bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurement of urinary retinol-binding protein by enzyme-linked immunosorbent assay, and its application to detection of tubular proteinuria.
Authors: Topping M D , Forster H W , Dolman C , Luczynska C M , Bernard A M
Published: January 2020

An enzyme-linked immunosorbent assay (ELISA) for urinary retinol-binding protein (RBP) has been developed and compared with urinary beta 2-microglobulin for the detection of tubular proteinuria. The assay has a working range of 10 to 250 micrograms of RBP per liter of urine. The within-assay CV was 3.2-7.1%, the between-assay CV 12.5%. A control population of 118 male subjects gave a geometric mean urinary RBP concentration of 7.7 micrograms per millimole of creatinine and a 95th centile of 22 micrograms per millimole of creatinine. Comparison of urinary RBP and beta 2-microglobulin concentrations in 80 control subjects and 117 subjects exposed to cadmium fumes gave correlations of r = 0.59 and 0.91, respectively. Of the 117 subjects exposed to cadmium fumes, 103 gave both RBP and beta 2-microglobulin concentrations on the same side of the upper 95th centile values of 22 and 38 micrograms per millimole of creatinine for RBP and beta 2-microglobulin respectively (Chi-square analysis p less than 0.001), demonstrating that RBP and beta 2-microglobulin detect tubular proteinuria with equal sensitivity and specificity. ELISA and an established latex immunoassay gave well-correlated results.
Liquid-chromatographic determination of vitamin K1 in plasma, with fluorometric detection.
Authors: Haroon Y , Bacon D S , Sadowski J A
Published: January 2020

This assay for phylloquinone (vitamin K1) in plasma requires a single liquid-chromatographic step. Much smaller volumes of plasma (0.5-1.0 mL) are required than in previous assays. Before liquid chromatography, we purified crude lipid extracts by conventional chromatography on silica, then extracted the lipid fraction by dissolving it in an acidic mixture of hexane/acetonitrile (1/4 by vol) containing 70 mmol of zinc chloride per liter. The vitamin K1 was selectively extracted into acetonitrile after being converted to vitamin K1 hydroquinone by addition of zinc metal. This procedure removes greater than 99% of contaminating lipids. We injected the lipid extract directly onto a reversed-phase column after re-converting the vitamin K1 hydroquinone to vitamin K1. Vitamin K1 was quantified by comparison with the internal standard (dihydro-vitamin K1) and detected fluorometrically after post-column "on-line" reduction to the hydroquinone with zinc metal. The lower limit of detection for vitamin K1 in the final reversed-phase system was about 0.05 microgram/L plasma; CVs for replicates were less than 10%. The mean concentration of vitamin K1 in plasma from 22 healthy fasting adults was 0.55 (range 0.09-2.12) micrograms/L.
A multilayer element for determining hemoglobin in whole blood: principles and analytical performance.
Authors: Burdick B A , Hilborn D A , Wu T W
Published: January 2020

We described the structure and analytical performance of a thin-film element for determining total hemoglobin in whole blood. The element can be used with the Kodak Ektachem DT60 analyzer. The method, analogous to the standard Drabkin's method, provides accurate and precise results on 10-microL samples of undiluted whole blood. Dynamic range is linear from 0 to 200 g/L, and the method is relatively free from interference.
Activity concentration and mass concentration (monoclonal antibody immunoenzymometric method) compared for creatine kinase MB isoenzyme in serum.
Authors: Murthy V V , Karmen A
Published: January 2020

Results of the "Tandem-E CKMB" immunoenzymometric procedure (y) for creatine kinase (CK; EC 2.9.3.2) were compared with electrophoresis (x) for 160 serum samples from patients suspected of having sustained myocardial infarctions. The results correlated well: y, microgram/L (Tandem assay) = 1.3x-6.3 U/L(electrophoresis) (r = 0.95). CK-MB mass measurement was more stable than enzyme activity after storage and appeared to be more sensitive. Sera from 86 other people, which had no detectable CK-MB upon electrophoresis, gave a mean CK-MB value of 1.1 microgram/L (SD 1.3, range 0-8) with the Tandem assay. To determine whether these low values represented actual isoenzyme, we tested for possible interference by heterophile antibodies in the patients' sera by preincubating the samples with mouse serum before the Tandem assay. The mouse serum did not interfere with the assay of sera that had substantial quantities of CK-MB by electrophoresis. However, in five of six samples that were negative by electrophoresis, the CK-MB values were substantially smaller, indicating that the values measured were false-positives caused by the presence of heterophile antibodies directed against mouse proteins, an interference that could be eliminated by pretreatment with mouse serum.
Clinical and laboratory evaluation of a radioimmunoassay kit for measuring the mid-molecule fraction of parathyrin.
Authors: Haver V M , Rinker A D , Ko B M , Tietz N W
Published: January 2020

We report the results of a laboratory and clinical evaluation of a commercial kit procedure (Immuno Nuclear Corp.) for measuring the mid-region of the parathyrin molecule. The estimated dose at 50% binding averaged 130 pmol/L, and the minimum detectable concentration was 14 pmol/L. The within-assay CV was less than or equal to 6.6%, the between-assay CV less than or equal to 12.5%. Relative analytical recoveries of various parathyrin fragments averaged 93% (intact, amino acid residues 1-84), 100% (midregion, 44-68), less than 0.01% (N-terminal, 1-34), and less than 0.01% (C-terminal, 69-84). Correlation of results obtained for 59 patients' samples with a radioimmunoassay for midregion/C-terminal parathyrin performed by the Mayo Medical Laboratory yielded the equation INC = 1.16 (Mayo)-1.94 pmol/L (r = 0.971). Clinical evaluation indicates that the INC results for parathyrin correlate with the diagnoses of patients at least as well as the results obtained with the Mayo assay; in some cases, the INC procedure better distinguishes hyperfunction from normal parathyroid function. The INC procedure can be easily performed in hospital laboratories, with a 4-h turnaround time for results.
Pronounced increases in the concentration of an ovarian tumor marker, CA-125, in serum of a healthy subject during menstruation.
Authors: Mastropaolo W , Fernandez Z , Miller E L
Published: January 2020

CA-125 is a glycoprotein associated with various ovarian tumors. A commercial radioimmunoassay involving a monoclonal antibody is available for it. In our laboratory, a normal-value study was conducted as part of a routine evaluation of this assay. One healthy subject had a serum CA-125 concentration greater than 300 kU/L, more than eightfold the upper limit of normal (35 kU/L). This increase, which coincided with the onset of the menstrual period, subsided to within the normal range by the end of the menstrual cycle. The half-life of CA-125, calculated from this decrease, was 6.4 days. Similar observations were made in the same subject over several menstrual cycles. Results of clinical and ultrasound examinations of the subject for ovarian tumors were negative. No clinical evidence of malignancy was present eight months after the initial discovery of an increased CA-125. None of the other 39 healthy subjects had a CA-125 value greater than 51 kU/L. Five of these subjects had CA-125 determined several times during their menstrual cycles; none exhibited pronounced variations in CA-125 concentrations. Evidently CA-125 can be extremely increased in a healthy woman, and possible effects of the menstrual period on serum CA-125 concentrations should be considered in pre-menopausal patients.
Affinity chromatographic separation of alpha-fetoprotein variants: development of a mini-column procedure, and application to cancer patients.
Authors: Chan D W , Miao Y C
Published: January 2020

This is an affinity chromatographic procedure for separating variants of alpha-fetoprotein in 0.1 mL of serum on a disposable mini-column containing 0.9 mL of concanavalin A-Sepharose, at pH 6.4 and with a flow rate of 5 mL/h. The binding capacity of the column is 52 micrograms. Analytical recovery was 90%. Between-run CVs varied from 2.0% for samples with a high percentage of concanavalin A nonreactive fraction to 13% for samples with a low percentage. The mean proportion of nonreactive alpha-fetoprotein was 13.4% for patients with hepatocellular carcinoma, 62.2% for testicular carcinoma, and 8.9% for nonmalignant liver diseases. This suggests that the alpha-fetoprotein variants could be used to distinguish hepatocellular carcinoma from other carcinomas. Serial determination of the concanavalin A nonreactive fraction in such patients showed a relatively constant percentage. This indicates that monitoring variants in patients with hepatocellular carcinoma throughout their clinical course may not provide useful information additional to that provided by the values for total serum alpha-fetoprotein.
Nephelometry of the kappa/lambda light-chain ratio in serum of normal and diseased children.
Authors: Renckens A L , Jansen M J , van Munster P J , Weemaes C M , Bakkeren J A
Published: January 2020

We determined, immunonephelometrically, the ratio between the two types of light chains of immunoglobulins, kappa and lambda, in serum of 94 children, ages 0.4 to 14 years, with no manifest immunological disorders. Children with an abnormal protein pattern by immunoelectrophoresis show other values for this ratio than do children in this reference group. We also determined the ratios for children with IgA deficiency (I), juvenile rheumatic arthritis (II), and acute lymphoblastic leukemia (III). Children with I show the same kappa/lambda ratios as for the children in the reference group. Children with II also show the same mean kappa/lambda ratios, but a significantly wider range of ratios. In children with III, the ratio during chemotherapy is slightly depressed, significantly lower than after cessation of therapy. All groups--healthy children and patients--show an increase in kappa-chain-bearing immunoglobulins with age, but the concentration of lambda-chain-bearing immunoglobulins remains relatively constant.
SimulTRAC simultaneous radioimmunoassay of thyrotropin and free thyroxin evaluated.
Authors: Gow S M , Caldwell G , Toft A D , Beckett G J
Published: January 2020

We assessed the analytical and diagnostic performance of a dual-isotope RIA for thyrotropin (TSH) and free thyroxin (FT4) in serum. The mean interassay CVs for these analytes were 7.9% and 5.0%, respectively. The mean minimum detection limit for TSH was 0.25 milli-int. unit/L, the mean analytical recovery 110%. There was good agreement with values for TSH and FT4 measured by alternative RIA procedures. Euthyroid patients were well distinguished from those with overt thyroid disease, although there was a small overlap in the case of TSH. Combining the two results better discriminated these categories and identified those patients with subclinical thyroid disease who had abnormal TSH concentrations but FT4 concentrations within reference limits. Euthyroid women taking estrogen-containing oral contraceptives had normal results for TSH and FT4, as did most pregnant women studied. During the third trimester of pregnancy, TSH concentrations of women with low FT4 concentrations were within reference limits. Similarly, euthyroidism was confirmed in patients with low FT4 due to nonthyroidal illness by the simultaneous finding of a normal TSH concentration.
Reversed-phase liquid-chromatographic determination of purine compounds in serum applied to studies of hypouricemia.
Authors: Kojima T , Nishina T , Kitamura M , Kamatani N , Nishioka K
Published: January 2020

With this rapid (20 min), sensitive (0.1 mumol/L), and reproducible reversed-phase "high-performance" liquid-chromatographic (HPLC) method hypoxanthine, xanthine, uric acid, allopurinol (4-hydroxypyrazolo[3,4-d]pyrimidine), and oxypurinol(4,6-dihydroxypyrazolo[3,4-d]pyrimidine) concentrations can be determined simultaneously in de-proteinized serum. We use an Amicon MPS-1 centrifugal ultrafiltration device, then inject the de-proteinized ultrafiltrate into a Toyosoda ODS-120A HPLC column and elute with a mobile phase consisting of potassium phosphate buffer (40 mmol/L, pH 2.2) containing 20 mL of methanol per liter. We used this method to investigate purine metabolism in 10 cases of hypouricemia (uric acid concentration in serum less than 60 mumol/L).
Analytical variance and definition of a reference change as a function of calcium concentration.
Authors: Caudill S P , Boone D J
Published: January 2020

Using data from the Centers for Disease Control (CDC) Proficiency Testing (PT) Surveys, we obtained estimates of repeatability (intralaboratory variability between results on the same material) and reproducibility (interlaboratory variability between results on the same material) for the Technicon SMA 6 (or 12/60) and SMAC 1 (or II) systems used with cresolphthalein complexone methodology to measure serum calcium. The two systems were comparable in terms of short-term (within-day) repeatability, long-term (three to six months) repeatability, short-term (one to two weeks) reproducibility, and long-term (three to six months) reproducibility. The long-term repeatability was essentially the same as the long-term reproducibility. Short-term repeatability, long-term repeatability, and long-term reproducibility increased linearly with increased calcium concentration over the range 1.75 to 2.95 mmol/L; short-term reproducibility showed no significant change over this range. The effect of analytical variance on the definition of a reference change in semiannual calcium measurements was demonstrated.
Measurement of total bilirubin by use of bilirubin oxidase.
Authors: Perry B , Doumas B T , Buffone G , Glick M , Ou C N , Ryder K
Published: January 2020

We used an enzymatic method for measuring total bilirubin in serum. Results by this method varied linearly with bilirubin concentrations to at least 300 mg/L. The day-to-day precision (CV) of the method ranged from less than 1% to about 11% at bilirubin concentrations of 183 and 12 mg/L, respectively. Commonly used anticoagulants, serum preparation materials, and selected drugs had no effect on the apparent bilirubin concentration, but turbidity caused a slight increase and hemoglobin concentrations of 2 g/L resulted in lower values, by as much as 17 mg/L at a bilirubin concentration of 95 mg/L. Patients' results obtained with this enzymatic method were slightly lower than those obtained with methods based on the Jendrassik-Grof principle. The largest differences, seen in samples with high "direct" bilirubin concentrations, can be decreased by measuring the absorbance at 425 nm instead of at 465 nm as recommended by the supplier of the bilirubin oxidase method.
A possible etiological role for ascorbate in calculi formation.
Authors: Chalmers A H , Cowley D M , Brown J M
Published: January 2020

Studies of recurrent stone formers indicated that they have significantly increased urinary oxalate and decreased ascorbate excretions. Results of oral and intravenous administration of ascorbate indicate an enhanced production of oxalate from ascorbate in recurrent calcium stone formers as compared with normal persons and that most of this oxalate is generated in the gut.
Detection of serum proteins by high-pressure gel-permeation chromatography with low-angle laser light scattering, compared with analytical ultracentrifugation.
Authors: Flapper W , van den Oetelaar P J , Breed C P , Steenbergen J , Hoenders H J
Published: January 2020

Human sera were subjected to analytical ultracentrifugation and "high-pressure" gel-permeation chromatography on a system of combined TSK Gel G5000 PW and G3000 SW columns. The chromatographic method produced remarkably superior resolution of the proteins, especially those exceeding 100 000 Da. We calculated the molecular masses of eluted fractions on the basis of their detection by low-angle laser light scattering and their differential refractive index. We discuss the results in relation to the clinical data.
Homogeneous substrate-labeled fluorescent immunoassay for disopyramide in human serum: semi- and fully automated procedures.
Authors: Johnson P K , Messenger L J
Published: January 2020

In this immunoassay for disopyramide in serum, to form the label, disopyramide is covalently attached to a fluorogenic enzyme substrate, 7-beta-galactosylcoumarin-3-carboxylic acid, which is nonfluorescent under the conditions of the assay. Hydrolysis, catalyzed by beta-galactosidase, yields a fluorescent product. As a result of competitive protein binding reactions between drug and label for limited antibody binding sites, this fluorescence is proportional to disopyramide concentration. The assay is sensitive to less than 0.5 mg of disopyramide per liter. Results obtained with either the semi-automated procedure (Ames TDA) or the fully automated (Optimate) procedure correlated well with those obtained by liquid chromatography (r = 0.98 and 0.99). For commercial controls containing various concentrations of the drug, the respective coefficients of correlation were 1.00 and 0.99 for the TDA and Optimate procedures. Within-run CVs for the two procedures were less than or equal to 5.1%, overall CVs less than or equal to 6.5%.
The Coomassie Brilliant Blue method underestimates drug-induced tubular proteinuria.
Authors: Goren M P , Li J T
Published: January 2020

The sensitivity of the Coomassie Brilliant Blue (CBB) procedure differs for different proteins. To investigate the impact of this variability on clinical measurements of proteinuria, we collected urine specimens from patients with glomerular disease or drug-induced tubular nephrotoxicity and compared urinary protein concentrations as determined by the CBB method with those obtained by the biuret method. The CBB method underestimated the protein concentration predicted by the biuret method by as much as eightfold in specimens with low proportions of albumin. Determinations of protein by the CBB method in serially diluted urine specimens from patients with tubular damage also deviated from linearity by as much as twofold for protein concentrations less than 1 g/L. We conclude that CBB binding is relatively insensitive for measuring proteins specifically associated with tubular damage and hence may be inappropriate for clinical use.
Creatinine clearance: enzymatic vs Jaffé determinations of creatinine in plasma and urine.
Authors: Apple F , Bandt C , Prosch A , Erlandson G , Holmstrom V , Scholen J , Googins M
Published: January 2020

We measured creatinine in plasma and urine samples from 17 normal subjects and 10 renally impaired subjects by four different methods: two enzymatic--Ektachem iminohydrolase and Boehringer Mannheim amidohydrolase--and two Jaffé reaction based--Beckman Astra 8 and Technicon AutoAnalyzer I. Creatinine clearances, standardized for body surface area, were also calculated. In both groups of subjects plasma creatinine values were significantly (p less than 0.05) lower, by 3 to 4 mg/L, when measured enzymatically than when measured by the Jaffé reaction. Additionally, creatinine clearances were significantly (p less than 0.05) greater by at least 30 mL/min when calculated from enzymatically measured creatinine values vs Jaffé method values for creatinine. The benefits of lack of interference with enzymatically measured creatinine concentrations and clearances should be assessed in relation to the lack of agreement with long-established (Jaffé) methods for determining creatinine (and inulin) clearances.
Use of 125I-labeled-histamine-cyclosporin C for monitoring serum cyclosporine concentrations in transplantation patients.
Authors: Wong P Y , Cheung M , Yip T K , Mee A V
Published: January 2020

We synthesized an 125I-labeled-histamine-cyclosporin C tracer, to obviate the use of tritiated tracer in radioimmunoassay of cyclosporine. With this tracer, the assay results varied linearly with concentration up to at least 800 micrograms/L. The within-assay CV was 6.6% at 39 micrograms/L, 4.2% at 100 micrograms/L, and 7.0% at 300 micrograms/L (n = 15). The between-assay CV was 10.0, 6.4, and 7.8% for the same respective concentrations. Comparison with an assay involving tritiated tracer (x) showed good agreement of results: y = 3.81 + 0.927x (r = 0.975, n = 604). Analytical recovery ranged from 100 to 106%. We also compared another commercially available radioiodinated tracer ("125Iodocyclosporin"; Immunonuclear Corp.). Our tracer appeared to be more specific for cyclosporine, as determined by assaying chromatographic fractions of bile extract from a patient being treated with cyclosporine. Results with use of our tracer compared favorably with those obtained with the tritiated tracer, and our assay has the advantages of gamma counting vs liquid-scintillation counting.
Mass-spectrometric assay of tocainide in serum.
Authors: Jones C W
Published: January 2020

This mass-spectrometric method for assaying tocainide in serum is specific, reproducible, and sensitive, and sample preparation is rapid. The drug is isolated from serum by liquid-solid extraction on a Baker C18 disposable column. Underivatized drug is separated by elution on a 0.20 mm X 25 m fused-silica capillary column coated with 5% phenylmethylsilicone, then quantified by mass-selective detection (selected ion monitoring). Sample size is 1 mL of serum, but smaller volumes may be used. Mean analytical recovery of the drug from the disposable columns is 75%. Commonly used antiarrhythmics, sedatives, or hypnotics do not interfere. The run-to-run CV at 3.7 mg/L is 4.0%, 3.0% at 10.5 mg/L.
New immunoenzymatic assay for human thyrotropin compared with two radioimmunoassays.
Authors: Pekary A E , Turner L F , Hershman J M
Published: January 2020

We have evaluated a new immunoenzymatic assay for human thyrotropin involving three monoclonal antibodies (Abbott HTSH EIA) and compared the results with those of two conventional nonequilibrium double-antibody radioimmunoassay (RIA) methods: Clinical Assays' RIA and a research RIA (J Clin Endocrinol Metab 1975; 41:676). Mean values for thyrotropin in 100 euthyroid serum samples were similar in the Abbott and Clinical Assays methods, but both sets were significantly higher than those by the research RIA. By all methods, values for hypothyroid patients were clearly higher than values for euthyroid subjects. Results for hyperthyroid and euthyroid subjects were resolved slightly better with the research RIA than with the Abbott kit. The new Abbott assay was far more sensitive than either the Clinical Assays RIA or our research RIA. The correlation of results of the Abbott assay with those of the Clinical Assays and the research RIA exceeded 90% for samples from hypothyroid patients. The Abbott assay replaces radioisotope counting with spectrophotometric detection.
Bilirubin interference with determination of uric acid, cholesterol, and triglycerides in commercial peroxidase-coupled assays, and the effect of ferrocyanide.
Authors: Spain M A , Wu A H
Published: January 2020

We examined the effect of bilirubin on measurement of serum uric acid, cholesterol, and triglyceride in the BMD-8700, Hitachi 705, Multistat III, CentrifiChem, Cobas-Bio, Ektachem 400, aca II, and RA-1000 analyzers. In general, bilirubin interferes more in peroxidase-coupled assays than in either direct methods or those coupled to NAD(P)+. The degree of interference, which can be spectral or chemical, varies and depends on the chromogen and wavelength used. We present data to show the origin and extent of the interference by bilirubin in these systems and demonstrate how ferrocyanide can provide a satisfactory resolution in some cases.
Different diuresis-dependent excretions of urinary enzymes: N-acetyl-beta-D-glucosaminidase, alanine aminopeptidase, alkaline phosphatase, and gamma-glutamyltransferase.
Authors: Jung K , Schulze G , Reinholdt C
Published: January 2020

We studied how much of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) and of the brush-border enzymes alanine aminopeptidase (EC 3.4.11.2), alkaline phosphatase (EC 3.1.3.1), and gamma-glutamyltransferase (EC 2.3.2.2) was excreted in urine over 8 h after a high intake of fluid (22 mL per kilogram of body weight). The hourly excretion of all four enzymes increased with the increasing urine flow rate. The excretion rate of the brush-border enzymes was more markedly influenced than that of N-acetyl-beta-D-glucosaminidase. By relating the enzyme excretion to urinary creatinine we could reduce the variability of brush-border enzyme output and could completely compensate for the effect of diuresis on the excretion of N-acetyl-beta-D-glucosaminidase.
An enzymatic method for calibration of serum lipase assays.
Authors: Hoffmann G E , Neumann U , Hoffmann S , Kaspar P , Weiss L
Published: January 2020

An enzymatic method for the calibration of a turbidimetric lipase assay is described, based on measurement of free fatty acids liberated by the action of lipase. The substrate of the turbidimetric assay is a colipase-containing triolein emulsion. For determination of the free fatty acids a commercial test kit including acyl-CoA synthetase, acyl-CoA oxidase, and peroxidase is used. Intra- and interassay imprecision (CV) is about 5% at above-normal lipase activities, about 10% at normal values. Temperature coefficients are 1.24 and 1.45, respectively, for measurements at 30 and 37 degrees C vs 25 degrees C.
Salivary amylase and pancreatic enzymes in serum after total body irradiation.
Authors: Junglee D , Katrak A , Mohiuddin J , Blacklock H , Prentice H G , Dandona P
Published: January 2020

Amylase increases in serum after total body irradiation (TBI). To investigate whether this increase is ascribable to pancreatic or salivary amylase, or both, we measured the post-irradiation activity concentration of the three major pancreatic enzymes (trypsin, amylase, and lipase) and of salivary-type amylase in serum from six leukemic patients given TBI before bone-marrow transplantation. Salivary amylase increased by as much as 50-fold after TBI, peaking on the first day after the irradiation. The increase was associated with the development of clinical parotitis. Pancreatic trypsin, total amylase, and lipase increased inconsistently and to a much smaller degree. There were no associated clinical features of pancreatitis. Our data show that, at the dose of radiation given to our patients, there is no danger of pancreatitis as a complication, but parotitis is a consistent development after TBI.
Direct measurement of creatine kinase-MB activity in serum after extraction with a monoclonal antibody specific to the MB isoenzyme.
Authors: Vaidya H C , Maynard Y , Dietzler D N , Ladenson J H
Published: January 2020

Fusion of splenocytes from A/J mice immunized by creatine kinase (EC 2.7.3.2)-MB isoenzyme (CK-MB) with SP2/0-Ag14 myeloma cell line generated hybridomas producing monoclonal antibodies specific to CK-MB. One of these monoclonal antibodies ("Conan-MB") was used to develop a direct assay for CK-MB activity. In the assay, serum is incubated for 30 min at room temperature with "Conan-MB" immobilized on latex beads. The beads are then washed, and CK-MB activity bound to the antibody is measured after incubation with CK enzyme reagent for 30 min at 37 degrees C. Results with the assay correlated well (r = 0.997) with those for CK-MB concentration as measured by a two-site immunoassay. Neither CK-MM, CK-BB, mitochondrial CK, nor a hemolysate of erythrocytes interfered. Use of this unique monoclonal antibody allows rapid, precise, and direct determination of CK-MB activity.
Simple method for preparing the cellular intermediate EAC14, and its use for estimation of the second component of complement.
Authors: Asghar S S , Out J , van der Helm H J
Published: January 2020

Sensitized erythrocytes carrying the first and fourth components of complement (EAC14) were prepared by incubating optimally sensitized sheep erythrocytes with normal serum appropriately diluted in Mg2+-free diethylbarbiturate buffer containing Ca2+. EAC14 cells so prepared were found to be suitable for use in estimating the second component of complement (C2) in human serum, and the method is described here.
Drug use in the neonate: interrelationships of pharmacokinetics, toxicity, and biochemical maturity.
Authors: Warner A
Published: January 2020

With advances in technology, increasing numbers of premature and very ill neonates are surviving and being referred for treatment to neonatal intensive-care units. A major feature of the treatment they receive is therapy with drugs. However, because of relatively limited information available in the area of neonatal pharmacokinetics, the few drugs that currently can be monitored directly, and the lack of an effective mechanism for measuring the biochemical and functional maturity of the neonate, especially as it relates to drug clearance, therapy of neonates with drugs is certainly more hazardous and possibly less effective than in adult patients. Toxic reactions of neonates to drug therapy can usually be related to the unique pharmacokinetic processes seen in this group, particularly to the maturity of clearance mechanisms. This is the basis for a link between maturity and drug efficacy or toxicity. In this usage, maturity refers to the functional capacity of organs and biochemical pathways. Of particular concern in this regard is kidney function and the activity of drug-metabolizing enzyme systems. Because direct assessment of these functions in the neonate is difficult, other types of maturity markers that can be easily measured and which relate to drug clearance need to be identified. Such markers could serve as a guide to the physician who is planning drug therapy for a neonatal patient. Several studies looking for a gestational age marker have provided some indication that biochemical maturity markers do exist and simply await discovery, thus affording an integration of pharmacokinetics and pathophysiology to achieve a more rational and effective therapeutic approach.
A sensitive radioimmunoassay for human proinsulin, with sequential use of antisera to C-peptide and insulin.
Authors: Ward W K , Paquette T L , Frank B H , Porte D
Published: January 2020

In this assay for immunoreactive human proinsulin (IRPI), it first is separated from plasma by use of an antiserum to human C-peptide. An immunoprecipitate is then formed by using a precipitating antiserum and polyethylene glycol, after which IRPI is dissociated from the antiserum by incubation in warm HCl, pH 2.0. The resulting mixture is assayed for insulin immunoreactivity by a double-antibody tracer-competition method involving incubation for four days with a high-affinity anti-insulin antiserum. Human proinsulin of recombinant-DNA origin is used as the standard. Added C-peptide at supraphysiological concentrations did not interfere with or react in the assay. Human insulin cross reacted by 1.5%. The detection limit for IRPI (2 SD from zero-dose binding) is 3 pmol/L. Proinsulin conversion intermediates are measured nearly as well as intact proinsulin. IRPI concentrations in 10 nondiabetic human subjects averaged 12.0 (SEM 1.6) pmol/L. The ratio of proinsulin to immunoreactive insulin averaged 14.3 (SEM 2.2)%. After intravenous arginine, the increase in proinsulin was less than that of insulin, and it declined more slowly.
Clinical utility of measuring free thyroxin and free triiodothyronine in serum of critically ill patients by ultrafiltration.
Authors: Lee L A , Mooney R A , Woolf P D
Published: January 2020

Free thyroxin (FT4) measurements by ultrafiltration were compared with results obtained by equilibrium dialysis and commercial RIA methods. In a group of critically ill patients, the mean +/- 2 SD FT4 value by ultrafiltration (26.5 +/- 21.4 ng/L) was higher than in the healthy reference population (19.3 +/- 9.8 ng/L, p less than 0.0001), and correlated well with equilibrium dialysis (23.8 +/- 20 ng/L, r = 0.87). By a two-step RIA method (Clinical Assays), the FT4 value was greater in the critically ill (14.7 +/- 9.6 ng/L) than in the reference population (12.8 +/- 5 ng/L, p less than 0.001) and was also correlated with ultrafiltration results (r = 0.71). FT4 values for the ill patients were lower than for the reference population in two one-step RIA methods (Clinical Assays: 8.6 +/- 7.1 ng/L; Corning Medical: 12.7 +/- 9.4 ng/L), neither of which gave results correlated with ultrafiltration (r = 0.16 and 0.22, respectively). The concentration of free triiodothyronine (FT3), also measured by ultrafiltration, was 4.5 +/- 2.2 ng/L in healthy subjects, 12.5 +/- 14.2 ng/L in hyperthyroidism, 1.7 +/- 2.0 ng/L in hypothyroidism, 2.2 +/- 1.9 ng/L in the critically ill subjects, and 3.8 +/- 0.9 ng/L in pregnancy. Thus, FT4 and FT3 measured by ultrafiltration accurately affects assessment of the thyroid status of the patient except in critical illnesses in which FT3 values are indistinguishable from those in hypothyroidism.
Progesterone in saliva: pitfalls and consequent implications for accuracy of the determination.
Authors: Lequin R M , van den Boogaard A , Vermeulen J , Danhof M
Published: January 2020

The concentration of steroid hormones in saliva is believed to reflect the concentration of free hormone in blood. Because the assay for progesterone in saliva has not been rigorously validated, we investigated some of the analytical variables involved. Saliva samples were divided into two portions. One was centrifuged and the supernate used for extraction; the other was homogenized by sonication and used as such for extraction. Progesterone concentrations in homogenized whole saliva were double or triple those in supernates. By equilibrium dialysis we established that 85% of progesterone was in the free form in supernates but only 60 to 70% in homogenized whole saliva, depending upon the phase of the cycle of the subjects. Salivary flow was stimulated by crystals of citric acid; unstimulated and stimulated saliva samples were obtained from the same person. Progesterone concentrations were significantly (p less than 0.02) higher in stimulated samples than in unstimulated ones, particularly in samples collected during the luteal phase.
Error in laboratory reference limits as shown in a collaborative quality-assurance program.
Authors: Compton P J , Stuart M C , Lazarus L
Published: January 2020

Laboratories participate in collaborative quality-assurance programs to maintain and improve the quality of their diagnostic assays, but little attention has been paid to diagnostic quality in these programs. We used a national quality-assurance program to assess the quality of laboratory reference intervals as exemplified by triiodothyronine, thyroxin, and thyrotropin immunoassays. The limits of the reference intervals used by laboratories bear virtually no relationship to laboratory bias, i.e., whether assays read "high" or "low." Further, correcting assay results from different laboratories for the reference limits used increases rather than decreases interlaboratory scatter. We conclude that the analytical quality of immunoassays now exceeds the quality of the reference limits supplied to clinicians to assist diagnosis, and that nationally or internationally defined reference limits would therefore provide more information at less cost than do individual laboratory reference limits.
Evaluation of a cystic fibrosis screening system incorporating a miniature sweat stimulator and disposable chloride sensor.
Authors: Warwick W J , Huang N N , Waring W W , Cherian A G , Brown I , Stejskal-Lorenz E , Yeung W H , Duhon G , Hill J G , Strominger D
Published: January 2020

A new sweat test (CF Indicator; Medtronic, Inc.) for cystic fibrosis (CF) features a compact, portable configuration of electrodes that dispense pilocarpine for iontophoresis. A disposable chloride sensor patch absorbs a specified volume of sweat, in which the chloride concentration is immediately determined as less than 40, 40-60, or greater than 60 mmol/L. We assessed the performance of the system in a five-center study, in relation to the clinical diagnosis and to the Gibson-Cooke sweat test (GCST) as a control test. With sweat chloride concentrations of less than or equal to 40 mmol/L defined as normal and greater than 40 mmol/L as indicating persons at risk for CF, the new system showed 91% specificity and 100% sensitivity for CF, as compared with 92.8% and 100%, respectively, for the GCST. When we used sweat chloride concentrations of less than or equal to 60 mmol/L as probably normal and greater than 60 mmol/L as probably indicative of CF, the new system showed a 99.1% specificity and 98.6% sensitivity, vs 97.8% specificity and 97.9% sensitivity for the GCST test. In both procedures, occasionally insufficient sweat was collected, and this appeared related to the age of the subject. We conclude that the new sweat test system is potentially useful in physicians' offices, in clinics, and similar settings.
Evaluation of a direct fluorometric method for determination of serum retinol.
Authors: Driskell W J , Hewett J S , Bashor M M
Published: January 2020

We evaluated the usefulness of a fluorometric method for determining serum retinol (Futterman et al., Invest Ophthalmol Vis Sci 1975;14:125-30) in which fluorescence (excitation 335 nm; emission 460 nm) of retinol is directly measured in unextracted, diluted serum. Using serum from 466 individual donors, we compared values so obtained with those by a "high-performance" liquid-chromatographic method. The correlation coefficient (r) was 0.74. When we compared fluorometric retinol values with retinol-binding protein values for the 466 samples, r was 0.71. About 1% of the 466 samples had markedly higher values by fluorometry than by chromatography, the result of positive interferences. For two serum pools, we obtained CVs of 1.58% (n = 57) and 1.79% (n = 57) in long-term precision studies lasting 60 days. Although the fluorometric method of Futterman et al. has not been widely adopted, we find that it is simple to perform and that results compare favorably with the chromatographic method in precision and accuracy. It is unique among commonly used serum retinol methods in that the serum need not be extracted with organic solvents.
Retinol, alpha-tocopherol, lycopene, and alpha- and beta-carotene simultaneously determined in plasma by isocratic liquid chromatography.
Authors: Milne D B , Botnen J
Published: January 2020

Retinol, alpha-tocopherol, lycopene, and alpha- and beta-carotene can be simultaneously determined in human plasma by reversed-phase liquid chromatography. Plasma--0.5 mL plus added internal standard, retinyl acetate--is deproteinized with 0.5 mL of ethanol, then extracted with 1.0 mL of petroleum ether. The organic layer is removed and evaporated, the residue is redissolved in 0.25 mL of ethanol, and 8-microL samples are injected into a 60 X 4.6 mm column of Hypersil ODS 3-microns particles at 35 degrees C. An isocratic methanol mobile phase, flow rate 0.9 mL/min, is used for the 9-min run. Retinol and retinyl acetate are monitored at 305 nm, the tocopherols at 292 nm, and the carotenoids at 460 nm. Between-run CVs were 3.1, 6.9, 6.1, and 6.5% for retinol, alpha-tocopherol, lycopene, and beta-carotene, respectively. Small sample requirement, simplicity of extraction, short run time, and good reproducibility make this procedure ideal for clinical or research use.
Stability of catecholamines in whole blood, plasma, and platelets.
Authors: Weir T B , Smith C C , Round J M , Betteridge D J
Published: January 2020

Checking catecholamine stability in whole blood, plasma, and platelets, we found that specimens stored at room temperature or frozen for periods ranging from 1.5 h to three weeks show no significant difference in measured catecholamine concentration. The implications of these findings are discussed.
Automated kinetic assay of angiotensin-converting enzyme in serum.
Authors: Beneteau B , Baudin B , Morgant G , Giboudeau J , Baumann F C
Published: January 2020

We have developed and validated an automated kinetic method for angiotensin-converting enzyme (EC 3.4.15.1) on the Olli C + D analyzer, modified from that of Ronca-Testoni (Clin Chem 1983;29:1093-6) with N-[3-(2-furyl)-acryloyl]-L-phenylalanylglycylglycine used as substrate. We have determined appropriate reaction conditions for the assay, verified the principal analytical reliability criteria (repeatability, reproducibility, sensitivity), and established normal reference intervals (mean +/- SD) for the enzyme's activity, using serum of normal adults (100 +/- 35 U/L, n = 150), newborns (130 +/- 27 U/L, n = 10), women taking oral contraceptives (103 +/- 30 U/L, n = 10), smokers (109 +/- 38 U/L, n = 27), and patients with sarcoidosis (220 +/- 48 U/L, n = 15).
Evaluation of simultaneous measurement of lutropin and follitropin with the SimulTROPIN radioimmunoassay kit.
Authors: Wians F H , Dev J , Powell M M , Heald J I
Published: January 2020

We evaluated the analytical performance of the "SimulTROPIN" (Becton-Dickinson Inc.) radioimmunoassay for the simultaneous measurement of lutropin (LH) and follitropin (FSH) in human serum. Dose response, linearity, analytical recovery, sensitivity, and reagent stability were all acceptable. Cross reactivity with other structurally related hormones [choriogonadotropin (CG) and thyrotropin (TSH)] was minimal. A normal reference interval was established for nonmidcycle, ovulatory women. We confirm the low degree of CG and TSH cross reactivity with LH and FSH reported by Becton Dickinson and conclude that this assay is an acceptable method for laboratory use in the simultaneous quantification of these analytes.
Simplified liquid-chromatographic assay of amiodarone and desethylamiodarone after solid-phase extraction.
Authors: Pollak P T , Carruthers S G , Freeman D J
Published: January 2020

We describe a rapid, simplified isocratic "high-performance" liquid-chromatographic method for simultaneous measurement of the antiarrhythmic drug amiodarone and its major metabolite, desethylamiodarone, in small volumes of sera (100 microL). Compared with liquid-liquid extraction, the solid-phase method of extraction saves time and glassware and improves reproducibility for small sample volumes. Amiodarone and desethylamiodarone could be measured at concentrations as low as 250 micrograms/L. Standard curves for the drug and metabolite are linear over the range of concentrations found in our patients. Within-run CVs (n = 6) ranged from 2.7% to 4.5% for amiodarone and from 4.0% to 5.7% for desethylamiodarone over the range of 250 to 4000 micrograms/L. Between-run CVs (n = 12) were 8.3% and 5.7% for amiodarone and desethylamiodarone, respectively. Commonly used cardiovascular medications do not interfere with the assay.
Thyroid screening of neonates without use of radioactivity: evaluation of time-resolved fluoroimmunoassay of thyrotropin.
Authors: Torresani T E , Scherz R
Published: January 2020

We evaluated the usefulness, in routine newborn screening for congenital hypothyroidism, of a time-resolved fluoroimmunoassay kit (DELFIA Neonatal TSH) for the determination of thyrotropin (TSH) in dried blood spots. A total of 11 531 dried blood samples from newborns were tested in parallel in each of two Swiss screening laboratories, by RIA and DELFIA. Six cases of confirmed congenital hypothyroidism were detected during the study period. The rate of false-positive results, after single TSH determination in the DELFIA assay, was 0.16%. Correlation of RIA and DELFIA results for TSH was very good in both laboratories (0.959 and 0.97, respectively). The new method fulfills the criteria for precision and sensitivity of a screening assay. Screening results are usually available the day after the sample arrives in the laboratory, thus favoring early diagnosis and allowing treatment to begin by the seventh or eighth postnatal day.
Thrombin specificity with tripeptide chromogenic substrates: comparison of human and bovine thrombins with and without fibrinogen clotting activities.
Authors: Sonder S A , Fenton J W
Published: January 2020

To assess the thrombin specificity of tripeptide chromogenic substrates, we determined the Michaelis--Menten (Km), catalytic (kcat), and specificity (kcat/Km) constants for S-2238 (H-D-Phe-Pip-Arg-p-nitroanilide), Chromozym-TH (Tos-Gly-Pro-Arg-p-nitroanilide), and Spectrozyme-TH (H-D-hexahydrotyrosyl-Ala-Arg-p-nitroanilide) with high-purity thrombin preparations. Human and bovine alpha-thrombins, prepared by essentially the same procedure, were each greater than 95% in the form of the enzyme (alpha-thrombin) and had a specific fibrinogen-clotting activity greater than 2000 kilo-clotting units per gram of protein (kcu/g). In contrast, human gamma- and bovine beta-thrombins, made by controlled passage of alpha-thrombin through trypsin agarose, were greater than 98% of their respective forms and had fibrinogen-clotting activity less than 1 kcu/g. With the tripeptide chromogenic substrates these four thrombin forms at pH 7.8 and 23 degrees C had Km values of 1.6 to 16 mol/L, kcat values of 35 to 130 s-1, and kcat/Km ratios of 4.7 to 52 L X mol-1 X s-1. Although Km for an individual substrate was slightly higher for bovine than for human alpha-thrombins and although Km values for the nonclotting forms (human gamma- and bovine beta-thrombins) were higher than for clotting forms (alpha-thrombins), we found no major differences among the kinetic values for the three substrates.
Determination of indoxyl sulfate in plasma of patients with renal failure by use of ion-pairing liquid chromatography.
Authors: Stanfel L A , Gulyassy P F , Jarrard E A
Published: January 2020

Using our newly developed ion-pairing reversed-phase liquid-chromatographic method for assay of indoxyl sulfate, we measured its concentration in plasma of normal subjects and patients in various degrees of renal failure. Response was linear over the range of 50 to 25 000 pmol of indoxyl sulfate injected into the chromatograph. We demonstrated the specificity of the assay for azotemic plasma by using enzymatic conversion with a sulfatase. For a moderately above-normal indoxyl sulfate concentration in azotemic plasma of 134 mumol/L (29 mg/L), the within-day CV was 1.6%, the day-to-day CV 2.8%. Mean analytical recovery was 101.0% (CV = 2.8%). Over a range of 29 to 192 mg of creatinine per liter of plasma (x), indoxyl sulfate (y) concentration (in mumol/L) was positively correlated (y = 1.30x + 0.43). This method should prove valuable for further study of uremic toxins.
Enzyme-linked immunoabsorbant assay of apolipoprotein AII in plasma, with use of a monoclonal antibody.
Authors: Stein E A , DiPersio L , Pesce A J , Kashyap M , Kao J T , Srivastava L , McNerney C
Published: January 2020

We produced a monoclonal antibody (C2-22) to human apolipoprotein (Apo) AII and describe its use in an enzyme-linked immunoabsorbant assay (ELISA) for Apo AII in human plasma and lipoprotein subfractions. No cross reactivity of the antibody with Apo CI, CII, CIII, E, or ablumin was detected. Apo AI and low- and very-low-density lipoprotein cross reacted by 0.25%, less than 0.2%, and less than 0.3%, respectively. Whole plasma high-density lipoprotein (HDL) and HDL subfractions (HDL2 and HDL3) produced parallel displacement curves. This quantitative ELISA is based on competition between solid-phase-bound Apo AII and free Apo AII. Bound C2-22 is detected by alkaline-phosphatase-labeled second antibody. The standard curve for the assay is linear for plasma diluted 500-fold originally containing 140 to 1140 mg of Apo AII per liter. Delipidation of plasma samples exposed no additional antigenic sites. Within- and between-run CVs were respectively 8.4% and 8.7% at 327 mg/L of Apo AII, and 6.8% and 7.4% at 587 mg/L. Results correlated well with those by a polyvalent-antisera-based RIA procedure: r = 0.916, p less than 0.01, RIA = 0.896 ELISA -19.1 mg/L.