bluuebunny/crossref_metadata_2025 huggingface dataset:
High-purity 4-nitrophenol: purification, characterization, and specifications for use as a spectrophotometric reference material.
Authors: Bowers G N , McComb R B , Christensen R G , Schaffer R
Published: January 2020

We describe specifications for high-purity 4-nitrophenol, which is suitable for spectrophotometric standardization. Such a reference material is needed in clinical enzymology to establish the proper molar absorptivity of 4-nitrophenol under final reaction conditions, particularly for measuring alkaline phosphatase activity in human serum. Some lots of 4-nitrophenol available commercially met these specifications, but several did not. The latter can be purified to meet our specifications by recrystallization or sublimation. The molar absorptivity of 4-nitrophenol (35 μmol/L) IN 10 mmol/L NaOH at 25 °C at 401 nm is 18380 +/− 90 L.mol−1.cm−1.
Cortisol directly determined in serum by fluoroimmunoassay with magnetizable solid phase.
Authors: Pourfarzaneh M , White G W , Landon J , Smith D S
Published: January 2020

We developed a direct fluoroimmunoassay for cortisol in serum. This method involves cortisol labeled at the 3 position with fluorescein, and antibodies to cortisol coupled to magnetizable cellulose/iron oxide particles. Sodium salicylate is used as a blocking agent to prevent interference from endogenous binding proteins in serum. Serum sample and labeled cortisol are incubated with the antibody-coupled solid phase for 30 min. The solid phase is then separated and washed to remove free labeled cortisol and endogenous fluorophores of the sample. Finally we elute the antibody-bound fraction of the labeled cortisol into an alkaline methanolic medium and measure its fluorescence. The separation, wash, and elution steps are facilitated by magnetic sedimentation. The assay is sufficiently sensitive, specific, and reliable for routine use and correlates acceptably (r = 0.92) with an established radioimmunoassay. Precision (CV) ranged from 4 to 10% in experiments on three pooled sera; analytical recovery for sera supplemented with as much as 360 μg of cortisol per liter was 91 to 109%.
Measuring thyroxine and thyrotropin simultaneously in a dried blood sample on filter paper, to screen for neonatal hypothyroidism.
Authors: Nagataki S , Ishibashi K , Ohsawa R , Suwa S , Tsukamoto N , Takahashi T , Obara Y , Liao Y Y
Published: January 2020

We have developed a highly sensitive radioimmunoassay of thyroxine and thyrotropin for mass screening for neonatal hypothyroidism. This assay involves a single disc (3 mm diameter) of dried blood on filter paper. The minimum detectable concentrations are 15 pg/tube (10 microgram/L) for thyroxine and 15 nano-int. units/tube (10 milli-int. units/L) for thyrotropin; intra- and interassay CV’s are < 15% in both assays. The high sensitivity of this method is due to use of labeled thyroxine with high specific activity (3 kCi/g) and of an anti-thyrotropin serum with high affinity (Keq = 7.8 × 10(11) L/mol). With this method, 11337 newborns were screened; a follow-up study revealed that only newborns with both high thyrotropin and low thyroxine concentrations had permanent hypothyroidism. We conclude that this method is sensitive, simple, and reliable and that the recall rate with this method is much lower than that of tests for measuring thyroxine or thyrotropin alone.
A coupled-enzyme equilibrium method for measuring urea in serum: optimization and evaluation of the AACC study group on urea candidate reference method.
Authors: Sampson E J , Baird M A , Burtis C A , Smith E M , Witte D L , Bayse D D
Published: January 2020

We describe a coupled-enzyme equilibrium method for measuring urea in serum, which is performed on supernates prepared by treating each specimen with Ba(OH)2 and ZnSO4 (Somogyi reagent). Analytical recovery of [14C]urea added to a variety of matrices was essentially complete (mean, 100.6%) for the supernates after precipitation. Nine variables were univariately examined in arriving at the reaction conditions for the method: glutamate dehydrogenase, urease, 2-oxoglutarate, ADP, Tris . HCI, NADH, EDTA, pH, and temperature. The reagent is stable for at least 48 days at--20 degrees C and for 23 days at 4 degrees C. Mean analytical recovery of urea (14 mmol/L) added to seven different specimens (three different matrices) was 100.8%. The analytical linear range of the method extends to 30 mmol of urea per liter. Of 22 potential interferents, only bilirubin at 1 mmol/L (580 mg/L), hemoglobin at 10 g/L, and hydroxyurea at 6 mmol/L showed more than 2% interference. We discuss precision and effects of specimen dilution, and compare results for 100 human serum specimens with those measured for the same specimens with four other urea methods. We examined the effects of measuring a blank, consisting of sample and reagent without urease, with each specimen.
Recent advances in measurement of amylase activity--a comparative study.
Authors: Kaufman R A , Tietz N W
Published: January 2020

We evaluated four kinetic amylase procedures with respect to kinetics, analytical range, blank rates, reagent stability, reagent impurities, interfering substances, and intrinsic sensitivities. Each of the methods is shown to have its own unique advantages and disadvantages. A preliminary discussion of some alternative methods, in which glycosidic p-nitrophenyl alpha-oligosaccharides are substrates, is included.
Creatine kinase radioimmunoassay and isoenzyme electrophoresis compared in the diagnosis of acute myocardial infarction.
Authors: Homburger H A , Jacob G L
Published: January 2020

We compared, in 116 patients, the relative usefulness of results of tests for creatine kinase B isoenzymes, as measured by radioimmunoassay, and the MB isoenzyme, as measured by electrophoresis, in diagnosis of acute myocardial infarction. The radioimmunoassay was specific for isoenzymes of creatine kinase containing the B subunit. All patients with acute transmural infarcts had positive test results by both techniques, but concentrations of B-isoenzymes were more frequently above normal than were MB bands in the case of patients with acute subendocardial infarcts and in the case of all patients with acute myocardial infarcts from whom sera were collected more than 24 h after onset of chest pain. Concentrations of B-isoenzymes also were increased, even when MB bands were not electrophoretically detectable, in specimens from several patients without documented actue myocardial infarcts. These abnormal results presumably were caused by increased concentrations of the BB isoenzyme in serum. Accordingly, an increased concentration of B-isoenzymes had less diagnostic specificity and predictive value for acute myocardial infarction than did a detectable MB band. Results of isoenzyme electrophoresis were more reliable for establishing this diagnosis, but the results of radioimmunoassay were more reliable for excluding it in patients with chest pain as the primary symptom.
A semi-automatic enzymic method for estimating urinary oxalate.
Authors: Yriberri J , Posen S
Published: January 2020

A two-enzyme method is presented for estimating urinary oxalate by continuous-flow analysis. Oxalate is decarboxylated, yielding formate, which is subsequently oxidized in the presence of NAD+, yielding NADH. The NADH, measured colorimetrically, is proportional to the amount of oxalate originally present in the sample. Urine is pretreated by precipitation of the oxalate and extraction of the precipitate in citrate buffer. A correction factor for the extraction efficiency is given by the recovery of [U-14C]oxalic acid added to aliquots of the 24-h urine specimen. The normal range obtained by this method was 0.20 to 0.52 mmol/24h (18 to 47 mg/24h) with a mean value of 0.37 mmol/24 h (33 mg/24h). The between-assay CV was 7.6% (n = 12), within-assay CV 3.2% (N = 25). Unlabeled oxalate (0.5 and 1.0 mol/L) added to 28 urine samples gave a mean analytical recovery of 95% (SD 10%).
Acid Phosphatase in Gaucher's Disease
Authors: Robinson Douglas B , Glew Robert H
Published: January 2020

Vol. 26

p 374: Second column, line 1: "designated," not "designed."

p 457: First line of abstract: correct number is "2.7.3.2"; second paragraph under "Serum Samples": "mammary artery," not "mammary vein."

p 458: Under "Column Chromatography," line 9 to read "buffer (200 mmol/L NaCl, 50 mmol/L Tris-HCl)..." Sublegend to Figure 1: add "a, concentration gel; b, separation gel." Under "Isoelectric Focusing," line 6 should read "7 mg of ammonium persulfate, 0.1 mL of pH 4-6 carrier ampholyte, and 0.1 mL of pH 5-7 ampholyte."

p 485:last paragraph: insert "98" before "(29.4%)," delete "low value," change "(6.5%)," to "(6.3%)," and change "µ/L" to "µg/L."
Effect of cations on the human creatine kinase isoenzymes.
Authors: Nealon D A , Henderson A R
Published: January 2020

We report our findings on the effect of Ca2+, K+, Na+, Fe2+, Mn2+, Zn2+, and Cu2+ on the activity of creatine kinase isoenzymes derived from human tissues. The only inhibitory cation was Ca2+, and Dixon plots of the data indicate the inhibition to be competitive for the Mg2+ in the reaction assay mixture. This effect of Ca2+ on all three isoenzymes is completely reversed by including Mg2+ (10 mmol/L) and ethylenediaminetetraacetate (2 mmol/L) in the assay mixture. Our findings suggest that it is unnecessary to add chelators to the patient's sample.
Estrogen radioimmunoassay suitable for the monitoring of ovulation induction.
Authors: Ashby C D , Danzer H C , Swerdloff R S
Published: January 2020

A rapid radioimmunoassay for estrogen in serum was characterized and validated. Analytical recoveries of estrone and estradiol were essentially 100%; estriol was not measured because it was eliminated by sample-handling procedures. Nonspecific interference from serum extracts, which was initially reflected by positive blanks, was eliminated by a sodium hydroxide wash of the extract. Validation of the procedure included determination of a reference interval for estrogen that agreed with the sum of estradiol and estrone concentrations reported in the literature for eugonadal women. Clinical applicability of the assay was demonstrated by monitoring estrogen concentrations during the menstrual cycle and during urogonadotropin (Pergonal)-induced follicular maturation. Because of its rapid turnaround time, the assay is ideally suited for monitoring the frequency with which urogonadotropin should be administered to the infertile patient, and the dosage, thus decreasing the likelihood of multiple pregnancies or ovarian hyperstimulation.
Muscle protein analysis. II. Two-dimensional electrophoresis of normal and diseased human skeletal muscle.
Authors: Giometti C S , Bárány M , Danon M J , Anderson N G
Published: January 2020

We used high-resolution two-dimensional electrophoresis to analyze the major proteins of normal and pathological human-muscle samples. The normal human-muscle pattern contains four myosin light chains: three that co-migrate with the myosin light chains from rabbit fast muscle (extensor digitorum longus), and one that co-migrates with the light chain 2 from rabbit slow muscle (soleus). Of seven Duchenne muscular dystrophy samples, four yielded patterns with decreased amounts of actin and myosin relative to normal muscle, while three samples gave patterns comparable to that for normal muscle. Six samples from patients with myotonic dystrophy also gave normal patterns. In nemaline rod myopathy, in contrast, the pattern was deficient in two of the fast-type myosin light chains.
Quantitative analysis for tricyclic antidepressant drugs in plasma or serum by gas chromatography-chemical-ionization mass spectrometry.
Authors: Chinn D M , Jennison T A , Crouch D J , Peat M A , Thatcher G W
Published: January 2020

We describe a gas chromatographic-chemical-ionization mass spectrometric procedure for determining concentrations of the tricyclic antidepressant drugs and their demethylated metabolites in plasma. The procedure requires 2.0 mL of plasma or serum and involves internal standard addition, extraction with a mixed organic solvent, and separation on a column packed with 0.5% potassium hydroxide and 2% Carbowax 20M. Between-assay and within-assay (n = 10) CVs were less than 11.0%. The procedure has been applied to several interesting cases, three of which are summarized.
Two new non-barbiturate buffers for electrophoresis of serum proteins on cellulose acetate membranes.
Authors: Ambler J , Rodgers M
Published: January 2020

Two new non-barbiturate buffers have been formulated for serum protein electrophoresis on cellulose acetate membranes. Both buffers give five distrinct fractions and are suitable for all systems, but have been primarily designed to meet the needs of the Gelman "Sepratek" system. One buffer is a tris(hydroxymethyl)aminomethane (Tris)/hippurate formulation; the other is composed of Tris/N-[tris(hydroxymethyl)methyl]glycine (Tricine)/sodium chloride/sodium salicylate. Both buffers allow reliable quantitation of protein fractions, giving results that correlated well with those obtained from barbiturate separations. The major advantage of these buffers is that they do not contain drugs of abuse.
Serum enzymes and isoenzymes in the diagnosis and differential diagnosis of myocardial ischemia and necrosis.
Authors: Lott J A , Stang J M
Published: January 2020

Diagnosis of injury to the myocardium is facilitated by information on the activities of creatine kinase (EC 2.7.3.2) MB isoenzyme (CK-MB) and lactate dehydrogenase (EC 1.1.1.27) isoenzyme 1 in serum, thee isoenzymes being present in higher activities in the myocardium than in other tissues or in normal serum. The temporal relationships of these isoenzymes, total creatine kinase, total lactate dehydrogenase, and aspartate aminotransferase (EC 2.6.1.1) are highly sensitive and specific for acute injury to the heart, particularly acute myocardial infarction. Chronic heart diseases, electric cardioversion for heart rhythm disturbances, coronary catheterization, and exercise usually do not produce increases of CK-MB, although abnormal aspartate aminotransferase, creatine kinase, lactate dehydrogenase, and lactate dehydrogenase isoenzyme 1 activities are seen in some individuals. Many other causes of increased activities of these enzymes and isoenzymes in serum are unrelated to injury to the heart. Because CK-MB is present in the skeletal muscle in low activities, substantial injury to skeletal muscle can increase CK-MB activities in the blood to abnormal values. Pulmonary embolism can mimic myocardial infarction in its clinical presentation. In patients with an accurately known time of onset of symptoms and serial enzyme analysis every 12 h during the first 48 h, acute myocardial infarction can be distinguished from pulmonary embolism by determinations of creatine kinase, CK-MB, aspartate aminotransferase, and lactate dehydrogenase isoenzyme 1 in serum.
An optimized assay of alanine aminopeptidase activity in urine.
Authors: Jung K , Scholz D
Published: January 2020

Elaboration of an optimized kinetic test for urinary alanine aminopeptidase at 37 degrees C is described. As final concentrations, 50 mmol/L tris(hydroxymethyl)aminomethane x HCl buffer, pH 7.8, and 2 mmol/L alanine-4-nitroanilide are proposed. Five- to sixfold activitiy is obtained as compared with a generally used test procedure. Coefficients of variation for the method (within-run and between-day precision) ranged from 1.56 to 3.45%. Men excreted more alanine aminopeptidase than did women if urinary volume or urinary timed volume was used as basis of reference. We saw no sex-related differences in excretion of alanine aminopeptidase when enzyme excretion was related to urinary creatinine.
Kinetic studies of a quantitative single-tube enzyme-linked immunosorbent assay.
Authors: Tsang V C , Wilson B C , Maddison S E
Published: January 2020

We describe a single-cuvette enzyme-linked immunosorbent assay (ELISA) based on enzymic rate kinetics. This kinetic assay can yield linear quantitative data on immunoglobin concentrations. Optimum assay conditions, component concentrations, reaction intervals, and pH are described. Assay linearity and sensitivity are demonstrated in systems involving immunoglobulin G and antibodies to it, and with pooled sera from monkeys infected with Schistosoma mansoni.
Kinetic ethylene glycol assay with use of yeast alcohol dehydrogenase.
Authors: Eckfeldt J H , Light R T
Published: January 2020

We describe a rapid, accurate, and precise two-point kinetic assay for ethylene glycol. The method involves use of a standard kit for ethanol determination with yeast alcohol dehydrogenase, and of a centrifugal analyzer. Alcohol dehydrogenase catalyzes the oxidation of ethylene glycol in a trichloroacetic acid-precipitated specimen; the resulting NADH production is monitored at 340 nm. The reaction rate is linear with concentration to 1.5 g of ethylene glycol per liter. Interference from methanol, ethanol, and isopropanol was easily recognized after a 30-min incubation at 100 degrees C. We believe that the method can be readily adaptable to any narrow-bandwidth, stable, temperature-controlled spectrophotometer and so should provide more widely for the prompt assessment of patients in whom ethylene glycol poisoning is suspected.
Commercially available blood-gas quality controls compared with tonometered blood.
Authors: Leary E T , Graham G , Kenny M A
Published: January 2020

We compared three commercially supplied blood-gas quality control solutions ("contrIL," "G.A.S.," and "Quantra") and tonometered whole blood under research and service conditions in four hospital laboratories. In some situations Quantra and tonometered blood were comparably sensitive to problems with blood-gas instrument malfunctions (e.g., hydraulics or temperature control) but the aqueous buffer-based materials were less sensitive. Within-day precision for all control materials was similar. The precision of pCO2 determinations in long-term studies was also similar for all control materials, as were pH determinations on commercial controls. For pO2 (especially at O2 < 50 mmHg), sample handling technique influenced the precision and accuracy of Quantra more than the other materials. The variability in pO2 determination with Quantra seems primarily to result from sensitivity in incubation temperature (3% change per degree C at 145 mmHg and 7% change per degree C at 43 mmHg). This study identifies some of the limitations of blood-gas control materials per se and some limitations related to specific blood-gas instruments that affect interpretation of results from them.
Reappraisal of the uroporphyrinogen I synthase assay, and a proposed modified method.
Authors: Chamberlain B R , Buttery J E
Published: January 2020

Problems encountered in the uroporphyrinogen I synthase assay were found to be due to the Triton reagent, slit-width and excitation wavelength settings of the spectrophotofluorometer, and leg-phase in the enzyme reaction. Of Triton X-100 from four suppliers, only three were found suitable. Differences in enzyme activity owing to slit-width adjustments were resolved by measuring the fluorescence of the sample (uroporphyrin) at 405 nm and the standard (coproporphyrin) at 401 nm. The lag-phase in the activated-enzyme assay is incorporated in the 30-min preincubation of the blood specimen in our modified method, thus giving higher enzyme activity than found with the original assay. The range for our method is 7.3--15.8 nmol s-1 L-1 (mean = 10.9; SD = 2.07).
Liquid-chromatographic measurement of urinary monosaccharides.
Authors: D'Amboise M , Hanai T , Noël D
Published: January 2020

We describe "high-performance" liquid chromatography of urinary monosaccharides on a macroporous anion-exchange resin (Hitachi 3013N), with acetonitrile/water as eluent. The separated xylose, arabinose, D-fructose, mannose, galactose, and D-glucose were detected by means of a post-column reduction reaction of tetrazolium blue. Absolute limits of detection were about 10 ng of monosaccharide. Lactose can also be measured.
Effect of in vitro hemolysis on assay of plasma catecholamines and DOPA.
Authors: Smith R T
Published: January 2020

I analyzed for free and sulfate-conjugated catecholamines and DOPA (3,4-dihydroxyphenylalanine) in four aliquots of a single plasma pool with use of Upjohn's "Cat-A-Kit" (catecholamines radioenzymatic assay kit [3H]. One aliquot was a clear plasma control; the others were supplemented with different concentrations of lysed human erythrocytes, to investigate possible interference by hemolysis with analysis for norepinephrine, epinephrine, dopamine, or DOPA in plasma. I observed no statistically significant difference that could be attributed to the degree of hemolysis between control and treatment groups for DOPA or any catecholamine fraction. Hemolysis in plasma from improper collection or processing techniques apparently does not preclude accurate and precise quantitation of free or sulfate-conjugated catecholamines or DOPA with the Cat-A-Kit.
Simplified method for detecting anti-insulin antibodies and insulin-anti-insulin immune complexes.
Authors: Virella G , Russell D , Laimins M , Colwell J
Published: January 2020

A simplified approach for estimating free and total anti-insulin antibodies and soluble insulin-anti-insulin immune complexes in serum has been developed and evaluated. For determination of free anti-insulin antibodies, a binding ratio for 125I-labeled insulin at fivefold serum dilution is calculated; the binding ratios obtained are reproducible (run-to-run coefficient of variation, 7.3%) and correlate well with titration of anti-insulin antibodies by other techniques (correlation coefficient, 0.9327). Total anti-insulin antibody concentrations are determined by calculating the [125I]insulin binding ratio for a sample previously acidified, adsorbed with acid dextran-coated charcoal, and neutralized. The difference in binding ratios between two aliquots of the same serum, one diluted 10-fold without manipulation and the second studied at an identical dilution after acidification and removal of free insulin, is taken as an index of the presence of soluble insulin-anti-insulin immune complexes. Because the tests can be performed at a single dilution, both time and materials are conserved without apparent loss of discrimination between sera with high antibody titers and high concentrations of insulin-anti-insulin immune complexes and sera with negative titers or low concentrations.
Detection of pentazocine and tripelennamine in urine.
Authors: Reid R W , Gerbeck C M
Published: January 2020

Pentazocine and tripelennamine can be identified in urine as a part of routine screening for drug abuse. Pentazocine, pentazocine hydrate, and tripelennamine can be detected by thin-layer chromatography of chloroform/isopropanol extracts of heat- and acid-hydrolyzed urine. Gas chromatography is used for confirmation. Gas chromatographic/mass spectrometric studies (chemical ionization mode) show that such extracts contain major molecular (M+) ions of m/e 256, 286, and 304, indicating the presence of tripelennamine, pentazocine, and pentazocine hydrate, respectively.
4-nitrophenyl phosphate--characterization of high-purity materials for measuring alkaline phosphatase activity in human serum.
Authors: Bowers G N , McComb R B , Upretti A
Published: January 2020

We studied 53 lots of 4-nitrophenyl phosphate (I), obtained from 20 different commercial suppliers, and used this information to set specifications for it. Using these well-defined specifications, we classified 21 lots of I as "unacceptable," 26 lots as "borderline," and six as "acceptable." All lots were shown to contain some 4-nitrophenol and inorganic phosphate. However, "acceptable" I had < 0.3 mmol of 4-nitrophenol and < 10 mmol of inorganic phosphate per mole of I. The mole concentration of I (based on disodium hexahydrate, formula weight 371) was determined by enzymic conversion to 4-nitrophenol in five lots of "acceptable" materials. The mole fraction of I ranged from 0.982 to 0.998. From these measurements and from estimates of impurities that absorb at 311 nm, as determined by liquid chromatography and spectrophotometry at other wavelengths, our best estimate of the molar absorptivity of I at 311 nm in 10 mmol/L NaOH at 25 degrees C was 9867 L x mol-1 x cm-1, with a total uncertainty of 76 L x mol-1 x cm-1. We recommend that I used in clinical laboratories for measurement of alkaline phosphatase activity in serum meet the specifications given in this paper: I content > 98%, maximum activity > 98% in comparative testing with other "acceptable" lots of I, and impurities not to exceed the values cited above.
Effect of outlying standard points on curve fitting in radioimmunoassay.
Authors: Hawker F J , Challand G S
Published: January 2020

Outlying points often appear in standard curves for radioimmunoassay. We have examined the effect of outlying standard points on the ability of various automated data-processing routines, and of manual operators, to position a radioimmunoassay standard curve correctly. Manual operators were found to be highly subjective in their handling of a standard curve containing outlying points. Automated methods without outlier rejection capability produced standard curves that were significantly erroneous. In contrast, a data-processing method with automated outlier rejection capability successfully identified outliers, but occasionally rejected valid points--and consequently misplaced the standard curve. Visual identification of outliers is unsatisfactory. Automated identification can be more satisfactory, but some patterns of outliers make it less so. We conclude that any automated data-processing method should contain an outlier rejection facility, but its results should be treated with caution.
Evaluation of a new valproic acid enzyme immunoassay and comparison with a capillary gas-chromatographic method.
Authors: Braun S L , Tausch A , Vogt W , Jacob K , Knedel M
Published: January 2020

A new homogeneous immunoassay (EMIT) for valproic acid was evaluated. Besides testing the manual version of this enzyme immunoassay, we also developed two mechanized procedures for centrifugal analyzers (the CentrifiChem and the COBAS system), which take less time and are more precise than the manual method. Within-assay precision (CV) was 4.5% with the manual technique and 2% with the analyzers. Between-assay precision (CV) ranged from 4 to 13% for all three techniques. Accuracy of th manual method was checked by dilution and analytical recovery experiments. Our comparison of the EMIT results with those obtained by a comparison method (capillary gas chromatography) showed no significant difference. No interference from hemolysis, hyperbilirubinemia, or aliphatic amino acids was observed. At high concentrations of bile acids and with lipemic sera the analytical recovery rates decreased slightly, to 87% and 92%, respectively.
Liquid chromatography at 3-methoxy-4-hydroxyphenylethylene glycol in urine with fluorescence detection.
Authors: Taylor J T , Freeman S , Brewer P
Published: January 2020

We describe a liquid-chromatographic procedure for determining low concentrations of 3-methoxy-4-hydroxyphenylethylene glycol in urine. After liquid-liquid extraction and cleanup of the sample, an aliquot of the extract is injected onto a reversed-phase chromatographic column, with isocratic elution with an acetonitrile/acetate buffer mobile phase. An on-line fluorescence detector is used for measuring the natural fluorescence of the compound. The method gives a relatively clean extract, which is suitable for measuring the less-than-normal concentrations associated with mentally depressed patients. Although a liquid-chromatographic technique has been recently described for measuring this compound in urine, the method involves electrochemical detection, which may not be available in many clinical laboratories, and, because of its nonspecificity, the method is only suitable for detecting the increased amounts of 3-methoxy-4-hydroxyphenylethylene glycol excreted by patients with pheochromocytoma.
A cellulose acetate electrophoretic procedure evaluated for quantitation of high-density lipoprotein cholesterol.
Authors: Schifman R B , Brumbaugh P F , Grover D , Finley P R , Harrow E J
Published: January 2020

We evaluated the performance of a commercially available cellulose acetate electrophoretic method for quantitating high-density lipoprotein cholesterol (I) in serum by comparing it to a method involving precipitation with dextran sulfate-500/Mg2+. In both methods, enzymic reagents are used for cholesterol measurement. For electrophoretic measurement of I the mean intramembrane CV ws 4.1% (at 220 to 360 mg/L) and the intermembrane CV ranged from 12.2 to 21.0% (at 220 to 880 mg/L). Interassay precision was significantly better for the precipitation method (CV = 3.9% at 390 mg/L). The electrophoretic procedure demonstrated significant measurement bias, both at high and low I concentrations. However, low-density lipoprotein cholesterol, measured electrophoretically correlated well with its calculated concentrations obtained by the precipitation method. Measurement of I by this electrophoretic procedure did not achieve the accuracy and reproducibility that have been demonstrated for precipitation methods and that are necessary for reliable clinical interpretation of results for I.
Homogeneous substrate-labeled fluorescent immunoassay for theophylline in serum.
Authors: Li T M , Benovic J L , Buckler R T , Burd J F
Published: January 2020

A substrate-labeled fluorescent immunoassay for theophylline in serum is described. 8-(3-Aminopropyl)-theophylline is covalently attached to a fluorogenic enzyme substrate, 7-beta-galactosylcoumarin-3-carboxylic acid. Hydrolysis of this theophylline-labeled substrate by beta-galactosidase yields a fluorescent product. When antibody to theophylline interacts with this substrate, the resulting complex is inactive as an enzyme substrate. For measuring theophylline, competitive protein-binding reactions are set up, with the theophylline in the sample competing with the substrate for the antibody-binding sites. The substrate not bound to antibody is hydrolyzed by beta-galactosidase, producing fluorescence that is proportional to the theophylline concentration. Results for theophylline determined by this method in clinical samples of serum correlated well (r > 0.96) with results obtained by gas-chromatographic or enzyme immunoassay procedures. The within-run CV for three control samples ranged from 1.1 to 2.8%, the between-run CB from 2.3 to 4.5%.
Double-antibody radioimmunoassay of thyroid microsomal antibody in serum.
Authors: Kung V T , Weber P M , dos Remedios L V
Published: January 2020

We describe a double-antibody radioimmunoassay for antimicrosomal antibodies in serum. Antigen in centrifuged pellets of microsome is solubilized by treatment with sodium deoxycholate solution and radiolabeled (125I] by the Chloramine T method. A 10-microL aliquot of human serum or antimicrosomal antibody standard is incubated overnight at room temperature with 125I-labeled microsome. Bound radiolabeled microsome is separated by precipitation with goat antihuman IgG antiserum. The percentage of 125I-labeled microsome bound by each serum is calculated, and its concentration of antimicrosomal antibody interpolated from a standard curve. The prevalence of abnormal antimicrosomal antibody was 16% in a self-referred asymptomatic or not acutely ill group of patients having a periodic multiphasic health examination. Values for patients with certain thyroid diseases generally agreed with other published data. We conclude that our assay is sensitive, precise, and accurate, and is more efficient for routine measurement of antimicrosomal antibody than the currently used solid-phase radioimmunoassay.
Lectins used to prepare serum free of glycoprotein hormones for use as a matrix in radioimmunoassay.
Authors: Stuart M C , Ellis S , Gowlland L , Tuff S
Published: January 2020

Hormone-free serum is required for use as a matrix for standards in the radioimmunoassay of hormones. The glycoprotein hormones are difficult to remove from serum by conventional techniques. We exploited the specific binding of carbohydrates by lectins to extract glycoprotein hormones from human serum. Passing serum over Concanavalin A Sepharose efficiently removed lutropin and the beta subunit of choriogonadotropin. Wheat-Germ Lectin Sepharose completely removed these, and also follitropin and thyrotropin. The latter treatment is shown to provide a suitable matrix for standards in radioimmunoassays of the four hormones.
Acetylcholinesterase and fetal malformations: modified qualitative technique for diagnosis of neural tube defects.
Authors: Haddow J E , Morin M E , Holman M S , Miller W A
Published: January 2020

A single-blind study involving amniotic-fluid samples from 214 pregnancies of known outcome confirms that an electrophoretically distinct isoenzyme of acetylcholinesterase is associated with fetal open neural tube defects. Furthermore, only one of 13 amniotic-fluid samples with false-positive results for alpha-fetoprotein showed the characteristic isoenzyme, indicating that qualitative acetylcholinesterase assessment can decrease the proportion of false positives from the alpha-fetoprotein assay. We have also identified this characteristic isoenzyme in amniotic fluids from pregnancies in which other serious fetal defects occurred. A detailed electrophoresis protocol for identifying this characteristic isoenzyme is described.
Sources of variation in the column-chromatographic determination of glycohemoglobin (HbA1).
Authors: Schellekens A P , Sanders G T , Thornton W , van Groenestein T
Published: January 2020

We studied the influence of many variables on the cation-exchange chromatographic determination of glycohemoglobin was minicolumns (HbA1) and with macrocolumns (HbA1a+b and HbA1c). Minicolumns are most sensitive to buffer composition. The best conditions are pH 6.78 and sodium, potassium, and phosphate concentrations of 54.8, and 36 mmol/L, respectively. In fact, there is no clear cut-off point for elution. A cyanide concentration of 1.5 mmol/L has no negative effects. For macrocolumns, the influence of temperature is demonstrated; 20 degrees C is optimal. A higher temperature caused results for HbA1 to be higher. For minicolumns, the temperature effect corresponds to 1% HbA1 per 1 degrees C, necessitating rigorous temperature control. The amount of sample applied also influences the results. Therefore a constant column load, chosen between 1.3 and 2.0 mg of Hb permilliliter of resin, is advised. Heparinized blood can be kept for three days at 4 degrees C; hemolyzed samples are best stored at -70 degrees C.
(omega-1)-Hydroxymonocarboxylic acids in urine of infants fed medium-chain triglycerides.
Authors: Shigematsu Y , Momoi T , Sudo M , Suzuki Y
Published: January 2020

Organic acids in the urine of infants fed a formula containing medium-chain triglycerides (octanoic acid 49% and decanoic acid 26% of total fatty acids) were determined by gas chromatography-mass spectrometry. We found a considerable amount of 5-hydroxyhexanoic acid and 7-hydroxyoctanoic acid, but no detectable 9-hydroxydecanoic acid, as well as a large amount of C6-C10-dicarboxylic acid. We believe that such acids are derived, via (omega-1)-hydroxylation, from medium-chain monocarboxylic acid or medium-chain acyl-CoA in the liver cell. The degree of (omega-1)-hydroxylation of medium-chain fatty acids relative to omega-hydroxylation apparently increases as the chain length of the fatty acids decreases.
Increased inactive creatine kinase B protein in the plasma of patients with malignancy.
Authors: Burnam M H , Barstis J L , Stern C S , Rose D M , Crouch M A , Haskell C M , Singh B N
Published: January 2020

Using a specific radioimmunoassay for enzymically inactive creatine kinase (EC 2.7.3.2) B protein (CK-Bi), we studied 139 patients with various malignancies. We found it to be increased in certain adenocarcinomas, including colorectal, lung, and prostate, as has also been reported for enzymically active CK-BB. Of 15 patients with leukemia or lymphomas with active disease, 12 also had increased values for CK-Bi in plasma. In these patients, concentrations of CK-Bi in plasma tended to correlate with disease activity, increasing with aggressive malignancy and decreasing on successful treatment. Thus, measurement of CK-Bi may constitute a sensitive means for detecting malignancy and, in certain subsets of patients, for monitoring therapeutic response.
Factors affecting enzyme-linked immunosorbent assay (ELISA) for insulin antibodies in serum.
Authors: Flynn S D , Keren D F , Torretti B , Dieterle R C , Grauds S
Published: January 2020

We have evaluated the factors affecting an enzyme-linked immunosorbent assay (ELISA) for circulating insulin antibodies. Dilutions of patients' sera were incubated in polystyrene tubes coated with procine insulin. The second incubation was with alkaline phosphatase-conjugated anti-human Fab. Each of these reactions was complete after 4 h. Specificity of the reaction for insulin antibodies was demonstrated by removal of anti-insulin activity after preincubation with insulin, but not with glucagon at similar concentrations. Sensitivity of the ELISA system, assessed by performing the reaction with affinity-column purified insulin antibodies, was 10 microgram of specific antibody per liter. Using this system, we examined sera from 22 patients who had been determined by radioimmunoassay to have insulin antibodies, and sera from 23 normal individuals. The ELISA results correlated reasonably well with those of RIA (r = +0.84). Besides detecting insulin antibodies in diabetic patients who are being treated with insulin, we use this ELISA test as a screening procedure to be certain insulin antibodies are not present when we use indirect immunofluorescence methods of fixed pancreatic substrate to detect islet-cell antibodies.
Measurement of "lamellar body phospholipid" in amniotic fluid as a method for assessing fetal lung maturity.
Authors: Duck-Chong C G , Henderson-Smart D J , Gupta J M , Hensley W J
Published: January 2020

A simple, rapid micro-method, suitable for use in a routine clinical laboratory, is described for isolating a surfactant fraction from 0.1 mL of human amniotic fluid and measuring its phospholipid content. We determined the phospholipid content of this fraction, referred to as "lamellar body phospholipid," in 451 samples of amniotic fluid collected within two days of delivery and related the data to the respiratory performance of the newborn in every case; 112 of the infants were delivered at 28-37 weeks gestation. The incidence of hyaline membrane disease was inversely related to the concentration of lamellar body phospholipid in the amniotic fluid. Eleven of 12 infants with lamellar body phospholipid values less than 25 mg/L and four of 44 infants with lamellar phospholipid values between 25 and 50 mg/L developed hyaline membrane disease or other serious respiratory problems possibly related to lung immaturity, whereas all of 395 infants with lamellar body phospholipid values of 50 mg/L or more were free from respiratory problems of this nature. The incidence of transient tachypnea was greatest when the lamellar body phospholipid value was between 25 and 50 mg/L, suggesting that this condition may be related to a degree of lung maturity.
A model for validation of radioimmunoassay kit reagents: measurement of follitropin and lutropin in blood and urine.
Authors: Santner S J , Santen R J , Kulin H E , Demers L M
Published: January 2020

We measured lutropin and follitropin in blood and urine with radioimmunoassay kits from Diagnostic Products Corporation and compared the results with those obtained by use of reagents from the National Institutes of Health (NIH) and the World Health Organization (WHO). The urine standard (second IRP-HMG) from WHO, the blood standard (LER-907) from NIH, and the commercial standards all effected similar displacement of trace material when the commercial gonadotropin kit reagents were used. Highly significant correlations were achieved for these hormones in blood or urine on comparing commercial and NIH/WHO reagents. Serial dilutions of urine samples produced similar relative potencies with the commercial reagents. Conversion factors are presented to relate results for LER-907, second IRP, or commercial standards. Commercially available reagents can provide a practical and reliable means of gonadotropin radioimmunoassay in blood or urine.
Liquid-chromatographic determination of guanidino compounds in plasma and erythrocyte of normal persons and uremic patients.
Authors: Kikuchi T , Orita Y , Ando A , Mikami H , Fujii M , Okada A , Abe H
Published: January 2020

We describe a method for determination of guanidino compounds in plasma and erythrocytes. The compounds are separated by liquid chromatography, the concentrations in the effluent being estimated fluorometrically (Trans. Am. Soc. Artif. Intern. Organs 24: 61, 1978). Guanidinoacetic acid, guanidinosuccinic acid, guanidinobutyric acid, guanidine, methylguanidine, taurocyamine, and arginine can be detected and quantitated in both plasma and erythrocytes from healthy individuals. The method was also applied to patients in chronic renal failure. Guanidinosuccinic acid, creatinine, guanidine, and methylguanidine are substantially increased in the plasma and erythrocytes of the uremic patient. Guanidino compounds can be effectively extracted from plasma with 0.5 volume of 300 g/L trichloroacetic acid, from erythrocytes with five volumes of 120 g/L trichloroacetic acid.
Internal sample attenuator counting (ISAC). A new technique for separating and measuring bound and free activity in radioimmunoassays.
Authors: Thorell J I
Published: January 2020

I describe a new method for the separation and counting of bound and free activity in radioimmunoassays. Particles containing a radiation-absorbing (attenuating) material are added to the assay. They shield the radiation from either the antibody-bound or the free radioligand. This obviates such manipulations conventionally involved in the separation and counting steps of radioimmunoassays as centrifugation and decanting. Bismuth oxide is used as the attenuator. Particles with different properties are described. In one type, bismuth oxide is combined with active charcoal in an agarose matrix and serves as an adsorbant for the free radioligand. In another type bismuth oxide is trapped within a polyacrylamide matrix to which antibodies are coupled. This particle can be used with a first- or a second-antibody bound activity. Application of the technique is illustrated with radioimmunoassays for thyroxin, triiodothyronine, human choriogonadotropin, and lutropin (luteinizing hormone).
One-minute electrochemical enzymic assay for cholesterol in biological materials.
Authors: Clark L C , Duggan C A , Grooms T A , Hart L M , Moore M E
Published: January 2020

In this rapid and specific micro-scale electrochemical enzymic assay for cholesterol and cholesterol esters, 10 microL of standard or sample is injected directly into a heated (50 degrees C) thermostated, oxystated cuvet containing pH 7.25 buffer, cholesterol oxidase (EC 1.1.3.6), and cholesterol esterase (EC 3.1.1.13). The cholesterol esters are hydrolyzed by the esterase, and the cholesterol is simultaneously oxidized by the oxidase. The hydrogen peroxide produced from oxidation of the unesterified cholesterol is measured by a polarographic anode covered with an acetate/polycarbonate membrane. The membrane allows hydrogen peroxide to diffuse to the platinum anode, where it is oxidized, but prevents the diffusion of ascorbic acid, uric acid, and bilirubin to the electroactive surface. Turbidity does not interfere. The correlation (r) between results by our method and the Abell-Kendall method for 105 samples of serum was 0.9994 and for 105 samples of plasma was 0.9997. Our method is convenient for the analysis of high-density lipoprotein cholesterol in plasma and serum supernates and in many kinds of tissue homogenates. Its limitations are also described.
Variability of total cholesterol concentrations in serum by repeated measurements in a large pediatric population--limitations of quality controls for laboratory analyses.
Authors: Sklov M C , Srinivasan S R , Webber L S , Berenson G S
Published: January 2020

We evaluated the variability in total cholesterol concentrations in serum in a large population of children over a period of time, to help us discern the limitations in reliability of current clinical laboratory methods for its analysis. We analyzed sera from a population of approximately 4000 children over a four-year period, quality-control sera, pooled sera, and surveillance samples from the Centers for Disease Control over a seven-year period, with a Technicon AutoAnalyzer II. Two methods of correction were suggested to adjust deviations in yearly serum total cholesterol means: yearly screening data were corrected on the basis of monthly variability in pooled-serum determinations and on the basis of deviations from values suggested by the Centers for Disease Control. Both correction methods were insufficient. These changes occur very slowly, and unless there is close monitoring and frequent comparison with a known standard, changes in laboratory results still can occur.
Effect of variations in globulin concentrations on serum radioimmunoassay results, as exemplified by choriogonadotropin.
Authors: Jawad M J , Wilson E A
Published: January 2020

Variations in serum protein concentrations can affect results of radioimmunoassay of human choriogonadotropin involving either polyethylene glycol or double-antibody methods of separation. Relative to the maximal binding of choriogonadotropin in the presence of normal (30 g/L) globulin concentrations, globulin concentrations of 10 to 60 g/L were associated with maximal binding ranging from 84.9 to 115.6% in the polyethylene glycol system, and from 109.9 to 92.5% in the double-antibody system. This effect was quantitatively much the same throughout the standard curve in the polyethylene glycol system, but diminished with the higher standard concentrations in the double-antibody system. In assays involving polyethylene glycol separation, an increase in globulin was associated with a linear increase in nonspecific binding, but no detectable effect of globulin on nonspecific binding was observed in the double-antibody system. Determination and compensation for nonspecific binding of individual samples (rather than for mean assay nonspecific binding) eliminated the globulin effect in the polyethylene glycol system but not in the double-antibody system.
Convenient radioimmunoassay for urinary human choriogonadotropin without interference by urinary human lutropin.
Authors: Wehmann R E , Harman S M , Birken S , Canfield R E , Nisula B C
Published: January 2020

We have devised a radioimmunoassay (RIA) for human choriogonadotropin (hCG) in first morning-voided urine specimens. Concanavalin A, a lectin, is used to extract and concentrate the hCG from urine. A high-affinity antiserum is used, directed to the hCG beta carboxy-terminal peptide, a unique immunological determinant not shared by the beta subunit of human lutropin. This ensures that urinary human lutropin-related molecules, which interfere with RIAs involving antisera to the intact hCG beta subunit, will not cross react in this assay. A concentrations of hCG as low as 0.4 microgram/L can be detected in the first morning-voided urine. The effective sensitivity of this assay for the unequivocal detection of hCG production is somewhat better than that achieved with the serum hCG RIA involving antisera to the hCG beta subunit. The improved specificity and sensitivity of this assay, and the greater convenience of collecting samples of urine rather than blood, are clinical useful advantages of this approach to assessing hCG production in humans.