bluuebunny/crossref_metadata_2025 huggingface dataset:
Beta-lipoprotein cholesterol quantitation with polycations.
Authors: Heuck C C , Schlierf G
Published: January 2020

We compared direct determination of beta-lipoprotein cholesterol after selective extraction of very-low-density and high-density lipoproteins from serum with poly(ethyleneimine) and a cation-exchange resin with the classical quantitation after lipoprotein fractionation with the ultracentrifuge. At beta-lipoprotein cholesterol concentrations between 1.50 and 5.00 g/liter the correlation is linear (r = 0.95). The precision for the extraction procedure is as good (CV 2.4-2.8%) as for the quantitation by ultracentrifugation (CV 3.2-6.0%). From solutions of isolated lipoproteins, very-low-density lipoproteins are 93% extracted and high-density lipoproteins 60%, but low-density-lipoproteins only 5%. The molecular mechanism of the extraction is supposed to be due to both hydrophobic interaction of long-chain fatty acid residues and ionic interaction of phospholipids located at the surface of very low-density and high-density lipoproteins and the lipophilic polycation.
Determination of cadmium in blood by use of atomic absorption spectroscopy with crucibles--and a rational procedure for dry-ashing.
Authors: Vesterberg L , Bergström T
Published: January 2020

A simple, minimal-reagent procedure for analysis of cadmium in blood is introduced. Blood samples from workers occupationally exposed to cadmium were collected with heparin. Titon X-100 surfactant was added, and 15-mul aliquots of the blood were pipetted into small nickel sampling cups and placed in a holder for 100 cups, which was enclosed in a stainless steel box. After the samples were dried and ashed in a muffle oven at various times and temperatures, cadmium was determined in an air/acetylene flame in an atomic absorption instrument with a deuterium arc background corrector. Absorbance measurements were made by signal-averaging facilities in the instrument. The conditions for analysis are specified. The method appears to be accurate and reproducible, with a CV of about 6% in the range 35-160 nmol/liter. The detection limit was 3.0 nmol/liter of blood. With the new procedure, many blood samples can be dried and ashed in parallel, which favors both simplicity and precision.
Creatine kinase isoenzyme MB in myocardial infarction: methods compared.
Authors: Griffiths J , Handschuh G
Published: January 2020

Several methods are available for specifically identifying and assessing the activity of MB isoenzyme of creatine kinase in serum. We compared two electrophoretic techniques with three column-chromatographic ones for efficacy in the diagnosis of myocardial infarction. No technique was considered absolute, but we found surprisingly large errors of diagnosis in the column-chromatographic techniques, attributable to incomplete elution.
Nonenzymic spectrophotometric determination of potential poison ivy cross-reactors.
Authors: Quattrone A J
Published: January 2020

I describe an inexpensive, nonenzymic analytical system for prescreening substances that might cross-react as Rhus toxing (e.g., poison ivy, poison oak, and sumac allergens) on human skin. By spectrophotometric assay after incubation with an oxidizing mixture of Cu(II)ammine complex and ammonium persulfate, I could accurately and reproducibly determine o-quinoidal products of several potential synthetic cross-reactors and native poison ivy allergen, and could distinguish these from catecholamines, resorcinol, p-hydroquinone, and a closely related phenol. A good correlation was obtained between this nonenzymic technique and an enzymic assay. This Cu(II)ammine/persulfate oxidative assay, however, is inexpensive and obviates any spectral interference from enzymic proteins.
Interference in a radioimmunoassay for human thyrotropin.
Authors: Spencer C A , Challand G S
Published: January 2020

Abnormally high plasma thyrotropin values were found by radioimmunoassay in some patients when an antiserum to porcine thyrotropin was used, normal results being obtained with an antiserum raised to human thyrotropin. These discrepancies were found in some subjects with no biochemical or clinical evidence of hypothyroidism and occasionally in sera from patients with unequivocal hyperthyroidism. We found a substance in serum that cross reacts with the anti-porcine antiserum, is stable on repeated freezing and thawing, and is independent of the 125I-labeled tracer preparation. It is unlikely that this substance is a separation-stage artefact related to immunoglobulins. Its apparent molecular weight (gel filtration) is 114 000, as compared with apparent molecular weights for standard thyrotropin and endogenous thyrotropin (as found in idiopathic hypothyroidism) of 34 700 and 38 000, respectively. We believe the substance is a normal serum constituent that is present in large quantities in a minority of subjects. Apparently unrelated to TSH, its exact chemical nature remains unidentified.
System for simplified discontinuous-gradient polyacrylamide-gel electrophoresis.
Authors: Zais D P , Roberts R C
Published: January 2020

We describe a modified polyacrylamide gel electrophoresis method with which plasma proteins are excellently resolved in 4 h. We made a three-layer discontinuous gradient gel with concentrations of 80,60, and 45 g of polyacrylamide per liter of tris(hydroxymethyl)aminomethane/ethylenediaminetetraacetic acid/borate buffer, pH 9.2. Time and material needed in the preparation of the gel was greatly decreased, a well-resolved (22-25 bands) pattern was obtained in 1 h, and the gel slabs were easier to remove from the cell than were gels prepared according to the manufacturer's specifications. We saw a marked shift in the higher polymers of haptoglobin type 2-2 toward the cathode in this system, and also a clearly resolved double band of alpha2-macroglobulin in whichthe more anodal band appears to represent alpha2-macroglobulin/protease complex.
Improved electroimmunoassay of factor VIII-related antigen.
Authors: Chute A , Haddow J E , Ritchie R F
Published: January 2020

Factor VIII-related antigen migrates poorly into gel during electrophoresis in agarose, probably in major part because of the relatively high sulfate content of the commonly used agars. A recently available low-sulfate residue agar, substituted for standard agar preparations in electroimmunoassay, allows Factor VIII antigen to travel more rapidly, producing "rockets" that are well defined and easily measured, making the assay easier and more reliable.
Creatine Kinase: Stability, Inactivation, Reactivation
Authors: Morin Leo G
Published: January 2020

I determined the in vitro biological half-lives or decay constants for creatine kinase isoenzymes at various temperatures. Values at 37 °C are consistent with values reported by others in vivo, which suggests that in vivo ir¬reversible inactivation is primarily thermal. Reversible inactivation appears to be an oxidation-reduction phe¬nomenon. Proteins and some inactivators (urate, cate¬cholamines) retard irreversible inactivation and preserve isoenzyme integrity. Dilution and thiols promote reversal of inactivity. Mercaptoethanol is the preferred thiol, par¬ticularly for storage and reactivation of isoenzyme MB. MB is sensitive to light and to freeze-thawing. I recommend that specimens be cooled promptly after drawing, that mercaptoethanol (10 mmol/liter) be added, and that they be stored refrigerated. Avoid prolonged exposure to light and freezing. A model of inactivation is proposed, which is based on the assumed existence of four monomer types: active, denatured, oxidized, and insulated. The model is consistent with dilution and thiol reactivation, lag phase variations, and subtype heterogeneity.
The Immediate Reaction between Bromcresol Green and Serum as a Measure of Albumin Content
Authors: Webster David
Published: January 2020

I investigated the immediate (<30s) reaction between bromcresol green and serum, as a measure of albumin content. Fifty sera were analyzed in duplicate for albumin by electrophoresis on cellulose acetate by a procedure previously shown to give results in good agreement with two immunoprecipitation techniques, and by a bromcresol green method with the absorbance being read as soon as possible after mixing. Results by the dye method were 3.0 g/liter higher than those by electrophoresis; this difference was independent of the albumin content of the serum. Slight turbidity, jaundice, or added hemoglobin did, not significantly interfere with albumin determination. Ap¬parently the immediate reaction between serum and the dye reagent provides a simple, reliable measure of albumin content when 3.0 g/liter is subtracted from the result ob¬tained.
Ultrafiltration for Improved Assay of Urinary Enzymes
Authors: Werner Mario , Gabrielson Dale
Published: January 2020

Ultrafiltration of urine simply and rapidly separates urinary enzymes from inhibitors, activators, and heat-stable, spurious activities present in urine. Using eight enzymes of clinical interest as examples, we verified the reliability of results obtained after ultrafiltration by comparison with results obtained after gel filtration
Assay of Phenobarbital: Adaptation of "EMIT" to the Centrifugal Analyzer
Authors: Finley Paul R , Williams R Jane , Lichti Donald F , Byers James M
Published: January 2020

We have adapted the centrifugal analyzer to the homo-geneous enzyme immunoassay ("EMIT"®) for phenobar-bital. The assay was modified to give greater range and sensitivity, and less reagent is needed. The transfer disc is prepared with totally automatic pipetting. Results are calculated with a micro-computer that provides a logit transformation of absorbance data. Precision and accuracy are excellent, and results correlate well with those by gas—liquid chromatography.
Plasma Fibrinogen, Cholinesterase Activity, and Anemia: Utility of Fibrinogen in Multiphasic Screening and in Assessing the Activity of Diseases
Authors: Rice Eugene W
Published: January 2020

I present the general pattern relationships between (a) plasma fibrinogen and cholinesterase activity and (b) plasma fibrinogen and hemoglobin, in 250 and 310 hos-pitalized adults, respectively. Although responses of cholinesterase were often unrelated to fibrinogen, when its activity in plasma was depressed, above-normal fi-brinogen concentrations were present in about 90% of the subjects. Further, despite the observed independence of hemoglobin and fibrinogen concentrations, whenever frank anemia was present (hemoglobin < 110 to 120 g/liter), hyperfibrinogenemia was also present in about 75-85% of such anemic patients. Thus, these studies affirm that fibrinogen determination would be an appropriate com-ponent of tests on hospital admission and also that hyp-erfibrinogenemia is a very sensitive "acute phase" reactant, and an important reaction for gauging the course of numerous disorders.
Detection of errors in methylmalonyl-CoA metabolism by using amniotic fluid.
Authors: Morrow G , Revsin B , Lebowitz J , Britt W , Giles H
Published: January 2020

We report a method for rapid prenatal detection of methylmalonic acidemia, consisting of measuring methylmalonly-CoA mutase (EC 5.4.99.2) activity in non-cultured amniotic cells and measuring the concentration of methylmalonate in the amniotic fluid. Immediate stabilization of the mutase activity in the non-cultured amniotic cell by its coenzyme adenosycobalamin, and use of methylmalonyl-CoA with high specific activity gives mutase activity comparable to that of cultured amniotic cells or normal fibroblasts. Consequently, findings of low mutase activity and a hight concentration of methylmalonate in the amniotic fluid allows accurate diagnosis of the vitamin B12-nonresponsive form of methylmalonic acidemia. These results can be obtained in two days. For the vitamin B12-responsive form, the correct diagnosis depends upon finding amniotic fluid methylmalonate, because cells from these patients will display normal methylmalony-CoA mutase activity after adenosylcobalamin is added. Problems in interpreting data on bloody samples and the limitations of the method are discussed.
Evaluation of the radioimmunoassay for benzoylecgonine (a cocaine metabolite) in human urine.
Authors: Mulé S J , Jukofsky D , Kogan M , De Pace A , Verebey K
Published: January 2020

The 125I-radioimmunoassay (RIA) for benzylecgonine (cocaine metabolite) in urine was evaluated by comparison with gas-liquid chromatography (GLC) and thin-layer chromatography (TLC) and the Enzyme-Multiplied Immunoassay Technique (EMIT). By RIA, a statistically significant concentration, 2 microng/liter, was observed for urinary benzoylecgonine. The coefficient of variation for the RIA was 2.58+/-0.38% inter-assay and 2.20+/-0.14% intraassay. There was cross-reactivity with cocaine (more reactive than benzoylecgonine) and other members of the tropane family of alkaloids. There was agreement between results by RIA and GLC in 95.5% of the samples, between RIA and TLC in 87.0%, and between RIA and EMIT in 84.5%. The percentage of true false-positives was 3.5% for the RIA in comparison to GLC, 8.8% in comparison to TLC, and 9.1% in comparison to EMIT. True false-negatives were insignificant (0 to 1.0%). GLC and RIA results correlated highly (phi=0.908). GLC, therefore, was the best comparison method for this evaluation study. RIA for benzoylecgonine is sensitive, reproducible, and reliable for the detection of cocaine in urine.
New colorimetric method for quantitative determination of protein in urine.
Authors: Yatzidis H
Published: January 2020

Total urinary protein is rapidly precipitated at room temperature by tannic acid. The tannic acid/protein precipitate, dissolved in aqueous triethanolamine/ferric chloride solution, gives a purple-violet color of high absorptivity. Absorbance at 510 nm is linearly related to concentration from 0.05 to 1.50 A for a protein content of 0.05 to 1.50 g/liter, and less than 5 mg/liter can be detected. The CV and analytical recovery ranged from 0.5 to 1.8% and 98 to 103%, respectively. Nonprotein urinary constituents do not interfere.
The effects of ethanol (0.75 g/kg body weight) on the activities of selected enzymes in sera of healthy young adults: 1. Intermediate-term effects.
Authors: Freer D E , Statland B E
Published: January 2020

We report the intermediate-term effects of three consecutive evenings of moderate ethanol ingestion (0.75 g/kg body weight each evening) on activity values for alkaline phosphatase, gamma-glutamyltransferase, creatine kinase, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase in sera of nine apparently healthy young adults. We define "intermediate-term" effects as those occurring between 10 h and 100 h after completion of the ethanol consumption schedule. The most pronounced changes in enzyme activity for the group of volunteers were: gamma-glutamyltransferase, +25% at 60 h after ethanol ingestion; alanine aminotransferase, +12% at 60 h after ethanol; and aspartate aminotransferase,--12% at 60 h after ethanol. All three enzymes exhibited similar time courses, i.e., mean peak activity changes were observed at 60 h, and all three mean enzyme activity values returned to near baseline by 100 h. The possible explanations for the observed changes and the clinical significance are discussed.
A fully automated solid-phase radioimmunoassay for triiodothyronine.
Authors: Rugg N E , Hasler M J , Bjornsen R E , Painter K
Published: January 2020

We report the first solid-phase radioimmunoassay for triiodothyronine with use of antibody-coated tubes, which is designed specifically for the fully automated radioimmunoassay instrument, Micromedic Systems' "Concept 4 Automatic Radioassay." Antisera to triiodothyropropionic acid/bovine serum albumin were raised in rabbits, purified by ammonium sulfate precipitation, and coated onto polypropylene tubes. Analytical recovery of exogenous triiodothyronine added to sera from normal men and women and pregnant women was quantitative. Intra-assay and inter-assay coefficients of variation are 4-5 and 6-9%, respectively. Correlation coefficients (r) for comparison of sample values with those obtained by three commercial laboratories were 0.95, 0.84, and 0.91. The sensitivity of the assay is 0.5 microng/liter. The assay can be performed either manually or be fully automated on the "Concept 4."
Gas-chromatographic profiling of urinary acidic and alcoholic catecholamine metabolites.
Authors: Muskiet F A , Fremouw-Ottevangers D C , Wolthers B G , Vries J A
Published: January 2020

Urinary acidic and alcoholic catecholamine metabolites were isolated separately from 24-h urines by simple extraction procedures. After derivatization, extracts were analyzed by means of simultaneous gas chromatography on two different stationary phases. The quality of the assay was checked by running "normal" and enriched commercial (Hyland) control urines in each series. Normal values are given for vanilmandelic acid, homovanillic acid, and 3-methoxy-4-hydroxyphenylethylene glycol, expressed as a function of age. We present excretion patterns showing these metabolites and also 3,4-dihydroxyphenylacetic acid, vanillacetic acid, 3,4-dihydroxyphenylethylene glycol, and vanilethanol for 12 patients with neurogenic tumors. Values obtained for vanilmandelic acid by this method correlate well with those obtained by the electrophoretic/colorimetric method of Hermann [Am. J. Clin. Pathol. 41, 373 (1964)].
Nickel content of human palatine tonsils: analysis of small tissue samples by flameless atomic absorption spectrophotometry.
Authors: Torjussen W , Andersen I , Zachariasen H
Published: January 2020

We describe a method for determining the nickel content of small tissue samples by flameless atomic absorption spectrophotometry in this case biopsy specimens from human palatine tonsils. Contact between tissue samples and metallic objects was avoided, except for the use of biopsy forceps (Type No. 8150.00 Wolf, stainless steel) for collecting samples, to imitate the actual procedure when small biopsy specimens are removed from the nasal mucosal membranes in nickel workers for histopathological and chemical investigations. Nickel contamination from this instrument was insignificant at the precision of the present procedures. The mean concentration of nickel in 15 tonsils was 13.5 +/- 7.0 (SD) microng/100g (wet wt). The mean nickel concentration in eight different samples of the same tonsil was 5.6 +/- 2.7 (SD microng/100 g.
Relation between concentrations of free glycerol and triglycerides in human sera.
Authors: Stinshoff K , Weisshaar D , Staehler F , Hesse D , Gruber W , Steier E
Published: January 2020

The concentrations of total glycerol, unesterified glycerol, and glycerol deriving from triglycerides (triacylglycerols) were measured in 468 fresh human sera from unselected outpatients of a municipal hospital. The distribution mode of free glycerol was log-normal; that of triglycerides was neither normal nor log-normal. A weak but statistically approved correlation between these variables was demonstrated. A comparison of triglyceride concentrations corrected for unesterified glycerol with those based on total glycerol shows that, for the triglycerides determination, analysis for total glycerol and substraction of 0.11 mmol/liter from the resulting value appears to be justified in clinical routine. Subtraction of individual sample-blank values is unnecessary. In a triglyceride concentration range from 1.71 to 2.85 mmol/liter the 95 percentile for the possible error of this procedure ranges from + 0.04 to - 0.08 mmol/liter.
Vitamin B12 radioassay with oyster toadfish (Opsanus tau) serum as binder.
Authors: Kubiatowicz D O , Ithakissios D S , Windorski D C
Published: January 2020

We describe a vitamin B12 radioassay in which oyster toadfish (Opsanus tau) serum is used as the binding protein. The serum is quite stable and has a high capacity and high binding affinity (K greater than 10(12) liter/mol) for vitamin B12. The binding is not significantly affected by temperature, the presence of denatured proteins, or the amount of vitamin B12 present. The radioassay is reproducible (CV 4.4%) within assay, 7.8% between assays) and sensitive (12 ng/liter). Assay accuracy is unaffected by the protein in serum or by reasonable variations in incubation temperature and time.
Evaluation of two commercial kits for serum gastrin assay, and comparison with a conventional radioimmunoassay procedure.
Authors: Gibson R G , Hirschowitz B I , Mihas A A
Published: January 2020

We compared two gastrin radioimmunoassay kits ("Immutope" kit, Squibb & Co.; "Gastrin R.I.A." kit, Schwarz/Mann) to the conventional gastrin radioimmunoassay of Yalow and Berson [Gastroenterology 58, 1 (1970)] as run by us and by a second reference laboratory. Although both kits were found to effectively discriminate above-normal and normal values for serum gastrin, they significantly underestimated very high values (greater than 1500 ng/liter). The Schwarz/Mann kit clearly had a superior quality label (lower nonspecific binding and higher specific activity) and a shorter incubation time. However, the 90-min incubation period cited for their kit caused overestimation of gastrin values in the lower range (5-300 ng/liter), which could be corrected by prolonging the incubation to 24 h. The Squibb antibody had fairly good cross reactivity to all gastrin species tested; the Schwarz/Mann antibody had poor affinity for natural human gastrin G34-II. Good correspondence was found for sera run by both reference laboratories (y = 0.96x + 10, r = 0.997), and values obtained with the Schwarz/Mann kit correlated best (+ 0.815) with those from the conventional radioimmunoassay procedure.
Adaptation of the EMIT serum digoxin assay to a mini-disc centrifugal analyzer.
Authors: Brunk S D , Malmstadt H V
Published: January 2020

We have modified the EMIT (Syva) serum digoxin assay for use with a mini-disc centrifugal analyzer. A minicomputer is used to control data acquisition, calculations, and readout for 14 cuvettes simultaneously. Thus, it is not necessary for time each individual assay manually or to plot calibration curves on graph paper. We use incubation times and dilutions similar to those used for the manual procedure, but we have changed the buffer solution, decreased the measurment time, and decreased the required serum volume per assay fivefold. We evaluated day-to-day and within-run precision, using Syva calibrators and control sera. EMIT assay values for radioimmunoassay control sera correlate well with the supplier's stated values.
Plasma lactate dehydrogenase activity will be increased if detergent and platelets are present.
Authors: Bais R , Prior M P , Edwards J B
Published: January 2020

Plasma lactate dehydrogenase activity is artifactually increased when analyzed on the SMAC (Technicon) continuous-flow analyzer. The factors responsible are incomplete centrifugation and the presence of detergent in the dilution buffer. The contribution of platelet lactate dehydrogenase in the plasma is demonstrated with a case of thrombocytosis.
Calorimetry, a time-honored technique with a potential in analytical work and cellular biology.
Authors: Levin K
Published: January 2020

Different types of calorimeters are briefly reviewed, stressing those instruments likely to be of interest in the clinical chemistry laboratory. The unspecific nature of the measuring procedure is emphasized and various pitfalls likely to cause analytical errors are pointed out. Recent work is reviewed where calorimeters have been used as analytical tools for the determination of glucose, protein, enzymes, and other substances. The results generally compared favorably with those obtained by conventional analytical procedures. In recent reports the time per analysis has been brought down to 2 min, with sample volumes in the micro range. Valuable information on the coagulation process has been obtained by use of calorimetry. I also review studies showing that intact cellular elements such as human blood cells, bacteria, and spermatozoa can successfully be investigated with calorimetric techniques. In particular, studies on human blood cells stimulated with various agents appear to be able to give valuable diagnostic information. I believe that new designs of microcalorimeters have placed an easily handled tool at the disposal of the worker in a clinical laboratory and that use of this tool can contribute to the development of our discipline.
Automated fluorometric analysis of galactose in blood.
Authors: Mason G A , Summer G K , Dutton H H , Schwaner R C
Published: January 2020

In galactosemia, prevention of mental retardation depends on early recognition of the disorder and institution of dietary restriction of galactose. We describe an automated fluorometric micromethod for galactose in whole blood spotted on filter paper. Galactose is oxidized by galactose oxidase to D-galacto-hexadialdose and H2O2 and measured as the highly fluorescent condensation product of homovanillic acid formed when H2O2 is acted upon by horseradish peroxidase. The procedure is 10-fold more sensitive than colorimetric procedures for galactose and is not hampered by the nonspecific fluorescence from endogenous NADPH that is encountered in methods in which galactose dehydrogenase is used. At a sampling rate of 40/h with a sample-to-wash ratio of 1/2, carryover is negligible, reproducibility is excellent, and 80% of steady state is achieved. Analytical recovery of added galactose was 95%. The method has the requisite sensitivity and accuracy for quantification of galactosemia and galactosuria in milkfed newborn infants and genetic evaluation of families of patients.
Simple procedure for measuring total protein in urine.
Authors: Meola J M , Vargas M A , Brown H H
Published: January 2020

We describe a procedure for measuring total protein in urine. The method is simple, sensitive, and free of interference from drugs that are known to affect other commonly used methods. The CV for a 1.1 g/liter urine control in daily, routine use is 4.6%. The procedure involves adsorbing the protein onto cellulose powder, binding of Ponceau S dye by the protein, washing away excess dye, and eluting the bound dye into dilute NaOH for colorimetry. Comparison of results by this method with those by the biuret and turbidimetric procedures showed good agreement in those cases where the specimens were suitable for assay by the comparison methods.
The beta-lipoprotein doublet in type 3 hyperlipoproteinemia.
Authors: Papadopoulos N K , Herbert P N
Published: January 2020

We used a previously reported agarose-gel electrophoresis system to study the lipoprotein patterns in patients with type 3 hyperlipoproteinemia. All samples from 20 subjects tested revealed the presence of two bands with beta-lipoprotein mobility, irrespective of quantitative changes in plasma lipids induced by diet or by the administration of drugs. If these observations are confirmed, the method will permit the diagnosis of type 3 hyperlipoproteinemia without resort to preparative ultracentrifugation and chemical measurement of the very-low-density lipoproteins.
Effect of analytical factors on immunochemical reference limits for complement component C3 in serum of a reference pediatric population.
Authors: Buffone G J , Lewis S A
Published: January 2020

We evaluated analytical factors such as antibody specificity, standard materials, and methodology for the measurement of C3. Mancini-type radial immunodiffusion and immunonephelometry were shown to give comparable data if variables other than procedural variables are eliminated. The most significant analytical factors affecting the measurement were antiserum specificity and source of standard material.
Automation of radioimmunoassays for some sex steroids with use of both iodinated and tritiated ligands.
Authors: Hammond G L , Viinikka L , Vihko R
Published: January 2020

We describe an automated technique for estradiol, progesterone, and testosterone, in which System Olli 3000 pipetting and incubation units are used. After extraction or chromatography, steroids are redissolved in ethanol or buffer, and duplicate aliquots are arranged for radioimmunoassay in 24-tube blocks. Addition of antibodies, tracers (125I or 3H), dextran-coated charcoal for separating free and bound ligands, and removal of a portion of the supernate for counting are all performed by the pipetting instrument. Incubations are at 37 degrees C in the incubation unit, or at 4 degrees C. After counting, steroid concentrations are computed from punch tape records by a Nova 840 computer. The management of assays in 24-tube units, and accurate simultaneous pipetting has reduced experimental error, and because there is no carryover, many different assays can be performed concurrently or in rapid sequence. Various scintillation media are compared.
Determination of "true" serum creatinine by high-performance liquid chromatography combined with a continuous-flow microanalyzer.
Authors: Brown N D , Sing H C , Neeley W E , Koetitz S E
Published: January 2020

We coupled a high-performance liquid chromatograph with a continuous-flow microanalyzer to produce a system for specifically determinig "true" creatinine in urine and serum specimens. A selective microbore pellicular cation-exchange column and a single eluting sodium citrate buffer are used to separate the noncreatinine but Jaffé-positive constituents from the creatinine in normal and experimental specimens. The effluent is analyzed continuously, on-line, the alkaline picrate complex being developed and measured in the microanalyzer. Physiological samples, reference standards, and internal control specimens are assayed in 6-min intervals subsequent to the initial injection. The relationship between concentration and peak are is linear for creatinine standards between 5 and 10 mg/liter. Specimen volumes ranging from 1 to 25 microliters, and containing creatinine in amounts exceeding 5 ng per injected sample, can be assayed with this system.
Concentrations of serum protein fractions in white women: effects of age, weight, smoking, tonsillectomy, and other factors;.
Authors: Wingerd J , Sponzilli E E
Published: January 2020

We measured the concentrations of protein in electrophoretic fractions (albumin and alpha1-, alpha2-, beta, and gamma-globulins) of serum from 9547 white women to determine their relations to age, weight, smoking and other factors. Albumin concentration decreases with age, beta globulin concentration increases. Increasing weight is associated with a decrease in albumin, but with increases in all the globulins. Cigarrete smoking is associated with highly significant differences in all the protein fractions. Surprisingly, very significantly lower concentrations of beta- and gamma-globulins were also found in women who reported a history of tonsillectomy. Height, amenorrhea, education, and alcohol and coffee consumption have some small but statistically significant effects upon the various fractions. Diurnal variation was also slight.
Determination of plasma procainamide and N-acetylprocainamide concentration by high-pressure liquid chromatography.
Authors: Dutcher J S , Strong J M
Published: January 2020

We describe a routine method for determining concentrations of the antiarrhythmic drug procainamide and its active metabolite, N-acetylprocainamide, in plasma. A simple extraction of 1.0 ml of plasma is followed by separation and chromatographic analysis by use of a column containing microparticulate silica. p-nitro-N-(2-diethylaminoethyl)benzamide hydrochloride was synthesized and used as the internal standard. Total chromatographic time is only 7 min. The day-to-day CV during three months of daily use was less than 4% of the mean for each compound, and we saw no deterioration in column performance during this time. Phenobarbital, phenytoin, lidocaine, primidone, methsuximide, quinidine, and their metabolites do not interfere.
Improved colorimetric determination of serum zinc.
Authors: Johnson D J , Djuh Y Y , Bruton J , Williams H L
Published: January 2020

We show how zinc may easily be quantified in serum by first using an optimum concentration of guanidine hydrochloride to cause release of zinc from proteins, followed by complexation of released metals with cyanide. The cyanide complex of zinc is preferentially demasked with chloral hydrate, followed by a colorimetric reaction between zinc and 4-(2-pyridylazo)resorcinol. This is a sensitive water-soluble ligand; its complex with zinc has an absorption maximum at 497 nm. Values found by this technique compare favorably with those obtained by atomic absorption spectroscopy.
Micromethod for determination of diazepam by electron-capture gas-liquid chromatography.
Authors: Rey E , Turquais J M , Olive G
Published: January 2020

We used 100 microliters of plasma for the determination of diazepam. After the internal standard, prazepam, is added, the serum is directly extracted with diethyl ether, the extract is evaporated, the residue is dissolved in ethanol, and the drug is measured by gas-liquid chromatography, with use of an electron capture detector. With this procedure, 2.5 ng of diazepam in the sample can be speedily measured with specificity, accuracy, and reproducibility (CV = 4.5%).
Homogeneous reactant-labeled fluorescent immunoassay for therapeutic drugs exemplified by gentamicin determination in human serum.
Authors: Burd J F , Wong R C , Feeney J E , Carrico R J , Boguslaski R C
Published: January 2020

We applied a homogeneous reactant-labeled fluorescent immunoassay to the measurement of therapeutic drug concentrations in human serum, exemplified here by gentamicin. A derivative of umbelliferyl-beta-galactoside was coupled covalently to the drug and this conjugate was found to be nonfluorescent under assay conditions. The drug/dye conjugate was a substrate for bacterial beta-galactosidase and yielded a fluorescent product. When the drug/dye conjugate was bound to anti-gentamicin antibody it was inactive as an enzymatic substrate. This inactivation was relieved by the presence of gentamicin in competitive binding reactions. Hence, the rate of production of fluorescence was proportional to the gentamicin concentration. The fluorescent assay yielded values which compared favorably to a radioimmunoassay for gentamicin in clinical serum samples (r=0.94, standard error of estimate=0.66 mg/liter). The fluorescent assay requires only 1 microliter of serum and offers several advantages over existing techniques: sensitivity, specificity, simplicity, and the obviation of radioisotopes.
Flow cytometric measurement of peptidases with use of 5-nitrosalicylaldehyde and 4-methoxy-beta-naphthylamine derivatives.
Authors: Dolbeare F A , Smith R E
Published: January 2020

Enzyme activity can be measured in single cells or in cell suspensions by either static or flow microfluorometry when a fluorogenic substrate is used. We have used amino acid derivatives of arylamines as fluorogenic substrates for tagging cellular proteinases. The liberated aromatic amine, which can diffuse from the cell, is trapped as a fluorescent insoluble Schiff-base product with 5-nitrosalicylaldehyde, with the peak of fluorescence emission shifted from lambdaem 425 nm to lambdaem 530 and 595 nm. Although the reaction is faster at pH 4 than at higher pH's, the equilibrium during the assay of certain peptidase activities is such that the liberated aromatic amine is trapped in the cell at pH values as high as 7.5. 5-Nitrosalicylaldehyde causes almost no inhibition of substrate hydrolysis at 1 mmol/liter, a concentration exceeding that required for complete trapping of reaction product. The kinetics of enzymatic reactions with four synthetic substrates are demonstrated in intact Balb 3T3 cells and sonicated preparations in the presence of 5-nitrosalicylaldehyde. Hydrolytic rates for the substrate, CBZ-ala-arg-arg-4-methoxy-beta-naphthylamine, are given for single 3T3 cells by microfluorophotometry and for suspensions of 3T3 cells by flow cytometry. The clinical value of the method is demonstrated for differentiating mixed populations of leukocytes.
Some observations on the kinetics of the Jaffé reaction for creatinine.
Authors: Shoucri R M , Pouliot M
Published: January 2020

The Jaffé reaction for creatinine assay appears to follow pseudo-first-order kinetics; first-order rate constants are different for different samples. Rate constants for 10 different serum samples varied from a low value of 0.0040 +/- 0.0003 s-1 to 0.0084 +/- 0.0008 s-1. We describe an approach for determining first-order rate constants from kinetic data and discuss the effects of the above observations on the mathematical formulations required for reliable kinetic determinations of creatinine.
Temperature dependence of the absorbance of alkaline solutions of 4-nitrophenyl phosphate--a potential source of error in the measurement of alkaline phosphatase activity.
Authors: Burtis C A , Seibert L E , Baird M A , Sampson E J
Published: January 2020

The absorbance of an alkaline solution of 4-nitrophenyl phosphate is a function of temperature. Quantitative evaluation of this phenomenon indicates that it (a) depends on the concentration of the compound and is independent of source, buffer concentration, and pH above 9.0; (b) is reversible; (c) is not a result of alkaline hydrolysis or 4-nitrophenol contamination; and (d) correlates with a temperature-induced shift of its absorbance spectrum. The phenomenon may represent a potential analytical problem in methods for alkaline phosphatase in which this compound is the substrate. If thermal equilibrium is not reached and maintained during an alkaline phosphatase assay, the thermochromic response will be included in the measured rate. The magnitude of this error depends on the thermal response and control characteristics of each particular instrument and the reaction conditions under which such an analysis is performed.
Radioimmunoassay of human homologous prolactin in serum with commercially available reagents.
Authors: Kao P C , Jiang N S , Abboud C F
Published: January 2020

A clincally useful and reproducible radioimmunoassay for human homologous prolactin, established with commercially available reagents, was studied and validated. We present detailed conditions for iodination and purification of labeled prolactin and the optimal conditions for the assay. By the method, we found values (microgram/liter) as follows for serum prolactin: normal men. 8.9 +/- 5.2 (mean +/- SD); normal women, 11.8 +/- 5.5; normal women taking contraceptive pills, 9.2 +/- 5.0; pregnant women in the third trimester, 188 +/- 69.5; patients with various diseases other than of the hypothalamic-pituitary axis, 9.3 +/- 6.3; in some patients with amenorrhea and galactorrhea of diverse origin, 78.2 +/- 87.4; and in some patients with surgically proven pituitary tumor, 1414 +/- 1980. Results under provocative testing are also presented for a patient with normal hypothalamic-pituitary function.
Creatine kinase: re-examination of optimum reaction conditions.
Authors: Morin L G
Published: January 2020

I have re-examined optimum reaction conditions for measurement of creatine kinase (EC 2.7.3.2). The optimum pH is 6.45, and 2,2-bis(hydroxymethyl)-2,2',2''-nitrotriethanol acetate, 200 mmol/liter, is the buffer of choice. Thioglycerol, 20 mmol/liter, is superior for both in-assay reactivation and for storage stability of sera. Fluoride, 25 mmol/liter, a broad inactivator of adenylate kinase (EC 2.7.4.3), has little effect on creatine kinase and is superior to AMP for adenylate kinase inhibition in the assay of creatine kinase. Magnesium ion, ADP, and buffer concentrations are interdependent and their optima must be determined together. The hexokinase/glucose-6-phosphate dehydrogenase activity ratio should not exceed 1.6. The range of linearity is limited by the glucose-6-phosphate dehydrogenase and NAD+ concentrations. Glucose-6-phosphate dehydrogenase, ADP, and NAD+ are the constituents most likely to result in unacceptable blanks. Creatine kinase is inhibited noncompetitively by anions: acetate and fluoride inhibit slightly, but sulfates, nitrates, and excessive chlorides should be avoided.
Formation and properties of lactate dehydrogenase inhibitors in NADH.
Authors: Loshon C A , McComb R B , Bond L W , Bowers G N , Coleman W H , Gwynn R H
Published: January 2020

We describe some characteristics of the mode of formation of inhibitors of lactate dehydrogenase from commercial NADH. Inhibitor formation is time- and concentration-dependent and also varies with the commercial source of NADH. At least two inhibitory components can form in concentrated NADH solutions. One of these can be separated from NADH by chromatography on either diethylaminoethyl-celluose or diethylaminoethyl-Sephadex; the second cannot. The NADH-associated inhibitor appeared to be present in each of the three commercial NADH preparations studied. The 260 nm/340 nm absorbance ratio was of no help in locating this inhibitor during chromatography.
Detection of heterozygotes for phenylketonuria. Total body phenylalanine clearance and concentrations of phenylalanine and tyrosine in the plasms of fasting subjects compared.
Authors: Jagenburg R , Regårdh C G , Rödjer S
Published: January 2020

Two tests have been compared for detection of heterozygotes for phenylketonuria, one based on determination of plasma phenylalanine and tyrosine concentrations in fasting individuals and the other on kinetic evaluation of the plasma elimination curve after intravenous loading with L-phenylalanine. The plasma elimination curve was biexponential and the kinetics were evaluated according to the two-compartment model. The constant, beta, expressing the rate of elimination from plasma at pseudo-equilibrium, the rate constant for the elimination from the central compartment, and the total body clearance were determined. Of these three, total body clearance, which on the average was reduced by 32% in the phenylketonuric heterozygotes, showed the best discriminatory ability, but was not better than the information on concentrations of phenylalanine and tyrosine in detecting heterozygotes for phenylketonuria.
High-pressure liquid chromatography and enzyme immunoassay compared with gas chromatography for determing phenytoin.
Authors: Chamberlain R T , Stafford D T , Maijub A G , McNatt B C
Published: January 2020

We compared a gas-chromatographic method for determination of phenytoin with a high-pressure liquid chromatographic technique and with enzyme immunoassay by three instrumental procedures. More than 100 sera from patients being treated with this drug were assayed by all these techniques. The coefficient of variation was the lowest (4.0%) with liquid chromatography, but all methods gave a CV of less than 10%. The correlation coefficients for all methods exceeded 0.97 when compared to gas chromatography. Operation costs varied with the number of tests per batch, reagent costs, and operator labor costs. All assays gave comparable values for the therapeutic range, so it would be plausible to use more than one method in a situation where (e.g.) satellite laboratories handle different quantities of assays. In any of these techniques, interferences from carbamazepine, mephenytoin, phenobarbital, and primidone were negligible.
Determination of some L-3,4-dihydroxyphenylalanine and dopamine metabolites in urine by means of mass fragmentography.
Authors: Muskiet F A , Fremouw-Ottevangers D C , van der Meulen J , Wolthers B G , de Vries J A
Published: January 2020

We describe a mass-fragmentographic method for determination in urine of the following metabolites of L-3,4-dihydroxyphenylalanine and dopamine: vanillactic acid, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylethanol, and 3,4-dihydroxyphenylethanol. Deuterated analogs were used as internal standards. The method is fast, reproducible, sensitive, and selective, and does not require the use of time-consuming clean-up procedures. Normal excretion values in terms of creatinine, expressed as a function of age, as well as values obtained for patients with neurogenic tumors, a patient during therapy with L-3,4-dihydroxyphenylalanine, and a patient receiving dopamine are presented and discussed.
Results for serum and plasma compared in 15 selected radioassays.
Authors: Kubasik N P , Sine H E
Published: January 2020

We evaluated the results for serum vs. plasma samples for 15 selected radioassay procedures, using 19 manufacturers' kits. Blood samples were collected with heparin, oxalate-fluoride, or ethylenediaminetetraacetate anticoagulants and compared with serum samples. Differences were demonstrated between serum and plasma which may be of sufficient magnitude to alter clinical interpretation of the results. Assays also demonstrated significant differences based on the kit manufacturer and procedure used.
The peak monitor of the Technicon SMAC system.
Authors: Percy-Robb I W , Simpson D , Taylor R H , Whitby L G
Published: January 2020

The peak-monitor software of the Technicon SMAC system has been investigated by experiments designed to test its ability to detect abnormally shaped peaks, deliberately produced by presenting serum samples of inadequate volume for analysis. The software provided by the manufacturer is shown to detect faulty peaks inefficiently, and a set of modified parameters has been identified which greatly improves the performance of the peak-monitor software.
Simple determination of high urinary excretion of 5-hydroxyindole-3-acetic acid with ferric chloride.
Authors: Yamaguchi Y , Hayashi C
Published: January 2020

We describe a method for determination of urinary 5-hydroxyindole-3-acetic acid, a metabolite of serotonin and tryptophan and a useful indicator of metastasizing carcinoid tumor. The analysis is based on the color development with ferric chloride added to the residue obtained on evaporating a diethyl ether extract of the sample that has been acidified with sulfuric acid and saturated with sodium chloride. The color is measured by 510 nm. The reaction was made highly specific by the acidification so that nonspecific reactions with catecholamines and with other phenolic compounds such as salicylic acid could be eliminated.
Analysis for 1,3-bis(2-chloroethyl)-1-nitrosourea by chemical ionization mass spectrometry.
Authors: Weinkam R J , Wen J H , Furst D E , Levin V A
Published: January 2020

We used chemical ionization mass spectrometry to quantify the cancer chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea in biological fluids. A stable isotope-labeled internal standard was added to the fluid being analyzed. Protonated molecular ion intensity ratios were determined by selected ion monitoring of sample extracts inserted directly into the chemical ionization source. Peak ratios were calculated for chlorine isotope ratios in their natural abundance, to verify the integrity of the data acquired, and to compute the drug concentration. The assay was applied to measure the compound in rat blood and plasma and in human plasma, and to monitor in vitro metabolism in rat-liver microsomal preparations. For the analyses we used a quadrupole mass spectrometer interfaced to a commercial computer system. Data-system characteristics are described.
Homogeneous enzyme immunoassay for cannabinoids in urine.
Authors: Rodgers R , Crowl C P , Eimstad W M , Hu M W , Kam J K , Ronald R C , Rowley G L , Ullman E F
Published: January 2020

We describe a homogeneous enzyme immunoassay for measurement of cannabinoid metabolites, as well as delta9-tetrahydrocannabinol (I) in urine. Malate dehydrogenase from pig heart mitochondria was labeled with a derivative of I. The compound used to calibrate the assay was the I metabolite, 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (II). With 15 microgram of II per liter of urine as the cutoff concentration, the assay can detect 25 microgram of II per liter with greater than 95% confidence. A positive response was obtained for urine specimens assayed within 30 min after exposure to cannabinoids. However, the persistence of metabolites of I in urine indicates that assay of this fluid is useful as an indicator of cannabinoid use but not as an indicator of intoxication.