bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of acid phosphatase isoenzymes of human seminal fluid, prostate, and leukocytes.
Authors: Lam W K , Yam L T , Wilbur H J , Taft E , Li C Y
Published: January 2020

We used ion-exchange column chromatography and electrophoresis on polyacrylamide gel to compare the acid phosphatase isoenzymes of prostate and leukocytes. The major isoenzyme of the prostate is band 2A; only a trace of band 2B was observed. However, the major isoenzymes of leukocytes are band 4 and band 2B, and only a small amount of band 2A was observed. The three isoenzymes isolated from leukocytes or prostate gland react to the antiserum prepared against the aicd phosphatase isoenzyme of seminal fluid. Acid phosphatases of leukocytes other than the three isoenzymes mentioned above did not interact with the antiserum.
Micro-scale method for liquid-chromatographic determination of chloramphenicol in serum.
Authors: Petersdorf S H , Raisys V A , Opheim K E
Published: January 2020

We describe the use of "high-performance" liquid chromatography to measure chloramphenicol in as little as 25 microL of serum. Serum is treated to precipitate proteins with acetonitrile containing p-nitroacetanilide as an internal standard. Chloramphenicol is eluted with a mobile phase of methanol in pH 7.0 phosphate buffer (35/65 by vol). The drug is measured at 278 nm and simultaneously monitored at 254 nm; interfering substances are detected by examining the 278 nm/254 absorbance ratios. This method is sensitive to less than 0.5 mg/L and the standard curve is linear to at least 50 mg/L. Inter-day precision ranged between 3--6%. We encountered no interference from endogenous compounds or from other drugs we tested.
Evaluation of a solid-phase enzyme immunoassay for insulin in human serum.
Authors: Kato K , Umeda Y , Suzuki F , Hayashi D , Kosaka A
Published: January 2020

We describe a "sandwich-type" enzyme immunoassay for insulin in serum, in which antibody Fab'-beta-D-galactosidase conjugate and an antibody-immobilized silicone rubber solid-phase are used. The interference by serum factors with the solid-phase enzyme immunoassay can now be removed by using a buffer containing gelatin. Serum samples of 50 microL can be analyzed by the enzyme immunoassay, which is as sensitive as radioimmunoassay for human insulin. Our results correlate well with those for radioimmunoassay (r = 0.97, slope = 0.92, y-intercept = 4.6 milli-int. units /L for 181 samples). Between-assay and within-assay coefficients of variation are less than 15% over the useful ranges of the assay (5--160 milli-int. units/L).
Urinary excretion of thymine and uracil in a two-year-old child with a malignant tumor of the brain.
Authors: Berglund G , Greter J , Lindstedt S , Steen G , Waldenström J , Wass U
Published: January 2020

A two-year-old boy with a malignant tumor of the brain (medulloblastoma) excreted large amounts of thymine and uracil in his urine. The excretion was related to progress and regress of the disease, and reached a maximum of 3.0 mol of thymine per mole of creatinine and 2.6 mol of uracil per mole of creatinine. The excretion by 20 apparently normal children was less than 0.01 mol/mol of creatinine for each of the two pyrimidines. Three children with brain tumors, two with leukemias, and one with neuroblastoma were also studied; two of them had a moderate increase in urinary pyrimidine excretion, but only up to 0.07 mol/mol of creatinine. The activity of dihydrouracil dehydrogenase (NADP+) (EC 1.3.1.2) in cultured fibroblasts from the patient was somewhat lower than in control fibroblasts. The tumor was considered to be the likely cause of the increased excretion of pyrimidines, but an impaired degradation of pyrimidines in the liver could not be ruled out.
Diabetic ketoalkalosis.
Authors: Koett J , Howell J , Steinberg S , Wolf P
Published: January 2020

The usual metabolic derangement in uncontrolled diabetes mellitus is metabolic acidosis, with an increase in the anion gap because of increased ketoacids and lactate. However, diabetic ketoalkalosis may occasionally be encountered, the prominent clinical feature of which is vomiting, with depletion of potassium, chloride, and hydrogen ions. Self-medication with absorbabe alkali may also contribute to the alkalosis. It would be dangerous to treat hyperlgycemic patients with alkali if their condition is ketoalkalosis instead of ketoacidosis.
Enzymic assay for serum theophylline.
Authors: Vinet B , Zizian L
Published: January 2020

We describe an original procedure for determination of theophylline in serum. The drug is extracted from 0.4 mL of serum at pH 7.4 with chloroform/isopropanol (20/1 by vol) and back-extracted into sodium hydroxide (1 mmol/L). The inhibition of beef-liver alkaline phosphatases by theophylline in this alkaline phase is measured at 25 degrees C, with p-nitrophenyl phosphate as substrate, in 2-amino-2-methyl-1-propanol buffer, pH 9.4- The reciprocal of enzyme activity and theophylline concentration are linearly related in the range 2 to 60 mg/L. The maximum interference to be expected from 3-methylxanthine would increase apparent theophylline concentration by no more than 1 mg/L. The method is accurate, free of interference by other xanthines and often-coadministered drugs, and results correlate well with those by the immunoenzymic assay. Major advantages are reagent stability, low cost, and simplicity of instrumentation.
Spectrophotometric measurement of carboxyhemoglobin and methemoglobin in blood.
Authors: Rodkey F L , Hill T A , Pitts L L , Robertson R F
Published: January 2020

This paper describes separate spectrophotometric procedures for rapidly measuring carboxyhemoglobin (COHb) and methemoglobin (MetHb) in blood. Absorbance measurements are made in the Soret region at a blood dilution near 1000-fold. For COHb estimation, the diluent contains sodium hydrosulfite, providing the two-component system COHb--Hb for absorbance measurements at 420 and 432 nm. The NA2S2O4 effectively prevents dissociation of COHb by oxygen. For MetHb estimation, the diluent contains KCN and carbon monoxide, providing the two-component system COHb--CNMetHb. Absorbance measurements are made at 420 nm, before and after addiely analyzed for the total of all MetHb forms present. Each procedure requires about 3 muL of blood and can be applied to human or animal blood. Results obtained by the present methods are in satisfactory agreement with currently accepted procedures, which require much larger samples or more elaborate equipment.
Improved double-antibody enzyme immunoassay for methotrexate.
Authors: Al-Bassam M N , O'Sullivan M J , Bridges J W , Marks V
Published: January 2020

We report an enzyme immunoassay procedure for methotrexate measurement that takes less than 3 h to perform. beta-D-Galactosidase (EC 3.2.1.23) from Escherichia coli was conjugated to methotrexate by means of the mixed anhydride reaction. Bound and free labeled drug were separated by a preincubated cubic complex of first and second antibody. The enzyme activity of the bound fraction was measured with o-nitrophenyl-beta-D-galactopyranoside as substrate. The standard curve covered the range 1 to 10 micrograms of methotrexate per liter. One microgram of methotrexate per liter inhibited binding of the tracer by 17%. The assay is specific for methotrexate in the presence of folinic acid (citrovorum factor), folic acid, tetrahydrofolic acid, and other methotrexate metabolities. Intra- and inter-assay CVs were less than 5 and 10%, respectively. Results obtained with this enzyme immunoassay method agreed well with those obtained with an established radioimmunoassay method.
Four electrophoretic methods compared for diagnosis of type III hyperlipoproteinemia.
Authors: Kuba K , Lippel K , Frantz I D
Published: January 2020

Blood drawn from 192 probands and 1129 first-degree relatives who were participants in a collaborative family study of hyperlipoproteinemia at nine Lipid Research Clinics was used to prepare aliquots of whole plasma and top (d less than 1.006 g/mL) and bottom (d greater than 1.006 g/mL) ultracentrifugal fractions. Each aliquot was analyzed at a central laboratory by electrophoresis on paper, agarose, and polyacrylamide gel, and by a combined electrophoretic precipitation technique. The electrophoretograms were evaluated for the presence or absence of a "floating-beta" lipoprotein band. All four methods agreed completely for 92.3% of the samples. An additional 2.0% of the samples were in agreement for three electrophoretic methods, but the paper electrophoretic results were not interpretable. Another 1.9% were considered to be "floating-beta" positive by paper electrophoresis but negative by the other three electrophoretic methods.
A kinetic method for glucose that is insensitive to variations in temperature and enzyme activity.
Authors: Mieling G E , Pardue H L , Thompson J E , Smith R A
Published: January 2020

We have adapted for glucose determination a new approach to kinetic analyses [Anal. Chem. 50, 1611 (1978)]; it is 50-fold less dependent upon some experimental variables than is a more conventional rate method. Modification of a commercially available hexokinase/glucose-6-phosphate dehydrogenase reagent system for glucose provides that the rate of production of NADH be first-order in total glucose concentration within about 30 s after sample and reagent are mixed. In the kinetic method, absorbance vs. time data recorded after 30 s and a multiple-linear-regression program are used to compute the absorbance change that would occur if the reaction were monitored to completion. Results demonstrate a linear relationship between glucose concentration and computed absorbance change. Application of the method to 51 human sera without rigorous control of either temperature or reagent composition yielded a regression equation of y = 1.01x -0.3 when kinetic results (y) were compared with equilibrium results (x) for the same samples analyzed in a hospital laboratory.
Rapid analysis of bilirubin in neonatal serum. I. The binding of bilirubin to albumin.
Authors: Lu K C , Gooding K M , Regnier F E
Published: January 2020

Evaulation of the severity of jaundice in the neonate may be determined by measuring the reserve binding capacity of serum proteins for free bilirubin. Determination of protein-bound bilirubin has been labor intensive, necessitating multiple runs on gel-permeation chromatography columns or, more recently, enzyme assays or fluorescence quenching analysis. We present a method for quantitation of free bilirubin and of bilirubin-binding capacity of serum by liquid chromatography. A gel-permeation column binds free bilirubin while allowing passage and quantitation of protein-bound bilirubin. Subsequent injection of a desorbing agent releases the adsorbed bilirubin from the column, permitting quantitation of free bilirubin. Bound and free serum bilirubin may be determined directly in less than 15 min using 10 microL of serum. The binding of bilirubin to neonatal serum is seen to be quite different from the binding to adult serum. Ion-exchange chromatography of adult and neonatal serum samples shows that their protein profiles are radically different. This difference probably accounts for the binding characteristics.
Use of magnetic circular dichroism spectroscopy for biologic monitoring of occupational exposures to toxicants.
Authors: Zygowicz E R , Hollebone B R , Perkins H M
Published: January 2020

Zinc protoporphyrin is the predominent fluorescent porphyrin accumulating in erythrocytes as a result of chronic lead absorption or iron-deficiency anemia. Although diagnostic concentrations or thresholds for it in erythrocytes have been inferred, normal adult averages have not been established. We quantitatively assessed average zinc protoporphyrin values during an investigation into the usefulness of magnetic circular dichroism spectroscopy as an analytical technique for biologic monitoring of industrial toxicant exposures. Blood samples drawn from 55 employees not exposed to lead were analyzed for blood lead, hematocrit, and zinc protoporphyrin. Average concentrations of zinc protoporphyrin determined with a hematofluorometer were compared with results obtained by magnetic circular dichroism spectroscopy. The latter results were comparable in kind but superior in quality to those of fluorometry. The magnetized spectroscopy followed Beer’s Law at concentrations well below indigenous concentrations and was more sensitive than difference spectroscopy. Spectroscopic resolution of heme and zinc protoporphyrin moieties in a pyridine/NaOH mixture was complete.
New optical technique for measuring erythrocyte deformability with the ektacytometer.
Authors: Groner W , Mohandas N , Bessis M
Published: January 2020

The laser light scattered by erythrocytes subjected to a well-defined shear stess can be analyzed with the ektacytometer to obtain information regarding the changes in cell shape due to fluid shear. We describe an optical technique whereby an observed quantitative output derived from a mesurement of light intensity through a spatial filter is related to the change in cellular dimensions that were previously observed under similar fluid-shear conditions by use of microscopy and a cone-plate viscometer (rheoscope). We also present the predictions of a theoretical model (of the ektacytometer) based on approximations of light-scattering theory developed for nonspherical particles, and give preliminary results for the accuracy and sensitivity of this measurement of erythrocyte deformability. With this optical technique the instrumentation (ektacytometer) is made quite simple and suitable for use in the typical laboratory. This would allow a regular, quantitative assessment of this important blood cell quality, to supplement the data obtained from the complete blood count.
Application of "high-performance" liquid chromatography to the study of sphingolipidoses.
Authors: Ullman M D , Pyeritz R E , Moser H W , Wenger D A , Kolodny E H
Published: January 2020

Quantitative high-performance liquid chromatographic analysis of perbenzoylated sphingolipids has been used to study the correlations of body chemistry to clinical phenomena. Plasma sphingolipids were isolated from 32 Gaucher (β-glucosidase deficiency) and six Fabry (α-galactosidase deficiency) patients by solvent partition and chromatographic separation on silicic acid columns. Plasma sphingolipids from a patient undergoing plasma-exchange were separated from interfering lipids with reversed-phase columns. Liquid-chromatographic analysis of sphingolipids provides useful supportive information for diagnoses because affected individuals are shown to possess increased circulating concentrations of the pathognomonic sphingolipid. We also used this technique to monitor sphingolipid concentrations in plasma and urine sediment during plasma exchange of a p atient with Fabry’s disease. Regular plasma exchanges produced and maintained decreased concentrations of sphingolipids in plasma, but near pre-exchange concentrations were observed within days after the therapy was terminated.
Enzyme immunoassay of hepatitis B surface antigen with use of the "Hepanostika" test kit and a new photometer, "Photo-Elisa 1".
Authors: Tanaka F , Nishi K , Arisue K , Hayashi C , Miyai K
Published: January 2020

We describe an enzyme immunoassay for hepatitis B surface antigen, with use of a Hepanostika test kit and "Photo-Elisa 1," a photometer capable of measuring small quantities of test material. Because the length of the optical path in the "Photo-Elisa 1" is only 1.2 mm, volumes as small as 15 micro L can be can be measured. Accuracy is enhanced through cancellation of background absorbance by measurement at two wavelengths. Results obtained with the kit compare well with those by radioimmunoassay. By substituting photometric reading for visual reading, Hepanostika provides a more nearly accurate screening test.
Diagnostic utility of C-terminal parathyrin measurement as compared with measurements of N-terminal parathyrin and calcium in serum.
Authors: Simon M , Cuan J
Published: January 2020

We compared results obtained from two parathyrin (parathyroid hormone) assays with differing specificities, using sera from 172 normal donors and from 98 patients with disorders of calcium regulation. Intact parathyrin was measured in both assays; the C-parathyrin assay also measured the 53-84 amino acid C-terminal hormone fragment; the N-parathyrin assay also measured the 1-34 N-terminal fragment. The reference interval for the C-parathyrin assay (860-3720 int. units/L; 430-1860 ng/L) was markedly higher than for the N-parathyrin assay (460-1260 int. units/L; 230-630 ng/L), a finding consistent with the longer half-life of C-parathyrin fragments in human circulation. Mean C-parathyrin in primary hyperparathyroid sera--5720 (SD 2760) int. units/L or 2860 (SD 1380) ng/L--clearly exceeded the reference interval and values for sera from patients with non-parathyroid malignancy [1740 (SD 760) int. units/L; 870 (SD 380) ng/L]. Secondary hyperparathyroid patients also had supranormal C-parathyrin values: 6100 (SD 2720) int. units/L; 3050 (SD 1360) ng/L. We found no consistent correlation between parathyrin and serum calcium in any clinical group. The two parathyrin assays showed about equal diagnostic power, but their results could not be used interchangeably in sequential monitoring of patients.
An immunochemical procedure for determination of mitochondrial aspartate aminotransferase in human serum.
Authors: Rej R
Published: January 2020

An immunochemical procedure is described for quantitation of mitochondrial aspartate aminotransferase (m-AspAT; EC 2.6.1.1) activity in human serum specimens. Antibodies directed against purified soluble aspartate aminotransferase (s-AspAT) from human erythrocytes were produced in rabbits and partly purified. Antibody sufficient for analyses of > 6000 specimens could be obtained from 15 mL of rabbit antiserum; contaminant AspAT activity of the antibody preparation was < 0.4 U/L. Addition of antibody directly to purified AspAT isoenzymes resulted in inhibition of s-AspAT but had no measurable effect upon m-AspAT. Antibody is incubated with serum in the presence of polyethylene glycol for 60 min at room temperature, then 60 min at 4 degrees C, and centrifuged (7000 x g, 4 degrees C, 15 min). No detectable s-AspAT activity remains in the supernatant fluid; thus m-AspAT activity can be measured directly. Precision, both within-day and day-to-day, was < 1 U/L, or 3.0% of residual m-AspAT activity. The method completely removed 1200 U of purified s-AspAT activity per liter; addition of s-AspAT to serum in increasing concentrations of about 500 U/L had no effect upon the measurement of residual m-AspAT activity. Results of the procedure described showed excellent correlation with those by an alternative procedure involving antibodies directed against m-AspAT. Addition of both anti-s- and anti-m-AspAT antibodies resulted in complete removal of serum AspAT activity. Univalent Fab fragments prepared anti-s-AspAT antibodies were capable of directly inhibiting s-AspAT activity without precipitation. Although a homogeneous immunoinhibition assay was possible, the greater precision of the precipitation assay made it preferable.
Decrease in plasma glucose concentration during storage at -20 degrees C.
Authors: Giampietro O , Navalesi R , Buzzigoli G , Boni C , Benzi L
Published: January 2020

We measured glucose concentrations with the Beckman Glucose Analyzer, before and after storage at -20 degrees C for various intervals, in 228 plasma samples from oral glucose tolerance tests on 17 normal subjects and 21 patients with impaired carbohydrate tolerance, and also in 82 control samples collected daily, after overnight fast, from seven insulin-dependent diabetics. We found a significantly (p < 0.001) positive relation between storage interval and percentage decrease in glucose concentration. Evidently, the longer plasma is stored, the greater the decrease in glucose.
Electrothermal atomic absorption spectrophotometry of cadmium in semen.
Authors: Wetzel L T , Bell J U
Published: January 2020

We describe the electrothermal atomic absorption spectrophotometry of cadmium in rabbit semen collected before and after seven days of subcutaneous administration of 0.5 mg of cadmium per kilogram body weight per day. The analytical technique involves combining an aliquot of an acid-digested semen sample with an equal volume of an (NH4)2HOP4 solution (50 g/L), to allow an increase in charring temperature, which results in more nearly complete destruction of matrix. Cadmium values as determined by this method correlated well with those determined by the method of standard additions. The dosing regimen resulted in a statistically significant (p < 0.01) increase in cadmium concentrations in semen (and whole blood) sampled just after the last day of cadmium administration.
Counting efficiency of some commercially available liquid scintillators compared.
Authors: Gray P , van Reenen O , Potgieter G M
Published: January 2020

We investigated four commercially available liquid scintillators as to overall counting efficiency and quenching effect of added phosphate-buffered isotonic saline. They differed significantly in these respects. Evidently, characteristics of the scintillator used in radioimmunoassays where β-particle emitters are counted should be carefully examined if results are to be optimal.
Evaluation of enzyme immunoassay, radioassay, and radioimmunoassay of serum methotrexate, as compared with liquid chromatography.
Authors: Buice R G , Evans W E , Karas J , Nicholas C A , Sidhu P , Straughn A B , Meyer M C , Crom W R
Published: January 2020

We evaluated three commonly used clinical methods for measuring serum methotrexate: enzyme immunoassay (EMIT), radioassay, and radioimmunoassay. Because potentially interfering compounds can be resolved by liquid chromatography, this method was selected as the comparison method. Patients’ serum samples, taken during 24 h after 6-h high-dose infusions, contained methotrexate in concentrations ranging from 10(-7) to 10(-4) mol/L. Chromatograms revealed substantial amounts of 7-hydroxymethotrexate in all samples, actually exceeding methotrexate by 12 and 24 h. Nevertheless, analysis of variance revealed no significant differences in results by any of the four methods, nor did results by the three test methods differ significantly after being adjusted by analysis of covariance with liquid chromatography as the covariate. Evidently any of the four techniques is suitable for monitoring serum methotrexate for 24 h after high-dose therapy.
Simplified radioimmunoassay of bradykinin in human plasma.
Authors: Verma P S , Lorenz P E , Sander G E
Published: January 2020

A greatly simplified radioimmunoassay for bradykinin in human plasma is described. Current techniques require multiple chromatographic steps or extraction procedures with analytical recoveries of bradykinin of often less than 60%. We present a method in which bradykinin is separated from components of higher relative molecular mass (including kininogens) in a single step, by use of a column of Sephadex G-25 medium (PD-10). The mean analytical recovery of tritiated bradykinin added to plasma is 85.5% (SD, 3.5%). The sensitivity of this radioimmunoassay is 25 pg per assay tube, equivalent to 125 ng per liter of plasma. Twenty to 30 blood samples may be completely processed and assayed within 6 h. As determined with this technique, concentrations of bradykinin in plasma from apparently normal subjects ranged from 2.5 to 5.2 microgram/L (mean 4.2, SD 1.1 microgram/L); these values are consistent with previously reported normal values.
Methods for ligand-receptor assays in clinical chemistry.
Authors: Smith R G , Sestili M A
Published: January 2020

This is intended to be a practical review for the clinical chemist of the laboratory procedures most commonly used to quantitate hormone receptors in various cellular fractions. These procedures include use of charcoal adsorption and hydroxylapatite for intracellular receptors and of centrifugation and filtration for membrane receptors. We discuss the use of the Scatchard analysis to establish the steroid-receptor affinity and the quantity of steroid-receptor binding sites. Both pre- and post-labeled sucrose density gradient methods are outlined. One section is devoted to the direct and indirect methods used in nuclear “exchange” assays. Basic theory underlying each assay is presented, but, more importantly, we assess the advantages and disadvantages of each procedure. On the basis of this information, one may decide which assay is best suited for a particular laboratory and (or) specimen.
Properties and clinical significance of an alkaline phosphatase-lipoprotein-X complex.
Authors: Weijers R N
Published: January 2020

The existence of an alkaline phosphatase-lipoprotein-X complex was demonstrated as an abnormally moving band in the agar-agarose gel alkaline phosphatase pattern of sera from 58 patients in a group of 72 patients whose sera contained lipoprotein-X. The position of the abnormally moving band in the agar-agarose gel alkaline phosphatase pattern and the percentage of alkaline phosphatase activity in the complex in relation to the total serum alkaline phosphatase activity depend on the serum lipoprotein-X concentration. The presence of the complex is a reliable indicator for cholestasis and of limited value in the differentiation between intrahepatic and extrahepatic cholestasis.
Enzyme-linked immunoassay for placental lactogen in human serum.
Authors: MacDonald D J , Nicol K M , Belfield A , Shah M M , Mack D S
Published: January 2020

We describe an enzyme-linked immunoassay for measuring human placental lactogen (HPL) in serum. After suitable dilution, sera are pipetted into the wells of polyvinyl microtitre plates previously coated with anti-HPL. After incubation the sera are decanted, and replaced by a conjugate of horseradish peroxidase and anti-HPL. An amount of enzyme proportional to the concentration of HPL in the serum is bound to the wells via an antibody—HPL—antibody bridge. Peroxidase activity is measured by oxidation of o-phenylenediamine, the resulting color being related to the concentration of HPL. Results correlate well with those by radioimmunoassay (r = 0.96). Our between-assay coefficient of variation was 13%; no discernible effect of protein was observed. This 4-h assay can be used to monitor placental function in pregnancy.
Spectrophotometric determination of urinary oxalate with oxalate oxidase prepared from moss.
Authors: Laker M F , Hofmann A F , Meeuse B J
Published: January 2020

A novel spectrophotometric enzymic procedure for estimating oxalic acid in urine is described. Oxalate oxidase, prepared from moss species, converts oxalic acid to hydrogen peroxide and carbon dioxide. Hydrogen peroxide is determined enzymatically with horseradish peroxidase, by oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone with N,N-dimethylaniline; the resulting indamine due is determined spectrophotometrically at 595 nm. Interfering substances are removed by adsorption to ion-exchange resins and oxidation with charcoal, thus avoiding oxalate recovery problems accompanying oxalate isolation. The procedure is rapid, sensitive, linear, and precise. Results agreed well with those obtained with a widely used chemical technique.
Hypophosphatasia (adult form): quantitation of serum alkaline phosphatase isoenzyme activity in a large kindred.
Authors: Millán J L , Whyte M P , Avioli L V , Fishman W H
Published: January 2020

We used heat inactivation, L-phenylalanine inhibition, and electrophoresis on polyacrylamide gel and cellulose acetate membranes--with and without use of specific antisera against the liver-bone, intestinal, and placental isoenzymes--to distinguish and quantitate the different alkaline phosphatase isoenzymes in sera from 23 adult members of a kindred affected by the adult form of hypophosphatasia. Nine subjects had values for total activity more than two standard deviations below the mean values for age- and sex-matched normal persons. Bone isoenzyme was diminished in all nine, whereas liver isoenzyme was subnormal in only four. Phosphoethanolamine and phosphoserine in the urine of eight hypophosphatasemic individuals correlated inversely with both total and liver alkaline phosphatase activity in their serum, but not with the activity of the bone isoenzyme. Total activity in the serum of adult kindred members correlated best with the circulating liver isoenzyme activity. The findings suggest that altered hepatic metabolism is responsible for the increased urinary excretion of phosphoethanolamine, and perhaps phosphoserine, in hypophosphatasia.
Creatinine: a review.
Authors: Narayanan S , Appleton H D
Published: January 2020

Measurement of creatinine has many applications. We review the determination of urinary creatinine as a valid index of completeness of 24-h urine collection, the clinical utility of the determination of creatinine clearance ratios, and measurement of the ratio of the clearance of specific analytes, such as amylase, to the ratio of clearance of creatinine. The chemistry and variables that affect the Jaffé reaction are reviewed, and attempts at improvement of specificity are discussed. We also review and assess techniques other than the Jaffé reaction for measurement of creatinine.
Identification of fluorescent Pseudomonas species.
Authors: Shelly D C , Quarles J M , Warner I M
Published: January 2020

Fluorescent pseudomonads may be identified through examination of the fluorescence "profiles" of diffusible pigments released into the growth medium. The profiles are obtained in the form of an emission-excitation matrix, the elements of which correspond to fluorescence intensity as a function of multiple exciting and emitting wavelengths. Use of a rapid scanning fluorometer to acquire these data provides a relatively fast method for identification of the microorganisms after incubation. This procedure can potentially be expanded for the identification of other microorganisms.
Magnetizable solid-phase fluoroimmunoassay of phenytoin in disposable test tubes.
Authors: Kamel R S , Landon J , Smith D S
Published: January 2020

We developed a fluoroimmunoassay for phenytoin in serum or plasma, based on the magnetic separation technique. The method involves sheep anti-phenytoin serum coupled to magnetizable cellulose/iron oxide particles, with a fluorescein-labeled phenytoin analog as tracer. After magnetic sedimentation of the solid phase from assay mixtures, the free fraction of the tracer is aspirated, removing endogenous fluorophores and other interfering components of the sample. The antibody-bound tracer is then eluted from the solid phase into a methanolic buffer medium and quantitated fluorometrically. The entire procedure, including fluorometry, is performed within disposable polystyrene test tubes. The assay involves only simple reagents and equipment, and correlates closely with established radioimmunoassay (r = 0.97) and gas-liquid chromatographic (r = 0.98) techniques.
beta-Lipoprotein quantitation in cord blood spotted on filter paper: a screening test.
Authors: Vladutiu G D , Glueck C J , Schultz M T , McNeely S , Guthrie R
Published: January 2020

We describe an electroimmunodiffusion technique for measuring beta-lipoprotein in cord blood spotted on filter paper. A series of cord-blood samples, taken from 916 consecutive live-birth infants, was spotted directly onto filter paper and assayed for beta-lipoprotein. Eleven had above-normal beta-lipoprotein. Of these 11, seven were tested two to six months later, along with their parents, for total cholesterol and triglyceride concentrations. Five infants had increased cholesterol values, and four parents of these infants had either increased cholesterol or triglyceride values. We also measured beta-lipoprotein concentrations in 63 paired samples of dried cord-blood and three-day post-delivery blood specimens, routinely collected for phenylketonuria screening. We saw a significant correlation between results for the specimens, but detected no cases of increased beta-lipoprotein. beta-Lipoprotein assay in dried specimens of cord blood is simple, inexpensive, and potentially is useful in mass screening of newborns for familial type II and combined hyperlipidemia.
Assay of delta-aminolaevulinate dehydratase in 10 microL of blood.
Authors: Wigfield D C , Farant J P
Published: January 2020

A method is presented for the assay of delta-aminolaevulinate dehydratase (EC 4.2.1.24) to be performed on 10 microL of whole blood. Results compare favorably with those obtained with an accepted method (J. Clin. Chem. Clin. Bio-chem. 12: 389-390, 1974) that involves use of 200 microL of blood.
Colorimetric vs liquid-chromatographic determination of urinary 5-hydroxyindole-3-acetic acid.
Authors: Tracy R P , Wold L E , Jones J D , Burritt M F
Published: January 2020

The popular colorimetric assay for urinary 5-hydroxyindole-3-acetic acid based on the reaction with 1-nitroso-2-naphthol has been criticized for lack of specificity. Goldenberg (Clin. Chem. 19: 38-44, 1973) proposed a modification of this method, in which 2-mercaptoethanol is used to discharge unwanted chromophores. We compared results by both the unmodified and modified colorimetric methods with those by a "high-performance" liquid-chromatographic method, with fluorometric detection. Agreement among all three assays was excellent in the range of 50 to 600 mg/24 h, but there were considerable differences in the range 0 to 30 mg/24 h between results by the unmodified colorimetric assay and the other two. In this range, results by the modified assay (y) correlated well with those by the chromatographic method (x), although the values were slightly lower (y = 0.85x + 0.11, r = 0.9303). Using the modified colorimetric assay to analyze 40 normal specimens, we defined a normal range of 0 to 6 mg/24 h). We recommend the modified colorimetric assay for use in the high-volume laboratory and either this assay or the liquid-chromatographic method for the smaller laboratory.
Measurement of serum lipase activity with the oxygen electrode.
Authors: Griebel R J , Knoblock E C , Koch T R
Published: January 2020

We describe the measurement of lipase (triacylglycerol lipase; EC 3.1.1.3) in serum by continuous monitoring of relative rates of oxygen consumption with a polarographic oxygen electrode. The reactions described by Proelss and Wright (Clin. Chem. 23: 522-531, 1977) are used: trilinolein, with lipase catalysis, yields linoleic acid, which reacts with oxygen in the presence of lipoxygenase. Lipase activity is measured by comparing the zero order reaction rate of the specimen with that obtained with linoleic acid standards. Results for lipase (y) correlated well with those by the copper-soap method of Myrtle and Zell (x) (Clin. Chem. 21: 1469-1473, 1975); y = 0.33x - 11; r = 0.98; n = 34. Results are unaffected by specimen dilution, and the standard curve is linear to 520 U/L. Day-to-day precision (CV) is 10.4% for normal activities, 6.1% for above-normal activities. We believe the method offers a precise, practical approach to lipase analysis.
Combined enzymic-Jaffé method for determination of creatinine in serum.
Authors: Masson P , Ohlsson P , Björkhem I
Published: January 2020

Concentrations of creatinine, as determined in serum by a method involving the combined use of creatinine amidohydrolase (EC 3.5.2.10) and alkaline sodium picrate were found to be factitiously low, owing to a reversal of the enzyme reaction. This effect could be eliminated by converting creatine, the product of the enzymic reaction, to creatine phosphate. The combined enzymic-Jaffé method was therefore modified to include creatine amidohydrolase, creatine kinase, ATP, and Mg2+ in the reaction mixture. The modified method has good precision. We saw no significant interferences by relatively high concentrations of acetone, acetylacetone, ADP, creatine, creatine phosphate, glucose, glycocyamine, or pyruvate. Likewise, no interferences were evident with icteric, lipemic, or hemolytic serum samples. There was an excellent agreement between creatinine values obtained with our method and by a reference method based on isotope dilution-mass fragmentography. Our method is considerably simpler than the fully enzymic method for determination of creatinine and might be a method of choice if a high accuracy is desired.
Creatine kinase B-subunit activity in human sera: temporal aspects of its sensitivity after myocardial infarction.
Authors: Elser R C , McKenna K
Published: January 2020

We assessed the changes in sensitivity and specificity of creatine kinase (CK) isoenzymes at various times after acute myocardial infarction. CK-B was measured by an immunoinhibition technique. CK-MB was measured both by column chromatography on Sephadex DEAE A-50 and by electrophoresis on agarose gel. For CK-B, the sensitivity in detecting infarct varied from 11% in the first 8 h after onset of symptoms to 80% at 32 h after the infarct. Sensitivities for CK-MB by column and by electrophoresis were 25 and 45% for the first 8-h period and 80 and 89% at the 32nd hour, respectively. Comparison of values for CK-B with values for CK-MB by column and by electrophoresis gave correlation coefficients of 0.935 and 0.896, respectively. Patients having macro CK-BB exhibit anomalously high values for CK-B with respect to total CK. Patients having a mild infarct may show no increase in CK-isoenzymes.
Purification of lactate dehydrogenase isoenzyme-5 from human liver.
Authors: Pettit S M , Nealon D A , Henderson A R
Published: January 2020

We present a method for preparing human liver lactate dehydrogenase (L-lactate:NAD+ oxidoreductase; EC 1.1.1.27) isoenzyme-5 by sequential ion-exchange chromatography, general-ligand (AMP analog) affinity chromatography, and preparative isoelectric focusing. The yield ws 40%, with a 493-fold purification. The final specific activity was 458 kU per gram of protein. The preparation contained less than 0.2% of lactate dehydrogenase isoenzyme-4, was homogeneous by agarose gel electrophoresis and also by polyacrylamide gel electrophoresis at pH 8.9 and 6.9, and showed one major protein band (containing all the enzyme activity) and one minor anodic contaminant (containing no enzyme activity) by analytical isoelectric focusing. The enzyme had a mean pI value of 9.59 (SD 0.04) (n = 5) at 5 degrees C. By comparison, the pI value of a preparation of rabbit lactate dehydrogenase-5 was 9.16 (5 degrees C).
A candidate reference method for determination of total protein in serum. I. Development and validation.
Authors: Doumas B T , Bayse D D , Carter R J , Peters T , Schaffer R
Published: January 2020

We developed a candidate Reference Method for measuring total serum protein by use of the biuret reaction. The method involves a previously described biuret reagent (Clin. Chem. 21: 1159, 1975) and Standard Reference Material (SRM) 927 bovine albumin (National Bureau of Standards) as the standard. At 25 degrees C, color development for 30 or 60 min provides identical serum protein values. Glucose (up to 10 g/L) and bilirubin (up to 300 mg/L) do not interfere. Hemoglobin, at 3 g/L, increases apparent serum protein by 0.4 g/L. The presence of dextran in serum causes easily detected turbidity, but this interference can be eliminated by centrifuging the reaction mixture. Therapeutic concentrations of ampicillin, carbenicillin, penicillin, oxacillin, nafcillin, chloramphenicol, cephalothin, and methicillin in blood do not interfere, nor do triglycerides up to 10 g/L. Within-run and day-to-day standard deviations of the method are 0.1 and 0.4 g/L, respectively.
Immunoreactive trypsin and pancreatic isoamylase activity in serum of patients with chronic renal failure or hepatic cirrhosis.
Authors: Fahrenkrug J , Staun-Olsen P , Magid E
Published: January 2020

In 121 patients with either liver cirrhosis or chronic renal failure, abnormal values for the concentrations of two pancreatic enzymes in serum were a frequent finding. In renal insufficiency a decreased rate of enzyme elimination is the most likely cause of the above-normal values we observed for serum immunoreactive trypsin and pancreatic isoamylase activity. As for patients with liver cirrhosis, we believe that changes in entrance rates into the blood--i.e., an affected pancreas--is a likely explanation of the abnormally high values we often found for these serum enzymes.
[14C]Oxalate absorption by normal persons, calcium oxalate stone formers, and patients with surgically disturbed intestinal function.
Authors: Tiselius H G , Ahlstrand C , Lundström B , Nilsson M A
Published: January 2020

Intestinal absorption of oxalate can be judged from the urinary excretion of orally administered [14C]oxalate. Fifteen normal subjects, 21 patients with "idiopathic" calcium oxalate stone disease and a high oxalate excretion, four patients operated with ileocecal resection, and seven patients operated with jejunoileal bypass were so investigated. We saw no significant difference in the amount of isotope excreted by normal subjects and idiopathic stone formers; 13.6% (SD 5.9%) and 14.4% (SD 6.5%), respectively, of the administered dose was accounted for in the urine. The patients with resection or bypass showed a quite different pattern of isotope excretion, and 18.3% (SD 7.0%) and 36.8% (SD 14.0%), respectively, of the isotope was accounted for in the urine.
Monoclonal antibodies in clinical immunology.
Authors: Sevier E D , David G S , Martinis J , Desmond W J , Bartholomew R M , Wang R
Published: January 2020

Advances in clinical immunodiagnostics resulting from the application of hybridoma technology are starting to appear. Monoclonal antibodies are beginning to displace their antiquated progenitors, polyclonal antisera, in many facets of immunology. Their homogeneity, specificity, and availability make hybridoma-derived antibodies the immunological reagents of the future in immunoassays, immuno-affinity chromatography, immunotherapy, and areas yet to be defined.
Lactate dehydrogenase isoenzyme-1: changes during the first day after acute myocardial infarction.
Authors: Bruns D E , Emerson J C , Intemann S , Bertholf R , Hill K E , Savory J
Published: January 2020

We studied the time course of change of lactate dehydrogenase isoenzyme-1 (LD-1) in serum of patients suspected of having had an acute myocardial infarction. LD-1 was measured at intervals of 4-8 h during the first and second hospital days, by an immunochemical method. Of the 65 patients in this study, 26 had acute myocardial infarctions by traditional criteria. The ratio of LD-1 to total LD had greater diagnostic value than did LD-1 alone. In 90% of patients with myocardial infarction this ratio was increased within 12 h of admission, and all had increased ratios within 24 h. The false-positive rate was less than 1%, and an increased LD-1/total LD ratio had a predictive value of 96% for myocardial infarction. These results suggest that LD-1 is useful in the diagnosis of myocardial infarction on the first day of hospitalization.
Low-density-lipoprotein apoprotein B in plasma as measured by radial immunodiffusion and rocket immunoelectrophoresis.
Authors: Havekes L , Hemmink J , de Wit E
Published: January 2020

Radial immunodiffusion (RID) and rocket immunoelectrophoresis (RIE) are compared with respect to determination of LDL-bound apo B in plasma. Isolated VLDL could not enter a 15 g/L agarose gel when either technique was used. However, in the presence of plasma proteins, migration of VLDL into agarose was enhanced. Only when plasma samples were kept frozen before the assay was plasma VLDL unable to enter the agarose gel when RID was used. With RIE the contribution of plasma VLDL to the apo B determination under these conditions was not always negligible. Besides enhancing the entry of VLDL into the agarose, the presence of proteins also influences apo B immunoreactivity of LDL and VLDL. For measuring LDL-bound apo B directly in unfractionated plasma we recommend: (a) RID in 15 g/L agarose gel; (b) freezing the plasma samples before assay; (c) diluting the plasma samples in saline supplemented with protein in the same concentration as is present in plasma (70 g/L); and (d) using plasma as the assay standard.
A direct radioimmunoassay for aldosterone in unextracted serum and plasma.
Authors: Lee T P , Tan C H
Published: January 2020

We describe a novel radioimmunoassay procedure for the direct estimation of aldosterone in unextracted plasma and serum samples, in which interfering binding proteins are digested by Proteinase K (Tritirachium alkaline proteinase, EC 3.4.21.14), a powerful proteolytic enzyme. Heating at 75 degrees C for 15 min inactivates the enzyme before radioimmunoassay. Alternatively, EDTA may be used to inactivate the enzyme. The antibody-bound fraction is then precipitated with polyethylene glycol and isolated by centrifugation. This easy method eliminates extraction and purification and gives accurate and reliable results. The total time required for 100 estimations, including counting time, is about 6 h.
Simultaneous quantitation of zinc protoporphyrin and free protoporphyrin in erythrocytes by acetone extraction.
Authors: Hart D , Piomelli S
Published: January 2020

Erythrocyte protoporphyrin content is increased in several human and veterinary disorders in which heme synthesis is disrupted. The nature of the defect determines whether zinc protoporphyrin, unchelated (free) protoporphyrin, or both are present in the erythrocytes. Extraction of the protoporphyrin into acetone/water (80/20 by vol) is a simple, rapid technique by which each protoporphyrin species is delivered into solution without removing zinc from zinc protoporphyrin. The ratio of zinc protoporphyrin to total protoporphyrin concentration correlated significantly with the ratio of fluorescence emission intensities at the zinc protoporphyrin peak and at an isosbestic point. This concentration ratio may be used with a quantitative measure of total erythrocyte protoporphyrin to define the absolute zinc protoporphyrin and free protoporphyrin content of erythrocytes.
Serum iron and total iron-binding capacity compared with serum ferritin in assessment of iron deficiency.
Authors: Peter F , Wang S
Published: January 2020

Ferritin values for 250 selected sera were compared with values for iron, total iron-binding capacity (TIBC), and transferrin saturation, to assess the potential of the ferritin assay for the detection of latent iron deficiency. The specimens were grouped (50 in each group) according to their values for iron and TIBC. In Group 1 (low iron, high TIBC) the saturation and ferritin values both indicated iron deficiency in all but one. In the 100 specimens of Groups 2 (normal iron, high TIBC) and 4 (normal iron, high normal TIBC), the saturation values revealed 16 iron-deficient cases, the ferritin test 55. For Groups 3 (low iron, normal TIBC) and 5 (low iron, low TIBC), the ferritin test revealed fewer cases of iron deficiency than did the saturation values (37 cases vs 51 cases, in the 100 specimens). Evidently the ferritin test detects iron deficiency in many cases for whom the serum iron and TIBC tests are not positively indicative. The correlation of serum ferritin with iron, TIBC, and transferrin saturation in the five groups was good only in the case of specimens for which the TIBC was normal; if it was abnormal the correlation was very poor.