bluuebunny/crossref_metadata_2025 huggingface dataset:
Simultaneous measurement of phenobarbital, phenytoin, primidone, ethosuximide, and carbamazepine in serum by high-pressure liquid chromatography.
Authors: Kabra P M , Stafford B E , Marton L J
Published: January 2020

We present a method for simultaneously determining five anticonvulsants [phenobarbital, phenytoin (diphenylhydantoin), primidone, ethosuximide, and carbamazepine] in as little as 25 microliters of serum. The proteins are precipitated with an acetonitrile solution containing hexobarbital as an internal standard. The anticonvulsants are eluted from a reversed-phase column with a mobile phase consisting of an acetonitrile/phosphate buffer (19/81 by vol) at a flow rate of 3.0 ml/min. The eluted drugs are detected by their absorption at 195 nm, and quantities estimated from their peak heights. Each analysis requires about 14 min. at an optimum column temperature of 50 degrees C. The lower unit of detection for all of these drugs is less than 10 ng. Sensitivities, for serum samples, of 1.0 mg/liter for all the drugs analyzed are attained routinely. Analytical recoveries for the five drugs varied from 97 - 107%, with good day-to-day precision (CV between 3.9 and 5.9%). Of more than 30 drugs tested for possible interference, only ethotoin interferes with the analysis of phenobarbital.
Creatine kinase isoenzyme activity in human placenta and in serum of women in labor.
Authors: Laboda H M , Britton V J
Published: January 2020

Creatine kinase (EC 2.7.3.2) isoenzymes in extracts of human placenta and in serum from nonpregnant women and women in labor were separated on columns containing diethylaminoethyl-cellulose and assayed. The distribution of the isoenzymes in placenta (n = 10) was 80% BB (200 +/- 66 U/g (wet weight), 19% MM (49 +/- 30 U/g), and 1% MB (2.6 +/- 1.7 U/g); The geometric mean for the serum BB activity of the nonpregnant women (n = 50) was 0.6 +/- 1.5 U/liter, as compared to 3.0 +/- 1.4 U/liter for patients in labor who had normal deliveries (n = 92). The arithmetic mean for serum BB activity of labor patients with induced labor (n = 20), premature labor (n = 7), cesarian section (n = 6), or hypertension and pre-eclampsia (n = 6) did not differ significantly from the arithmetic mean BB activity for serum of labor patients with normal deliveries. However, the arithmetic mean serum BB activity of patients with stillbirths (n = 7) was significantly smaller than the arithmetic mean for normal labor patients.
Measurement of free thyroid hormones in serum by dialysis and gas chromatography.
Authors: Petersen B A , Giese R W , Larsen P R , Karger B L
Published: January 2020

We describe a simultaneous, absolute analysis for free (dialyzable) triiodothyronine and thyroxine in serum by the sequence: dialysis, internal standard addition to the dialysate, cation-exchange chromatography, derivatization, and gas chromatography with electron capture detection. The detection limit is 0.2 pg and the precision (repeatability) in the range of 4 to 20 ng/liter is 1.6%. Mean values for free T3 and T4 in 29 euthryoid sera were 4.2 and 18.9 ng/liter, respectively, in close agreement with accepted values. Correlation coefficients for the two compounds were 0.99 and 0.97, respectively, when this procedure was compared with a method involving tracer dialysis and radioimmunoassay. A chromatographic peak was seen for free 3,3',5'-triiodothyronine in the analyses of two of eight hyperthyroid sera, and, with a larger sample volume, from a euthyroid serum. The new method is a potential reference and research procedure for analysis of free iodothyronines.
Recent developments in the clinical assessment of the metabolism of aromatics by high-performance, reversed-phase chromatography with amperometric detection.
Authors: Kissinger P T , Bruntlett C S , Davis G C , Felice L J , Riggin R M , Shoup R E
Published: January 2020

High-performance liquid chromatography with electrochemical detection has developed into a tool with excellent capability to monitor picomole amounts of individual metabolites in both tissue and body-fluid specimens. Significant technical advances in the area of microparticle chemically-bonded stationary phases have led to dramatic improvements in both sensitivity and resolution. Reversed-phase systems can be modified to include charged exchange sites by addition of detergents to the mobile phase. Adjustment of the surface charge in this manner permits capacity factors for ionic sample components to be increased or decreased. This concept is quite compatible with electrochemical detection and has provided the foundation for several clinical assays now in routine use. Examples of additional applications are presented for the determination of catecholamines in tissue, 3-methoxy-4-hydroxyphenylglycol in urine, and dopamine-beta-hydroxylase activity in human serum.
A rate nephelometer for measuring specific proteins by immunoprecipitin reactions.
Authors: Sternberg J C
Published: January 2020

A kinetic nephelometric method and instrument have been developed for the rapid determination of specific serum proteins by means of immunoprecipitin reactions. The maximum rate of change of scattered light intensity in an antigen-antibody reaction can be made to occur within 60 s after initiation of the reaction and provides a measure of the antigen concentration under antibody excess conditions. A mathematical relationship has been found for the conversion of the nonlinear maximum rate data directly into a linear concentration read-out, making possible the use of single-point calibration. Instrument operating parameters and computations are programmed for a particular analysis by means of machine-readable cards. Antigen-excess samples are detected rapidly by injection of calibrator into the reaction mixture after the rate signal has dropped to a pre-selected level. The method correlates well with both radial immunodiffusion and end-point nephelometric methods.
Assay procedures for polyamines in urine, serum, and cerebrospinal fluid.
Authors: Seiler N
Published: January 2020

Putrescine (1,4-diaminobutane) and the polyamines spermidine and spermine have attracted interest as possible markers of malignancy and of response to chemotherapy. The assay methods currently in use for these amines are reviewed and compared in respect to sensitivity, reproducibility, and applicability to routine analysis for them in urine, blood, and cerebrospinal fluid.
Determination of acetaminophen concentrations in serum by high-pressure liquid chromatography.
Authors: Horvitz R A , Jatlow P I
Published: January 2020

We describe a method for determination of serum acetaminophen concentrations in serum by reversed phase high-pressure liquid chromatography. The homolog N-propionyl-p-aminophenol was used as an internal standard. The procedure, which requires only a single extraction with diethyl ether, can be optimized to be linear over the ranges of 10 to 100 or 1 to 20 mg/liter. Within-run CV was 1.2%; between-run CV was 4.4% and 4.9% at two different concentrations. Many commonly used drugs were tested and found not to interfere. The procedure is simple and rapid enough for use on an emergency basis in cases of overdosage, and can be optimized for measurement of either therapeutic or toxic concentrations.
An automated competitive binding procedure for measuring thyroxine in serum.
Authors: Hofman L F , Bouley A M , Barron E J
Published: January 2020

A competitive binding assay for serum thyroxine has been automated, with the use of small, reusable Sephadex columns to separate thyroxine from endogenous thyroid-binding globulin and later to separate the bound and free thyroxine. Sixty samples an hour are run through columns, which are held in an aluminum turntable rotated by a fraction collector motor. Reagents and samples are fed to the columns by a proportioning pump. Waste eluates are collected and drained to the sink by a Teflon tray positioned between the columns and counting tubes, also held by the turntable. A cut out area in the tray allows one to collect the bound fraction, which can then be counted in a scintillation counter. Values are calculated and printed by a desk calculator interfaced with the scintillation counter. The "day-to-day" CV for this method is 5%. The accuracy is satisfactory when tested by comparison with other methods, by recoveries, and by linearity of dilutions.
Simultaneous radioimmunoassay of thyrotropin and thyroxine in human serum.
Authors: Bluett M K , Reiter E O , Duckett G E , Root A W
Published: January 2020

The importance of early diagnois and treatment of congenital hypothyroidism has been well established, and several screening programs have been undertaken to detect neonates with this disorder by measurement of concentrations of thyrotropin or thyroxine in the serum. However, measurement of either hormone alone may fail to identify all affected patients. Accordingly, we have established a simultaneous double-antibody, dual-isotope radioimmunoassay for both. Sensitivity, slope, analytical recovery, and precision characteristics of the simultaneous assay do not differ from those of each assay performed separately. Values for the two analyses in the single and simultaneous assays correlate well (r = 0.951 for thyroxine, 0.983 for thyrotropin). This assay system permits determination of both hormones within 72 h after specimen collection and thus should allow more rapid evaluation, diagnosis, and treatment of infants with congenital hypothyroidism.
Plasma free amino acid patterns in pregnancy: relationships to gestational age and applications to detection of fetal distress.
Authors: Schoengold D M , deFiore R H
Published: January 2020

Data on free amino acids in serial samples of plasma from 15 pregnancies from 14 women have been compared to data from normal nonpregnant women by use of pattern-recognition program. The overall amino acid patterns were found to be a reproducible function of gestational age. Two samples from a woman with a pregnancy terminated by a spontaneous abortion were clearly resolved from all the other samples by this procedure.
High-performance liquid chromatographic assay for 25-hydroxyvitamin D3 in serum.
Authors: Gilbertson T J , Stryd R P
Published: January 2020

We describe a precise and specific method for measuring 25-hydroxyvitamin D3 in 1 ml of human serum. Extraction with chloroform/methanol followed by chromatography on a 0.5 X 2-cm silica gel column yields a sample that is sufficiently free of extraneous material for high-performance liquid chromatography on a column of microporous silica gel (10 micron average particle diameter). The measurement is not influenced by vitamins D2 or D3, 25-hydroxyvitamin D2, or any of the more hydroxylated metabolites of the vitamin D group. Results by this method correlate well with a competitive protein-binding assay (r = 0.961), but with a negative bias of 6.9 +/- 3.3 microgram/liter. We measured concentrations of 25-hydroxyvitamin D3 in serum drawn during February from 24 persons who were judged normal by physical examinations. The range was 5.5-23.8 microgram/liter (mean, 15.0 +/- 5.2 microgram/liter). The day-to-day CV for the assay was 5.46%.
Evaluation of ampouled tonometered buffer solutions as a quality-control system for pH, pCO2, and pO2 measurement.
Authors: Maas A H , Veefkind A H , Van den Camp R A , Teunissen A J , Winckers E K , Jansen A P
Published: January 2020

In response to the need for an adequate quality-control system for blood-pH and blood-gas analyzers, we investigated the practical application of ampouled phosphate-bicarbonate-chloride solutions tonometered with mixtures of carbon dioxide, oxygen, and nitrogen. This system offers three discrete sets of pH, pCO2, AND PO2 values, which are consistent with normal and pathophysiologically high and low values. The stated values were based on the U.S. National Bureau of Standards scale for pH and on gas analysis for pCO2 and pO2. Influence of temperature, air contact, calibration gas, and storage was established. Internal and external quality control by means of these ampoules is presented. The system is stable, accurate, precise, and suitable for simultaneous quality control of pH, pCO2, and pO2 measurements.
Measurement of antitryptic activity of serum with a centrifugal analyzer.
Authors: Konings C H
Published: January 2020

The Selected Method [Clin. Chem. 20, 396 (1974)] for the enzymatic assay of alpha1-antitrypsin in serum has been adapted for use with the E.N.I.-GEMSAEC. With alpha-N-benzoyl-D,L-arginine-p-nitroanilide as substrate, the difference between the tryptic activity measured with and without addition of serum in the same run has been used to calculate the trypsin-inhibitory capacity. The rate of increase in absorbance at 400 nm of the p-nitroanilide formed, has been evaluated during a reaction time of 140 s. Results correlated well (r = 0.986) for 54 human sera analyzed as described here and by the Selected Method. The adaptation on GEMSAEC can be used in detecting alpha1-antitrypsin deficiency in the newborn.
Semiautomated spectrophotometry of total phospholipids in plasma.
Authors: Wootton D G
Published: January 2020

A one-stage semiautomated method for estimating total phospholipids in plasma is reported. The phospholipids are extracted into aqueous ethanol, combined with a Lewis acid, and the change in pH is monitored colorimetrically. The method, compared with the acid digestion method of Gomori [J. Lab. Clin. Med. 27, 955 (1942)], gives results that are about 0.25 mmol/liter lower, but seems to be a suitable and less hazardous alternative.
Gas-liquid chromatographic determination of nicotine and cotinine in plasma.
Authors: Hengen N , Hengen M
Published: January 2020

We report a procedure for determining nicotine and cotinine in plasma. Nicotine is extracted from 1 ml of plasma with diethyl ether, back extracted, and analyzed by gas-liquid chromatography with a nitrogen/phosphorus detector. Nicotine and its internal standard, modaline, had retention times of 1.9 and 2.9 min, respectively. Cotinine is then extracted from the same plasma with dichloromethane and similarly analyzed. Cotinine and its internal standard, lidocaine, had retention times of 3.8 and 4.9 min. Day-to-day reproducibilities (CV) within 14% for nicotine and within 6% for cotinine are attainable for the respective concentration ranges 1-100 microgram/liter and 1-200 microgram/liter. Nornicotine and related alkaloids do not interfere. The sensitivity was such that less than 0.1 microgram (0.62 nmol) of nicotine and 0.1 microgram (0.62 nmol) of nicotine and 0.1 microgram (0.57 nmol) of cotinine could be detected per liter.
Optimization of methods for aspartate aminotransferase and alanine aminotransferase.
Authors: Bergmeyer H U , Scheibe P , Wahlefeld A W
Published: January 2020

Conditions for accurate measurement of catalytic activity of aspartate aminotransferase and alanine aminotransferase in human serum have been reinvestigated. The basic variables (kind of buffer, buffer concentration, pH, ion effects, and the influence of pyridoxal-5-phosphate) can now be considered optimized. On this basis, the kinetic parameters of both aminotransferases were determined, i.e., Michaelis and inhibitor constants for substrates and reaction products. With a mathematical approach for two-substrate enzyme reactions the substrate concentrations were calculated from the viewpoints "most economical," "most convenient," and "lowest variability." Also the conditions for the indicator reactions have been newly defined with respect to a kinetic model. All calculated data were rechecked experimentally and it can be shown that both approaches fully agree. Furthermore, we show that the mathematical approach allows more precise recommendations for optimized methods. For technical reasons, the catalytic activity of aspartate aminotransferase in human serum can only be measured as a 0.96 fraction of its theoretical maximum velocity, the catalytic activity of alanine aminotransferase as a 0.91 fraction. The assay conditions for a Reference Method are finally described and recommendations are made for optimized routine methods for determination of the catalytic activity of these transferases in human serum.
Liquid-chromatographic procedure for tricyclic drugs and their metabolites in plasma.
Authors: Vandemark F L , Adams R F , Schmidt G J
Published: January 2020

We describe a procedure for determining amitriptyline and imipramine and their active metabolites nortriptyline and desipramine, respectively, at therapeutic concentrations in human plasma by use of liquid chromatography. The drugs are extracted at pH 10.5 into hexane/isoamyl alcohol, which is evaporated and the residue chromatographed. Protriptyline is used as the internal standard. As little as 10 microgram of each drug per liter could be detected in plasma, the limit being established by variability in drug-free plasmas. The day-to-day coefficient of variation for each drug at a concentration of about 100 microgram/liter was about 7%. Doxepin and diphenhydramine interfere with the analysis of amitriptyline. Total analysis time for a single sample is 20 min.
Kinetics of the reactions of unconjugated and conjugated bilirubins with p-diazobenzenesulfonic acid.
Authors: Landis J B , Pardue H L
Published: January 2020

We report the kinetics of the reactions of unconjugated bilirubin and conjugated bilirubin with p-diazobenzene sulfonic acid in aqueous media. Our studies confirm that each reaction proceeds in two steps and that the second step is catalyzed by sulfanilic acid. In the presence of an excess of p-diazobenzenesulfonic acid and in the absence of sulfanilic acid, the reaction for either unconjugated or conjugated bilirubin proceeds in two successive first-order steps, the second step being much the slower. This study emphasizes the first step in the reaction for each species and includes data on the effects of p-diazobenzenesulfonic acid, albumin, benzoate, and caffeine concentrations, pH in the range from 4 to 12, and temperature. Mechanisms proposed for reactions with and without caffeine are used to develop rate equations, and the kinetic data are used to evaluate rate constants, acid dissociation constants for the different bilirubin species, and formation constants for bilirubin-caffeine complex species that are proposed.
Improved micromethod for determination of underivatized clonazepam in serum by gas chromatography.
Authors: Edelbroek P M , De Wolff F A
Published: January 2020

We describe a gas-chromatographic micromethod, with use of a solid injection system and an electron capture detector, for determination of underivatized clonazepam in serum. Vigorous mixing of 100 microliter of serum with 20 microliter of borate buffer (pH 9.0) and 100 microliter of a cyclohexane/dichloromethane mixture (4/6 by vol) containing methyl nitrazepam as internal standard suffices to extract 86% of the clonazepam into the organic layer. We obtained a linear response curve for clonazepam in serum in the concentration range 5 to 150 microgram/liter. The lowest detectable concentration by our method is about 1 microgram/liter of serum. Interference by several anticonvulsant drugs, metabolites, and related compounds was investigated. The assay is simple, rapid, and suitable for clinical routine determination of clonazepam in serum in therapeutic concentrations. We illustrate the steady-state concentration of clonazepam in serum during chronic oral administration to 28 patients.
Drug screening by enzyme immunoassay with the American Monitor KDA.
Authors: Pearson J R
Published: January 2020

The methods for barbiturate, opiate, methadone, and amphetamine have been modified for use with the American Monitor KDA. The modification, which incorporates automatic correction for endogenous lysozyme activity, was evaluated by comparing results obtained with the KDA for human urine samples containing known amounts of drug(s) with results obtained with the procedure recommended by Syva for the Gilford 3500. There was 98% agreement bewteen the two systems. Six calibrators and 40 samples can be assayed for all four drugs in about 2.5 h. The procedure has proven to be reliable for screening urine samples obtained from clients at a methadone treatment center.
Additive, multiplicative, and mixed analytical errors.
Authors: Werner M , Brooks S H , Knott L B
Published: January 2020

Depending on the change in magnitude of analytical error occuring with change in concentration of analyte, two limiting types of error properties can be defined: additive and multiplicative. We investigated whether one of these two error types also characterizes the overall error of methods involving multiple procedural steps, or whether mixed error properties result in these cases. Using "open" quality-control data (i.e., and analyst identifies controls) from each of two hospitals and "blind" quality-control data from one hospital for 11 different assays, we found: (a) With current methodology, overall errors typically are mixed, though predominantly additive and multiplicative overall errors exist as well. (b) "Blinding" the quality-control system typically augments the multiplicative but not the additive error component.
Radioenzymatic assay of DOPA (3,4-dihydroxyphenylalanine).
Authors: Johnson G A , Gren J M , Kupiecki R
Published: January 2020

We modified the single-isotope radioenzymatic assay for catecholamines [Life Sci. 21, 625 (1977)] to assay 3, 4-dihydroxyphenylalanine (DOPA). DOPA decarboxylase is used to convert DOPA to dopamine, which concurrently is converted to [3H]-3-O-methyldopamine in the presence of catechol-O-methyltransferase and [methyl-3H]-S-adenosylmethionine and assayed radioenzymatically. For assay of plasma DOPA, 50 microliter of untreated plasma is added directly into the incubation mixture. A duplicate mixture containing an internal standard requires a second 50-microliter aliquot of plasma. Because the assay measures both DOPA and endogenous dopamine, two additonal aliquots of plasma must be assayed for dopamine in the absence of the decarboxylase by the differential assay; DOPA is estimated by difference. The assay is sensitive to 25 pg (500 ng/liter of plasma). Analysis of DOPA (DOPA plus dopamine) and the concurrent differential assay of catecholamines in at least 10 samples can be done in a single working day. Plasma DOPA concentrations for 42 normotensive adults were 1430 +/- 19 ng/liter (mean +/- SEM). In contrast, do-pamine concentrations for these same subjects averaged 23 +/- 20 ng/liter. Values for the 24 women subjects (15 10 +/- 62 ng/liter) significantly (P = 0.04) exceeded those for the men (1320 +/- 75 ng/liter).
Aspartate aminotransferase activity and isoenzyme proportions in human liver tissues.
Authors: Rej R
Published: January 2020

Aspartate aminotransferase (EC 2.6.1.1) activity and the distribution of its isoenzymes in human liver were examined. Rabbit antiserum against porcin soluble (i.e., non-mitochondrial) enzyme cross-reacted with the soluble enzyme of human origin and was used in an immunoprecipitation assay to quantitate the soluble and mitochondrial isoenzymes. These were separated by rapid, semiquantitative electrophoresis on cellulose acetate and by three other quantitative techniques: isoelectric focusing and anion-and cation-exchange chromatography. The mitochrondrial enzyme averaged 81% of the total activity in normal adult human liver (n = 4). Its contribution was dramatically reduced in single specimens of human fetal liver (56% of total activity) and hepatoblastoma tissue (38%). Total enzyme activities (mumol min-1 per gram of tissue) were: adult, 150; fetal, 38; tumor, 6. Total enzyme concentrations (micromoles of enzyme per kilogram of tissue) found were: adult, 10.8; fetal, 2.7; tumor, 0.4. The concentrations and isoenzyme distribution in human liver are compared to those in various animal model systems. Other methods for quantitative estimation of the isoenzymes and their adaptability for use in estimating concentrations in serum are reviewed.
Enzyme immunoassay and gas--liquid chromatography compared for determination of lidocaine in serum.
Authors: Pape B E , Whiting R , Parker K M , Mitra R
Published: January 2020

Lidocaine, an anti-arrhythmic 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 5.0 and a between-assay coefficient of variation of less than 6.5. Regression analysis of 87 serum samples analyzed by this technique (y) and by gas-liquid chromatography (x) gave the equation y = 0.96x - 0.03 (r = 0.99). Clinical evaluation of the results indicates the enzyme immunoassay technique to be highly specific and sensitive for lidocaine.
Improved liquid chromatographic determination of placental estriol in urine.
Authors: Gotelli G R , Wall J H , Kabra P M , Marton L J
Published: January 2020

Placental estriol in urine can be determined, after enzymatic hydrolysis, by liquid chromatography. A single-step extraction technique results in rapid processing with minimal sample manipulation. Analytical recovery of estriol added to urine was 96 to 106%. Day-to-day precision (CV), established by 10 replicate analyses at a mean concentration of 15.9 mg of estriol per liter, was 3.7%.
Radioimmunoassay for urinary lysozyme in human serum from leukemic patients.
Authors: Peeters T L , Depraetere Y R , Vantrappen G R
Published: January 2020

We present a radioimmunoassay for lysozyme in human serum, based upon human lysozyme isolated from the urine of leukemic patients and antiserum prepared against this lysozyme in the goat. In the separation step, a second antibody is used. By properly adjusting the concentrations of unlabeled and 125I-labeled lysozyme and of the antibodies, maximal precision (SD, 0.04 mg/litre) was obtained in the range 0.00 to 2.00 mg/litre. In 20 normal volunteers the lysozyme concentration was 4.6 +/- 0.8 mg/litre (mean +/- SD), in 13 patients with monocytic leukemia 34.4 +/- 8.6 mg/litre. Correlation with lysoplate determinations was excellent in leukemic sera (r = 0.97) but was poor in normal sera (r = 0.35), possibly owing to the existence of isoenzymes.
High-performance liquid chromatography of quinidine in plasma, with use of a microparticulate silica column.
Authors: Peat M A , Jennison T A
Published: January 2020

We describe a routine method for determining plasma concentrations of quinidine by liquid chromatography. The procedure requires 1.0 ml of plasma (or serum) and involves internal standard addition, extraction with ether, and separation on a column of microparticulate silica. Day-to-day CV (15 days) was less than 5% and no deterioration in column performance has been observed during 12 months. Comparison with a fluorometric procedure gave a correlation coefficient of 0.995.
Modified micro-scale enzymatic method for plasma ammonia in newborn and pediatric patients; comparison with a modified cation-exchange procedure.
Authors: Wu J , Ash K O , Mao E
Published: January 2020

We modified two "kit" procedures for plasma ammonia to make them suitable for use with newborn and pediatric patients. Available methods either require too large a specimen, are insufficiently sensitive in the normal range, or are too cumbersome. One was an enzymatic (Sigma Chemical Co., St. Louis, Mo. 63178), the other a cation-exchange (Hyland, Costa Mesa, Calif. 92626) method. The modified micro-scale enzymatic method requires 100 microliter of plasma (CV at 38 micronmol/liter, 8%). Reagent costs per patient assay, including controls and standards, are currently $0.53. An assay run can be completed in 30 min. The modified cation-exchange method requires 200 microliter of plasma; the within-run CV is 12% for a concentration of 34.2 micronmol/liter. Reagents currently cost $0.74 per assay and each run requires less than 2 h to complete. The correlation coefficient for the two methods was 0.98.
Radial diffusion assay of NAD- and NADP-dependent oxidoreductases.
Authors: Shih J C , Teulings E P
Published: January 2020

With a radial diffusion assay we measured the activity of six NAD- and NADP-dependent oxidoreductases: alcohol, glucose-6-phosphate, hydroxysteroid, lactate and malate dehydrogenases, and glutathione reductase. The enzyme was allowed to diffuse for 24h in an agarose gel in which the substrate was incorporated, then reacted the pyridine nucleotide coenzyme. The size of an enzyme diffusion zone could be made visible by the change of the fluorescence of the coenzyme against the background when the coenzyme was either oxidized or reduced. The procedure for each enzyme is reported. The results indicate that this new technique may be applicable to all NAD- and NADP-dependent enzymes. Because of its simplicity and potentiality for screening many samples, we think this method has applications in the clinical laboratory and in nutrition studies.
Assay of vitamins D2 and D3, and 25-hydroxyvitamins D2 and D3 in human plasma by high-performance liquid chromatography.
Authors: Jones G
Published: January 2020

I describe a new assay that is capable of measuring vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in 2 ml of plasma or serum. Plasma is extracted by the Bligh and Dyer technique [Can. J. Biochem. Physiol. 37, 911 (1959)], the lipid component is fractionated by two high-performance liquid-chromatographic systems based upon adsorption and reversed-phase chromatography, and each of the four vitamin D metabolites is measured by its absorbance at 254 nm. The method has a sensitivity limit of 0.5 mug/liter of plasma. The identity of metabolite peaks was confirmed by mass spectrometry, ultraviolet absorption spectrophotometry, and rechromatography, and there was good correlation (r=0.84) between plasma 25-hydroxyvitamin D as measured by the present method and by a protein binding assay developed in our laboratory. Mean concentrations of vitamin D and 25-hydroxyvitamin D in normal adults (n=25) in December were 2.2 +/- 1.1 (SD) and 16 +/- 3.9 (SD) mug/liter, respectively. 25-Hyroxyvitamin D2 made up 31% of the total 25-hydroxyvitamin D. Patients receiving pharmacological doses of vitamin D had values for vitamin D and 25-hydroxyvitamin D that were 10- to 100-fold normal. This method provides a rapid, reliable physico-chemical assay that appears to have advantages over existing protein binding assays and can be used to measure circulating vitamin D.
Simplified two-step column-chromatographic determination of taurine in urine.
Authors: Anzano M A , Naewbanij J O , Lamb A J
Published: January 2020

A two-step column-chromatographic procedure for accurate and rapid determination of taurine in urine is described. Sulfosalicyclic-acid deproteinized samples are chromatographed on a 0.9 X 10 cm column of cation-exchange resin (AG 50W-XB), with use of a pH 2.2 sodium citrate eluting buffer such that taurine and the more highly acidic compounds in urine are eluted in the void volume, and then on a 0.9 X 8 cm column of anion-exchange resin (AG 2-X8), from which taurine is preferentially eluted with 1 mol/liter acetic acid. The color developed with ninhydrin is directly proportional to taurine amounts as low as 0.01 mumol/sample. The method is highly reproducible, with analytical recoveries greater than 95%. The presence of 333 mumol of urea and 1 mumol of cysteic acid did not interfere in the analysis. When a mixture of C14-labeled amino acids other than taurine were co-chromatographed with taurine, less than 2% of the total counts loaded were located in the taurine fraction. Values for urinary taurine excretion by rats according to the present method agreed well with values obtained with an automated amino acid analyzer. Advantages of the present method for the determination of taurine are discussed.
D(-)-N-Methylglucamine buffer for pH 8.5 to 10.5.
Authors: Chromy V , Kulhánek V , Fischer J
Published: January 2020

A new amine buffer for the pH range 8.5 to 10.5 based on D(-)-N-methylglucamine and its hydrochloride is described. Important physical and chemical constants characterizing this buffer system have been determined: thermodynamic pKa = 9.52 (25 degrees C), buffer capacity = 0.055, dilution value pH 1/2 = -0.02, temperature coefficient dpKa/dT = 0.023, the heat of ionization delta H degrees = 36.93 kj mol-1. D(-)-N-Methylglucamine, which is available in highly purified form, is highly soluble in water. Thus one can prepare either buffer solutions or stable solid mixtures when combined with D(-)-N-methylglucaminium chloride. D(-)-N-Methylglucamine buffers are suitable for both biochemistry and pH control.
Radioimmunoassay of creatine kinase isoenzymes in human serum: isoenzyme BB.
Authors: Zweig M H , Van Steirteghem A C , Schechter A N
Published: January 2020

We describe a sensitive, specific radioimmunoassay for the BB isoenzyme of creatine kinase (CK-BB) in serum. A sequential saturation assay was used to achieve sufficient sensitivity to detect the isoenzyme in 100-microliter serum samples of all healthy persons and patients tested. Bound and free antigen were separated by a second antibody system. Large excesses of purified isoenzyme MM did not react in the assay. Cross reactivity of two preparations of CK-MB was only 1 to 7+. The 95th percentile of serum CK-BB in 208 healthy adults was 6.2 microgram/liter. Within-assay and between-assay precision ranged from 5.5 to 11.9% and 9.7 to 13.6%, respectively.
A solid-phase radioimmunoassay for vitamin B12 in serum, with use of radioiodinated tyrosine methyl ester of vitamin B12.
Authors: Endres D B , Painter K , Niswender G D
Published: January 2020

Although radioassays for vitamin B12 with use of any of several binding proteins have been available for many years, a radioimmunoassay for B12 has not been reported. We describe here such a radioimmunoassay, incorporating, for the first time, a radioiodinated tyrosine methyl ester of B12 as the radioactive tracer. Polypropylene tubes are coated with antiserum raised in a rabbit against B12/bovine serum albumin to simplify the separation of bound and free radioactivity. Factors affecting the preparation of coated tubes are described. The assay is accurate, sensitive, precise, and specific for vitamin B12. Accuracy of the assay is unaffected by the presence of denatured protein. The advantages of this radioimmunoassay over conventional radioassays are discussed.
Flameless atomic absorption spectrophotometric determination of manganese in rat brain and other tissues.
Authors: Bonilla E
Published: January 2020

I describe a method for determining manganese in biological tissues by flameless atomic absorption spectrophotometry, It is simple to perform and does not require prior extraction with solvents. Small tissue samples are dried for 48 h at 110 degrees C and dissolved in concentrated nitric acid; the clear acid solution is introduced into the furnace and the sequential drying (120 degrees C, 20 s), charring (800 degrees C, 70 s), and atomization (2400 degrees C, 8 s) are automatically performed. The technique of standard additions was used ot overcome matrix effect. The detection limit is 0.86 X 10(-11) g of Mn. The CV is 4.7%. Only sodium and potassium interfere at 500-fold concentrations, and their interferences were overcome by using the Deuterium Arc Background Corrector.
Toxicology test-ordering patterns in a large urban general hospital during five years: an update.
Authors: Walberg C B , Pantlik V A , Lundberg G D
Published: January 2020

Analytical data from the clinical toxicology laboratory of a large urban hospital, the Los Angeles County--University of Southern California Medical Center, are reported for the year 1976 and are compared to similar data previously documented for the year 1972. Drugs assayed, number of tests requested, and number of positive results are collated. Data on 58 assays show that the overwhelming majority of the requests continue to be for those tests that were originally classified as tests with 4-h turn-around time in the patient-focused concept for a clinical toxicology service in 1972. Total workload increased by 70%. The number of patients on whom some toxicologic assay was requested doubled in spite of a decrease in the number of patients admitted to the hospital during this five-year period. The data show that assays for some socially and clinically significant drugs--ethanol diazepam, tricyclics, and phencyclidine--increased disproportionally while others remained relatively constant, or even decreased.
Comparison of double- and single-isotope enzymatic derivative methods for measuring catecholamines in human plasma.
Authors: Evans M I , Halter J B , Porte D
Published: January 2020

We directly compared the reliability of a single-isotope enzymatic derivative technique for measurement of plasma catecholamines with that of the well-established double-isotope method. A significant (p less than 0.001) correlation was observed between measurements (n = 52) in the two assays, both for norepinephrine (r = 0.97) and epinephrine (r = 0.80). Means and coefficients of variation for the two analytes in a pooled specimen of plasma, measured repeatedly during six months, were virtually identical by each assay method. Basal plasma catecholamine concentrations in two different groups of apparently healthy subjects were also similar by each method. Dopamine concentrations in plasma were consistently below the limits measurable by either technique. The single-isotope assay requires half the assay time and 1/200th the sample as the double-isotope method. We conclude that this assay is just as reliable as the double-isotope technique and gives virtually identical values for norepinephrine and epinephrine concentrations in the physiological range.
The "Southmead System," a simple, fully-automated, continuous-flow system for immunoassays [Appendix: application to serum thyroxine radioimmunoassay].
Authors: Ismail A A , West P M , Goldie D J
Published: January 2020

A simple, fully-automated, continuous-flow system suitable for radioimmunoassay, enzyme immunoassay, and fluoro-immunoassay is described. The system is versatile, inexpensive, and requires only equipment and skills that already are available in most clinical chemistry laboratories. In this system, the antibody is covalently linked to solid-phase, porous particles of agarose (Sepharose, size 40-70 micrometer in the dry form). After sampling and mixing the assay reactants, the assay "cocktail" containing the solid-phase-bound antigen and the unbound fraction is directed to the separation block. Separation is achieved by use of a highly porous membrane (pore size, about 10 micrometer) and by controlling the volume of inflow and outflow a fixed and precise amount of fluid is filtered through the membrane containing only the free (unbound fraction), which can then be quantitated. The percentage of unbound fraction misclassified is negligible (less than 1.0%). Application of these principles to the assay of serum thyroxine is described in an appendix.
Free thyroxine in serum as estimated by polyacrylamide gel filtration.
Authors: McDonald L J , Robin N I , Siegel L
Published: January 2020

In this new method for estimating free thyroxine concentration in serum by use of polyacrylamide gel filtration, the prolonged incubation that is characteristic of equilibrium dialysis is obviated. Although the values so obtained for the percentage of free thyroxine are severalfold higher than those obtained by dialysis, the results of the two procedures correlate well over a wide range of thyroid states. In addition, the precision of the polyacrylamide method compares well with that for equilibrium dialysis. The method is convenient for use in clinical laboratories and values for free thyroxine in serum of patients with metabolic thyroid disorders are diagnostically discriminatory.
Screening for urinary oligosaccharides: a quantitative procedure.
Authors: Purkiss P , Hughes R C , Watts R W
Published: January 2020

A method for screening urine for abnormalities in glycoconjugate excretion is presented. An oligosaccharide-containing fraction is isolated from urine by gel chromatography and the carbohydrate-containing components in this fraction are degraded to monosaccharides, suitable derivatives of which are analyzed by gas-liquid chromatography. A pattern which characterizes the monosaccharides originating in complex carbohydrates in urine is thus obtained. Data obtained by applying this method to normal subjects in various age groups are presented and the applicability of the method and its limitations are illustrated by the use of examples from patients with previously diagnosed disorders of glycoconjugate metabolism.
Improved technique for developing creatine kinase isoenzyme bands by using a substrate in gelatin matrix.
Authors: Mickle D A , Washington G A , Porter C J
Published: January 2020

We describe a simple technique for reacting CK isoenzymes with a substrate in gelatin to make them visible after they have been separated by cellulose acetate electrophoresis. The electrophoresed strip requires only 5 min of contact with the substrate in gelatin and about 12 min of drying in an oven at 55 degrees C. The developed isoenzyme bands are discrete, intense, and rectangular in shape. The technique has a sensitivity of 2 to 5 U/liter, and results for isoenzyme MB can be reported about 30 min from the time a specimen is received.
Creatine kinase isoenzyme variants in human serum.
Authors: Ljungdahl L , Gerhardt W
Published: January 2020

In serum from about 800 patients, total creatine kinase and its subunit B activities were determined by the recommended Scandinavian creatine kinase method in the absence and presence of a creatine kinase M subunit inhibitory antibody. Eight patients had supranormal subunit B activities, but normal or near-normal values for total creatine kinase activity. Electrophoresis of sera from these eight patients showed, in addition to the normally migrating isoenzyme MM, one or two abnormally migrating creatine kinase isoenzyme bands, located between normally migrating isoenzymes MM and MB. Experimental data suggest that these abnormal bands may be isoenzyme BB with changed electrophoretic mobility. The eight patients had no particular disorder in common.
Atomic absorption spectrophotometry of free circulating platinum species in plasma derived from cis-dichlorodiammineplatinum(II).
Authors: Bannister S J , Chang Y , Sternson L A , Repta A J
Published: January 2020

We describe a method of analysis for free circulating platinum species derived from cis-dichlorodiammineplatinum(II) in blood plasma. Protein-bound and free platinum species were separated from each other by centrifugal ultrafiltration. Platinum in the ultrafiltrate was converted to a cationic complex by reaction with ethylenediamine, and the product was collected on paper impregnated with cation-exchange resin, where it could be stored indefinitely without loss. The platinum was eluted from the disk with 5 mol/liter hydrochloric acid, and an aliquot of this solution was then analyzed by flameless atomic absorption spectrophotometry. The overall analytical recovery of platinum was 80 +/- 2%. The minimum quantity of cis-dichlorodiammineplatinum detectable was 35 microgram/liter of plasma at the 99% confidence level. Detector response was linearly related to drug concentration in the range from 80 microgram to 290 mg of Pt per liter of plasma. Reaction variables were made optimal, so as to yield maximum sensitivity and reproducibility (+/- 2%) consistent with minimal sample transfers and manipulations.
Increased renin concentration in plasma and amniotic fluid during storage.
Authors: Craven D J , Symonds E M
Published: January 2020

Renin concentration was determined in plasma and amniotic fluid samples immediately after collection, after storage at -18 degrees C for up to 48 h, and after storage at 5 degrees C for as long as 48 h. Renin concentration increased, both in plasma (p less than 0.05) and amniotic fluid (P less than 0.02), after the storage at 5 degrees C, but no significant alteration occurred on storage at -18 degrees C. Partial activation of inactive renin could explain the observed increase in renin concentration at 5 degrees C.
A totally automated system for enzyme immunoassay of theophylline in serum.
Authors: Castro A , Ibanez J , Voight W , Noto T , Malkus H
Published: January 2020

We describe a procedure for enzyme immunoassay of theophylline (1,3-dimethylxanthine) in which all phases of the assay are totally automated in a Kinetic Analyzer (KA-150). This system permits assay of 75 10-mul samples per hour, with results available at 30-s intervals after initial sample preparation and preincubation. We compared results for 138 clinical samples by an ultraviolet method (x) and the present method (y). The slope of the comparison curve was 0.902, the y-intercept 0.402, and the correlation coefficient 0.984. The coefficient of variation for samples run in duplicate on the same day was 4.9%; it was 8.1% for samples run on different days. Specificity, sensitivity, simplicity, speed, and small reagent requirement all make this an attractive alternative to chromatographic procedures.
Enzymic determination of cholesterol in serum lipoproteins separated by electrophoresis.
Authors: Cobb S A , Sanders J L
Published: January 2020

We describe a reliable, straightforward procedure for measuring the cholesterol content of serum lipoproteins separated by electrophoresis on cellulose acetate. The lipoprotein fractions are made visible by use of an enzymic reagent and then quantitated by densitometry. Standard electrophoresis equipment is used and the procedure requires just over 1 h for completion. All major fractions except chylomicrons are detected readily. Accuracy and precision agree favorably with results reported for generally accepted methods. Concentration and color development are linearly related to cholesterol concentrations up to 1.5 g/liter in a given lipoprotein fraction. We recommend this method for use in assessing high-density lipoprotein cholesterol and as an aid in lipoprotein phenotyping.
Laboratory standardizatin in an international study: the Kaunas--Rotterdam intervention study (KRIS).
Authors: Boerma G J , Toleikis A , Mather A , Bartels C T , Margeviciene L , Glasunov I S , Leijnse B
Published: January 2020

Laboratory results obtained in different laboratories over lengthy periods of time usually are difficult to compare. In cooperative long-term studies where such results must be pooled, thorough standardization of methods is vital. We describe a program in which comparable plasma cholesterol and glucose analyses have been obtained, by simple methods. In the Netherlands and the Soviet Union in close collaboration with the Center for Disease Control, Atlanta, Ga., U.S.A. The two laboratories produced glucose values (direct o-toluidine reaction) within 2% of the target reference values and cholesterol results (direct Liebermann-Burchard reaction) with a consistent 6-8% positive bias over the reference method values. Intralaboratory precision was subject to preset acceptance limits. The use of common control materials, exchange of patient samples, and on-site comparison of all details of laboratory procedures are vital tools in standardization efforts. A laboratory protocol that included quality requirements and rejection criteria was developed and proved to be indispensable. The experience gained should be useful in standardizing inter-laboratory results in similar studies.
Analysis for glutathione in blood by high-performance liquid chromatography.
Authors: Rabenstein D L , Saetre R
Published: January 2020

A high-performance liquid chromatographic method is presented for determination of glutathione in whole blood. Sample preparation involves hemolysis, protein precipitation, centrifugation, and filtration. The glutathione in the filtrate is then separated from other sulfhydryl-containing molecules by liquid chromatography with Zipax SCX cation-exchanger followed by detection with a mercury-based electrochemical detector. The liquid-chromatographic analysis time is approximately 5 min. Because of the chromatographic separation and the selectivity of the detector, the detection step is free from interferences from other components of blood. The method has been checked by comparison with the colorimetric assay based on reaction with 5,5'-dithiobis(2-nitrobenzoic acid). The chromatographic results are consistently slightly lower, presumably because of the greater selectivity of this method.