bluuebunny/crossref_metadata_2025 huggingface dataset:
Measuring water content of feces by the Karl Fischer method.
Authors: Jensen R , Buffangeix D , Covi G
Published: January 2020

We describe a technique for measuring the water content of stools by the Karl Fischer method. The analysis is based on removal of water into a mixture of methanol/chloroform (1/2), after dispersion of the stool by sonication in presence of solvent. An aliquot of the solution thus obtained is placed in themeasuring cell of a Karl Fischer apparatus and then analyzed in the classic way. We further describe the advantages of this method (odorless, precise, reproducible) in contrast to other current methods. In addition the same organic solution can also be used in determining the lipid content of stools.
Factors influencing results for thyroxine concentration in blood, as measured in paper filter spots in a screening program for neonatal hypothyroidism.
Authors: Dussault J H , Morissette J , Fiset P , Laberge E , Laberge C
Published: January 2020

To reduce the number of false-positives in our screening program for neonatal hypothyroidism, we investigated the effect of age, body weight, and interval between sampling and analysis on results for filter paper spot thyroxine determination. The statistical analysis included an analysis of covariance. The age of the infant did not influence the results, the interval influenced them minimally, but weight did require a correction factor of about 0.16 ng/kg below the mean weight. This correction factor should reduce our false-positives from 0.75% to 0.60%. Furthermore, because all hypothyroid infants had results that fell more than 2.8 SD below the geometric mean, we intend to measure thyrotropin concentrations in the blood eluate of all the spots with a thyroxine concentration below that cut-off point.
An immobilized-enzyme flow-enthalpimetric analyzer: application to glucose determination by direct phosphorylation catalyzed by hexokinase.
Authors: Bowers L D , Carr P W
Published: January 2020

A novel flow-enthalpimetric analyzer is described and its use demonstrated by an analysis in which glucose is determined by its hexokinase-catalyzed phosphorylation reaction. The method depends on measurement of the temperature differential across a column packed with glass-supported immoblized enzyme. Sample volumes of 120 mul can be used to obtain a calibration curve that is linear up to 25 mmol of glucose per liter. A precision (within-day) of 5% is generally observed in the optimum concentration range where glucose is quantitatively phosphorylated. Results by the technique correlate reasonably with those by the o-toluidine and the hexokinase/glucose-6-phosphate dehydrogenase methods: Other sugars--including fructose, glucosamine, and mannose--will interfere; galactose does not. The technique is amenable to both routine and emergency analyses.
Automatic Quantitative Radiometric Assay of Bacterial Metabolism
Authors: Buddemeyer Edward , Hutchinson Robert , Cooper Malcolm
Published: January 2020

In a two-compartment scintillation vial, suspensions of bacteria were cultured with 1 µCi of [U-14C] glucose and the released 14CO2 was measured continuosly, cumulatively, and automatically in a liquid-scintillation counter modified to maintain sample temprature at 37 °C. We could follow the metabolism of bacterial populations through their early phase of exponential growth with good precision. The data were obtained conveniently, with use of conventional regents, glassware, and counting equipment. From analysis of the exponential portion of the curves for cumulative activity vs. time, we could measure cell replication rate precisely in units of time. The resulting values were demonstrably independent of some common experimental variables, including the number of bacteria in the inoculum and counting system sensitivity. Sensitivity of the bacteria to antibiotics was measured to within a few percent by noting the relative prolongation of replication time in the presence of those inhibitors. The digital data from the scintillation counter are susceptible to on- or off-line computer analysis, thus providing the prospect for a totaly-automated analytical system. The method shows promise for the mechanized quantitative analysis of bacterial growth, and its inhibition.
Use of laser nephelometry in the measurement of serum proteins.
Authors: Deaton C D , Maxwell K W , Smith R S , Creveling R L
Published: January 2020

Measurement of immunoglobulins (Ig) and of complement component C3 in human serum by automated and manual nephelometric techniques is tedious, and the effective linear range is too narrow. We describe a laser nephelometer/reagent system for measuring serum proteins by the use of forward light scatter (which enhances the ratio of reaction/blank), electronic blank subtraction, laser light (632 nm), and electronic signal selection. We report data establishing the range of linearity for immunoglobulins IgG, IgA, IgM, and complement C3 with this system, and correlations with results by a radial immunodiffusion. We also compared an electroimmunodiffusion system for quantitation of albumin and IgG in cerebrospinal fluid to our technique. The nephelometric system described provides a rapid, accurate, precise, and objective way to measure immunoglobulins and C3.
A minicomputer-automated array spectrometer for liquid-chromatographic detection of metabolites.
Authors: Dessy R E , Reynolds W D , Nunn W G , Titus C A , Moler G F
Published: January 2020

A third-generation multiwavelength array spectrometer was developed as a detector for the high-resolution liquid-chromatographic characterization of metabolites. Components include a PDP-8/e minicomputer, matched pair of linear photodiode arrays, holographically-ruled gratings, fiber optics, flow cells, and high intensity xenon light source. The wavelength range is 256 nm differential with 1-nm resolution and can be adjusted from 200 to 800 nm. The system is capable of storing 20 spectra per second (200-456 nm) in a dual-beam mode. Special features include minicomputer-driven signal enhancement via integration as a function of signal strength. The display output includes presentation of the total absorption chromatogram vs. elution time in both real and post-run time as well as selectable single absorption band vs. elution time (post-run time). Application of this dedicated system is illustrated by the separation and charcterization of the metabolites of a carcinogen, 4-ethylsulfonyl-1 napthalenesulfonamide.
Reverse-phase chromatography of polar biological substances: separation of catechol compounds by high-performance liquid chromatography.
Authors: Molnár I , Horváth C
Published: January 2020

Catecholamines and their metabolites have been separated isocratically by reverse-phase chromatography with aqueous (no organic solvent admixed) eluents. Unlike ion-exchange or ion-pair chromatography, mixtures of both acidic and basic substances can be separated in a single chromatographic run, because the retention is governed by hydrophobic interactions between the nonpolar moiety of the solute molecules and the octadecyl-silica stationary phase. The relative retention values strongly depend on the pH of the eluent, which governs the degree of dissociation of ionogenic solutes. The reproducibility of the results and the stability and efficiency of the chromatographic systems make this approach particularly attractive for use in clinical analysis.
Anticonvulsants in serum, determined with a fully mechanized enzyme analyzer.
Authors: McClean S W , Young D S , Yonekawa W
Published: January 2020

The "EMIT" enzyme immunoassay system (Syva) that involves use of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) as the enzyme label has been adapted to a fully mechanized kinetic enzyme analyzer for analysis of phenobarbital, phenytoin, and primidone. This procedure, compared with the gas-chromatographic procedure of Kupferberg [Clin. Chim. Acta 29, 283 (1970)], gave a weighted regression line--forced through the origin--of y=(1.01+/-0.04) x for phenobarbital, y=(0.95+/-0.04) x for primidone. Within-run coefficients of variation based on single determinations were 9,11, and 22% for primidone, phenobarbital, and phenytoin, respectively. Run-to-run assay CV was 6% for primidone and phenobarbital and 13% for phenytoin, based on the means of triplicate determinations of a sample with a mid-range concentration.
Automated turbidimetry of amylase activity by use of a discrete kinetic analyzer.
Authors: Malkus H , Ibanez J , Castro A , DiCesare J L
Published: January 2020

We adopted an automated turbidimetric rate method for determining amylase activity to the KA-150 Kinetic Analyzer. In the method, an insoluble amylopectin substrate is used with activity determined by the rate of decrease in turbidity. Run-to-run CV for 59 samples with activities up to 400 units (arbitrary amylase units per 100 ml of sample) was 2.8%. A comparison with a similar method, performed by nephelometry, for 104 sera, showed a correlation coefficient (r) of 0.992, with a slope of 1.02. In an additional comparison with an amyloclastic method, for 52 sera r was 0.997, with a slope of 1.01. Day-to-day precision for control sera with activities near the upper limit of normal (279 and 216 units) averaged 2.5% during two months. Measured and calculated activity were linearly related to well above the upper limit of normal (normal range, 60-200 arbitrary units), showing a deviation from linearity of about 10% at 450 units. Commercial reagents available for the Perkin-Elmer Model 91 Amylase Lipase Analyzer can be used with the KA-150.
Quantitative alkaline phosphatase isoenzyme determination by electrophoresis on cellulose acetate membranes.
Authors: Siede W H , Seiffert U B
Published: January 2020

We present a new method for quantitative determination of alkaline phosphatase isoenzymes. This method consists of electrophoretic separation on cellulose acetate membranes, special fixation technique to avoid elution and diffusion of enzyme protein during incubation, specific staining, and quantitative evaluation by densitometric measurement. We highly recommend the precedure for routine clinical laboratory use. In all normal individuals we observe two isoenzymes of hepatic origin and one isoenzyme each of osseous, intestinal, and biliary origin. Quantitative normal values are presented. Precision of the method is calculated, the CV being less than 10%. The exactness of densitometric quantification is proved by comparison with kinetic assay of alkaline phosphatase isoenzymes by use of an elution method. Clinical implications of alkaline phosphatase isoenzymograms are reported and discussed in detail.
Fetal lung maturity, as assessed by gas-liquid chromatographic determination of phospholipid palmitic acid in amniotic fluid.
Authors: Ip M P , Draisey T F , Thibert R J , Gagneja G L , Jasey G M
Published: January 2020

We describe a new and specific method for measurement of lecithin palmitic acid in amniotic fluid. Dipalmitoyl lecithin, the major alveolar surfactant, has previously been estimated by measuring the lecithin-sphingomyelin ratio, total lecithin, total phospholipid phosphorus, and (or) total palmitic acid. Our method is more specific for estimation of dipalmitoyl lecithin, because nonphospholipid sources of palmitic acid are removed by solvent extraction. Using a hexane/2-propanol/sulfuric acid system, we obviated the major interferences from triglycerides and free fatty acids. The palmitic acid derived from the phospholipid fraction is measured by gas-liquid chromatography of its methyl ester. No contribution appears to be made by sphingomyelin palmitic acid--probably owing to the mild hydrolysis conditions. The measured palmitic acid therefore appears to be derived from lecithins, principally dipalmitoyl lecithin. The value for palmitic acid determined by this method correlates well with the lecithin-sphingomyelin ratio and total phospholipid phosphorus. Infants are unlikely to develop respiratory distress syndrome when the measured palmitic acid in amniotic fluid exceeds 8.0 mg/liter, which corresponds to an lecithin-sphingomyelin ratio of 2.0.
Determination of plasma and erythrocyte lithium concentrations by atomic absorption spectrophotometry.
Authors: Hisayasu G H , Cohen J L , Nelson R W
Published: January 2020

We describe a method for determining plasma and erythrocyte lithium concentrations by atomic absorption spectrophotometry. Plasma and hemolyzed whole-blood are diluted and analyzed, with use of a lithium hollow-cathode lamp, at 670.8 nm. Erythrocyte lithium concentrations are calculated indirectly from the hematocrit. The standard deviation for a 0.43 mmol/liter pool of whole blood, run daily over 11 months, was +/-20 mumol/liter (CV=5.1). The lithium concentration of a lyophilized pool assayed periodically over the same period (n=127) was 1.84+/-0.05 mmol/liter (CV=2.7%). The relatively low erythrocyte/plasma lithium ratio and the microhematocrit centrifugation force (9600 to 13 600 X g) make corrections for trapped plasma insignificant. Problems with matrix matching and viscosity are overcome by using a plasma pool standard for calculations. Values for erythrocyte lithium concentrations were unchanged in samples stored at room temperature up to 24 h. Hemolysis appears to be of possible clinical significance. This method is useful as a routine clinical laboratory procedure for monitoring patients with affective disorders, who are undergoing therapy with lithium.
Gas-chromatographic/mass-spectrometric determination of theophylline in whole blood.
Authors: Sheehan M , Hertel R H , Kelly C T
Published: January 2020

We describe a combined gas-chromatographic mass-spectrometric method for determination of theophylline in whole blood. With use of a probability-based matching approach, the extensive gas-chromatographic interference did not affect mass-spectrometric quantitation of theophylline. Concentrations expected and found were linearly related from 1 to 60 mg/liter; the run-to-run coefficient of variation was less than 8%, within-run less than 6%. Some other xan-thines and drugs frequently co-administered with theophylline did not affect results. Only 0.2 ml of blood is required, which makes the procedure useful for monitoring theophylline in newborns and pediatric populations.
Estimation of maprotiline in serum by gas-chromatography, with use of a nitrogen-specific detector.
Authors: Gupta R N , Molnar G , Gupta M L
Published: January 2020

We describe a gas-chromatographic procedure for estimating therapeutic concentrations of maprotiline, a new tetracyclic antidepressant, in serum by use of a nitrogen-specific detector. Desmethyldoxepin, a secondary amine similar in structure to maprotiline, is added as a mass internal standard to the specimen before extraction, to obviate the need for accurate measurements of volumes during extraction and analysis. Both maprotiline and the internal standard are converted to acetyl derivatives, to avoid the adsorption of secondary amines on the column. A highly selective liquid phase is used, which allows a very good separation of desmethyldoxepin, maprotiline, desmethylmaprotiline, and the interferences from serum and reagents.
Calorimetry of cholinesterase activity in serum.
Authors: O'Farrell H K , Chattopadhyay S K , Brown H D
Published: January 2020

Cholinesterase activity in human serum was measured calorimetrically and compared to values obtained with the spectrophotometric method of Rappaport et al. [Clin. Chim. Acta 4, 227 (1959)]. The response of the calorimeter upon mixing serum with acetylcholine corresponded to the cholinesterase activity of the serum, the thermogram peak height being linearly related to enzyme activity at serum dilution volumes at which interference heat was negligible. The interference heat contributed by the dilution mixing characteristics of serum, studied separately, was found to be similar for heat-denatured, aged, or lyophilized samples. In all cases, heat of mixing was negligible when 1 ml of serum in buffer (2/3 by vol) was mixed with 1 ml of buffer. When this amount of serum was assayed with acetylcholine, cholinesterase activities of 2 and 3 kU/liter were recorded as thermograms with peak heights of 2.3 and 3.5 cm, respectively.
Protective effect of glycerol against the increase in alkaline phosphatase activity of lyophilized quality-control serum.
Authors: Tanishima K , Minamikawa Y , Yokogawa N , Takeshita M
Published: January 2020

We studied the effectiveness of glycerol or ethylene glycol in preventing the increase in alkaline phosphatase activity of lyophilized or refrigerated quality-control serum after reconstitution or repeated freezing and thawing. Control serum reconstituted completely from the lyophilized state with subsequent storage at -20 degrees C showed a considerable decrease in alkaline phosphatase activity immediately after thawing, and a gradual increase in activity on allowing it to stand at room temperature. Adding glycerol or ethylene glycol to the reconstituted serum obviated these changes in activity, glycerol being more effective than ethylene glycol. Reconstituted serum with added glycerol maintained maximum activity before refrigeration during either storage for 30 days or on repeated freezing and thawing. Practical applications of this glycerol-supplemented control serum are discussed.
Creatine kinase in serum: 3. Further study of adenylate kinase inhibitors.
Authors: Szasz G , Gerhardt W , Gruber W
Published: January 2020

In search of an appropriate inhibitor to suppress the interference of adenylate kinase with the creatine kinase assay, we found that the combination diadenosine pentaphosphate (10 mumol/liter) and AMP (5 mmol/liter) is a better inhibitor than is fluoride (25 mmol/liter). The latter inhibits adenylate kinase uncompetitively and weakly (Ki = 2.5 mmol/liter), and must be incorporated in the starting reagent, and at 30 degrees C it becoms fully effective only after a lag phase of 6 min. In this concentration, fluoride inhibits adenylate kinase from erythrocytes, muscle, liver or platelets by 94, 92, 88, and 87%, respectively, and creatine kinase by 8%. Bromide and chloride also inhibit creatine kinase. Attempts to replace AMP by a specific inhibitor of liver adenylate kinase failed. Homologs of diadenosine pentaphosphate with either fewer or more phosphoryl groups in the polyphosphate bridge inhibited even more weakly than did the pentaphosphate. Platelets can significantly contribute to adenylate kinase activity in serum. The inhibitor combination inhibited adenylate kinase from platelets by 90%.
Direct potentiometric measurement of sodium and potassium in whole blood.
Authors: Ladenson J H
Published: January 2020

I compared results for sodium and potassium in whole blood and plasma as measured with a newly available potentiometric analyzer, the "Orion SS-30". No significant difference was found for either sodium or potassium in 207 such comparisons. With use of the flowing, high mixing-velocity liquid junction of the Orion SS-30, the residual liquid junction potential due to blood cells was found to be less than 0.1 mV and to be independent of the hematocrit. This is in contrast to the hematocrit-dependent residual liquid junction potential of approximately 0.6 mV noted by others at normal hematocrit values with the open capillary liquid junctions now commonly used in pH instruments. I also found that the potassium concentration can increase significantly during the mixing of whole blood, and such samples should be mixed gently, if at all. Evidently sodium and potassium can be accutately and easily measured directly in heparinized blood.
Design of quality-control specimens for use with a small multi-channel analyzer.
Authors: Caragher T E , Grannis G F
Published: January 2020

Specimens having linear relationships between concentration and instrument response for seven analytes were prepared for use with a small multi-channel analyzer. The specimens had inter-specimen and inter-constituent relationships that facilitated the performance evaluation of all channels simultaneously with each individual specimen. Techniques are described for use of the specimens in various aspects of quality control.
Specific serum quinidine assay by high-performance liquid chromatography.
Authors: Crouthamel W G , Kowarski B , Narang P K
Published: January 2020

The cardioactive drug quinidine has a narrow therapeutic index; consequently, determination of serum quinidine concentrations can be important. We describe a relatively rapid and specific assay for quinidine in serum by high-performance liquid chromatography. It is suitable for use in patient monitoring or pharmacodynamic studies. Alkalinized serum is extracted with benzene, which is evaporated under nitrogen and reconstituted with methanol; an aliquot is chromatographed. Quinidine is separated from its metabolites and dihydroquinidine (a contaminant in quinidine raw materials). The retention time for quinidine is 4 min 10 s, for dihydroquinidine it is 5.5 min. Results for patients' sera by this assay method and the double-extraction method of Cramer and Isaksson [Scand. J. Clin. Lab. Invest. 15, 553 (1963] correlate well (r = .975).
Binding proteins from fish sera and intrinsic factor compared in vitamin B12 radioassay.
Authors: Ithakissios D S , Kubiatowicz D O , Windorski D C , Wicks J H
Published: January 2020

We compare serum proteins from rainbow trout, chinook salmon, coho salmon, and oyster toadfish with intrinsic factor as binding proteins in a simplified radioassay for B12. Regression analysis of B12 values, determined in 21 serum samples, shows good correlation (r greater than .975) between results for the fish sera and intrinsic factor. The accuracy of the five assays, as evaluated by analytical recovery of B12 added to pooled human serum, ranges from 90 to 110%. Intra-assay precision ranges from 2.6% for coho salmon serum to 5.5% for intrinsic factor, Ionic strength and variations in pH influence binding of [57Co]vit B12 to the fish sera. Maximum binding occurs from pH 6 to 10 at an ionic strength of 0.1 for all sera. The sera are stable for longer than two years when stored at -20 degrees C. Important advantages of fish sera are their high binding capacity (typical assay dilutions range from 1500-fold for trout serum to more than 50 000-fold for chinook salmon); high affinity for B12 (K greater than 10(12) liter/mol); their relative constant binding characteristics as compared to commercial intrinsic factor preparations; and the finding that the accuracy of radioassays with use of fish sera is not significantly affected by the amount of B12 or human serum proteins present.
Electrophoretic assay of specific estrogen receptors: a contribution to methodology.
Authors: van Netten J P , Algard F T , Montessori G , Weare B
Published: January 2020

Experimental evidence is presented that supports the use of the cold agar-gel electrophoretic method for the clinical quantitation of specific estrogen-binding protein present in some human mammary carcinomas. It is necessary to dilute tumor extracts to avoid interference by serum-borne, non-relevant hormone-binding proteins such as albumin, which migrates to the same anodal region as does the binding protein. Dilution to 2.5 mg or less of total protein per milliliter circumvents such interference while still permitting reliable quantitation of the binding protein. Seventy-two mammary carcinomas were compared for binding-protein content by both the cold agar-gel electrophoresis and a single-point dextran-coated charcoal assay. The correlation coefficient (0.96) indicated excellent agreement between results by the two methods. In addition results are presented which indicate that the preparation of tumor extracts for electrophoresis does not require the use of an ultracentrifuge.
Methotrexate assay by enzymatic inhibition, with use of centrifugal analyzer.
Authors: Finley P R , Williams R J
Published: January 2020

We present a method for measuring methotrexate by enzymatic inhibition, with use of the centrifugal analyzer. In the presence of dihydrofolate reductase (EC 1.5.1.3), dihydrofolic acid is converted to tetrahydrofolate. NADPH acts as a coenzyme. Methotrexate inhibits the action of dihydrofolate reductase. Increasing concentrations of methotrexate depress the conversion of NADPH to NADP+, and the reaction may be followed at 340 nm. Precision, accuracy, and sensitivity are satisfactory. A comparison to the radioimmunoassay method (x) showed the following least-squares regression: y = 0.9813x + 5.6 microgram/liter; r = .9812; Sy = 6.3 microgram/liter. Some interference was noted from bilirubin, lipermia, and hemoglobin. Up to 60 patients' samples per hour may be estimated by this technique.
Method for measuring increased plasma hemoglobin in the presence of erythrocytes.
Authors: Kruszyna R , Smith R P , Ou L
Published: January 2020

Despite its many drawbacks the classic benzidine technique is the method most widely used for determining plasma hemoglobin. An additional disadvantage, reported here, is that the method measures hemoglobin in erythrocytes as well as in plasma. Thus, if plasma specimens are accidentally contaminated with erythrocytes, apparent free hemoglobin in plasma will be artifactually high. Although the present method lacks the sensitivity of the benzidine technique, it self-corrects for the presence of erythrocytes. We have found it particularly useful for specimens of rodent (rat mouse) blood, where some cells appear to resist centrifugation, where the small sample sizes frequently result in accidental contamination of plasma with cells, and where normal values may be higher than those for human plasma.
A comprehensive classification of kinetic methods of analysis used in clinical chemistry.
Authors: Pardue H L
Published: January 2020

This paper presents a comprehensive classification of kinetic methods of analysis used in clinical chemistry. Objectives of the presentation are to place kinetic methods into perspective with other analytical appraoches, to identify concisely what is and is not a kinetic method, to group different kinetic methods into categories that identify important differences in operational and performance characteristics, and to suggest terminology that can identify the different approaches without ambiguity. In addition to these major objectives, the paper includes a brief historical development of the kinetic approach that covers the period from 1881 to the present, and discusses some current terminology that is believed to be ambiguous and misleading. I suggest that the detection step in analytical methods can be divided into two general categories, called equilibrium and kinetic methods. The kinetic methods are then subdivided into two general categories called fixed sensor-signal and variable sensor-signal methods. Each of these groups is then subdivided into one-point, two-point, and multi-point methods, which are then further subdivided according to what types of blanks are used, what variables are measured, and how the measured data are used to compute enzyme activity or analyte concentration. The paper identifies some commercial instruments that represent different approaches and briefly discusses relative merits of different approaches.
Mechanisms of cellular enzyme release. I. Alteration in membrane fluidity and permeability.
Authors: Verrill H L , Pickard N A , Gruemer H D
Published: January 2020

Above-normal plasma enzyme activities resulting from increased release of intracellular macromolecules are an important diagnostic feature of Duchenne muscular dystrophy. These and other biochemical and histological characteristics of this disease are faithfully duplicated when imipramine and serotonin are administered to the rat. Imipramine, but not serotonin alone, causes release of enzymes from rat diaphragms and human lymphocytes and release of hemoglobin from erythrocytes in vitro. Ouabain causes neither. Imipramine-induced enzyme release is decreased by adding ATP to the specimen in vitro or in hypertonic solution. Imipramine inhibits the capping phenomenon (an aggregation of antigen/antibody complexes of the membrane) of human B lymphocytes labeled with fluorescein-conjugated antihuman immunoglobulins. Serotonin alone has no such effect, but, administered together with imipramine, it potentiates the inhibition of capping by imipramine.
Criteria for establishing a standardized method for determining alkaline phosphatase activity in human serum.
Authors: Bretaudière J P , Vassault A , Amsellem L , Pourci M L , Thieu-Phung H , Bailly M
Published: January 2020

We investigated factors influencing alkaline phosphatase activity in the course of developing criteria for the establishment of a standardized method for its determination in human serum at 30 degrees C. The effects of pH, phosphorylatable acceptor (2-amino-2-methyl-1-propanol and diethanolamine), 4-nitrophenyl phosphate, magnesium ion, zinc ion, temperature, volume fraction of specimen, and details of initiation of the reaction have been studied, with use of partly purified enzymes from bone, intestine, liver, and placenta, and sera from patients with a predominant characterized isoenzyme. The purity of the diethanolamine was examined and contaminant monoethanolamine was characterized as a competitive inhibitor. Two sets of recommended conditions are: 2-amino-2-methyl-1-propanol, 0.9 mol/liter; 4-nitrophenyl phosphate, 16 mmol/liter; magnesium ion, 1 mmol/liter; volume fraction of specimen 1/30, and pH30 degrees C 10.5; diethanolamine, 1.8 mol/liter; 4-nitrophenyl phosphate, 18 mmol/liter; magnesium ion, 1 mmol/liter; volume fraction of specimen 1/60, and pH30 degrees C 10.1. Serum is preincubated with all reagents but 4-nitrophenyl phosphate, which is used as the reaction-initiating substrate.
4-Nitrophenol in 4-nitrophenyl phosphate, a substrate for alkaline phosphatase, as measured by paired-ion high-performance liquid chromatography.
Authors: Culbreth P H , Duncan I W , Burtis C A
Published: January 2020

We used paired-ion high-performance liquid chromatography to determine the 4-nitrophenol content of 4-nitrophenyl phosphate, a substrate for alkaline phosphatase analysis. This was done on a reversed-phase column with a mobile phase of methanol/water, 45/55 by vol, containing 3 ml of tetrabutylammonium phosphate reagent per 200 ml of solvent. At a flow rate of 1 ml/min, 4-nitrophenol was eluted at 9 min and monitored at 404 nm; 4-nitrophenyl phosphate was eluted at 5 min and could be monitored at 311 nm. Samples of 4-nitrophenyl phosphate obtained from several sources contained 0.3 to 7.8 mole of 4-nitrophenol per mole of 4-nitrophenyl phosphate.
Inhibition by concanavalin A as the basis for a specific assay of serum 5'-nucleotidase activity.
Authors: Zygowicz E R , Sunderman F W , Horak E , Dooley J F
Published: January 2020

Concanavalin A inhibits serum 5'-nucleotidase activity, without causing significant inhibition of alkaline phosphatase activity. This observation serves as the basis for a new method for assaying the 5'-nucleotidase activity in serum, which depends upon the difference between the enzymic hydrolysis of adenosine-5'-monophosphate in the presence and absence of concanavalin A. A denosine released by the 5'-nucleotidase reaction is deaminated by a coupled reaction with adenosine deaminase to liberate inosine and ammonia, and ammonia is measured colorimetrically by the Berthelot reaction. In sera from 40 healthy adult persons, 5'-nucleotidase activity averaged 6.4 U/liter (SD, +/-2.0; range, 3-12). In sera from 100 patients, measurements of 5'-nucleotidase activity by the new assay averaged 8% lower than by a generally accepted method in which phenyl phosphate is used to suppress hydrolysis of adenosine-5'-monophosphate by alkaline phosphatase activity. The clinical validy of the new assay was tested by measuring serum 5'-nucleotidase activities in rats with bile duct ligation and in rats treated with thioacetamide to induce hepatocellular injury.
Radioimmunoassay for normalized thyroxine with use of Sephadex columns.
Authors: Goedemans W T , Bartels P C , Roijers A F
Published: January 2020

We describe a reproducible radioimmunoassay, with use of Sephadex columns, for measuring normalized thyroxine. In comparison with a competitive protein-binding procedure, the present method is more specific because antibody rather than thyroxine-binding globulin is responsible for the competition between endogeneous and tracer thyroxine. In barbital buffer, various concentrations of thyroxine binding pre-albumin and albumin had no influence on the results. Values obtained by the present method correlated well with those by the free thyroxine index and a generally accepted thyroxine normalized method.
Diminished cap formation in lymphocytes from patients and carriers of Duchenne muscular dystrophy.
Authors: Verrill H L , Pickard N A , Gruemer H D
Published: January 2020

Currently, the most useful clinical laboratory aid in establishing the carrier state of Duchenne muscular dystrophy is to determine creatine kinase (EC 2.7.3.2) activity in the plasma. The considerable overlap between plasma creatine kinase activities of controls and of carriers at the childbearing age contributes appreciable difficulty to genetic counseling of potential carriers. The consistent failure of lymphocyte cap formation in Duchenne muscular dystrophy patients and carriers in this study suggests a valuable tool for the confirmation of the carrier state.
Activation of alanine aminotransferase in serum by pyridoxal phosphate.
Authors: Lustig V
Published: January 2020

Alanine aminotransferase activity in serum increases significantly when serum is incubated with pyridoxal phosphate. The increase depends on the L-alanine concentration in the final assay mixture, being greatest at 800 mmol/liter. Preincubation of 22 normal sera, in a 10:1 ratio with an 8.09 mmol/liter pyridoxal phosphate solution, resulted in an increase in the alanine aminotransferase activity from 10.5 +/- 4.9 U/liter (mean +/- SD) to 28.4 +/- 5.3 U/liter, an increase of 170%. The absolute amount of apoalanine aminotransferase is relatively constant over a wide range of enzyme activities.
Quantitative gas-chromatographic flame-ionization method for chloramphenicol in human serum.
Authors: Least C J , Wiegand N J , Johnson G F , Solomon H M
Published: January 2020

We describe a flame-ionization gas-chromatographic procedure for determination of the potentially toxic antibiotic, chloramphenical, in serum. The serum (500 mul) is extracted into ethyl acetate and nonpolar impurities are subsequently partitioned into hexane. The drug is chromatographed as its bis-trimethylsilyl derivative, with the analog thiamphenicol as the internal standard. Within-run precision (CV) is 4.4% at a serum concentration of 41 mg/liter and 9.2% at a concentration of 5 mg/liter. Over a six-month period, the run-to-run variation was 5.1% at 40 mg/liter (n = 24). Results by the gas-chromatographic method compared well with those by an established colorimetric procedure; mean concentrations for the comparison samples in the two procedures were 18.4 mg/liter and 17.6 mg/liter, respectively (n = 27), with a coefficient of correlation of 0.998. The gas-chromatographic method is more precise and specific than classical microbiological procedures and is suitable for routine therapeutic monitoring of serum chloramphenicol concentrations.
Purification and iodination of antibody for use in an immunoradiometric assay for serum ferritin.
Authors: Gonyea L M
Published: January 2020

Antibodies were purified by affinity chromatography and radioiodinated by a conjugation method for use inthe immunoradiometric assay for serum ferritin. These procedures are simpler and more reproducible than those described earlier, and the radiolabeled preparations were usable for at least 13 weeks.
Urinary excretion of immunologically reactive metabolite(s) after intranasal administration of cocaine, as followed by enzyme immunoassay.
Authors: Van Dyke C , Byck R , Barash P G , Jatlow P
Published: January 2020

Using the enzyme immunoassay technique (EMIT), we determined the time course of urinary excretion of benzoylecgonine in 16 surgical patients and three volunteers who received intranasal cocaine. After doses varying from 13 to 130 mg, the test for benzoylecgonine was positive in 1 to 4 h, peaked at about 10 to 12 h, remained positive for 18 to 27 h, and became negative after about 17 h. This information should be considered by drug dependency treatment programs in which the EMIT procedure is used to screen urines for cocaine use.
Glyceraldehyde-phosphate dehydrogenase (total and isoenzyme activity) in the early diagnosis of myocardial infarction.
Authors: Griffiths J , Shaw S
Published: January 2020

Enzyme "panels," in which creatine kinase and lactate dehydrogenase activities in serum are measured, are useful indicators of myocardial infarction. We examined a further enzyme, glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.12), by comparison with creatine kinase (EC 2.7.3.2), in the early diagnosis of such infarctions. Results indicate that this total dehydrogenase appears in the serum before total creatine kinase activity; however, the lack of cardio-specificity relating to the dehydrogenase isoenzyme fraction 2 in comparison to the creatine kinase MB band is a major disadvantage, as is its relatively poor in vitro stability. We conclude that measurement of this dehydrogenase does not allow a substantially earlier diagnosis of myocardial infarction.
Clinical aspects of porphyrin measurement, other than lead poisoning.
Authors: Lamon J M
Published: January 2020

Measurement of porphyrins and porphyrin precursors has clinical significance beyond the detection or confirmation of lead poisoning. The porphyric diseases of man are characterized by the presence of a specific clinical syndrome and demonstration of abnormal porphyrin excretion or accumulation in the tissues. In addition, porphyrins accumulate in other disorders that are unrelated to lead poisoning or the porphyrias. These causes of erythrocyte porphyrin accumulation, porphyrinuria, and the porphyrias are discussed.
Free erythrocyte porphyrins in the detection of undue absorption of Pb and of Fe deficiency.
Authors: Piomelli S
Published: January 2020

A fluorometric assay for free erythrocyte porphyrins (FEP) is described in detail, the direct test being used for the analysis of blood specimens collected with anticoagulants and the spot test for analysis of blood specimens collected on filter paper. A method for determining the amount of blood absorbed by different lots of filter paper is described.
The hematofluorometer.
Authors: Blumberg W E , Eisinger J , Lamola A A , Zuckerman D M
Published: January 2020

A portable filter fluorometer, the hematofluorometer--which utilizes "front face" optics, internal standards, and digital computation capabilities--has been specifically designed for the rapid (approximately 5 s) assay of erythrocyte zinc protoporphyrin in unprocessed blood. A small (unmeasured) drop of blood, such as is obtained from a finger puncture, is placed on disposable cover slip and inserted in the sample holder of the instrument. The operator pushes the holder of the instrument. The operator pushes the holder, which also holds the permanent blank and standard, into the instrument. Zinc protoporphyrin concentration is automatically and instantaneously computed and the value is displayed on a digital readout as micrograms of zinc protoporphyrin per deciliter of blood for some standard hematocrit. No calibration adjustment need be performed by the operator. The hematofluorometer is designed for "field" use as well as for laboratory assays.
Increased serum carnitine concentration in renal insufficiency.
Authors: Chen S H , Lincoln S D
Published: January 2020

We measured serum carnitine in groups of patients with various diseases. The concentration was frequently above normal in patients with renal insufficiency. The clinical significance of such an increase in renal insufficiency may be analogous to that of increases in urea nitrogen, creatinine, or uric acid in the serum.
Enzymic measurement of cholesterol in serum with the CentrifiChem centrifugal analyzer.
Authors: Pesce M A , Bodourian S H
Published: January 2020

Cholesterol is measured by mixing 5 mul of sample with 350 mul of a reagent consisting of phenol, 4-aminoantipyrine, and the enzymes cholesterol oxidase, cholesterol esterase, and peroxidase. After 12 min, the resulting quinoneimine is measured at 520 nm. Readings and cholesterol concentrations are linearly related up to 4.0 g/liter. Lipemic sera and samples containing uric acid (up to 200 mg/liter), hemoglobin (up to 1 g/liter), and certain drugs (clofibrate, phenobarbital, nicotinic acid, Ketochol, Ovral-28), gave no interference. Abnormally high concentrations of bilirubin and ascorbic acid in serum lowered the cholesterol values. This enzymic assay, compared with the method of Abell and with a rate method that uses the Hantzsch reaction, gave correlation coefficients of 0.987 and 989, respectively.
Transient hyperphosphatasemia of infancy--an insufficiently recognized syndrome.
Authors: Posen S , Lee C , Vines R , Kilham H , Latham S , Keefe J F
Published: January 2020

Spectacular transient increases in serum alkaline phosphatase were observed in five infants in the absence of demonstrable pathology.
L-p-Bromotetramisole, a new reagent for use in measuring placental or intestinal isoenzymes of alkaline phosphatase in human serum.
Authors: Van Belle H , De Broe M E , Wieme R J
Published: January 2020

L-p-Bromotetramisole is proposed as a new reagent for use in determining intestinal and (or) placental isoenzymes of alkaline phosphatase in human serum. Its main advantage over L-phenylalanine is its high discriminating potency at very low concentrations. For highest accuracy, the samples may even be "titrated" with the inhibitor. Analytical recoveries of intestinal and placental isoenzymes in serum are complete, and results correlate well with those by other methods. At the appropriate concentrations, L-p-bromotetramisole gives nearly identical results on a variety of sera, irrespective of the buffer used. Within-day CV's were 6.1 and 2.1 for 20% and 60% of the resistant isoenzyme activity, respectively. The day-to-day CV was 3.45 for a sample containing 53.3% of the resistant isoenzyme (n=15). The well-known increase in placental isoenzyme activity with time of gestation could be confirmed on 612 sera from pregnant women. The activities of the intestinal isoenzyme in sera from healty blood donors (n = 126) and hospitalized patients (n = 135) agree with previous findings obtained by the other techniques.
Ion pair extraction technique with azure A, for differentiating biles of normal subjects and patients with liver disease from patients with Crohn's disease with small bowel involvement.
Authors: Webb G R , Macdonald I A , Williams C N
Published: January 2020

The ion pair extraction technique with Azure A solution, chloroform/methanol (equal volumes), and dilute sulfuric acid, was used to study bile-rich duodenal aspirates of (a) healthy volunteers and patients with (b) cholestatic liver disease, (c) Crohn's disease with small bowel involvement, and (d) those with primarily colon involvement. Duplicate aliquots of extracted duodenal aspirates from these four groups were subjected to ion pair extraction, with and without prior acetylation, and the absorbance ratios at 645 nm without acetylation/with acetylation ("acetylation index") compared; values (+/- SD) obtained were 0.052 +/- 0.0098, 0.038 +/- 0.014, 0.136 +/- 0.048, and 0.077 +/- 0.056, respectively, for the four groups. The acetylation index for group c was significantly (P less than 0.001) different from that of groups a and b. All other intergroup comparisons were not significant. The acetylation index correlated positively (r = 0.922) with the ratio of glycine- to taurine-conjugated bile acids, measured independently, implying that it reflects this ratio.
Use of equilibrated blood for internal blood-gas quality control.
Authors: Leary E T , Delaney C J , Kenny M A
Published: January 2020

We have used equilibrated human blood for blood-gas quality control since 1970. In blood equilibrated 24 h after shedding, gas tensions are stable for 4 to 6 h at 0 to 4 degrees C; each control specimen is analyzed several times during that period to resolve malfunctions, etc. Three-fourths of all errors in gas-tension measurement detected with equilibrated blood were detected with the highest-tension controls. Equilibrated blood controls signal about one error every 14 d on each instrument. For more complete quality control, we supplement analysis of equilibrated blood with other sorts of controls, comparing results obtained by assaying each patient's specimen on two instruments being our most effective adjunct. Such comparisons have identified erroneous assays in 3.9% of the specimens tested. The magnitude of interinstrument discrepancies (random errors) have ranged from 9 to 100% of the appropriate determinations. We use control data derived from equilibrated blood analysis for special management purposes (evaluating instruments, quantitating micro- vs. macro-sampling discrepancies, and decreasing instrument-repair costs).
Intra-individual variation in the concentrations of IgA, IgG, IgM, and complement component C3 in serum of a normal adult population.
Authors: Butts W C , James G E , Keuhneman M
Published: January 2020

A recent study [Clin. Chem. 22, 1635 (1976)] reported intra-individual variation in 10 serum proteins to be much smaller than interindividual variation. We report results of a similar study involving about 700 apparently healthy adults in whom we estimated the relative magnitude of the intra- and interindividual variation in serum IgA, IgG, IgM and complement component C3. Specimens were collected from each subject weekly for as long as 10 weeks (average, four weeks). The four serum proteins were quantitated by radial immunodiffusion by the maximal-diffusion technique. Traditional 95% reference intervals were computed relative to WHO reference preparations for the immunoglobulins. For C3, the reference interval was computed relative to a commercial reference preparation. We, too, found the ratios of intra-individual to interindividual variation for adults to be so small that the traditional reference intervals do not have the assumed diagnostic sensitivities. Furthermore, these ratios did not change after dividing the study population into subgroups according to sex and age; evidently such subgrouping do not improve the diagnostic sensitivity. The relatively small intra-individual variations were also observed at the extremes of protein concentration ranges.
Cortisol production rate determined by radio gas-chromatography.
Authors: Derks H J , Muskiet F A , Wolthers B G , Thijssen J H , Drayer N M
Published: January 2020

We describe a ratio gas-chromatographic method for determination of cortisol production rate by measuring the isotope dilution of urinary cortisol metabolites. The method was calibrated by analyzing [3H]tetrahydrocortisol and [3H]tetrahydrocortisone of known specific activities. Results are reasonably well reproducible, the coefficients of variation ranging from 8-15% of allotetrahydrocortisol/tetrahydrocortisol and from 9-19% for tetrahydrocortisone. Correlation coefficients were 0.966 and 0.998 for tetrahydrocortisone and allotetrahydrocortisol/tetrahydrocortisol, respectively, when the method was compared with a method involving thin-layer chromatography and colorimetry. Only one chromatographic step is needed for both purification and quantitation, thus time and effort are saved.