Showing papers in "Clinical Chemistry in 1964"

An automated procedure for the simultaneous determination of calcium and phosphorus.

Abstract: A completely automated method for the simultaneous or individual determination of calcium and phosphorus in serum and urine without preliminary sample treatment is reported. Separation of calcium and phosphorus from interfering materials is achieved by dialysis under acid conditions. The dialyzed calcium is determined colorimetrically using a metal complexing dye (cresolphthalein complexone) in an alkaline medium. Magnesium, phosphate, citrate, sulfate, etc., did not introduce significant errors under the test conditions. Inorganic phosphorus is determined by a modified Fiske-SubbaRow procedure. Reproducibility and recovery data as well as comparisons with manual methods are presented.

A simple serum iron method using the new sensitive chromogen tripyridyl-s-triazine.

Abstract: A simple, specific and reproducible serum iron method is described using the recently synthesized and highly sensitive chromogen, tripyridyl-S-triazine. The absorbance of this chromogen is compared to other iron reagents. Procedural modifications are given for using the Ramsay iron-binding capacity method with this serum iron procedure. The normal values found agree with most of those in the literature which has been reviewed.

Micromethods for Measuring Phenylalanine and Tyrosine in Serum

Abstract: Spectrophotofluorometric micromethods for the determination of phenylalanine and tyrosine on 25 µl of serum are described. These methods were applied to detect phenylketonuria among homozygotes and heterozygotes and in the newborn population. The data presented agree with those previously reported. (1) Phenylketonurics have markedly elevated serum phenylalanine and lower serum tyrosine than the controls. (2) The heterozygotes have a higher fasting serum phenylalanine and, after a standard oral phenylalanine test, show a higher and more prolonged rise of serum phenylalanine than the controls. There was a lesser increase of serum tyrosine after phenylalanine loading in the heterozygotes than the controls. (3) Premature infants of low birth weight have higher serum phenylalanine and tyrosine levels than normal birth weight infants, presumably due to enzyme immaturity. Simultaneous determinations of serum phenylalanine and tyrosine will differentiate newborn infants who are suspected to be phenylketonurics from the homozygotes (phenylketonurics).

Multiple Automatic Sequential Analysis

Abstract: A multiple automatic analyzer has been constructed which determines albumin, total protein, chloride, carbon dioxide, sodium, potassium, glucose, and urea nitrogen. The results for each of these 8 determinations are obtained in sequence for an individual sample and are recorded on a single sheet of paper. The entire system is calibrated by means of a standard solution containing all 8 of the components under test. Results appear on the record in a directly readable and immediately usable form. The actual record is used as the laboratory report sent to the requesting physician.

Method for the Determination of Vitamin E (Total Tocopherols) in Serum

Abstract: The use of tripyridyltriazine has been investigated as a chromogenic reagent for the colorimetric determination of vitamin E in serum. The advantages of using this re-agent have been elucidated. Comparison is made with a method employing terpyridyl.

Application of Atomic Absorption Spectrophotometry in the Determination of Calcium in Serum

Abstract: Atomic absorption spectrophotometry (AAS) was applied to the determination of serum calcium. A special diluent was developed to abolish the effect of absorption depressors. This made possible measurement of calcium in directly diluted serum samples without prior removal of any serum constituents. Values obtained by AAS on calcium oxalate precipitates from serum were identical with those found by the direct dilution technique.

A rapid method for cholesterol determination with a single reagent.

Abstract: A method for the determination of serum cholesterol, using a single reagent, is described. This method is simple, rapid, and economical, and prevents some of the errors inherent in the manipulative process of earlier procedures.

A colorimetric method for determination of serum chloride.

Abstract: A procedure is outlined for the determination of body fluid chloride levels from as little as 0.2-ml. of fluid. The results are highly reproducible and compare very favorably with the widely used method of Schales and Schales. The color produced is quite stable, and the interference of extraneous compounds has been found to have no effect on the accuracy of the method.

Determination of uric acid: an automated analysis based on a carbonate method.

Abstract: An automated method is described for the determination of uric acid by a carbonate method. Since uric acid is separated from proteins by dialysis, the loss of uric acid due to protein precipitation in manual methods is avoided. There is no appreciable interference from salicylates, high levels of blood sugar, or ascorbic acid. The method shows an acceptable correlation with the uricase method.

Rapid Determination of Plasma Ammonia by an Ion-Exchange Technic

Abstract: A method is described for the colorimetric determination of ammonia in plasma by a batch ion-exchange procedure. The method combines separation of ammonium ion, using a strongly acidic cation-exchange resin, with a sensitive colorimetric procedure which simultaneously elutes and colorimetrically reacts with the ammonium ion to produce a stable color. Interference by basic amino acids is negligible, and the great sensitivity of the color reaction permits the measurement of ammonia of the order of 0.5 µg./ml. The effects of variations in the experimental conditions are examined, and the results of recovery experiments and other tests of precision and accuracy are reported. In comparison with other methods in current use, this method has distinct advantages in sensitivity, simplicity, and rapidity of determination, provided the conditions and methods of measurement are controlled carefully.

The Precision, Accuracy, and Inherent Errors of Automatic Continuous Flow Methods

Abstract: An automatic continuous-flow system was evaluated under routine daily use for six constituents, sodium, potassium, chloride, carbon dioxide, urea, and glucose. Two parameters have been quantified, interaction between samples and instrumental drift. Results also were influenced by the available amount of sample. Application of correction technics is shown to improve the accuracy of the automatic-analysis system.

A study of the starch-iodine complex: a modified colorimetric micro determination of amylase in biologic fluids.

Abstract: The limitations, inaccuracies, and practical difficulties of saccharogenic methods are discussed. A modified colorimetric microdetermination of amylase is described in which the digestion of starch is measured by the decrease in the starch-iodine color. Experimental data show that there are two other serum factors that can also cause a fall-off in color: (1) an immediate 10-15% depression of color, probably due to serum proteins and countered by using serum in the control; (2) an acid-serum factor causing a progressive fall-off in color subsequent to the initial depression. Iodine prevents this, and must be added as soon as the acid has been added to stop the enzyme activity. Results of 189 consecutive assays of human sera are presented. Amylase activity of duodenal aspirate has been determined simultaneously by the method described and the Lagerlof method. Results are compared. Changes in serum amylase and lipase levels in artificially produced pancreatitis in dogs are presented. Optimal conditions for amylase activity are reviewed, and in the light of these, different amyloclastic methods and their results compared. Achroic-point technics are briefly evaluated.

Fluorometric Determination of Urinary Metanephrine and Normetanephrine

Abstract: A detailed procedure for the quantitative determination of metanephrine and normetanephrine is given. Epinephrine and norepinephrine are eliminated by selective adsorption on an alumina column. Metanephrine and normetanephrine are isolated with a cation-exchange resin. Fluorescence is developed by a two-step ferricyanide oxidation at two different pH values followed by tautomerization. The concentrations of both metanephrine and normetanephrine are quantitated by differential fluorometry.

Simplified estimation of leucine aminopeptidase (lap) activity.

Abstract: An accepted colorimetric assay for the end product of the enzymic hydrolysis of L-leucyl-β-naphthylamide by leucine aminopeptidase in serum and urine has been modified. This modification has been shown to result in enhanced facility of handling large numbers of assays of this enzyme with considerably improved precision.

A quantitative assay for urinary porphobilinogen.

Abstract: A procedure has been developed for the rapid, quantitative determination of urinary porphobilinogen. Interfering substances are removed by selectively adsorbing the porphobilinogen with an ion-exchange resin using a batch method. After acid elution of the porphobilinogen, Ehrlich9s reagent is added to produce the chromophore, which is then measured spectrophotometrically. Obtaining absorption at two wavelengths permits a differentiation of porphobilinogen from interfering reactants not removed by the resin treatment. This assay is relatively simple, dependable, highly reproducible for increased amounts, and more specific than the qualitative assay.

Commonly used medications that interfere with routine endocrine laboratory procedures.

Abstract: Ten groups of commonly utilized medications were examined both in vitro and in vivo for their ability to interfere with optical density measurement and/or color development in such routine endocrine laboratory tests as those for 17-ketosteroids and 17-hydroxycorticosteroids. Certain antihypertensives, tranquilizers, psychic energizers, monoamine oxidase inhibitors, and soporifics were found to create the most problems in interference. The importance of removing these medications from the patients9 therapeutic regimes prior to obtaining specimens for such endocrine procedures is stressed.

Complexometric Titration of Calcium and Magnesium by a Semiautomated Procedure

Abstract: A semiautomatic procedure for the direct estimation of both serum calcium and magnesium is described. The indicator used, Eriochrome blue SE (EBSE), has the advantage of stability and freedom from interference by naturally occurring substances. The procedure requires 3-5 min. per sample and is very reproducible. The values obtained agree with those obtained by back-titration of excess chelator and are consistently 0.3 mg./100 ml. less than those obtained by a standard oxalate precipitation method.

A stable murexide reagent for the estimation of calcium in micro quantities of serum.

Abstract: The effects of various solvents on the fading rate of alkaline murexide were studied. Glycerol, methanol, and propylene glycol were found to retard the fading rate of murexide at pH 12.0 to a considerable extent. Propylene glycol was the most satis-factory of these solvents. It was further noted that the fading rate of alkaline murexide in propylene glycol-water solution was directly proportional to the amount of water contained in the reagent, and that a reagent stable for at least 1 week at room temperature could be prepared.

Plasma amino acids in young and older adult human subjects.

Abstract: Nineteen amino acids were determined in the plasma of young and older individuals by the chromatographic procedure of Moore, Spackman, and Stein (7,8). Of the essential amino acids, valine, methionine, isoleucine, leucine, phenylalanine, and lysine were found to be lower in older subjects. In all, 11 of the 19 amino acids studied showed lower values in these individuals, indicating some differences in protein metabolism between the elderly and younger adults.

A Microprocedure for the Determination of 4-Pyridoxic Acid in Urine

Abstract: A microprocedure for the estimation of urinary 4-pyridoxic acid which is apparently specific, yet simple enough for routine clinical use, has been adapted from the Fujita et al. (16-17) and Reddy et al. (22) procedures. Average values for urinary 4-pyridoxic acid for 7 women ranged from 3.8 to 6.9 µM/24 hr. by this method. These values are compatible with average daily intakes of vitamin B6 and indicate that by this method interfering fluorescent substances have been removed by chromatography. Good agreement was obtained with replicate analyses.

Cholesterol measurement in serum and in plasma.

Abstract: The effect on the plasma cholesterol concentration of various anticoagulants added to the blood in dry form, as compared with that of serum, has been studied. The amounts of anticoagulants used, per 5 ml. of blood, were: heparin, 2.5 mg.; oxalate mixture, 12 mg.; tetrasodium EDTA, 5 mg.; sodium oxalate, 12mg.; and ACD mixture, 5.3 mg. citric acid, 15.8 mg. Na citrate, and 17.6 mg. dextrose. All these anticoagulants caused a decrease in the plasma cholesterol concentration. The mean cholesterol decrease (serum minus plasma) produced by heparin was only 4 mg./100 ml. (S.E., ± 1.24), but this difference was statistically significant. When the heparin was prepared by drying a solution of heparin in 0.9% NaCl a greater difference between serum and plasma was observed. The cholesterol difference (serum minus plasma) producd by Na oxalate increases with increasing amounts of anticoagulant. The decrease in plasma cholesterol concentration produced by the anticoagulants can be explained by changes of water distribution between cells and plasma, reflected by changes of hematocrit produced by the anticoagulant.

Studies on the Determination of Bile Pigments V. Comparison of Some Methods for Determination of Total, Free, and Conjugated Bilirubin in Serum

Abstract: Tests for total bilirubin on 10 icteric sera by 5 diazo procedures and a spectrophotometric method showed fair agreement with most samples but an occasional grossly discrepant result. Results for free and conjugated bilirubin by diazo methods, acetone precipitation, and extraction of serum with ethyl acetate and chloroform were widely discrepant. Problems in documentation of accuracy of such procedures are discussed. It is concluded that unavailability of reference standards of bilirubin conjugates poses a serious obstacle to solution of the analytical problems.

Adaptation of the Berthelot Color Reaction for the Determination of Urea Nitrogen in Serum and Urine to an Ultramicro System

Abstract: Existing methods for the estimation of urea and ammonia nitrogen in biologic fluids by means of the Berthelot color reaction have been adapted to an ultramicro system for routine clinical analysis. The resulting procedure is convenient and accurate, and can be carried out using 5 µl. of sample or less.

Photometric Determination of Urinary Proteins

Abstract: Previous reports have dealt with application of a tryptophan method for determination of serum total (19) and gamma (20) globulins and CSF total globulins (21) . The method has now been extended to the determination of urinary globulins. Total urinary protein (including mucoprotein) was determined with the Lowry procedure after perchloric acid-acetone precipitation. Normal values on morning samples (32 subjects) are: T.P., 24.0 ± 19.6 mg./100 ml.; protein (albumin plus globulin), 6.9 ± 4.3 mg./100 ml.; mucoprotein, 17.1 ± 16.3; globulin, 4.4 ± 2.4, albumin, 2.5 ± 2.4; and A/G ratio, 0.58 ± .4 S.D. Preliminary data on patients with a variety of kidney ailments indicate that an increase in the urinary globulin fraction is a sensitive indicator of early renal damage.

A Method for Determination of Magnesium in Serum and Urine

Abstract: A method is described for a determination of magnesium in serum and urine. This method is based on the formation of a red color produced by a thiazole yellow-Mg(OH)2 complex in alkaline solution.

Direct automated determination of glucose by a glucose oxidase-peroxidase system.

Abstract: A simple, accurate, automated method for the determination of glucose in plasma, urine, and spinal fluid, which does not require dialysis or protein-free filtrates is presented. Only unusually high uric acid levels in the plasma interfere with the procedure described.

Distribution of ammonia in cells and plasma.

Abstract: A method for the determination of plasma and whole blood ammonia is outlined which incorporates the desirable features of several older methods. By means of this method it has been demonstrated that the distribution of ammonia in erythrocytes and plasma of normal individuals is nearly equal to that of hydrogen ions. The ratio remains constant in cirrhosis, alkalemia, and ammonemia. It was observed in 82 consecutive admissions of cirrhotic patients that alkalemia was present in only a small percentage of the cases.

An Automated Determination of Alkaline Phosphatase Utilizing p-Nitrophenol Phosphate

Abstract: A technic is described for the automated determination of serum alkaline phosphatase. This method permits a production rate of 60 determinations per hour on 0.2 ml. of serum per determination. Calculations are simplified since no serum blank is required.

The photometric microdetermination of tryptophan in biologic materials.

Abstract: A modification of the Fischl (6) glyoxylic acid method for tryptophan was developed which permits the reaction to occur quantitatively in aqueous solution by substitution of 70% HCIO4 (w/v) for concentrated H2S04. Spectral analysis of the chromogen formed showed a strong adsorption band at 365 mµ for pure tryptophan which was 17 times more sensitive than that at 560 mµ. Factors were studied which influence the absorbance of the tryptophan chromogen at 365 mµ and the most favorable conditions for its formation. A new method for hydrolyzing proteins and peptides was evolved using 3.5% HCIO4 (w/v) which results in complete rupture of the peptide bond while retaining intact the indole nucleus. Application of the modified glyoxylic acid method to perchloric acid hydrolyzates of various proteins and peptides of known tryptophan content gave experimental values in close agreement with those calculated from the structural formulae. The over-all reproducibility of the procedure for separate hydrolyzates, run in duplicate, of the same protein or peptide sample was about ±3.0%. Comparison of the tryptophan values obtained for 19 different "purified" peptides and proteins checked rather well with some of the results reported in the literature for the same materials using other procedures, except for trypsin and ovomucoid.

Tris (hydroxymethyl) aminomethane as a standard for kjeldahl nitrogen analysis.

Abstract: Solutions of tris (hydroxymethyl) aminomethane (Tris) have been shown to be a convenient primary standard for the Kjeldahl analysis. This compound is readily available in analytical reagent grade and has all the desirable properties of a primary standard. It is unnecessary to protect the Tris solution from C02, as any reaction between Tris and this gas cannot be detected by the titrimetric procedures involved.