bluuebunny/crossref_metadata_2025 huggingface dataset:
A coupled NAD+-peroxidase spectrophotometric assay for cholesterol.
Authors: Avigad G , Robertson B
Published: January 2020

We describe a modified enzymic reagent for determination of cholesterol and cholesterol ester in serum and in high-density lipoprotein. In this new procedure, the hydrogen peroxide produced by the action of cholesterol oxidase is used as a substrate for NAD+ peroxidase. Spectrophotometric determination of the NADH consumed in this coupled reaction provides a direct and absolute measure of the cholesterol originally present in free form and that liberated by the action of cholesterol ester hydrolase.
Monoclonal antibodies to human IgG: reaction characteristics in the centrifugal analyzer.
Authors: Deverill I , Jefferis R , Ling N R , Reeves W G
Published: January 2020

We used monoclonal antibodies to human IgG in a kinetic turbidimetric method with a centrifugal analyzer. Results for total IgG with monoclonal and polyclonal reagents correlated well (r = 0.979). Within- and between-batch CVs compared favorably (3.1 and 4.8%, respectively, for polyclonal antisera and 3.5 and 4.0%, respectively, for monoclonal reagents). The monoclonal antibody combination used was shown to be of high titre, with an avidity toward the lower end of the range seen with conventional antisera to IgG. It displayed reaction characteristics similar to those observed with polyclonal antisera, although the time scale of reaction was extended and the effective concentration range was narrowed. Single monoclonal antibodies may not produce complexes of a size exhibiting measurable turbidity when reacted with soluble antigen. However, evidence is presented for the incorporation of such complexes into precipitates produced by other related antigen-antibody systems.
Unexplained increase in serum creatine kinase isoenzyme MB activity in a lung cancer patient.
Authors: Goffman T , Cantrell J , Schein P
Published: January 2020

In this case of mixed small cell--large cell cancer of the lung in an elderly woman, creatine kinase (EC 2.7.3.2) isoenzymes were assayed serially because of chest pain. The proportions of serum CK-BB and CK-MB isoenzyme activities were persistently above normal (CK-MB 10-18%, normal less than 5%). Electrocardiograms revealed no signs of ischemia or infarction. At autopsy no gross or microscopic infarction or inflammation of the heart was seen. There was also no infarction of smooth or skeletal muscle. The tumor was the probable source of most of the circulating CK-MB isoenzyme. Future cases may pose a similar diagnostic dilemma: differentiating creatine kinase that is present as a result of myocardial infarction from tumor-related CK-MB. Whether or not CK-MB assay could be useful in detecting tumors remains to be investigated.
Simultaneous determination of the four major catecholamine metabolites and estimation of a serotonin metabolite in urine by capillary gas chromatography of their tert-butyldimethylsilyl derivatives.
Authors: Muskiet F A , Stratingh M C , Stob G J , Wolthers B G
Published: January 2020

We have developed a routine capillary gas-chromatographic profiling method for simultaneous quantitative determination of the tert-butyldimethylsilyl derivatives of homovanillic acid, vanilmandelic acid, 3-methoxy-4-hydroxyphenylethylene glycol, and 3,4-dihydroxyphenylacetic acid and the estimation of 5-hydroxyindole-3-acetic acid in urine. The method is useful for diagnosis and followup of patients with functional tumors characterized by increased urinary excretion of metabolites originating from the metabolism of tyrosine and tryptophan--e.g., neuroblastoma, pheochromocytoma, carcinoid, and melanoma. It may also be applicable in pharmacokinetic studies of administered aromatic amino acids (parkinsonism, mental diseases, loading tests) and for diagnosis and followup of patients with inborn errors of metabolism that are characterized by organic aciduria (for instance, tyrosyluria and phenylketonuria).
Double-antibody fluorescence immunoassay of tobramycin.
Authors: Karnes H T , Gudat J C , O'Donnell C M , Winefordner J D
Published: January 2020

This heterogeneous assay for tobramycin involves fluorescein-labeled tobramycin, which competes with native unlabeled tobramycin for anti-tobramycin binding sites. Bound and free labeled antigen are separated by precipitation with a second antibody. Fluorescence intensity of the resuspended precipitate is inversely proportional to native tobramycin concentration. Background interference was consistently about 10% of the total fluorescence precipitated. Assay sensitivity was sufficient to detect nanogram quantities of tobramycin per assay tube. Correlation coefficients (r) were 0.96 and 0.98 for comparisons of this assay with a microbiological assay and a radioimmunoassay, respectively. Mean analytical recovery was 101% and the CV was less than 10% throughout the therapeutic range.
Radioassay for cobalamin (vitamin B12) requiring no pretreatment of serum.
Authors: Houts T M , Carney J A
Published: January 2020

We describe a radioassay for cobalamin (vitamin B12) in human serum or plasma that requires no boiling or other pretreatment of the sample. Normal chicken serum covalently coupled to magnetizable particles is used as the binding agent. The assay is performed at pH 12.9, at which pH all cobalamin in human serum is released from its binding proteins, whereas the binding agent maintains a high affinity for cobalamin (Ka 1.7 x 10(10) L/mol). Under these assay conditions the binding protein shows a specificity for cobalamin similar to that of purified intrinsic factor. The assay is simple, rapid, and precise, and results correlate well with those of the Euglena gracilis microbiological assay and an intrinsic-factor binding assay.
Rapid immunochemical identification of enterococci with use of a centrifugal analyzer after a brief antigen-extraction step.
Authors: Levinson S S
Published: January 2020

The precipitin reaction between antigens from enterococci and group-specific antisera was monitored turbidimetrically with a centrifugal analyzer. Linear standard curves (absorbance vs antigen concentration) were developed for studying optimal conditions for enzymic extraction of the antigen by use of an enzyme-containing filtrate from Streptomyces albus. This extraction was optimized by adding to the filtrate 1 mg of lysozyme per milliliter and incubating the bacteria for 40 min at 52 degrees C. The assay was adjusted so that detection of enterococci was indicated by positive absorbance changes near 0.1 A. When the concentration of bacteria from which the antigen was extracted was within a standardized range, non-enterococci produced no positive absorbance changes. Reproducibility of standard antigen and antisera solutions was excellent during three months. The procedure provides an objective, well-controlled means for identifying clinically important enterococci from pure cultures in about 1 h. In contrast, current methods involving biochemical media require as long as 48 h.
Four methods for determining albumin in azotemic sera evaluated.
Authors: Gulyassy P F , Depner T A , Ma W K
Published: January 2020

The methods most commonly used for determination of albumin in serum depend on its binding of dyes. The binding of many drugs as well as of several dyes is impaired in azotemic sera from patients with renal failure. We therefore evaluated four methods used to measure albumin concentration in sera of patients with various degrees of renal failure, comparing results with those by the most specific method, radial immunodiffusion. The automated bromcresol green, manual immediate bromcresol green, cellulose acetate electrophoresis, 2-(4'-hydroxyazobenzene)benzoic acid binding, and Na2SO3 precipitation/biuret methods were evaluated. The correlations with results of radial immunodiffusion for azotemic sera differed from method to method but were approximately the same for each method, as in previous published reports for samples from a heterogeneous population of patients. The ratios of serum albumin concentration by these methods to albumin concentration as measured by radial immunodiffusion ranged from 0.96 to 1.13 for 29 to 41 normal and azotemic sera, but none of the ratios showed any variation beyond random scatter over a range of serum creatinine from 6.0 to 180 mg/L. Thus the choice of method to apply to azotemic sera will depend on the relative importance of accuracy, speed, cost, and technical complexity.
Liquid-chromatographic analysis for urinary porphyrins.
Authors: Ford R E , Ou C N , Ellefson R D
Published: January 2020

Urinary porphyrins are separated, according to number of carboxyl groups, in a system consisting of a mu-Bondapak C18 stationary phase and a mobile phase of methanol and aqueous sodium phosphate (pH 3.5) in a linear gradient. The specimen is prepared simply by adjusting the pH of a 5-mL sample to 2.0 and removing solids by centrifugation. The eluted porphyrins are measured fluorometrically. Naturally occurring non-porphyrin fluorescent substances are eluted ahead of the porphyrins. Chromatography requires about 20 min, and a re-establishment of initial conditions requires an additional 15 min.
Activation of human creatine kinase isoenzymes by pH and various sulfhydryl and chelating agents.
Authors: Nealon D A , Pettit S M , Henderson A R
Published: January 2020

We report the effect of serum pH and of the presence or absence of mercaptoethanol, N-acetyl-L-cysteine, monothioglycerol, ethylenediaminetetraacetate, and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate on the activation of the human creatine kinase isoenzymes. At the serum pH giving maximal enzyme stability and minimal assay lag phase (Nealon et al., Clin. Chem. 26: 1165-1169, 1980) thiol activation of CK-1 and CK-3 is nearly maximal with monothioglycerol in an optimized creatine kinase assay (Szasz et al., Clin. Chem. 22: 650-656, 1976). However, CK-2 is maximally activated at pH 8.5, a pH at which this isoenzyme is least stable on storage and its assay lag phase is prolonged. These findings suggest irreconcilable problems in the storage, activation, and assay of CK-2.
Rapid liquid-chromatographic determination of metformin in plasma and urine.
Authors: Charles B G , Jacobsen N W , Ravenscroft P J
Published: January 2020

We describe an analysis for metformin in plasma and urine by use of cation-exchange "high-performance" liquid chromatography. The assay requires only 0.5 mL of sample and involves pretreatment with trichloroacetic acid containing an internal standard (1-propylbiguanide), followed by centrifugation and injection into the chromatograph. The column eluent is monitored at 230 nm. Metformin and 1-propylbiguanide give retention times of 8 and 10 min, respectively. Within-day and between-day precision (CV) for the assay of plasma containing 2 mg (1.2 X 10(-5) mol) of metformin hydrochloride per liter was 3.6 and 3.9%, respectively. Similar precision was obtained for urine. The limit of detection is about 0.1 mg (6.0 X 10(-7) mol) of metformin hydrochloride per liter. Several commonly used drugs, including other oral hypoglycemic agents, do not interfere with this method.
Liquid-chromatographic determination of 4-aminopyridine in serum, saliva, and urine.
Authors: Uges D R , Bouma P
Published: January 2020

We have developed "high-performance" liquid-chromatographic methods for determining 4-aminopyridine, an acetylcholine-releasing drug, in serum, saliva, and urine. As little as 1 microgram/L can be detected by extracting the alkalinized sample plus the internal standard (3,4-diaminopyridine) into dichloromethane, mixing the organic phase with 1-pentanol, evaporating the dichloromethane, and injecting the residue onto a reversed-phase column, where it is eluted with acetonitrile/methanol/aqueous ammonium carbonate, with detection at 245 nm. Analytical recoveries from serum averaged 86.7%. The CV at 50 micrograms/L was 2.9% (n = 8). For urine samples containing very high concentrations of 4-aminopyridine, we mixed urine and potassium carbonate in an automatic injector vial, extracted the drug into dichloromethane, centrifuged, and injected an aliquot of the extract into the chromatograph. Analytical recoveries averaged 92%, and the CV was about 2% for drug concentrations of 0.1-8 mg/L of urine.
Analysis for 1,25-dihydroxyvitamin D in human plasma, after a liquid-chromatographic purification procedure, with a modified competitive protein-binding assay.
Authors: Jongen M J , van der Vijgh W J , Willems H J , Netelenbos J C
Published: January 2020

1,25-Dihydroxyvitamin D in plasma is measured by competitive protein-binding assay after isolation from plasma. The present method is improved, as compared with those hitherto described, with regard to receptor preparation, isolation of vitamin D metabolites from plasma, and procedure for the competitive protein binding. Receptor preparation from healthy rather than rachitic chicks, together with a fast isolation procedure, results in a high yield of active receptor protein: 12 animals provide receptor for about 4000 incubations. No loss of binding activity was observed during one year. 1,25-Dihydroxyvitamin D is isolated from plasma by a single extraction and a one-step chromatographic purification. Analytical recovery for the entire procedure averaged 78.1% (SD 4.7%). Other vitamin D metabolites (25-hydroxyvitamin D and 24,25-dihydroxyvitamin D) can also be separated with this procedure. The main features of the modified binding assay are the use of a stabilized cytosol receptor and dextran-coated charcoal instead of polyethylene glycol. The smallest detectable amount in the binding assay is 1-2 pg (2.4-4.8 fmol). Intra- and interassay CVs are 7.0% and 4.8%, respectively. 1,25-Dihydroxyvitamin D concentrations in plasma of 20 healthy subjects averaged 51.7 (SD 10.8) ng/L [124 (SD 26) pmol/L]. Three anephric patients showed values of 3, 6, and 7 ng/L (7, 14, and 17 pmol/L).
Agar gel electrophoretic determination of glycosylated hemoglobin: effect of variant hemoglobins, hyperlipidemia, and temperature.
Authors: Aleyassine H , Gardiner R J , Blankstein L A , Dempsey M E
Published: January 2020

We investigated the effect of temperature, variant hemoglobins, and hyperlipidemia on determination of glycosylated hemoglobin by an electrophoretic method (Clin. Chem. 26: 1598-1602, 1980). We found that: (a) temperature variations ranging from 4 to 30 degrees C were without effect on results obtained by electrophoresis; (b) concurrent determination of glycosylated hemoglobin by electrophoresis and column-chromatography in blood specimens from 150 diabetic patients yielded almost identical mean values for both procedures when operations were carried out at 22 degrees C; (c) electrophoretic determination of glycosylated hemoglobin in whole-blood hemolysate was not affected by concentration of triglycerides; and (d) unlike column-chromatographic procedures, which underestimate the percentage of glycosylated hemoglobin in patients with hemoglobin S and C, the electrophoretic method accurately determined the proportion of glycosylated hemoglobin in these hemoglobinopathies. Evidently, electrophoresis on agar gel is an excellent alternative to cation-exchange column-chromatographic methods for glycosylated hemoglobin.
Low results for inorganic phosphorus with the SMAC continuous-flow analyzer.
Authors: Robertson E A , Elin R J , Johnson E
Published: January 2020

Serum inorganic phosphorus concentrations as measured with the SMAC are lower than those found with other methods. To resolve this problem we analyzed patients' specimens and performed analytical recovery studies with four different systems (SMAC, AutoAnalyzer II, aca, and the Fiske--SubbaRow method). With the SMAC, results for patients' specimens are significantly lower (p less than 0.0001) than with any of the other three methods. The SMAC recovered only about 87% of the added inorganic phosphorus. The value assigned to SMAC Reference I for inorganic phosphorus was 0.876 of the value obtained when the material was analyzed by the reference method. Thus there is a significant systematic error in the SMAC method for inorganic phosphorus determination, attributable to an erroneous inorganic phosphorus concentration assigned the SMAC calibration material by the supplier (Technicon).
Esterolytic method for determination of heparin in plasma.
Authors: Kapke G F , Feld R D , Witte D L , Owen W G
Published: January 2020

This assay for heparin is based on the heparin-accelerated rate of alpha-thrombin III. The rate or product formation from the residual active thrombin is inversely proportional to plasma heparin content. The assay can be performed manually, but our results were obtained with a discrete analyzer, the ABA-100. The assay is insensitive to concentrations of antithrombin III in plasma. Precision studies gave CVs of less than 10%. This assay was compared to the Dade Protopath heparin assay and a correlation coefficient of 0.90 was obtained (n = 62). The correlation between activated partial thromboplastin times and heparin concentrations (r = 0.67) was calculated frm results on 78 plasma specimens from 10 patients.
Characterization of a spectrophotometric assay for angiotensin converting enzyme.
Authors: Ryder K W , Jay S J , Jackson S A , Hoke S R
Published: January 2020

Although serum angiotensin converting enzyme (EC 3.4.15.1) activity has generally been shown to be increased in patients with sarcoidosis, considerable variation in the diagnostic usefulness of this test has been reported. We investigated the possibility that this variation may be the result of inhibition of the widely used spectrophotometric assay by various substances. We also prospectively examined the predictive value of measurements of this enzyme in serum from 100 patients being evaluated for sarcoidosis. The following did not significantly affect the results: storage at 4 or 25 degrees C for one week, hemoglobin, lipoproteins, or corticosteroid medications. Bilirubin, at concentrations of 20 mg/L of serum or move, significantly inhibited the assay. Sera with increased activity showed nonlinear reaction rates during the usual 60-min reaction interval, This was corrected by shortening the reaction time to 30 min. We found a predictive value of 88% when the serum angiotensin converting enzyme test was applied to the diagnosis of active sarcoidosis.
Kinetic measurement of guanine deaminase in serum with a centrifugal analyzer.
Authors: Nishikawa Y , Fukumoto K
Published: January 2020

We describe an enzymic, one-step kinetic method for determination of guanine deaminase (guanase, EC 3.5.4.3) in serum with a centrifugal analyzer. A combined enzyme-substrate system consists of the enzymes xanthine oxidase, catalase, and aldehyde dehydrogenase, the coenzyme NAD+, the substrate guanine, and ethanol in tris(hydroxymethyl)methylamine buffer, with KCl added as activator for aldehyde dehydrogenase. The method requires only 40 microL of sample. Guanase activity in 28 samples can be determined within 10 min by setting a 4-min lag period. The increase in absorbance at 340 nm is linearly proportional to the activity of guanase to 60 U/L. Within-run precision (CV) was 1.32 to 4.50% over the range studied. Day-to-day precision corresponds to CVs of 4.8 to 7.2% over the same range of guanase activity. The reference interval, as calculated from data on 25 healthy humans, was 0 to 1.02 U/L. The enzymic automated method shows good correlation with Caraway's (Clin. Chem. 12: 187, 1966) method (r = 0.949).
Simultaneous measurement of estrogen and progesterone receptors in tumor cytosols with use of 125I-labeled estradiol and of 3H-R5020.
Authors: Thibodeau S N , Freeman L , Jiang N S
Published: January 2020

We describe a dual-isotope assay for measuring the concentration of estrogen receptors and progesterone receptors in tumor cytosols. The concentrations of these receptors are derived from separate Scatchard-plot analyses of a single dextran-coated charcoal assay that incorporates both radioiodinated estradiol and tritiated R5020 as the labeled ligands. The two isotopes, radioiodine and tritium, are easily measured with a liquid-scintillation counter. The concentrations of estrogen and progesterone receptors derived from the dual-label assay are identical to values derived from the respective single-label assays. Moreover, values obtained from an assay with dextran-coated charcoal that incorporates radioiodinated estradiol are identical to those obtained from an assay in which tritiated estradiol is used as the labeled ligand. The dual-isotope assay requires both less time and less tissue.
Albumin and beta 2-microglobulin radioimmunoassays applied to monitoring of renal-allograft function and in differentiating glomerular and tubular diseases.
Authors: Woo J , Floyd M , Cannon D C
Published: January 2020

Blood and urine were samples daily from 11 renal-allograft recipients from one to six weeks after the transplant. Clearances of both albumin (Calb) and beta2-microglobulin (C beta 2 mu) were significantly increased in all 11 patients. Five patients (Group I) with acute allograft rejection showed markedly increased Calb and moderately increased C beta 2 mu, concurrent with decreased creatinine clearance (CCr). Five other patients (Group II) with no evidence of rejection demonstrated episodes of grossly increased C beta 2 mu with minimally increased but stable Calb and normal CCr. One patient had no evidence of rejection nor indications of glomerular or tubular proteinuria. While changes in serum beta 2-microglobulin concentration closely paralleled those of serum creatinine in the Group II patients, the results diverged in the Group I patients because the increase in serum beta 2-microglobulin exceeded that of serum creatinine and preceded the increased in creatinine by one to five days, suggesting that measurement of serum beta 2-microglobulin might afford earlier indication of the nature and extent of renal damage in the allograft recipients.
Hydrophobic compounds interfere in radioimmunoassay for basic protein in myelin.
Authors: Macklin W B , Lees M B , Cohen S R , Ayella S B
Published: January 2020

Hydrophobic compounds influenced the accuracy of the radioimmunoassay for myelin basic protein when lipids (stearic acid, phosphatidylcholine, cholesterol, cerebroside, sulfatide, or GM1 ganglioside) or proteolipids (white-matter proteolipid apoprotein, kidney proteolipid apoproteins, or heart proteolipid apoproteins) were added to a known amount of basic protein and the samples assayed. All of these interfere with the assay, but the direction of the error depends on the quantity added: low concentrations of lipid decrease apparent basic protein, high concentrations enhance it. Obviously, results of basic-protein assays must be interpreted carefully.
The role of organic volatile profiles in clinical diagnosis.
Authors: Zlatkis A , Brazell R S , Poole C F
Published: January 2020

The organic volatile constituents of biological fluids contain clinically useful diagnostic information for the recognition of metabolic disorders in man. To gain access to this information, it was necessary to develop the methodology for reproducibly stripping the trace concentrations of volatiles from biological fluids (dynamic headspace, gas phase-stripping, solvent extraction, and the transevaporator technique), to separate the complex extracts by high-resolution capillary column gas chromatography, and to develop computer-aided data-handling and pattern-recognition techniques for analyzing the immense amount of information generated. The normal and pathological organic volatiles identified by gas chromatography--mass spectrometry in urine, serum, and breast milk are tabulated. Clinical applications of the above techniques to the study and diagnosis of diabetes mellitus, respiratory virus infection, renal insufficiency, and cancer are described.
Immunochemical determination of CK-MB isoenzyme in human serum: a radiometric approach.
Authors: Usategui-Gomez M , Wicks R W , Farrenkopf B , Hager H , Warshaw M
Published: January 2020

The presence of creatine kinase isoenzyme MB (CK-MB) in human serum is an indicator of myocardial injury. In this assay for CK-MB, reagent containing both rabbit antibodies to CK-MM and 125I-labeled sheep antibodies to CK-BB is added to the patient's serum and incubated for 1 h at ambient temperature. Goat anti-rabbit immunoglobulin, conjugated to a mixture of amylose and polyvinylidene fluoride floccules, is then added. After a 15-min incubation, the mixture is centrifuged, isolating the two M-subunit-containing isoenzymes as insoluble complexes. Because the B-subunit portion of MB is labeled with 125I, the radio-activity in the pellet will be proportional to the amount of MB present. The discarded supernate contains excess sheep 125I-labeled BB antibodies, free or bound to BB isoenzyme if it is present. The preparation and characterization of the isoenzymes and antibodies is explained. Concentrations of CK-MB is sera of patients with and without an acute myocardial infarction were assayed in serial specimens obtained in 103 consecutive admissions to a coronary care unit. The performance of this radiometric procedure compared well with CK-MB electrophoresis, giving a sensitivity of 100% and a specificity of 92%.
High-molecular-mass alkaline phosphatase in serum and bile: nature and relationship with lipoprotein-X.
Authors: Crofton P M , Smith A F
Published: January 2020

Using immunoelectrophoresis and other techniques, we have demonstrated an association between lipoprotein-X and (a) alkaline phosphatase and (b) other enzymes originating from the hepatocyte membrane, namely gamma-glutamyltransferase and leucine aminopeptidase. The high-molecular-mass forms of these enzymes, in both serum and bile, were precipitated by lipoprotein-X antiserum but not by antisera to other plasma proteins. The activity of high-molecular-mass alkaline phosphatase in serum was positively correlated with lipoprotein-X and with lipoprotein-X-associated alkaline phosphatase, both assessed semi-quantitatively. On the other hand, many sera possessed high activities of high-molecular-mass alkaline phosphatase but no detectable lipoprotein-X. Incubation of serum with conjugated bile salts and with synthetic detergents, at concentrations which did not dissociate the high-molecular-mass enzymes, caused parallel alterations in the electrophoretic mobility of serum lipoprotein-X and its associated enzyme activity. Incubation of normal dialyzed hepatic bile with normal, lipoprotein-X-negative serum produced an alteration in electrophoretic mobility of biliary lipoprotein and its associated enzyme activity from anodal to cathodal in agar gel. Digestion with papain had a variable effect on the different enzymes in the complex, without affecting the lipoprotein moiety. Leucine aminopeptidase was removed most readily from the complex to give the low-molecular-mass form present in normal serum; gamma-glutamyltransferase dissociated somewhat less readily, and alkaline phosphatase was completely resistant to dissociation from the complex. These results are discussed in the light of current knowledge, and a hypothesis is proposed for the nature of the high-molecular-mass enzymes in serum and bile.
Studies on the quality of specimens obtained by skin puncture of children 1. Tendency to hemolysis, and hemoglobin and tissue fluid as contaminants.
Authors: Meites S , Lin S S , Thompson C
Published: January 2020

When preferred skin-puncture and blood-collecting techniques were used, the mean artifactually produced hemoglobin in 417 samples of plasma from pediatric patients was 260 mg/L. Highest values occurred exclusively in newborns, 0 to 13 days old (n = 176; mean, 390 mg/L), one-third of whom were premature. The highest value was 1470 mg/L. Only 3% of the samples exceeded 1000 mg/L. After 13 days, technically produced hemolysis is about the same as that for adult plasma (less than 200 mg/L). When skin-puncture and blood-collecting techniques are faulty--e.g., from excessive squeezing at the puncture site--plasma K and hemoglobin may be increased in the newborn to 13-day-old infant, and occasionally in older subjects. By determining plasma hemoglobin, K, blood hemoglobin, and hematocrit the contribution of "tissue fluid" is readily calculated. With excessive squeezing at the sampling site, tissue fluid sometimes contaminates plasma with hemoglobin and K, particularly in the youngest group. Values for alanine and aspartate aminotransferases, lactate dehydrogenase, inorganic P, and Na were little altered, even with squeezing, a fact particularly surprising for the enzymes, which are known to be present in markedly higher concentrations in erythrocytes and other tissues. Our data validate the Elson--Ivor--Gochman method (Am. J. Clin. Pathol. 69: 354-355, 1978) for quantitation of hemoglobin in plasma.
Simple extraction method and radioimmunoassay for somatostatin in human plasma.
Authors: Peeters T L , Depraetere Y , Vantrappen G R
Published: January 2020

Interference by human plasma proteins in the radioimmunological determination of somatostatin was eliminated by subjecting plasma samples to acid--ethanol precipitation. The assay was performed on the lyophilized supernate from 300 microliters of human plasma, with reagents that are commercially available. Sensitivity was 2.6 pg, corresponding to 8.7 ng/L of plasma. The intra-assay CV was 8%; the inter-assay CV was 12% for a low (30 ng/L) and 15% for a high (100 ng/L) standard. The mean analytical recovery of exogenous somatostatin from plasma was 95%, and the standard synthetic cyclic somatostatin showed parallel dilution curves with extracted plasma samples. Neither gastrin HG-17 and HG-34, pentagastrin, secretin, glucagon, vasoactive intestinal polypeptide, substance P, nor pancreatic polypeptide interfered in the assay system. Mean immunoreactive somatostatin in 20 normal fasting subjects was 31.5 (SD 15.6) ng/L and ranged from 14 to 67 ng/L.
Response monitoring and action limits: use of ADC numbers in understanding the operational characteristics of the Beckman ASTRA chemistry system.
Authors: Kessner A , Burnett R W , Bowers G N
Published: January 2020

The Beckman ASTRA is a microprocessor-controlled multichannel chemistry analyzer. The output of each module is available to the operator as analog-to-digital conversion (ADC) numbers, which we record during each calibration (Response Monitoring). After so studying three instruments for a total of 33 months, we have established limits for Calibrator ADC numbers that indicate possible operator action (Action Limits). These Action Limits are tighter than the microprocessor's programmed limits, and alert the operator to short- and long-term drift. These tighter limits warn of (a) impending failure of the instrument to calibrate or (b) possible inaccuracies in results for patients. We have instituted changes in preventive maintenance based on our studies of each module's operational characteristics, and have replaced electrodes that failed to meet our Response Monitoring specifications. Response Monitoring and Action Limits based upon ADC numbers have significantly enhanced our understanding of the ASTRA system and thus improved its operational efficiency and analytical reliability. Estimates of precision and accuracy (true value) were satisfactory in comparison to our prior single-channel continuous-flow and flame photometry analytical measurement systems.
Evidence that the low-affinity folate-binding protein in erythrocyte hemolysate is identical to hemoglobin.
Authors: Hansen S I , Holm J , Lyngbye J
Published: January 2020

Gel filtration studies on erythrocyte hemolysate demonstrated the presence of a folate binding protein, apparently of the low-affinity type, that co-elutes with hemoglobin. Further, the folate binder eluted with a low salt concentration after DEAE-Sepharose CL-6B anion-exchange chromatography of erythrocyte hemolysate at pH 6.3. The chromatographic behavior of hemoglobin labeled with [3H]folate was so similar to that of the present binder as to suggest that the folate binder in erythrocytes is in fact hemoglobin.
3H and 125I radioimmunoassays of haloperidol compared with fluoroimmunoassay involving antibody coupled to magnetizable solid phase.
Authors: Rowell F J , Hui S M , Kamel S R
Published: January 2020

Radioimmunoassays for haloperidol are described, involving use of tritium- or 125I-labeled drug or tritium-labeled spiroperidol, and a rabbit antiserum to a drug/bovine serum albumin conjugate. The 125I-labeled drug was prepared by the Chloramine T iodination technique. A fluoroimmunoassay for haloperidol is also described in which the antiserum is coupled to magnetizable solid-phase medium, and fluorescein-labeled haloperidol is used. The assays have acceptable accuracy, precision, and reproducibility, and are specific for haloperidol and similar butyrophenones, with no significant interference from known metabolites and other drugs. Only the radioimmunoassays have sufficient sensitivity to cover the whole range of haloperidol concentrations in serum. The fluoroimmunoassay can be used to monitor high concentrations of haloperidol in 150-microL samples or the complete concentration range of 1-mL serum samples that are extracted and concentrated before assay.
Ionized calcium analyzer with a built-in pH correction.
Authors: Fogh-Andersen N
Published: January 2020

We describe a new semi-automated apparatus for simultaneously measuring the concentration of free calcium ion and of hydrogen ion (pH) at 37 degrees C. The sample volume is 110 microL. In addition to the actual values for these concentrations in the sample, the apparatus calculates the concentration of free calcium ion at pH 7.40. Mean values for serum from 51 fasting bedridden patients without calcium metabolic disorders and 64 fasting hospital employees were 1.192 and 1.232 mmol/L, respectively, with SD of 0.042 and 0.040 mmol/L, respectively. The within-series analytical SD was 12 mumol/L and the day-to-day SD of the pH-corrected concentration of free calcium ion was 21 mumol/L, as calculated from measurements made on a serum pool after equilibration with a CO2--air mixture. The mean dependency on pH as determined in 120 consecutive patients' sera equalled the built-in pH correction. The accuracy was evaluated by comparison with other calcium ion-selective electrodes.
The triiodothyronine uptake test: an assessment of methods.
Authors: Witherspoon L R , Shuler S E , Garcia M M
Published: January 2020

How well the free thyroxine index reflects thyroid functional status depends on the degree to which the triiodothyronine uptake test normalizes the effects of thyroxine binding protein concentrations on the total thyroxine concentration. We examined eight triiodothyronine uptake tests in which were used different secondary binders representative of those available in commercial kits. The relation between triiodothyronine uptake and thyroxine-binding globulin concentrations was established by use of sera from euthyroid individuals. We examined the effects of both high (greater than 20 mg/L) and low (less than 10 mg/L) thyroxine-binding globulin concentrations on triiodothyronine uptake. The precision of each assay, expressed as within- and between-run coefficient of variation, was calculated from multiple measurements on high, low, and midrange triiodothyronine uptake serum pools. The effects of variation in temperature and in exposure times were examined. The clinical most useful assays exhibited the ability to reflect a wide range of thyroxine-binding globulin concentrations and demonstrated little or no time or temperature effects.
Simplified method for measuring sex-hormone binding globulin.
Authors: Fattah D I , Chard T
Published: January 2020

We describe a simple, rapid method for measurement of sex-hormone binding globulin. Serial dilutions of pregnancy serum are prepared in serum from males that has been pre-treated by heating to 60 degrees C for 1 h to destroy endogenous binding globulin, which is then determined by a long-used technique to yield a set of "standards." In the assay itself, a fixed amount of [3H]-labeled and unlabeled dihydrotestosterone is incubated with standard or unknown, and the bound fraction precipitated with saturated ammonium sulfate. A plot of percent of the steroid bound vs standard dilution yields a sigmoid curve, from which the results in unknowns can be read by simple extrapolation. Within-assay CVs for pools of serum from men, women, and women in late pregnancy were 6.56, 9.59, and 8.4%, respectively. Between-assay CVs for the same pools were 8.05, 9.5, and 11.5%, respectively. The correlation between results obtained by this method and those of the older technique was 0.95 for samples from non-pregnant subjects and 0.73 for those from pregnant women. Our procedure is simpler and faster than previous methods and accurately measures the differences in the globulin in sera from men, women, and pregnant women. Forty to 50 samples can be assayed in a working day.
Assay for 3-methoxy-4-hydroxyphenylethylene glycol in human urine by gas chromatography, with electron capture detection.
Authors: Kim C , Van Loon G R
Published: January 2020

We describe a gas-chromatographic method, with electron capture detection, for determining 3-methoxy-4-hydroxyphenylethylene glycol in human urine. High sensitivity (5 pg per injection), recovery (93-97%), and reproducibility (CV 4.3%) are obtained. By this method, the 24-h urinary excretion of the free compound, the sulfate conjugate, the glucuronide conjugate, and the total in six normal male subjects average 0.15 (SEM 0.015), 1.09 (SEM 0.10), 1.22 (SEM 0.10), and 2.34 (SEM 0.21) mg, respectively. In three young women with amenorrhea/hirsutism, administration of dexamethasone decreased the total excretion of the compound.
Two-dimensional analysis of human lymphocyte proteins: I. An assay for lymphocyte effectors.
Authors: Willard K E , Anderson N G
Published: January 2020

We describe an assay for lymphocyte effectors that is capable of establishing the existence of regulators of lymphocyte gene expression (including post-transcriptional control and protein processing) and has the ability to characterize the response at the molecular level. The hypothesis that circulating effectors substances excreted through the kidney can be actively present in human urine was tested with this assay. Thus, biologically active protein molecules in urine were detected at concentrations of less than 1 mg/L and over a wide range of dilutions. Activities were detected and quantitated by culturing human lymphocytes with human urinary proteins in the presence of [35S]methionine and subsequently analyzing the labeled lymphocyte proteins by two-dimensional gel electrophoresis. Thus, protein analysis by two-dimensional gels was used to indirectly detect changes produced in cultured lymphocytes after exposure to regulatory molecules. Proteins or sets of lymphocyte proteins appeared or disappeared after exposure to normal or pathological human urinary proteins. Normal human urinary proteins triggered the appearance of sets of proteins referred to by number as the "Urocon" proteins and suppressed the synthesis of protein sets referred to as "Urocof" proteins. In addition to the normal alterations described, urinary proteins from individuals with influenza or acute leukemia and after renal transplantation were capable of inducing unique alterations in lymphocyte patterns.
Enzyme immunoassay of antibodies to Sjögren's syndrome B antigen.
Authors: Teppo A M
Published: January 2020

I describe a solid-phase enzyme immunoassay of IgG-IgA-, and IgM-type antibodies to Sjögren's syndrome B antigen. Polystyrene tubes are coated with the purified antigen. The antibodies are allowed to bind with their antigens, and then detected with alkaline-phosphatase-conjugated anti-human IgG, IgA, or IgM sera. The amount of alkaline phosphatase fixed to the tubes is determined in pH 10.0 diethanolamine buffer at 37 degrees C with p-nitrophenyl phosphate as substrate. The absorbance of the p-nitrophenolate ion liberated in 1 h at 37 degrees C is measured at 406 nm. When rheumatoid factor is present, the values obtained for IgG antibodies are too low, and those for IgM antibodies are too high.
14C-labeled proteins as markers for gradient analysis of steroid-hormone receptors.
Authors: Ledden D J , Feldhoff R C , Feldhoff P W , Heidemann P H , Wittliff J L
Published: January 2020

Two useful [14C]marker proteins--[14C]human serum albumin (4.6S) and [14C]glucose oxidase (7.9S)--can be simply prepared. Both may be used as molecular-mass standards in polyacrylamide gel electrophoresis as sucrose density-gradient centrifugation. The utility of these markers for estrogen receptor studies was investigated under a variety of conditions, to ensure that they do not interfere with current assay procedures. Their use as internal markers allows more samples to be analyzed per rotor, a significant factor because each centrifugation run requires 16 h and two bucket spaces for each sample assayed; improves accuracy and overall quality control by eliminating any problems resulting from variations among individual gradients; and facilitates evaluation of changes in gradient profiles, which may provide clinically and biochemically relevant information concerning the microheterogeneity of estrogen receptors.
Spectrophotometric and liquid-chromatographic studies of thymolphthalein monophosphate. Specifications for high-quality substrate for the measurement of prostatic acid phosphatase activity.
Authors: Bowers G N , Onoroski M , Schifreen R S , Brown L R , Klem R E , Ewen L M
Published: January 2020

Fourteen lots of thymolphthalein monophosphate (TMP), disodium salt, obtained from 10 commercial suppliers were compared spectrophotometrically at 445 and 595 nm, liquid-chromatographically with monitoring at 254 nm, and enzymically by measurements of activity of prostatic acid phosphatase in human serum. Eight lots were classified as "unacceptable," six as "acceptable." Spectrophotometric testing revealed four lots with excessive thymolphthalein and three lots with grossly deficient amounts of TMP. In general, the chromatographic results paralleled those obtained by spectrophotometry, and both results correlated well with enzymic activity. Changing water content in this hygroscopic salt was a major problem, which resulted in great uncertainty as to the formula weight and therefore as to the moles of TMP actually taken. From these studies, specifications for high-quality TMP were determined. The critical importance of simultaneous enzymic activity measurements in comparisons with other "acceptable" lots in defining an adequate TMP substrate is stressed. Use of these specifications for selecting TMP for acid phosphatase activity measurements should improve intra- and inter-laboratory analytical performance.
Automated two-site immunoradiometric assay of human alpha-fetoprotein in maternal serum.
Authors: Kemp H A , Simpson J S , Woodhead J S
Published: January 2020

Immunoassays of alpha 1-fetoprotein in maternal serum are now in widespread use as a means of antenatal detection of neural tube defects. For population screening to be effective the initial test must be capable of promptly producing reliable results. We describe an automated two-site immunoradiometric assay based on 125I-labeled antibodies and cellulose-coupled antibody, with which up to 100 patients' samples can be assayed in 3 h. The assay is precise, and results obtained agree closely with those produced by conventional radioimmunoassay procedures.
Jendrassik--Grof analysis for total and direct bilirubin in serum with a centrifugal analyzer.
Authors: Garber C C
Published: January 2020

Total bilirubin is determined here with a multipoint calibration approach and with use of Standard Reference Material No. 916 bilirubin from the National Bureau of Standards. The bilirubin calibration solutions prepared in solutions of Cohn Fraction V human albumin were stable for at least one year when stored at -80 degrees C. Studies for direct bilirubin analysis show that the serum sample should be preincubated with the HCI reagent before the diazo reagent is added to minimize the false reaction with unconjugated bilirubin. With a preincubation of 15 min and a reaction interval of 15 min a 250 mg/L unconjugated bilirubin standard gave only 1 mg of apparent conjugated bilirubin per liter. Day-to-day precision studies gave CVs of 3.3% at 12.4 mg/l, 2.1% at 41.2 mg/l, and 1.0% at 197.4 mg/L for total bilirubin and 1.6% at 61.2 mg/L for direct bilirubin, the latter based on a serum pool stored at -80 degrees C. Lipemic sera caused negligible errors. Hemolysis caused slightly larger errors for total bilirubin but much smaller errors for direct bilirubin when measured at 600 nm rather than 550 nm. Comparison of results with the Rotochem (y) to those with a manual Jendrassik--Grof method (x) for total bilirubin gave y = 0.9009x - 0.04 mg/L, Sy/x = 1.3 mg/L, r = 0.9987 when different calibrators were used for each method and y = 0.9813x - 2.9 mg/L, Sy/x = 1.5 mg/L, r = 0.9997 when the same calibrators were used. For direct-bilirubin comparisons, y = 1.2762x + 0.2 mg/L, Sy/x = 1.3 mg/L, r = 0.9926.
Automated determination of copper in undiluted serum by atomic absorption spectroscopy.
Authors: Weinstock N , Uhlemann M
Published: January 2020

An injection method has been adapted for the determination of copper concentration in untreated, undiluted serum by flame atomic absorption spectroscopy. Serum, 50 or 100 microliter, is automatically injected by a commercial microprobe system into a plastic cone connected to the capillary tube of the burner, at a rate of 240 samples per hour. The required sample volume is considerably decreased, and sensitivity is increased 20- to 40-fold. After 500 measurements we observed no memory effects, carryover, or clogging of the burner. We discuss common difficulties with calibration standards due to viscosity and other physicochemical interferences, and suggest the use of pooled human serum as a secondary standard. Within-run CV was 1.8%, the day-to-day CV 2.2%. Comparison with a dilution method gave a correlation coefficient exceeding 0.98.
Long-term stability of a stabilized liquid quality-control serum.
Authors: Hartmann A E , Juel R D , Barnett R N
Published: January 2020

To evaluate the long-term stability of a new liquid quality-control serum ("Decision", Beckman Instruments, Inc.) stabilized with ethylene glycol (330 mL/L), we analyzed it for 22 commonly measured analytes during storage at 2--8 degrees C for 24 days or -15 to -20 degrees C for 55 weeks. Three separate laboratories replicated the analyses, using various analytical methodologies. The data were subjected to linear regression analysis, regressing concentration on time. Analytes were considered unstable when the linear regression coefficient was unequal to zero with 95% or greater probability in all three laboratories. By this criterion all of the analytes were stable for at least 24 days when the control serum was stored at refrigerator temperature and for at least 55 weeks at freezer temperature. We conclude this material is a satisfactory substitute for existing lyophilized quality-control materials and offers certain advantages: stability, vial-to-vial uniformity, decreased waste, and eliminated reconstitution.
Quantitation of urinary uric acid by reversed-phase liquid chromatography.
Authors: Hausen A , Fuchs D , König K , Wachter H
Published: January 2020

The proposed isocratic liquid-chromatographic method for uric acid has good performance characteristics. Results by this procedure and by an enzymic method for uric acid correlated well (r = 0.986). Our method is inexpensive, rapid, and considerably simpler than other techniques for determining uric acid in urine.