxidase-coupled assay system and alcohol oxidase (EC 1.1.3.13) from Pichia species. Absorptivity is low enough to give useful results without sample dilution. The procedure is also applicable to saliva samples and utilizes only one working reagent. The absorbance of the blue dye that is formed is measured at 600 nm. The standard curve for the method is linear for ethanol concentrations up to 4 g/L. Average analytical recovery of ethanol in human plasma exceeded 99%. Within-run and between-run CVs were less than 2.45% and less than 1.92%, respectively. Results correlate very well with those by gas chromatography (r = 0.9977). The method is adaptable to automation.
Further investigations regarding the toxicity of sodium nitroprusside.
Authors: Butler A R , Glidewell C , McGinnis J , Bisset W I
Published: January 2020

Sodium nitroprusside is a valuable vasodilator, but its use has been curtailed because of numerous reports that, in the presence of blood, nitroprusside decomposes with release of toxic cyanide. We have examined the release of cyanide in terms of the known chemistry of nitroprusside and suggest that photochemical decomposition of nitroprusside and (or) its metabolism in vivo invalidates the analytical procedure used by previous workers. We also present evidence for the stability of nitroprusside in blood, based on 13C nuclear magnetic resonance studies.
Rapid determination of alpha-amylase activity by use of a new chromogenic substrate.
Authors: Dupuy G , Hilaire G , Aubry C
Published: January 2020

A new chromogenic substrate that is blocked at the nonreducing end, 4,6-benzylidene-alpha-D-4-nitrophenylmaltoheptaoside, is used to determine alpha-amylase (EC 3.2.1.1) activity in serum in a coupled assay with alpha-glucosidase (EC 3.2.1.20) and glucoamylase (EC 3.2.1.3) as auxiliary enzymes. The duration of the lag phase between 25 and 37 degrees C is less than 90 s, and the molar absorptivity of 4-nitrophenol is constant. The main cleavage product of the substrate by human pancreatic and salivary alpha-amylase is 4-nitrophenylmaltoside; in the presence of the auxiliary enzymes, greater than 95% of hydrolyzed substrate is accounted for as 4-nitrophenol. The combined reagent is stable for at least 20 days at 2-8 degrees C; precision is good, with CVs ranging from 1.7 to 3.3%; and the correlation of results with those by the 4-nitrophenylmaltoheptaoside method is excellent. Heparin (40 kilo-int. units/L), ascorbic acid (2.8 mmol/L), bilirubin (430 mumol/L), hemoglobin (170 mumol/L), glucose (55 mmol/L), and triglycerides (11 mmol/L) do not interfere in the assay.
Methods compared for determining glutathione peroxidase activity in blood.
Authors: Faraji B , Kang H K , Valentine J L
Published: January 2020

We compared four methods for determination of glutathione peroxidase (EC 1.11.1.9) activity, using blood samples from 52 healthy volunteers. Two methods depended on direct assay of the amount of glutathione remaining at specific time intervals; the two indirect methods involved measuring the rate of disappearance of NADPH. We assessed the precision and reproducibility of each method. One of the indirect assays proved to be far superior to the other methods. Results of each of the methods were correlated with one another. We present the normal reference intervals for glutathione peroxidase activity for all four methods.
Direct determination of angiotensin-converting enzyme inhibitors in plasma by radioenzymatic assay.
Authors: Reydel-Bax P , Redalieu E , Rakhit A
Published: January 2020

We describe a simple, rapid, specific radioenzymatic assay for "CGS 16617," a new, potent inhibitor of angiotensin-converting enzyme (ACE; EC 3.4.15.1) in human plasma. This assay is based on the principle that the inhibitor (i.e., the drug) binds to the ACE in plasma and hence the amount of free ACE in plasma is inversely related to the amount of active inhibitor present. Free enzyme is reacted with a radiolabeled substrate, and the radioactive product is selectively extracted into the scintillation cocktail for quantification. Fivefold-diluted plasma samples are incubated with [3H]hippuryl-glycyl-glycine enzyme substrate at 37 degrees C for 30 min and the liberated [3H]hippuric acid is selectively extracted into scintillation cocktail. The radioactivity is counted in a liquid scintillation counter. Both within-run and between-run, the variability (CV) of the assay is less than 10%. As little as 200 ng of the drug per liter can be quantified in 50-microL plasma samples. The method can also be used to assay two other ACE inhibitors, pentopril and CGS 14831, demonstrating that the method can be readily adapted to any ACE inhibitor having a single active component in plasma. The ester prodrug pentopril can also be assayed after ester hydrolysis. This method is suitable for analysis of large numbers of samples in clinical laboratories for routine monitoring of the concentrations of active ACE inhibitors in blood.
Prospective clinical evaluation of an improved fluorescence polarization assay for predicting fetal lung maturity.
Authors: Talt J F , Foerder C A , Ashwood E R , Benedetti T J
Published: January 2020

We performed a prospective clinical evaluation of our newly developed fluorescence polarization procedure to predict fetal lung maturity (Clin Chem 1986;32:248-54). Net fluorescence polarization was measured at 34 degrees C after a 6.5-min incubation of amniotic fluid with fluorophore. For the 26 cases of neonatal respiratory distress syndrome encountered in 196 deliveries, the net polarization exceeded 0.287 for 22 (85%) of these, and exceeded 0.260 for all 26. The specificity of the polarization assay equaled or exceeded the specificity of the lecithin/sphingomyelin ratio for all sensitivities greater than 70%. Neither assay was a good predictor of the clinical severity of respiratory distress. For a separate group of 21 amniotic fluid specimens clinically contaminated with blood or meconium, the discriminatory power of the polarization assay was decreased, but six of seven respiratory-distress cases still had polarization values greater than 0.260. We conclude that this fluorescence polarization assay is a better overall predictor of fetal lung maturity than is the lecithin/sphingomyelin ratio, and that polarization values less than 0.260 are associated with little risk of respiratory distress.
Discrepancies in carcinoembryonic antigen measurements: survey and control values vs values for patients.
Authors: Klee G G , Dodge L A , Reynoso G
Published: January 2020

We analyzed the carcinoembryonic antigen (CEA) test results reported in the College of American Pathologists' (CAP) surveys to determine the relationship between the source of CEA used to manufacture the survey specimens and the discrepancies among analytical methods. With the 1983 survey specimens, which were prepared from metastatic colon carcinoma, laboratories using Roche RIA with Clinetics columns reported results that were only one-half the values reported by laboratories using the Abbott polyclonal enzyme immunoassay. With the 1984 specimens, prepared from a different metastatic colon carcinoma, and the 1985 specimens, prepared from a tissue-culture source of CEA, the Roche results were about one-sixth as large as the Abbott results. These differences are larger than the reported assay differences for patients' specimens. In addition, twofold proportional differences were found when survey and control specimens were tested with different lots of Abbott polyclonal reagent, whereas only random differences were found with 102 patients' specimens. Evidently, assay systems perform differently with proficiency-testing and control specimens than with patients' specimens.
Clinical value of immunoradiometric assay of thyrotropin for patients with nonthyroidal illness and taking various drugs.
Authors: John R , Evans P E , Scanlon M F , Hall R
Published: January 2020

Using a two-site immunoradiometric assay, we measured concentrations of thyrotropin (TSH) in serum of 134 clinically euthyroid subjects, 93 patients with nonthyroidal illness, and 80 patients who were being treated with various drugs. Abnormal concentrations of TSH, free thyroxin, and free triiodothyronine, respectively, were recorded in serum of three (3.2%), 19 (20.4%), and 37 (39.8%) of the patients with nonthyroidal illness and in three (3.8%), five (6.3%), and 10 (12.5%) of the patients taking drugs. TSH could be detected in all patients' serum samples. We conclude that, for most patients without thyroid disease, a basal (i.e., unstimulated) measurement of their TSH concentration in serum will indicate their thyroid status more reliably than will assay of free thyroxin or free triiodothyronine.
Ascorbic acid as an antioxidant in measurements of catecholamines in plasma.
Authors: Hugh D , Grennan A , Abugila M A , Weinkove C
Published: January 2020

Sodium metabisulfite, commonly used to prevent the oxidation of catecholamines during extraction from plasma onto alkaline alumina, does not prevent their subsequent degradation in acetic acid eluates. However, ascorbic acid, a potent antioxidant, is extracted with the catecholamines onto the alumina and prevents such destruction. However, ascorbic acid may interfere with the electrochemical measurement of catecholamines, unless sequential oxidation and reduction are used. Other methods of minimizing catecholamine oxidation in acetic acid eluates include refrigerating at 4 degrees C and capping the sample vials to exclude atmospheric oxygen.
Erythrocytic glucose-6-phosphate dehydrogenase measured by a differential pH technique.
Authors: Barenghi L , Ceriotti F , Ripamonti M , Luzzana M , Bonini P
Published: January 2020

We propose a new quantitative electrochemical method for determining glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity in purified erythrocytes or in whole blood, based on measurement of the pH change caused by oxidation of glucose 6-phosphate to 6-phosphogluconic acid, with simultaneous reduction of NADP+ to NADPH + H+. No sample pretreatment (e.g., preparation of hemolysate) is needed, and the automatic correction for sample blanks obviates interference from 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The method is simple and fast, and the standard curve is linear to at least 2200 U/L at 37 degrees C. The within-day CV was 3.9% for activities in healthy individuals (mean value 1204 U/L), and 10% for deficient ones (classified as belonging to class II, mean value 407 U/L). Results (y) correlated well with those obtained with the WHO-recommended method (x): y = 1.13x + 0.02 (r = 0.971) for purified erythrocytes. Normal reference intervals are reported for the enzyme in purified erythrocytes and in whole blood.
Improved radioimmunoassay of melatonin in serum.
Authors: Sieghart W , Ronca E , Drexler G , Karall S
Published: January 2020

Melatonin was extracted from serum by using Baker reversed-phase C-18 columns. More than 99% of the applied melatonin was retained by the columns, and more than 97% was eluted from the columns in 300 microL of methanol. We then determined melatonin in the serum extract by a modification of a standard radioimmunoassay, using filtration instead of centrifugation to collect the [3H]melatonin-antibody complex precipitated by saturated ammonium sulfate. These modifications allow more rapid, accurate, and reproducible determination of melatonin than do previously published procedures.
Cardiac enzyme changes in myxedema coma.
Authors: Hickman P E , Silvester W , Musk A A , McLellan G H , Harris A
Published: January 2020

A 74-year-old man with myxedema and hypothermia had increased activities in plasma of creatine kinase (CK; EC 2.7.3.2), aspartate aminotransferase (AST; EC 2.6.1.1), and lactate dehydrogenase (LD; EC 1.1.1.27) and increased proportions of CK-MB (up to 20% of total CK) and LD1 isoenzymes, but no clinical or investigational evidence of associated myocardial infarction. This case illustrates that plasma enzyme activity and isoenzyme profiles in such clinical settings should be interpreted with caution, because increases in CK-MB and LD1 may relate to myxedema coma or hypothermia (or both) rather than to myocardial infarction.
Cross reactions in radioimmunoassay: a mathematical model for correcting assay results, as exemplified by eliminating the interference of intact thyrotropin in an assay of its beta subunit.
Authors: Dotti C , Ceda G P , Denti L , Cavalieri S , Borasi G , Valenti G
Published: January 2020

To correct the results of a radioimmunoassay for beta-thyrotropin (TSH) subunit by eliminating the proportion ascribable to intact TSH, we have devised a method that experimentally reproduces the conditions under which the interference develops. Beta-TSH subunit was assayed in several preparations containing known concentrations of both beta-TSH and TSH. The TSH-induced overestimation of beta-TSH was linearly related to the concentration of antigen in the sample. At a constant concentration of TSH, therefore, the following equation is applicable: F = aE + b, where F is the measured (but overestimated) concentration of beta-TSH, E is the actual concentration of beta-TSH, and a and b are the slope and the intercept of the regression line, respectively. a and b, once expressed as a function of TSH, allow the correction of the overestimation. The analysis of the results according to the mass action and conservation laws shows that the antiserum is more avid for the interferent (intact TSH) than for the antigen (beta-TSH).