bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of free thyroxin measurements in patients with non-thyroidal illnesses.
Authors: Lawlor J F , Blaustein M
Published: January 2020

Sera from 51 euthyroid patients with various non-thyroidal illnesses were used for the evaluation of free thyroxin measurements by use of two commercially available radioimmunoassay kits. A derived value for the percentage of free thyroxin was then calculated as the ratio of the free and total thyroxin concentrations. Values for the percentage of free thyroxin as measured directly via equilibrium dialysis were compared with those derived from both the Corning Medical (r = 0.56) and the Clinical Assays (r = 0.85) kit procedures. The Clinical Assays procedure, in which a solid-phase "immunoextraction" is used, demonstrated a large dynamic range of response for this patient population, in contrast to the narrow response seen with the Corning Medical procedure. Results for euthyroid patients with significant systemic illness by the Clinical Assays procedure compared favorably with those by equilibrium dialysis.
Enzyme immunoassay of gentamicin with use of a centrifugal analyzer.
Authors: Pesce M A , Bodourian S H
Published: January 2020

Gentamicin was determined in serum by the homogeneous enzyme immunoassay system (EMIT) adapted to the Multistat centrifugal analyzer. The standard curve can be extended to 16 mg/L; however, poor precision is obtained at concentrations greater than 10 mg/L because a small difference in change in absorbance there will produce a significant variation in gentamicin concentration. For example, the within-run precision of the method (CV) was 3.4% for a 5.0 mg/L sample (range 4.6--5.4 mg/L) and 11.1% for a 12.2 mg/L sample (range 10.4--15.4 mg/L). We recommend that all samples containing gentamicin at concentrations exceeding 10 mg/L be diluted with Tris buffer. Recovery of gentamicin added to four serum specimens averaged 102%. There is no interference from bilirubin at concentrations up to 200 mg/L or from moderately lipemic samples. Hemoglobin in serum in excess of 625 mg/L results in lower gentamicin values. Gentamicin is stable for six days in serum stored at 23, 4, or -20 degrees C. Comparison of gentamicin results by the proposed method with those by a manual EMIT procedure, and with those by a homogeneous fluorescence immunoassay method gave correlation coefficients of 0.986 and 0.947.
Enzymic analysis for rapid detection of microbial infection in human body fluids: an overview.
Authors: Yolken R H
Published: January 2020

One possible means of rapidly detecting microorganisms in patients with suspected infectious diseases is the direct measurement of microbial enzymes in body fluids. This technique is based on the fact that bacterial, fungal, and viral organisms possess enzymes that are not produced by mammalian cells and are thus not found in uninfected human body fluids. Detection of one of these microbial enzymes in blood, cerebrospinal fluid, or other body fluids would thus be indicative of microbial infection. Potentially useful enzymes for this purpose include bacterial beta-lactamases, fungal adenine deaminases, and viral thymidine kinases. In addition, glycosidases such as neuraminidases and galactosidases can be used as markers for microbial infection, provided that the enzymic activity can be appropriately identified as being of microbial origin. The direct measurement of microbial enzymes offers great potential for the rapid diagnosis of infectious diseases.
Immunochemical evaluation of the organ specificity of prostatic acid phosphatase.
Authors: Shaw L M , Yang N , Brooks J J , Neat M , Marsh E , Seamonds B
Published: January 2020

In a survey of normal and cancerous human tissues we determined the distribution of immunoreactive prostatic acid phosphatase, using rabbit antiserum to acid phosphatase purified from prostatic fluid. In all normal tissues and blood cells studied except leukocytes we found less than 0.1% (expressed as micrograms per gram of wet weight of tissue) of the quantity of immunoreactive prostatic acid phosphatase detected in normal prostate tissue by radioimmunoassay. A small quantity of cross-reactive antigen (2.5 microgram/10(8) cells) was found in leukocytes. In all normal and cancerous nonprostate tissues surveyed by an immunohistochemical technique we detected no immunoreactive prostatic acid phosphatase, except in kidney tissue. Faint but reproducible staining was detected in the lumen of distal tubules and collecting ducts and within interstitial capillaries. Immunoreactive prostatic acid phosphatase was detected in the urine of pre- and post-pubertal males and females. We propose that this material is from serum (low concentrations of immunoreactive prostatic acid phosphatase are present in the serum of men and women) and that it is excreted into urine by the kidneys. Full proof of this must await future experimentation. The specificity of our antiserum for prostatic acid phosphatase was demonstrated by the fact that the Mr 100 000 and 20 000 liver acid phosphatase isoenzymes did not cross with our antiserum in either the radioimmunoassay or double-diffusion analysis. Similarly, preparations of isoenzymes 5A and 5B are human serum albumin did not cross react.
Use of a flow-injection sample manipulator as an interface between a "high-performance" liquid chromatograph and an atomic absorption spectrophotometer.
Authors: Renoe B W , Shideler C E , Savory J
Published: January 2020

We describe an integrated, molecular-absorbance, atomic absorption instrument for studying metal/ligand binding in clinical samples. For an interface between the "high-performance" liquid chromatograph and the atomic absorption instrument we used a flow-injection sample manipulator, thus allowing both the chromatograph and the atomic absorption detector to operate at their separate optimum conditions. After specimen separation with a gel permeation column, we measured the molecular components of the column eluate by molecular absorbance spectrometry and the atomic components (calcium and magnesium) by flame atomic absorption spectrophotometry. This instrument system is capable of separating and analyzing multiple components within 20 min of injection of the sample on the column. The chromatograms presented demonstrate the utility of the system for investigating metal binding to a variety of ligands in clinical samples.
Direct immunological determination of aspartate aminotransferase isoenzymes.
Authors: Rej R , Keese C R , Giaever I
Published: January 2020

We examined the suitability of a rapid immunological technique to determine the amount of aspartate aminotransferase (EC 2.6.1.1) isoenzymes in human serum or tissue extracts. Purified isoenzymes were absorbed as a monolayer to the surface of an indium metal film on glass. The enzyme retains immunological reactivity, allowing the specific binding of aspartate aminotransferase antibodies at the surface. The amount of isoenzyme in a specimen is estimated from the competition for the antibody between the free isoenzyme in the specimen and that at the surface. The surface is further incubated with goat antibodies to rabbit IgG, and the extent of antibody binding is determined by densitometry. There is no cross reactivity between the cytoplasmic and mitochondrial forms, so these two isoenzymes can be determined simultaneously. The minimum detectable concentration by this technique is about 50 micrograms of enzyme protein per liter. The within-day coefficient of variation for determination of either isoenzyme was about 20%. Our results suggest that normal and patients' sera contain considerably more immunologically active than catalytically active isoenzymes.
Enzyme-enhancement immunoassay: a homogeneous assay for polyvalent ligands and antibodies.
Authors: Gibbons I , Hanlon T M , Skold C N , Russell M E , Ullman E F
Published: January 2020

A homogeneous enzyme immunoassay for proteins has been developed that avoids the need for a labeled antigen. The technique involves antibody labeled with beta-galactosidase (EC 3.2.1.23), succinylated antibody, and a macromolecular o-nitrophenyl-beta-galactoside substrate. The enzyme-labeled antibody and the succinylated antibody form an immune complex in the presence of sample antigen. An enzyme within this negatively charged microenvironment produces a product that forms a second light-scattering phase, whereas the product produced by free enzyme remains soluble. Thus the antigen modulates the rate of increase in light scattering. The technique has been applied to assays for human immunoglobulin G and C-reactive protein as well as for specific antibodies.
A proposed simple method for detection and measurement of antibodies to insulin in serum by use of Staphylococcus aureus containing protein A.
Authors: Oliver J R , Hakendorf P , Zeegers P , Ross W
Published: January 2020

A method is proposed for using Staphylococcus aureus containing protein A for the detection and measurement of insulin antibodies in the sera of insulin-requiring diabetics.
Reference intervals for serum IgG, IgA, IgM, C3, and C4 as determined by rate nephelometry.
Authors: Jolliff C R , Cost K M , Stivrins P C , Grossman P P , Nolte C R , Franco S M , Fijan K J , Fletcher L L , Shriner H C
Published: January 2020

We report reference intervals for IgG, IgA, IgM, C3, and C4 for a population of 750 well children and 120 healthy adults. Ranges were established by rate nephelometry (previous studies have been based on immunodiffusion). Our results generally agree with previously established immunoglobulin ranges, except for some disagreement as to ages when adult values are attained.
Enzymic measurement of urinary pyrophosphate with a centrifugal analyzer.
Authors: Roullet J B , Lacour B , Ulmann A , Bailly M
Published: January 2020

We describe a simple, rapid, and fully automated technique for measuring urinary pyrophosphates with a centrifugal analyzer (the ENI GEMSAEC). This technique depends on the enzymic magnesium-dependent reaction with UDPG pyrophosphorylase (UTP: alpha-D-glucose-1-phosphate uridylyl transferase, EC 2.7.7.9) and spectrophotometry of the NADPH formed in a combined system of phosphorylation and reduction. Many samples of urine can be analyzed quickly without pretreatment, with high sensitivity (1.3 mA/mumol of substrate) and good reproducibility. The mean within-run coefficient of variation for a 50 mumol/L pyrophosphate solution was 1.4%. We determined the optimum enzyme and magnesium concentrations necessary for use in a 4-min reaction. Because there is no inhibitory effect of chloride and phosphate ions, pyrophosphate can be measured directly in urine, without prior extraction. With this technique, the mean value (and SD) for urinary pyrophosphate excretion by 30 healthy subjects was 39.3 (SD 17.2) mumol/24 h.
Gas-chromatographic/mass spectrometric detection of 3-hydroxy-3-methylglutaryl-CoA lyase deficiency in double first cousins.
Authors: Norman E J , Denton M D , Berry H K
Published: January 2020

Gas chromatography/mass spectrometry was used for the detection of 3-hydroxy-3-methylglutaryl-CoA lyase (EC 4.1.3.4) deficiency in double first cousins. This enzyme is in the last step of leucine catabolism and is also involved in ketogenesis. Quantitation of urinary organic acids as their cyclohexyl esters demonstrated increased concentrations of 3-hydroxy-3-methylglutaric acid, 3-methylglutaconic acid, 3-methylglutaric acid, and 3-hydroxyisovaleric acid. The procedure is more rapid, sensitive, and specific than previously reported gas-chromatographic methods for acid quantitation. The affected children initially presented with symptoms similar to Reye's syndrome; the acids were quantitated during periods of altered intake of protein and fat. Both leucine and fat intake contributed to increased acid excretion. These studies suggest that life-threatening episodes of hypoglycemia are best prevented with a low-protein, low-fat diet.
cAMP radioimmunoassay without interference from calcium or EDTA.
Authors: Chiang C S , Kowalski A J
Published: January 2020

Calcium and EDTA interfere with radioimmunoassay of cAMP or acetylated cAMP. Both interferences with the assay of acetylated cAMP can be obviated by including in the buffer 15 mmol of CaCl2 per liter, which at the same time increases the affinity of the antibody by 89%. As a result, antibody can be decreased by 65%. This method requires no prepurification (extraction or chromatography), and no correction for analytical recovery is necessary. This assay can detect 12 fmol of cAMP per tube, so only microliter quantities of plasma are needed.
Influence or age and sex on the kinetics of intravenously administered L-phenylalanine.
Authors: Jagenburg R , Regårdh C G , Rödjer S
Published: January 2020

We studied the kinetics of intravenously administered L-phenylalanine with respect to the effect of age and sex, using a two-compartment model. We found that the volume of the peripheral compartment and total body clearance decrease with age. The sex-related influence was less obvious when distribution volumes and total body clearance were corrected for differences in body size. We emphasize the necessity of having age-matched control subjects in kinetic studies.
Improved one-dimensional thin-layer chromatography of phospholipids in amniotic fluid.
Authors: Pappas A A , Mullins R E , Gadsden R H
Published: January 2020

With this extraction procedure and chromatographic method, six amniotic fluid phospholipids [phosphatidylcholine (lecithin), sphingomyelin, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and phosphatidylglycerol] are completely resolved in less than 70 min from the time the specimen is received. The mobile phase (chloroform/petroleum ether/methanol/acetic acid, 5/3/1.6/1, by vol) is used with commercially available 7.5 X 10 cm silica-gel plates. The phospholipids are made visible by immersing the plate in cupric acetate/phosphoric acid. Lecithin/sphingomyelin (L/S) ratios are determined densitometrically. Rf values are well reproducible for both samples and standards, as are L/S ratios, and the method is simple and inexpensive. The relation between L/S ratios (for 100 specimens of amniotic fluid measured by this method) and fetal status is consistent with other reports. We confirm phosphatidylglycerol to be a sensitive indicator of fetal lung maturity: when it was detectable in amniotic fluid, the newborn invariably showed no symptoms of the respiratory distress syndrome.
Immunochemical determination of CK-MB isoenzyme in human serum. II. An enzymic approach.
Authors: Wicks R , Usategui-Gomez M , Miller M , Warshaw M
Published: January 2020

A novel immunochemical technique for a specific enzymic determination of the myocardial isoenzyme of creatine kinase, CK-MB, involves determination of B-subunit activity of a specimen in which the M-subunit activity has been inhibited by specific antibodies to the M-subunit. Interfering activities from CK-BB isoenzyme, atypical forms of creatine kinase, and adenylate kinase are eliminated by using a blank tube in which all the M-subunit-containing isoenzymes have been removed by a specific immunoprecipitation step. The assay is convenient, linear, and reproducible, and results compare well with those by agarose electrophoresis.
Radioimmunoassay of serum parathyrin: comparison of five commercial kits.
Authors: Gorog R H , Hakim M K , Thompson N W , Rigg G A , McCann D S
Published: January 2020

We compared serum parathyrin radioimmunoassay data obtained with five commercially available kits for normal subjects, patients on dialysis, and patients with hyperparathyroidism. The Cambridge Nuclear kit gave low results, did not distinguish the dialysis-group sera from the normal sera, and had the greatest CV (56%) both inter- and intra-assay. The Immuno Nuclear PTH-II kit gave more satisfactory clinical data than their PTH-C-terminal kit; however, as with most procedures involving a substantial and variable nonspecific binding correction for each serum, parallelism between samples and the standard curve was poor, turnaround time slow, and inter-assay CV high (39%). Unlike the other kits, the Nichols and the Iso-Tex products depend on a direct methodology; they displayed good parallelism and distinguished among our three populations. A Scatchard plot of results with the Nichols kit was closest to the ideal linear model; the Iso-Tex kit had the shortest turnaround time.
Improved method for determining erythrocyte creatine by the diacetyl-alpha-naphthol reaction: elimination of endogenous glutathione interference.
Authors: Li P K , Lee J T , Li C S , Deshpande G
Published: January 2020

We describe a simple, accurate, and reproducible method for determining erythrocyte creatine. The method is free from glutathione inhibition and is adaptable to use with standard spectrophotometers as well as centrifugal analyzers. A clear filtrate, essentially free of protein, hemoglobin, and glutathione, is prepared from 0.1 mL of packed erythrocytes by treatment with Ba(OH)2 and ZnSO4, then reacted with diacetyl-alpha-naphthol. The standard curve for this method is linear from 10 to 500 mg/L. We show that endogenous sulfhydryl species such as erythrocyte glutathione will interfere with the creatine-diacetyl-alpha-naphthol reaction. This observation confirmed a suspicion of underestimation of erythrocyte creatine by the method of Griffiths (Clin. Chim. Acta 9: 210, 1964). Added p-chloromercuribenzoic acid did not completely eliminate this inhibition. In the present method these interfering sulfhydryl species are eliminated from the reaction mixture, thus obviating the need for p-chloromercuribenzoic acid and dialysis. The reference interval for this method is 42-80 mg/L.
Beta 2-microglobulin determined by radioimmunoassay with monoclonal antibody.
Authors: Swanson R A , Tracy R P , Katzmann J A , Wilson D M , Young D S
Published: January 2020

In this sensitive radioimmunoassay for beta 2-microglobulin (beta 2-M) involving a commercially available monoclonal antibody, we used a second monoclonal antibody, produced in our laboratory, for affinity-chromatographic purification of beta 2-M. Both monoclonal antibodies bound to all of the charge isomers of beta 2-M identified by two-dimensional gel electrophoresis. Polyethylene glycol was used to separate the phases after incubation for 3 h at room temperature. Sensitivity was 0.25 ng of beta 2-M. Within-assay CVs were less than 5.4%, between-assay CVs less than 7.3%. Analytical recovery was 95-102%. The reference interval was 1.2-2.2 mg/L for 49 healthy subjects. The correlation coefficient for comparison with a polyclonal antibody assay was 0.997 for 44 patients. In a study correlating serum beta 2-M and creatinine concentrations with glomerular filtration rate for 50 patients, correlation coefficients for log-log transformed data were -0.94 and- 0.95, respectively. Concentrations of beta 2-M were increased in serum of patients with imparied renal function and also in patients with normal renal function who had disorders involving the immunologic response. We found no clear-cut advantage of measuring serum beta 2-M over serum creatinine in the estimation of glomerular filtration rate.
Determination of glycosylated hemoglobin by affinity chromatography: comparison with colorimetric and ion-exchange methods, and effects of common interferences.
Authors: Klenk D C , Hermanson G T , Krohn R I , Fujimoto E K , Mallia A K , Smith P K , England J D , Wiedmeyer H M , Little R R , Goldstein D E
Published: January 2020

An affinity-chromatographic method for determination of glycosylated hemoglobin (Anal. Lett. 14: 649-661, 1981) is compared with the thiobarbituric acid colorimetric (I) (Clin. Chem. 27: 669-672, 1981) and the ion-exchange liquid-chromatographic (II) (Diabetes 29: 623-628, 1980) methods. A correlation of 0.98 was obtained for the affinity method vs II and 0.97 for affinity vs I (n = 51). The within-run CV was 1.9% for specimens from non-diabetic individuals and 1.0% for those from diabetics. The respective between-run CVs were 3.4% and 2.4%. Failure to remove "labile" glucose adducts by 5-h incubation of erythrocytes in isotonic saline (37 degrees C) contributed an average error of 13.1% for II, 5.4% for I, and 1.6% for the affinity method. Affinity chromatography gave a decrease of 0.1-0.2% glycosylated hemoglobin for each 1.0 degree C temperature increase between 18 and 27 degrees C. Varying the pH of the wash buffer used in the affinity procedure from 7.75 to 8.25 (pH 8.0 optimum) produced at net change of 0.5% in glycosylated hemoglobin with one diabetic specimen. Using the affinity method, we determined the reference interval for glycosylated hemoglobin in 124 apparently healthy individuals to be 5.3 to 7.5% (mean 6.36%, SD 0.55%). Rechromatography by II and isoelectric focusing analysis of the fractions obtained by the affinity separation revealed a substantial population of glycosylated hemoglobins not measured by II. The affinity method offers a rapid, simple, precise, and accurate alternative to methods currently in use and gives substantial freedom from many common interferences.
An improved assay of mammalian collagenase activity, and its use to determine hepatic extracellular matrix susceptibility to degradation.
Authors: Lindblad W J , Fuller G C
Published: January 2020

This rapid, sensitive method of determining collagenase (EC 3.4.24.7) activity incorporates several advantages of previous methods. Soluble [14C]acetylated collagen is prepared as the enzyme substrate and collagen-cleavage products are separated from noncleaved collagen by precipitation with dioxane/methanol. The assay is more reproducible than previous methods and has a lower detection limit, 15 mU of enzyme activity. We used the method in a competitive substrate assay with isolated extracellular hepatic matrix from cirrhotic and normal rat liver. Purified collagenase was consistently bound to normal rat matrix to a greater extent than to cirrhotic matrix, suggesting that in hepatic fibrosis the extracellular matrix is not as susceptible to collagenase degradation as that in normal liver.
Improved determination of aluminum in serum and urine with use of a stabilized temperature platform furnace.
Authors: Leung F Y , Henderson A R
Published: January 2020

This method for determining aluminum in serum and urine is essentially free from matrix interference and gives a linear response with concentration to at least 500 micrograms/l. Use of a stabilized temperature platform (L'vov platform, Perkin-Elmer Corp.) to approach a "steady-state" temperature, addition of matrix modifiers [especially Mg(NO3)2], and the use of peak area integration all helped substantially diminish spectral interference. With the platform furnace, serum protein concentrations as great as 260 g/L did not interfere with the determination of Al. The within- and between-assay precision (CV) was less than or equal to 3.5% and less than or equal to 7.4%, respectively. Analytical recovery of Al added to serum ranged between 95 and 101% throughout the linear calibration range (to 500 micrograms/L), either when measured directly from the standard curve or by the method of standard additions. The reference interval for Al in 28 healthy subjects was 2-14 micrograms/L (mean 6.5, SD 4.1 micrograms/L), and for 130 patients on hemodialysis, 20-550 micrograms/L (mean 87.5, SD 62.5 micrograms/L).
Clinical evaluation of immunochemical assay of lactate dehydrogenase isoenzyme 1 in early detection of acute myocardial infarction.
Authors: Wang T Y , Godfrey J H , Graham L G , Haddad M N , Hamilton T C
Published: January 2020

We immunochemically measured lactate dehydrogenase isoenzyme 1 (LD-1), calculated LD-1/LD ratios (% LD-1) for 122 specimens from 60 patients, and compared the results with those for the conventional cardiac profile and other findings such as clinical presentation and electrocardiogram. Results for LD-1 and % LD-1 could be classified into three groups: group I, with LD-1 less than 64 U/L; group II, with LD-1 greater than 64 U/L and % LD-1 between 17 and 37%; and group III, with LD-1 greater than 64 U/L and 5 LD-1 greater than 38%. These three groups correlated closely and consistently with three patients of cardiac profile, i.e., those of no acute myocardial infarct, myocardial ischemia, and acute myocardial infarct, respectively.
A continuous-flow (AutoAnalyzer II) procedure for measuring erythrocyte transketolase activity.
Authors: Waring P P , Fisher D , McDonnell J , McGown E L , Sauberlich H E
Published: January 2020

In this semiautomated method, an AutoAnalyzer II is used to measure the enzymic production of glyceraldehyde 3-phosphate in hemolysates, to assay erythrocyte transketolase (EC 2.2.1.1) activity. Hemolysate and indicator reactions are separated by dialysis to eliminate hemoglobin interference and increase sensitivity. Internal standards of glyceraldehyde 3-phosphate in hemolysate carriers were quantitatively measured with good precision and accuracy in the presence or absence of the transketolase substrate, ribose 5-phosphate. Chart-recorder values for these standards were used to calibrate the AutoAnalyzer output in IUB units (U) of transketolase activity. Substrate-product relationships were examined to characterize reaction kinetics and optimize assay conditions. AutoAnalyzer transketolase results correlated well with those from two manual procedures.
Quantitative determination of lecithin and sphingomyelin in amniotic fluid.
Authors: Yee H Y
Published: January 2020

A quantitative method for lecithin and sphingomyelin is presented. In this method, which may be calibrated with a primary standard phosphate salt or pure phospholipids, 2 mL of amniotic fluid is extracted, and the solvent passed through a column of hydroxyapatite to remove all phospholipids except lecithin and sphingomyelin. After evaporation, the residue is treated with methanolic sodium hydroxide, which hydrolyzes lecithin to inorganic phosphate and a methyl ester. The unreacted sphingomyelin is removed by extraction, and the solvent evaporated. Both fractions are digested and assayed for phosphate by reduced phosphomolybdate. The total time required is 2 h. Analytical recoveries were 91-97%, and excellent agreement was found for controls. Amniotic fluid specimens were assayed to illustrate application of the proposed method. If this method is used for obtaining a lecithin/sphingomyelin ratio, a new ranges must be established, as this method is not equivalent to thin-layer chromatographic ones.
Two automated methods for plasma antithrombin III compared, and the clinical significance of the results.
Authors: Prellwitz W , Schmitt K F , Machner M , Schuster C J , Weilemann L
Published: January 2020

Antithrombin III (AT III) activity was determined with two different new chromogenic substances--Chromozym-TH (Tos-Gly-Arg-p-nitroanilide; Boehringer Mannheim) and alpha-N-carbobenzyl-oxy-L-lysine-thiobenzyl ester (Du Pont)--with both a discrete (aca) and a centrifugal analyzer (COBAS BIO). The correlation between the Chromozym-TH/centrifugal analyzer and Du Pont ester/aca methods was good (r = 0.9890). Precision within and between runs was similar to that for typical enzymic determinations. AT III in plasma of 226 healthy men and women ranged from 76.6 to 141.1% (100% = "normal"). We found no significant differences ascribable to oral contraceptives. AT III activity was decreased in 27% of patients with acute thromboembolic diseases (n = 62), in 48% of patients the first day after abdominal operations without complications (n = 78), and in 100% of patients with reversible or irreversible shock (n = 58). In patients receiving continuous therapy with heparin (1500 USP units/h) we saw no decrease in AT III within 96 h of beginning treatment. Plasma from 14 of 16 patients with disseminated intravascular coagulopathy showed a decrease in AT III of from 17 to 51% of normal before and during heparin therapy. We then treated all 16 patients with AT III concentrate. During such treatment, AT III in plasma must be monitored over short intervals to assure that sufficiently high proportions of AT III (greater than 70% of normal) are reached.
Liquid-chromatographic procedure for simultaneous analysis for eight benzodiazepines in serum.
Authors: Lensmeyer G L , Rajani C , Evenson M A
Published: January 2020

We describe an efficient extraction and liquid-chromatographic method for separating commonly encountered benzodiazepine drugs and their pharmacologically active metabolites. After a single extraction of the drugs from serum, chlordiazepoxide, demoxepam, N-desmethyl-chloriazepoxide, diazepam, N-desmethyldiazepam, N-desalkylflurazepam, oxazepam, and prazepam can be resolved and quantified by using a C18 reversed-phase "high-performance" column and a ternary-solvent gradient system. Three separate solutions [60 mmol/L ammonium acetate (pH 7.69), 60 mmol/L acetic acid (pH 2.8), and acetonitrile] were incorporated into a gradient mobile phase such that changes in pH and solvent composition occur. Complete chromatographic resolution of the benzodiazepines resulted, permitting quantification of all within 15 min. The standard curve is linear to at least 8 mg/L for each drug, and the detection limit for each was 0.05-0.10 mg/L. The day-to-day precision for both high and low concentrations yielded CVs of 5 to 9%. Extraction of each drug from serum was 95 to 100% complete. Exogenous and endogenous interferences are minimal. Finally, we circumvented the instability problem of benzodiazepine standards in solution by using a simple reduced-pressure drying process that produces a working standard that is stable for at least nine months.
Urine contains an inhibitor for turbidimetric determinations of protein.
Authors: Nakamura J , Yakata M
Published: January 2020

Our examination of urine components separated by gel filtration revealed the presence of an inhibitor that decreases the analytical recovery of protein in a turbidimetric assay involving sulfosalicylic acid as reagent (Proc. Soc. Exp. Biol. Med. 92: 748, 1956). The apparent relative molecular mass of this inhibitor was in the range 160 000-240 000. A study with purified proteins showed a similar inhibition by gamma-globulin, glycoprotein, and beta-lipoprotein in the assay of albumin by the same turbidimetric method. In contrast, measurement of protein by a dye binding method was not affected by these materials. The low values for apparent urinary protein given by the turbidimetric method as compared with those by the dye-binding method are at least partly ascribable to the inhibitor.
High-resolution zone electrophoresis, combined with immunofixation, in the detection of an occult myeloma paraprotein.
Authors: Reichert C M , Everett D F , Nadler P I , Papadopoulos N M
Published: January 2020

A high-resolution agarose gel electrophoretic technique, coupled with immunofixation, was used to follow paraprotein concentrations retrospectively in a patient with multiple myeloma of nine years' duration. Although the patient's IgA lambda gammopathy "disappeared" shortly after the initiation of therapy, as judged by routine cellulose acetate electrophoresis and immunoelectrophoresis, high-resolution zone electrophoresis demonstrated a monoclonal band that we identified by immunofixation as the IgA lambda paraprotein. The combination of the two simple, inexpensive, and reliable techniques of high-resolution agarose electrophoresis and immunofixation thereby permitted detection and identification of a myeloma protein in a patient otherwise thought to be in complete remission. We believe this approach is useful in assessing persistent or recurrent disease in patients with a known history of myeloma; this combination of techniques may also prove beneficial in the early diagnosis of multiple myeloma.
Protein A-bearing Staphylococcus aureus as the solid phase in an enzyme immunoassay and its application to determination of urinary albumin.
Authors: Harper J R , Mahmoudi N , Orengo A
Published: January 2020

Protein A-bearing Staphylococcus aureus was used as a solid-phase matrix in a sandwich-type enzyme immunoassay for urinary albumin. Heat-inactivated, formalin-fixed bacteria were coated with affinity-purified goat anti-human albumin, exposed to solutions containing standard or unknown concentrations of albumin, then challenged with an alkaline phosphatase/anti-human albumin conjugate obtained by periodate oxidation. Alkaline phosphatase activity bound to the bacteria was a function of albumin concentration from 25 to 1000 micrograms/L. This assay was applied to determinations of urinary albumin concentrations between 1.25 and 1000 mg/L. Between-run CV was 2.55 (63.9 mg/L concentration). Within-run CVs for albumin concentrations of 1.9, 38.1, and 638.0 mg/L were 3.7, 3.7, and 2.4%, respectively. Analytical recovery was 95 to 107% across the full working range of the assay. Bence Jones proteins and hemoglobin had no significant effect on the assay. Nonspecific binding of the enzyme-antibody conjugate was 1.3% (SD = 0.7%). Values agreed well with those by radial immunodiffusion.
Anatomically-related variations in trace-metal concentrations in hair.
Authors: DeAntonio S M , Katz S A , Scheiner D M , Wood J D
Published: January 2020

Scalp-hair analysis is used as an indicator of trace-metal concentrations in the human body. The major shortcoming of this approach is the inability to differentiate between endogenous and exogenous trace metals. Our analyses show no correlation between the concentrations of calcium, magnesium, copper, iron, or zinc in scalp hair and pubic hair in 67 donor-matched hair samples. We interpret this as a strong indication that exogenous trace metals make a significant contribution to the results for trace metals in human hair.
Transformation of creatine kinase-BB isoenzyme in vitro: effect of carboxylic acids, thiols, pH, cations, and chelators.
Authors: Lott J A , Heinz J W
Published: January 2020

Creatine kinase isoenzyme BB from rat brain was incubated with serum, dialyzed serum, or plasma, with added carboxylic acids, thiols, buffers, or cations. It was found to be very unstable at 37 degrees C under all these conditions, being transformed to CK-BB', a form that migrates like CK-MB on agarose electrophoretic plates. This transformation is enhanced at alkaline pH and, especially, by Zn2+. CK-BB' is quite stable, and probably results from binding of cations to CK-BB, because the transformation is prevented by EDTA and citrate.
A more specific, liquid-chromatographic method for free cortisol in urine.
Authors: Canalis E , Reardon G E , Caldarella A M
Published: January 2020

Currently used assays for urinary cortisol reportedly overestimate it, owing to cross-reacting substances. We describe here a method for separating and measuring by liquid chromatography cortisol extracted from urine. The method is specific for cortisol and as little as 5 ng per sample can be measured. Mean analytical recovery of added cortisol was 98.8% (SD 6.1%) and the coefficients of variation ranged from 3.1 to 4.7% (within-day) and from 7.1 to 14% (between-day). Mean (and SD) urinary excretion of cortisol for 45 normal men and women was 20.1 (SD 7.6) micrograms/24 h; for 29 children it was 14.1 (SD 6.0) micrograms/24 h. Results by radioimmunoassay were 1.4- to 4.3-fold greater than by this method, and results of the two assays did not correlate well (r = 0.59, p less than 0.01). We consider the present method to be a practical and specific assay for three cortisol in urine.
Falsely negative laboratory diagnosis for myocardial infarction owing to the concurrent presence of macro creatine kinase and macro lactate dehydrogenase.
Authors: Pudek M R , Jacobson B E
Published: January 2020

Macro creatine kinase (CK, EC 2.7.3.2) and macro lactate dehydrogenase (LD, EC 1.1.1.27) were both present in the serum of a 70-year-old woman with myocardial infarction. This interfered with the interpretation of the CK and LD isoenzyme analyses. Gel filtration and immunoprecipitation showed that the macro CK consisted of IgG and CK and the macro LD of IgG and LD. The IgG in this patient bound both MB and BB isoenzymes of CK, resulting in a macro CK complex that co-migrated with CK-MM on cellulose acetate electrophoresis. This situation led to a falsely negative laboratory diagnosis for myocardial infarction.
Effectiveness of analysis of free thyroxin concentration in serum for diagnosis of covert thyroid disease.
Authors: Symons R G , Walichnowski C M , Murphy L J
Published: January 2020

The indirect indices of free-thyroxin concentration (fT4) are of limited effectiveness in diagnosing covert (mild) thyroid disease. We compared measurements of fT4 and of normalized T4 (NT4, the effective thyroxin ratio) in a group of patients being treated for thyroid disease, most of whom were clinically euthyroid. Although fT4 values correlated well with NT4, many of the patients (34% in one of the comparisons) had fT4 and NT4 values that gave different indications of thyroid status. A review of the patients' treatment and other biochemical indications revealed that measurements of fT4 were more effective in detecting mild disturbances of thyrometabolic status than were assays for NT4, total T4, total triiodothyronine, or thyrotropin. This study suggests that direct fT4 assays are particularly useful in diagnosing covert thyroid disease.
A candidate reference method for uric acid in serum. II. Interlaboratory testing.
Authors: Duncan P , Gochman N , Bayse D , Burnett R , Carey N , Carter R , Fellows W D , Garber C , Kessler G , McComb R , Miller W , Nast P , Ryan W , Schaffer R , Tejeda B , Vanderlinde R , Widdowson G
Published: January 2020

We describe the interlaboratory testing of a candidate Reference Method (Part I) for uric acid in serum. The method is based on the ultrasound spectrophotometric quantitation of uric acid before and after incubation with uricase. A comprehensive investigation involving 12 laboratories was organized to document the transferability, intra- and interlaboratory precision, and general reliability of the candidate Reference Method. The interlaboratory CV with this method was about 2 to 6% for uric acid concentrations ranging from 0.12 to 0.60 mmol/L. The results detailed here demonstrate that the method can be successfully duplicated among different laboratories.
Clinical significance of plasma galactose and erythrocyte galactose-1-phosphate measurements in transferase-deficient galactosemia and in individuals with below-normal transferase activity.
Authors: Pesce M A , Bodourian S H
Published: January 2020

We correlated the clinical symptoms of transferase-deficient galactosemia with the plasma galactose and erythrocyte galactose-1-phosphate concentrations in six galactosemic patients during dietary treatment, in a child before treatment, and in 12 individuals with below-normal erythrocyte hexose-1-phosphate uridylyltransferase activity. All the treated patients were asymptomatic. Normal galactose and either normal or above-normal galactose-1-phosphate concentrations were found. Three of these patients were clinically normal as newborns while ingesting galactose-containing foods and may resemble the asymptomatic Negro galactosemic. The clinical symptoms of galactosemia were observed in the untreated patient, who showed markedly above-normal concentrations of galactose and galactose-1-phosphate, protein and reducing substances in the urine, above-normal bilirubin and alkaline phosphatase in the plasma, with normal values for glucose, aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyltransferase. Clinical improvement in this patient paralleled the decline in erythrocyte galactose-1-phosphate. The individuals with below-normal hexose-1-phosphate uridylyltransferase activity (range 7--17 U/g of hemoglobin) had normal galactose and galactose-1-phosphate concentrations and were asymptomatic.
Dissociation of mannan--serum complexes and detection of Candida albicans mannan by enzyme immunoassay variations.
Authors: Reiss E , Stockman L , Kuykendall R J , Smith S J
Published: January 2020

Candida albicans mannan was added to normal human sera and the resulting complexes were dissociated by boiling (boil) with EDTA or by alkali treatment (bead method). The mannan released was detected by "sandwich" enzyme immunoassay (EIA) or by EIA inhibition. Each EIA took 2.3 h to perform. The total time for the boil-EIA combination was 2.7 h and for the bead-EIA, 3.8 h. The temperature favorable for antigen--antibody incubation was 4 degrees C. The sandwich EIAs were preferable to EIA inhibition because absorbance was directly proportional to mannan concentration, within-run variation was decreased, and accuracy was increased. The boil-sandwich EIA had the highest sensitivity in the 12.5 to 200 micrograms/L range.
Relationships of bilirubin binding parameters.
Authors: Wells R , Hammond K , Lamola A A , Blumberg W E
Published: January 2020

The apparent unbound bilirubin concentration by the "peroxidase" method (U) and the total unconjugated bilirubin in blood (T), albumin-bound bilirubin (B), and reserve bilirubin binding capacity (R) by the bilirubin hematofluorometer were measured in 164 specimens from 98 neonates and in a series of artifactual specimens, made by adding bilirubin to the blood of a single adult donor. Linear correlations between U and (B/R) were found for both the prepared specimens (r = 0.99) and the clinical specimens (r = 0.87), with the slopes of the regression lines being close to the reciprocal of the albumin-bilirubin binding constant, a prediction of the mass action law. An excellent linear correlation was observed for the prepared specimens (r = 0.96) between U and (T--B), the concentration of bilirubin bound to low-affinity secondary sites ("loosely bound bilirubin"). A simple model for low-affinity binding of bilirubin in blood predicts this simple relation. A significant linear correlation between U and (T--B) was found for the clinical specimens, although the correlation was less good (r = 0.72), as one would expect. The demonstrated simple linear relationships between U, (B/R), and (T--B) support the hypothesis that both the hematofluorometer and peroxidase methods provide valid measurements of bilirubin binding status.
Sodium ion binding in human serum.
Authors: Kissel T R , Sandifer J R , Zumbulyadis N
Published: January 2020

The amount of sodium ion binding in human sera and in dialyzed human sera was estimated from standard-addition titrations with an ion-selective electrode and from measurements of 23Na nuclear magnetic resonance (NMR) linewidth. For the untreated sera, maximum binding was 1% (1.4 mmol/L) as indicated by NMR; virtually no binding was found via the titration method. For dialyzed sera with low-sodium, normal-protein content, NMR indicated that sodium binding was less than 1.3% (0.14 mmol/L). The same dialyzed fluid analyzed with ion-selective electrodes shows no sodium binding, within the limits of experimental error (+/- 4%). Sodium ion binding to serum protein thus contributes only minimally to differences in sodium measurements observed between the direct (undiluted) ion-selective electrode and flame-photometric methods.
Direct radioimmunoassays for "aldosterone" and "18-hydroxycorticosterone" in unprocessed urine, and their use in screening to distinguish primary aldosteronism from hypertension.
Authors: Vecsei P , Benraad T J , Hofman J , Abdelhamid S , Haack D , Lichtwald K
Published: January 2020

For distinguishing primary aldosteronism from essential hypertension, we use simple direct radioimmunoassays for "aldosterone" (aldosterone and other materials that react with the antibody to aldosterone) and "18-hydroxycorticosterone" (similarly) in unprocessed urine. Patients with primary aldosteronism have high values for "aldosterone." This diagnosis can be validated by assays of further urine samples from the same person and by additional direct assays for "18-hydroxycorticosterone." In none of 65 urine samples from 26 patients with primary aldosteronism were both "aldosterone" and "18-hydroxycorticosterone" values within their reference intervals. However, a few "aldosterone" and "18-hydroxycorticosterone" values for patients with essential hypertension and normal aldosterone excretion were also (moderately) increased. Thus, when high values are found, true aldosterone values must be estimated by extraction and chromatography, to eliminate false positives. The "aldosterone" and "18-hydroxycorticosterone" values by our procedure are much higher than the corresponding values for urinary free aldosterone and 18-hydroxycorticosterone. Although not identified, the immunoactive materials are probably metabolites of aldosterone and 18-hydroxycorticosterone.
Measuring hemoglobin in plasma by reaction with tetramethylbenzidine.
Authors: Levinson S S , Goldman J
Published: January 2020

The reaction of hemoglobin with tetramethylbenzidine was studied with a centrifugal analyzer. We found that the hemochromogen reaction was significantly influenced by albumin. Increasing amounts of albumin apparently stimulate the hemochromogen reaction during the earlier part of an incubation and inhibit it later in the incubation. However, during an intermediate period, when the crossover from stimulation to inhibition occurs, the assay is independent of albumin and bilirubin concentrations. Increasing the temperature of incubation or the hydrogen peroxide concentration, or decreasing the tetramethylbenzidine concentration, shortens the crossover time. By adjusting these variables the method can be used with most instrumentation. We developed procedures for use on a centrifugal analyzer or for manual assay (reaction times, 4 and 20 min, respectively). Accuracy, as indicated by a comparison with a direct hemoglobin method (r = 0.990) and by recovery experiments, was excellent. The CV for the automated assay was less than 3%. Plasma collected in citrate and heparin showed good recovery of hemoglobin; recovery in EDTA was poorer.
Purification of tracer for somatomedin C radioimmunoassay by hydrophobic interaction chromatography.
Authors: Baxter R C , Brown A S
Published: January 2020

We have purified tracer for use in the somatomedin C radioimmunoassay by hydrophobic interaction chromatography. Material showing greatest immunoreactivity binds to Octyl Sepharose CL-4B (Pharmacia) in a buffer mixture consisting of 130 mL of acetonitrile and 870 mL of 0.1 mol/L NH4HCO3, pH 7.8, but is eluted by increasing the acetonitrile content to 180 mL/L. As compared with tracer purified by binding to specific antiserum in liquid phase, precipitating the complex with second antibody, and then dissociating by gel chromatography at acid pH, this tracer shows equal immunoreactivity against specific somatomedin C antiserum. Either preparation allows excellent discrimination between extracts of normal, acromegalic, and hypopituitary plasma samples; thus either is suitable for use in the somatomedin C radioimmunoassay. Tracer purification by hydrophobic interaction chromatography is rapid and inexpensive. It may be useful in preparing highly immunoreactive tracers for other peptide radioimmunoassays.
Rapid method for eliminating labile glycosylated hemoglobin from the assay for hemoglobin A1.
Authors: Nathan D M , Avezzano E , Palmer J L
Published: January 2020

The irreversible formation of stable glycosylated hemoglobin proceeds through a labile intermediate that is indistinguishable, by most methods, from the stable glycosylated product. The inclusion of the labile intermediate, which changes acutely with acute blood glucose changes, detracts from the utility of the assay as an index of chronic glucose concentration. We have developed a rapid, reliable chemical method for eliminating the labile intermediate: a 30-min incubation of whole-blood samples with semi-carbazide (30 mmol/L) and aniline (12 mmol/L) at pH 5 and 38 degrees C. The semicarbazide serves as a glucose trap; the transfer of glucose from the labile glycosylated hemoglobin to the semicarbazide is catalyzed by the acidic pH and aniline. This treatment is effective in the three most commonly used assays: "high-performance" liquid chromatography, electrophoresis, and a minicolumn kit.