bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct radioimmunoassay of 6-sulfatoxymelatonin in plasma with use of an iodinated tracer
Authors: Harthé Catherine , Claustrat Bruno , Brun Jocelyne , Chazot Guy
Published: January 2020

We describe here a direct a radioimmunoassay (RIA) for the determination of 6-sulfatoxymelatonin (aMT6s) in plasma, with iodinated aMT6s as tracer. The aMT6s antiserum was raised in rabbit by immunization with a bovine serum albumin conjugate, giving negligible cross-reactivities for related compounds. The low limit of detection (15 pmol/L) allowed a direct assay that required only a 100-microL plasma sample. Dilutions of plasma and of synthetic aMT6s gave the same parallel response in the RIA. A preliminary study showed a circadian variation in healthy volunteers, with mean concentrations ranging from 52 (at 1600-2100 h) to 378 pmol/L (at 0400 h), whereas this rhythm was abolished in pinealomectomized patients. After administration of melatonin orally, or by infusion, the aMT6s concentrations in plasma concorded with previous data on aMT6s production and pharmacokinetics, with aMT6s being cleared from plasma more slowly than melatonin. This assay should have practical application in the development of new pharmaceutical formulations that minimize the hepatic metabolism of melatonin.
Sequential concentrations of copper and ceruloplasmin in serum from preterm infants with rickets and fractures
Authors: Koo Winston W , Succop Paul , Hambidge K Michael
Published: January 2020

Concentrations of copper (Cu) and ceruloplasmin in serum were measured serially in 49 preterm infants with mean (+/- SEM) birth weights of 979 +/- 33 g and gestational ages of 28.4 +/- 0.3 weeks at three, six, nine, and 12 months postpartum. Serial radiographic studies showed 17 infants with (group A) and 32 infants without (group B) rickets or fractures. Cu and ceruloplasmin concentrations in serum also were measured in 21 healthy term infants (group C) with birth weights 3668 +/- 98 g at three, six, and 12 months postpartum. Analyses of covariance of serial changes in these serum variables--taking into account such potential covariates as differences in gestational age, birth weight, initial weight and length, changes in weight and length during the study, the duration of parenteral nutrition, and increased enteral copper intake--showed both groups of preterm infants had significantly lower concentrations of Cu in serum up to age six months and ceruloplasmin up to age three months (P less than 0.001) when compared with term infants. By one year of age, Cu and ceruloplasmin concentrations in serum in all groups had increased significantly (P less than 0.001), into the adult range, and were not significantly different among groups. These data document a maturational lag in copper metabolism in small, preterm infants. Changes in concentrations of Cu and ceruloplasmin in serum were significantly correlated (r = 0.92, P less than 0.001) but were not significantly different between preterm infants with and without rickets or fractures at each age.
Overestimation of albumin in heparinized plasma
Authors: Hallbach Jɒrgen , Hoffmann Georg E , Guder Walter G
Published: January 2020

Monochromatic measurement of albumin by the bromcresol green method leads to overestimation of albumin in the presence of heparin. The interference seems to be caused by fibrinogen, which, in the presence of heparin, produces an almost constant increase of the measuring signal over the whole range of suitable wavelengths (550 to 700 nm). The albumin overestimation can be eliminated by using a bichromatic modification. An application of this procedure for the automated analyzer RAXT, RA-1000 (Bayer Diagnostik, Technicon) is presented. We recommend using only bichromatic methods for albumin determination with bromcresol green to avoid unexpected analytical artifacts.
Albuminuria vs urinary total protein for detecting chronic renal disorders
Authors: Shihabi Z K , Konen J C , O'Connor M L
Published: January 2020

A literature review and our own data are presented to demonstrate that urinary albumin (UA) excretion increases in many renal disorders and that it offers a far more sensitive indicator than the commonly used urinary total protein (UTP) for the early detection of renal involvement in many chronic diseases such as diabetes mellitus, hypertension, and systemic lupus erythematosus. In many individuals with these disorders, UA increases severalfold, even while UTP remains within the reference interval. UA is also more suited than UTP for following therapeutic responses in these slowly progressive renal disorders. Increases in UA are associated with increased mortality. UTP measurements are plagued with many analytical problems, whereas UA is much easier to standardize. We recommend that both UA and UTP be measured when quantitative urine protein assays are ordered, especially when the UTP is less than 300 mg/g of creatinine.
Rapid liquid-chromatographic method for simultaneous determination of plasma prednisone, prednisolone, and cortisol in pediatric renal-transplant recipients
Authors: McBride J H , Rodgerson D O , Park S S , Reyes A F
Published: January 2020

A rapid liquid-chromatographic method is described for simultaneously determining prednisone, prednisolone, and cortisol in plasma. Before chromatography, samples containing an internal standard (methylprednisolone) were extracted with use of Chem Elut (Analytichem) columns. After elution of the glucocorticosteroids, the eluates were dried and reconstituted in a mobile phase of tetrahydrofuran/water (25/75 by vol). Samples were injected onto a reversed-phase C18 column, and analysis time was 15.5 min. Measures of analytical performance were all acceptable, and the method was used to assess noncompliance in pediatric renal-transplant recipients who were receiving prednisone and cyclosporine. Of the 37 pediatric patients tested, five (13.5%) were identified as noncompliant. The method is simple, accurate, and precise, and other steroids and medications commonly given to transplant recipients do not interfere with it.
Effect of diabetes on the free polyol pattern in cataractous lenses
Authors: Yoshioka S , Kameyama K , Sanaka M , Sekine I , Kagimoto S , Fujitsuka S , Saitoh S
Published: January 2020

To obtain information about the effects of lenticular polyols on the prevention, initial stages, and development of diabetic cataracts, we identified and determined with gas-liquid chromatography or gas-liquid chromatography/mass spectrometry eight polyols in cataractous lenses of non-insulin-dependent diabetes mellitus patients and nondiabetic subjects. In the diabetics' lenses, the concentrations of polyols (e.g., sorbitol, fructose, mannitol, and adonitol) were higher than in the nondiabetics' lenses, whereas the concentration of 1-deoxyglucose was lower. The mean concentration of myo-inositol in lenses of diabetics was lower than that of nondiabetics, but this difference was statistically not significant. The total content of eight polyols in the diabetics' lenses did not differ significantly from that in the nondiabetics. In the lenses of diabetics, the content of glucose correlated positively with that of adonitol, fructose, and sorbitol. In the lenses of nondiabetics, the content of glucose correlated positively with that of mannitol and inversely with that of 1-deoxyglucose and myo-inositol. In diabetics, hemoglobin A1 (%) correlated positively with the concentration of adonitol in the lenses and inversely with the concentration of lens myo-inositol; however, it did not correlate with the concentration of glucose in lenses. Regulation of both the metabolism of lenticular polyols and the pattern of polyols in serum may be necessary for normalizing lenticular polyol content.
Immunoturbidimetric method for routine determinations of apolipoproteins A-I and B
Authors: Brustolin D , Maierna M , Aguzzi F , Zoppi F , Tarenghi G , Berti G
Published: January 2020

A simple immunoturbidimetric method for quantifying apolipoproteins (apo) A-I and B in serum or plasma is described. A special reagent formulation, including large amounts of suitable detergents, obviates the need for a sample blank even with grossly lipemic specimens. The assay is rapid, easily automated, and thus convenient for routine work. For both apo A-I and apo B, the assay range is about 0.2-3.5 g/L. The performance characteristics were assessed with discrete (Optimate and Olli CD) and centrifugal analyzers (Cobas Fara and IL Monarch 2000). Average analytical recovery was 101.5% for apo A-I and 99.4% for apo B. Dilution tests showed found/expected ratios of 101.2% (apo A-I) and 101.0% (apo B). Overall precision (CV) ranged from 1.4% to 3.3% for apo A-I and from 1.1% to 8.3% for apo B. Comparisons with commercially available rate nephelometry, radial immunodiffusion, and immunoturbidimetric methods gave good correlations (r greater than or equal to 0.938). Using the immunoturbidimetric method, we also established the relationships between apolipoproteins and lipids and determined the reference intervals. We conclude that the proposed method is suitable for routine use in clinical laboratories.
Detecting multiple cystic fibrosis mutations by polymerase chain reaction-mediated site-directed mutagenesis
Authors: Friedman K J , Highsmith W E , Silverman L M
Published: January 2020

The polymerase chain reaction (PCR) has been applied in a novel manner to detect the multiple mutations causing cystic fibrosis (CF). PCR-mediated site-directed mutagenesis (PSM) has been applied to create allele-specific restriction enzyme cutting sites for three of the more common mutations. Two other mutations after cutting sites on their own. We discuss the implications for the expedient detection of five different CF-causing mutations.
Prevalence of non-A, non-B hepatitis/hepatitis C virus antibody in laboratory quality-assurance sera
Authors: Dodd Leslie G , McBride James H , Gitnick Gary L , Howanitz Peter J , Rodgerson Peter J
Published: January 2020

Quality-assurance sera (QAS) are prepared from pooled sera composed of thousands of individual donations. Previous studies documented that a substantial percentage of individual QAS test positive for viral disease markers, including antibodies to human immunodeficiency virus and to hepatitis B surface antigen. We tested 239 QAS from various proficiency programs and commercial sources to determine the prevalence of hepatitis C virus (HCV) antibody. We tested samples for anti-HCV by using an enzyme immunoassay (EIA; Abbott Labs.) and an enzyme-linked immunosorbent assay (ELISA; Ortho Diagnostics). We observed an overall positive rate of 49% by one or both assays in all categories of sera tested. In addition, we found a greater rate of positivity (58%) in proficiency program samples than in commercial samples (43%). We found discrepant results between the two assays for 15 of 239 samples (6%). In the discrepant samples, the EIA result was positive, whereas the ELISA result was negative. Anti-HCV positivity in QAS has important implications for laboratory personnel handling these samples.
Enzyme immunoassay screening of alpha 1-antitrypsin in dried blood spots from 39 289 newborns
Authors: Schoos R , Dodinval-Versie J , Verloes A , Lambotte C , Koulischer L
Published: January 2020

We present a new, simple, and inexpensive sandwich-type double-antibody enzyme immunoassay for alpha 1-antitrypsin in dried blood collected on the fifth day post-partum. The method is very sensitive, having a detection limit of 2.84 fmol/well. Intra- and interassay CVs are 6.1% and 10.3%, respectively, for assay of 5-mm-diameter blood spots eluted into 7 mL of phosphate buffer. Since February 1984, we have used this method to systematically screen 39 289 consecutive births: 336 of these newborns (0.085%) showed values for alpha 1-antitrypsin below the cutoff value of 800 mg/L (50th percentile, 1470 mg/L). Of these 336 we were able to obtain 0.5 mL of serum from 161 for further testing. Four presented with a ZZ phenotype and 15 with a SZ phenotype, which indicates a deficiency in alpha 1-antitrypsin. Our data suggest a prevalence of 1.4% and 3.6% of Z and S alleles, respectively, in the French-speaking community of Belgium.
Micro-quantification of cyclosporine and its metabolites and determination of their spectral absorptivities
Authors: Fu Irong , Bowers Larry D
Published: January 2020

We report a micromethod for the analysis of cyclosporine (CsA), based on quantification of its constituent amino acids. The amino acids were released by gas-phase hydrolysis, derivatized with fluorenylmethyl chloroformate, and separated and analyzed in a reversed-phase HPLC system. The imprecision (CV) of the amino acid analysis was less than 4%, and several determinations of the amount of standard CsA were within 1% of the weighed material. The detection limits (signal-to-noise ratio = 2) were 500 fmol for ultraviolet detection and 100 fmol for fluorescence detection. We also used this method to determine the ultraviolet absorptivities of CsA and five metabolites at 210, 214, and 230 nm. The molar absorptivity of most metabolites was about 10% higher than that of CsA, although the metabolite that was oxidized to a carboxyl group on the terminal carbon of N-methyl-butenyl-methyl-threonine (AM1A) had a molar absorptivity about 40% higher than that of CsA.
Rapid detection and initial characterization of genetic variants of human serum albumin
Authors: Sheat J Merle , Peach Robert J , George Peter M
Published: January 2020

We have studied the detection and classification of genetic variants of human serum albumin by electrophoresis. Samples from 10 patients who were heterozygous for eight different albumin variants were studied by two methods. In agarose gel electrophoresis, each of these variants has an abnormal mobility and can be classified on the basis that structural changes at the N-terminus abolish 63Ni binding. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole serum, glycosylated variants are easily detected because of their greater apparent molecular mass.
Stabilization of turbidimetric immunoassay by covalent coupling of antibody to latex particles
Authors: Thakkar H , Davey C L , Medcalf E A , Skingle L , Craig A R , Newman D J , Price C P
Published: January 2020

Turbidimetric immunoassay is commonly used to quantify serum proteins. Latex-particle enhancement of this type of assay has been primarily associated with increasing assay sensitivity. However, covalent coupling of an antibody to a latex particle can offer other advantages that are also pertinent in measurement of high concentrations of analytes. By using a common antibody with IgG as a model analyte, we describe the development of a nonenhanced and a latex-particle-enhanced turbidimetric assay for measuring serum IgG. Both assays show adequate analytical recovery and parallelism, and results compare well with those by rate nephelometry. The latex-enhanced assay has equivalent sensitivity, working range, and interassay precision, but much greater signal change and calibration stability than the nonenhanced assay. In addition, with latex particles, less antiserum is needed. Coupling antibodies to latex particles offers considerable advantages, even when an improved assay detection limit is not required.
Detection of human apolipoprotein E3, E2, and E4 genotypes by an allele-specific oligonucleotide-primed polymerase chain reaction assay: development and validation
Authors: Green E K , Bain S C , Day P J , Barnett A H , Charleson F , Jones A F , Walker M R
Published: January 2020

A polymerase chain reaction (PCR) assay has been developed and validated by using allele-specific oligonucleotide (ASO) primers to specifically amplify E3, E2, and E4 polymorphic sequences of the human apolipoprotein E (apo E) genes. Degenerate ASOs containing one or two additional 3' mismatches provided greater specificity than did ASOs containing a single mid-sequence or 3' allele-specific mismatch with plasmid pEB4 or genomic DNA as template. Optimal specificity and efficiency of amplification did not correlate with primer annealing conditions, whether determined theoretically or via oligo-melting experiments. Pre-cycling denaturation times and high cycling denaturation temperatures were also required for optimal amplification, presumably because of the high G:C content (75-85%) of apo E gene sequences. Conditions permissive for amplification and discrimination with plasmid DNA did not transpose favorably to amplification from human genomic DNA from peripheral blood leukocytes; the latter required nested primer reactions. These data may be valuable in predicting PCR assay conditions for other G:C-rich sequences containing polymorphic sequence differences. The assay described is both more accurate and rapid (24 h) than previously described methods for phenotyping or genotyping human apo E from blood specimens.
Improved fluorometric determination of malonaldehyde
Authors: Conti M , Morand P C , Levillain P , Lemonnier A
Published: January 2020

Lipoperoxidation is implicated in various pathological conditions. Malonaldehyde (MDA) is the most commonly used marker of this process. We propose simple modifications to Yagi's fluorometric assay for MDA determinations, to avoid long and tedious manipulations by eliminating the first precipitation and washing steps, analogous to HPLC methods, and to increase both the sensitivity and the specificity of the assay by measuring synchronous fluorescence. The proposed technique is easier, faster, and more sensitive than Yagi's method (Academic Press, 1982: Lipid peroxides in biology and medicine). The results obtained with the novel method correlate with those from the HPLC method described by Therasse and Lemonnier (J Chromatogr Biomed Appl 1987;413:237-41).
Drug analysis at the 1988 Olympic Winter Games in Calgary
Authors: Chan S C , Torok-Both G A , Billay D M , Przybylski P S , Gradeen C Y , Pap K M , Petruzelka J
Published: January 2020

A comprehensive drug testing program was carried out during the 16 days of the 1988 Olympic Winter Games in Calgary, Canada. State-of-the-art technology was applied, involving high-resolution gas chromatography, high-performance liquid chromatography, gas chromatography/mass spectrometry, fluorescence polarization immunoassay, and radioimmunoassay. Samples from selected athletes were screened for five drug classes: stimulants, narcotic analgesics, anabolic steroids, beta-blockers, and diuretics. In addition, samples were also screened for local anesthetics, corticosteroids, beta-human choriogonadotropin, and cannabinoids. During the 16-day event, 428 urine samples were processed and 3090 screening procedures performed. We describe the methods for analysis at the Olympic Games and present the results.
Characterization of sodium-selective optode membranes based on neutral ionophores and assay of sodium in plasma
Authors: Seiler K , Wang K , Bakker E , Morf W E , Rusterholz B , Spichiger U E , Simon W
Published: January 2020

Plasticized poly(vinyl chloride) membranes that incorporate a highly lipophilic sodium-selective neutral ionophore (ETH 4120) and novel H(+)-selective chromo-ionophores, lipophilic isologs of Nile Blue, are used in the competitive binding of Na+ and H+ for a reversible, optical determination of sodium activities in buffered solutions at different selected pH values. These optode membranes are used to assay total sodium concentrations in pH-buffered human blood plasma.
Carboxyterminal propeptide of type I procollagen in cerebrospinal fluid in childhood and in children with leukemia undergoing intrathecal treatment
Authors: Vainionpää L , Risteli L , Lanning M , Risteli J
Published: January 2020

We determined the reference interval for the carboxyterminal propeptide of type I procollagen (PICP), an indicator of the synthesis of type I collagen, in cerebrospinal fluid (CSF) by studying 32 infants and children, ages less than or equal to 15 years. The concentration of PICP is age dependent, with particularly high concentrations occurring in children younger than 1.5 years. In older children the concentration is stable (reference interval 20-92 micrograms/L). We also investigated the possibility that PICP in CSF could reflect local fibroproliferative changes in the arachnoid in a cohort of 42 children with acute lymphoblastic leukemia who were monitored by repeated sampling in connection with intrathecal therapy. Initially, there was no difference in PICP between the children with newly diagnosed leukemia and the controls. PICP concentrations were significantly higher (P less than 0.01) during intrathecal methotrexate therapy, with median values above the reference interval. Continuous corticosteroid treatment was associated with a significant decrease in PICP (P less than 0.02 and P less than 0.01, respectively, in two groups treated according to different protocols), close to the lower limit of the reference interval. Intrathecally administered methotrexate and systemic corticosteroid treatment are known to be associated with the development of arachnoiditis and with general repression of collagen synthesis, respectively. We conclude that PICP in CSF is a sensitive indicator of local fibroproliferation and ongoing collagen synthesis.
Analysis of ion behavior and potentials in a Na+ ion-selective membrane containing methyl monensin
Authors: Fujiwara M
Published: January 2020

Ion-selective electrodes (ISEs) are widely used in clinical examinations. The mechanisms of ISEs have been studied to develop a disposable ISE for "dry chemistry" analyses. The behavior of Na+ and Ag+ in an ion-selective polymer liquid membrane (ISM) containing methyl monensin (MMO), which is selective for Na+, has been clarified by x-ray microanalysis and measurement of electrical potential. The results indicate that Na+ is not transported across the membrane because of a stability of the Na(+)-MMO complex, whereas Ag+ (from the Ag/AgCl electrode) is transported with Cl- across the membrane by diffusion. The difference in potential, which follows a Nernst gradient, is generated by the formation of a double layer of electrons at the interface between the ISM and the electrolyte solution. These results are explainable by the stability of the trap in the polymer liquid membrane. MMO acts as a stable trap of Na+ and as a metastable site of Ag+ for movement.
Micro method of analysis for magnesium in myocardial biopsies
Authors: Millane T , Devarajan S , Fashola T , Bush R , Holt D W
Published: January 2020

We developed a single-stage technique involving a proteolytic enzyme (pepsin) for the solubilization of cardiac muscle to measure magnesium in tissue. This new method has been developed specifically for use with very small cardiac biopsy samples (less than 1 mg) obtained with modern myocardial biopsy forceps. Pepsin digestion of the tissue releases magnesium ions into solution and, after centrifugation and dilution with lanthanum chloride, the resulting supernate is suitable for analysis by atomic absorption spectrometry. Recovery of magnesium after a single digestion approaches 99%. In direct comparison studies of pepsin digestion and a traditional method involving nitric acid extraction, the pepsin digestion consistently yielded more magnesium. In contrast to traditional methods of tissue solubilization, pepsin digestion is well suited to extraction of magnesium from the small biopsy samples commonly presented for analysis in clinical practice.
Time-resolved immunofluorometric assay of 17 beta-hydroxysteroid dehydrogenase in plasma
Authors: Mäentausta O , Menjivar M , Vihko R
Published: January 2020

We describe a time-resolved immunofluorometric assay (TR-IFMA) for human 17 beta-hydroxysteroid dehydrogenase (17HSD) in which antibody-coated microtiter strip wells and europium chelate-labeled polyclonal antibodies are used. In preparing the label, a polyclonal antibody is affinity-purified and derivatized with diethylenetriamine-pentaacetic acid. With this derivative, five to eight europium ions can be combined with one antibody molecule without decreasing the antibody's immunoreactivity. The minimum detectable concentration of 17HSD is 0.13 microgram/L; the intra- and interassay CVs are less than 8% and less than 15%, respectively, for concentrations between 0.3 and 100 micrograms/L. There is no difference between the concentrations of 17HSD in plasma specimens taken during the proliferative and luteal phases of the menstrual cycle, the measured mean concentration being 0.22 microgram/L. We found no correlation between plasma 17HSD and progesterone concentrations. The plasma concentrations of 17HSD increase during pregnancy, the mean concentrations being 1.5, 4.4, and 12.5 micrograms/L, during the first, second, and third trimesters of pregnancy, respectively. In the specimens from 18 men, the mean concentration was 0.18 microgram/L. In six plasma specimens from patients with endometrial adenocarcinoma, the mean concentration was 0.20 micrograms/L. Pre-analytical aspects are important in the assay of 17HSD because of the lability of the enzyme protein. Preferably, blood should be sampled into EDTA-containing tubes, plasma should be separated within 15 min, and glycerol must be added without delay to a final volume of 200 mL/L.
The "ladder light chain" or "pseudo-oligoclonal" pattern in urinary immunofixation electrophoresis (IFE) studies: a distinctive IFE pattern and an explanatory hypothesis relating it to free polyclonal light chains
Authors: Harrison H H
Published: January 2020

I previously reported the occurrence of a pattern of multiple, equally spaced, low-concentration light chain bands in urine in immunofixation electrophoresis (IFE) studies (Clin Chem 1990;36:1526-7). Although the pattern varies somewhat among patients with regard to the number of bands, light chain type, and concentration, it is relatively invariant with regard to the location and spacing of three prominent kappa bands. The regular spacing of these urinary light chain bands imparts a "ladder rung" or "pseudo-oligoclonal" appearance. High-resolution, two-dimensional electrophoresis of these urine specimens reveals a regularly spaced, isomassic, restricted zonal distribution in the light chain region. These findings may be explained as the results of expression of superfamilies of related, polyclonal, free light chains, which co-migrate because of the limited variety of amino acid substitutions allowed by recombination constraints on light chain genes. This urinary IFE phenomenon is called the ladder light chain (LLC) pattern or pseudo-oligoclonal free light chain pattern to avoid implications of origin from specific clonal proliferation.
Restricted electrophoretic heterogeneity of immunoglobulin light chains in urine: a cause for confusion with Bence Jones protein
Authors: MacNamara E M , Aguzzi F , Petrini C , Higginson J , Gasparro C , Bergami M R , Bianchi G , Whicher J T
Published: January 2020

The detection of Bence Jones protein, an important part of the investigation of suspected myeloma, is most commonly done by agarose or cellulose nitrate electrophoresis followed by immunofixation. Bence Jones protein is recognized as single or multiple bands of one type of light chain. Unfortunately, improvements in sensitivity of these techniques (use of high-affinity antisera and higher resolution electrophoresis) frequently allow detection of multiple light chain bands in the urine of patients who do not have a B-cell dyscrasia. The bands are usually kappa, although they may be accompanied by lambda bands. This pattern may lead to the misdiagnosis of Bence Jones protein and oligoclonal light chain production in patients. Here we show that this pattern is produced by polyclonal light chains; it is present in the urine of all patients with a tubular proteinuria of any etiology and may be induced in healthy individuals by blocking their renal tubular protein reabsorption. Polyclonal light chains separate into monomers and dimers on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and into four major bands with many minor bands by isoelectric focusing. This difference in charge and possibly size results in the banding pattern seen on good-quality electrophoresis and immunofixation.
Statistical criteria for separate reference intervals: race and gender groups in creatine kinase
Authors: Harris E K , Wong E T , Shaw S T
Published: January 2020

Previously published data confirming differences in creatine kinase (EC 2.7.3.2) among various race and gender subgroups in the Los Angeles area have been re-examined with use of recently proposed statistical criteria for defining separate reference intervals. Results indicate that one criterion may be too lenient, whereas another is clearly too restrictive in suggesting the need for separate intervals. Further experience with other analytes in both large and small population samples would be helpful.
Monitoring Cyclosporine in Liver-Transplant Recipients: Effects of Clinical Status on the Performance of Two Monoclonal Antibody-Based Methods
Authors: Tredger Michael J , Gonde Christopher E , Williams Roger
Published: January 2020

The characteristics of the Abbott selective fluorescence polarization immunoassay for cyclosporine in whole blood (TDXSP) were compared with the Incstar radioimmunoassay by using kit standards (INCSP) or TDXSP standards (INCTDX) with samples from adult (48) and pediatric (24) liver-transplant recipients and a further 32 control specimens. The TDXSP assay was faster, less labor intensive, and more precise at higher concentrations; it gave results a median of 5 micrograms/L lower than INCSP values, irrespective of biliary T-tube status, patient's age, or severity of cholestasis in nine patients, comparisons being made for serum bilirubin more than eight times normal or less than 50 mumol/L. However, both assays overestimated INCTDX values by approximately 10%. There was a progressive increase in the ratio of TDXSP to both INCSP and INCTDX results in 57 samples stratified in relation to serum bilirubin concentrations. Cross-reactivity of TDXSP with cyclosporine metabolites M1 (23%) or M17 (12%) was greater than for INCSP (15% and 5%, respectively) or INCTDX (22% and 4%, respectively), but the converse was true with M21 (2% for TDXSP vs 4% and 8%, respectively). Some overestimation of cyclosporine whole-blood concentrations (relative to INCTDX results) may be detected with either the TDXSP (because of cross-reactivity with metabolites) or the INCSP assays (probably because of matrix effects). The similar magnitude of these overestimations suggests that alternative factors such as speed, precision, and cost may influence which method one selects for use in routine monitoring.
Monoclonal Fluorescence Polarization Immunoassay Evaluated for Monitoring Cyclosporine in Whole Blood After Kidney, Heart, and Liver Transplantation
Authors: Winkler M , Schumann G , Petersen D , Oellerich M , Wonigeit K
Published: January 2020

In a prospective study we evaluated a novel fluorescence polarization immunoassay (FPIA) for determining cyclosporine (CsA) in whole blood. FPIA uses a monoclonal antibody and is performed on the TDx (Abbott). The within-series (CV less than 2%) and between-days (CV less than 3.3%) precision of the assay was excellent. The results obtained by the monoclonal FPIA in samples from transplant patients (n = 100) averaged 31.9% and 20.2% higher than those by HPLC and a specific radioimmunoassay (INCStar), respectively. Results by all three methods correlated well. Follow-up studies during the early course after liver transplantation, however, suggested that high metabolite concentrations affect FPIA results. This is explained by previously described cross-reactions of the monoclonal antibody with some CsA metabolites. The FPIA results in samples of such patients should be interpreted cautiously.
Age-Correlated Modifications of Copper-Zinc Superoxide Dismutase and Glutathione-Related Enzyme Activities in Human Erythrocytes
Authors: Ceballos-Picot Iréne , Trivier Jean-Marc , Nicole Annie , Sinet Pierre-Marie , Thevenin Marc
Published: January 2020

To obtain a comprehensive profile of the erythrocyte antioxidant defense potential during aging, we investigated copper-zinc superoxide dismutase (CuZn-SOD), seleno-dependent glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-RD), and glutathione-S-transferase (GSH-S-T) activities in human erythrocytes from 167 apparently healthy subjects, ages one month to 63 years (102 females, 65 males). We found a negative correlation between age and activities of CuZn-SOD (r = 0.362, P less than 0.001), GSSG-RD (r = 0.549, P less than 0.001), and GSH-S-T (r = 0.575, P less than 0.001). In contrast, we found a positive correlation between age and GSH-Px activity (r = 0.401, P less than 0.001). To evaluate aging changes, we divided the subjects into five groups: Group 1 (newborn-age one year), Group 2 (1-11 years), Group 3 (11-25 years), Group 4 (25-40 years), and Group 5 (40-63 years). Significant age-related modifications in erythrocyte enzyme activities appeared in Group 3 for CuZn-SOD, GSSG-RD, and GSH-Px activity, whereas for GSH-S-T activity age-related modifications appeared in Group 2. We found no sex-related differences in erythrocyte CuZn-SOD, GSSG-RD, GSH-Px, and GSH-S-T activities.
Simultaneous Quadruple-Label Fluorometric Immunoassay of Thyroid-Stimulating Hormone, 17 α-Hydroxyprogesterone, Immunoreactive Trypsin, and Creatine Kinase MM Isoenzyme in Dried Blood Spots
Authors: Xu Y Y , Pettersson K , Blomberg K , Hemmilä I , Mikola H , Lövgren T
Published: January 2020

We describe a quadruple-label fluorometric immunoassay for simultaneously measuring four analytes: thyroid-stimulating hormone (TSH), 17 alpha-hydroxyprogesterone (17 alpha-OHP), immunoreactive trypsin (IRT), and creatine kinase MM (CK-MM). The assay is based on immunoreagents labeled with four different lanthanide ions (Eu3+, Tb3+, Sm3+, and Dy3+), on dissociative fluorescence enhancement applying the principle of co-fluorescence, and on time-resolved fluorometry. The monoclonal anti-alpha-TSH and anti-IRT antibodies and the polyclonal anti-CK-MM antibody were labeled with Eu3+, Sm3+, and Dy3+, respectively; 17 alpha-OHP was labeled with Tb3+. The assay was performed in microtitration strip wells coated with a mixture of monoclonal antibodies against beta-TSH, IRT, and CK-MM and a polyclonal goat anti-rabbit IgG for capture of the rabbit anti-17 alpha-OHP antibodies. After completion of the immunoreactions, the bound fractions of the lanthanides were dissociated into the co-fluorescence enhancement solution, creating highly fluorescent chelates. The four lanthanide-specific signals were subsequently measured in a time-resolved fluorometer. The detection limits of the assay were 0.1 mIU/L for TSH, 2 nmol/L for 17 alpha-OHP, 2 micrograms/L for IRT, and 4 U/L for CK-MM.
Homologous Radioimmunoassay of Human Osteocalcin
Authors: Bouillon R , Vanderschueren D , Van Herck E , Nielsen H K , Bex M , Heyns W , Van Baelen H
Published: January 2020

Osteocalcin or bone gamma-glutamic acid-containing protein (GLA protein) was isolated from human bone and used to develop a homologous radioimmunoassay of human osteocalcin. The effect of age on serum osteocalcin was studied in 380 normal children and adolescents and 330 normal adults. The mean (+/- SD) values in adults were higher in men [25 +/- 5 micrograms/L (4.3 +/- 0.8 nmol/L)] than in premenopausal women [20 +/- 6 micrograms/L (3.4 +/- 1.0 nmol/L); P < 0.01], but both were lower than in postmenopausal women [29 +/- 2 micrograms/L (5.0 nmol/L)]. The highest concentrations were seen in girls [ages 10-12 years: 99 +/- 38 micrograms/L (17.0 nmol/L)] and boys [ages 14-16 years: 107 +/- 57 micrograms/L (18.4 nmol/L)]. These mean values were substantially higher than those previously reported for results of heterologous osteocalcin radioimmunoassays but the correlation (r = 0.87, n = 77, P < 0.001) between both sets of results was excellent. In patients with metabolic bone diseases characterized by high or low bone turnover, the increase or decrease in serum osteocalcin observed was as expected. This homologous radioimmunoassay of human osteocalcin thus reflects bone turnover but reports serum concentrations higher than previously suspected.
Flow-Injection Analysis for Malondialdehyde in Plasma with the Thiobarbituric Acid Reaction
Authors: Ikatsu H , Nakajima T , Murayama N , Korenaga T
Published: January 2020

A simple, precise, and rapid method to measure plasma malondialdehyde (MDA) was developed by use of solvent extraction--flow-injection analysis. The reagent solution, containing thiobarbituric acid (TBA), 5 g/L in 100 mL/L phosphoric acid, and extraction solvent (methylisobutyl ketone, MIBK) were propelled with a double-plunger micropump at a flow rate of 0.3 mL/min, and 20 microL of sample was introduced into the reagent stream. After TBA-MDA reactant was extracted into MIBK, the organic phase was continuously separated by a successive phase-separation system equipped with two phase separators, and the absorbance of the TBA-MDA reactant was measured at 532 nm. This approach resulted in excellent sensitivity, a CV of < 1.5%, a good correlation with the conventional manual method, and a sampling frequency of 7 samples/h, suggesting that this semiautomated method is suitable for measuring plasma MDA.
Nonimmunological Assay of Urinary Albumin Based on Laser-Induced Fluorescence
Authors: Kessler M A , Hubmann M R , Dremel B A , Wolfbeis O S
Published: January 2020

We describe the first nonimmunological assay of albumin in urine with a detection limit of 1 mg/L. The method is simple, rapid, and accurate. It is based on the probe Albumin Blue 670, which becomes highly fluorescent on binding to albumin. An inexpensive diode laser was used as the light source for measurement of laser-induced fluorescence. The assay was coupled to a flow-injection analysis system capable of running 20 samples per hour. The working range was 1-100 mg/L, which covered albumin concentrations found in nonpathological urine and in urine with slightly increased albumin. This range makes prediction of nephropathy possible at an early stage. Other serum proteins and hemoglobin do not interfere. The coefficients of variation were < 4% and < 7% within one day and from day to day, respectively. A correlation coefficient of 0.990 (n = 100) was obtained for comparison with the Behring nephelometric assay.
Iohexol in Serum Determined by Capillary Electrophoresis
Authors: Shihabi Z K , Constantinescu M S
Published: January 2020

Iohexol, a nonionic compound used as a contrast medium for angiography and as a measure of the glomerular filtration rate, was quantified in serum by capillary electrophoresis. Comparable results were obtained for serum samples deproteinized with acetonitrile or analyzed directly after 50-fold dilution with borate buffer. Serum samples were electrophoresed for 2.6 min at 12 kV in a borate buffer with detection at 254 nm and with 3-isobutyl-1-methylxanthine as internal standard. Acetonitrile deproteinization gave a greater sensitivity than did sample dilution. Between-run CVs were between 4.7% and 6.7%, and within-run CVs were between 2.5 and 3.2%. Analytical recoveries were 95-105%. Results of the method compared well with those by high-performance liquid chromatography (slope 0.96, intercept 0.005 g/L). This method demonstrates the potential of capillary electrophoresis for rapid and simple quantification of small molecules.
Development of Monoclonal Antibodies for an Assay of Cardiac Troponin-I and Preliminary Results in Suspected Cases of Myocardial Infarction
Authors: Bodor G S , Porter S , Landt Y , Ladenson J H
Published: January 2020

To improve the specificity of biochemical markers of myocardial infarction (MI), we have developed a double monoclonal "sandwich" enzyme immunoassay to measure cardiac troponin-I (cTnI) in serum. We produced eight IgG monoclonal antibodies against human cardiac troponin-I (cTnI) and tested them against human and animal (canine, bovine, and rabbit) troponins. Five antibodies were cardiac-specific; none of the antibodies were species-specific. Two of the five cTnI-specific monoclonal antibodies were utilized in an immunoassay. Standards were made by adding purified human cTnI to affinity-stripped cTnI-free human sera to cover the range 0-100 micrograms/L for cTnI. The dose-response curve was nonlinear but reproducible. Total assay imprecision (CV) varied between 11% and 21%. The upper limit of the reference range (nonparametric 95% interval) was established as 3.1 micrograms/L by measuring cTnI concentration in sera of 159 hospitalized patients without evidence of cardiac disease. Purified human skeletal TnI up to 10,000 micrograms/L did not affect the assay (calculated cross-reactivity < 0.1%). Diagnostic sensitivities of creatine kinase MB isoenzyme (CK-MB) and cTnI were evaluated retrospectively in 49 consecutive patients with proven MI. In the 30 patients for whom sufficient information was available to establish an accurate time course, CK-MB was more sensitive during the first 4 h after the onset of chest pain, but thereafter the sensitivities were similar up to 48 h. However, cTnI is more cardiac-specific than is CK-MB and remains increased longer than does CK-MB.
Ratio of Creatine Kinase 2 Mass Concentration to Total Creatine Kinase Activity not Altered by Heavy Physical Exercise
Authors: Ordóñez-Llanos J , Serra-Grima J R , Mercé-Muntañola J , González-Sastre F
Published: January 2020

Serum creatine kinase isoenzyme 2 concentrations (CK 2 mass) were measured in marathon runners during training and 1 and 2 days after a race and compared with values from 36 acute myocardial infarction (AMI) patients whose total CK and (or) CK 2 activities were similar to those of runners in the basal state. During training, runners had CK and CK 2 activities 53% and 43% above reference values, respectively, and 36% had CK 2 activity > 5% of total CK. Nine runners (26%) showed CK 2 mass values > 6 micrograms/L but < or = 10 micrograms/L; 35 of the AMI subjects, despite having CK activities similar to those of runners, had values > 10 micrograms/L. The ratio of CK 2 mass to total CK activity was significantly (P < 0.0002) different between sexes for runners. At 1 and 2 days after racing, 100% of CK and CK 2 activities and 71% and 57% of the percentages of CK 2 activity, respectively, were abnormally high; 57% and 43% of CK 2 mass values were > 10 micrograms/L, being comparable with those observed for the AMI group. Basal CK 2 mass values of the runners appeared only slightly higher than that for sedentary subjects, but after exercise half the subjects presented increased values similar to those observed for AMI subjects. The ratio of CK 2 mass to total CK activity appeared unaltered by exercise in all but one of the samples assayed, indicating its utility in evaluating CK 2 mass increases originating in skeletal muscle.
Effects of Angiotensinase Inhibitors on Plasma Protein Binding and IC50 Determinations of Renin Inhibitors
Authors: Dayton B D , Stein H H , Cohen J , Baker W R , Boyd S A , Condon S L , Donner B G , Fung A K , Luly J R , Rosenberg S H
Published: January 2020

To establish whether the use of proteinase inhibitors in the routine determination of in vitro plasma renin activity overestimates the potency of renin inhibitors in vivo, we examined the effects of phenylmethylsulfonyl fluoride and 8-hydroxyquinoline sulfate on the binding to plasma proteins and the respective IC50 values (50% inhibiting concentrations) of three renin inhibitors. All three renin inhibitors, A-64662, A-65317, and A-74273, bound (> 60%) to plasma proteins at both pH 6.0 and 7.4, with slightly greater binding at pH 7.4. Phenylmethylsulfonyl fluoride (1.45 mmol/L) had no significant effect on the protein binding at either pH 6.0 or 7.4; 8-hydroxyquinoline sulfate (3.4 mmol/L) caused a modest dissociation (10-30%) of the renin inhibitors from plasma proteins at both pH values; and the effects of both proteinase inhibitors together were similar to those of 8-hydroxyquinoline alone. At pH 7.4, phenylmethylsulfonyl fluoride increased the potencies of the three renin inhibitors slightly (< or = 43%), whereas IC50 values determined in the presence of 8-hydroxyquinoline decreased by 1.5- to 3.7-fold. The greatest increase in potency occurred with the most hydrophilic compound, and with both angiotensinase inhibitors the effect was no greater than that of 8-hydroxyquinoline alone. The results show that any dissociation of the hypotensive activity measured in vivo from the plasma renin activity measured in vitro is not simply an artifact in the plasma renin activity assay stemming from the use of these angiotensinase inhibitors, especially if only phenylmethylsulfonyl fluoride is used.
Pre-Beta High-Density Lipoprotein Determined by Immunoblotting with Chemiluminescent Detection
Authors: O'Kane M J , Wisdom G B , McEneny J , McFerran N V , Trimble E R
Published: January 2020

We describe a novel assay of pre-beta high-density lipoprotein (HDL), a unique apolipoprotein A-I (apo A-I)-containing lipoprotein particle. The pre-beta and alpha lipoproteins are separated by electrophoresis in agarose and transferred onto a membrane by capillary blotting. The membrane blot is sequentially incubated with sheep anti-human apo A-I antiserum and then with a conjugate of rabbit anti-sheep immunoglobulin and horseradish peroxidase. Chemiluminescence formed by the peroxidase-catalyzed oxidation of luminol in the presence of an enhancer is captured on photographic film, and the pre-beta HDL band is quantified by transmission densitometry. The assay is calibrated with standards prepared from a reference serum diluted in 9 mol/L urea. Within-batch precision (CV) at pre-beta HDL concentrations of 22.1 and 44.3 mg/L was 7% and 4.9% respectively. Pre-beta HDL contained 1.6% (0.65-2.6%, mean and range) of total serum apo A-I in 30 normolipidemic subjects.
Abbott IMx and Serono MAIAclone Assays Compared for Lutropin Determinations in Urine
Authors: Weiss P , Zech H , Schönholzer H P , Fritzsche H
Published: January 2020

The urinary lutropin (luteinizing hormone; LH) profiles of 20 patients were determined in 10 consecutive urine samples (n = 200) with a manual technique (MAIAclone, Serono; y) and a fully automated (Abbott IMx; x) technique. For 132 single determinations within the range of detection, the regression line was y = 1.39 x - 0.07 (r = 0.85). Retrospective analysis of the LH profiles obtained by the IMx yielded the classification "not pregnant" (negative) in 14 cases, of which 13 were correctly classified; 6 profiles were classified as "pregnant" (positive), which correlated with the clinical findings in 4 cases. Nine of the LH profiles analyzed with the MAIAclone method were classified as negative, which was correct in seven cases. Retrospective analysis of 11 LH profiles established with the MAIAclone assay gave positive results, which agreed with the clinical outcome in three cases. The diagnostic sensitivity of the IMx and MAIAclone methods was 87% and 47%, respectively; diagnostic specificity was 80% and 60%, respectively. In smaller series (< or = 40 samples), the IMx method has considerable advantages over the MAIAclone method in cost and convenience, because standard curves can be stored and assays can be performed as single determinations.