bluuebunny/crossref_metadata_2025 huggingface dataset:
An automated approach to characterize, in simple kinetic terms, the generation and inactivation of thrombin and the interaction of fibrinogen with thrombin.
Authors: Hoak D R , Banerjee S K , Kaldor G
Published: January 2020

Here, we used a fully automated, computer-directed centrifugal analyzer (which permitted simultaneous turbidimetry and calculation of results) and purified thrombin, fibrinogen, and various inhibitors to study clot formation. The Km and Vm for these reactions were useful in detecting and partly characterizing anticoagulants. We also explored the generation and inactivation of thrombin, using the two-stage prothrombin time and antithrombin activity tests. The amount of thrombin instantaneously generated and inactivated was monitored under artificially created pathological conditions. The pseudo-first-order rate constant for thrombin generation and inactivation and the instantaneous concentration of enzymatically active and inactive thrombin were used in the characterization of these conditions. We believe this approach is suitable for routine clinical use.
Non-resolving jaundice: bilirubin covalently attached to serum albumin circulates with the same metabolic half-life as albumin.
Authors: Reed R G , Davidson L K , Burrington C M , Peters T
Published: January 2020

In hepatobiliary disease and biliary obstruction, bilirubin often becomes covalently bound to albumin circulating in serum, producing a nondissociable complex. To determine how long this complexed bilirubin remains in the circulation, we compared the metabolic clearance of bilirubin-albumin complexes with the clearances of free bilirubin and unmodified albumin. Radiolabeled bilirubin, albumin, and covalent bilirubin-albumin were injected into the circulation of Sprague-Dawley rats and serial samples of plasma were analyzed for the injected compounds. The half-life of bilirubin was 6.2 min. The half-life of bilirubin covalently bound to rat serum albumin was 1.9 to 2.1 days, identical to that of unmodified rat albumin. We conclude that bilirubin covalently attached to albumin is maintained in the circulation with the long half-life of albumin rather than the short half-life of bilirubin. Because albumin in humans has a half-life of 19 days, covalent attachment of bilirubin to human albumin could result in persistence of hyperbilirubinemia long after the resolution of disease.
Some thermodynamic parameters and temperature-conversion factors for determining alpha-amylase concentration in serum.
Authors: Hafkenscheid J C , Hessels M
Published: January 2020

We measured the relationship between measured activity concentration and temperature for alpha-amylase (EC 3.2.1.1), using 10 different substrates. At 25-37 degrees C the Arrhenius plot was linear. The activation energy ranged from 33.3 kJ.mol-1 with maltoheptaose as substrate to 54.4 kJ.mol-1 with the Blue-Starch method. Activation energy was lowest for substrates having seven glucose moieties, the ones most suitable for determining alpha-amylase--i.e., the presence of more or fewer glycosyl units increased the activation energy. Substrates with blocked groups at the nonreducing end of the oligosaccharide chain were not considered here, because the relative reaction rates obtained with these substrates were less than those obtained with nonblocked substrates. We also determined temperature-conversion factors for alpha-amylase reaction with the various substrates. The results are discussed in relation to thermodynamic parameters of some other enzymes, e.g., creatine kinase and alkaline phosphatase.
Information induction for predicting acute myocardial infarction.
Authors: Rudolph R A , Bernstein L H , Babb J
Published: January 2020

We show how to make an unsupervised discrimination of disease and nondisease states by measuring information and using newer notions of inductive reason. We also present a new theory of group-based reference values that is based on measuring information uncertainty. We use data on the isoenzymes creatine kinase-MB (CK-MB) and lactate dehydrogenase-1 (LD1) and on the percentage of LD1 from 101 patients with acute myocardial infarction (AMI) and from 41 patients with suspected, but unfounded, infarction (non-AMI). Calculating the Shannon entropy, a concept from information theory, of the data base allows determination of a difference in entropy values ("effective information"), which determines decision cutoff values that produce binary-base patterns yielding the fewest classification errors. Redundancy in testing is important because it provides the information to approach a goal of errorless discrimination by coding the test results and meeting the conditions of the "Noisy Channel Theorem" of information theory. This redundancy improves the predictive value of diagnosis by isolating the area of equivocation to evident patterns. Results for CK-MB and LD1 are 99% correct in assigning cases to AMI and non-AMI categories; adding %LD1 increases the proportion of errorless binary patterns from 25% to 90%.
High-performance liquid affinity chromatography: rapid immunoanalysis of transferrin in serum.
Authors: Ohlson S , Gudmundsson B M , Wikström P , Larsson P O
Published: January 2020

We describe a new method for quantitatively measuring substances of clinical interest by high-performance liquid affinity chromatography (HPLAC). As a model system we selected analysis for transferrin in human serum with immobilized antibodies in a high-performance liquid chromatographic system. SelectiSpher-10 Activated Tresyl columns (5 or 10 x 0.5 cm) were used for in situ coupling of polyclonal antibodies to transferrin. The amount of transferrin eluted was determined by integrating the eluted peak at 280 nm. The whole analytical procedure--including injection of sample, washing, elution, and analysis of data--takes only 7 min. We characterized the HPLAC system for analysis of transferrin in several ways: intra-assay CV approximately 3%; inter-assay CV 2-9%; linear response up to 1 mg/mL column volume; detection limit approximately 3 micrograms; analytical recovery 98% +/- 2%; purity of eluted sample greater than 95% (SDS-PAGE). The HPLAC method was compared with "rocket" immunoelectrophoresis, a commonly used method of analysis for transferrin, and there was excellent correlation between the two methods (r = 0.96, n = 60). Benefits of this HPLAC technique include high precision, rapid analysis, and simplified sample handling.
A new approach to the quality control of steroid receptor assays.
Authors: Borsotti M , Quercioli M , D'Ettole R , Piffanelli A , Messeri G
Published: January 2020

Lyophilized cytosols prepared from calf uterus and human breast tumor tissue are commonly used to assess the reliability of routine steroid receptor assays. However, preanalytical error (sample preparation, storage, homogenization) cannot be detected in this way. Participating laboratories were asked to mail us all their receptor results obtained over a four-month interval, and to include some information about the patients involved (age, menopausal status, nodal status). After verifying the homogeneity of the populations investigated, we computed consensus means for the percentages of positive samples and for their absolute value. Despite the homogeneity of the characteristics of the populations studied, results from some laboratories systematically differed from the consensus mean. This approach to the quality control of steroid receptors allows evaluation of the whole procedure, from sample preparation to analysis, and thus may be helpful as an addition to the usual practice of distribution of cytosols and tissue powders for assay.
Use of the Reflotron system for cholesterol assay in capillary blood, venous blood, and serum--evaluation of accuracy and lot-to-lot reagent comparability.
Authors: Boerma G J , Gelderland J , van Gorp I , Leijnse B
Published: January 2020

Since its recalibration in 1987, the Reflotron "dry-chemistry" cholesterol method can produce accurate results. Here we report how cholesterol concentrations in venous blood, plasma, and serum determined with Reflotron compare with values determined for fingerstick samples. When no EDTA is used in the latter, accurate results may meet the WHO/CDC criterion of a maximum bias less than or equal to 5%. Reflotron users should avoid use of EDTA. Lot-to-lot variation among the solid-phase reagent carriers was evaluated in four lots of these strips. The largest difference, 0.4 mmol/L, was seen in a commercial quality-control serum with a cholesterol concentration of approximately 7 mmol/L. The maximum differences in results for several serum pools were not always found between the same reagent lots, although the highest results in five of six materials occurred with a single lot of strips. As in all clinical chemical testing, regular quality control is necessary.
Determination of 4,4'-methylenebis(2-chloroaniline) in urine by liquid chromatography with ion-paired solid-phase extraction and electrochemical detection.
Authors: Okayama A , Ichikawa Y , Yoshida M , Hara I , Morimoto K
Published: January 2020

This highly specific and sensitive method for measuring urinary 4,4'-methylenebis(2-chloroaniline) (MBOCA) involves liquid chromatography with electrochemical detection. Before chromatography, urine samples are prepared by ion-paired solid-phase extraction on a disposable octadecylsilica column with acidic methanol solution containing 1-heptanesulfonic acid. This enhances the specificity of the method. Mean overall recovery ranged from 97.1% to 99.5% at added MBOCA concentrations of 20 and 100 micrograms/L in urine. Sensitivity for urine was 1 microgram/L. The intra-assay CV was 2.2% at a MBOCA concentration of 100 micrograms/L. We believe that this method is acceptable for routine measurement of MBOCA in urine from individuals exposed to this industrial chemical.
Automating the quantification of heme in feces.
Authors: van den Berg J W , Koole-Lesuis R , Edixhoven-Bosdijk A , Brouwers N
Published: January 2020

We present a modification of the HemoQuant assay, a good but lengthy and tedious method for determining heme in feces by means of its transformation to porphyrins. The laborious extraction procedure was replaced by a simple centrifugation procedure. The nonhomogeneous hot oxalic acid suspension was replaced by acetic acid. We observed no significant difference in results between samples analyzed by the older method vs the present modification (r = 0.996, n = 52). Mean (and SD) analytical recoveries of added hemoglobin and protoporphyrin were 99% (7%) and 93% (6%), respectively. The analytical procedure can now be automated by using discrete samplers and a flow-through fluorometer. Initial sampling and dilution of feces are still done manually, however. The excellent specificity, sensitivity, and overall analytical performance of the original method are retained, while circumventing the practical inconveniences of this reliable screening test for occult blood in feces.
Physiological changes in human erythrocyte cholinesterase as measured with the "pH-stat".
Authors: Garcia-López J A , Monteoliva M
Published: January 2020

Cholinesterase activity in human erythrocytes was determined in 1903 blood samples by the "pH-stat" method. Differences in activity were examined as a function of sex, age, and pregnancy. Reliability intervals for the population average and approval or normality intervals for individual values were established. Sex- and age-related differences were very significant.
Kinetic rate assay of urinary N-acetyl-beta-D-glucosaminidase with 2-chloro-4-nitrophenyl-N-acetyl-beta-D-glucosaminide as substrate.
Authors: Makise J , Saito E , Obuchi M , Kanayama M , Ichikawa K , Harakawa K , Yoshida K
Published: January 2020

In this new procedure for determining the activity of N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30; NAG) in urine, a new synthetic substrate, 2-chloro-4-nitrophenyl-N-acetyl-beta-D-glucosaminide is used. No sample blank is required because the activity is determined kinetically. Very high precision is possible, with CVs of less than 3.8% both within-run and between-run. Glucose, ascorbic acid, bilirubin, bromosulfonphthalein, and phenosulfonphthalein do not interfere. The reference interval (mean +/- 2 SD) for healthy subjects was 1.9-11.1 U per gram of creatinine. NAG activity can be determined over a wide range (2-500 U/L) with high sensitivity. The method is suitable for use with various automated analyzers, and it should be useful for screening for renal diseases.
Measurement of parathyrin in blood from thyroid veins: two radioimmunoassays compared in patients with primary hyperparathyroidism.
Authors: Muñoz-Torres M , Diaz J , Escobar-Jimenez F , Gonzalez-Calvin G , Vara C , Ruiz Requena M E , Vara R , Morales I
Published: January 2020

We measured parathyrin (PTH) in peripheral venous blood samples and in thyroid veins (both homolateral and contralateral to the lesion) in 13 patients with surgically confirmed parathyroid adenomas. Two different RIAs were used, one specific to the mid-region of the molecule (44-68, M-PTH), the other specific to the carboxy-terminal region (65-84, C-PTH). With the M-PTH assay we established a statistically significant multiple correlation (P less than 0.05) between the PTH concentrations in blood from the peripheral and thyroid veins; no significant correlation was found when we used the C-PTH assay. Our results confirm the superiority of the M-PTH RIA over the C-PTH RIA for study of hormonal secretion in primary hyperparathyroidism.
Influence of timing in the fructosamine assay.
Authors: Frandsen E K , Bacchus R A
Published: January 2020

The fructosamine assay, based on the measurement of the reducing activity in serum at alkaline pH, provides an index of protein glycation. The reducing activity is expressed in equivalents of 1-deoxy-1-morpholinofructose (DMF) by direct comparison with the activity either of this synthetic compound or with a secondary protein standard calibrated against DMF. This study reports the influence of assay timing on the apparent serum fructosamine concentration. The kinetics of alkaline reducing activity in serum differed from that in both DMF and a secondary protein standard. When compared with DMF, activity in serum increased but decreased relative to the protein standard as the pre-incubation interval of the assay was shortened. The use of secondary protein standards results in underestimation of serum fructosamine concentrations when the pre-incubation phase of the assay is shorter than that used for the calibration of the secondary standard. Ascorbate exerted an inhibitory effect in fructosamine assays with pre-incubation times exceeding 5 min. The inhibition increased with both the concentration of ascorbate and the duration of the pre-incubation.
A model for the effect of bias for cholesterol on the population at risk.
Authors: Kroll M H , Ruddel M , Elin R J
Published: January 2020

The location of the Reference Value for an analyte within the population distribution affects the magnitude of error due to methodological bias. Using the gaussian distribution, we evaluated the effects of systematic and proportional biases of the method (positive and negative), mean value, and standard deviation on the magnitude of error. We chose four Reference Values for cholesterol as a model. For a population with a mean of 2.0 and SD of 0.36 g of cholesterol per liter, a 3% positive proportional bias causes sixfold more error at the 50th percentile than at the 97.5th. In general, the error for a given bias (proportional or systematic) is greater for a Reference Value within the body than at the tails of the distribution. Further, the magnitude of the error varies as a function of the mean and standard deviation of the population.
Qualitative study of fecal alpha 1-proteinase inhibitor in normal subjects and patients with Crohn's disease.
Authors: Mizon C , Becuwe C , Balduyck M , Colombel J F , Cortot A , Mizon J , Degand P
Published: January 2020

We applied sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting to analyze fecal alpha 1-proteinase inhibitor (alpha 1 PI) from healthy subjects and patients with Crohn's disease. A component with Mr 38,000 was characterized in normal fecal extracts as well as in six pathological samples. In these cases, the Crohn's disease activity index (CDAI), a clinical index of severity of the disease, was 170 (SEM 47). In contrast, alpha 1 PI of Mr 51,000 was detected in fecal extracts from eight patients with active Crohn's disease (CDAI = 287, SEM 39). We conclude that fecal alpha 1 PI can be considered a marker of intestinal disease activity.
Adulterants causing false negatives in illicit drug testing.
Authors: Mikkelsen S L , Ash K O
Published: January 2020

Illicit-drug users may attempt to falsify results by in vitro adulteration of specimens. We investigated eight additives (NaCl, Visine, handsoap, Drano, bleach, vinegar, golden-seal tea, and lemon juice) claimed by drug users to invalidate enzyme immunoassay (EIA) drug assays. We also analyzed adulterated urine specimens to determine if they could be identified, adding adulterants at several concentrations to 222 EIA-positive specimens confirmed by gas chromatography and mass spectrometry (GC/MS) to contain illicit drugs. To identify adulterated urines, we monitored pH, relative density, and urine color and turbidity at adulterant concentrations that falsified EIA results. Specimens contaminated with NaCl had relative densities greater than 1.035. Liquid Drano, bleach, and vinegar shifted urine pH outside the physiological range. Golden-seal tea caused a dark appearance, and specimens containing liquid soap were unusually cloudy. Lemon juice had no effect on the assays. Visine was the only adulterant not detected. The adulterants interfered somewhat differently with each of the drug assays. EIA assays for illicit drugs can be invalidated by specimen adulteration producing false-negative results. Therefore, if urine drug testing is to be conducted, pH, relative density, and appearance should be assessed and suspect specimens should be rejected. Not all adulterants can be detected, so observed collection is strongly recommended.
"Flipped" patterns of lactate dehydrogenase isoenzymes in serum of elite college basketball players.
Authors: Rotenberg Z , Seip R , Wolfe L A , Bruns D E
Published: January 2020

We kinetically measured total lactate dehydrogenase (LD, EC 1.1.1.27), total creatine kinase (CK, EC 2.7.3.2), and aspartate aminotransferase (AST, EC 2.6.1.1.) in 16 elite college basketball players, before the competition season and not in close temporal relation to near-maximal exercise, and in 17 healthy non-athlete controls. LD isoenzymes were determined by both electrophoretic and immunoprecipitation methods. CK-MB isoenzyme was measured electrophoretically. We found significantly higher mean LD-1 values and LD-1/LD-2 ratios in the players than the controls: 31.6 (SD 3.7)% vs 25.8 (SD 3.2)% (P less than 0.005) and 1.1 (SD 0.13) vs 0.87 (SD 0.16) (P less than 0.001), respectively. A "flipped" LD pattern (LD-1 greater than LD-2) was found in half the players and in six of the eight black athletes, but in only two of the control group and in none of the black controls. Mean CK activity in serum exceeded normal values in the serum of the athletes and was higher in comparison with the control group [274 (SD 156) vs 103 (SD 82) U/L]. Mean CK was significantly higher in the eight athletes with the flipped LD pattern than in those with LD-1 less than LD-2 [322 (SD 163) vs 180 (SD 98) U/L; P = 0.05], and also in comparison with CK in the two controls with flipped LD pattern. We saw no significant difference in mean CK between the nine players with normal immunochemical LD-1/LD ratios and the seven players with above-normal ratios. CK-MB was not detected in either athletes or controls. None of the players had any clinical or electrocardiographic evidence for myocardial ischemia or infarction. Evidently the flipped LD pattern usually found in patients with acute myocardial infarction and reported in some athletes after extreme exercise such as ultra-marathon running may also be found in athletes who are in their "basal fitness shape" but who are not involved in competitive physical activity.
Sensitive, practical bioassay of thyrotropin, with use of FRTL-5 thyroid cells and magnetizable solid-phase-bound antibodies.
Authors: Tokuda Y , Kasagi K , Iida Y , Hatabu H , Hidaka A , Misaki T , Konishi J
Published: January 2020

We have developed a new method for assessing the bioactivity of thyrotropin (TSH) in human serum by measuring cAMP production in FRTL-5 thyroid cells as an index of stimulation. To eliminate serum inhibitors of the cAMP response to TSH, we purified samples by mixing them with anti-TSH antibodies coupled to magnetizable particles. This method of immunoaffinity purification was simple and suitable for the measurement of a large number of samples. The detection limit of the bioassay was 3.13 milli-int. units of human TSH per liter. Intra-assay and interassay coefficients of variation ranged from 5.4 to 8.6% and from 12.6 to 21.5%, respectively. The concentrations of bioassayable TSH closely correlated with those of immunoassayable TSH, both in the immunoaffinity-purified samples (r = 0.946, P less than 0.001) and in the serum samples (r = 0.945, P less than 0.001) from 14 euthyroid subjects and 53 hypothyroid patients with Hashimoto's thyroiditis. The bioactivity-to-immunoactivity ratio was almost constant (0.84 +/- 0.30; mean +/- SD) over the range of concentrations of immunoassayable TSH in serum, 6.3 to 177 milli-int. units/L. We also demonstrate that the technique is applicable to measurements of bioactivities of circulating forms of immunoreactive TSH in different pathophysiological conditions.
Clinical and analytical evaluation of two immunoassays for direct measurement of creatine kinase MB with monoclonal anti-CK-MB antibodies.
Authors: Apple F , Preese L , Bennett R , Fredrickson A
Published: January 2020

We examined the clinical and analytical performance of two immunoassays (Becton Dickinson CK-MB; Ciba-Corning Magic Lite CK-MB) in which monoclonal anti-CK-MB antibodies are used for directly measuring creatine kinase (EC 2.7.3.2) isoenzyme MB (CK-MB) in serum, and also one electrophoretic method (Ciba-Corning). Within- and between-assay precision for both immunoassays was good at the upper reference limits (less than 10% CV). Analytical recoveries ranged from 102 to 114%. Both immunoassays were free from interference by CK-BB, mitochondrial-CK, macro-CK, adenylate kinase, and CK-MM. Retrospectively, we evaluated four categories of patients, using both immunoassays and electrophoresis: normal controls, acute myocardial infarction (AMI) patients, severe skeletal muscle trauma patients, and acutely ill patients known not to have AMI. In general, there were excellent correlations among all three methods. CK-MB activity (U/L) measured by the Becton Dickinson immunoassay was approximately 50% of the mass concentration (microgram/L) of the Magic Lite immunoassay and 50% of the activity concentration (U/L) determined by electrophoresis. Both immunoassays were easy to perform and sensitive to the low CK-MB concentrations often found with low total-CK activities.
Serum lipoperoxidation products in an infant with hemolytic-uremic syndrome.
Authors: Brown R E , Alade S L , Knight J A , Evans B J
Published: January 2020

We detected increased concentrations of lipoperoxidation products, as malondialdehyde, in the serum of an infant with classic hemolytic-uremic syndrome. The concentrations declined when peritoneal dialysis was initiated and eventually returned to normal after clinical recovery. Our observation adds to the existing body of evidence that links the pathogenesis of hemolytic-uremic syndrome to peroxidative damage and to the rationale for using antioxidant agents as part of the therapy for this disease process.
Alkaline phosphatase isoenzymes in serum determined by high-performance anion-exchange liquid chromatography with detection by enzyme reaction.
Authors: Parviainen M T , Galloway J H , Towers J H , Kanis J A
Published: January 2020

This rapid, reproducible method for separating and determining individual alkaline phosphatase (EC 3.1.3.1) isoenzymes in serum is based on high-performance liquid chromatography with a weak anion-exchange column (SynChropak AX 300). The isoenzymes so resolved are detected by using an on-line enzyme reaction followed by spectrophotometric monitoring at 405 nm of the 4-nitrophenol formed. Complete diagnostic profiles of the various isoenzymes present in normal and pathological sera are obtained within 20 min. The mean (and SD) normal concentrations of the bone B1 and intestinal isoenzymes in serum of adults were 3.7 (4.3) and 4.5 (3.9) U/L, respectively (n = 14), and of the bone isoenzyme B2 and liver isoenzymes L1 and L2, 5.8 (8.6), 33.0 (10.6), and 12.0 (4.8) U/L, respectively (n = 17). Concentrations of the B2 and L1 isoenzymes in adults over age 40 years differed significantly from those in adults younger than 40 years, that of bone isoenzyme being lower (P less than 0.05) and that of the liver isoenzyme being higher (P less than 0.001) in the younger adults.
Neopterin estimation compared with the ratio of T-cell subpopulations in persons infected with human immunodeficiency virus-1.
Authors: Fuchs D , Banekovich M , Hausen A , Hutterer J , Reibnegger G , Werner E R , Gschnait F D , Dierich M P , Wachter H
Published: January 2020

We measured neopterin, a biochemical indicator for the activation of cell-mediated immune reactions, in urines from 105 individuals at risk of infection with human immunodeficiency virus-1 (HIV-1), 83 of whom were seropositive for antibody to HIV-1. We compared absolute numbers of T-cell subsets (CD4+ helper/inducer T-cells, CD8+ suppressor/cytotoxic T-cells), and the ratio of CD4+ T-cells to CD8+ T-cells with the urinary neopterin concentrations. Concentrations of neopterin in urine were inversely correlated with absolute numbers of CD4+ T-cells and with CD4+/CD8+ ratios in anti-HIV-1 seropositive subjects but not in those seronegative. Various statistical comparisons of the data further demonstrated that neopterin concentrations showed larger differences between anti-HIV-1 seronegative and seropositive subjects than absolute numbers of CD4+ T-cells or CD4+/CD8+ ratios. These results seem to indicate that neopterin concentrations increase earlier in the course of HIV-1 infection, before effects on T-cell subpopulations are detectable, and may further support the suggestion that neopterin measurement could be of use for monitoring infected subjects or predicting the progression of disease.
Quantitative precipitin reaction and enzyme-linked immunosorbent assay of mannans of Candida albicans NIH A-207 and NIH B-792 strains compared.
Authors: Tojo M , Shibata N , Osanai T , Mikami T , Suzuki M , Suzuki S
Published: January 2020

We assessed the difference between results by enzyme-linked immunosorbent assay (EIA) in plastic support wells and the quantitative precipitin reaction (QPR) in glass test tubes for antigenic mannans and antibodies of two representative Candida albicans strains, NIH A-207 and NIH B-792. We investigated each of four mannan subfractions, with different phosphate contents, for their reactivities to the homologous polyclonal rabbit antiserum. Each series of mannan subfractions showed a reactivity proportional to their phosphate content in EIA, in a similar manner as observed in QPR. Moreover, in EIA, the cross-reactivities between the bulk mannans of the two C. albicans strains and the polyclonal antiserum of a Saccharomyces cerevisiae wild-type strain containing specific antibodies to the non-reducing terminal alpha-1,3-linked D-mannopyranose unit resembled those of the same antigen-antibody reactions in QPR. However, the mannan of C. albicans NIH A-207 strains, a weak antigen in the cross-QPR system, reacted fairly strongly in EIA in its high concentration range, indicating that EIA can be used to detect such an epitope in these mannans in concentrations undetectable by QPR. We conclude that EIA is a useful technique for immunochemical assay of yeast mannans and their antibodies on a smaller scale than with QPR.
Fibronectin concentrations in plasma in peripheral vascular disease.
Authors: Ejim O S , Fonseca V , Coumar A , Mathur S , Bell J L , Dandona P
Published: January 2020

Fibronectin (Fn) concentrations were measured immunoturbidimetrically in plasma of normal subjects and patients with peripheral vascular disease (PVD) before and after venous compression, which caused Fn concentrations to increase in both normal subjects and PVD patients. Basal Fn concentrations and those after 10-min compression were not significantly different in normal subjects and PVD patients. Five minutes after the release of compression, Fn had consistently declined in normal subjects and reverted to baseline values; in contrast, in PVD patients values either increased further or decreased inconsistently. Thus the Fn concentration at 15 min was significantly (P less than 0.001) greater in PVD patients than in normal subjects. Plasma albumin concentrations, measured in parallel to ensure that changes in Fn concentrations were not nonspecific, increased to a greater extent in normal subjects than in PVD patients and reverted to normal after the removal of compression. The Fn/albumin ratio remained unchanged in normal subjects after venous compression, whereas that in PVD patients increased and remained higher, even after decompression. The sustained increase in plasma Fn concentrations and in the Fn/albumin ratio in PVD patients after venous compression may indicate endothelial injury.
Simultaneous quantification by double rocket immunoelectrophoresis of apolipoproteins A-I and B in blood spotted on filter paper.
Authors: Micic S , Arends J , Nørgaard-Pedersen B , Christoffersen K , Andersen G E
Published: January 2020

We describe double rocket immunoelectrophoresis for simultaneous quantification of apolipoprotein A-I (apo A-I) and B (apo B) in blood on filter paper. The apolipoproteins from blood spots on filter paper were eluted with detergents (sodium dodecyl sulfate and Triton X-100). The eluates were subjected to electrophoresis on agarose gel containing antisera against both apolipoproteins. Within- and between-assay CVs for apo B/A-I ratios were less than 5.5% and 7.2%, respectively. The apo B/A-I ratio was influenced by length and temperature of storage. In results for 121 venous blood samples, the apo B/A-I ratios in dried blood spots correlated well with those in serum (r = 0.92) and correlated somewhat with the ratios for low-density lipoprotein/high-density lipoprotein cholesterol in serum (r = 0.87). Of these specimens, 68 were from patients with known familial hypercholesterolemia, all of whom had an apo B/A-I ratio greater than 0.90. We think this method will be of value for detecting familial hypercholesterolemia and possibly familial hyperapobeta- and hypoalphalipoproteinemia.
Direct, rapid assay of pancreatic isoamylase activity by use of monoclonal antibodies with low affinity for macroamylasemic complexes.
Authors: Rosenblum J L
Published: January 2020

Two Monoclonal Antibodies produced against Pancreatic Amylase have been produced and used to develop immunoassays (MAPA assays) for serum isoamylases. One antibody binds pancreatic isoamylase specifically and neither antibody inactivates amylolytic activity. MAPA assays that measure pancreatic and total enzymatic activities directly and rapidly in hyperamylasemic serum are described in this report. The influence of isoamylase determination on diagnosis is also investigated in 35 hyperamylasemic patients. The percentage for pancreatic amylase in serum as determined by immunoassay correlates well with results obtained by conventional electrophoresis (r = 0.90). Chart review indicates that use of MAPA assays would have potentially changed the diagnosis in 27% of patients. Five of six additional patients with hyperamylasemia caused by macroamylasemia had normal immunoreactive amylase values, and in all six patients the values were strikingly lower than predicted. The ability to assay organ-specific pancreatic isoamylase directly and early in the clinical course should reduce the risks of misdiagnosis and inappropriate therapy, especially in salivary-type hyperamylasemia or macroamylasemia.
Catabolism of circulating enzymes: plasma clearance, endocytosis, and breakdown of lactate dehydrogenase-1 in rabbits.
Authors: Smit M J , Beekhuis H , Duursma A M , Bouma J M , Gruber M
Published: January 2020

Lactate dehydrogenase-1 (EC 1.1.1.27), intravenously injected into rabbits, was cleared with first-order kinetics (half-life 27 min), until at least 80% of the injected activity had disappeared from plasma. Radioactivity from injected 125I-labeled enzyme disappeared at this same rate. Trichloroacetic-acid-soluble breakdown products started to appear in the circulation shortly after injection of the labeled enzyme. Body scans of the rabbits for 80 min after injection of 131I-labeled enzyme revealed rapid accumulation of label in the liver, peaking 10-20 min after injection. Subsequently, activity in the liver declined and radioactivity (probably labeled breakdown products of low molecular mass) steadily accumulated in the bladder. Tissue fractionation of liver, 19 min after injection of labeled enzyme, indicated that the radioactivity was present both in endosomes and in lysosomes, suggesting uptake by endocytosis, followed by breakdown in the lysosomes. Measurements of radioactivity in liver and plasma suggest that the liver is responsible for the breakdown of at least 75% of the injected enzyme. Radioautography of tissue sections of liver and spleen showed accumulated radioactivity in sinusoidal liver cells and red pulpa, respectively. These results are very similar to those for lactate dehydrogenase-5, creatine kinase MM, and several other enzymes that we have previously studied in rats.
Chemometrics: measurement reliability.
Authors: Castañeda-Méndez K
Published: January 2020

Applying the principles of chemometrics can lead to development of a powerful, simple, comprehensive system for characterizing measurement reliability of analytical processes. The system described here consists of a format for specifications, definitions, and decision rules for evaluating and comparing analytical processes. Two cases illustrate its advantages over the use of precision, accuracy, total error, and regression statistics for such evaluations.
Simultaneous liquid-chromatographic determination of urinary vanillylmandelic acid, homovanillic acid, and 5-hydroxyindoleacetic acid.
Authors: Gironi A , Seghieri G , Niccolai M , Mammini P
Published: January 2020

We describe a liquid-chromatographic method for quantifying, simultaneously by a single procedure, vanillylmandelic acid (VMA), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in urine. After solvent extraction of acidified urine, the analytes were chromatographed on a C8 column, with use of a mobile phase of phosphate buffer (20 mmol/L, pH 4.0) and methanol with a variable gradient elution, and detected fluorometrically. We report the analytical recovery, sensitivity, precision, working linear range, and potential for interference from similar molecules or drugs. The results of such tests demonstrate that the proposed method is sensitive and reproducible. It is, furthermore, easy to perform, and thus is suitable for use in the clinical laboratory.
Pyrochemiluminescence: real-time, cost-effective method for determining total urinary nitrogen in clinical nitrogen-balance studies.
Authors: Konstantinides F N , Boehm K A , Radmer W J , Storm M C , Adderly J T , Weisdorf S A , Cerra F B
Published: January 2020

Total urinary nitrogen (TUN) determinations for nitrogen-balance studies were traditionally performed by the Kjeldahl method, but this method is laborious, hazardous, prone to error, and no longer widely available in most clinical laboratories. During the last several decades, urinary urea nitrogen (UUN) determinations have replaced TUN as an index of urinary nitrogen excretion in many clinical laboratories, owing to its ease of determination, decreased cost, and wide availability. However, the validity of using UUN for estimating nitrogen loss has been questioned in many disease states, owing to wide variations in the proportional amount of urea found in TUN. Chemiluminescence has been proposed as an alternative to the Kjeldahl method for TUN. TUN values obtained from 24-h urine collections measured by both micro-Kjeldahl (x) and Pyrochemiluminescence (y) (Antek Instruments, Inc.) techniques were comparable by linear regression analysis: n = 97; r = 0.996; r2 = 0.992; y = 1.048x - 0.606; P less than 0.001. Automated induction of samples and calculation of results allows up to 42 samples to be run unattended. This dramatically reduces labor and overall costs for TUN determinations, while providing a more accurate and economical assessment of nitrogen excretion than UUN in a clinical setting.
An improved solid-phase enzyme and luminescent immunoassay system for steroid hormones and digoxin.
Authors: Hubl W , Daxenbichler G , Meissner D , Thiele H J
Published: January 2020

A uniform solid-phase system has been developed for enzyme (ELISA) or luminescent (LIA) immunoassays for steroids. These assays were improved by (a) irradiating microtiter plates or polystyrene tubes before coating with antibody or Protein A, (b) coating the plastic trays with nonspecific anti-gamma-globulin or Protein A instead of the steroid-specific first antibody, and (c) partial denaturation of the second antibody before coating the plates or tubes with it. Specific antibodies were raised against cortisol, aldosterone, 17-hydroxyprogesterone, and digoxin. Horseradish peroxidase was used as label for the ELISA and aminoethylisoluminol for the LIA. In comparison with the first (specific) antibody coating method we observed some advantages: From 10- to 33-fold lower concentrations of first antibodies were necessary to bind the same amount of steroids; precision was better (CV, 3.8-7.5% vs 6.9-15.5%). The high sensitivity of these assays (0.5-2.0 pg per tube for the steroids) also allows determination of the steroids and digoxin in plasma and saliva.
Immunoenzymometric assay of creatine kinase with monoclonal antibodies to the MB isoenzyme compared with electrophoresis.
Authors: Hall M , Neisler M , Johnson J T
Published: January 2020

We compare a "second-generation" immunoenzymometric assay (Tandem-E CKMB II) for creatine kinase (EC 2.7.3.2) MB with its electrophoretic (Beckman Paragon system) determination. In the former, two monoclonal antibodies are directed against the B and M subunits. We evaluated 502 samples from 253 patients. Precision, linearity, and analytical recovery for both assays were excellent. The two methods correlated well (r = 0.936). The reference interval for individuals with no suspected cardiac disorder was 0-6.0 micrograms/L; that for non-infarct patients was 0-18.0 micrograms/L. Peak CK-MB values determined by the two assays agreed for 95% of the patients, in terms of exceeding the normal reference interval or not. Diagnostic efficiencies were 86% (Tandem) and 88% (electrophoresis). The immunoenzymometric assay showed no cross reaction with other CK isoenzymes. Both assay methods performed well in detecting CK-MB, although there were some false positives by both methods, as judged from electrocardiographic results. When total CK for the Tandem assay exceeds 2000 U/L, we recommend calculation of a ratio (CK-MB, micrograms/L:total CK, U/L).
One-step chemiluminescent immunoassay of free thyroxin with acridinium-ester-labeled thyroxin evaluated and compared with a two-step radioimmunoassay.
Authors: Bounaud J Y , Bounaud M P , Begon F
Published: January 2020

This new one-step chemiluminescent immunoassay of free thyroxin (FT4) involving a thyroxin-immunoglobulin conjugate labeled with acridinium ester (Magic Lite System; Ciba Corning Diagnostics Corp., Medfield, MA) is rapid (one 1-h incubation), requires two calibrators per run, and takes 10 s per sample for the quantification step. Analytical performances were excellent: within- and between-run CVs of less than 10% in the working range, no significant effect of hemolysis, bilirubin, or lipemia, and no significant interaction between the conjugate and the thyroxin-binding proteins. Magic Lite results (y) correlated well with those obtained by the Sclavo (x) two-step radioimmunoassay (Sclavo, Siena, Italy): y = 1.35x + 1.32 (r = 0.94, n = 267, P less than 0.001, Sxy = 6.29). Clinical sensitivities (diagnostic efficiencies) for hypothyroidism and hyperthyroidism were 0.91 and 0.98 for normal interval limits of 12 and 21.5 pmol/L (95% confidence interval). Magic Lite results in situations where patient therapy, treatment, or unusual conditions can result in a lack of correlation between the clinical status and the FT4 values were qualitatively the same as those obtained by the Sclavo assay.
Subtyping of erythrocyte phosphoglucomutase-1 as a genetic marker for bone-marrow engraftment and hematopoietic chimerism after allogeneic bone-marrow transplantation in a patient with acute lymphoblastic leukemia.
Authors: Grahovac B , Labar B , Stavijenić A
Published: January 2020

We report an effective follow-up of the establishment of bone-marrow function after an allogeneic bone-marrow transplantation in a patient with acute lymphoblastic leukemia, by means of a suitable genetic marker, phosphoglucomutase-1 (EC 5.4.2.2) isoenzyme. A patient with acute lymphoblastic leukemia received allogeneic bone-marrow graft from a sibling who was of the same sex and blood group, HLA-identical, and mixed-lymphocyte-culture nonreactive. To monitor the bone-marrow engraftment and the type and degree of chimerism established, we used a genetic marker, the phosphoglucomutase-1 isoenzyme system, to reveal the difference between the bone-marrow host and donor. We did phosphoglucomutase-1 isoenzyme subtyping of the host's and donor's erythrocytes before transplantation, and isoenzyme phenotyping of the host's erythrocytes during a year after transplantation. Establishment of bone-marrow graft function, a period of temporary mixed chimerism with a population of both host's and donor's erythrocytes, a period of the exclusive presence of donor's erythrocytes, and the resumed appearance of host's erythrocytes after eight months, with no signs of relapse of leukemia, were all observed by analysis of phenotypes. These isoenzymes served as a significant and practical genetic marker, which could be successfully used in studies on bone-marrow transplantation.
An unusual case of toluene-induced metabolic acidosis.
Authors: Jone C M , Wu A H
Published: January 2020

We used gas chromatography-mass spectrometry to analyze serum specimens from a patient whose metabolic ketoacidosis resulted from sniffing a toluene-containing substance; we also analyzed a sample of the abused substance itself and a serum specimen from a normal subject. The blood samples from the patient showed abnormally high concentrations of acetoacetic, azelaic, benzoic, and 3-hydroxybutyric acids, and a smaller amount of hippuric acid than in the control serum. The ketone bodies and benzoic acid appeared to be factors contributing to her acidosis and ketosis. Analysis for volatile solvents in the patient's serum revealed a low concentration of methyl acetate, in contrast to the high concentrations of toluene and bis(2-ethylhexyl) phthalate and the trace concentration of xylene in the abused substance. We consider this case unusual because the presence and concentration of specific ketoacids was unexpected in light of what was known about the patient.
A monoclonal-antibody-based radioimmunoassay for measurement of protein C in plasma.
Authors: Howard P R , Bovill E G , Mann K G , Tracy R P
Published: January 2020

A monoclonal-antibody-based competitive radioimmunoassay for measuring human protein C is reported. With use of a purified protein C standard, the solid-phase assay was sensitive to less than 80 micrograms of protein C per liter. Intraassay CVs ranged from 5% to 8%; the inter-assay CV was 5.4%. Analytical recovery averaged 104% for purified protein C added to 10 samples of normal plasmas. The assay antibody could deplete plasma of all protein C, as determined by immunoaffinity chromatography followed by polyclonal Western blot analysis. Liquid-chromatographic gel permeation of plasma indicated a single immunoreactive species that had an appropriate molecular mass for monomeric protein C. Studies of monoclonal-antibody specificity showed no significant interferences by other vitamin K-dependent proteins. The mean protein C antigen concentration in plasma of 54 healthy subjects was 3.21 (SD 0.56) mg/L and was 1.51 (SD 0.52) mg/L for 22 patients on long-term warfarin therapy. Results of the monoclonal RIA correlated well with those by a polyclonal RIA also developed in our laboratory (r = 0.93) and an amidolytic functional assay (r = 0.88) for both normal plasma and plasma from patients on long-term warfarin therapy.
Microassay for nuclear binding of steroid receptors with use of intact cells from small samples of avian and human tissue.
Authors: Colvard D S , Jankus W R , Berg N J , Graham M L , Jiang N S , Ingle J N , Spelsberg T C
Published: January 2020

A micro version of a nuclear binding assay to assess the biological activity of receptors for steroid hormones was developed for application to small (needle) biopsies of human tumors for the purpose of predicting responses to steroid therapy. This easier assay requires 10-fold less tissue than the original nuclear binding assay described for progesterone receptors in the avian oviduct, endometrium, and endometrial carcinomas (Spelsberg TC, et al., Endocrinology 1987;121:631). We describe the application of this micro assay to normal avian oviduct and cancers of the human breast, and we demonstrate a tissue specificity and saturation of nuclear binding. The micro assay reliably measured as little as 0.5 mg equivalents of tissue per assay tube. Results for breast tumors determined to be estrogen-receptor-positive by the standard dextran-coated charcoal method were also determined with this nuclear binding assay. As described previously for progesterone receptors in endometrial carcinomas, some receptor-positive breast biopsies displayed negligible capacity for nuclear binding. Therefore, with the present assay we have identified nonfunctional receptors in these biopsies, which may be useful for accurate prediction of patients' responses to therapy with hormones.
Adaptations to keep a thyrotropin immunoradiometric assay "supersensitive" with automated pipetting.
Authors: Surmont D W , Alexandre J A
Published: January 2020

The sensitivity of the immunoradiometric kit "RIA-gnost hTSH" (Behring) is poorer with a common automated pipetting station than with careful manual pipetting, principally because of tracer contamination of the inner wall of the tube, above the level reached by the wash solution. This can be eliminated by adapting the wash buffer volume so as to fill the entire tube. In this way, one can detect as little thyrotropin as 0.02 milli-int. unit/L, which is better than the 0.03 milli-int. unit/L claimed by the manufacturer. Cross-contamination from pipetting can induce overestimation of "suppressed" thyrotropin values. Prolonging the counting time to obtain an optimal counting error is of no practical value in the thyrotropin range where activity is low.