bluuebunny/crossref_metadata_2025 huggingface dataset:
Complexes of immunoglobulins A and G with aspartate aminotransferase isoenzymes in serum.
Authors: Nagamine M , Okochi K
Published: January 2020

We report the presence of complexes between aspartate aminotransferase (AST, EC 2.6.1.1) and immunoglobulin (Ig) in the serum of a patient suffering from lung cancer with metastasis to the liver. After fractionation of the serum by gel filtration, AST-Ig complexes (AST-IgA, AST-IgG) were demonstrated by counterimmunoelectrophoresis. Dissociating the complexes and recombining them with purified isoenzyme fractions, s-AST (cytoplasmic) and m-AST (mitochondrial), revealed that only s-AST binds to IgG, whereas IgA binds to both s-AST and m-AST. Although the association of AST with IgG has been reported, to our knowledge this is the first finding of both AST-IgA and AST-IgG complexes in a patient's serum. Serum AST-IgG complexes have been demonstrated in both healthy and diseased individuals; in the latter category, as reported here and by others, the liver is implicated.
Column deactivation in analysis for underivatized tricyclic antidepressants by gas chromatography with use of a nitrogen detector.
Authors: Vinet B
Published: January 2020

I describe deactivation treatment of the OV-17 chromatographic column to minimize adsorption of tricyclic antidepressant drugs on the solid support of the column. The procedure involves heat treatment at 399 degrees C under a low flow of nitrogen, with bleeding of OV-17 liquid phase from the injector tube into the column. The column is then conditioned with vapors of phenyldiethanolamine succinate, added to the carrier gas stream by bleeding from a coated injector glass tube. This deactivation process much improves the chromatographic performance of the column, allowing a sensitivity at the nanogram level with a nitrogen-sensitive detector. Determinations of tricyclic antidepressants in plasma with such a deactivated column results in a low CV and a linear calibration curve, reflecting the effectiveness of the deactivation.
Effects of contaminants in blood-collection devices on measurements of therapeutic drugs.
Authors: Ji S Q , Evenson M A
Published: January 2020

Substances in evacuated blood-collection devices produce gas-chromatographic peaks with retention times similar to those of drugs. All tubes tested except the serum separator tubes contained tris(2-butoxyethyl) phosphate, as determined by mass spectrometry. In addition, the partition coefficient during the solvent-extraction step increased for some drugs by as much as 40%, while for other drugs it decreased by as much as 30%, depending on the drug and the collection tube. The Becton Dickinson serum separator tube also contains several other compounds identified by mass spectrometry to be dibasic esters used in the preparation of the polyester gel. The Corning serum separator tube and the Venoject serum tube contain still other impurities. The Becton Dickinson royal-blue-stoppered tube contained the least amount of impurities of the tubes tested. CVs of 25 commonly measured drugs in plasma pools increased from 5% for samples collected in glass tubes to greater than 20% for samples collected in evacuated blood-collection tubes. Clearly, accuracy and precision of measurements of therapeutic drugs can be seriously compromised if an inappropriate blood-collection device is used.
Short-term biological variation of plasma analytes in renal disease.
Authors: Fraser C G , Williams P
Published: January 2020

Analytical, intra-individual, and inter-individual components of variation were estimated from duplicate analyses of 11 plasma analytes in an average of 13 specimens obtained, over a two-day period, from nine patients with impaired renal function. Analytical variance was 17.3% of the total variance for sodium; less than 5% of the total variance for potassium, chloride, bicarbonate, albumin, and calcium; and less than 1% for urea, creatinine, glucose, creatine kinase (EC 2.7.3.2), and alanine aminotransferase (EC 2.6.1.2). Average intra-individual variances were of the same order as those found in healthy individuals. All analytes except glucose showed strong individuality. We postulate that, in nonacute pathological processes where new homeostatic steady states are reached, biological variations around the new set points are of the same order as those found in healthy individuals.
Radioimmunometric assay for a monoclonal antibody-defined tumor marker, CA 19-9.
Authors: Del Villano B C , Brennan S , Brock P , Bucher C , Liu V , McClure M , Rake B , Space S , Westrick B , Schoemaker H , Zurawski V R
Published: January 2020

We describe a solid-phase radioimmunometric sandwich assay for a new tumor marker defined by a monoclonal antibody (19-9). This antibody reacts with a carbohydrate antigenic determinant (CA 19-9) found at low concentrations in sera from healthy individuals but frequently increased in sera from patients with adenocarcinomas. The assay is sensitive and simple to perform. It requires duplicate 100-microL samples and may be performed in 6 h. The concentration of CA 19-9 in samples is determined by reference to a standard curve, which is essentially linear from 0 to 120 arbitrary units/mL. The average CV is approximately 10% in the range of 5.8 to 120 units/mL. The minimum detectable dose is 1.4 units/mL and analytical recovery of CA 19-9 is 97.6 to 100.6%. The average concentration of CA 19-9 in sera from 1020 healthy individuals was 8.4 (SD 7.4) units/mL; only 0.6% of such sera had concentrations greater than 37 units/mL. The assay has high specificity (98.5%), even among patients with benign diseases, and has high sensitivity (up to 79%) for patients with gastrointestinal adenocarcinomas, especially those of the pancreas.
Six methods for determining urinary protein compared.
Authors: Dilena B A , Penberthy L A , Fraser C G
Published: January 2020

Inter-laboratory surveys have shown that "routine methods" of urinary protein determination are often unsatisfactory. Therefore, we compared six frequently used methods for determination of protein in urine with respect to linearity, within-batch and between-batch precision, comparative bias, and practicability. We assayed dilutions of human and bovine albumin and serum, and fresh and lyophilized human urine. We find that the AACC Selected Method has poor practicability and poor precision under routine conditions, but good linearity. The sulfosalicylic acid/biuret technique is also impracticable, requires a large sample, and is not linear at low concentrations of urine protein. The Coomassie Brilliant Blue technique has a narrow range of linearity and poor precision. The sulfosalicylic acid/sodium sulfate turbidimetric method is not precise and cannot be standardized with bovine materials. The Ponceau-S technique has good performance characteristics and practicability, and we recommend it for routine laboratory use.
Total bone and liver alkaline phosphatases in plasma: biological variations and reference limits.
Authors: Schiele F , Henny J , Hitz J , Petitclerc C , Gueguen R , Siest G
Published: January 2020

We have studied factors affecting biological variation in total plasma alkaline phosphatase in a population of 32 329 apparently healthy subjects four years old or older. Quantification of the bone and liver isoenzymes after thermal denaturation made it possible to specify the contributions of each isoenzyme to variations in the total activities. The main factors that modify plasma alkaline phosphatase activity are age, sex, hormonal state (puberty or menopause), and morphometric parameters (height, body weight, or degree of overweight). The bone isoenzyme is mainly responsible for the variations associated with age, sex, and puberty and to some extent with the menopause. Activity of the liver isoenzyme was also altered at the menopause and by certain drugs, such as oral contraceptives and blood-lipid-lowering agents. These data allow us to propose reference limits for total plasma, bone, and liver alkaline phosphatases according to age and sex.
An enzymic creatinine assay and a direct ammonia assay in coated thin films.
Authors: Sundberg M W , Becker R W , Esders T W , Figueras J , Goodhue C T
Published: January 2020

We developed a thin-film enzymic assay for creatinine that makes use of creatinine iminohydrolase (EC 3.5.4.21) to convert creatinine to N-methylhydantoin and ammonia. The ammonia diffuses through a semipermeable layer and is quantitated by reaction with bromphenol blue. A paired analysis of the sample on a separate coating without the enzymic reaction measures endogenous ammonia and, for samples with normal concentrations of ammonia, allows accurate determination of serum creatinine to 150 mg/L without dilution. Results of this assay (y) compare well with those by a liquid-chromatographic comparison assay (x) by linear regression (slope = 0.935, intercept = 1.13 mg/L, r2 = 0.995). It is insensitive to many substances, such as ketones and keto acids, that interfere with conventional assays. Results of the ammonia assay (y) correlate well with those by a semi-automated enzymic assay (x) based on glutamate dehydrogenase (slope = 1.068, intercept = 17.3 mumol/L, r2 = 0.985).
Liquid-chromatographic determination of tobramycin in serum with spectrophotometric detection.
Authors: Kabra P M , Bhatnagar P K , Nelson M A , Wall J H , Marton L J
Published: January 2020

We describe a simple, precise, accurate, and specific liquid-chromatographic procedure for determination of tobramycin in 50 microL of serum. Tobramycin and the internal standard (sisomicin) are quantitatively converted into their trinitrophenyl derivatives by reaction with a water-soluble derivatizing agent (2,4,6-trinitrobenzenesulfonic acid) at 70 degrees C for 30 min. The derivatives are extracted from the crude reaction mixture by using a reversed-phase Bond-Elut C18 column, and separated on a reversed-phase octyl column with a mobile phase consisting of an acetonitrile/phosphate buffer (70/30 by vol) at a flow rate of 3.0 mL/min. The eluted compounds are detected at 340 nm, and quantified from their peak areas. Chromatography is complete in less than 4.5 min at the optimum column temperature of 50 degrees C. The lower limit of detection for tobramycin is less than 0.2 mg/L. Analytical recoveries for tobramycin varied from 94 to 99%, linearity extended to 25 mg/L, and day-to-day precision (CV) was between 4.6 and 5.1%. Numerous drugs and antibiotics tested do not interfere. Results correlate well (r greater than 0.95) with those by radioimmunoassay and EMIT.
"Sandwich" enzyme immunoassay for serum ferritin with polypropylene test tubes as the solid phase.
Authors: Revenant M C
Published: January 2020

A "sandwich" enzyme immunoassay for ferritin in human serum is described, in which horseradish peroxidase-labeled antibody and a very sensitive chromogen, 2,2'-azino-di(3-ethyl-benzthiazolin-6-sulfonate), are used. The solid phase is polypropylene test tubes treated with glutaraldehyde; immunologic reactions are developed during constant rotation of the tubes. The assay requires 20 microL of serum per assay and takes less than 3 h. The standard curve is linear between 0 and 400 micrograms of serum ferritin per liter. Analytical recoveries of various amounts of standard added to a serum sample ranged from 92 to 98.6%. The sensitivity is 2 micrograms/L, which translates to 0.4 ng per assay tube. Within-run CV ranges from 1.6 to 5.4% and between-run from 4.2 to 7.8% according to the ferritin concentration. Results by competitive radioimmunoassay correlated well (r = 0.98). No "hook effect" was observed for concentrations up to 13 mg/L. The reference interval is 40-240 micrograms/L for men, 8-100 micrograms/L for women.
Laboratory evaluation of the Boehringer Mannheim "Hitachi 705" automatic analyzer.
Authors: Kineiko R W , Floering D A , Morrissey M
Published: January 2020

We evaluated a new multi-channel chemistry analyzer, the Hitachi 705 Automatic Analyzer, marketed by Boehringer Mannheim Diagnostics, Inc. The instrument is a computer-controlled discrete analyzer, which can be run in a combination profile mode and single-test mode. Sixteen different tests per sample may be performed at the rate of 180 tests per hour. The Hitachi 705 is especially suitable for use in hospitals that do not perform profile testing. Precision and linearity were excellent and the instrument was relatively trouble-free, with little operator attention required during operation. The Hitachi 705 was easily interfaced to our laboratory computer. We compared the performance of the instrument with that of the Du Pont aca; the two instruments compared favorably.
Proportion of glycosylated hemoglobin in erythrocytes of a person with hemoglobins A, S, and G Philadelphia.
Authors: Krauss J S , Jonah M H , Yau S J , Trinh M
Published: January 2020

The apparent proportion of column-chromatographically measured glycosylated hemoglobin in erythrocytes from an individual with hemoglobins A, S, and G was only 2.3% because the slow glycosylated variant hemoglobins were retarded in the column. In contrast, the value for glycosylated hemoglobin was 7.8% by a new cellulose acetate electrophoretic method that includes use of dextran sulfate buffer. The erythrocyte metalloprotein, carbonic anhydrase B, was shown to co-migrate with glycosylated hemoglobin by this technique, however. Thus carbonic anhydrase B, HbF, and HbA all have weak attraction for negative charges at acid pH. We believe that carbonic anhydrase B should contribute significantly (at least 10-20% absorbance) to the HbA1 by this electrophoretic method. We conclude that microcolumn chromatography should remain the method of choice for HbA1 determination, that subject-based reference intervals should be used for HbS or HbC heterozygotes, and that electrophoretic HbA1 methods should be reserved for use with both HbS and HbC homozygotes and HbSC disease.
A reference method for measurement of alkaline phosphatase activity in human serum.
Authors: Tietz N W , Burtis C A , Duncan P , Ervin K , Petitclerc C J , Rinker A D , Shuey D , Zygowicz E R
Published: January 2020

We present an official AACC reference method for the measurement of alkaline phosphatase, the culmination of optimization experiments conducted by a group of independent laboratories. The details of this method and evaluation of factors affecting the measurement are described. A metal ion buffer has been incorporated that maintains optimal and constant concentrations of zinc(II) and magnesium(II) ions. Final reaction conditions are: pH (30 degrees C), 10.40 +/- 0.05; 2-amino-2-methyl-1-propanol buffer, 0.35 mol/L; 4-nitrophenyl phosphate, 16.0 mmol/L; magnesium acetate, 2.0 mmol/L; zinc sulfate, 1.0 mmol/L; and N-(2-hydroxyethyl)ethylenediaminetriacetic acid, 2.0 mmol/L.
Improved dialysis method for free thyroxin in serum compared with five commercial radioimmunoassays in nonthyroidal illness and subjects with abnormal concentrations of thyroxin-binding globulin.
Authors: Helenius T , Liewendahl K
Published: January 2020

We describe a method for free thyroxin (FT4) in serum, based on radioimmunoassay of T4 in serum dialysate, FT4(D). The method is analytically accurate, sensitive, reproducible, and suitable for routine use in the clinical laboratory. We compared results by this method with those obtained with five commercial FT4 assays (Amerlex, GammaCoat, ImmoPhase, LiquiSol, Spiria) and the free T4 index (FT4I). In several of the patients with nonthyroidal illness FT4(D) was above normal and FT4 as measured with the commercial assays was subnormal. In the third trimester of pregnancy all the FT4 methods gave decreased mean values, though the decreases were of various magnitudes: FT4(D) 32%, Amerlex 44%, GammaCoat 40%, Spiria 19%, LiquiSol 16%, and ImmoPhase 13%, respectively. FT4I was significantly higher than normal during late pregnancy. In women taking oral contraceptives the mean FT4I was increased, but there was no significant effect on the results obtained with the various FT4 methods. All subjects with hereditarily high or low serum thyroxin-binding globulin had normal FT4(D) and abnormal FT4I. Amerlex, ImmoPhase, and LiquiSol misclassified some of the subjects with hereditarily abnormal thyroxin-binding globulin.
Capillary gas-chromatographic determination of urinary homovanillic acid and vanillylmandelic acid.
Authors: Tuchman M , Crippin P J , Krivit W
Published: January 2020

This relatively simple, rapid method for quantification of homovanillic acid (HVA) and vanillylmandelic acid (VMA) involves solvent extraction and capillary gas chromatography. With use of this extraction method, the overall analytical recovery from an aqueous solution is 92.5% for HVA and 79.2% for VMA. 3,4-Dihydroxybenzoic acid is the internal standard. Minimal detectable quantities with this method are less than 1 mg/g of creatinine. Capillary gas chromatography produces a chromatogram that is superior to that of packed-column gas chromatography. We also report quantitative results for urinary HVA and VMA in neuroblastoma patients and in normal individuals, demonstrating the diagnostic value of this method.
Radioimmunoassay for human prothrombin.
Authors: Krishnan G
Published: January 2020

I describe a radioimmunoassay for human prothrombin, with use of a double-antibody technique. Antiserum raised in rabbits was absorbed with Al(OH)3 and heated to 56 degrees C for 30 min. 125I-labeled prothrombin retaining more than 90% of its biological activity was prepared by the iodine monochloride method. The mean concentration of prothrombin in plasma of 12 normal individuals was 100 +/- 29.4 mg/L (2 SD). Prothrombin values were somewhat lower than those obtained by the Laurell electroimmunoassay or by two-stage biological assay of the same plasma, done the same day. The biological values were converted to protein on the basis of 1960 int. units/mg by comparison with the other two assays. The ability of activation fragments of human prothrombin to inhibit binding of labeled prothrombin to its antibody was evaluated by competitive radioimmunoassay. Although precipitin lines formed with undiluted antiserum against all the fragments tested (F-1, F-1.2, prethrombin-1, and thrombin), none of the fragments competed well with prothrombin, even in 10-fold molar excess. Evidently, the structural integrity of the prothrombin molecule is essential for its maximum binding to the antiserum, and antigenic sites are lost during its activation.
Micromethod for determining plasma ammonia nitrogen with use of an ion-selective electrode.
Authors: Cooke R J , Jensen R L
Published: January 2020

A micromethod for measuring the ammonia nitrogen content of plasma by use of an ion-selective electrode is evaluated and described. The effect of storing plasma for 24, 48, and 72 h at -70 degrees C was evaluated. Values for newborns and fasting adults were 880 micrograms/L (SD, 210 micrograms/L) and 620 micrograms/L (SD, 170 micrograms/L), respectively.
Reversal of changes in lipoprotein A and lipoprotein B cholesterol during and for a year after a detoxication treatment program in chronic alcoholism.
Authors: Perier C , Janin J , Pierre-Louis S , Frey J
Published: January 2020

We studied the individual and occasional changes in lipid metabolism induced by chronic alcohol abuse. In addition, the influence of a detoxication treatment program on the evolutionary changes in some serum lipidic components was studied for a one-year period. Before this program, total cholesterol was above normal, with high values for LP-A cholesterol, whereas for some patients LP-B cholesterol was increased. After the program, there was an increase in total cholesterol, LP-B cholesterol, and apolipoprotein B, with a decrease in LP-A cholesterol. These evolutionary changes continued during the one-year period after the end of the inpatient program.
Determination of urinary vanillylmandelic acid by liquid chromatography with electrochemical detection.
Authors: Moleman P , Borstrok J J
Published: January 2020

We describe an improved method for the assay of urinary vanillylmandelic acid by "high-performance" liquid chromatography, with electrochemical detection. A 1-mL aliquot of urine is acidified and extracted with ethyl acetate, then the ethyl acetate extract is extracted with phosphate buffer, pH 8.5. An acidified aliquot of the phosphate extract is injected into a reversed-phase column and vanillylmandelic acid is detected electrochemically. The between-day CV is 5-6% for concentrations ranging from 10 to 75 mumol/L. We also discuss the critical steps for extraction and calibration.
Radioimmunoassay for arginine-vasopressin in cold ethanol extracts of plasma.
Authors: Camps J , Martínez-Vea A , Pérez-Ayuso R M , Arroyo V , Gaya J M , Rivera-Fillat F
Published: January 2020

A radioimmunoassay for arginine-vasopressin in human plasma with use of a commercially available antibody was developed and evaluated. The hormone was extracted from plasma with cold 98% ethanol, which showed a significantly higher (p less than .001) and more precise recovery than with the acetone-ether procedure (65.3 +/- 3.7% vs 50.8 +/- 6.0%, respectively). The sensitivity was 0.31 pg per tube. Results for normal subjects in different physiological conditions and in patients with diabetes insipidus and inappropriate antidiuretic hormone secretion showed the good reliability of the method.
Continuous-flow system for automation of latex immunoassay by particle counting.
Authors: Bernard A M , Lauwerys R R
Published: January 2020

Latex immunoassay is a nonisotopic method based on agglutination, by protein, of calibrated latex particles coated with a specific antibody. The assay has been automated in a simple continuous-flow system by incubating the reaction mixture in a heated mixing coil for 25 min and measuring the agglutination with a cell counter. No external shaking of the latex suspension and no additional reagent is required for the agglutination. The method can accurately and precisely quantify a wide variety of proteins in plasma and urine, including human ferritin, beta 2-microglobulin, retinol-binding protein, and albumin. Depending on the antigen-antibody system, the detection limit ranges from 10(-10) to about 10(-12) mol/L. Within- and between-assay CVs are less than 10%. In the assay of ferritin, sera are pretreated to eliminate interferences from chylomicrons, complement, and rheumatoid factor.
Evaluation of serum gamma-glutamyltransferase by electrofocusing, and variations in isoform patterns.
Authors: Lilja H , Jeppsson J O , Kristensson H
Published: January 2020

Serum gamma-glutamyltransferase (EC 2.3.2.2) showed microheterogeneity on electrofocusing, owing to variations in sialic acid content. We investigated the isoform patterns of papain-treated serum samples on agarose gels containing nonionic detergent and ampholytes in the low pH range. Serum from cases of cholestasis show seven bands with gamma-glutamyltransferase activity. These same bands were also found in liver tissue similarly treated, and they had pl values ranging from 3.8 to 4.2. Papain-treated sera that also had been neuraminidase digested showed only a single band, still enzymatically active and with a pl of 5.9. Increased gamma-glutamyltransferase in serum as a result of alcohol abuse was combined with a abnormally high degree of sialylation of the enzyme, giving more anodal isoforms. The decline in the concentration of this enzyme during several weeks of abstinence was accompanied by a gradual decrease in gamma-glutamyltransferase sialyation and the appearance of more cathodal fractions.
Immunoquantification of total apolipoprotein B in serum by nephelometry: influence of lipase treatment and detergents.
Authors: DaCol P , Kostner G M
Published: January 2020

The immunoquantification of total apolipoprotein B in human serum has been evaluated by rate and equilibrium nephelometry. The presence of triglyceride-rich lipoproteins spoiled all immunochemical assays and yielded too-high values for apolipoprotein B. The use of detergents improved the results substantially, but results were inaccurate at high triglyceride concentrations. Of many detergents investigated, only Thesit, Kryo Ebo, and Apovax were useful, decreasing the light-scatter signals almost linearly with increasing detergent concentrations. The regression lines, however, were not parallel among the different apo B-containing lipoproteins. Incubating sera or apo B-containing lipoproteins with bovine milk lipoprotein lipase or bacterial triacylglycerol lipase, at concentrations of 100 kU/L, hydrolyzed all of the triglycerides and most of the phosphatidylcholine within 18 h at 37 degrees C Lipase-pretreatment of samples gave optimal correlation between apo B values as determined by nephelometry with those obtained gravimetrically. We also assessed the influence of sample storage, freezing, and thawing on the nephelometric apo B assays.
Liquid-chromatographic study of fluorescent materials in uremic fluids.
Authors: Swan J S , Kragten E Y , Veening H
Published: January 2020

Using reversed-phase "high-performance" liquid chromatography with fluorescence detection, we separated and identified some naturally fluorescent compounds in uremic serum and hemodialysate from patients with chronic renal disease. Several of the naturally fluorescent compounds were identified as indole derivatives by co-chromatography with authentic standards. In one specific case, the identity was confirmed by an enzymic peak-shift method. Compounds identified included indican, kynurenic acid, tryptophan, and 5-hydroxy-indole-3-acetic acid. Comparison of normal and uremic serum showed that the fluorescent materials are present in significantly greater concentrations in samples from uremic patients.
Improved determination of aluminum in serum by electrothermal atomic absorption spectrophotometry.
Authors: Bertholf R L , Brown S , Renoe B W , Wills M R , Savory J
Published: January 2020

This method for determining aluminum in human serum involves electrothermal atomic absorption spectrophotometry. A longer drying time allows less pre-dilution of the sample, and the method also includes a flush cycle after atomization. Standard-addition methodology is used to eliminate matrix effects and the need for a standard curve. We used this method on serum from 50 normal persons and from 34 patients with chronic renal failure who were on long-term intermittent hemodialysis. The mean normal serum aluminum concentration was 6.1 micrograms/L (CV 2.7%), and values for the patients ranged from 13 to 475 micrograms/L.
Evaluation of a direct potentiometric method for sodium and potassium used in the Du Pont aca.
Authors: Koch D D , Parrish D , Ladenson J H
Published: January 2020

We evaluated an accessory that enables the Du Pont aca discrete analyzer to measure Na+ and K+ by direct potentiometry (aca/ISE). Na+ and K+ gave linear responses in both the blood and urine modes, with no carryover. No interfering species were identified in the blood mode. Intra-assay and interassay precision for Na+ and K+ were more than adequate and analytical recoveries comparable to those with flame photometry were found for plasma. The Na+ and K+ values for plasma were comparable with those obtained by use of another direct potentiometric analyzer (the Orion SS-30), but slightly higher than flame-photometric values. Samples from patients with hyperlipemia and multiple myeloma gave clinically relevant values with the aca/ISE, but the flame photometric values were artefactually low. Whole blood could not be accurately analyzed. The aca/ISE is a precise, easy-to-use instrument, which gives values for plasma similar to those obtained with direct potentiometric analyzers. For analysis of urine, certain precautions are necessary.
Reversed-phase liquid-chromatographic simultaneous analysis for thiopental and pentobarbital in serum.
Authors: Kelner M , Bailey D N
Published: January 2020

We describe a reversed-phase liquid-chromatographic assay suitable for therapeutic monitoring of thiopental and pentobarbital simultaneously in human serum. The drugs are extracted from serum at pH 6.6 into n-butyl chloride containing thiamylal and barbital as the respective internal standards. The compounds are back-extracted into dilute sodium hydroxide, an aliquot of which is submitted to chromatography. The lowest measurable concentrations are 1.0 mg/L for thiopental and 2.0 mg/L for pentobarbital. The standard curve is linear from 0 to 100 mg/L for both. Between-run CVs are: at 25 mg/L, 4.1% (thiopental) and 3.2% (pentobarbital); at 50 mg/L, 2.8% (thiopental) and 3.4% (pentobarbital). Data on patients receiving thiopental and pentobarbital illustrate use of the method.
Metabolism of creatinine in vivo.
Authors: Boroujerdi M , Mattocks A M
Published: January 2020

Two forms of radiolabeled creatinine were used in this study, [carbonyl-14C]- and [amidino-14C]creatinine, the latter being synthesized in this laboratory. These labeled compounds were given intravenously to rabbits. Specimens of plasma, urine, feces, and selected tissues were collected. These were liquid-chromatographed and the separated components compared for radioactivity and identity. This first use of amidino-labeled creatinine revealed the metabolic conversion of small amounts of creatinine to an unidentified non-basic compound and guanidinobutyrate in normal rabbits and to guanidinopropionic acid and arginine in a rabbit that had a low creatinine clearance. A mechanism is suggested for this metabolic process. The proposed pathway may be a major route of elimination of creatinine in renal failure.
The succinonitrile triple-point standard: a fixed point to improve the accuracy of temperature measurements in the clinical laboratory.
Authors: Mangum B W
Published: January 2020

In an investigation of the melting and freezing behavior of succinonitrile, the triple-point temperature was determined to be 58.0805 degrees C, with an estimated uncertainty of +/- 0.0015 degrees C relative to the International Practical Temperature Scale of 1968 (IPTS-68). The triple-point temperature of this material is evaluated as a temperature-fixed point, and some clinical laboratory applications of this fixed point are proposed. In conjunction with the gallium and ice points, the availability of succinonitrile permits thermistor thermometers to be calibrated accurately and easily on the IPTS-68.
Sensitive colorimetric assay for angiotensin converting enzyme in serum.
Authors: Neels H M , van Sande M E , Scharpé S L
Published: January 2020

A sensitive colorimetric procedure has been developed for the assay of angiotensin converting enzyme (EC 3.4.15.1) in serum. Serum (10 microL) is incubated for 30 min with hippuryl-glycyl-glycine as described earlier (Clin Chem 28: 1352-1355, 1982). After a Folin-Wu deproteinization, the liberated glycyl-glycine is derivatized with a borate-buffered (pH 9.3) trinitrobenzenesulfonate solution (60 mmol/L) to form trinitrophenyl-glycylglycine, the absorbance of which is read at 420 nm vs a serum blank. The linear range extends to an activity of more than 900 U/L of serum and the detection limit is less than 4 U/L. The mean activity for serum from 50 blood bank donors and 25 patients with active sarcoidosis was 281 (SD 77) and 693 (SD 81) U/L, respectively. The method demonstrates good precision (CVs less than 2.8%) and correlates well (r = .99) with results from a "high-pressure" liquid chromatographic procedure for determining hippuric acid. In addition, the proposed method is widely applicable, involving only commonly available apparatus.
Gas-chromatographic determination of cholesterol sulfate in plasma and erythrocytes, for the diagnosis of recessive X-linked ichthyosis.
Authors: Muskiet F A , Jansen G , Wolthers B G , Marinkovic-Ilsen A , van Voorst Vader P C
Published: January 2020

We describe a rapid method for determining cholesterol sulfate in plasma and erythrocytes. After its single-step isolation by means of anion-exchange chromatography cholesterol sulfate is hydrolyzed, trimethylsilylated, and determined by gas chromatography with flame ionization detection. 5 beta-Cholestan-3 alpha-ol sulfate is used as internal standard. The method enables simultaneous determination of dehydroepiandrosterone sulfate in plasma. We applied it for the diagnosis of seven patients with recessive X-linked ichthyosis. Concentrations are given for plasma and erythrocytes from four unaffected relatives of patients with X-linked ichthyosis, a patient with placental sulfatase deficiency, two patients with other types of ichthyoses, and 20 controls. The method may also be of use for the rapid isolation of other organic sulfates from biological material, as illustrated by a comparison of gas chromatograms of urine from a normal pregnant woman and that from a patient with placental sulfatase deficiency.
A macro creatine kinase isoenzyme in serum of apparently healthy individuals.
Authors: Whelan P V , Malkus H
Published: January 2020

Activity of creatine kinase B subunit, as measured by an immunoinhibition assay, is persistently increased in certain healthy, asymptomatic adults, whose values for total CK activity are within normal limits. Serum samples from two such individuals were investigated by electrophoresis, heat inactivation, determination of activation energies, and immunosorption (on protein A). The results demonstrated the presence of a circulating complex of IgG and CK-BB, which has been described as macro CK, type I. To our knowledge, this is the first report of such macro CK isoenzymes in healthy individuals.
Liquid-chromatographic assay of cefmenoxime in serum and urine.
Authors: Reitberg D P , Schentag J J
Published: January 2020

This is a simple, precise liquid-chromatographic procedure for determining cefmenoxime in patients' serum and urine. p-Anisic acid is used as the internal standard. Protein is precipitated from 0.5 mL of serum or dilute urine with 100 microL of perchloric acid. The clear supernate is injected directly onto a mu-Bondapak CN reversed-phase column. The mobile phase is acetate buffer, pH 3.8 (25 degrees C). The flow rate is 2.5 mL/min. Column effluent is monitored at 254 nm. Extraction recovery from serum averaged 74.6%. Calibration curves were linear from 0.5 mg/L, the lower limit of quantification, to 100 mg/L. We present cefmenoxime concentrations in serum from a patient being treated for pneumonia. The procedure was evaluated in the clinical setting to determine its applicability to the study of cefmenoxime pharmacokinetics in critically ill patients.
The Kodak Ektachem 400 Analyzer evaluated for automated enzymic determination of plasma creatinine.
Authors: Smith C H , Landt M , Steelman M , Ladenson J H
Published: January 2020

We have evaluated an automated creatinine iminohydrolase procedure for plasma creatinine analysis, as used in the Kodak Ektachem 400 Analyzer. The correlation with kinetic and end-point (equilibrium) alkaline picrate procedures is generally excellent. The enzymatic procedure has the advantage of being unaffected by bilirubin, cephalosporins, and acetoacetate, all of which interfere in alkaline picrate procedures. Its accuracy, however, greatly depends on calibration values provided by the manufacturer. Storage of samples at -20 degrees C produced little change in measured creatinine. The Ektachem procedure has a greater range of linearity (140 mg/L) than the alkaline picrate procedures, but it shares with them the disadvantages of suboptimal precision in the normal range. The increased specificity, ease of performance, and other advantages of this use of creatinine iminohydrolase make it an attractive procedure.
Densitometry of phosphatidylcholine and sphingomyelin in high-density lipoproteins.
Authors: Schmitz G , Jabs H U , Assmann G
Published: January 2020

We describe the quantitative densitometric determination of phosphatidylcholine (PC) and sphingomyelin (SP) in human serum after precipitation with phosphotungstic acid/MgCl2 and use of thin-layer chromatography. After development, chromatographic plates were charred with methanolic sulfuric acid and MnCl2 and scanned by direct reflectance densitometry in an automated densitometric system interfaced to a basic programmable computing integrator. The method is sensitive enough to detect abnormally low concentrations of PC and SP in high-density lipoproteins. The accuracy of the method was tested either with the Bartlett phosphorus assay or with enzymatic methods for PC and SP; correlations of the described method with the enzymatic determinations were r = 0.93 and 0.88, respectively. Day-to-day precision (CV) for the phospholipid determination was 8.6% for PC and 12.2% for SP. The major advantage of this inexpensive technique is that native plasma or serum or the serum supernate after precipitation can be used without prior delipidation. With this technique serum high-density lipoproteins had PC values of 1.08 (SD 0.32) mmol/L in men (n = 158) and 1.12 (SD 0.37) mmol/L in women (n = 192); similarly, SP values were 0.23 (SD 0.07) mmol/L in the men and 0.23 (SD 0.08) mmol/L in the women. The differences by sex are not significant.
Heterogeneity of lipoprotein electrophoretic patterns in patients with type IIa hyperlipoproteinemia.
Authors: Hoeg J M , Papadopoulos N M , Gregg R E , Brewer H B
Published: January 2020

Previous evaluations of plasma lipoproteins from patients with type IIa hyperlipoproteinemia have demonstrated quantitative but not qualitative differences from lipoproteins isolated from normal subjects. Using a reproducible, sensitive, high-resolution agarose gel electrophoretic system, we evaluated the plasma lipoprotein of 22 consecutive, untreated, unrelated patients with type IIa hyperlipoproteinemia. Five distinct electrophoretic patterns were observed. This electrophoretic heterogeneity in type IIa hyperlipoproteinemia reflects subtle charge differences in the lipoproteins from these patients, which were not detected with paper electrophoresis or with lipoprotein quantification. There was little, if any, difference in total plasma cholesterol, low- or very-low-density lipoproteins, plasma triglycerides, or concentrations of apolipoproteins A-I, A-II, B, C-II, or E in these subgroups. However, unique clinical features were correlated with the electrophoretic patterns. These findings support the concept that the type IIa hyperlipoproteinemic phenotype includes several molecular defects that are reflected in subtle differences in lipoprotein charge.
Acridinium esters as high-specific-activity labels in immunoassay.
Authors: Weeks I , Beheshti I , McCapra F , Campbell A K , Woodhead J S
Published: January 2020

A chemiluminescent acridinium ester has been synthesized that reacts spontaneously with proteins to yield stable, immunoreactive derivatives of high specific activity. The compound has been used to prepare chemiluminescent monoclonal antibodies to human alpha 1-fetoprotein having average incorporation ratios as great as 2.8 mol of label per mole of antibody, which corresponds to a detection limit of approximately 8 X 10(-19) mol. These antibodies have been used in the preliminary development of a two-site immunochemiluminometric assay for human alpha 1-fetoprotein, which requires only a 30-min incubation and a quantification time of 5 s per sample.
Enzymatic colorimetry of lecithin and sphingomyelin in aqueous solution.
Authors: McGowan M W , Artiss J D , Zak B
Published: January 2020

A procedure for the enzymatic determination of lecithin and sphingomyelin in aqueous solution is described. The phospholipids are first dissolved in chloroform:methanol (2:1 by vol), the solvent is evaporated, and the residue is redissolved in an aqueous zwitterionic detergent solution. The enzymatic reaction sequences of both assays involve hydrolysis of the phospholipids to produce choline, which is then oxidized to betaine, thus generating hydrogen peroxide. The hydrogen peroxide is subsequently utilized in the enzymatic coupling of 4-aminoantipyrine and sodium 2-hydroxy-3,5-dichlorobenzenesulfonate, an intensely red color being formed. The presence of a non-reacting phospholipid enhances the hydrolysis of the reacting phospholipid. Thus we added lecithin to the sphingomyelin standards and sphingomyelin to the lecithin standards. This precise procedure may be applicable to determination of lecithin and sphingomyelin in amniotic fluid.
Micromethod for determination of lactate dehydrogenase isoenzyme C4 activity in human seminal plasma.
Authors: Butrimovitz G P , Farina F , Sharlip I
Published: January 2020

We have developed an automated enzymatic assay for LDH-C4, a lactate dehydrogenase (EC 1.1.1.27) isoenzyme found exclusively in spermatozoa, as a marker of human spermatogenesis. LDH-C4 activity is assayed bichromatically in the Abbott ABA-100 spectrophotometer, with 2-oxohexanoate as substrate. As many as 27 samples (2.5 microL) of human seminal plasma can be analyzed sequentially, with apparent zero-order kinetic conditions and with a precision (CV) of 5%. The apparent Km for the 2-oxohexanoate reaction is 11.2 mmol/L. Electrophoretic and enzymatic studies indicate that LDH-C4 acts on this substrate to produce hydroxyhexanoate, in contrast to lactate dehydrogenase isoenzymes 1 and 5--further evidence for the uniqueness of LDH-C4. The sensitivity, specificity, and speed of this assay system makes it practicable for studies on, and evaluation of, male fertility.
Viscosity adaption for an automated micromethod of flame atomic absorption spectrometry, and intracellular trace-element analysis after pressure decomposition: zinc determination in plasma and erythrocytes.
Authors: Sprenger K B , Franz H E
Published: January 2020

We measured zinc in the plasma of 50 normal persons, and in erythrocytes of six, by an automated micromethod of atomic absorption spectrometry. Using a rotation viscometer, we measured the viscosity and flow behavior of plasma, undiluted and equi-volume diluted (with water), from 12 patients, and, for comparison, of two- to 20-fold dilutions of an 870 mL/L glycerol solution. Erythrocytes were washed three times, lyophilized, and wet-ashed in a pressure decomposition device. The mean viscosity of equi-volume diluted plasma is about the same as that of the glycerol solution diluted 7.5-fold. The observed concentration of zinc in normal plasma was 13.8 (SD 1.9) mumol/L, with intra-assay CV (n = 30) of 2%, day-to-day CV (n = 22) 3.8%, and accuracy (measurement of an aqueous std.) 2.5%. The mean normal value for erythrocyte zinc was 34.7 micrograms/g, with intra-assay CV (n = 8) 5%, day-to-day CV (n = 6) 5.9%, and accuracy 4.6%. Our results compare well with those by neutron activation analysis, the differences in values being 3% for plasma, 1% for erythrocytes.
Test-request patterns for clinical chemistry in a British and a Canadian renal dialysis unit.
Authors: Gardner M D , Henderson A R
Published: January 2020

We recorded, during four months, the total number of routine clinical chemistry tests requested for patients with end-stage renal failure who were on regular maintenance hemodialysis during the study: 20 in the Renal Dialysis Unit of a British and 20 in a Canadian teaching hospital. The pattern of tests ordered was substantially the same in each unit, but the frequency of testing was not. The total number of tests (and tests per patient per month) was 1616 (20.2) for the British and 6939 (86.7) for the Canadian unit (significant at p less than 0.001). This study suggests a significant difference between the two countries in the utilization of laboratory services, and supports our earlier findings on total clinical chemical workloads in hospitals.
Simplified methods for measuring enzyme kinetics in a single cell.
Authors: Marković N S , Marković O T , Young D S
Published: January 2020

We discuss techniques for measuring myeloperoxidase activity cytochemically in isolated polymorphonuclear neutrophils. We have used a sophisticated scanning microdensitometer as a reference device and compared results obtained by Zeiss Ultraphot III B and Reichert Zetopan microscopes with those of the scanning microdensitometer. We made both end-point and kinetic measurements, using conventional microscope slides and a special incubation chamber. Some resolution is lost with simpler microscope systems, but they provided results sufficiently like those obtained with the scanning microdensitometer that they could be used in the clinical laboratory to allow investigation of enzyme activity in single cells.
Automated fluorometer/photometer system for homogeneous immunoassays.
Authors: Li T M , Robertson S P , Crouch T H , Pahuski E E , Bush G A , Hydo S J
Published: January 2020

A fully automated bench-top clinical analyzer (OPTIMATE TM; Ames/Gilford) performs homogeneous fluorescent immunoassays, colorimetric immunoassays, and determinations of routine blood analytes; drugs, enzymes, metabolites, specific proteins, and hormones in serum. Unique features include a combination fluorescence/absorbance aspirating thermocuvette, a photon-counting fluorometer/photometer, a multi-reagent distribution valve to dispense as many as three reagents plus buffer, and a user-replaceable programmable memory cartridge for software updates. We have evaluated the performance of OPTIMATE substrate-labeled fluorescent immunoassays for gentamicin, tobramycin, amikacin, theophylline, phenytoin, phenobarbital, primidone, carbamazepine, and quinidine with this automated system. A sample throughput of 92 samples per hour is achieved by reading fixed-point fluorescence results every 39 s after an initial 4-min reaction period. Precision studies indicate typical CVs of less than or equal to 6% for mid-range controls. Standard curves can be reused for as long as two weeks before recalibration. With clinical samples, results by the OPTIMATE procedure correlated well (r greater than or equal to 0.97) with those by a reference method.
Pyruvate kinase isoenzymes in human serum.
Authors: Ngo J L , Garratt K N , Eversole P , Orlando R A , Ibsen K H
Published: January 2020

Pyruvate kinase activity in serum from presumably healthy individuals is labile, but can be maintained for a week when samples are stored frozen. Contrary to published data our electrophoretic and isoelectrofocusing studies with fresh sera show the presence of both K and M isoenzymes; the latter predominates. In freezer-stored serum the putative M isoenzyme showed a progressive decrease in pl value as well as a decreased electrophoretic mobility.
Freeze/thaw stability of transferrin, and reference values obtained with kinetic nephelometry.
Authors: Stoddard L , Dennis W , Parvin R M , van Assendelft O W
Published: January 2020

To determine whether transferrin is stable to repeated freezing and thawing, we used blood specimens from 100 normal adults. Their transferrin concentrations were determined by a laser kinetic nephelometric immunoassay. The sera were stored at 4 degrees C and analyzed the next day (day 1). Specimens 1-25 were stored at -20 degrees C and assayed again on days 3, 4, 8, 16, 22, and 43. Specimens 26-100 were stored at 4 degrees C and analyzed again on day 2. Transferrin proved to be stable to repeated freezing and thawing. Normal reference values with respect to sex and age are given, as determined with the Baker 420 Immunological Nephelometer.
Comparison of the Du Pont aca and Dow methods for determination of high-density lipoprotein cholesterol.
Authors: Rehak N N , Elin R J , Chesler R , Johnson E
Published: January 2020

We compared the Du Pont aca (phosphotungstate-enzymic cholesterol) and the Dow (dextran sulfate/Mg2+-enzymic cholesterol) methods for the determination of high-density lipoprotein cholesterol (HDLC) and total cholesterol in serum from 113 patients. The aca results for both total cholesterol and HDLC were significantly greater (p less than 0.0001) than the Dow results, the aca method overestimating the HDLC concentration (mean recovery 107.2% in serum samples with values assigned by the Centers for Disease Control). The precision of the aca method for HDLC was essentially the same as that of the Dow method. Bilirubin (up to 0.17 g/L), hemoglobin (up to 4 g/L), and slight lipemia (triglycerides up to 5.4 g/L) did not interfere with the aca method.