bluuebunny/crossref_metadata_2025 huggingface dataset:
Cofactor in serum for high-affinity folate binding in milk.
Authors: Holm J , Hansen S I , Lyngbye J
Published: January 2020

Affinity-chromatographic purification of the folate-binding protein from whey of cow's milk results in the removal of a heat-resistant cofactor that is important in high-affinity folate binding (Biochim, Biophys. Acta 579: 479, 1979). Thus a shift from negative to positive cooperativity occurred in the presence of cofactor (Biochim, Biophys. Acta 579: 479, 1979). We demonstrate the presence of a similar cofactor in sera from men, pregnant women, and umbilical cords. Even highly diluted (10 000-fold) serum samples were active, and the cofactor was also present in erythrocyte hemolysates (10 000-fold diluted). The identity of the cofactor, which may act as an overall modulator of high-affinity folate binding, is still obscure.
Quantitation of serum gentamicin concentration by a solid-phase immunofluorescence method.
Authors: Tsay Y , Wilson L , Keefe E
Published: January 2020

We describe the use of a solid-phase immunofluorescence method (FIAX system) for measuring drug concentrations, specifically gentamicin, in serum or plasma. Antibody to gentamicin is immobilized on a polymeric surface. This Sampler is immersed in a mixture of serum standard or unknown and a buffered solution of fluorescent-labeled gentamicin. After 30 min, the fluorescence of the specifically bound labeled gentamicin is measured with a fluorometer. The assay detects gentamicin in concentrations as low as 50 mug/L. It is specific for gentamicin, cross reactivity to some other therapeutic drugs, except netilmicin and sisomicin, being low. Results by this assay compare well with those by a radioimmunoassay (r = 0.94) and a radioenzymic assay (r = 0.95). The assay is specific, economical, simply performed, requires little pipetting, and does not involve the use of radioisotopes.
Trimethoprim interferes with serum methotrexate assay by the competitive protein binding technique.
Authors: Hande K , Gober J , Fletcher R
Published: January 2020

Administration of Bactrim (a combination of trimethoprim and sulfamethoxazole) to a patient who also was receiving methotrexate caused a significant increase in apparent plasma methotrexate concentrations as determined by competitive protein binding assay with use of dihydrofolate reductase (EC 1.5.1.3) from Lactobacillus casei as the binding protein. This spurious increase was caused by trimethoprim in the patient's plasma. A plasma trimethoprim concentration of 0.1 mg/L inhibited binding of radiolabeled methotrexate to dihydrofolate reductase by 50%. In contrast, radioimmunoassay for methotrexate was not affected by concomitant administration of trimethoprim. The competitive protein binding assay for methotrexate should not be used in patients being treated with Bactrim or Septra (a similar combination). However, the L. casei competitive protein binding assay technique can be used to assay plasma trimethoprim concentrations with sensitivity to 0.02 mg of trimethoprim per liter.
Zinc concentrations in serum as related to myocardial infarction.
Authors: Lekakis J , Kalofoutis A
Published: January 2020

Zinc concentrations in serum from 99 patients with acute myocardial infarction were correlated with the incidence of further complications and with activities in serum of the "cardiac" enzymes aspartate aminotransferase and lactate dehydrogenase. A significantly subnormal zinc concentration was observed for the patients, the lowest values being observed on the second and third days after infarct, particularly in patients with serious complications. Moreover, a linear correlation was observed between zinc values and enzyme activities until the fourth day after infarct. We conclude that measurement of zinc in the serum may have diagnostic value for acute myocardial infarction, although its prognostic value is still speculative.
Photometric determination of glycosylation of hemoglobin in diabetes mellitus.
Authors: Subramaniam C V , Radhakrishnamurthy B , Berenson G S
Published: January 2020

We evaluated glycosylation of hemoglobin (HbA + HbA1) in 25 control subjects and in 133 diabetic patients who were in various stages of blood glucose control, by measuring ketoamine-linked hexoses in hemoglobin. These hexoses were converted by digestion with 10 mol/L acetic acid for 16 h at 100 +/- 5 degrees C to 5-hydroxymethylfurfuraldehyde, which was quantitated by reaction with 2-thiobarbituric acid. Glycosylation of hemoglobin was expressed as micromoles of hydroxymethylfurfuraldehyde per gram of globin protein (the "HMF index"). A mean HMF index of 1.67 (SD = 0.23) was obtained for controls; that for diabetic patients was 2.93 (SD 0.95). The index correlated well (r = 0.83, p < 0.001) with average blood glucose concentration as measured during the preceding 16 weeks, over a wide range of glucose values (1 to 6 g/L). It correlated even better (r = 0.92, p < 0.001) when corrected for variations in hemoglobin concentration. Thus measurement of ketoamine-linked hexoses of hemoglobin or HMF index provides an independent and useful alternative to the currently used methods that measure only HbA1 or HbA1c.
Direct, fixed-time kinetic assays for beta-hydroxybutyrate and acetoacetate with a centrifugal analyzer or a computer-backed spectrophotometer.
Authors: Li P K , Lee J T , MacGillivray M H , Schaefer P A , Siegel J H
Published: January 2020

In the course of studying the control of blood glucose in juvenile onset diabetics, we developed convenient methods for determining beta-hydroxybutyrate and acetoacetate. Here we describe fixed-time, enzymic, reaction-rate procedures for directly measuring these organic acids with a centrifugal analyzer (Rotochem IIA/36) or a computer-backed spectrophotometer (Gilford 102 system). In either case, the method requires only 20 micro L of plasma; is rapid, accurate, and precise; and analytical recovery is quantitative. Data are presented comparing results obtained with both instruments. Metabolic acidosis can be rapidly assessed and monitored with these methods, as illustrated by an example.
Automated "Gamma-flo" radioimmunoassay of urinary cyclic AMP.
Authors: Brooker G , Murad F
Published: January 2020

The "Gammaflow" automated assay concept of Brooker et al. (Science 194:270, 1976) has been adapted to the assay for urinary cyclic AMP in patients' samples on the now commercially available Squibb Gamma-FloTM system, a totally automated continuous-flow immunoassay instrument. The instrument aspirates the unknown sample, combines it with radioligand and specific antiserum, incubates the mixture, separates antibody-bound radioligand from free radioligand, counts the radioactivity of the bound ligand, computes the standard curve, and determines subsequent unknown samples by comparison to the standard curve. The urinary cyclic AMP assay we report here is rapid, accurate, and concentrations of cyclic AMP as low as 15 nmol/L can be detected. The assays, which can be done at 60 determinations per hour, are accurate at all dilutions of urine tested and inter-assay (day-to-day) CVs were 3.9% at the midpoint of the standard curve (B/B0 = 0.49).
Effect of dimethyl sulfoxide on serum osmolality.
Authors: Runckel D N , Swanson J R
Published: January 2020

Consecutive values for serum osmolality measured in a patient exceeded (548 mOsm) the previously reported pathological range and, indeed, exceeded that generally considered compatible with life. The patient was receiving large amounts of intravenous dimethyl sulfoxide as part of an experimental protocol to control intracranial pressure resulting from trauma. This compound increases serum osmolality linearly with respect to concentration over the range considered therapeutically significant, 0 to 10 mL/L. The mechanism of its effect on osmolality readings is discussed. It does not interfere with any values for those routine measurements made by continuous-flow (SMAC, Technicon).
A study of the use of polyethylene glycol in estimating cholesterol in high-density lipoprotein.
Authors: Demacker P N , Hijmans A G , Vos-Janssen H E , van't Laar A , Jansen A P
Published: January 2020

We studied polyethylene glycol 6000 precipitation of lipoproteins other than high-density lipoproteins, before cholesterol is estimated in the supernate. Other lipoproteins in the supernatant fractions were detected by using rocket immunoelectrophoresis. A polyethylene glycol concentration of 75 g/L in the final mixture appeared to be optimal, and results agreed with those obtained by ultracentrifugation. Differences in serum pH, use of polyethylene glycol from different suppliers, or the presence of ethylenediaminetetraacetate resulted in values that differed significantly (by 40 to 60 mumol/L) from the reference values. Polyethylene glycol did not interfere in four different methods for determination of cholesterol. In combination with an enzymic cholesterol method, the polyethylene glycol method appeared to be very precise, even when lipemic sera (triglycerides up to 5.5 mmol/L) were analyzed that had diminished high-density lipoprotein cholesterol values. We consider this method a method of choice, especially when lipemic sera are tested and enzymic cholesterol analysis is used.
A digitally linearized thermistor thermometer referenced to IPTS-68.
Authors: Berger R L , Clem T , Gibson C , Siwek W , Sapoff M
Published: January 2020

We describe a digitally linearized thermistor thermometer that is accurate to ±0.01 °C from 0 to 60 °C at a wattage of 50 microW and a sampling time of 1 s. It is traceable to the International Practical Temperature Scale of 1968 (IPTS-68). Analog and digital outputs are provided, the latter as ASCII RS-232C. The thermistor is a 10-cm long immersible probe, 1.5 mm in diameter. Only 0.5 cm of the probe need be immersed, with proper heat sinking of at least 1 cm of the remaining 9.5 cm, for accurate readings. Error from self-heating of the thermistor is less than 5 × 10(-3) °C at all power settings from 50 to 1 microW.
Radioimmunoassay of creatine kinase B-isoenzymes in serum of patients with azotemia, obstructive uropathy, or carcinoma of the prostate or bladder.
Authors: Homburger H A , Miller S A , Jacob G L
Published: January 2020

We measured the concentrations of creatine kinase B-isoenzymes by radioimmunoassay in 271 serum specimens from patients with azotemia, benign prostatic hyperplasia, adenocarcinoma of the prostate, and transitional cell carcinoma of the bladder. There was no correlation between the concentrations of B-isoenzymes and creatinine in the sera of azotemic patients. Above-normal concentrations of B-isoenzymes were found in sera from three patients with acute renal failure, but in only two of 28 specimens from patients with chronic renal failure. Above-normal concentrations of B-isoenzymes also were found in sera from three of 18 patients with untreated carcinoma of the prostate, 10 of 25 patients with treated carcinoma, 20 of 135 patients with benign prostatic hyperplasia, and 10 of 33 patients and with transitional cell carcinoma of the bladder. An above-normal concentration of B-isoenzymes in serum has a low predictive value for adenocarcinoma of the prostate, was not a sensitive indicator of the presence of carcinoma, and was noted paradoxically in six patients with treated carcinoma who had normal acid phosphatase activities in serum. We conclude that routine measurement of B-isoenzymes is not useful to establish the diagnosis of adenocarcinoma of the prostate.
Gas-chromatographic method of analysis for urinary organic acids. II. Description of the procedure, and its application to diagnosis of patients with organic acidurias.
Authors: Tanaka K , West-Dull A , Hine D G , Lynn T B , Lowe T
Published: January 2020

A gas-chromatographic method for urinary organic acid analysis is described, designed to be used routinely for the diagnosis of organic aciduria. It involves extraction of urine with ethyl acetate, dehydration of extract residues, and trimethylsilylation. Organic acids are identified by using an extensive list of retention indices published in the accompanying paper (this issue). Quantitative values are given for organic acids in urines from 50 ostensibly normal subjects. Typical chromatograms of urinary organic acids from patients with eight well-established organic acidurias are also shown.
Clinical chemical values for some common laboratory animals.
Authors: Caisey J D , King D J
Published: January 2020

In the course of evaluating the suitability of the Becton Dickinson “Microtainer” for the collection of blood samples from laboratory animals, we collected a large quantity of reference data. These data are presented and compared with published work on the effect of blood-collection methods in man and some laboratory animals.
Determination of beta-choriogonadotropin in human plasma; evaluation and comparison of five "kit" methods.
Authors: Knox B S , McKee J W , Hair P I , France J T
Published: January 2020

We evaluated five quantitative, commercially available radioimmunoassay methods (Beta-Tec, BIO-RIA, Cambridge Nuclear, Mallinckrodt, and Serono) for rapid determination of the beta-subunit of human choriogonadotropin in serum or plasma. Only three of the kits (Beta-Tec, Mallinckrodt, and Serono) were found suitable for reliable determination of low concentrations of this analyte, which is of importance in the early diagnosis of trophoblastic disease in men and monitoring effectiveness of chemotherapy of trophoblastic tumors in men and women.
Interlaboratory evaluation of four RIA kits for determination of plasma cortisol, with special reference to accuracy: influence of matrix in calibration standards.
Authors: Lantto O , Björkhem I , Blomstrand R , Kallner A
Published: January 2020

We used four RIA kits for determination of plasma cortisol to assay 12 plasma samples in four laboratories, and compared the results with those obtained with a highly accurate method based on isotope dilution/mass spectrometry. All kits (Farmos, NEN, Sorin, and Clinical Assays) showed 10-50% deviation from the comparison method in the normal range (200-700 nmol/L). The inter- and intralaboratory imprecision (CV) ranged between 8-27% and 5-11%, respectively. Possible explanations for this low accuracy are discussed. Retention of an intact biological matrix in the calibration standards seems to be one prerequisite for accuracy.
Apparent normal leukocyte acid maltase activity in glycogen storage disease type II (Pompe's disease).
Authors: Potter J L , Robinson H B , Kramer J D , Schafter I A
Published: January 2020

We present a case of glycogen storage disease type II (Pompe’s disease) with the classical clinical presentation and characteristic electrocardiographic changes of this disorder. An acid maltase (EC 3.2.1.20) determination in the peripheral leukocytes revealed normal activity; however, acid maltase activity was completely absent in a pre-mortem skeletal muscle biopsy. Post-mortem studies showed acid maltase activity to be absent in all tissues examined, including cultured skin fibroblasts. Massive glycogen deposition corresponded to the localization of the enzymic deficiency, except in the brain, where glycogen content was within the normal range. The acid maltase activity in mixed peripheral leukocytes was due to an isoenzyme of acid maltase in the granulocyte series. Antenatal diagnosis was accurate in a subsequent pregnancy, but discordance between enzyme activity in different cell lines in an individual with a genetic disease is a conceivable source of error in both prenatal and postnatal diagnoses.
Isolation and characterization of a human pancreas-specific protein.
Authors: Nerenberg S T , Prasad R , Pedersen L , Biskup N S , Faiferman I
Published: January 2020

Homogenates of human pancreas in saline were centrifuged at 27 000 X g and the supernates were fractionated by preparative polyacrylamide gel electrophoresis. The gels were divided into sections and each section was injected into rabbits; after absorption with polymerized serum from apparently normal humans, the antiserum obtained by injecting one of the sections was tested against a variety of human tissue extracts but reacted only with saline extracts of human pancreas. The absorbed antiserum, polymerized and made insoluble with glutaraldehyde, was used to purify a pancreas-specific antigen by immunoaffinity batch technique. The purified antigen proved to be a protein with some carbohydrate content (180 mg/g by weight) and a molecular mass of about 2.25 X 10(5) daltons. The antigen is relatively thermostable, and precipitates in the range of 245.64-340.2 g/L saturated ammonium sulfate; its antigenic activity is not affected by incubation with ribonuclease or deoxyribonuclease, but is destroyed by incubation with trypsin or neuraminidase and by extraction with perchloric acid. Immunofluorescence studies show that the antigen is diffusely present in the cytoplasm of pancreatic acinar cells.
Radioimmunoassay for detection of latent chronic alcoholic pancreatitis, an unrecognized clinical syndrome.
Authors: Nerenberg S T , Prasad R , Pedersen L D , Biskup N S
Published: January 2020

We developed a radioimmunoassay procedure in which we used an antibody monospecific for pancreatic acinar antigen, to sensitively and specifically test for the presence of subclinical (latent) alcoholic pancreatitis. The antigen was isolated, purified, and partially characterized. Results of testing appropriate populations of patients support the idea that chronic alcoholism is associated with chronic, subclinical damage to the pancreas and concomitant continuous release of a pancreatic acinar protein into the peripheral circulation, where it can be detected and quantitated. About 40% of the samples from chronic alcoholics (whether during a bout of acute alcoholism or during abstinence) demonstrated the circulating antigen, strongly suggesting that those alcoholics who will develop symptomatic, chronic alcoholic pancreatitis can be readily detected during the non-symptomatic stage.
Use of 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone chromogenic system in direct enzymic assay of uric acid in serum and urine.
Authors: Fossati P , Prencipe L , Berti G
Published: January 2020

A new direct colorimetric procedure for uric acid assay in serum or urine is described, utilizing a 3,5-dichloro-2-hydroxybenzene sulfonic acid/4-aminophenazone chromogenic system in the presence of horseradish peroxidase and uricase from Aspergillus flavus. This chromogen system has a high absorptivity, affording useful results with sample/reagent volume ratios as low as 0.025. The procedure is applicable to serum, plasma, or diluted urine. A single working reagent is used; the reaction is complete in less than 15 min at room temperature. The red dye formed is measured at 520 nm; a blank sample measurement is not needed. The standard curve for the method is linear for uric acid concentrations up to 1500 mumol/L. Average analytical recovery of uric acid in human sera and urine exceeded 99%; within-run and between-run precision studies showed CV's of less than or equal to 1.2 and less than or equal to 2.2%, respectively. The new procedure correlated well with the uricase/catalase and uricase/ultraviolet methods. The method is suitable for automation.
Effects of centrifugation, storage, and contamination of amniotic fluid on its total phospholipid content.
Authors: van Voorst tot Voorst E J
Published: January 2020

The total phospholipid content of the supernatant fluid decreases rapidly with increasing relative centrifugal force when amniotic fluid is centrifuged. Possible explanations for this are discussed. The effect of storage on the total phospholipid content of amniotic fluid at different temperatures and the influence of contamination with blood and meconium are also described.
Hepatic function in mononucleosis induced by Epstein-Barr virus and cytomegalovirus.
Authors: Horwitz C A , Burke M D , Grimes P , Tombers J
Published: January 2020

We evaluated serial enzyme and bilirubin determinations as aids to diagnosis of Epstein-Barr virus-induced infectious mononucleosis (121 cases) and the heterophil-negative mononucleosis-like illness due to cytomegalovirus (33 cases). Laboratory evidence for either type of mononucleosis includes mild to moderate hepatic dysfunction, with aspartate aminotransferase activity increased, but lower than commonly encountered in active viral hepatitis. Of the enzymes commonly assayed in evaluating liver function, aspartate aminotransferase activity was the most commonly abnormal: in 96.7% of those with Epstein-Barr virus disease and 87.9% with cytomegalovirus disease. Values for alkaline phosphatase were increased in 94.2% of the Epstein-Barr virus cases and 63.6% of the cytomegalovirus cases, and gamma-glutamyltransferase values were increased in 90.9% and 75.8%, respectively. We conclude that, in serially studied patients, normal results for liver-function studies or very high aspartate aminotransferase activities (greater than 1000 U/L) eliminate, for practical purposes, both Epstein-Barr virus and cytomegalovirus as diagnostic considerations.
Specific lymphocyte phospholipid changes in chronic renal failure.
Authors: Kalofoutis A
Published: January 2020

I describe phospholipid fluctuations in lymphocytes of peripheral blood of patients with chronic renal failure who were undergoing maintenance hemodialysis. The findings of decreased concentration of total phospholipids, phosphatidylcholine, and phosphatidylethanolamine, and increased concentrations of phosphatidylinositol and diphosphatidylglycerol are discussed, in relation to the lymphocyte membrane enzymic systems, and immune response.
Continuous-flow fluorometry of low galactose concentrations in blood or plasma.
Authors: Henderson J M , Fales F W
Published: January 2020

Clearance of 0-100 mg/L concentrations of galactose from the blood depends on nutrient hepatic blood flow. We can measure such concentrations, which was not previously possible, by a continuous-flow method involving the use of galactose oxidase and peroxidase, the latter being coupled to a fluorogenic substrate, p-hydroxyphenylacetic acid. Interfering substances in the peroxidase reaction are removed by zinc/alkali precipitation. Sensitivity is maximized by using saturating concentrations of the enzymes and substrate. In prepared plasma test samples with galactose concentrations of 10, 40, 70, and 100 mg/L, the within-run CV's ranged from 2.1 to 8.6%, and day-to-day CV's from 2.2 to 17.2%, the largest CV's being for the 10 mg/L concentration. Normal subjects are shown to clear galactose more efficiently than subjects with moderate cirrhosis.
Improved protein-binding radioassay for plasma cortisol.
Authors: Novacenko H , Halpern F , Sachar E J
Published: January 2020

We describe an improved competitive protein-binding radioassay for total plasma cortisol, which permits rapid analysis of many samples. Plasma, 0.1 mL, is directly applied to a Whatman 3MM filter-paper strip, which is extracted once with dichloromethane. The extract is evaporated and the residue is assayed directly by the micro-method of Murphy (J. Clin. Endocrinol. Metab. 27: 973, 1967). Comparison with the original Murphy method (ethanol extraction) yielded excellent agreement (r = 0.988). One hundred samples, in duplicate, can easily be assayed at a time.
Digitalis glycosides in serum, urine, and cerebrospinal fluid, determined with a commercial radioimmunoassay.
Authors: Nore A K , Storstein L , Larsen A , Amlie J P
Published: January 2020

We evaluated a commercial (Clinical Assays, Inc.) 125I radioimmunoassay for digitoxin for use in measuring cardiac glycosides in serum, buffer, urine, and cerebrospinal-fluid samples. We also assessed interference of seven cardioactive metabolites of digitoxin, including digoxin, with the assay. The precision of the assay was adequate for all matrixes studied for glycoside concentrations less than 5 microgram/L. Reactivity in the assay was dependent on the nature of the matrix: buffer and serum samples containing digitoxin showed a stronger reactivity than did samples of urine and cerebrospinal fluid. Patients' samples must therefore be analyzed with a standard curve prepared by use of the same matrix. The digitoxosides of digitoxin showed a slightly higher reactivity in the assay than did digitoxin itself, but digitoxigenin showed a 50% lesser reactivity. Digoxin, its digitoxosides, and digoxigenin hardly react at all in the assay and so do not interfere with digitoxin determinations.
Improved direct specific determination of serum iron and total iron-binding capacity.
Authors: Ceriotti F , Ceriotti G
Published: January 2020

Serum iron is released from transferrin and reduced at pH 1.7 by treating serum with a 10 g/L ascorbic acid solution in 0.1 mol/L HCl. When ferrozine is added to this reagent, it forms a complex with iron that is as intensely colored as at higher pH values, and under these conditions no turbidity is produced. The second major interference, that from copper, is eliminated by adding 1 g of thiosemicarbazide per liter, which at a low pH forms a stable, uncolored complex with copper without affecting the reaction of ferrozine with iron.
Evaluation of an immobilized-enzyme analyzer for measuring galactose in serum.
Authors: Buffone G J , Johnson J M , Lewis S A , Sparks J W
Published: January 2020

Reportedly, galactose provides an alternative carbohydrate source and improved homeostatic regulation of glucose in the premature infant. Because of its potentially toxic effects, sensitive methods are needed for monitoring its concentration during therapy. We evaluated an immobilized galactose oxidase/hydrogen peroxide electrode system and a modified homogeneous enzymic method. Both methods are suitable for measuring galactose in a small sample and are comparably precise. The latter method gives superior analytical recoveries below 100 mg/L, but is linear in absorbance response to only 300 mg/L. We find the immobilized-enzyme method superior for monitoring treatment of neonates with galactose, because it requires only a few minutes and 25 microL of serum, and the analytical procedure is simpler.
Adaptation of the enzyme-multiplied immunoassay for methotrexate to the centrifugal analyzer.
Authors: Finley P R , Williams R J , Griffith F , Lichti D A
Published: January 2020

We adapted the homogeneous enzyme-multiplied immunoassay for methotrexate to the centrifugal analyzer. The sensitivity of the assay has been extended from 200 nmol/L down to 12.5 nmol/L. Several protocols may be followed, depending on whether the samples to be tested are from patients on low- or high-dose methotrexate regimens. Precision, accuracy, and sensitivity were satisfactory. Comparison with the enzymic-inhibition method (x) gave the following least-squares regression: y = 1.002 x - 0.013 mumol/L; r2 = 0.9721; Sy/x = 0.014 mumol/L.
Incidence of a split alpha 2-glycoprotein band in the electrophoretic pattern for serum of adenocarcinoma patients.
Authors: Dermer G B , Silverman L M , Gendler S J , Tökés Z A
Published: January 2020

We electrophoresed serum samples on Mylar-backed cellulose acetate membranes and stained for glycoproteins with the periodic acid—Schiff reagent. The samples were from untreated adenocarcinoma patients, adenocarcinoma patients receiving chemotherapy, and patients with other malignancies, and also from patients with benign proliferative diseases, inflammatory diseases, and other non-malignant conditions. Forty-five per cent of the sera from untreated adenocarcinoma patients and 80% of those from adenocarcinoma patients with progressive systemic disease exhibited a splitting of the alpha 2-glycoproteins into a fast and slow band. Such a pattern was seen in only 4% of the non-adenocarcinoma cancer patients and 4% of the control group. Serial studies indicated that electrophoretic patterns of alpha 2-glycoproteins change with clinical status. Non-cancer patients with high concentrations of acute-phase proteins in their serum did not exhibit two alpha 2-glycoprotein bands. Further characterization of serum proteins from the fast alpha 2 region suggest that alpha 1-acid glycoprotein and haptoglobin beta chains are the principal components staining with periodic acid—Schiff reagent. These components are markedly less apparent in, or are absent from, the fast alpha 2 region of normal sera.
Qualitative testing for circulating immune complexes by use of zone electrophoresis on agarose.
Authors: Kelly R H , Scholl M A , Harvey V S , Devenyi A G
Published: January 2020

On binding of antibody to antigen an immune complex is formed that has a net surface charge different from that of either of the two components. This, together with clonal restriction of the antibody response, gives rise to distinctive patterns that are readily apparent in stained agarose gels after routine zone electrophoresis. Most circulating immune complexes appear as a rectangular pattern, with well-defined edges, located in the gamma-region. The identity of the material responsible for these patterns has been established by three different experimental approaches: analysis of tetanus/anti-tetanus complexes formed in vitro, analysis of sera from rabbits with experimental immune complex disease, and analysis of human type II and type III cryoglobulins. Studies of reproducibility, interfering substances, and correlation with other assays for detecting immune complexes indicate that zone electrophoresis in agarose gel is a sensitive, highly specific technique for immune complex detection, of potential value as a screening tool.
Taurine transport in epilepsy.
Authors: Goodman H O , Connolly B M , McLean W , Resnick M
Published: January 2020

Previous studies of plasma taurine concentrations in epileptics have yielded equivocal results. We measured plasma and urinary taurine in 41 epileptic and 68 control subjects and found plasma concentrations among epileptics to be comparable in general to those of controls, but that two or three classes of plasma taurine concentrations, possibly genetically regulated, occur in both epileptic and control subjects. Our previous studies and data from the present study on taurine excretion revealed three excretion classes under genetic control. The principal finding is that epileptics include disproportionate numbers of low excretors (high reabsorbers), who are presumptive homozygotes for the allele effecting higher reabsorption. If confirmed, these findings suggest that the transport of taurine, rather than absolute taurine concentration, may explain the efficacy of taurine administration in some epileptics but not in others. The locus involved may be one component in the polygenic diathesis to the idiopathic epilepsies.
A simplified assay for dihydroxylated vitamin D metabolites in human serum: application to hyper- and hypovitaminosis D.
Authors: Mason R S , Lissner D , Grunstein H S , Posen S
Published: January 2020

We describe a simplified assay for 24,25-and 1.25-dihydroxyvitamin D in human serum. It involves two preparative steps, and normal chick intestine is used in preparing cytosol-binding protein. Our results for 24,25-dihydroxyvitamin D include a reference interval of 2.9—16 nmol/L (1.2—6.7 microgram/L), a mean of 6.7 nmol/L (2.8 microgram/L), an intra-assay CV of 11%, and an interassay CV of 22%. For 1,25-dihydroxyvitamin D, these data were 29—168 pmol/L (12—70 ng/L), 86 pmol/L (36 ng/L), 12%, and 22%, respectively. In hypoparathyroid patients with vitamin D intoxication, mean concentrations of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D in serum were significantly above normal; the 1,25-dihydroxyvitamin D concentrations were significantly below normal. Patients with malabsorption and/or post-gastrectomy states had significantly subnormal values for both 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D in serum, and there was a significantly negative correlation between each of these biochemical values and the severity of osteomalacia. We also discuss cost effectiveness of assaying vitamin D metabolites in human serum.
Thyroxine-binding globulin capacity and concentration evaluated from blood spots on filter-paper in a screening program for neonatal hypothyroidism.
Authors: Dussault J H , Morissette J , Letarte J , Guyda H , Laberge C
Published: January 2020

We describe a simple method for evaluating thyroxine-binding globulin capacity and concentration from a single 1-cm blood spot on filter-paper used in a screening program for neonatal hypothyroidism. This method permits prompt diagnosis of about 90% of the infants with thyroxine-binding globulin deficiency in our abnormal low-thyroxine, low-thyrotropin population. There was excellent equivalence between results obtained by our method and by the method of Chopra et al. (J. Clin. Endocrinol. Metab. 35:565, 1972), and minimal overlap between the population with low thyroxine-binding globulin and the low-thyroxine, normal thyrotropin population. We recommend this method to all programs in which a primary thyroxine measurement is used in screening for congenital hypothyroidism.
Enzymic color development of urinary 3 alpha-hydroxysteroids on thin-layer chromatograms.
Authors: Yamaguchi Y
Published: January 2020

I describe the use of 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) for enzymic color development of 3 alpha-hydroxysteroids. Urinary 3 alpha-hydroxysteroids are converted to 3-ketosteroids by the enzyme, and the NADH formed is reacted with 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl-tetrazolium chloride and "diaphorase" to form the colored formazan. The dye so formed on a thin layer chromatographic plate is measured by densitometoric scanning. I illustrate use of this method by showing results for urinary excretion of 3 alpha-hydroxysteroids by some various sorts of patients.
Measurement of anticonvulsants in serum by reversed-phase ion-pair liquid chromatography.
Authors: Christofides J A , Fry D E
Published: January 2020

We describe a method for the simultaneous liquid-chromatographic determination of ethosuximide, ethylphenacemide, primidone, phenobarbital, carbamazepine, and phenytoin in serum. The drugs, together with an internal standard, are extracted into ethyl acetate at pH 7.0. The extract is analyzed isocratically at ambient temperature on a reversed-phase column of "SAS Hypersil" with a mobile phase of acetonitrile/tetrabutyl ammonium phosphate solution (2/8 by volume). The eluted drugs are detected by their absorption at 200 nm, and quantitated from their peak heights as compared with those of extracted standards. The day-to-day CV of the method varied between 5.1 and 9.6% for concentrations ranging from less than therapeutic to toxic. The results, when compared with those by gas chromatography, gave correlation coefficients of 0.936 for phenytoin, 0.977 for phenobarbital, and 0.939 for primidone. No drug interference was found except that amobarbital and ethylphenacemide co-chromatographed.
Pre-precipitated and solid-phase second antibody compared in radioimmunoassay.
Authors: Brown T R , Bagchi N , Ho T T , Mack R E
Published: January 2020

We describe simple methods for preparing and determining the titer of pre-precipitated second antibody. We compared the performance of this type of insolubilized second antibody with that of a commercially available solid-phase second antibody (DASP) for separation of reverse triiodothyronine (rT3) and for thyrotropin radioimmunoassays. Equilibrium time for both reagents was about 30 min, considerably briefer than for the usual second-antibody procedure. Within-assay precision was improved and percentages of nonspecific binding (mean and SD) were lower if pre-precipitated second antibody was used in both assays [rT3 pre-precipitate = 1.48 (SD 0.16), rT3 DASP = 3.76 (SD 0.22); thyrotropin pre-precipitate = 1.45 (SD 0.18), thyrotropin DASP = 3.45 (SD 0.26)]. The cost of DASP was three to 10 times that of pre-precipitated second antibody prepared with commercially available products.
Transient increase in serum creatine kinase isoenzyme BB activity after prostatectomy in a patient with massive benign prostatic hyperplasia.
Authors: Goodman Z D , Hamilton S R
Published: January 2020

The activity of creatine kinase isoenzyme BB (CK-BB) in serum is rarely abnormally high (i.e., detectable). An increase in immunoreactive CK-BB or CK-BB activity in patients with prostatic disease has been proposed as an indication of prostatic adenocarcinoma. Here we report the case of an elderly man with massive benign prostatic hyperplasia but no clinical or pathological evidence of prostatic adenocarcinoma, whose serum CK-BB activity was found by agarose gel electrophoresis to be 1 U/L (normal: 0%), 10% of his total CK activity. Serum CK-BB activity was further increased to 16 U/L (20% of total CK activity) 1 h after prostatectomy, but became undetectable by the second day after the operation. The findings suggest that: (a) the source of the serum CK-BB activity was the enlarged prostate gland; (b) abnormally high CK-BB activity in serum of men with prostatic disease does not necessarily indicate the presence of prostatic adenocarcinoma; and (c) myocardial injury could be erroneously diagnosed postoperatively in prostatectomy patients if CK isoenzyme methods are used that do not consistently separate "heart-specific" CK-MB from CK-BB.
Automated creatine kinase-MB estimation by immuno-inhibition: a clinical evaluation.
Authors: Delahunty T J , Foreback C C
Published: January 2020

We detected the presence of creatine kinase (EC 2.7.3.2) MB isoenzyme (CK-MB) in serum by using an immunoinhibition technique to inactivate the creatine kinase isoenzyme of muscle origin (CK-MM). The GEMSAEC centrifugal analyzer is ideally suited for this procedure because it rapidly mixes reagents and has a throughput of 60 samples/h. The within-run CV was acceptable when the CK-MB value was 7.5 U/L or more. CK-MM was totally inactivated up to 5000 U/L, a value well above that usually seen in myocardial-infarct patients. We assessed the predictability of the assay for detecting myocardial infarction among 120 coronary-care patients when 2% of total CK activity was considered diagnostic and compared the results with those obtained with an electrophoresis technique. The positive predictive value of the immunoinhibition assay was 97.8%, as compared to 92% with the CK electrophoresis technique, when lactate dehydrogenase (EC 1.1.1.27) isoenzyme results were included in both cases. The optimal negative predictive value was 95.1% with the immunoinhibition assay vs 98.2% with electrophoresis. We conclude that the immunoinhibition technique for estimating CK-MB can be automated to assess myocardial status rapidly, precisely, inexpensively, and sensitively.
Determination of urinary amino acids by liquid chromatography with “dabsyl chloride".
Authors: Lin J K , Wang C H
Published: January 2020

We describe a “high-performance” liquid-chromatographic procedure for measuring amino acids in 0.5 mL of urine. The procedure includes direct derivatization of amino acids in urine samples with “dabsyl chloride" (4-dimethylaminoazobenzene-4’-sulfonyl chloride). An aliquot of this dabsylated amino acid solution is analyzed on a muBondapak C18 column with ethanol/sodium acetate (20 mmol/L, pH 4.0) 4/6 (by vol.), as mobile phase. Dabsylated amino acids are detected by their absorbance at 425 nm and quantitated by measuring peak heights. The procedure allows for the reliable analysis of amino acids in urine at concentrations near 16 mg/L. The sensitivity of this analysis on column approaches 5 ng/sample. Higher urinary tryptophan concentrations were found in the urines of some cancer patients, whereas we saw no significant difference in urinary glycine between cancer patients and control subjects. The present method was shown to be a straightforward procedure for detecting phenylalanine in phenylketonuric urine. Extension of this procedure to screening for other inborn errors of aminoaciduria is recommended.
Fluorometric and colorimetric enzymic determination of triglycerides (triacylglycerols) in serum.
Authors: Winartasaputra H , Mallet V N , Kuan S S , Guilbault G G
Published: January 2020

We describe two fully enzymic methods, fluorometric and colorimetric, for determination of triglycerides (triacylglycerols) in serum. Samples are incubated with microbial lipase for 10 min, and the glycerol released from the triglycerides is oxidized by NAD+ in the presence of glycerol dehydrogenase. In the fluorometric method, the resulting NADH is in turn oxidized by resazurin as catalyzed by diaphorase to form resorufin, a highly fluorescent compound. In the colorimetric method, the NADH is oxidized by coupling with a tetrazolium salt/diaphorase system to form formazan, a highly colored compound. Calibration curves, constructed by plotting change in fluorescence or absorbance vs concentration of triglycerides, were linear up to 6 and 5 g of triglycerides per liter of serum for the fluorometric and colorimetric methods, respectively. The assays require only 5 and 15 microL of serum for fluorometry and colorimetry, respectively. The CV was 0.59% for the fluorometric method, 0.91% for the colorimetric procedure. The time for analysis for either method is less than 15 min. The results correlate well with those obtained by the Dow Diagnostic Kit method, a colorimetric method in which glycerol kinase and glycerol-1-phosphate dehydrogenase form NADH from ATP and NAD+ in the presence of glycerol and glycerol 1-phosphate.
Restoration of lead-inhibited 5-aminolevulinate dehydratase activity in whole blood by heat, zinc ion, and (or) dithiothreitol.
Authors: Sakai T , Yanagihara S , Ushio K
Published: January 2020

We examined effects of heat, zinc, ion, and dithiothreitol in restoring the activity of lead-inhibited-5-aminolevulinate dehydratase (EC 4.2.1.24). The ratio of non-activated to activated activity produced by dithiothreitol correlated well with blood lead concentration among 35 lead workers. The individual effects of heat, zinc, or dithiothreitol differ from each other in the shift of pH optimum as well as in the extent to which activity is restored. Dual or triple combinations of these agents show additive or complementary restoration of activity. The combination of heat and zinc or zinc and dithiothreitol expands the range of optimum concentration of zinc in restoring activity. Using these combinations of agents, we can expect more accurate evaluation of lead exposure than by measuring only activation of zinc. Although dithiothreitol most powerfully restores activity, it restores not only the activity inhibited by lead exposure but also the activity removed by oxidation of SH-groups in the enzyme molecule.
Albumin-bound fluorescence in serum of patients with chronic renal failure.
Authors: Schwertner H A , Hawthorne S B
Published: January 2020

A band that is strongly fluorescent and migrates electrophoretically with serum albumin is commonly found in electrophoretograms of sera from patients with chronic renal failure. We sought to determine whether the fluorescence originates from binding of certain still-unidentified metabolites or drugs, from an abnormal albumin species, or from some other protein entity. Molecular-exclusion column chromatography, polyacrylamide gel isoelectric focusing, and cellulose acetate electrophoresis, along with results of charcoal treatment and alcohol extraction, provided evidence that the fluorescence comes from fluorescent ligands tightly bound to albumin. The fluorescent intensity of the albumin fraction, isolated by molecular-exclusion chromatography, coincides with the albumin-associated fluorescence determined electrophoretically and with the intensities of the fluorescence emission spectrum for serum. A fluorescent species with an emission maximum of 415 ± 5 nm, separated by thin-layer chromatography, appears to account for the increased serum fluorescence.
Stability of standard curves prepared for EMIT homogeneous enzyme immunoassay kits stored at room temperature after reconstitution.
Authors: Bach P R , Larsen J W
Published: January 2020

We examined the stability of standard curves obtained with use of homogeneous enzyme immunoassay reagents (EMIT; Syva Corp., Palo Alto, CA) for assay of lidocaine, procainamide, N-acetylprocainamide, gentamicin, and theophylline, when stored at room temperature (23-24 degrees C) after reconstitution of the lyophilized materials. Standards were run and curves obtained for as long as 16 days after reconstitution. All standard curves were acceptable, according to the criteria specified in Syva product literature. Absorbance changes for all calibrators, including the zero calibrator, increased gradually with time. Increases of non-zero calibrators were largely offset by a parallel increase in the zero calibrator, such that the quantity delta A--delta A0 was very nearly constant with time. Standard curves based on the quantity delta A--delta A0 can be used for longer than curves based only on delta A.
Method for determination of hydrogen peroxide, with its application illustrated by glucose assay.
Authors: Graf E , Penniston J T
Published: January 2020

We describe a simple colorimetric method for determining micromolar quantities of hydrogen peroxide, based on the oxidation of iodide in the presence of ammonium molybdate and photometry of the resulting blue starch-iodine complex. Color development is linearly dependent on analyte concentration, but only slightly time dependent, and the color of the complex formed is stable for several hours. In the range of wavelengths that may be used (570 to 630 nm), lack of interference from other biological compounds makes this method seem suitable for routine analyses. As one illustrative application of the method we quantitated glucose by measuring hydrogen peroxide produced from it by glucose oxidase catalysis. This method of quantitating glucose is more than five times as sensitive as the commonly used dianisidine method. With the appropriate hydrogen peroxide-producing oxidases, this method may be used to directly measure amino acids, purines, uric acid, xanthine, and hypoxanthine.
Interferences appearing in fluorometrically measured liquid-chromatographic profiles of creatine kinase isoenzymes in serum.
Authors: Schlabach T D , Fulton J A , Mockridge P B , Toren E C
Published: January 2020

We observed nonenzymic peaks when serum isoenzymes of lactate dehydrogenase (EC 1.1.1.27; LD) and creatine kinase (EC 2.7.3.2.; CK) were separated by “high-performance” liquid chromatography and detected by continuously monitoring the column effluent for enzyme activity. Such background peaks were particularly apparent in CK isoenzyme profiles obtained from human sera. We observed two nonenzymic peaks with fluorescence detection, one in the CK-MB region, the other in the CK-BB region. Serum albumin was a major component in the artifactual CK-MB peak, with lipoprotein as a minor component. We present evidence that the material responsible for the other peak fluoresced quite strongly and is mostly pre-albumin.
Liquid-chromatographic separation and on-line bioluminescence detection of creatine kinase isoenzymes.
Authors: Bostick W D , Denton M S , Dinsmore S R
Published: January 2020

We separated isoenzymes of creatine kinase by anion-exchange chromatography, with use of an elution of gradient containing lithium acetate (0.1 to 0.6 mol/L). A stream splitter was used to divert a 5% side stream of column effluent, which was subsequently mixed with the reagents necessary for bioluminescence assay of the separated isoenzymes. The use of the stream splitter greatly decreased the rate of consumption of reagent and, when combined with a peristaltic pumping system, permitted independent control of the side-stream flow rate. Thus both the residence interval in a delay coil in which the ATP reaction product is formed and the bioluminescence response could be increased. Bioluminescence emission was monitored in a flow-through fluorometer without use of an external light source or filters. Separation and detection of the isoenzymes of creative kinase were rapid, sensitive, and highly selective. The incremental decrease of bioluminescence response owing to inhibition by the ions in the eluent was less than 31% across the entire gradient.