bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzymic assay of creatinine in serum and urine with creatinine iminohydrolase and glutamate dehydrogenase.
Authors: Tanganelli E , Prencipe L , Bassi D , Cambiaghi S , Murador E
Published: January 2020

We describe an assay for creatinine in which it is converted by creatinine iminohydrolase (EC 3.5.4.21) into ammonia and N-methylhydantoin. The ammonia is subsequently assayed by use of alpha-ketoglutarate and glutamate dehydrogenase (EC 1.4.1.3). Use of NADPH as coenzyme eliminates all interferences from endogenous reactions. Endogenous ammonia in the sample is eliminated during a preincubation. The reaction reaches the endpoint in 15 min at working temperatures of 20-37 degrees C. No sample blank or reagent blank is needed. The standard curve is linear at least to 884 mumol (100 mg) of creatinine per liter. Average analytical recovery of creatinine in serum and urine is 99%. Within-run and between-run CVs are less than or equal to 2% and less than or equal to 6% for creatinine values of 335 mumol/L (38 mg/L) and 80 mumol/L (0 mg/L), respectively. Results by the described method (y) compare well with those by Jaffé's kinetic test (y = 1.01x -- 12.8), Berthelot/AutoAnalyzer method after treatment with immobilized creatinine iminohydrolase (y = 0.987x -- 13.2), Jaffé's test run on the SMA 12/60 (y = 1.011x -- 5.8), the Wahlefeld method (y = 1.014x -- 0.88), and Jaffé's test after deproteinization and absorption on fuller's earth (y = 0.985x -- 3.08). The method may be suitable for discrete, including centrifugal, automation.
Simultaneous enzyme immunoassay of two thyroid hormones.
Authors: Blake C , Al-Bassam M N , Gould B J , Marks V , Bridges J W , Riley C
Published: January 2020

We describe an enzyme immunoassay in which the two thyroid hormones, triiodothyronine and thyroxin, are measured simultaneously in a single tube. The method involves labeling the two with separate enzymes (beta-galactosidase and alkaline phosphatase, respectively), whose catalyzed reactions can easily be distinguished from each other by absorption spectrophotometry, with o-nitrophenyl-beta-galactoside and phenolphthalein monophosphate as substrates. Performance of this dual assay method compares well with that of conventional single-hapten enzyme-labeled assays, and results compare well with those by two single-hapten radioimmunoassays. The dual assay has certain advantages over single-hapten methods: smaller sample volume, lower reagent cost, and shorter overall assay time. As presented here, the use of enzyme labels to measure two (or more) haptens simultaneously represents a significant advance in the use of immunoassay techniques.
Plasma hormone concentrations and serum 17 beta-hydroxysteroid dehydrogenase activity in molar pregnancy.
Authors: Massart C , Le Pogamp C , Nicol M
Published: January 2020

Concentrations of unconjugated 17 beta-estradiol, estrone, and estriol, and of progesterone, 17 alpha-hydroxyprogesterone, testosterone, delta 4-androstenedione, dehydroepiandrosterone sulfate (DHEAS), choriogonadotropin beta-subunit, and prolactin were measured by radioimmunoassay in plasma of six patients with molar pregnancies of seven to 15 weeks of gestation. Serum 17 beta-hydroxysteroid dehydrogenase (EC 1.1.1.6.2) activity was also determined. Estriol was not detected in any of the six women. Values for DHEAS and progesterone were normal, except for one patient who had above-normal plasma progesterone. Values for the other hormones and the enzyme were within normal limits in very early intact molar pregnancies, but were above normal limits in more advanced cases. A close correlation was seen between 17 beta-hydroxysteroid dehydrogenase activity and concentrations of estradiol, estrone, testosterone, and 17 alpha-hydroxyprogesterone; the last four also correlated significantly with beta-choriogonadotropin in molar pregnancies, whether benign or invasive. Benign trophoblastic disease showed significant correlation of progesterone with beta-choriogonadotropin and with estradiol. We conclude that the estradiol, estrone, testosterone, and 17 alpha-hydroxyprogesterone were principally derived from molar tissue.
Two-cycle liquid-chromatographic quantitation of cortisol in urine.
Authors: Nakamura J , Yakata M
Published: January 2020

When the free cortisol in urine was analyzed by liquid chromatography after pretreatment with the usual organic extraction, interfering substances having chromatographic behavior similar to that of cortisol and the internal standard precluded accurate measurement. To remove these materials, we processed the organic extract by liquid chromatography with a normal-phase column for clean-up; the specific cortisol fraction and the internal standard collected in the effluent were then subjected to liquid chromatography with a reversed-phase column. The "two-cycle" chromatograms thus obtained were sufficiently specific for the quantitation of urinary cortisol. The method clearly detects cortisol in urine at a minimum concentration of about 5 micrograms/L when 1 mL of urine is extracted. The mean urinary free cortisol in apparently healthy individuals was 40.9 (SD 17.7) micrograms/day (n = 32) by this method.
Improved liquid-chromatographic assay of quinidine and its metabolites in biological fluids.
Authors: Rakhit A , Kunitani M , Holford N H , Riegelman S
Published: January 2020

We describe a simple, rapid, and specific assay for quinidine and its known metabolites in plasma, urine, and bile. Plasma proteins are precipitated by adding acetonitrile, which also contains the internal standard. The supernatant fluid is evaporated and the reconstituted residue is separated on a reversed-phase column, with fluorescence detection. The standard curve is linear and results are reproducible over the clinical concentration ranges: quinidine 0.4 to 8.0 mg/L and the three metabolites (quinidine 10,11-diol, 3-hydroxyquinidine, and quinidine-N-oxide) 0.05 to 1.5 mg/L. As little as 10 micrograms of the N-oxide metabolite per liter and 1 microgram of the other analytes per liter can be quantitated in 0.5 mL of plasma, urine, or bile. With the use of an automated chromatographic system, many samples can be analyzed in a continuous run.
Heparin interferes with the radioenzymatic and homogeneous enzyme immunoassays for aminoglycosides.
Authors: Krogstad D J , Granich G G , Murray P R , Pfaller M A , Valdes R
Published: January 2020

Heparin interferes with measurement of aminoglycosides in serum by biological, radioenzymatic, and homogeneous enzyme immunoassay techniques, but not with radioimmunoassay. At concentrations greater than or equal to 10(5) and greater than or equal to 3 X 10(6) USP units/L, respectively, it interferes with the radioenzymatic assay by inhibiting the gentamicin 3-acetyltransferase (EC 2.3.1.60) and kanamycin 6'-acetyltransferase (EC 2.3.1.55) enzymes used in the assay. It interferes with the homogeneous enzyme immunoassays for gentamicin and tobramycin (at concentrations greater than or equal to 10(5) and greater than or equal to 10(4) USP units/L, respectively), but not with the commercially available homogeneous enzyme immunoassays for other drugs. Heparin interference with the homogeneous enzyme immunoassay for aminoglycosides requires both the heparin polyanion and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) bound to a cationic aminoglycoside. This interference can be reproduced with dextran sulfate (but not dextran), and does not occur with free enzyme (glucose-6-phosphate dehydrogenase) alone. We describe how heparin interferes with these two assays and demonstrate interference at heparin concentrations that may be present in intravenous infusions or in seriously underfilled blood-collection tubes.
Single-stage automated assay for heparin.
Authors: Kapke G F , Feld R D , Witte D L , Johnson G F
Published: January 2020

We have developed a single-stage assay for heparin, using reagents modified from the two-stage Dade Protopath heparin synthetic substrate assay. The single-stage assay involves simultaneous mixing of a plasma sample, an antithrombin III source, alpha-thrombin, and the alpha-thrombin fluorogenic substrate. The synthetic substrate, antithrombin III, and heparin-antithrombin III complex compete for the alpha-thrombin active site. The alpha-thrombin is inactivated by the heparin-antithrombin complex while substrate is being hydrolyzed, so that total product formation decreases with heparin concentration. Day-to-day CV was 9.3% at a heparin concentration of 246 USP units/L. Comparison of results of the single-stage heparin assay with those of a two-stage esterolytic assay yielded the linear regression equation: esterolytic = 0.834 (single-stage)--7 USP units/L (r = 0.94, n = 47). Bilirubin interfered with the single-stage assay, resulting in an apparent increase in sample heparin concentration. The single-stage heparin assay can be automated for centrifugal analyzers capable of double-reagent addition and fluorometric detection, substantially decreasing reagent requirements and therefore costs.
Homologous radioimmunoassay for human parathyrin (residues 53-84).
Authors: Hitzler W , Schmidt-Gayk H , Spiropoulos P , Raue F , Hüfner M
Published: January 2020

We describe a sequential saturation double-antibody radioimmunoassay for carboxyl-terminal fragments of human parathyrin (hPTH) in serum. Standards are prepared with synthetic hPTH (residues 53-84) in hPTH-free serum. Antisera are obtained by immunizing guinea pigs with partly purified hPTH extracted from adenomatous glands. Tracer is prepared by labeling hPTH (53-84), presumably at the histidine residue, with 125I by the Chloramine T method at pH 8.6. Dilution curves for hPTH extracted from adenomas are superimposable on dilution curves for the synthetic 53-84 fragment. Dilution of sera from hyperparathyroid patients showed linearity of response with concentration in the present assay, but non-linearity in the heterologous radioimmunoassay. In contrast to the heterologous system, which discriminated 28 of 32 patients with primary hyperparathyroidism from 32 normals (normal range: undetectable to 54 pmol/L, omitting the highest and lowest values from controls), the present assay separated these groups without overlap.
Fetal lung maturity in complicated pregnancy, as predicted from microviscosity of amniotic fluid.
Authors: Simon N V , Hohman W A , Elser R C , Levisky J S , Carp M J , Ashton S S
Published: January 2020

We measured the microviscosity of amniotic fluid between 28 and 40 weeks of gestation in 252 normal pregnancies and in 172 pregnancies complicated by factors known to influence fetal lung maturation, including chronic high blood pressure, pregnancy-induced hypertension, diabetes mellitus, and therapy with betamethasone. Comparison of the microviscosity value distributions and regression analysis indicated significantly lower microviscosity values in hypertensive disorders, in Class D and Classes F or R diabetes, and after 48 h of treatment with betamethasone. Few changes were observed in Classes A, B, or C diabetes. These observations are consistent with the accelerated maturation of surfactant observed in chronic intrauterine stress and the lower incidence of hyaline membrane disease reported after glucocorticoids.
Micromethod for lipid-chromatographic determination of cholesterol in lipemic sera.
Authors: Webb L E , Lengle E , Izzo R , Woods E
Published: January 2020

A micromethod involving "high-performance" lipid chromatography was developed for determining cholesterol in sera obtained from animals that has been infused with high doses of lipid emulsions. Only 50 micro L of serum is required, and there is no interference from turbidity, bilirubin, fat-soluble vitamins, and sterols. Correlation with the commonly used Abell-Kendall method was good at cholesterol concentrations as great as 10.0 g/L (r = 0.998). The assay is ideal for determinations on samples from pediatric and hyperlipidemic patients. It is particularly useful for monitoring patients on lipid-emulsion therapy and for toxicological studies with small animals.
p-Nitrophenylglycosides as substrates for measurement of amylase in serum and urine.
Authors: McCroskey R , Chang T , David H , Winn E
Published: January 2020

Pantrak E.K. (endpoint and kinetic) Amylase reagent (Calbiochem-Behring) is the first commercially available alpha-amylase reagent in which p-nitrophenyl-d-glycosides are used as the substrate. We describe the effect of reagent composition on reagent performance. The reagent performance compares well with that of Amylochrome reagent (Hoffmann-La Roche), Du Pont aca, and Beckman D.S. amylase reagents in assays of sera and urines. We detected no interference from increased concentrations of glucose or pyruvate in the sample. The reagent can be used in either a manual fixed-time or an automatable kinetic assay.
Functional activity of intrinsic factor measured by using solubilized receptor protein.
Authors: Marcoullis G , Rothenberg S P
Published: January 2020

The traditional radioimmunoassay for gastric intrinsic factor, in which this protein is measured on the basis of immunoreactivity rather than function, is of no value for identifying intrinsic factor that binds cobalamin but does not bind to the ileal receptor site, or for detecting animal intrinsic factor, which does not cross react with human intrinsic factor. Accordingly, we have applied a radioassay for the intrinsic factor receptor protein to measure the functional activity of intrinsic factor in gastric juice. The receptor protein reagent was partly purified from guniea pig ilea and its interaction with intrinsic factor--CN[57Co]-cobalamin was determined by precipitation with sodium sulfate at a final concentration of 150 g/L. Results of this assay were comparable with results obtained for intrinsic factor by radioimmunoassay. The receptor protein did not bind immunoreactive intrinsic factor that was functionally abnormal. This functional radioassay for intrinsic factor is not species specific and will be of value when specific antiserum to intrinsic factor is not available and when cobalamin malabsorption is to be evaluated in patients who are secreting normal amounts of immunoreactive intrinsic factor.
More SensitiveFlamelessAtomicAbsorptionAnalysisof Vanadiumin Tissueand Serum
Authors: Stroop Steven D , Helinek Gerard , Greene Harry L
Published: January 2020

Vol. 28

p 79: See response in Letter of Cornelisand Versieck, p 1709.

p 91: In the note added in proof, "131" should read "1.31."

p 241: In Table 1, "6.5" should read "65"; in the footnote to Table 2, "0.05" should read "0.5" and the next to last paragraph should read: "Finally, in a few instances we have compared the glucose values obtained by using a drop of sample according to the instructions, versus dipping the strips into the sample, and found some variance between the values obtained by the two methods."

p 344: Under "staining reagent," the correct concentration of orthophosphoric acid is 160 g/L (not 80 g/L). It is prepared by diluting 95 mL of the concentrated acid (850 g/kg, relative density 1.69 g/mL) to 1 L with distilled water.

p 418: ref. 94, cited in the first full paragraph, should read "84."

p 419: The volume number of ref. 1 should be 50, not 19.

p 502: Corrections to two equations: the term (S2y - S2x) should read (S2y - S2x)2 and in the third equation in the second column delete the repetition of the term "(wx4)," which should only appear once.

p 531: In last full paragraph in column one, the word "propionate" in the next to last line should instead read "dimethyl sulfoxide."

p 614: References 21 and 22 are missing

21: Gerhardt, W., Ljungdahl, L., Borjesson, J., et al., Creatine kinase B-subunit activity in human serum. I. Development of an iminunoinhibition method for routine determination of S-creatine kinase B-subunit activity. Clin. Chim. Acta78, 29-41 (1977).

22: Neumeier, D., Prellwitz, W., Wurzburg, U., et al., Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity: Activity kinetics and diagnostic significance in myocardial infarction. Clin. Chim. Acta73, 445-451 (1976).

p 727: With regard to this Letter, the authors later pointed out that although the first sentence states that glycosylated hemoglobins have a higher isoelectric point, glycosylation actually decreases the isoelectric point. Later (p 728) they speculated that the fast hemoglobin variant might be HbN-Baltimore (α2β295Lys→Glu), but two reference laboratories identified it as the slightly slower HbJ-Baltimore (α2β216Gly→Asp).

p 735: In the middle column, the third full paragraph, "Department of Internal Medicine" should read "Department of Laboratory Medicine."

p 736: In column one, under Awards, the citation should have stated "in recognition of his contribution in the, field of clinical chemistry."

p 1199: Table 1 folate concentrations should be "mg/L," not rg/L.
Enzyme immunoassay of the glycoprotein tropic hormones--choriogonadotropin, lutropin, thyrotropin--with solid-phase monoclonal antibody for the alpha-subunit and enzyme-coupled monoclonal antibody specific for the beta-subunit.
Authors: Wada H G , Danisch R J , Baxter S R , Federici M M , Fraser R C , Brownmiller L J , Lankford J C
Published: January 2020

Monoclonal antibody technology has made it possible to produce homogeneous populations of antibodies to discrete determinants on an antigen surface. We have produced monoclonal antibodies to the alpha-subunit and beta-subunits of the glycoprotein hormones choriogonadotropin, thyrotropin, and lutropin, and developed two-site simultaneous enzyme-linked immunospecific assays for these hormones. The anti-alpha-subunit monoclonal antibody was used as the solid-phase (coated tube) capture antibody for all three hormones; the anti-beta-subunit monoclonal antibodies were coupled to horseradish peroxidase (EC 1.11.1.7). Cross reactions between the closely related choriogonadotropin and lutropin were apparently greater in this method than in RIA, with use of the same antibodies. Ka of the antibodies did not appear to be as critical to sensitivity of the sandwich assay as it was for RIA. The lower limit of detection was 0.2 microgram/L after a 2-h incubation with serum sample at room temperature and a 30-min incubation with enzyme substrate at room temperature after washing away excess enzyme conjugate. Within-assay precision (CV) was very good, less than 6%.
Analysis of serum by high-field proton nuclear magnetic resonance.
Authors: Bock J L
Published: January 2020

Improvements in nuclear magnetic resonance (NMR) technology are generating an expanding variety of medical applications. In this investigation I have used high-field proton NMR to identify and quantity endogenous and ingested substances in human serum. After addition of a small amount of 2H2O and a reference compound to a 0.4-mL specimen, spectra were recorded for 3 min in Fourier-transform mode, with use of presaturation to suppress the extremely intense H2O peak. Compounds detected at clinically significant concentrations include glucose, alcohols, acetone, organic acids, and salicylate. Less than 1 mmol/L of some of these substances could be detected. For serum containing 20--500 mg of added methanol per liter, peak area was a linear function of concentration (r = 0.998). High-field proton NMR, despite the drawback of expensive, sophisticated instrumentation, offers some unique advantages for clinical chemistry: it permits rapid, specific, nondestructive measurement of several compounds simultaneously, including some that may be inconvenient to measure by conventional means.
Potential clinical applications of photoacoustics.
Authors: Rosencwaig A
Published: January 2020

Photoacoustic spectroscopy offers the opportunity for extending the exact science of noninvasive spectral analysis to intact medical substances such as tissues. Thermal-wave imaging offers the potential for microscopic imaging of thermal features in biological matter.
Semiautomated microfluorometric instrument and reagent system for monitoring disease-related immunosuppression.
Authors: Ranney D F , Quattrone A J
Published: January 2020

Several common metabolites and drugs in the serum in of patients with inflammatory, infectious, autoimmune, immunodeficient, neoplastic, and toxicant-induced diseases can produce artifactual suppression of the [methyl-3H]-thymidine assay, which is widely used to evaluate lymphocyte responsiveness. We have developed a sensitive, semiautomated, fluorescence-enhancement assay in which true immunosuppressors are measured in the presence of absence of such interfering substances. Peripheral blood lymphocytes are activated with mitogens in standard microtiter culture trays. Changes in lymphocyte DNA content are quantified with a reagent formulation containing mithramycin, the fluorescence of which is enhanced on binding to DNA in the presence of MgCl2. We solubilize cells within the intact microtiter tray by using an automated, inverted "Array Sonicator," and measure fluorescence with an automated, photon-counting fluorometer. With this system, immune response modulation can be accurately assessed in the presence of patients' sera and other complex test substances (e.g., supernates from hybridomas, fermentation vats, viral preparations, and macrophage cultures.
Establishing the direct-potentiometric "normal" range for Na/K: residual liquid junction potential and activity coefficient effects.
Authors: Czaban J D , Cormier A D , Legg K D
Published: January 2020

The observed reference ranges for sodium and potassium as determined by direct potentiometry vary from instrument to instrument, depending on the composition of the calibration standards. To resolve the existing confusion as to which reference intervals are most appropriately considered "normal," we propose a straightforward convention (based on plasma-water concentration units) in which the difference between direct (undiluted sample) and indirect (diluted sample) methodologies is accounted for by the volume displacement effect of proteins, lipids, and other dissolved substances in a typical plasma sample. Thus, the proposed reference intervals for sodium and potassium are approximately 7% greater by direct potentiometry than by procedures involving dilution. Data consistent with this convention can be obtained with a variety of aqueous-based calibrants, provided care is taken to minimize the errors resulting from activity coefficient and liquid junction potential effects. Additional experimental results are presented to show that these effects also account for the apparent suppression of the sodium ion concentration observed in the presence of bicarbonate ion.
Electrochemical investigation of hapten-antibody interactions by differential pulse polarography.
Authors: Wehmeyer K R , Halsall H B , Heineman W R
Published: January 2020

The binding of electroactively labeled estriol with estrogen-specific antibody and its subsequent displacement by unlabeled estriol have been monitored by differential pulse polarography. Estriol was found to be electro-inactive in the potential range -200 mV to -1000 mV vs a silver/silver chloride electrode. Estriol labeled in the 2 and 4 position with nitro groups was electroactive, giving two reduction waves at -422 mV and -481 mV vs a silver/silver chloride electrode. The peak current was linear with concentration over the range 60 micrograms/L to 3.7 mg/L. The addition of aliquots of estrogen-specific antibody reduced the peak current proportionately, indicating the binding of ligand to specific antibody. Subsequent addition of unlabeled estriol produced incremental increases in peak reduction current, indicating competitive and reversible binding of the two ligands for the antibody. Separation of bound from free labeled hapten was not necessary because reduction of the antibody-bound labeled estriol is attenuated at the electrode.
Separation of alkaline phosphatase isoenzymes from human kidney and comparison with alkaline phosphatases from other human tissues, urine, and Escherichia coli.
Authors: Hodson A W
Published: January 2020

Human kidney isoenzymes of alkaline phosphatase (EC 3.1.3.1) after extraction with butan-1-ol were separated by ammonium sulfate precipitation, gel filtration, and chromatofocusing fractionation methods. The separation at each fractionation step was monitored by starch gel and equilibrium-gradient-pore electrophoresis, the latter technique also being used to determine molecular mass. The determined molecular mass (daltons) of alkaline phosphatase from human placenta was 132 000, from urine 95 000, and three isoenzymes from kidney were 195 000, 140 000, and 95 000, respectively. The mass of Escherichia coli alkaline phosphatase was 80 000 daltons, and that of human liver alkaline phosphatase was assumed to be 160 000 daltons. The urinary isoenzyme and the electrophoretically fastest migrating kidney isoenzyme were similar with regard to pH optima, charge, and molecular mass as well as response to L-phenylalanine, L-homoarginine, heat, and urea. Bacterial alkaline phosphatase could be distinguished from the alkaline phosphatases in human tissues and urine by differences in the response to changes in pH and several other physicochemical properties.
Multivariate analysis of plasma enzyme profiles in severe head injury.
Authors: Bourguignat A , Ferard G , Jung G , Klumpp T , Metais P
Published: January 2020

We followed the changes in the activities of four enzymes (aldolase, aspartate aminotransferase, creatine kinase, and lactate dehydrogenase) in plasma for four days after head injury. The progression of the changes differs significantly between survivors and nonsurvivors. Stepwise discriminant analysis, involving the four enzymes, allowed us to divide 81 to 92% of head-injured patients (n = 280 selected without conscious bias) correctly into the two groups as early as 72 h after trauma. Most of the patients who were misclassified according to our biochemical criteria had received phenobarbital for sedation. Valuable prognostic information in head injury evaluation may thus be obtained by daily determination of enzymatic activities of these four enzymes.
Effects of substrate concentration on results of determination of prostatic acid phosphatase with thymolphthalein monophosphate.
Authors: Kessner A , Woodard E J , Bowers G N
Published: January 2020

The relation between concentration of thymolphthalein monophosphate substrate and catalytic activity was investigated for the determination of prostatic acid phosphatase. This study, an extension of previously reported work (Clin. Chem. 27: 1372, 1981), shows that lot-to-lot variation in purity of thymolphthalein monophosphate preparations is reflected in substrate-velocity curves. Plateau regions in these curves at 1.5-2.5 g/L result from the combined effects of (a) substrate concentrations that are an order of magnitude below Km and (b) a further decrease in available substrate caused by formation of substrate aggregates in the presence of serum. To simplify the identification of superior lots of thymolphthalein monophosphate, we give a mixed-substrate protocol for testing different lots.
Simple, reproducible procedure for selective measurement of lipoprotein lipase and hepatic lipase.
Authors: Blache D , Bouthillier D , Davignon J
Published: January 2020

Existing methods for determining the release of lipoprotein lipase (EC 3.1.1.34) and hepatic lipase (EC 3.1.1.3) into plasma after heparin injection give highly variable results, primarily traceable to errors in the isolation of labeled oleate from the substrate, triolein. Methods involving anion-exchange resin to bind oleate show high variability and have a low yield. Introducing a strong base in the last step of the assay may spuriously increase the counts from oleate, whereas a detergent such as Triton X-100, used to minimize this problem, has a strong quenching effect. We report a simple and rapid method in which we eliminate rather than correct for the sources of variation. The substrate, tri[1-14C]oleoyl-glycerol, is sonicated under strictly standardized conditions with gum arabic, 50 g/L. Incubation is stopped by addition of a benzene/chloroform/methanol mixture and NaOH, 0.2 mol/L. Labeled oleic acid is extracted with hexane after acidification of the alkaline aqueous (upper) phase, so that no alkali is introduced into the scintillation liquid. For lipoprotein lipase measurement, hepatic lipase is inactivated by a specific antiserum, whereas hepatic lipase is measured after lipoprotein lipase is inactivated by NaCl, 1.0 mol/L. The method is efficient and specific, and quenching and chemiluminescence artifacts are avoided.
Urinary oxalate determination by use of immobilized oxalate oxidase in a continuous-flow system.
Authors: Potezny N , Bais R , O'Loughlin P D , Edwards J B , Rofe A M , Conyers R A
Published: January 2020

In this procedure, oxalate oxidase (EC 1.2.3.4) immobilized in a continuous-flow system is used to determine oxalate in urine. The hydrogen peroxide formed from oxalate is detected by use of a color reaction with peroxidase (EC 1.11.1.7), 3-methyl-2-benzothiazoline hydrazine, and N,N-dimethylalanine. However, urine contains an oxalate oxidase inhibitor, which cannot be removed by heating, ion-exchange resins, or cellulose columns. This makes it necessary to precipitate the oxalate before assay. The overall assay system is accurate (oxalate recovery, 95.9%), sensitive (less than or equal to 5 mumol/L), precise (within-batch CV less than 1.25%, between-batch CV less than 5%), and relatively rapid (60 samples per working day). The assay system has better accuracy than an established chemical method and a gas-chromatographic method, and is considerably less arduous than and correlates well (r = 0.94) with a modified chemical method. The reference interval for urinary oxalate excretion is 0.16-0.56 mmol per day (n = 97). Only nonphysiological concentrations of ascorbate interfere with the assay, by increasing the oxalate result in the overall assay, presumably by post-micturition formation of oxalate from ascorbate in the urine samples.
Effect of protein on the determination of total bile acids in serum.
Authors: Hanson N Q , Freier E F
Published: January 2020

We measured total serum bile acids on a fluorescence-light-scattering micro centrifugal analyzer by the direct enzymatic method with 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) and with resazurin as a fluorogenic electron acceptor. We found that serum protein has an inhibitory effect on the measurement of bile acids, but this effect was eliminated by adding bovine serum albumin to the reaction mixture in a final protein concentration (12.2 g/L) that was high compared with that contributed by a normal serum specimen. The assay is a sensitive method that reaches equilibrium in 5 min. The method is microscale (5 microL of sample, 150 microL of working reagent), is easy to perform, and is accurate (analytical recovery = 104.1%) and precise (CV = 11.1 and 5.7% on specimens with bile acid concentrations of 7.6 and 35.4 mumol/L, respectively). Normal values are 1-12 and less than 9 mumol/L on nonfasting and fasting individuals, respectively. Pure 3 alpha-hydroxysteroid dehydrogenase must be used: we found several enzyme preparations that gave falsely high values for bile acid.
Simplified liquid-chromotographic analysis for cyclosporin A, and comparison with radioimmunoassay.
Authors: Carruthers S G , Freeman D J , Koegler J C , Howson W , Keown P A , Laupacis A , Stiller C R
Published: January 2020

We describe a simplified isocratic "high-performance" liquid-chromatographic method for measuring a new immunosuppressive drug, cyclosporin A, in biological fluids with use of its analogs cyclosporin C and cyclosporin D as internal standards. The method is reproducible and accurate and appears to be specific for cyclosporin A; the detection limit is 31 micrograms/L. The chromatographic measurements of the concentration of cyclosporin A in serum of patients receiving the drug were invariably lower than those by radioimmunoassay and the difference became more pronounced the greater the period of time after dosing. Because measurements of cyclosporin A in serum standards were almost identical with both techniques, the differences between the two sets of results for patients' samples suggests that the radioimmunoassay is nonspecific and measures metabolites of cyclosporin A.
Assessment of the fundamental accuracy of the Jendrassik-Gróf total and direct bilirubin assays.
Authors: Lo D H , Wu T W
Published: January 2020

Using unconjugated bilirubin (Bu) and authentic human disconjugated bilirubin (dBc) supplemented in low-bilirubin serum pools, we have directly verified for the first time that the Jendrassik-Gróf total bilirubin assay modified after Doumas et al. (Clin. Chem. 19: 984-993, 1973) detects Bu and dBc quantitatively, in Bu-equivalent concentrations relative to gravimetric concentration assignment reinforced with quantification by nuclear magnetic resonance (Wu et al., Clin. Chem. 26: 1323-1335, 1980). By contrast, the Jendrassik-Gróf direct bilirubin assay (also modified after Doumas) quantifies only 70 +/- 5% of the gravimetrically determined dBc as Bu equivalents. By using Bu and dBc (instead of Bu only, as is routinely done) to calibrate the responses of the total and direct bilirubin assays, the analyses of Bu and dBc become much more quantitative. We mathematically explored the meanings of the conventional terms total, direct, and indirect bilirubins. The clinical and diagnostic implications of our observations are discussed.
Evolutionary changes in acute-phase proteins in alcoholic hepatocellular diseases.
Authors: Perier C , Chamson A , Engler R , Frey J
Published: January 2020

We studied the pattern of acute-phase proteins (orosomucoid, C-reactive protein, and haptoglobin) in hepatocellular deficiency due to chronic alcohol consumption, characterized by a decrease in serum transferrin concentration. We found that their patterns could vary independently of hepatocellular deficiency, but depend on the progression of hepatic disease. The most useful protein for discriminating the stage of inflammatory reaction is orosomucoid. In moderate hepatocellular deficiency, acute-phase proteins are increased independently of the decrease in transferrin, whereas in severe hepatocellular deficiency the acute-phase proteins are also decreased. Thus, it is possible to distinguish the two stages of hepatocellular deficiency by following changes in the concentration of orosomucoid.
Soluble forms of gamma-glutamyltransferase in human adult liver, fetal liver, and primary hepatoma compared.
Authors: Selvaraj P , Balasubramanian K A
Published: January 2020

We isolated the soluble forms of gamma-glutamyltransferase (EC 2.3.2.2; gamma-GT) from adult and fetal human liver and primary hepatoma and compared their properties. The Km value for L-gamma-glutamyl-p-nitroanilide and glycylglycine, the Ki for anthglutin, and the pH optimum were identical for the enzyme from all three sources. Nor were significant differences observed among the three in their heat stability, inhibition by serine and borate, or ability to transfer the gamma-glutamyl moiety to various amino acids and dipeptides. Unlike membrane-bound gamma-GT, the soluble form from all three sources entered polyacrylamide gel and showed identical electrophoretic mobilities. Treatment with neuraminidase decreased the electrophoretic mobilities to a similar extent. The relative molecular mass of the enzyme from each of the three sources is about 84 000. Immunoinhibition and immunoprecipitation of gamma-GT from the three sources by antibody to fetal liver gamma-GT followed an identical pattern. Gamma-GT from fetal liver and hepatoma differed significantly from that of adult liver in affinity for wheat-germ agglutinin and Ricinus communis agglutinin (RCA-120). In many of the properties studied, soluble gamma-GT resembles the papain-digested form of membrane-bound gamma-GT.
Assay of urinary N-acetyl-beta-glucosaminidase in a centrifugal analyzer.
Authors: Powell S C , Scaro J , Wilson E , Shihabi Z K
Published: January 2020

We describe a kinetic, fluorometric assay for urinary N-acetyl-beta-glucosaminidase (EC 3.2.1.30) by measurement of the release of 4-methylumbelliferone (7-hydroxy-4-methylcoumarin). The method is simple, involving only a single reagent, and is applicable to the assay of other enzymes that hydrolyze similar fluorescent-labeled substrates. The enzyme distribution in human tissues and fluids is described. The enzyme is present in high concentrations in human kidney, liver, and lung. Its concentration in urine is shown to be a sensitive indicator of early renal damage, which precedes changes in serum creatinine and urinary protein. Assay of the enzyme is quite useful in monitoring renal damage due to myoglobinuria.
Cathodal bands in electrophoretograms of creatine kinase isoenzymes in serum collected after cardiac surgery: a poor prognostic sign.
Authors: Keshgegian A A , Marchant B L
Published: January 2020

Of 708 patients who had undergone cardiac surgery, the serum of 23 showed one or two enzyme bands cathodal to CK-MM in creatine kinase (CK) isoenzyme electrophoretograms. Postoperative mortality rate during hospitalization was: no bands, 8.8%; one band, 13%, two bands, 63% (p less than 0.001). Patients whose sera showed cathodal bands were slightly older than those without, and their postoperative serum lactate dehydrogenase (LD) activity was greater, with very high proportions of LD 5. The two cathodal bands in patients who died differed in cathodal electrophoretic mobility from the two bands in survivors, implying that different enzyme forms were involved. All cathodal bands were inhibited by reagent containing diadenosine pentaphosphate, as was adenylate kinase from erythrocytes and liver cytoplasm. Mitochondrial CK from liver and presumed mitochondrial CK in serum from a patient with malignancy were not inhibited. We conclude that the appearance of two enzyme bands cathodal to CK-MM, probably representing adenylate kinase and possibly originating from various tissues, is associated with a poor prognosis in patients after cardiac surgery.
A "column-batch" method for separating MB and LD1 in a single fraction.
Authors: Morin L G , Barton E G
Published: January 2020

In this "column-batch" method for separating the MB and BB isoenzymes of creatine kinase and the LD1 isoenzyme of lactate dehydrogenase, one can, alternatively, separate MB from BB or obtain a combined fraction containing MB, BB, and LD1. The principal advantage is that the resulting fractions are twofold as concentrated as was the applied sample. Thus, activity can be measured by conventional automated methods, with no need for the modifications to compensate for diluted fractions that are required by other ion-exchange methods. Another advantage is the total absence of interference by the MM isoenzyme. A strong anion exchanger (AG-MP1, Bio-Rad) is used in the acetate form at pH 6.3. There is no retention of MM; retained MB, BB, and LD1 are eluted with a solution of magnesium acetate. Results are compared with those obtained for subunit B and LD1 by immunoinhibition. Results with patients are considered consistent with myocardial infarction if MB exceeds 20 U/L and 3% of the total CK and LD1 exceeds 130 U/L or 28% of the total LD activity.
Purification and characterization of human pancreatic phospholipase A2.
Authors: Eskola J U , Nevalainen T J , Aho H J
Published: January 2020

We purified human pancreatic phospholipase A2 from postmortem pancreatic tissue by elution of the semi-purified enzyme on CM-Sephadex C-25 with a linear NaCl gradient at pH 6.0. The enzyme appeared as a single polypeptide chain with an isoelectric point of 9.2 +/- 0.1. The relative molecular mass of the enzyme was estimated to be 15 800 +/- 1000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme is resistant to heating and to a 25 g/L concentration of sodium dodecyl sulfate. It is inhibited by Ca2+ ions in the presence of ovolecithin and deoxycholate. By immunohistochemical methods we showed the enzyme to be localized in the apical zymogen granule portion of pancreatic acinar cells.
Five recent urinary tests for early pregnancy evaluated.
Authors: Ryder K W , Munsick R A , Oei T O , Young P C , Blackford H F
Published: January 2020

We evaluate five tests developed for the earlier detection of pregnancy, either in the clinical laboratory or by the patient in her home. These tests offer no advantage over other urinary pregnancy tests, and the results are distinctly inferior to those reported (Clin. Chem. 29: 561-563, 1983) for some serum tests for pregnancy.
Quantification of urinary oxalate with oxalate oxidase from beet stems.
Authors: Obzansky D M , Richardson K E
Published: January 2020

We describe an automated (ABA-100) enzymic method for determination of urinary oxalate by use of oxalate oxidase (EC 1.2.3.4) isolated from beet stems. The H2O2 produced by the oxidation of oxalate by oxalate oxidase is measured by coupling with oxidation and conjugation of 3-methyl-3-benzothiazolinone hydrazone with N,N-dimethylaniline with catalysis by horseradish peroxidase. The resulting indamine dye is measured spectrophotometrically by the difference in absorption at 500 and 600 nm. Interfering substances are removed by oxidation with acidic ferric chloride and by cation-exchange chromatography. The assay is sensitive to 5 mg of urinary oxalate per liter, the standard curve is linear to 70 mg/L, and the procedure requires less than 3 h for completion. The within-run CV was less than 1.6%, the between-day CV less than 5.6%. The oxalate oxidase method results in a mean and reference interval for oxalate excretion that are comparable with those by isotope dilution, gas-chromatographic, colorimetric, and other enzymic procedures.
Salivary urea nitrogen as an index to renal function: a test-strip method.
Authors: Akai T , Naka K , Yoshikawa C , Okuda K , Okamoto T , Yamagami S , Inoue T , Yamao Y , Yamada S
Published: January 2020

To investigate the feasibility of using salivary urea nitrogen as an index of renal glomerular filtration rate, we developed and applied a new analytical system consisting of a urease-containing test strip and an automatic reflectance spectrometer. The concentrations of urea nitrogen so determined correlate well (r = 0.93) with concentrations in serum. These preliminary data suggest that our method can be used routinely as a simple and reliable means of detecting abnormalities of renal function.
An enzyme-linked immunosorbent assay for lactose synthase (galactosyltransferase) in serum and its application as a tumor marker in ovarian carcinoma.
Authors: Verdon B , Berger E G , Salchli S , Goldhirsch A , Gerber A
Published: January 2020

This assay for lactose synthase (galactosyltransferase, EC 2.4.1.22) in serum involves two sequential incubations: serially diluted standard or sample antigen is reacted with a fixed amount of antibody; unbound antibody is then adsorbed to wells of antigen-coated microtiter plates and determined by a second antibody directed against the first antibody and coupled to phosphatase. The standard curve is linear for galactosyltransferase concentrations of 10 to 600 micrograms/L. The within-assay CV of a serum sample was 9.3% (SD 4.1%), the between-assay was 3.8% (SD 2.4%). Serum galactosyltransferase concentrations computed from three different dilutions yielded CVs of 6.5% (SD 5.7%, n = 14). We evaluated the method's accuracy by recovery analysis and by comparing enzyme activity in serum with that of purified galactosyltransferase from human milk. The normal reference interval, as estimated from data on 27 healthy blood donors, was 60-436 micrograms/L (mean 224, SD 101 micrograms/L). We applied the assay to samples of serum from ovarian carcinoma patients grouped according to tumor burden. We also determined galactosyltransferase in ascites fluid and found these values useful for diagnosis, whereas determinations in serum may serve mainly for patient monitoring.
Spectrophotometric method for selective assay of the five isoenzymes of human lactate dehydrogenase, based on their different stabilities at alkaline pH.
Authors: Takizawa N , Yamauti J , Marui Y , Hayashi C , Yamashita J , Horio T
Published: January 2020

After preincubation of human lactate dehydrogenase (EC 1.1.1.27) isoenzymes 1 through 5 with lactate at pH 9.4, 9.8, 10.25, and 10.6 at 30 degrees C for 10 min, the reduction of NAD+ was measured at the same pH values and temperature during the interval 1-2 min after adding the coenzyme. Relative to the reference activities measured at pH 8.7 by the method of Buhl et al. (Clin Chem 23: 1289-1295, 1977), the respective activities of isoenzymes 1 through 5 thus measured were 111, 104, 96, 68, and 0% at pH 9.4; 123, 108, 88, 0, and 0% at pH 9.8; 140, 100, 0, 0, and 0% at pH 10.25; and 138, 0, 0, 0, and 0% at pH 10.6. These relations allow selective assay of the respective isoenzymes in samples containing mixtures of them. Optimal conditions for such selective assay at 37 degrees C are reported.
(3-sn-Phosphatidyl)cholines (lecithins) in amniotic fluid.
Authors: Touchstone J C , Levin S S , Dobbins M F , Matthews L , Beers P C , Gabbe S G
Published: January 2020

Many currently used thin-layer chromatographic methods for phospholipid assay rely on charring the developed plate in the presence of cupric acetate. Saturated acyl phospholipids do not react. We find that substitution of cupric sulfate results in detection of both saturated and unsaturated phospholipids. By exploiting the difference with the two reagents, one can separately estimate the amounts of saturated phospholipid. The method described here is reproducible, and we illustrate its use in determinations of (3-sn-phosphatidyl)cholines (lecithins) in amniotic fluid from problem pregnancies.
Time-course of cigarette smoke contamination of clinical hydrogen breath-analysis tests.
Authors: Rosenthal A , Solomons N W
Published: January 2020

The time-course of the contamination of exogenous hydrogen from cigarette smoke on postprandial breath hydrogen concentration was evaluated in 10 subjects, six regular smokers and four occasional smokers. Breath hydrogen values were determined by gas chromatography 10 min, 5 min, and immediately prior to smoking a filter cigarette; during smoking from a sample of exhaled air containing smoke; and 5, 10, and 15 min after extinguishing the cigarette. A three- to 137-fold increase above basal hydrogen concentrations was produced by exhaled cigarette smoke, but most subjects had re-equilibrated to baseline values within 10 to 15 min after the cigarette. If subjects undergoing clinical hydrogen breath tests cannot refrain from smoking during the duration of the test, one should allow an interval of at least 15 min from the end of smoking to the collection of a breath sample.
Enzyme immunoassay for carcinoembryonic antigen.
Authors: Smith A M , Macdonald D J
Published: January 2020

In this rapid, simple enzyme immunoassay for carcinoembryonic antigen (CEA) in serum the "sandwich" system is used. The CEA is first extracted from the serum into acetate buffer by heating at 70 degrees C, then incubated in a polyvinyl microtitration plate previously coated with anti-CEA. The bound CEA is then reacted with anti-CEA conjugated to horseradish peroxidase and the activity of the peroxidase bound to the wells of the plate is quantified by adding the enzyme substrate. The assay is sensitive to 3.1 micrograms/L (which can be improved to 0.39 microgram/L by prolonging the incubation times to 120 min), precise (between-assay CV 8.0%), and shows adequate analytical recoveries. Results compared favorably with those by a radioimmunoassay kit (Abbott Diagnostics), r = 0.99. The reference interval for serum CEA in normal adults is shown. This assay is appropriate for use in monitoring patients with colorectal tumors after surgery.