bluuebunny/crossref_metadata_2025 huggingface dataset:
Absorbance nephelometry of immune complexes by reaction with anti-IgG after treatment with polyethylene glycol.
Authors: Levinson S S , Goldman J
Published: January 2020

We describe absorbance nephelometry of immunoglobulin G (IgG) in immune complexes (IC) with use of anti-IgG after precipitating the IgG from serum by three treatments with polyethylene glycol, 25 g/L final concentration. The three-step procedure removed all of the monomeric IgG, eliminating interference with the IC assay. Analytical recovery of IC was nearly 100%. Both the standard curve and results for control sera showed good day-to-day reproducibility, with CVs of less than 8%. Eighteen of 20 patients with diseases involving IC showed distinctly increased concentrations of IC; no increases were seen in 15 normal persons. Measurement of IC in 44 samples by this assay and a C1q assay gave a Spearman rank correlation coefficient of 0.76. The assay appears suited to routine use with manual double-beam spectrophotometers and stable nephelometers. The procedure is inexpensive, straightforward, and easy to perform.
Determination of thiamin and thiamin phosphate esters in blood by liquid chromatography with post-column derivatization.
Authors: Kimura M , Itokawa Y
Published: January 2020

We demonstrate a liquid-chromatographic method involving post-column derivatization for determining the concentration of thiamin and its phosphate esters in human blood. Blood, erythrocytes, or plasma is deproteinized and centrifuged. Aliquots of the samples are applied to a mu Bondapak C18 column attached to a "high-performance" liquid chromatograph. Addition of potassium ferricyanide/sodium hydroxide solution to the column effluent with a proportioning pump converts thiamin phosphates into fluorophores, the intensities of which are measured with a spectrofluorophotometer. Thiamin, thiamin monophosphate, thiamin pyrophosphate, and thiamin triphosphate eluted as single peaks; no coeluting substances were detected. Thiamin pyrophosphate was the ester present in greatest concentration, followed by thiamin triphosphate; thiamin monophosphate and thiamin were present in slight amounts. This method allows easy determination of thiamin and its phosphate esters in 0.1 mL of blood.
Kinetic methods that are independent of the rate of reaction.
Authors: Harris R C , Hultman E
Published: January 2020

Kinetic methods for the quantification of substrates offer a number of advantages over equilibrium methods, such as speed and economy. However, in their most commonly practiced form they are liable to error from various sources in addition to those ordinarily encountered in equilibrium methods, owing to variations in enzymic activity between test and standard assays. In certain circumstances the differences in enzymic activity may be such as to completely invalidate the method. Such difficulties may, however, be avoided if three or more measurements are made during the reaction of the sample and are evaluated by one of five means. In each case data from the early course of the reaction are used to predict the total change in sensor signal at infinite time, from which the concentration of the substrate may be calculated as for an equilibrium method.
Discrepancies in measurement of aspartate aminotransferase by continuous-flow analysis.
Authors: West P
Published: January 2020

I compared results for aspartate aminotransferase (EC 2.6.1.1) obtained with a reaction-rate analyzer (LKB 2086 Mark Two), based on IFCC methodology, and a continuous-flow analyzer (the Technicon SMA 2) for 115 patients' sera and seven commercial quality-control sera. The data from the SMA 2 showed a clear positive bias in those sera with activities exceeding 40 U/L (the upper limit of the reference range). Independent data to support the bias of the SMA 2 and other continuous-flow analyzer systems are presented. Application of a correction factor to the SMA 2 data above the upper limit of the range significantly decreased this bias. Failure to apply such a factor to data obtained from continuous-flow analyzers could lead to serious clinical misinterpretation.
Accuracy of lactate dehydrogenase isoenzyme estimations by a thin-layer agarose fluorescent technique: experience with ternary and quaternary mixtures of purified human lactate dehydrogenase isoenzymes.
Authors: Pridgar E M , Leung F Y , Henderson A R
Published: January 2020

We have further assessed the accuracy of the thin-layer agarose fluorescent technique of Elevitch et al. [Am J Clin Pathol 46, 692 (1966)]. Previously, we used semi-purified human lactate dehydrogenase isoenzyme-1 and -5 [Clin Chem 22, 1995 (1976)] and isoenzyme-1 and -2 [Clin Chem 27, 1708 (1981)] to show that this assay accurately measures the proportions of these binary mixtures. In the present study, using ternary and quaternary mixtures of isoenzyme- 1, -2, -3, and -5, we show that the assay gives accurate estimations of all of these isoenzymes, within the errors of the techniques used. We also show that peak area (integration) is more nearly accurate, but less precise, than peak height (amplitude) measurements.
Influence of phospholipid saturation on classical thin-layer chromatographic detection methods and its effect on amniotic fluid lecithin/sphingomyelin ratio determinations.
Authors: Spillman T , Cotton D B , Lynn S C , Bretaudiere J P
Published: January 2020

We compared seven techniques, commonly used for detection of amniotic fluid phospholipids in thin-layer chromatography, with respect to their sensitivity to saturation of the fatty acid carbon-chain of lecithin. The techniques fell into two classes: sensitive and insensitive; those classed as saturation sensitive were less than or equal to 10% as sensitive to fully saturated lecithin as to lecithin with singly unsaturated acid moieties. Color development increased with the number of carbon-carbon double bonds per molecule but required only a single unsaturated acid ester in either the alpha or the beta position. Mixtures of lecithins with defined saturation, when detected by saturation-sensitive methods, mimicked the uneven coloration of amniotic fluid lecithin bands, supporting the hypothesis that this uneven coloration results from natural saturation heterogeneity. Detection techniques representative of these classes are shown to give widely differing values for the lecithin/sphingomyelin ratio for actual specimens of amniotic fluid.
Partial characterization, properties, and clinical significance of a lactate dehydrogenase-immunoglobulin A kappa complex in serum.
Authors: Weijers R N , Mulder J , Kruijswijk H
Published: January 2020

The existence of a lactate dehydrogenase-immunoglobulin A kappa complex was demonstrated as a marked increase of lactate dehydrogenase activity at the lactate dehydrogenase-3 band in the agar-agarose gel-electrophoresis pattern of sera from seven patients from an unselected group of 21 800 patients. The complex was isolated, in an almost pure form, by gel filtration and affinity chromatography, from the serum of a patient with circulating hepatitis B surface antigen. The complex had a relative molecular mass (Mr) of approximately 445 000 as determined by gel filtration. Electrophoresis in sodium dodecyl sulfate in the absence of reducing agents showed the presence of two subunit bands with Mr 170 000 and 34 000. We therefore propose that the native complex consists of one monomeric immunoglobulin A kappa linked to two tetrameric lactate dehydrogenase molecules. The enzymic activity of the complex is probably from lactate dehydrogenase isoenzyme-5.
Characterization of bacteria by mixed-dye fluorometry.
Authors: Shelly D C , Warner I M , Quarles J M
Published: January 2020

We describe an indirect detection method for bacterial identification and differentiation, based on selective adsorption of several fluorescent dyes. The lipid, protein, and nucleic acid components of fixed whole cells are stained with two mixtures, each containing two fluorochromes. The unadsorbed dyes were measured simultaneously with a video fluorometer [Clin Chem 22: 1483, 1976]. A dye-absorption matrix of the response can be generated, and we did so for each of nine bacteria. These responses were compared to a control or "complete" response matrix, and the response ratios of each bacterial species for each of the four dyes were calculated and plotted to obtain a characteristic pattern. From the response-ratio plots, plus simple pattern-recognition techniques, we could differentiate among all the bacteria. This rapid, sensitive technique is potentially applicable to a wide variety of bacteria.
Controlled dispersion analysis: flow-injection analysis without injection.
Authors: Riley C , Aslett L H , Rocks B F , Sherwood R A , Watson J D , Morgon J
Published: January 2020

Flow-injection analysis is a precise, elegant, and economical technique, but its most troublesome feature is the mode of injection of the sample slug. We describe an alternative approach in which the sample is aspirated by the sample probe. In the simplest version the probe normally rests in reagent; when sampling is to take place, the pump is stopped and the probe is transferred to the sample container. The pump makes a predetermined angular movement, the probe is returned to reagent, and the pump is restarted. In more advanced versions the same approach is combined with the merging zone technique. The system is economical, precise, and capable of full automation in a multichannel discretionary analyzer.
Liquid-chromatographic separation and determination of coproporphyrins I and III in urine.
Authors: Sakai T , Niinuma Y , Yanagihara S , Ushio K
Published: January 2020

We describe a method of determining coproporphyrin I, III, and I plus III in urine by "high-performance" liquid chromatography. Urine is simply injected after dilution with an equal volume of glacial acetic acid. Some urinary coproporphyrin apparently binds zinc without acetic acid treatment. The working linear range of coproporphyrin concentrations is 10 to 2000 micrograms/L of urine. The sensitivity of the method is sufficient to detect as little as 10 micrograms of coproporphyrins per liter of urine. Analytical recoveries for both coproporphyrins were 96.7-106%. Results by the present method and those by an extraction method (Br J Ind Med 31:72-74, 1974) correlate well (r = 0.975). Mean (and range) coproporphyrin I, III, and I plus III concentrations in urine from normal subjects are 33.7 (7-75), 28.6 (0-130), and 62.2 (7-174) micrograms/L, respectively.
Clinical evaluation of immunoinhibition determination of creatine kinase B subunits in coronary care.
Authors: Booth J , McCarthy P J , Walmsley R N
Published: January 2020

One hundred patients with chest pain of cardiac origin were evaluated on the basis of clinical findings, electrocardiograph results, and total creatine kinase (CK) and creatine kinase B-subunit (CK-B) activity (as determined by immunoinhibition with the Boehringer CK-MB kit) in serum. All patients diagnosed as having had an acute myocardial infarction had increased values for both CK-B and total CK. In no case was normal total CK activity associated with an increased CK-B, nor was normal CK-B associated with an increased total CK. During collection of data for reference ranges, we found 10 patients who had no evidence of cardiac disease but had various other diseases, who exhibited high values for CK-B in serum; four of these had normal values for total CK. We conclude that estimations of CK-B in serum by this method added no more diagnostic information than did data on total CK in the evaluation of chest pain.
Fluorometric procedure for determining "lamellar body" phospholipids in the particulate fraction of amniotic fluid after filtration.
Authors: Egberts J , Soederhuizen W , Gebhardt D O
Published: January 2020

A rapid (30-min) semiautomated continuous-flow procedure is described for use in assessing the phospholipids of the particulate ("lamellar body") fraction of human amniotic fluid. The method is based on measuring the difference in fluorescence of 1,6,-diphenyl-1,3,5-hexatriene added to amniotic fluid before and after micropore filtration. The filtration step removes "lamellar body" particles, which are considered to contain the fetal lung surfactant. The phospholipid values for the filtered particles are independent of background fluorescence, which increases when amniotic fluid is contaminated by bilirubin pigments or blood components. Over a wide range (3-150 mumols/L) the fluorescence increases linearly with the phospholipid concentration of the amniotic fluid. There is a good agreement between the value for particulate "lamellar body" phospholipid, the ratio of the "lamellar body" phospholipids to total amniotic fluid phospholipids, and the lecithin/sphingomyelin ratio.
Radioimmunoassay for bupropion in human plasma: comparison of tritiated and iodinated radioligands.
Authors: Butz R F , Smith P G , Schroeder D H , Findlay J W
Published: January 2020

We evaluated the potential usefulness of 125I-labeled p-hydroxybupropion in a direct radioimmunoassay for bupropion in human plasma as compared with a currently used [3H]bupropion dextran-coated charcoal method. In both radioimmunoassay methods succinoylpropylbupropion antiserum was used that was highly specific for unchanged drug, cross reactivities with known bupropion metabolites being less than 0.3%. However, the use of 125I-labeled p-hydroxybupropion afforded greater sensitivity (0.3 microgram/L vs 0.6 microgram/L with [3H]bupropion) and was readily adaptable to the more convenient polyethylene glycol separation method. Between-assay CVs were 3.8 to 12.2% (mean 7.6%) with the 125I-based radioimmunoassay and 5.1 to 11.5% (mean 7.5%) with the 3H-based assay. Agreement between the two radioimmunoassay determinations of buproprion in human plasma samples collected over a 60-h period after oral drug administration was excellent (slope = 1.086, r = 0.989). We find the 125I-based assay a convenient and suitable alternative to the [3H]bupropion assay in pharmacokinetic studies in humans.
Quantification of lipoprotein cholesterol in serum from children with different lipoprotein profiles: heparin-calcium precipitation and ultracentrifugation compared.
Authors: Srinivasan S R , Webber L S , Whitaker C F , Berenson G S
Published: January 2020

We compared the serum lipoprotein cholesterol concentrations in subgroups of children (n = 360), ages 5-17 years, as measured by the heparin-Ca2+ and preparative ultracentrifugation methods. Children were grouped from the total population on the basis of their previous results for serum beta- and pre-beta-lipoprotein cholesterol (Group I: low beta- and low pre-beta-; Group II: high beta- and low pre-beta; Group III: high beta- and high pre-beta-; Group IV: low beta- and high pre-beta-). The values for very-low-density (VLDL) cholesterol by ultracentrifugation method were 44, 53, 15, and 10 mg/L greater than the values for pre-beta-lipoprotein cholesterol by the heparin-Ca2+ method in Groups I, II, III, and IV, respectively; the differences were not significant in Group IV. The values of low-density (LDL) cholesterol were 64, 137, 144, and 73 mg/L less than the values for beta-lipoprotein cholesterol in Groups I, II, III, and IV, respectively (p less than 0.005). On the other hand, high-density (HDL) cholesterol concentrations in the respective four groups were 10, 37, 93, and 52 mg/L greater than alpha-lipoprotein cholesterol concentrations; the differences were significant for Groups II, III, and IV (p less than 0.005). Overall, the values for LDL-cholesterol correlated highly with beta-lipoprotein cholesterol (r = 0.94), whereas correlations for VLDL- and HDL-cholesterol values with pre-beta-lipoprotein cholesterol (r = 0.76) and alpha-lipoprotein cholesterol (r = 0.77) were somewhat lower. The differences between these two methods may result from their different operational definitions for measuring serum lipoproteins and the possibility that without appropriate corrections the values obtained by preparative ultracentrifugation do not serve as reference values.
Ultrafiltrable calcium and magnesium in ultrafiltrates of serum prepared with the Amicon MPS-1 system.
Authors: D'Costa M , Cheng P T
Published: January 2020

We evaluated the Amicon micropartition system (MPS-1) for preparing ultrafiltrates of serum for use in evaluating ultrafiltrable Ca and Mg. We found no adsorption of either to the filter and 99.6% retention of serum proteins on the membrane. Ultrafiltrate volumes recovered (100-450 microL) varied with centrifugation time (10-30 min) and temperature. Centrifugation time did not affect the measured concentration of ultrafiltrable calcium and magnesium, and pH change in the 1-mL serum specimen during a 30-min centrifugation at room temperature was negligible. There was an inverse relationship between temperature and ultrafiltrable Ca and Mg concentrations. The precision (CV) between filters ranged from 1.2 to 5.1% for ultrafiltrable Ca and 1.5 to 2.7% for ultrafiltrable Mg. The correlation between ultrafiltrable Ca (y) and ionized Ca (x) in samples from 115 patients with calcium-related metabolic disorders was good (y = 1.04x + 0.18; r = 0.9128). We find the MPS-1 to be a simple and convenient tool for the rapid production of serum ultrafiltrates.
Development and clinical evaluation of a microcentrifugal analyzer method for determining creatine kinase MB isoenzyme.
Authors: Massey T H , Butts W C
Published: January 2020

We have adapted to a microcentrifugal analyzer an immunoinhibition assay for measuring the activity of creatine kinase MB by using an inhibitory antibody for the M monomer. The method actually measures half the MB activity, but results are not multiplied by two because atypical isoenzymes of creatine kinase, including BB, IgG-BB, and the isoenzyme derived from mitochondria, are also detected, if they are present. Results correlated well with an electrophoresis method for 36 serum samples. Myocardial infarction was assessed in 175 patients admitted to our coronary-care unit, with respect to sensitivity (100%) and specificity (98%) when a decision point of 100 U/L (30 degrees C) was chosen for total creatine kinase activity (dithiothreitol-activated) and 6 U/L (30 degrees C) for the isoenzyme (by immunoinhibition). Atypical isoenzymes are easily recognized and confirmed by electrophoresis when the MB activity (by immunoinhibition) exceeds 6 U/L and 20% of the total creatine kinase activity.
Interference of fetal hemoglobin and labile glycosylated hemoglobin with measurements of glycosylated hemoglobin.
Authors: Yatscoff R W , Tevaarwerk G J , Clarson C L , Warnock L M
Published: January 2020

We examined the effect of fetal hemoglobin and labile glycosylated hemoglobin on a number of diverse methods used to measure glycosylated hemoglobin. Samples were supplemented with various amounts of cord blood to give proportions of fetal hemoglobin ranging from 1 to 20% of total hemoglobin concentration. Procedures in which the separation of hemoglobin A1 from the major hemoglobin A fraction is based on differences in ionic properties (cation-exchange chromatography and electrophoresis) are subject to interference by fetal hemoglobin, whereas procedures that base the quantitation on other properties (colorimetry and affinity column chromatography) are not. The same procedures that are affected by the presence of fetal hemoglobin are also subject to interference by labile glycosylated hemoglobin. We conclude that the affinity chromatographic and colorimetric methods may give a more nearly accurate determination of glycosylated hemoglobin.
Automated rate-immunonephelometric determination of serum prealbumin (transthyretin).
Authors: Jacob R A , Gorman N
Published: January 2020

We describe an automated determination of serum prealbumin (transthyretin), based on the maximum rate of light scatter of an antigen-antibody precipitate. Optimal dilutions of antibody reagent and of 100-microL serum samples are given. Within- and between-assay CVs are less than 5%. Results by this method are nearly identical with those obtained by a radial-immunodiffusion technique. Measuring the rate of light scatter provides a considerably faster test than equilibrium scatter, electrophoretic, or radial-immunodiffusion methods. Treatment before analysis to clear lipemic sera obviates falsely high results.
Optimization of enzyme-based assays in coagulation testing.
Authors: Coleman P L , Perry J F , Wehrly J A
Published: January 2020

Optimized assays for antithrombin III and plasminogen have been developed based on a study of the kinetic parameters Km and Kcat for four commercially available substrates: the p-nitroanilide derivatives of D-Phe-pipecolyl-Arg (S-2238), and toluenesulfonyl-Gly-Pro-Arg (Chromozym TH), which are thrombin substrates; D-Val-Leu-Lys (S-2251), a plasminogen/streptokinase substrate; and alpha-N-carbobenzoxy-L-lysine thiobenzyl ester, a substrate for both enzymes. We used a centrifugal analyzer system for rapid data acquisition and interactive analysis. Optimized conditions for assay of a particular enzyme are not constant for different substrates in the same buffering agent. For example, in 1,4-piperazine diethanesulfonic acid buffer at 37 degrees C, thrombin-catalyzed hydrolysis of Chromozym TH is optimal at 125 mmol/L buffer, 100 mmol/L NaCl, and pH 8.2, whereas substitution of S-2238, also a tripeptide p-nitroanilide, yields optimal hydrolysis at 85 mmol/L buffer, 300 mmol/L NaCl, and pH 7.2. We conclude that optimized assay conditions are best obtained by an extensive survey of available buffers and a detailed investigation of the effects of variation in pH and in the concentrations of the buffer and auxiliary reagents through use of both one-factor-at-a-time and multivariate response surface experimentation.
Performance of the Hitachi 705 evaluated.
Authors: Douville P , Forest J C
Published: January 2020

We evaluated a new discrete analyzer, the Hitachi 705. The instrument can process sequentially as many as 19 different enzymes or other analytes, with a throughput of 180 tests per hour. We evaluated the spectrophotometer, the sample and reagent probes, and the performance for 11 assays: creatine kinase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransférase, amylase, glucose, urea nitrogen, creatinine, albumin, calcium, and phosphorus. The total imprecisions (within-day plus day-to-day component of imprecision) (CV) were: for the photometer (six concentrations of potassium dichromate), 0.14 to 0.4%; for the serum probe (55 to 20 microL), 0.4 to 1.0%; for the reagents probes (50 to 350 microL), 0.3 to 1.2%; for the enzymatic procedures, 2.0 to 8.0%; and for the other tests (glucose, urea nitrogen, creatinine), 1.0 to 4.0%. Good linearity was obtained in the proposed range for the 11 tests, except for calcium. Comparison of the total analytical error with the medically allowable error at a decision value showed that the test results were acceptable, except for calcium and phosphorus. The instrument showed good reliability and practicability.
Studies of the Regan alkaline phosphatase isoenzyme in plasma from a lung-carcinoma patient.
Authors: Cleeve H J , Tua D C
Published: January 2020

We compared an alkaline phosphatase (EC 3.1.3.1) isoenzyme from a plasma sample of a patient with carcinoma of the lung, who was suspected of having the Regan-type isoenzyme, with placental serum alkaline phosphatase and with tissue preparations from placenta, liver, bone, and intestine. The placental and Regan phosphatases responded similarly to heat treatment and modification by various inhibitors, except that the Regan isoenzyme was more resistant to EDTA inactivation than was the placental serum alkaline phosphatase, but more sensitive than placental tissue alkaline phosphatase. Isoelectric focusing and Cellogel electrophoresis showed the Regan isoenzyme to have a mobility similar to that of the placental type. We conclude that the isoenzyme was of the Regan type and support the contention that the placental and Regan isoenzymes are products of the same structural gene, with small differences due to post-genetic modification.
Analysis of human leukemic cells by use of high-resolution two-dimensional electrophoresis. I: results of a pilot study.
Authors: Anderson N L , Wiltsie J C , Li C Y , Willard-Gallo K E , Tracy R P , Young D S , Powers M T , Anderson N G
Published: January 2020

We analyzed mononuclear leukocytes from patients with various human leukemias by high-resolution two-dimensional electrophoresis. Tumor cells of the granulocytic, monocytic, and lymphoid lineages [obtained from chronic granulocytic leukemia in blast transformation, acute monocytic leukemia, and chronic lymphocytic leukemia (CLL), respectively] can be easily recognized by using a series of cell-type marker proteins identified by comparison of fractionated normal cell populations. B and T cell types of CLL could be distinguished, the results correlating well with those obtained by use of monoclonal-antibody staining methods. In two cases representing almost pure B-cells (classical CLL; 0% T, 85% B) and T-cells (cutaneous T-cell leukemia; 77% T, 0% B), 27 of 29 marker proteins showed quantitative B/T differences comparable to those observed in comparisons of normal B-and T-lymphocytes prepared by cell sorting. These results indicate that cells from relatively well-differentiated leukemias show complex patterns of gene expression very similar to those of the corresponding normal cells and strongly support the use of large marker panels in cell-type determination. Less-well-differentiated acute leukemias [such as acute undifferentiated and acute granulocytic (FAB:M1)] appear to yield protein patterns corresponding less closely to recognizable mature cell types, and may show expression of novel proteins related to the state of differentiation.
Serum lipoprotein measurement--liquid chromatography and sequential floatation (ultracentrifugation) compared.
Authors: Okazaki M , Itakura H , Shiraishi K , Hara I
Published: January 2020

An HPLC method [J. Biochem. (Tokyo) 91:1381, 1982] was used for evaluating serum lipoproteins, with on-line monitoring of either cholesterol or phospholipids. Five well-distinguished lipoprotein fractions were observed, based on their particle sizes. Serum of 15 normal persons, 12 subjects with various types of hyperlipidemia, 20 patients with various liver diseases, and two cases of familial LCAT deficiency were examined and the results compared with those by a sequential ultracentrifugal floatation technique. In the normal group, the amounts of fractions 2, 3, and 4 by the HPLC method correlated well with concentrations of the LDL, HDL2, and HDL3 fractions as measured by the ultracentrifugal method, respectively. In the hyperlipidemic group, similar good correlations were observed between fractions 1, 2, 3, 4 and chylomicrons + VLDL, LDL, HDL2, and HDL3 fractions, respectively. For those with liver diseases or LCAT deficiency, the corresponding fractions correlated less well, and characteristically the elution profile of lipoproteins in these groups showed heterogeneity of particle size within each lipoprotein density class, especially in LDL and HDL2.
Enzyme-linked immunosorbent assay for detection of antibodies to extractable nuclear antigens in systemic lupus erythematosus, with nylon as solid phase.
Authors: Ishaq M , Ali R
Published: January 2020

In this enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against extractable nuclear antigens (ENA) in sera of patients with systemic lupus erythematosus (SLE), nylon is used as solid phase for antigen binding instead of the commonly used polystyrene surface. Optimal conditions for activation of the nylon beads, antigen coating, and other relevant factors have been investigated. We compared the incidence of anti-ENA antibodies in SLE, using chromogenic and fluorogenic enzyme substrates. Of SLE patients, 54% were positive for anti-ENA antibodies when chromogenic substrate was used as compared with 68% for fluorogenic substrate. Antibody activity against Sm and RNP antigens was distinguished on the basis of ribonuclease sensitivity of the RNP antigen. The method described offers advantages such as decreased background activity, increased surface area, facility for prolonged storage of antigen-coated solid phase, and miniaturization of the assay.
Creatine kinase and lactate dehydrogenase: stability of isoenzymes and their activity in stored human plasma and prostatic tissue extracts and effect of sample dilution.
Authors: Shain S A , Boesel R W , Klipper R W , Lancaster C M
Published: January 2020

We examined the effect of storing human plasma or extracts of prostate at -90 degrees C on the activity of creatine kinase and lactate dehydrogenase and isoenzyme distribution. Enzyme activities were unaltered during storage for as long as six weeks. If these preparations were thawed only once at 2 to 4 degrees C, they could be stored for as long as 165 days at -90 degrees C with no change in isoenzyme distribution. Inexplicably, apparent isoenzyme distribution of prostatic lactate dehydrogenase was sensitive to sample dilution, whereas the isoenzyme distribution of lactate dehydrogenase in plasma was not. Our observations emphasize the importance of validating details of analytical protocols that are to be used for quantification of new types of specimens.
Modified technique for periodic acid-Schiff staining of glycoproteins on agarose-film electrophoretograms.
Authors: Trivedi S M , Frondoza C G , Humphrey R L
Published: January 2020

We have modified the periodic acid-Schiff staining technique for glycoproteins for use with thin agarose-film electrophoresis membranes. With this procedure, carbohydrate-rich proteins can be detected with negligible background staining and insignificant staining of nonglycoproteins such as albumin and nonglycosylated Bence Jones proteins (free light chains). On the other hand, carbohydrate-rich M components of immunoglobulins M and A in serum and in cerebrospinal fluid from patients with plasma cell dyscrasia are readily detected. The technique is two- to threefold more sensitive than Ponceau S. Glycoproteins in serum and body fluids can be determined as a routine analytical procedure.
Clinical laboratory evaluation of Ames' Seralyzer Reflectance Photometer for total bilirubin and cholesterol.
Authors: Brown S , Savory J , Wills M R
Published: January 2020

We evaluated the dry-reagent technology for determining cholesterol and adult total bilirubin in serum with the Ames Seralyzer Reflectance Photometer. This instrument is designed to measure the intensity of light of a selected wavelength that is reflected from reagent-impregnated test strips saturated with sample. Regression analysis of the data for total bilirubin in adults (y) compared with corresponding values obtained with the Technicon SMAC (x) yielded the regression line: y = 1.07x + 2.5 (n = 128, r = 0.99). Day-to-day precision (CV) was 3.8% at 8.1 mg of bilirubin per liter, 5.34% at 48.3 mg/L. Regression analysis of the cholesterol data (y) vs cholesterol values obtained with an enzymatic cholesterol method (x) yielded the regression line: y = 0.91x + 191 mg/L (n = 100, r = 0.98). Day-to-day CVs for cholesterol were 3.6% at 1.62 g/L, 3.11% at 2.62 g/L.
Liquid-chromatographic measurement of creatinine in serum and urine.
Authors: Okuda T , Oie T , Nishida M
Published: January 2020

We describe the adaptation of a "high-performance" liquid chromatographic method for determination of creatinine in serum and urine. The proposed method is simple, rapid, precise, and accurate. The retention time for creatinine can be varied simply by changing the KH2PO4 concentration in the mobile phase: acetonitrile/aqueous KH2PO4 (1/4 by vol). Within-day precision (CV) was 1.2-3.6% in serum chromatographed with an internal standard, and 2.3-2.8% in serum when an external standard was used. Between-day precision (CV) was 1.3-2.1% in serum and 1.3-2.7% in urine (with an external standard). Analytical recoveries of creatinine added to serum were 94-100% for the method with an internal standard, 95-103% with an external standard.
Characteristics of high-affinity folate binding in human leukocytes.
Authors: Holm J , Hansen S I , Lyngbye J
Published: January 2020

High-affinity binding of [3H]folate in leukocytes from normal subjects was studied in equilibrium dialysis experiments (pH 7.4, 37 degrees C). Binding displayed positive cooperativity, and the binding affinity increased with decreasing concentration of the binding protein. Both phenomena could be interpreted in terms of ligand binding to a polymerizing system where the affinity of ligand for the oligomer is greater than its affinity for the polymer prevailing at higher concentrations of the binding protein.
Measurement of true salicylate concentrations in serum from patients with Reye's syndrome.
Authors: Kang E S , Todd T A , Capaci M T , Schwenzer K , Jabbour J T
Published: January 2020

Patients with Reye's syndrome who have been given aspirin are said to maintain higher-than-anticipated salicylate concentrations in blood, for longer than expected. We explored whether this could be attributed to spurious results from nonsalicylate compounds in the Trinder reaction for salicylates. All of 63 organic acids and amines examined that form colored complexes with Trinder's reagent had detectable absorbance at 540 nm at 0.2 g/L, including some endogenous compounds known to be increased in Reye's syndrome patients and many others endogenous in humans. By subjecting deproteinized sera to thin-layer chromatography and eluting the salicylate fraction before complexing it with ferric ion, true salicylate can be measured quantitatively and differentiated from interfering compounds. In addition, when we examined the effect of salicylate on palmitate binding to serum proteins, we found that salicylate concentrations of 0.2 g/L displaced [16-14C]palmitate binding to protein more in Reye's syndrome patients than in Reye's syndrome survivors or children with influenza. This suggests the presence of atypical binding characteristics for salicylate and palmitate in the acute disorder but not in survivors or children with influenza.
Enzyme inactivation in serum before determination of total bile acids.
Authors: Hanson N Q , Freier E F
Published: January 2020

Endogenous NADH-generating enzymes must be inactivated before total serum bile acids can be measured accurately by the direct enzymic method. To do this, we pretreat the sera with NaOH, in a final concentration of 0.1 mol/L. Consequently, lactate dehydrogenase activity at least as high as 30 000 U/L is destroyed, obviating blank determinations. Values for bile acid in serum, so obtained, agree with values obtained after pretreatment with heat, an alkali-methanol solution, or sodium pyruvate, but our pretreatment has the advantages of ease, speed, economy, and negligible blank values.
Reagent for the enzymatic determination of serum total cholesterol with improved lipolytic efficiency.
Authors: Siedel J , Hägele E O , Ziegenhorn J , Wahlefeld A W
Published: January 2020

We describe a sensitive method for quantifying the extent of cholesterol ester cleavage during enzymatic assay of total cholesterol in serum. Lipids are extracted from the assay mixture with chloroform/methanol (1/1 by vol), concentrated, then quantified by "high-performance" thin-layer chromatography. Although with conventional enzymatic reagents for determination of serum total cholesterol the hydrolysis of the cholesterol esters may be incomplete, a new enzymatic cholesterol reagent (Monotest Cholesterol, High Performance, Boehringer Mannheim) gives virtually complete cholesterol ester cleavage (i.e., greater than or equal to 99.5%). Use of this reagent with its improved lipolytic efficiency yields results for serum total cholesterol that are identical to those measured with a candidate reference procedure involving alkaline cholesterol ester saponification.
Glycosylated hemoglobin measured by affinity chromatography: micro-sample collection and room-temperature storage.
Authors: Little R R , England J D , Wiedmeyer H M , Goldstein D E
Published: January 2020

Under proper conditions, whole blood can be stored at room temperature for as long as 21 days before measurement of glycosylated hemoglobin by affinity chromatography. Whole blood (anticoagulated with EDTA or heparin) was placed in capillary tubes, which were then sealed at both ends and stored at room temperature. Just before assay, whole blood was rinsed from the tubes and diluted 10-fold with water. Samples of each patient's blood were assayed as whole-blood hemolysates by affinity chromatography after zero, seven, 14, and 21 days of storage. Values for glycosylated hemoglobin did not change over 21 days of storage and values for each storage day correlated well (r = 0.97, p less than .0001) with hemoglobin A1C measured in fresh erythrocyte hemolysates by "high-performance" liquid ion-exchange chromatography.
Liquid-chromatographic determination of aminoglutethimide in plasma.
Authors: Robinson B A , Cornell F N
Published: January 2020

A simple, rapid "high-performance" liquid-chromatographic procedure is presented for the determination of aminoglutethimide in plasma. After precipitation of the protein with acetonitrile, an aliquot of the supernate is injected directly onto a radially compressed, reversed-phase column. The aminoglutethimide is isocratically eluted with a mobile phase of acetonitrile/water/tert-butyl ammonium phosphate. The method is both accurate and precise and has been in routine use in our laboratory for more than 12 months.
Automated particle-counting immunoassay of C-reactive protein and its application to serum, cord serum, and cerebrospinal fluid samples.
Authors: Collet-Cassart D , Mareschal J C , Sindic C J , Tomasi J P , Masson P L
Published: January 2020

The assay for C-reactive protein has been fully automated as a particle-counting immunoassay. For cerebrospinal fluid, cord serum, and adults' serum its range of sensitivity extends from 1 microgram/L to 300 mg/L, with a minimal sample dilution of twofold and a maximal dilution of 50-fold being required. This range is so broad because free antibodies are added to the reaction medium. However, we have used Fab fragments rather than whole antibody to avoid too steep a standard curve and a decrease of agglutination at high concentrations of antigen. For 99 consecutive cord sera examined, the concentrations of C-reactive protein ranged from 7 micrograms/L to 1.750 mg/L. The geometric mean was 50 micrograms/L and the upper normal limit (geometric mean +/- 2 SD of the log values) was established at 525 micrograms/L.
Dipalmitoylphosphatidylcholine in amniotic fluid quantified by fast-atom-bombardment mass spectrometry.
Authors: Ho B C , Fenselau C , Hansen G , Larsen J , Daniel A
Published: January 2020

Dipalmitoylphosphatidylcholine (DPC) is quantified by taking advantage of stable-isotope-labeled d9-DPC as internal standard. Use of a mass spectrometer to measure the ratio of d9/d0 makes this procedure a quantitative one. d9-DPC was synthesized by refluxing dipalmitoylethanolamine with d3-methyl iodide in methanol in the presence of sodium bicarbonate for 26 h. The yield of d9-phosphatidylcholine (d9-lecithin) was 89% after column-chromatographic purification. Fast atom bombardment was used to desorb the preformed phosphatidylcholine ions in a mass spectrometer of Nier-Johnson geometry. In our assessment of accuracy and precision of this technique, we found a correlation coefficient of 0.9994 between signal and sample concentration. The method was less precise when the total d0- plus d9-DPC was less than 0.2 micrograms or when the ratio of d0- to d9-lecithin exceeded 100. The within-run CV was about 1.0%. The amount of DPC in amniotic fluid samples assessed by mass spectrometry was compared with results for total phosphatidylcholine quantified by thin-layer chromatography. The fate of DPC in various laboratory manipulations was also studied.
Detection of morphine in urine by hemagglutination inhibition, with use of lyophilized reagents.
Authors: Vanzetti G , Cassani M , Valente D
Published: January 2020

We describe a modified test of hemagglutination inhibition for the detection of morphine in urine, similar to the well-known test for pregnancy. The reaction takes place in test tubes or ampoules containing carefully matched amounts of lyophilized morphine antiserum and tanned human erythrocytes coated with morphine conjugated to rat serum albumin. The reagents are reconstituted by adding 100 microL of urine and 400 microL of water, and the result is read after 60 min. The detection limit, tested with the method of Gorodetzky (Clin Chem 19:753, 1973), was about 200 ng of total morphine per milliliter of urine. For more than 2000 samples, results by our test agreed satisfactorily with those obtained by an accepted RIA method. The test is suitable for rapid screening in field work, monitoring subjects during detoxication, and use in nonspecialized laboratories. Confirmatory analysis is needed for quantitative measurements, forensic purposes, and discrimination between morphine and cross-reacting opiates.
Radioimmunoassay for human corticosteroid-binding globulin.
Authors: Rosner W , Polimeni S , Khan M S
Published: January 2020

We isolated corticosteroid-binding globulin from human plasma and used the isolated protein as the antigen in raising antibodies in rabbits. The resulting antiserum was used to develop an RIA with which we can determine the globulin in as little as 0.007 microL of serum, although we routinely use, after appropriate dilution, 0.02 microL. The mean concentration of corticosteroid-binding globulin in the serum of 40 normal subjects was 32.4 (SD 4.2) mg/L. The correlation (r) between results with this RIA and those by an established binding assay was 0.94.
Effect of Fluosol-DA on radioimmunoassay results.
Authors: Hammarstrom M , Mullins R , Sgoutas D
Published: January 2020

Fluosol-DA, an emulsion of perfluorocarbons, is used as a blood substitute. Its presence in samples affects results of certain radioimmunoassays routinely done in our laboratory. In several, added Fluosol-DA caused an upward displacement of the standard curve, the extent of which depended on the structure and quantity of the assayed antigen, the concentration of Fluosol-DA, and the nature of the immunoreagents. Fluosol-DA markedly affected results of assays for triiodothyronine, thyroxin, and digoxin (by one method), but assays for aminoglycosides and protein hormones were unaffected. We determined that Fluosol-DA interacts with unbound labeled (and, presumably, with unlabeled) antigen to form a complex that coprecipitates with the bound antigen-antibody fraction during the separation step. In comparison with the idealized condition we observed an apparent increase in antibody-bound labeled antigen and consequently an apparent underestimation of the measured antigen. Depending on the nature of the immunoreagents, antibodies can extract more or less of their respective antigens from particles of Fluosol-DA emulsion.
Influence of age and sex on the concentration of free thyroxin in serum and on the free thyroxin: total thyroxin ratio.
Authors: Wilke T J
Published: January 2020

The influence of age and sex on the concentration of free thyroxin and on the free thyroxin/total thyroxin ratio was studied in 104 men and 89 women, ages 15 to 83 years. Free thyroxin gradually declined with age in men but not in women. In addition, sex- and age-related variations for free thyroxin were similar to those for total thyroxin and the two indirect assessments of free thyroxin: the free thyroxin index and the total thyroxin/thyroxin binding globulin ratio. Further, free thyroxin and the free thyroxin/total thyroxin ratio were higher in men than in women. However, the free thyroxin/total thyroxin ratio showed relatively little change with age in either sex. This study suggests that the free thyroxin/total thyroxin ratio is helpful in minimizing the age-related variations in both free and total thyroxin.
Enzyme immunoassay of thyroxin-binding globulin in dried blood samples on filter paper.
Authors: Hata N , Ito M , Mizuta H , Nose O , Miyai K
Published: January 2020

A double-antibody enzyme immunoassay was developed for determination of thyroxin-binding globulin in dried blood samples on filter paper. The measurable concentration range of thyroxin-binding globulin in two 3-mm blood discs was 3.3 to 52 mg/L equivalent of serum (i.e., equivalent to the concentrations in known serum standards). Thyroxin-binding globulin in dried blood samples on filter paper was stable for at least four weeks when kept dry at -20 degrees C, 4 degrees C, or room temperature. The mean coefficients of variation were 6.6% (within assay) and 5.9% (between assays). The concentrations of thyroxin-binding globulin in dried blood samples determined by this method correlated well with those in serum determined by radioimmunoassay (r = 0.95) and by enzyme immunoassay (r = 0.96). This method is applicable for detecting cases of thyroxin-binding globulin deficiency and avoids the false-positive results for neonatal hypothyroidism obtained by measuring thyroxin.
Unconjugated estriol in serum as measured by liquid chromatography with electrochemical detection, compared with radioimmunoassay.
Authors: Kaplan L A , Hohnadel D C
Published: January 2020

Concentrations of unconjugated estriol in maternal serum or plasma increase throughout pregnancy, particularly during the third trimester. We present and discuss the results of a comparative study of unconjugated estriol as measured by a new "high-performance" liquid-chromatographic assay and by a conventional semiautomated radioimmunoassay procedure. In the new method, serum is extracted and chromatographed on a reversed-phase mu Bondapak-C8 column under radial compression. The estriol peak is detected with a glassy-carbon electrochemical detector. The chromatographic results (y) correlated well with the RIA results (x) for 105 samples from 45 pregnant women in their third trimester (y = 1.07x - 0.55 micrograms/L, r = 0.933), with no significant difference between the means of the two methods.
Two-site immunochemiluminometric assay for human alpha 1-fetoprotein.
Authors: Weeks I , Campbell A K , Woodhead J S
Published: January 2020

In this two-site immunochemiluminometric assay for human alpha 1-fetoprotein, acridinium ester-labeled monoclonal antibodies are used that have an average incorporation ratio of 0.3 mol of acridinium ester per mole of antibody. The solid-phase antibody consists of sheep anti-alpha 1-fetoprotein IgG covalently coupled to a diazonium derivative of reprecipitated aminoaryl cellulose. The assay involves a 1-h incubation after the simultaneous incubation of labeled and solid-phase antibodies. The subsequent quantification time is 10 s per tube. The assay has a working range of 20 to 246 kilo-int. units/L for CVs less than or equal to 10% and a sensitivity of detection of 1.3 kilo-int. units/L. Experiments with monoclonal antibodies labeled to an average incorporation ratio of 2.8 mol of acridinium ester per mole of antibody suggest that assay sensitivity increases with increased specific activity. alpha 1-Fetoprotein concentrations in the sera of pregnant women at 14-20 weeks of gestation, as measured by the present assay, agreed with the results of a conventional radioimmunoassay.