bluuebunny/crossref_metadata_2025 huggingface dataset:
Results with six "kit" radioimmunoassays for primary bile acids in human serum intercompared.
Authors: Roda A , Roda E , Aldini R , Capelli M , Festi D , Sama C , Mazzella G , Morselli A M , Barbara L
Published: January 2020

We examined six radioimmunoassay procedures for measuring primary bile acids in human serum (two 3H-labeled and four 125I-labeled). A significant (p < 0.01) correlation was observed between measurements in the assay both for cholic acid and chenodeoxycholic acid, at low and high concentrations of serum bile acids. All kits were acceptable with respect to accuracy, precision, stability, and analytical recovery. All six procedures gave similar results for chenodeoxycholic and cholic acid in sera of 80 healthy subjects; the agreement was also close when the two primary bile acids were compared with their sum in serum. Normal values ranged from 0.4 to 2.5 mumol/L for conjugated chenodeoxycholic acid and from 0.3 to 1.5 mumol/L for conjugated cholic acid. The 125I assays do not require liquid-scintillation equipment but 125I induces a decrease in the affinity constant of antibody. The sensitivity of the assays was still adequate for measuring bile acids in the serum of healthy fasting persons and liver-disease patients.
Simultaneous liquid chromatography of 5-fluorouracil, uridine, hypoxanthine, xanthine, uric acid, allopurinol, and oxipurinol in plasma.
Authors: Wung W E , Howell S B
Published: January 2020

A reversed-phase "high-pressure" liquid-chromatographic method is described for simultaneous analysis for 5-fluorouracil, uridine, hypoxanthine, xanthine, uric acid, allopurinol, and oxipurinol. Separation was optimal with phosphate buffer (50 mmol/L, pH 4.60) as eluent. A simple acid extraction procedure yielded quantitative recoveries and permitted adequate separation for interfering peaks. Compounds were identified by their retention times, absorbance ratios, co-elution with standards, and enzymatic shifts. With a computerized integrator we quantitated these compounds in widely varying concentrations with a single injection. The limit of sensitivity was 0.1 mumol/L for the compounds studied. This method was applied to determine mean values for those compounds in normal human plasma. They are (in mumol/L): uric acid 276 (SD 55), hypoxanthine 0.46 (SD 0.21), xanthine 0.40 (SD 0.27), and uridine 4.50 (SD 1.70). Erythrocytes and platelets can continue to release hypoxanthine and xanthine into plasma or serum after a blood specimen has been drawn. We believe this explains the higher values previously reported for hypoxanthine and xanthine in serum.
Binding of enzyme--IgG complexes in human serum to Protein-A Sepharose CL-4B.
Authors: Bauer K , Bayer P M , Deutsch E , Gabl F
Published: January 2020

We describe a simple method for detecting enzyme--immunoglobulin G (IgG) complexes in human serum. Protein-A Sepharose CL-4B binds IgG and therefore also the enzyme--IgG complexes, which can then be separated easily from the serum by centrifugation. We demonstrate this separation in two patients, one with a complex of IgG and creatine kinase (EC 2.7.3.2) BB isoenzyme, the other with an IgG--alkaline phosphatase (EC 3.1.3.1) complex. Both patients had unexplainably high activities of the respective enzymes in their serum. The method we propose should be a useful, simple, routine method of detection in cases where IgG--enzyme complexes are suspected.
Two agarose electrophoretic systems for demonstration of oligoclonal bands in cerebrospinal fluid compared.
Authors: Gerson B , Krolikowski F J , Gerson I M
Published: January 2020

Demonstration of oligoclonal bands by electrophoresis of cerebrospinal fluid is an important aid in establishing the diagnosis of multiple sclerosis. Electrophoretic systems vary in their effectiveness in doing so. We compared two systems in this respect. For a thin-layer agarose system, sensitivity was less (47%) than for a high-resolution agarose system (87%). Each system had good specificity (92 and 85%, respectively). Interpretation of electrophoretic patterns for cerebrospinal fluid should be available in clinical laboratories. Further, the best available system should be used for demonstration of oligoclonal bands.
Mannitol estimation in biological fluids by gas-liquid chromatography of trimethylsilyl derivatives.
Authors: Laker M F , Mount J N
Published: January 2020

We describe a procedure for estimating mannitol concentrations in biological fluids. Samples are mixed with internal standard solution (alpha-methylglucose), deproteinized if necessary, desalted, and dried. Specimens are then derivatized by adding pyridine/bis(trimethylsilyl)acetamide/trimethylchlorosilane and heating at 60 degrees C for 30 min. Samples are chromatographed on a 275-cm column of 10% OV-17, operated at 190 degrees C, and quantitated by peak-height measurement. The technique is linear, accurate, precise, sensitive, and free from interference. It has been used to measure mannitol in plasma, urine, and bile.
Accuracy, precision, and stability in measurement of hemoglobin A1C by "high-performance" cation-exchange chromatography.
Authors: Schifreen R S , Hickingbotham J M , Bowers G N
Published: January 2020

We present a "high-performance" liquid-chromatographic method for determination of HbA1c with use of a carboxylate cation-exchange column, ethanolic mobile phases, and a hemolysate-storage reagent that stabilizes a sample for up to five weeks at 4 degrees C. Temperature control and the addition of ethanol to the phosphate step-gradient increase precision by stabilizing peak shape and column activity. Lengthy equilibration of the column between samples is not necessary, because the pKa of the cation-exchange resin is decreased from 6.10 in aqueous media to 5.59 in ethanol/water (5/95 by vol) at 25 degrees C. We identified interfering heme compounds, distinct from HbA1d or HbA1e, that elute with the glycosylated hemoglobins and developed a method of correcting for their presence. The day-to-day coefficient of variation was 3.5%, and the reference interval for a mixed group of 20 healthy adults between the ages of 20 and 56 was 3.2—5.2% HbA1c.
Conditions affecting the colorimetry of orotic acid and orotidine in urine.
Authors: Harris M L , Oberholzer V G
Published: January 2020

We studied conditions affecting a colorimetric assay of total orotic acid (orotic acid plus orotidine) in urine. Most interfering substances can be conveniently removed on a small column of cation-exchange resin, and an improved control reaction corrects for residual background color. Analytical recovery from urine is nearly complete (greater than 95%) and the absorption spectrum for analyte eluted from the column closely resembles that for an orotic acid standard. We determined reference intervals for total orotic acid, expressed as a molar ratio to creatinine, for neonates, children, and adults, and assessed the effect of age, protein intake, and pregnancy. The method is simple enough to use as a reliable and accurate urine-screening test.
Ion effects in measurement of ionized calcium with a calcium-selective electrode.
Authors: Kaufman R A , Tietz N W
Published: January 2020

We examined the performance characteristics of an improved electrode for measurement of ionized calcium (Orion Research, Inc.) with respect to interferences by Na+, K+, Li+, Mg2+, and H+, as well as the effect of changes in ionic strength and osmolality. At physiologically observed concentrations, all ions except Na+ insignificantly affected ionized-calcium measurements. The decrease in ionized Ca2+ with increased Na+ concentration is caused by the increase in ionic strength and the associated decrease in Ca2+ activity. Sodium had no significant direct effect in concentrations below 250 mmol/L. A previously published (Clin. Chem. 23: 690, 1977) equation for the Na+ correction is invalid for this improved electrode. Changes in osmotic pressure negligibly affect electrode response. Significant changes in ionic strength, as would be observed in severe hypo- and hypernatremic sera, increase or decrease, respectively, values for ionized calcium by changing Ca2+ activity. With aqueous Ca2+ solutions, changes of less than or equal to 3% were observed for Na+ concentrations that deviate from 140 mmol/L by not more than 20 mmol/L. Because the calcium ion activity is considered to be the physiologically important variable, we suggest that no correlation be made for ionic strength effects.
Case report: changes in creatine kinase isoenzymes after myocardial infarction in a patient with prostatic carcinoma.
Authors: Alexander R L
Published: January 2020

The detection of serum proteins that can serve as tumor markers has been reported with increased frequency in patients with various localized or metastatic cancers. Many of these reports have concerned patients with prostatic carcinoma, who had normal or increased concentrations of creatine kinase (EC 2.7.3.2) in serum. In many instances the creatine kinase BB isoenzyme was increased in these patients. I describe the case of a patient with carcinoma of the prostate (stage D), who had a myocardial infarction three to four days after admission. The serum activity of the MM, MB, and BB isoenzymes changed markedly after the infarction. These changes and the possible tissue source(s) of this isoenzyme are discussed in relationship to the clinical symptoms of the patient.
On-line liquid-chromatographic analysis for drugs in serum with the Technicon "FAST-LC" system: performance data for theophylline and for four commonly used anticonvulsants and their metabolites.
Authors: Dolan J W , van der Wal S , Bannister S J , Snyder L R
Published: January 2020

We describe a new instrument for use in assay of therpeutic drugs in serum by "high-performance" liquid chromatography, the "FAST-LC" system (Technicon). Serum samples are aspirated directly into the unit, extracted with solvent, and the evaporated and redissolved extract is injected onto a chromatographic column. We illustrate the performance of the system by assays in serum for theophylline and four anticonvulsants (primidone, phenobarbital, phenytoin, and carbamazepine) plus two of their active metabolites (phenylethylmalonamide and carbamazepine epoxide). For theophylline, final chromatograms are monitored at 270 nm, at analysis rates of 10/h. Concentration and absorbance are linearly related from 0 to 130 mg of theophylline per liter. For the anticonvulsants, chromatograms are monitored at 200 nm, at analysis rates of 7.5/h. The six individual determinations are each linear beyond the therapeutic range. For both drug panels, day-to-day CV's were 4 to 6%. Results correlate well with those by enzyme immunoassay. A total sample volume of 150 microL is required.
Mechanized amidolytic technique for determination of factor X and factor-X antigen, and its application to patients being treated with oral anticoagulants.
Authors: van Wijk E M , Kahlé L H , ten Cate J W
Published: January 2020

We describe a mechanized chromogenic assay for factor X, the results of which correlate well with those for the one-stage clotting assays for factor X in which it is activated either via the extrinsic pathway by thromboplastin or directly by Russell's viper venom. We purified human factor X and raised monospecific antibodies to it in rabbits. We used our chromogenic assay for factor X to develop a factor-X-inhibitor neutralization assay for determination of factor-X antigen. Patients receiving oral anticoagulant treatment had significantly different factor-X activities after activation via thromboplastin or with Russell's viper venom. The concentration of factor-X antigen, although decreased, significantly exceeded factor-X clotting activity or chromogenic activity in this group of patients. Results of the chromogenic assay for factor X correlated well with results of "Thrombotest," a clotting test introduced by Owren (Lancet ii: 754, 1959) to control anticoagulant therapy. For patients taking oral anticoagulant drugs, the therapeutic range by our assay is 180 to 300 units/L.
Continuous-flow enzymatic determination of total serum cholesterol and method standardization with CDC-calibrated pooled sera.
Authors: MacAulay M A , Jacklyn C L , Mathers J M , Storm V A
Published: January 2020

We compared Boehringer Mannheim's enzymatic kit for the continuous-flow (AutoAnalyzer II) determination of serum cholesterol with Technicon's N-24a extraction method. Results for patients' samples analyzed by the enzymatic method were higher than those by the comparison method. To evaluate accuracy in the cholesterol determinations, we enrolled the enzymatic method into the Center for Disease Control's (CDC) Lipid Standardization program. We calibrated the method by use of a pooled sera for which cholesterol content was assigned by CDC after analysis by their reference Abell-Kendall procedure. We discuss the difficulties with available calibration material and limitations in the application of some commercial control materials to the enzymatic cholesterol method. The continuous-flow variables, Michaelis-Menton constants, percent ester-hydrolase activity, and other factors affecting the performance of the enzyme-linked cholesterol method are evaluated. We believe pooled sera with an assigned value for cholesterol content is the best calibrator material.
A rapid electrothermal atomic absorption spectrophotometric method for cadmium and lead in human whole blood.
Authors: Subramanian K S , Meranger J C
Published: January 2020

A rapid graphite-furnace atomic absorption spectrophotometric procedure is described for determining cadmium and lead in heparinized human whole blood. A known aliquot of the blood sample is diluted fivefold with an aqueous solution composed of 5 g each of diammonium hydrogen phosphate and Triton X-100 per liter, the solution is vigorously agitated, and a 10-microL aliquot is injected into a pyrocoated graphite tube under optimized instrumental conditions. Values for Cd and Pb in the sample are obtained by direct comparison to linear working curves prepared from aqueous standards of the metals in the diammonium hydrogen phosphate-Triton medium; there is no need to use the method of standard addition or matrix-matched calibration curves. Also, the method is free of matrix effects. At least 30 samples can be analyzed per hour. The rapidity, simplicity, and sensitivity of the method make it attractive as a screening technique for routine environmental surveillance involving large throughput of samples.
Bilirubin binding and neonatal acidosis.
Authors: Perlman M , Kapitulnik J , Blondheim S H , Alayoff A , Russell A
Published: January 2020

Plasma of neonates with severe metabolic acidosis secondary to fetal hypoxia bound less bilirubin than that of neonates without acidosis, as determined by Sephadex gel filtration. There was a significant correlation between the amount of bilirubin adsorbed by Sephadex and the base deficit. The method used ruled out any influence of plasma pH per se on binding. Our results suggest that organic anions that accumulate in the plasma of asphyxiated acidotic neonates may compete with bilirubin for binding sites on albumin.
Enzyme immunoassay of disopyramide in serum.
Authors: Pape B E
Published: January 2020

Disopyramide, an antiarrhythmic drug, was quantitated in serum by a commercially supplied enzyme immunoassay procedure. Replicate analyses of serum controls resulted in a within-assay coefficient of variation of less than or equal to 5% and a between-assay coefficient of variation of less than or equal to 7%. Regression analysis of 105 serum samples analyzed by this technique (y) and by gas chromatography (x) gave the equation y = 0.99x + 0.04 (r = 0.97). Clinical evaluation of the results indicates the enzyme immunoassay technique to be highly specific and sensitive for disopyramide. The selectivity of the assay vs other drugs has been determined.
Liquid-chromatographic assay for urinary homovanillic acid, with fluorescent detection.
Authors: Rosano T G , Brown H H , Meola J M
Published: January 2020

We describe a procedure for measuring urinary homovanillic acid by "high-performance" liquid chromatography. The pH of urine samples is adjusted, and they are chromatographed directly on an octyl silica column. The effluent is reacted with a ferricyanide reagent, to convert homovanillic acid to a product that is fluorescent at 420 nm on excitation at 320 nm. The standard curve is linear up to a homovanillic acid concentration of 20 mg/L; the detection limit is 0.3 mg/L. The method is evaluated for precision, recovery, potential interference, and reference values, and compared (r = 0.99) with a published spectrophotometric method. The proposed method is suitable for the routine analysis for homovanillic acid in urine.
gamma-Glutamyltransferase in human serum: an analysis of kinetic models.
Authors: Solberg H E , Theodorsen L , Strømme J H
Published: January 2020

The purpose of the study was to elucidate details of the kinetic model for gamma-glutamyltransferase when assayed with gamma-glutamyl-3-carboxy-4-nitroanilide and glycylglycine as substrates. Data from several sets of initial velocity measurements were fitted by nonlinear regression to a set of different kinetic models. gamma-Glutamyltransferase acts by a "ping-pong bi-bi" mechanism. A model encompassing transfer and autotransfer, competitive inhibition by the acceptor substrate, and no inhibition by the donor substrate gives the best fit to the experimental data. Effects of spectrophotometric nonlinearity may simulate noncompetitive inhibition by the donor substrate. The nonlinearity is dependent on the absorption of the incubation mixture and therefore is related to the concentration of the donor substrate and the wavelength (405-412 nm) used to monitor the reaction. With decreasing pH the autotransfer fraction decreases and the binding of the donor substrate to the acceptor site increases, simulating an increased competitive inhibition by the donor substrate. These results are of importance when elaborating optimum assay conditions for gamma-glutamyltransferase in serum.
Spectrophotofluorometry of serotonin in blood platelets.
Authors: Vatassery G T , Sheridan M A , Krezowski A M
Published: January 2020

A rapid sensitive, and reproducible assay for determination of platelet serotonin is reported. Serotonin in extracted into an ascorbic acid solution by freezing and thawing and sonication. Ascorbic acid stabilizes the serotonin in the extract, and added ethanol enhances the final fluorescence of serotonin, which is measured in a concentrated hydrochloric acid medium (activation at 295 nm and emission at 540 nm). The method has an average coefficient of variation of 4.1%; 95% of added standards is accounted for. The average serotonin content of platelets from 15 men, ages 30-65 years, was 0.67 (SE 0.03) microgram/10(9) platelets, which compares favorably with previously reported values. 5-Hydroxyindole compounds such as 5-hydroxyindole-3-acetic acid and 5-hydroxytryptophan interfere with the assay, but there is relatively little of these compounds in platelets.
Rate-nephelometric inhibition immunoassay of phenytoin and phenobarbital.
Authors: Finley P R , Dye J A , Williams R J , Lichti D A
Published: January 2020

A proposed rate-nephelometric inhibition immunoassay of phenytoin and phenobarbital in human serum involves the sequential addition to buffer of 42-microL aliquots of sample containing the hapten (drug) of interest, a hapten conjugate (drug-equine apoferritin), and specific antibody to the hapten. The drug and the drug conjugate compete for the binding sites on the antibody. The free hapten-antibody complex is soluble and so does not scatter light, whereas the complex of antibody and drug conjugate is insoluble and thus scatters light. The latter immunoprecipitation is competitively inhibited by free hapten. Thus the higher the concentration of free hapten present, the fewer immunoprecipitin complexes are formed, and the less light scatter. Precision, accuracy, linearity, analytical recovery, and comparison with patients' samples assayed with the DuPont aca were excellent. There was no significant interference from hemolysis, icterus, or lipemia. Many potentially interfering drugs and metabolites were checked for cross reactivity, with negative results. Reaction times range from 30 to 50 s.
Kinetic potentiometric determination of creatinine in serum with a picrate ion-selective membrane electrode.
Authors: Diamandis E P , Hadjiioannou T P
Published: January 2020

We describe a new kinetic method for potentiometric determination of creatinine in serum, based on the creatinine--picrate reaction in alkaline medium (Jaffé reaction). The reaction is monitored with a picrate-selective electrode, and the increase in electrode potential during 270 s is measured and related directly to the creatinine concentration. Small cation-exchange columns are used to separate creatinine from interfering substances. Analytical recovery of creatinine added to serum was 100.7%. Results for a series of samples compared well with results obtained with a spectrophotometric method (r = 0.994).
Kinetic latex agglutinometry II: a rapid, quantitative assay for factor VIII antigen.
Authors: Crane L J
Published: January 2020

A rapid method for measuring factor VIII antigen in plasma is presented, based upon optical measurement of the rate of agglutination of a stirred suspension of latex particles coated with anti-factor VIII immunoglobulin, when mixed with plasma containing factor VIII antigen. A log-log relationship exists between factor VIII concentration and the reciprocal of the time required for a fixed absorbance change. The assay is rapid, requiring less than 3 min for normal plasma, involves stable reagents, and can be performed with conventional instrumentation. Antigen values in normal plasma a well as plasma from patients with von Willebrand's disease (angiohemophilia) correlate well with values measured by electroimmunoassay or by radioimmunoassay.
Continuous-flow enzyme immunoassay for thyroxine in serum.
Authors: Nolan J P , DiBenedetto G , Tarsa N J
Published: January 2020

We have evaluated a fully automated thyroxine assay involving the use of a homogeneous enzyme assay and a Technicon AutoAnalyzer II continuous flow system. Comparison by regression analysis with a thyroxine radioimmunoassay kit method gave a slope of 0.82 and a y-intercept of 7.81 micrograms/L (SE 0.86 microgram/L, r = 0.95). Within-run precision yielded CVs of 1.0-6.1%, between-day CVs were 2.0-14.4%; within-day precision showed a mean variance of 7.81 micrograms/L. Mean analytical recovery was 96.1%. Bilirubin, hemoglobin, and lipemia interfered with quantitation of results when their concentrations exceeded 50 mg/L, 300 mg/L, and 5 g/L, respectively. The reference interval for euthyroid status was calculated to be 50-110 micrograms/L. Sensitivity was 5.0 micrograms/L with a mean carryover of 1.65%. Current reagent and labor costs for enzyme immunoassay ($0.40) were less than for the manual radioimmunoassay procedure ($0.40) were less than for the manual radioimmunoassay procedure ($1.70). The assay is economical, simple to perform, and amenable to high-throughput thyroid screening in the routine laboratory.
Lactate dehydrogenase isoenzyme 1 with the centrifugal analyzer after immunochemical removal of other isoenzymes.
Authors: Bruns D E , Emerson J C , Bertholf R L , Hill K E , Savory J
Published: January 2020

We describe a centrifugal analyzer method for measuring the LD-1 isoenzyme of lactate dehydrogenase (EC 1.1.1.27) in serum, after immunochemical precipitation of the other four isoenzymes. Enzymic activity was measured kinetically at 30 degrees C with the pyruvate-to-lactate assay. The method for LD-1 was linear to 1000 U/L. The precision (CV) of the assay was 1.0--2.2% within-run and 3.1--4.5% day-to-day. The reference interval was 26--73 U/L (n = 51), corresponding to 21--35% of total LD activity.
Complementary use of amperometric and spectrophotometric detection for concurrent monitoring of serum tryptophan metabolites by reversed-phase liquid chromatography.
Authors: Krstulovic A M , Friedman M J , Sinclair P R , Felice J
Published: January 2020

We describe an improved reversed-phase liquid-chromatographic method for concurrent measurement of tryptophan and eight of its metabolites in serum. These were separated by gradient elution and detected both by ultraviolet absorption at 254 nm and amperometrically at an oxidation potential of +0.700 V vs Ag/Cl. Detection limits for the electrochemically-detected compounds were between 100 pg and 1.0 ng, and approximately 1-5 ng for the ultraviolet-absorbing compounds. Analytical recovery of the compounds after deproteinization (by ultrafiltration) varied between 65 and 95%. As little as 500 microL of serum is required. No interference(s) from other naturally occurring serum constituents were encountered. This highly sensitive method is relatively fast and offers a useful tool for diagnosis and treatment of diseases involving derangement in tryptophan metabolism.
Diagnosis of hemolytic disease by electrophoresis of erythrocyte lactate dehydrogenase isoenzymes on cellulose acetate or Agarose.
Authors: Van Lente F , Marchand A , Galen R S
Published: January 2020

We determined the LD-1/LD-2 isoenzyme ratio in hemolysates of erythrocytes by electrophoresis on cellulose acetate and on agarose. A ratio exceeding 1.0 was found with the former but not the latter. Results were similar for in vitro models of hemolytic disorders. Using cellulose acetate electrophoresis, we determined the predictive value of data on total LD activity and of the LD-1/LD-2 ratio in diagnosis of hemolytic disease in 100 patients. The sensitivity of the "flipped" LD-1/LD-2 ratio was only 58%, the specificity was 93%, and the predictive value was 74% for diagnosis of hemolytic disease. A normal total LD activity is highly predictive (92%) for ruling at the presence of hemolytic disease.
Circulating IgG-LD complex, dissociable by addition of NAD+.
Authors: Gorus F , Aelbrecht W , Van Camp B
Published: January 2020

Macromolecular LD (lactate dehydrogenase, EC 1.1.1.27) was present in the serum of a patient suffering from idiopathic fibrosis of the lung and presenting signs of autoimmune disease. By using gel filtration and affinity chromatography techniques, the vast majority of the patient's serum LD activity was shown to consist of LD-IgG complexes, which dissociated in the presence of added nicotinamide adenine dinucleotide (NAD+). Binding studies with tritiated NAD+ indicated that complex formation was not ascribable to a lack of circulating cofactor. The most likely explanation for the complex formation was the existence of LD binding sites on IgG molecules. The disruption of the complex by NAD+ might be explained by a competition between IgG molecules and NAD+ for the LD active site or by conformational changes induced in the LD molecules on binding of NAD+.
Direct determination of therapeutic concentrations of lithium in serum by flow-injection analysis with atomic absorption spectroscopic detection.
Authors: Rocks B F , Sherwood R A , Riley C
Published: January 2020

In this flow-injection system for direct determination of lithium in serum by atomic absorption spectroscopy, the 10-microL sample is manually injected into a continuously flowing non-segmented stream of de-ionized water, which is pumped, via a dispersion tube, to the spectrometer's nebulizer. Controlled dispersion of the sample zone, before it is introduced into the nebulizer, produces the required sample dilution. Effects of varying the length of the dispersion tube, the flow rate, and the sample size were studied. Analytical readout is obtained, in the form of transient peaks, 5 s after sample injection. It is necessary to include physiological concentrations of sodium and potassium in the standard because each of these cations enhances the lithium absorbance signal. Analytical recovery (98.5 to 101%) and CV (about 2%) are good, and results compare well with those obtained by aspiration of prediluted samples (n = 121, r = 0.99).
Isolation and preliminary characterization of a fraction of bilirubin in serum that is firmly bound to protein.
Authors: Lauff J J , Kasper M E , Wu T W , Ambrose R T
Published: January 2020

We have isolated from pathological sera a bilirubin fraction (delta) that is very tightly, if not covalently, bound to protein, most likely albumin. This delta fraction absorbed at a lambda max of 433 nm in the visible spectrum, between the lambda max of unconjugated (alpha) and that of conjugated (Bc) bilirubin when measured in solutions containing albumin. However, unlike the other bilirubin species, this fraction could not be separated from the proteins in serum by exhaustive ultrafiltration in the presence of caffeine/benzoate solution. In the Jendrassik-Grof diazo procedure for bilirubin analysis, the delta fraction gave a large direct reaction (76-89% of the total reaction). Yet, when relatively hydrophobic azo dyes were formed by reaction of the delta fraction with the diazonium salt of dichloroaniline, only 50% of the dyes were extractable from aqueous solution. On chromatography the rest remained associated with protein. Of the extractable dye, more than 70% was accounted for by two liquid-chromatographic peaks with retentions identical with those of azo dyes formed from unconjugated bilirubin. This delta fraction was not appreciably separated from protein by treatment with strong acid or base, or by prolonged digestion with various enzymes. Finally, in a highly denaturing solvent (urea/mercaptoethanol), this fraction was not dialyzable through a membrane with a 12 000-dalton cutoff.
Quantitation of haptens by homogeneous immunoprecipitation. 1. Automated analysis of gentamicin in serum.
Authors: Wu J W , Hoskin S , Riebe S M , Gifford J E , O'Neill S P
Published: January 2020

A new homogeneous immunoprecipitation inhibition assay has been developed to quantitate concentrations of hapten in human serum or plasma without the use of radioactive isotopes, enzymes, fluorescent markers, or laser nephelometers. This immunoprecipitation is based on spectrophotometric measurement of the inhibition by free hapten of the precipitating reaction between antihapten antibody and polyhaptenic antigen. The immunoprecipitation analysis of the antibiotic gentamicin in human serum is reported here. A serum test sample is mixed with gentamicin-human serum albumin polyhaptenic conjugate and rabbit antiserum to gentamicin on a centrifugal analyzer, and the subsequent reaction monitored for 3 min. No sample dilution or pretreatment is required. The within-run and between-run coefficients of variation are well below 10%. The results on patients' test samples correlate well with those obtained by commercially available radioimmunoassay and enzyme immunoassay kits.
Two-dimensional electrophoretic patterns of proteins of normal mucosa, polyps, and carcinomas of the large intestine.
Authors: Thorsrud A K , Vatn M H , Jellum E
Published: January 2020

Two-dimensional protein maps of biopsies from normal mucosa, tubular adenomas (polyps), and colonic carcinomas in humans have been studied in order to obtain information on the possible precancerous role of intestinal polyps. The maps are complex and several hundred protein spots are seen after staining with Coomassie Blue. Most of the spots are common to these three tissue types, but there are also differences. Polyps and carcinomas had a strikingly similar protein pattern, different from that of normal mucosa. There were negligible differences in the two-dimensional protein maps of tubular adenomas from patients who also had colonic cancer, as compared with adenomas from persons without carcinomas. The results lend further support to the hypothesis that polyps may be precursors of carcinomas of the large intestine.
Patterns for normal colon mucosa and colon adenocarcinoma compared by two-dimensional gel electrophoresis.
Authors: Tracy R P , Wold L E , Currie R M , Young D S
Published: January 2020

A two-dimensional gel "map" of the proteins in normal cells and transformed cells may well help establish the presence of putative tumor-specific protein antigens, which can be isolated and used as antigens. We so examined normal colon mucosa and colon adenocarcinoma, using a sensitive silver stain. Samples were promptly prepared after excision by rapidly trimming the tissue to a small, grossly homogeneous piece, which was frozen on solid CO2. Frozen histological sections and permanent mounts were also prepared for several areas, to document the cellular constitution and to grade the carcinoma. Sample preparation for electrophoresis is described. The DNA was pelleted by centrifugation (108 000 x g x 1.5 h). Little or no pellet was observed, indicating complete solubilization. Many of the tumors contained proteases, liberated during sample preparation, so protease inhibitors were included. A highly reproducible pattern was observed for normal mucosa. Comparison of the gels from paired samples with histological data allows many of the spots to be tentatively assigned to the different cell types present in the mucosa (epithelium, connective tissue, or muscularis mucosa) and, in most tumors analyzed, detection of a series of spots that are not detectable in the pattern for normal tissue. These apparently tumor-associated proteins may be of epithelial cell or stromal origin.
Characterization of allergen extracts by two-dimensional electrophoretic techniques: Micropolyspora faeni antigens.
Authors: Vesterberg O , Holmberg K
Published: January 2020

Thermophilic actinomycetes and saprobic fungi are important in the etiology of allergic occupational diseases such as "farmer's lung" disease. Each such organism produces several protein antigens. Inhaled, these antigens stimulate production of antibodies. Detection of precipitating antibodies has been useful in the diagnosis of diseases so induced. Characterization of allergen extracts from microorganisms associated with these diseases is important, to improve the sensitivity and precision of the precipitin analysis. For this purpose we submitted crude allergen extracts to electrophoresis and isoelectric focusing in agarose gels. Staining the gels revealed many protein components in each extract, especially after isoelectric focusing. After separation in one dimension, a lane of gel was cut out and the proteins were electrophoresed at right angles into another gel, which contained antibodies. Several arcs of immunoprecipitates, indicating different antigens, were seen. This technique ("crossed immunoelectrofocusing") has earlier been used with polyacrylamide in the first dimension, but it is improved by using instead agarose of a special quality. Further to improve the quantification, we isolated pieces of gel containing the proteins of interest and used them as samples in zone immunoelectrophoresis assay. This method is straightforward, easy to evaluate, and about 100-fold as sensitive as radial immunodiffusion. The amount of protein in each sample is usually proportional to the distance from the upper gel surface to the front of each immunoprecipitate. The increased sensitivity allows study of many hitherto unexamined antigens.
Chloroquine and its metabolites in human cord blood, neonatal blood, and urine after maternal medication.
Authors: Essien E E , Afamefuna G C
Published: January 2020

Chloroquine, its N-dealkylated metabolites, and chloroquine N-oxides were detected in the urine of pregnant women who were receiving chloroquine medication whereas chloroquine and its nonpolar metabolites, desethyl- and didesethylchloroquine and 7-chloro-4-aminoquinoline, have been found in the neonates' urine, blood, and cord blood. We used thin-layer chromatography to separate chloroquine and its metabolites after their extraction from biological fluids. These compounds were identified by comparing their chromatographic and ultraviolet spectrophotometric characteristics with those of reference compounds. That chloroquine and its relatively nonpolar metabolites (including one without the alkyl side-chain, 7-chloro-4-aminoquinoline) cross the placenta is demonstrated by the presence of these compounds in the cord blood, neonatal systemic blood, and neonatal urine. The selective transfer of the compounds in the cord blood, neonatal system blood, and neonatal urine. The selective transfer of the compounds across the placenta and the clinical implications of these findings are discussed.
Stability of immunologic activity of human prostatic acid phosphatase in serum.
Authors: Chen I W , Sperling M I , Maxon H R , Kaplan L A
Published: January 2020

The stability of the immunologic activity of human serum prostatic acid phosphatase (iPAP;EC 3.1.3.2) under various storage conditions was evaluated. Although more stable than the enzymic activity of serum PAP, serum iPAP was inactivated rapidly at room temperature at neutral and especially at alkaline pH. Purified iPAP in a phosphate buffer (pH 7.5) was stable for at least three days at room temperature but showed instability similar to that of serum iPAP when added to heat-treated serum (30 min at 56 degrees C, pH 8.3). Addition of a protease inhibitor, a sulfhydryl agent, or a chelating agent failed to preserve iPAP. Acidification of serum with an acetate buffer (5 mol/L, pH 5.0), 20 microliters/mL of serum, protected but could not restore serum iPAP activity. When stored at 24 degrees C, serum iPAP was most stable at pH 6.5. Acidification had little effect on PAP values by radioimmunoassay, iPAP values from acidified and unacidified serum samples being essentially the same. We recommend that blood samples drawn for PAP immunoassays be kept at 4 degrees C and that serum samples be separated and acidified as soon as practical. For long-term storage, acidified serum samples should be kept in small aliquots at -20 or -70 degrees C.
Liquid-chromatographic direct determination of phenylalanine and tyrosine in serum or plasma, with application to patients with phenylketonuria.
Authors: Hilton M A
Published: January 2020

With this method, picomole amounts of phenylalanine and tyrosine can be rapidly separated and directly measured by absorbance at 206 nm. As little as 25 microliters of serum or plasma suffices. Plasma is deproteinized and 20 microliters of the supernate, representing 1.8 microliters of plasma, is applied to a reversed-phase column and eluted isocratically with a solution containing methanol and H3PO4, pH 2.4. The tyrosine and phenylalanine peaks appear at 6 and 10 min, respectively. We used the method to measure phenylalanine in plasma from six children with phenylketonuria.
Why we need better test evaluations.
Authors: Zweig M H , Robertson E A
Published: January 2020

A laboratory test is clinically useful only if it successfully answers a question of consequence to patient management. Unfortunately, the results and conclusions of many published test evaluations are misleading or of uncertain validity because common-sense principles of study design are overlooked. This is illustrated by examples from recent literature. We suggest that tests should be evaluated with prospective studies of patients representative of the population for which the test will ultimately be used. The clinical question to be addressed by the test should be clearly stated, and then answered for each patient by means independent of the test being evaluated. When comparing tests with each other, decision levels should be chosen to give either the same sensitivity or specificity for each. The use of soundly designed protocols for the clinical evaluation of tests provides the information needed to select the most effective tests.
Enzyme immunoassay for factor VIII-related antigen.
Authors: Cejka J
Published: January 2020

I describe a simple enzyme-linked immunosorbent assay (ELISA) for the quantitation of Factor VIII-related antigen in plasma with use of commercially available peroxidase-labeled antiserum and solid-phase support. Regression analysis of 85 plasma samples analyzed by this technique (y) and by a commonly used electroimmunoassay (Anal. Biochem. 15: 45-52, 1966) (x) gave the equation y = 0.223 + 0.77x (r = 0.973). The present method was also compared with enzyme immunoassay in which a phosphatase-labeled antiserum prepared in our laboratory was used; the correlation between the two assays was very good. The simplicity and specificity of the ELISA technique should make it a useful alternative to the more difficult and time-consuming Laurell method.
Urinary urea: are currently available methods adequate for revival of an almost abandoned test?
Authors: Eckfeldt J , Levine A S , Greiner C , Kershaw M
Published: January 2020

Measurement of urinary urea excretion has been suggested as a means of estimating nitrogen balance in hospitalized patients who are malnourished. Because proficiency-testing surveys show gross variations in mean urea as determined by various automated methods and extremely poor precision occasionally, we compared urinary urea measurements and ammonia interference in three widely used methods. The coupled urease/glutamate dehydrogenase method (used in the DuPont aca) showed positive interference from ammonia, as expected; with the diacetylmonoxime (Technicon (12/60) and the urease conductivity (Beckman ASTRA) methods we saw no such interference. Generally, interference by ammonia is less than 10%, but (rarely) it may exceed 25%. However, if urine specimens are properly diluted and potential sources of interference recognized, all three methods appear capable of providing clinically useful data.
Idiopathic hemochromatosis: serum ferritin concentrations during therapy by phlebotomy.
Authors: Garry P J , Saiki J H
Published: January 2020

We report the case of a 54-year-old man who presented with symptoms of idiopathic hemochromatosis, an inherited disorder involving regulation of iron absorption. These symptoms usually do not appear until total body iron content reaches 15 g, about threefold normal. Therapy involves mobilization and removal of excess stored iron through weekly or twice-weekly phlebotomies of 500 mL, until the hemoglobin concentration becomes less than 110 g/L and remains there for several weeks, or until serum ferritin concentrations indicate that almost all the stored iron has been removed (ferritin less than 12 micrograms/L). Here, concentrations of ferritin in serum were used as an index to iron overload and removal of stored iron. We report changes in hemoglobin, serum ferritin, iron, and total iron-binding capacity during the course of removing by phlebotomy more than 20 g of iron from a patient with idiopathic hemochromatosis.
Multilayer film analysis: evaluation of ion-selective electrolyte slides.
Authors: Costello P , Kubasik N P , Brody B B , Sine H E , Bertsch J A , D'Souza J P
Published: January 2020

We evaluated the Kodak Ektachem multilayer ion-selective electrolyte slides. For various types and concentrations of control material the precision (CV) within- and between-day ranged from 0.5 to 1.3% (1.7-2.1%) for sodium, 1.2 to 2.2% (2.7-2.9%) for potassium, 2.9 to 4.6% (5.9-6.7%) for carbon dioxide, and 0.7 to 1.6% (1.3-1.4%) for chloride. For all these analytes, analytical recovery was about 100%, except in the supra-physiological ranges, for which carbon dioxide recovery was about 110-120%. Either serum or heparin-treated plasma can be used, interchangeably, for analysis; use of serum treated with lithium iodoacetate is unacceptable. Comparisons with results by continuous-flow procedures demonstrated good correlation for sodium, potassium, and chloride; carbon dioxide comparisons indicate an Ektachem calibrator change may be required. Abnormally low protein concentrations or lipemia had no observed effects on results for electrolytes. Abnormally high protein concentrations affect sodium results slightly (approximately 5 mmol/L).
Modified handling of internal catecholamine standards in radioenzymic assays.
Authors: Ellis J P , Burns J W
Published: January 2020

A departure from a radioenzymic procedure widely used for plasma catecholamine assay is reported, in which we use "mean net standards" instead of individual internal standards. Interlaboratory/intermethod analyses validated use of the modified principle and demonstrated its preferential use over the conventional principle. Also demonstrated is use of the modified procedure for calculating catecholamine concentrations of several plasma samples collected from an exercising man and an acceleration-stressed swine. Benefits already derived from use of the procedure include: (a) quantification of certain plasma samples that could not be satisfactorily quantified by conventional options, (b) an apparent gain in analytical precision in virtually all other analyses, and (c) costs of analysis reduced by about 25%.
Screening for neonatal Duchenne muscular dystrophy by bioluminescence measurement of creatine kinase in a blood sample spotted on paper.
Authors: Dellamonica C , Collombel C , Cotte J , Addis P
Published: January 2020

Neonatal screening for Duchenne-type muscular dystrophy is greatly simplified by use of a new bioluminescence procedure for creatine kinase. The blood of newborn myopathic children consistently showed increased activity. The improved method permits the analysis from a dried sample of whole blood spotted on filter paper; it shows high correlation with existing procedures and is highly specific and precise. The use of the improved method in a screening program involving 158 000 newborns is reviewed. We find a prevalence of 1/5929 living eighth-day boys.
Preparation of soluble apolipoproteins A-I, B, and C-II by a chromatofocusing column method, and evaluation of their concentrations in serum in pulmonary disease.
Authors: Jauhiainen M , Laitinen M , Marniemi J , Liippo K , Penttilä I , Hietanen E
Published: January 2020

A chromatofocusing column method for isolating ApoB is described. LDL is first isolated by sequential ultracentrifugation and delipidated with n-butanol/diisopropyl ether. Chromatofocusing of ApoLDL yielded a large ApoB peak at pI 5.0-5.3. ApoA-I and ApoC-II were prepared analogously, with HDL and VLDL as the source of apoprotein. Antisera were raised in rabbits, and electroimmunoassay techniques were used for determination. ApoB was water-soluble after chromatofocusing. Intra-assay precision (CV) was 4.7% for ApoA-I, 7.8% for ApoB in the "rocket" electrophoresis. Interassay precision (CV) was 6% for ApoA-I and 8% for ApoB. Apolipoprotein concentrations were measured in subjects who had undergone lung resection and patients with obstructive pulmonary disease. After lung resection, the concentration of ApoA-I in serum was significantly decreased (p less than 0.001) and that of ApoB significantly increased (p less than 0.001) as compared with controls. The ApoA-I/ApoB ratio was significantly lower in the lung-resection group. ApoA-I and ApoB concentrations were unchanged in chronic obstructive pulmonary disease. ApoC-II concentrations in each group were similar to those for control subjects. Of the lipids, values for total cholesterol were above normal after lung resection (p less than 0.002), as were those for triglycerides (p less than 0.02).
Creatine kinase activity and isoenzyme pattern in various normal tissues and neoplasms.
Authors: Tsung S H
Published: January 2020

I measured total creatine kinase (CK; EC 2.7.3.2) activity and isoenzyme pattern in normal and neoplastic tissues. CK activity was detected in all of them examined. In various tumors it was greater than, less than, or the same as that in normal tissue, no clear correlation being seen between total activity and growth rate or degree of differentiation. In several cases, there was a greater proportion of the CK-MM isoenzyme, and 15 of 53 cases showed an atypical CK-MM band. The atypical CK-MM band, also reported by others, might be an insensitive and nonspecific tumor marker. The CK-BB isoenzyme, ubiquitous in neoplastic tissues, might accordingly be a nonspecific marker. Total CK activity was very low in most tumor tissues. Presumably a bulky tumor or an advanced stage of malignancy is a requisite to release of routinely detectable CK-BB into the circulation.