bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of values for calcium and protein in serum, and of a derived index obtained from a probability population sample.
Authors: Kelly A , Munan L , PetitClerc C , Ho K P , Billon B
Published: January 2020

We measured serum protein and calcium concentrations in 2340 individuals between 10 and 96 years of age from 900 families chosen by probability methods to give a representative population. These values were used to calculate an index, based on a regression analysis of serum protein on calcium, which was then treated as a new variable. Age-sex specific reference values and frequency distributions are presented for this index as well as for protein and calcium calculated by both parametric and nonparametric methods.
Bile acid and detergent interaction with radioassays based on coated charcoal.
Authors: Andersen K J , Schjonsby H , Skagen K W , von der Lippe G
Published: January 2020

We tested the radioassays for cyclic AMP and human gastrin, both involving separation on coated charcoal, for interaction with bile acids and detergents, and found a concentration-dependent interaction of taurocholic and glycocholic acid as well as of the surfactant Triton X-100 and sodium laurylsulfate in both assays. The interaction was detectable from concentrations of 0.5 mmol of bile acid per liter or 625 mg of detergent per liter, giving rise to falsely decreased gastrin values and falsely increased or decreased values for cyclic AMP. The interactions demonstrated may be a general effect on all radioassays that are based on the use of coated charcoal in the separation of free from bound ligands.
Nature of the interaction between low-density lipoproteins and polyanions and metal ions, as exemplified by heparin and Ca2+.
Authors: Cham B E
Published: January 2020

I examined the effect of lipid concentration on the interaction of the very-low density and low-density lipoproteins in serum with heparin and calcium. A precipitate forms when partly delipidated serum is subjected to the polyanion-metal ion system. Although this precipitate is less turbid than the complexed lipoprotein-polyanion-metal ion in undelipidated serum, the two precipitates contain identical amounts of apolipoprotein. Totally delipidated serum produces only a slight precipitate with heparin and calcium, and this precipitate contains only a fraction of the appolipoproteins. Cholesterol and triglycerides are the major determinants of turbidity when serum is mixed with heparin and calcium, but have no effect on the precipitation of the protein moiety. Phospholipids contribute a small proportion of the turbidity in the lipoprotein-polyanion-metal ion interaction. Precipitation of the low-density lipoproteins by heparin and calcium depends on the protein moiety and on the chemical composition of the lipid moiety. Phospholipid is required, but cholesterol and triglyceride are not, for total precipitation of the complex.
Plasma high-density lipoprotein cholesterol concentrations determined after removal of other lipoproteins by heparin/manganese precipitation or by ultracentrifugation.
Authors: Bachorik P S , Wood P D , Albers J J , Steiner P , Dempsey M , Kuba K , Warnick R , Karlsson L
Published: January 2020

The widely used heparin/MnCl2 precipitation procedure for determination of plasma high-density lipoprotein cholesterol has been re-examined in light of recent reports that isolated preparations of the lipoprotein are only partly precipitated under the test conditions. In the present study, the procedure as applied to plasma tolerated rather wide variations in heparin and MnCl2 concentrations without significant effects on the assayed values in several plasma pools tested. The procedure was further tested on 129 individual samples by comparison with an ultracentrifugal method in which high-density lipoprotein-cholesterol is assumed to be represented by the cholesterol content of the plasma fraction of relative (to water) density greater than 1.063. Our results indicate that high-density lipoprotein is not precipitated under the test conditions when applied to unfractionated plasma.
Erythrocyte uroporphyrinogen I synthase activity in diagnosis of acute intermittent porphyria.
Authors: Peterson L R , Hamernyik P , Bird T D , Labbé R F
Published: January 2020

Measurement of the activity of uroporphyrinogen I synthase provides an excellent laboratory aid in the diagnosis of acute intermittent porphyria, particularly in those patients who are asymptomatic or in whom the disease is not biochemically manifested by porphyrin precursor excretion. We describe here a simplified fluorometric method for measuring the activity of this enzyme in whole blood. The assay is based upon a coupled-enzyme procedure in which added delta-aminolevulinic acid and the dehydratase that is present in erythrocytes are used to generate porphobilinogen as substrate for uroporphyrinogen synthase. After appropriate incubation the protein is removed we sensitivity, specificity, and precision of the assay compare well with previously described procedures. Activity in nonporphyric male subjects was 31 (SD, 6.0) nmol of porphyrin formed per milliliter of erythrocytes per hour at 37 degrees C. Application of the method for identifying gene carriers of acute intermittent porphyria is demonstrated in three generations of an affected family.
Application of a vidicon spectrometer for simultaneous multicomponent drug determinations.
Authors: McDowell A E , Harner R S , Pardue H L
Published: January 2020

A rapid scanning vidicon spectrometer has been evaluated for the simultaneous determination of drugs in mixtures, without a separation step. Spectral data in the ultraviolet region are collected on-line with a small computer at repetition rates of 100 scans per second. Absorbance data at several wavelengths are processed by matrix equations to resolve them into the concentration of each component in each mixture. Results are reported for two-component mixtures of procainamide and N-acetylprocainamide in serum, for two-component aqueous mixtures of butabarbital and seconbarbital, and for seven-component aqueous mixtures of phenobarbital, diphenylhydantoin, aminophylline, acetaminophen, salicylamide, phenylbutazone, and secobarbital. We conclude that the computer-inter-faced vidicon spectrometer is a viable tool for simultaneous multicomponent determinations.
An evaluation of the Beckman chloride/carbon dioxide analyzer.
Authors: Hicks J M , Aldrich F T , Iosefsohn M
Published: January 2020

We compared the Beckman Cl/CO2 Analyzer to the American Instrument Company chloride titrator for measurement of chloride in serum, sweat, and spinal fluid. We also compared it to the Natelson gasometer and a modified continuous-flow (AutoAnalyzer) technique for measurement of carbon dioxide in serum. Effects of bilirubin and hemoglobin on results for both the CO2, and Cl were negligible. The linearity, stability, and precision of the equipment was studied. We believe the Beckman Cl/CO2 Analyzer to be ideal for simultaneous measurement of chloride and carbon dioxide in a pediatric or emergency-determination setting.
A search for the best buffer to use in assaying human lactate dehydrogenase with the lactate-to-pyruvate reaction.
Authors: Buhl S N , Jackson K Y , Lubinski R , Vanderlinde R E
Published: January 2020

Highly purified human lactate dehydrogenases I and V were assayed in 17 different buffers, at a variety of reaction pH's. Diethanolamine and 2-amino-2-methyl-1,3-propanediol provided the best measurements of the enzyme, assayed lactate-to-pyruvate. However, the commercial preparation of 2-amino-2-methyl-1,3-propanediol contained insoluble matter and was relatively expensive. All of the four buffers nowmost commonly used were found to present difficulties. Glycine and pyrophosphate were inhibotory tolactate dehydrogenase activity with increasing buffer concentration. 2-Amino-2-methyl-1-propanol had three major disadvantages: it is chemically unstable during reagent preparation; activity is dependent on buffer concentration; and the pH optima for isoenzymes I and V are vastly different. The pKa of tris(hydroxymethyl)aminomethane is 8.0 at 30 degrees C, whereas to measure total activity the reaction pH should be greater than 8.5; thus tris(hydroxymethyl)aminomethane has limited buffering capacity at the reaction pH.
Aspartate aminotransferase activity in human serum. Factors to be considered in supplementation with pyridoxal 5'-phosphate in vitro.
Authors: Horder M , Moore R E , Bowers G N
Published: January 2020

The pyridoxal phosphate reactivation of the apo form of aspartate aminotransferase (EC 2.6.1.1) in human serum has been studied with "normal" and above-normal activity of this enzyme. The extent of the reactionation did not depend on the presence of the substrates, L-aspartate or 2-oxoglutarate. Reactivation was greatest with 110 mumol of added pyridoxal phsophate present per liter during a preinucation for 7 min in tris(hydroxymethyl)methylamine buffer wit;h serum volume fractions ranging from 0.017 to 0.267. In comparison with measurements prformed with no exogenous pyridoxal phosphate present, we found two potential sources of error when this cofactor was added: (a) reagent and sample blanks in the pyridoxal phosphate-supplemented system were two- to eightfold higher and (b) progress curves were nonlinear when L-aspartate rather than 2-oxoglutarate was used as the startin substrate. Aspartate aminotransferase measurement sith pyridoxal phosphate supplementation was slightly more precise than without.
Improved thyroxine radioimmunoassay, for filter paper discs saturated with dried blood.
Authors: Mitchell M L
Published: January 2020

I describe an improved procedure for estimating thyroxine in dried blood on 0.32-cm filter paper discs, by radioimmunoassay. A weak anion-exchange resin is used to separate free and antibody-bound radiothyroxine and, by measuring the resin-bound 125I-labeled thyroxine rather than the supernatant radioactivity, a tedious and cumbersome step is eliminated. The unique properties of the resin permit considerable latitude in the experimental conditions, in contrast with charcoal, without compromising sensitivity or precision. There is excellent correlation between thyroxine values obtained from discs and those of the corresponding plasmas. Application of this procedure for large-scale screening of newborns has proved to be highly successful in identifying thyroid insufficiency in several babies before clinical signs were apparent.
Negative anion gap in a young adult with multiple myeloma.
Authors: Gumprecht T , O'Connor D T , Rearden A , Wolf P L
Published: January 2020

A young patient with multiple myeloma was found to have a negative anion gap, with marked asymptomatic hyponatremia. The cause for his negative anion gap is thought to be the myeloma protein, which acts as a cation at physiological pH. Such a hyponatremia responds to reduction in serum concentration of paraprotein and should not be treated by sodium replacement.
Diazotization of catecholamines and their analogs and metabolites for urinary screening tests: chemical aspects.
Authors: Vauquelin G , Verheyden R , Ebinger G , Strosberg A D
Published: January 2020

Coupling of diazotized p-nitroaniline to catecholamines and their metabolites in urine has been proposed for use in screening for secreting neuroblastoma in childhood. We have coupled diazotized p-nitroaniline to catecholamines, derivatives, and metabolites and examined the reaction products by thin-layer chromatography and physico-chemical methods (ultraviolet spectra, mass spectroscopy, nuclear magnetic resonance). We conclude that during diazotization, products containing a p-hydroxybenzyl alcohol or a p-hydroxybenzoic acid structure (e.g., vanillic acid, vanilmandelic acid, 3-methoxy-4-hydroxyphenyl-ethyleneglycol, metanephrine, normetanephrine, synephrine, and isoproterenol) react with a diazonium cation, with release of their alcohol or acid moiety. Therefore the mentioned qualitative screening methods are very nonspecific. In contrast, thin-layer chromatographic screening methods provide complete separation and unambiguous identification of those metabolites and are to be preferred for use in detecting secreting neuroblastoma in childhood.
Microscale modification of a cation-exchange column procedure for plasma ammonia.
Authors: Oberholzer V G , Schwarz K B , Smith C H , Dietzler D N , Hanna T L
Published: January 2020

A column cation-exchange resin procedure for plasma ammonia was modified to require only 100 mul of plasma per determination. Mean analytical recovery of standard from resin (six samples) was 98% (range, 94-100%) as compared to a mean of 63% (range, 58-70) for nine samples when a batch cation-exchange procedure was used. Absorbance was proportional to sample concentration up to 800 mumol/liter. Analytical recovery of standard from plsma (six samples) was complete (mean, 103%; range, 90-113). Thirteen aliquots of a specimen of fresh plasma from a single adult individual gave a mean value of 20 mumol/liter (range, 11-26). The mean plasma venous ammonia concentration for 27 adults was 16 mumol/liter (range, 0-39), and for 15 newborns it was 60 (range, 34-102). Values for capillary plasma measured at the same time were higher, and we discuss possible explanations for this. Values are given for infants and children from one month to 14 years of age. Effects of storage time and temperature on plasma ammonia concentration are discussed.
An optimized continuous-monitoring procedure for semiautomated determination of serum acid phosphatase activity.
Authors: Bais R , Edwards J B
Published: January 2020

A continuous-monitoring method for measuring acid phosphatase activity with alpha-naphthyl phosphate as the substrate was critically evaluated and modified. Using partially purified prostatic acid phosphatase, we show that certain conditions for the assay must be satisfied to ensure linearity. These conditions include maintaining the pH between 5.6 and 5.9 and the addition of detergent to sustain linearity. The results obtained with alpha-naphthyl phosphate have been compared with those obtained by using p-nitrophenyl phosphate as substrate. When used with an automatic rate analyzer, the modified method is as sensitive but more reproducible.
Stabilization of blood glucose by cooling with ice: an effective procedure for preservation of samples from adults and newborns.
Authors: Lin Y L , Smith C H , Dietzler D N
Published: January 2020

Glycolysis causes a considerable decrease in blood glucose when whole blood is kept at room temperature without preservative. The most commonly used preservative, NaF, makes analysis of other serum constituents such as sodium and calcium and urea difficult or impossible, an especially serious limitation when sample size must be restricted. In samples at room temperature without preservative, plasma glucose decreased 36 mg/liter per hour in blood from adults and 60 mg/liter per hour in blood from newborns. Cooling on ice slowed these rates to 3.9 and 11, respectively. Plasma potassium increased 0.3 mmol/liter per hour in cooled specimens from both adults and newborns. Sodium, calcium, chloride and urea values were unaffected. We conclude that cooling effectively stabilizes plasma glucose for 4 h in samples from both adults and newborns and that potassium may be measured with negligible change for as long as 1 h and other constituents for the entire period.
Automated Enzymatic Determination of L-Lactate in Serum, with Use of a Miniature Centrifugal Anlyzer
Authors: Hadjiioannou T P , Hadjiioannou S I , Brunk S D , Malmstadt H V
Published: January 2020

We describe an automated enzymatic reaction-rate method for spectrophotometric determination of lactate in serum with a miniature centrifugal analyzer. The L(+)-lactate is selectively oxidized in the presence of lactate dehydrogenase (EC 1.1.1.27) and NAD+ to from NADH, whitch is measured from its absorption. Reaction rates are determined automatically, and unknown concentrations are calculated from a computer0generated calibration curve with aqueous lithium lactate standards. Lactte concentrations in the range 0.32-1.6 µg/4 µl (80-400 mg/liter) of sample were determined with relative errors and coefficient of variation of 4.8%. Analytical recovery of lactate added to pooled serum was 89-112% (average, 101%). Comparison with a kit ("Rapid Lactate") method gave a correlation coefficient squared of 0.979 over a concentration range of 39-779 mg/liter.
Enzymatic rate method for measuring cholesterol in serum.
Authors: Pesce M A , Bodourian S H
Published: January 2020

An enzymatic rate assay is described for measuring cholesterol in serum. Cholesterol is analyzed by mixing 5 mul of sample with a reagent consisting of cholesterol esterase, cholesterol oxidase, catalase, acetylacetone, methanol, and hydroxypolyethoxydodecane in a ammonium phosphate buffer at pH 7.0. The rate of increase in absorbance of the dihydrolutidine product is measured at 37 degrees C and 405 nm. The change in absorbance between 4 and 10 min is used to calculate the cholesterol concentrations by using simultaneously determined free cholesterol standards. The change is linearly related to cholesterol concentration up to 4 g/liter. Samples containing bilirubin up to 200 mg/liter, uric acid up to 200 mg/liter, and hemoglobin up to 1 g/liter, or certain drugs (clofibrate, phenobarbital, nicotinic acid, salicylate, Ketochol, and Ovral) gave no interference. Ascorbic acid added to serum caused a positive interference. Lipemic samples gave values that were slightly lower than did the method of Abell et al., used for comparison. Our kinetic assay, compared with the method of Abell et al., the enzymatic assay used with Abbott's Bichromatic Analyzer, and the Technicon SMA 12/60 enzymatic procedure gave correlation coefficients of 0.992, 0.985, and 0.986, respectively.
Determination of the molar absorptivity of NADH.
Authors: McComb R B , Bond L W , Burnett R W , Keech R C , Bowers G N
Published: January 2020

The molar absorptivity of NADH at 340 nm has been determined by an indirect procedure in which high-purity glucose is phosphorylated by ATP in the presence of hexokinase, coupled to oxidation of the glucose-6-phosphate by NAD+ in the presence of glucose-6-phosphate dehydrogenase. The average value from 85 independent determinations is 6317 liter mol-1 cm-1 at 25 degrees C and pH 7.8. The overall uncertainty is -4.0 to +5.5 ppt (6292 to 6352 liter mol-1 cm-1), based on a standard error of the mean of 0.48 ppt and an estimate of systematic error of -2.6 to +4.1 ppt. Effects of pH, buffer, and temperature on the molar absorptivity are also reported.
Double-antibody enzyme immunoassay applied to human alpha 1-fetoprotein.
Authors: Belanger L , Hamel D , Dufour D , Pouliot M
Published: January 2020

We report the development of a double-antibody system for enzyme immunoassay of human alpha 1-fetoprotein. Equilibrium and sequential saturation procedures were evaluated and compared with a similar double-antibody radioimmunoassay. With our method, 3 mug of alpha 1-fetoprotein per liter can be detected. The dose-response curve covers a 100-fold range of analyte concentrations. Within-run and between-run coefficients of variation are respectively 6.9% and 10.0%. Analytical recovery of added antigen is 99.5 +/- 10.6%. Results are routinely obtained in 24 h but can be obtained in less than 8 h. Alpha 1-fetoprotein was measured in a variety of clinical samples, including sera from 488 pregnant women. The advantages of the present method over radioimmunoassay and other enzyme immunoassay procedures are discussed.
Complement C3 in serum and plasma, as measured by radial immunodiffusion with four commercial kits.
Authors: Vladutiu A O , Winiarski B M
Published: January 2020

Quantitation of the C3 component of complement by single radial immunodiffusion is subject to error because the C3 molecule has several antigenic determinants and anti-C3 sera differ in their specificity to these determinants. C3 was measured in plasma (ethylenediaminetetraacetate anticoagulant) and serum, in five normal individuals, by use of four such commercial kits. The effects of ethylenediaminetetraacetate and of incubation at -20, 4, and 37 degrees C for 1, 2, and 7 days were investigated. We found wide variations in C3 in the same sample, as measured with different kits. Incubation for longer than 48 h at 4 degrees C changed C3 concentrations as compared to those in fresh samples. Reference sera for C3 assay and standardized procedures for sample processing are both essential to valid results.
Changes in electrophoretic mobilities of alpha-and beta-lipoproteins as a result of plasma delipidation.
Authors: Cham B E , Knowles B R
Published: January 2020

A two-phase system containing the ternary mixture butanol/disopropyl ether/plasma in different proportions yields ordered delipidation of alpha-lipoproteins, pre-beta-lipoproteins- and beta-lipoproteins in plasma, as quantitated by densitometry after electrophoresis. As a consequence of delipidation the electrophoretic mobilities of pre-beta-lipoproteins and beta-lipoprotein increased, that of alpha-lipoprotein decreased.
Simultaneous quantitation of 4-hydroxy-3-methoxymandelic (vanilmandelic) and 4-hydroxy-3-methoxyphenylacetic (homovanillic) acids in human urine.
Authors: Addanki A , Hinnenkamp E R , Sotos J F
Published: January 2020

We describe a simultaneous analysis for vanilmandelic and homovanillic acids in urine. An aliquot of a final extract, containing phenylpropriopionic acid as external standard, is dried, an internal standard (resorcinol) is added, and the mixture is silylated and chromatographed. The two compounds are identified from their characteristic retention times and from their elution behhavior in relation to four different marker compounds (the external and internal standards and two other naturally occurring compounds, p-hydroxyphenylacetic and hippuric acids) and are quantitated from their relative response factors. Within-run precision (CV) varied from 2 to 14% for vanilmandelic acid and 1 to 18% for homovanillic acid. The ratio of the amount of phenylpropionic acid extracted to the amount of added resorcinol found is a useful quality-control index. Values for vanilmandelic acid obtained by this method and by the technique of Pisano et al. [Clin. Chim. Acta 7, 285 (1962)] correlated well (r = 0.844).
Use of enzyme-loaded erythrocytes in in-vitro correction of arginase-deficient erythrocytes in familial hyperargininemia.
Authors: Adriaenssens K , Karcher D , Lowenthal A , Terheggen H G
Published: January 2020

The capacity of arginase-deficient erythrocytes of patients with familial hyperargininemia to produce urea and to catabolize arginine can be increased in vitro by introducing human liver arginase into their erythrocytes. The results of this study on a specific human model show that it is possible to change the metabolic function of a genetically defective erythrocyte by incorporating exogenous human enzyme. The in vivo application of enzyme-loaded erythrocytes for enzyme replacement therapy of inborn metabolic errors in humans must await in vivo studies on animal models.
Gas-chromatographic analysis for succinimide anticonvulsants in serum: macro- and micro-scale methods.
Authors: Bonitati J
Published: January 2020

A gas-chromatographic analysis for the succinimide anticonvulsant drugs--ethosuximide, methsuximide, and phensuximide--in 1.0 ml of serum was modified to improve its reliability, speed, and precision. A separate procedure for 10-100 mul of serum was also developed. Neither method requires an initial preparation of derivatives. The working range of each method is about 10-100 mg/liter for the macro-method, 2-100 mg/liter for the micro-method. The methsuximide metabolite, N-desmethylmethsuximide, is included in both methods. Concentrations of N-desmethylmethsuximide in the blood of two patients with petit mal epilepsy are reported.
Nonchromatographic radioimmunoassay of unconjugated estriol in plasma, with polyethylene glycol as precipitant.
Authors: Schiller H S , Brammall M A
Published: January 2020

We describe a rapid, reliable radioimmunoassay for unconjugated estriol in plasma. Polyethylene glycol (Carbowax 6000) is used to separate antibody-bound and free steroid. The assay is sensitive (25 pg for standards), precise, and accurate. At high and low concentrations of estriol, intra-assay coefficients of variation were 7.1% and 7.6%, respectively, and inter-assay coefficients of variation were 7.2% and 10.0 %, respectively. Free [3H] estriol is not precipitated by polyethylene glycol. This radioimmunoassay of estriol, with a highly specific antiserum and with polyethylene glycol as the antibody precipitant, is a reliable one-day assay that is practical both for the clinical laboratory and the obstetrician.
Measurement of 25-hydroxyvitamin D3 in serum.
Authors: Bouillon R , Kerkhove P V , De Moor P
Published: January 2020

We describe a method for measuring 25-hydroxyvitamin D3 in serum. Extraction with dichloromethane/methanol (2/1 by vol), followed by chromatography on a column of Sephadex LH-20, resulted in an overall analytical recovery of 82% +/- 3.5% (SD). Diluted normal rat serum was used as binding protein because it contains a transport protein that has both a high affinity (Ka = 2 X 10(10) liter/mol) and a high capacity (3 X 10(-6) mol/liter) for 25-hydroxyvitamin D3. There is no advantage in using more complex binding proteins derived either from rachitic animals or from cytosol preparations. Concentrations of 25-hydroxyvitamin D3 (13.4 +/- 4 mug/liter) in the serum of apparently normal Belgian subjects are lower than those reported for North Americans, but resemble those reported for the United Kingdom.
Gamma-glutamyltransferase: Substrate inhibition, kinetic mechanism, and assay conditions.
Authors: Stromme J H , Theodorsen L
Published: January 2020

Gamma-glutamyltransferase activity in serum is shown to be competitively inhibited by the two substrates gamma-glutamyl-4-nitroanilide and glycylglycine. Awareness of this is of importance when one is choosing final reaction conditions for the assay of the enzyme. Gamma-glutamyltransferase probably acts by a "ping-pong bi-bi" kinetic mechanism, which fits with the double competitive substrate inhibition demonstrated. The product, 4-nitro-aniline, appears to be an uncompetitive dead-end inhibitor of both substrates. Various amino acids, particularly glycine and L-alanine, inhibit the enzyme. Their inhibition patterns are uncompetitive with glycylglycine and competitive with gamma-glutamyl-4-nitroanilide. On the basis of the present and other studies, the Scandinavian Society for Clinical Chemistry and Clinical Physiology is going to recommend for routine use a gamma-glutamyltransferase method in which the final concentrations of gamma-glutamyl-4-nitroanilide and glycylglycine are 4 and 75 mmol/liter, respectively.
Clinical laboratory evaluation of the Perkin-Elmer KA-150 Enzyme Analyzer.
Authors: Hardin E , Passey R B , Gillum R L , Fuller J B , Lawrence D
Published: January 2020

We evaluated the Perkin-Elmer KA-150 Enzyme Analyzer in a clinical laboratory situation according to a rigid evaluation protocol. The factors evaluated for five commonly assayed enzymes were as follows: (a) rate of analysis of patient samples, (b) analytical linearity, (c) overall precision (within-run and between-day), (d) carry-over from a sample with activity near 1000 U/liter, (e) error detection at low and high concentrations, (f) correlation of values with those obtained by methods used in our laboratory and (g) normal-value ranges. We find the KA-150 to be an effective and rapid enzyme analyzer.
Electrophoretic pattern of amylase isoenzymes in serum and urine of normal persons.
Authors: Otsuki M , Saeki S , Yuu H , Maeda M , Baba S
Published: January 2020

We separated and measured amylase isoenzymes in the serum and urine of 3036 normal persons by electrophoresis on a thin layer of polyacrylamide gel. We wished to establish the normal pattern of these isoenzymes and to evaluate the usefulness of this method of electrophoresis in clinical diagnosis. Results for patients with hyper- or hypofunctioning pancreas and salivary glands suggested that essentially all the isoamylases in human serum and urine are derived from the salivary glands and the pancreas, and revealed that isoamylases of more than 98% of normal persons consisted of two major isoenzymes and two to three minor ones. Although these observations indicate that data on changes in the proportion of amylase activity of each isoenzyme can be useful in clinical medicine, the following points should be remembered: (a) quantitative differences in the isoenzyme pattern were observed, depending upon the condition of the samples; (b) because the proportion of isoamylase activity in serum of different normal persons differs, seriatim determination of amylase isoenzymes is necessary; and (c) because five different genetically controlled types of isoamylases were observed in normal persons, genetic investigations are also necessary.
Comparison of three procedures for isolating human ferritin, for use as a standard in an immunoradiometric assay.
Authors: Gonyea L M , Lamb C M , Sundberg R D , Deinard A S
Published: January 2020

We evaluated three methods for isolating ferritin for use as a standard, with respect to purity of the products, ease of preparation, and yield. Examination of the respective products by gel filitration on Sephadex G-200 and Sepharose 6B suggested that the preparations isolated by ammonium sulfate and cadmium sulfate precipitation (Method 1) and by ultracentrifugation (Method 2) were homogeneous, while the product of a procedure including precipitation with ammonium sulfate (Method 3) contained significant amounts of nonferritin protein. The ratios of ferritin as measured by immunoradiometric assay to the amount of protein in the product indicated the ferritin prepared by Method 1 to be the most highly purified. Methods 1 and 2 were both comparatively simple. Although the yield from Method 1 was lowest, it is probably the method of choice, on the basis of the ease of obtaining a highly purified product. The most appropriate method for estimating protein in the isolated preparations appears to be that of Lowry et al.
Theoretical approaches to estimation of plasma renin activity: a review and some original observations.
Authors: Oparil S
Published: January 2020

Performance of accurrate, reproducible, and interpretable assays for plasma renin activity and other components of the renin/angiotensin system in the clinical setting requires a clear understanding of the various reactions in the renin/angiotensin cascade and the nature of their interactions. Plasma renin activity, the rate of angiotensin generations from plasma incubated in vitro, is the most commonly used clinical index of function in the renin/angiotensin system. Renin activity is measured by radioimmunoassay of angiotensin I generated in vitro under carefully controlled conditions. The value obtained for plasma renin activity depends on pH and duration of incubation and on the method used to protect the angiotensin I generated. We recommend incubation at neutral pH in buffered plasma for three hours in the presence of either ethylenediaminetetraacetate + 8-hydroxyquinolone + dimercaprol or ethylenediaminetetraacetate + phenylmethylsulfonylfluoride. Addition of a standard preparation of human renin to the plasma incubation step of the renin activity assay serves the dual purpose of permitting measurement of renin activity and furnishing an internal standard for comparison of assay procedures. The many variables among renin assay methods can be cancelled by referring to a common internal renin standard.
Gas-liquid chromatographic profile of neutral and acidic metabolites in cerebrospinal fluid from newborns and infants.
Authors: Malcolm R D , Leonards R
Published: January 2020

A profile (chromatographic pattern) of the neutral and acidic metabolites present in cerebrospinal fluid from newborns and infants was obtained by gas-liquid chromatography. The metabolites are those extracted with ethyl acetate and diethyl ether. They are converted to trimethylsilyl ether derivatives, and chromatographed on a column containing 5% silicone OV-101 (methyl) packed on Chromosorb W. Several substances were tentatively identified from their methylene unit values. We established a control profile for infants, and compared profiles for infants and adults. Noteworthy qualitative and quantitative differences from the control were seen for cerebrospinal fluid from subjects with neurological disorders of infections. The technique may be of use in neurological diagnosis.
Improved determination of prostatic acid phosphatase (sodium thymolphthalein monophosphate substrate).
Authors: Ewen L M , Spitzer R W
Published: January 2020

We have modified a previously described method for determining acid phosphatase, with thymolphthalein monophosphate as substrate, to increase its sensitivity. We assessed effects of serum on variables influencing acid phosphatase activity as measured by this method. The method is shown to be not completely specific for prostatic acid phosphatase. The importance of standardizing methodology in measurement of enzyme activities is demonstrated.
Creatine kinase in serum: 1. Determination of optimum reaction conditions.
Authors: Szasz G , Gruber W , Bernt E
Published: January 2020

To establish optimum conditions for creatine kinase (EC 2.7.3.2) activity measurement with the creatine phosphate in equilibrium creatine reaction, we re-examined all kinetics factors relevant to an optimal and standardized enzyme assay at 30 and 25 degrees C. We determined the pH optimum in vaious buffers, considering the effect of the type and concentration of the buffer, as well as the influence of various buffer anions on the activity. The relation between activity and substrate concentration was shown and the apparent Michaelis constants of creatine kinase for creatine phosphate and ADP were evaluated. We tested the effect on creatine kinase measurement of the concentration of substrates (glucose and NADP+) in the auxillary and indicator reactions, especially the influence of the added auxiliary (hexokinase) and indicator (glucose-6-phosphate dehydrogenase) enzymes on the lag phase, at different temperatures. The NADP+ concentration proved to be the factor limiting the duration of constant reaction rate. We studied the inhibition of creatine kinase and adenylate kinase by AMP and established a convenient AMP concentration. For reactivation of creatine kinase, N-acetyl cysteine as sulfhydryl compound was introduced. Finally, we examined the relationship between activity and temperature.
Interference of fatty acids in the competitive protein-binding assay for serum thyroxine.
Authors: Shaw W , Hubert I L , Spierto F W
Published: January 2020

An increase in apparent thyroxine values obtained by competitive protein-binding assay on storage of sera is well documented. We find that the major source of this positive bias is probably the unsaturated nonesterified fatty acids. Nonesterified fatty acids cause a positive basis in the competitive protein-binding assay for serum thyroxine because they inhibit the binding of radioactive thyroxine by the serum-binding reagent (probably thyroxine-binding globulin). This inhibition by fatty acids may be due to the formation of a fatty acid/thyroxine complex. The degree of inhibition caused by the fatty acids depends on the length of the carbon chain of the fatty acid and the degree of saturation. Short-chain fatty acids are more potent inhibitors of thyroxine binding than those with a longer chain, and unsaturated fatty acids are more potent inhibitors of thyroxine binding than are saturated fatty acids. The polyunsaturated fatty acid, arachidonic acid, was the most potent inhibitor of all the fatty acids tested. Triglycerides (triacylglycerols) insignificantly inhibit thyroxine binding in this assay.
Four commercial urease reagents and a laboratory-prepared reagent compared for analysis of blood urea nitrogen with the Beckman analyzer.
Authors: Norkus N S , Kubasik N P , Sine H E
Published: January 2020

Three laboratory-prepared urease reagents were compared with a commercial preparation supplied for routine use on the Beckman Blood Urea Nitrogen Analyzer. There were discrepancies in results for urea nitrogen among the four urease reagents when matching serum and the corresponding oxalate/fluoride treated plasma were compared as measured with the Beckman Analyzer and continuous-flow (AutoAnalyzer) method. All four urease preparations were affected by fluoride, but to different extents. We believe that an effective laboratory reagent can be prepared in the laboratory at significantly lower cost.
Use of blood specimens collected on filter paper in screening for abnormal hemoglobins.
Authors: Schmidt R M , Brosious E M , Holland S , Wright J M , Serjeant G R
Published: January 2020

Both cellulose acetate electrophoresis and citrate agar electrophoresis were performed on 834 blood samples collected on filter paper in Jamaica and shipped for testing to the National Hemoglobinopathy Standardization Laboratory at the U.S. National Center for Disease Control. Additionally, 30 blood samples collected locally were stored on filter paper, in microhematocrit capillary tubes, and as whole blood specimens; at selected times the samples were tested for stability to determine the best sample-collection technique for hemoglobin electrophoresis. Results were most nearly accurate when both cellulose acetate electrophoresis and citrate agar testing were used. The methods are easy to perform, but results are unreliable if the blood samples on filter paper are stored at 4 degrees C for longer than two weeks before they are tested.
Automated enzymic determination of ethanol in blood, serum, and urine with a miniature centrifugal analyzer.
Authors: Hadjiioannou T P , Hadjiioannou S I , Avery J , Malmstadt H V
Published: January 2020

We describe an automated spectrophotometric reaction-rate method for determination of ethanol in serum and urine with a miniature centrifugal analyzer. The theanol is selectively oxidized in the presence of alcochol dehydrogenase and NAD+ to form NADH, which is measured by the rate of change of its absorbance. Reaction rates are determined automatically, and unknown concentrations are calculated from a computer-generated working curve based on aqueous ethanol standards. Blood, serum, or urine specimens need not be deproteinized. The method permits duplicate analysis of at least 30 samples per hour. Coefficients of variation and relative errors are about 2-3% for ethanol concentrations of 0.3-3.0 mug per 2 mul of sample. Analytical recovery of ethanol added to serum is 92-103% (average, 98.5%). Comparisons with distillation-oxidation, gas-chromatographic, and conventional enzymic procedures give satisfactory agreement.
Assessment of interference by aspirin with some assays commonly done in the clinical laboratory.
Authors: Routh J I , Paul W D
Published: January 2020

We have assessed the interference of acetylsalicylic acid (aspirin) with some common clinical laboratory assays, because of its widespread use and reported interference. The therapeutic regimens involved ingestion of 10 325-mg tablets daily for three days or eight tablets for five days or two weeks. Twenty-one commonly done determinations were run on control sera, and sera were collected during and after drug therapy. Significant changes in t-test values, indicating the significance of the standard deviation of the difference between controls and specimens drawn after drug therapy, were observed for chloride (increased), and for total protein, calcium, cholesterol, uric acid, bilirubin, and thyroxine (decreased). Aspirin therapy depressed the apparent concentrations of these constituents progressively in the three-day regimen and initially in the long-term regimens, followed by recovery toward zero t-values at the end of the longer regimens.
Serum theophylline analysis by high-pressure liquid chromatography.
Authors: Evenson M A , Warren B L
Published: January 2020

We have developed and evaluated a rapid, high-pressure liquid-chromatographic method for theophylline in serum. Only 0.2 ml of serum is required for each determination, and the sensitivity of this method is 0.5 mg/liter. This method, involving liquid extraction and silica adsorption chromatography, provides adequate selectivity, accuracy, and precision for routine or research applications. Little sample preparation is required before chromatography. We found no endogenous or exogenous interferences. Use of beta-hydroxypropyl theophylline as the internal standard provides reproducible results for this micro-scale method.
Gas-chromatographic method for acetaminophen (N-acetyl-p-aminophenol) based on sequential alkylation.
Authors: Dechtiaruk W A , Johnson G F , Solomon H M
Published: January 2020

A gas-chromatographic procedure for acetaminophen is described in which the drug is chromatographed as the O-heptyl-N-methyl derivative. This derivative is prepared by a sequential alkylation procedure in which the phenolic hydroxyl group of the parent compound is alkylated off-column with heptyl iodide and the amide group is derivatized on-column by reaction with trimethylanilinium hydroxide. The internal standard, N-propionyl-p-aminophenol, is subjected to the same derivatization procedure. This gas-chromatographic procedure correlates well with conventional colorimetric and spectrophotometric procedures for acetaminophen and is more sensitive. Within-run precision (CV) was 2.0% at a serum concentration of 10.0 mg/liter (n = 10) and between-run precision was 4.0% over a period of eight months. This method is particularly applicable to studies of the pharmacokinetics of acetaminophen.
Gas-chromatographic quantitation of theophylline in small volumes of plasma.
Authors: Perrier D , Lear E
Published: January 2020

We describe a rapid procedure for quantitating theophylline in 100-mul plasma samples by use of a gas-liquid chromatograph equipped with a flame ionization detector. This methos is especially useful for monitoring theophylline concentrations in serum or plasma of infants, because sufficiently large blood samples can be readily obtained from a heel prick. The method is specific for theophylline in the presence of caffeine, theobromine and phenobarbital. For plasma concentrations equal to or greater than 5 mg/liter the average daily coefficient of variation was less than 7% while the coefficient of variation from day to day was less than 11%. The same approach can also be used to measure concentrations of phenobarbital in small volumes of plasma or serum, and is readily adapted to determination of theophylline and phenobarbital in larger samples.
alpha1-Fetoprotein measurement in blood spotted on paper: discriminating test for hereditary tyrosinemia in neonatal mass screening.
Authors: Grenier A , Bélanger L , Laberge C
Published: January 2020

We describe an electroimmunodiffusion technique for measuring alpha1-fetoprotein in blood spotted on chromatography paper. The system is being used as a complementary test in a neonatal mass-screening program for detection of inborn metabolic diseases in the Province of Quebec. In a series of 102 cases of neonatal hypertyrosinemia, the test has proven to be highly discriminative for hereditary tyrosinemia. It has permitted early detection of eight cases of this disease, including two that would have been missed by the previously used screening procedure, tyrosine measurement only. The test not only virtually eliminates the risk of misdiagnosis or missed diagnosis, but also permits earlier diagnosis of hereditary tyrosinemia and considerably reduces the follow-up work required for newborns with transitory tyrosinemia. The AFP test is simple, fast, practical, and inexpensive. Combined with tyrosine determination, it constitutes an optimal device for mass screening of hereditary tyrosinemia.
Anomalous behavior of glutathione reductase on dilution.
Authors: Spooner R J , Delides A , Goldberg D M
Published: January 2020

Serum glutathione reductase activity decreases on standing at room temperature. Dilution of the sample slows this decay in sera from patients with a variety of clinical disorders, and reverses the inactivation in sera from patients with liver or biliary tract disease. Prolonged contact with dithioeythritol or acetate buffer, pH 6.4, at 4 degrees C prevents spontaneous inactivation of the native serum and abolishes the effect of dilution. These hitherto unreported phenomena are a source of potential inaccuracy in assays for this enzyme. The inactivation may be the result of conformational folding with "burying" of active sites, or of molecular aggregation brought about by hydrogen bonding or disulfide bond formation.
Evaluation of a new system ("Corvac") for separating serum from blood for routine laboratory procedures.
Authors: Spencer W W , Nelson G H , Konicki K A
Published: January 2020

We evaluated the suitability of a new system ("Corvac," Corning) for rapid, complete separation of cell-free serum from whole blood. Twenty-one different clinical chemical procedures were performed on about 1150 paired blood samples, several by more than one method, to afford a more comprehensive evaluation. The tube of clotted blood collected in the conventional manner ("Vacutainer") underwent a double centrifugation and served as the control sample, while its paired specimen collected in the new system was processed by a single centrifugation. We evaluated significant differences between the means of the paired samples, the standard deviations of the differences between the means, and the bias of the means for the values of each constituent for each collection method. For several tests we observed differences that were statistically but not clinically significant. Only in the case of lactate dehydrogenase were large differences consistently demonstrated.
Lactate dehydrogenase activities in serum and plasma.
Authors: Rothwell D J , Jendrzejczak B , Becker M , Doumas B T
Published: January 2020

We investigated lactate dehydrogenase activity in serum and plasma because of the conflicting data found in the literature. We assayed serum, platelet-rich, and platelet-poor plasma by two colorimetric endpoint methods and by an ultraviolet kinetic procedure. Platelet-poor plasma and serum had essentially the same activities by all three methods, whereas the activity in platelet-rich plasma plasma averaged fourfold that in platelet-poor plasma or serum when the assay was performed under conditions that result in lysis of platelets and release of their lactate dehydrogenase. When measurements were performed in platelet-rich plasma under conditions that prevented lysis of platelets, all three types of specimens gave the same results. This occurred when the osmolality of the reaction mixture was about 240 mOsm/kg of water. At an osmolality of about 120 mOsm, the activity of platelet-rich plasma was substantially lower than that of platelet-poor plasma or serum. If plasma must be used, the sample must first be centrifuged (3000 X g, 15 min) to provide a platelet-free plasma.
Simple determination of 3alpha-hydroxy fraction and 3beta-hydroxy fraction of urinary 17-ketosteroids.
Authors: Yamaguchi Y , Hayashi C , Kumahara Y
Published: January 2020

We describe a method for determination of the 3alpha-hydroxysteroid fraction and 3beta-hydroxysteroid fraction of 17-ketosteroids in urine. The method includes enzymatic hydrolysis with beta-glucuronidase and extraction with ethyl acetate and color development of 3beta-hydroxysteroids by 3beta-hydroxysteroid oxidase and of total 17-ketosteroids by the Zimmermann reaction. The value for 3alpha-hydroxysteroids is calculated by subtracting the value for 3beta-hydroxysteroids from the value for total 17-ketosteroids. Recovery was assessed and results compared with those by an accepted method. Results were satisfactory for measuring these fractions of 17-ketosteroids.