bluuebunny/crossref_metadata_2025 huggingface dataset:
Antidepressants in the treatment of post-psychotic depression in schizophrenia: drug interactions and other considerations.
Authors: Siris S G , Sellew A P , Frechen K , Cooper T B , Mandell J , Casey E
Published: January 2020

Adjunctive imipramine has been found to be useful in the treatment of a substantial number of patients with syndromally defined post-psychotic depressions. This paper examines the clinical effects of the combined anticholinergic activity of imipramine, when added to ongoing fluphenazine decanoate/benztropine treatment, in such patients. Little additional anticholinergic impact of the imipramine was observable beyond that already attributable to the benztropine, and no significant relationships were found between a clinical measure of peripheral anticholinergic activity and either global clinical outcome or antidepressive efficacy. This paper also reports on the concentrations of imipramine and its metabolites in plasma under the conditions of this therapeutic trial. The changes in relative concentrations of imipramine and metabolites with time were consistent with the concept that fluphenazine competes with tricyclic metabolism. The relationship of plasma imipramine and desipramine to clinical improvement in this group of secondary depressions did not parallel previously reported relationships of these antidepressant molecules to clinical outcome in primary depressions.
Compliance during tricyclic antidepressant therapy: pharmacokinetic and analytical issues.
Authors: Perel J M
Published: January 2020

Information on steady-state concentrations of parent tricyclic antidepressants (TCAs) and their major metabolites in plasma is useful in ascertaining compliance, for possible pharmacokinetic changes during longer treatment, and for prospective individualized dosing procedures. Adequate response can be maintained when there are relatively small fluctuations in drug concentration from visit to visit, but large fluctuations increase the liklihood of relapses during the acute-treatment phase and recurrences afterwards. Changes in the individualized in dosage regimens complicate measurement of concentrations in plasma at steady state. Longitudinal examinations of concentration/dosage (L/D) values in our three-year imipramine (IMI) maintenance study reveal that patients having large intra-individual fluctuations can be classified as noncompliers, likely to have recurrences. The time course and magnitude of L/D values reflect higher accumulation of both components, IMI and desipramine (DMI), in plasma. Evidently, dose-dependent kinetics lead to higher steady-state concentrations in plasma than previously observed with similar dosages in shorter-term treatment. With amitriptyline (AMI), in a 12-month study, the mean total concentrations of both AMI and nortriptyline (NT) progressively increased, by 22% and 33%, respectively. The pharmacokinetic linearity of AMI and (or) NT, i.e., (L/D)high/(L/D)low, is maintained over much wider dosage and age range than with IMI or DMI. We advocate concurrent use of the L/D method and co-administration of riboflavin for identification of noncompliers. We describe our current experience with various analytical procedures in this regard, concluding that high-performance liquid-chromatographic methods, with appropriate selection among ultraviolet, enhanced fluorescence, and electrochemical detectors for each TCA under specified therapeutic conditions, are most suitable and versatile, having superior analytical parameters. A suitable alternative procedure is gas-chromatography (N/P mode). Although immunoassays (EMIT, radiochemical) are most convenient for toxicological screens, their significant cross reactivities with several phenothiazines and the detection limits, requiring higher concentrations for EMIT, restrict their usage during research and (or) clinical monitoring.
Colorimetric assay of blood coagulation factor XIII in plasma.
Authors: Lee K N , Birckbichler P J , Patterson M K
Published: January 2020

In this new colorimetric assay for Factor XIII in plasma, 5-(biotinamido)pentylamine is used as the amine substrate. Factor XIII, a zymogen, is transformed by thrombin and Ca2+ to active Factor XIIIa, and the incorporation of 5-(biotinamido)pentylamine into N,N-dimethylcasein is used to measure catalytically active Factor XIIIa. The biotinylated enzymatic product is immobilized onto 96-well microtiter plates, complexed with streptavidin-beta-galactosidase, and the absorbance at 405 nm is monitored for production of p-nitrophenol from p-nitrophenyl-beta-D-galactopyranoside. Concentrations of N,N-dimethylcasein, 5-(biotinamido)pentylamine, Ca2+, and thrombin were chosen to allow near-maximum velocity of amine incorporation. A linear relationship was obtained between assay product and plasma volume, from 0.5 to 50 microL of plasma. Results correlated well (r greater than 0.924) with those from the most frequently utilized radiometric filter-paper assay for Factor XIII. The method appears to be ideal for routine diagnostic estimation of Factor XIII in plasma because of its simplicity, its lack of use of radioisotopes, and its potential for assay of large numbers of samples by use of microtiter plates and automated plate readers.
Isocratic separation of ATP and its degradation products from biological fluids by automated liquid chromatography.
Authors: Tekkanat K K , Fox I H
Published: January 2020

Two groups of metabolites (a) IMP, AMP, ADP, ATP, and cAMP in extracts of fibroblasts and erythrocytes and (b) hypoxanthine, xanthine, adenosine, and inosine in plasma and urine have been separated by ion-pairing reversed-phase chromatography on a microBondapak C18 column, with use of the following reagents: 60 mmol/L KH2PO4, 0.45 mmol/L tetrabutylammonium phosphate, and 1.26 mol/L acetonitrile, pH 3.2 (at 23 degrees C) (group a) and 20 mmol/L KH2PO4, 0.45 mmol/L tetrabutylammonium phosphate, and 0.35 mol/L acetonitrile, pH 2.70 (at 24 degrees C) (group b). Under both sets of conditions, the compounds are completely separated in less than 15 min. The separation is isocratic, so the method is easily adaptable to automation.
Determination of lamellar body size, number density, and concentration by differential light scattering from amniotic fluid: physical significance of A650.
Authors: Dubin S B
Published: January 2020

A new method for characterizing amniotic fluid is presented. The difference in optical absorbance between amniotic fluid diluted with distilled water or glycerol is measured. This differential approach is shown to determine the total light scattered by the surfactant-containing lamellar bodies and not to be affected by interfering chromogens. The lamellar body size, number density, and concentration are in turn quantified from the wavelength dependence of the absorbance difference, which is shown to be accurately predicted by light-scattering theory in the domain of "anomalous diffraction." The concentration of the lamellar bodies is demonstrated to be directly related to the absorbance difference at 650 nm. A maturity criterion, based on that generally accepted for the direct measurement of absorbance at 650 nm (that is, A650 greater than or equal to 0.15), is determined for the differential approach. Hence, measurement of the absorbance difference at 650 nm can be used to assess amniotic fluid both for its optical absorbance and its lamellar body concentration.
Autoantibodies to thyroxin and triiodothyronine.
Authors: Desai R K , Bredenkamp B , Jialal I , Omar M A , Rajput M C , Joubert S M
Published: January 2020

Two clinically euthyroid patients with multinodular goiter were found to have high "free" thyroxin (Amerlex-M, Amersham and Coat-a-Count, DPC) and triiodothyronine (Amerlex-M) concentrations (FT4 and FT3, respectively). The presence of antibodies to T4 and T3 was confirmed by the finding that polyethylene glycol precipitated a far greater proportion of radioactivity when radiolabeled FT4 or FT3 analog (Amerlex-M) was incubated with serum from these patients than was true for normal subjects. With this method we could not demonstrate antibodies to thyroid hormones in 116 healthy volunteers. Of 101 hyperthyroid patients tested, one had antibodies to T4 but none had antibodies to T3. One patient had antibodies to T4, and one to T3, of 36 hypothyroid patients tested. All patients with thyroid hormone antibodies also demonstrated antithyroglobulin antibodies (measured immunoradiometrically). Evidently, the presence of thyroid hormone antibodies should be suspected when results of thyroid-function tests are discordant with the clinical state, and we suggest that measurement of thyrotropin by an assay with improved detection limits will aid in correctly determining thyroid status.
Results for 74 substances tested for interference with determination of plasma catecholamines by "high-performance" liquid chromatography with electrochemical detection.
Authors: Koller M
Published: January 2020

Various catecholamine metabolites, catecholamine-related compounds, catechols, drugs, amines, and other nitrogen compounds were injected onto an HPLC system ("ClinRep Catecholamine-Plasma" assay kit with a reversed-phase C18 column) used for measuring catecholamines. None of the 74 substances tested co-eluted with any of the catecholamines--norepinephrine, epinephrine, or dopamine--or with the internal standard, 3,4-dihydroxybenzylamine.
More-sensitive enzyme-multiplied immunoassay technique for procainamide and N-acetylprocainamide in plasma, serum, and urine.
Authors: Henry P R , Dhruv R A
Published: January 2020

A commercially available (Syva Co.) enzyme-multiplied immunoassay technique (EMIT) for the quantitative determination of procainamide (PA) and N-acetylprocainamide (NAPA) was modified to allow automated quantitative analysis of approximately 100 samples per day, in a working range of 0.1 to 2.0 micrograms/mL. Such a test was needed to evaluate the pharmacokinetic characteristics of controlled-release dosage forms characterized by long half-lives at low plasma concentration. Analytical recovery of PA and NAPA from serum, plasma, and urine was satisfactory, but at extreme ratios for PA:NAPA the accuracy of determining the lower-concentration component became unsatisfactory. In fact, however, we found no such ratios in 5400 clinical samples assayed by this procedure.
Pseudouridine is unsuitable as an endogenous renal clearance marker.
Authors: Bernert J T , Bell C J , Guntupalli J , Hannon W H
Published: January 2020

We assessed the clearance of endogenous pseudouridine in humans to evaluate the potential use of this modified nucleoside as a marker of glomerular filtration rate. Pseudouridine concentrations in serum ultrafiltrates and in the corresponding 24-h urine specimens from 19 healthy men were determined by high-performance liquid chromatography. Mean (and SD) pseudouridine concentrations in serum and urine from this group averaged 2.77 (0.34) mumol/L and 203.2 (64.8) mumol/L, respectively. The calculated clearances of the nucleoside [87.3 (24.9) mL/min, n = 19], however, averaged approximately one-third lower than the corresponding creatinine clearances in the same individuals [131.8 (28.4) mL/min]. Measurement of simultaneous clearances of [3H]pseudouridine and [14C]inulin in rats also yielded a lower pseudouridine clearance, 0.78 relative to inulin. Our results are thus consistent with a partial net reabsorption of pseudouridine in both experimental animals and in humans, indicating that this compound would not be a suitable endogenous marker for routine estimation of the glomerular filtration rate.
Competitive enzyme inhibition immunoassay of apolipoprotein A-I: use of monoclonal antibodies.
Authors: Bekaert E D , Ayrault-Jarrier M , Petit E , Betourne C , Robin H , Polonovski J
Published: January 2020

An original competitive enzyme inhibition immunoassay has been developed for determination of total apolipoprotein A-I (apo A-I) in serum and in plasma. This specific assay involves a single monoclonal antibody (F59 4A12 2F4, directed to the -COOH terminal region of the apo A-I molecule) and a stable secondary plasma standard. Delipidation of serum samples exposed no additional antigenic sites, which suggests that all the apo A-I molecules express the epitope detected by Mab 4A12 on the surface of the apo A-I-containing particles. Within- and between-run CVs were, respectively, 6.1% and 7.5% at a 1.47 g/L concentration of apo A-I, 7.1% and 8.2% at 1.21 g/L, and 6.5% and 7.3% at 1.80 g/L. Results (y) correlated with those obtained with an electroimmunoassay (x), in which we used a mixture of three monoclonal antibodies, including Mab 4A12, as follows: y = 250x - 7, r = 0.744, P less than 0.001.
A method for direct estimation of imprecision profiles, with reference to immunoassay data.
Authors: Sadler W A , Smith M H , Legge H M
Published: January 2020

A three-parameter model for directly estimating imprecision profiles from replicated immunoassay results is compared with a six-parameter indirect profile model obtained by the usual method of combining error in the raw response measurements with the slope of the standard curve. Direct estimation is likely to be less reliable when based on the limited data collected from a single assay, and may underestimate variability at high concentrations when many results are clustered at the upper end of the concentration range. However, at concentrations near the assay detection limit (often a region of particular interest), direct estimation is superior to the indirect method if a logistic or related function is used as the standard curve model. Direct estimation of imprecision profiles has useful application whenever the internal details of an assay system are not readily available, for example, in analysis of data collected in external surveys.
Discriminant analysis of laboratory tests in patients admitted to a coronary care unit.
Authors: Lacher D A , Paolino M J
Published: January 2020

Discriminant analysis of chemistry and hematology laboratory test results was used to classify patients with and without myocardial infarction in a coronary care unit. We studied 64 patients with myocardial infarction and 70 patients without infarction, using logistic regression, linear and quadratic discriminant analyses on untransformed and logarithmically transformed data. Serum aspartate aminotransferase (AST, EC 2.6.1.1), the best single discriminating test, classified 73% of patients correctly. Quadratic discriminant analysis on log-transformed data had a 98.5% classification accuracy when all variables were used in the discriminant function and had the highest classification accuracy and precision. All of the discriminant methods had acceptable cross-validation.
Time series modeling for quality control in clinical chemistry.
Authors: Alwan L C , Bissell M G
Published: January 2020

Autocorrelation of clinical chemistry quality-control (Q/C) measurements causes one of the basic assumptions underlying the use of Levey-Jennings control charts to be violated and performance to be degraded. This is the requirement that the observations be statistically independent. We present a proposal for a new approach to statistical quality control that removes this difficulty. We propose to replace the current single control chart of raw Q/C data with two charts: (a) a common cause chart, representing a Box-Jenkins ARIMA time-series model of any underlying persisting nonrandomness in the process, and (b) a special cause chart of the residuals from the above model, which, being free of such persisting nonrandomness, fulfills the criteria for use of the standard Levey-Jennings plotting format and standard control rules. We provide a comparison of the performance of our proposed approach with that of current practice.
Sialic acid in sickle cell disease.
Authors: Ekeke G I , Ibeh G O
Published: January 2020

Neuraminic (sialic) acid concentrations in serum from normal and sickle cell (HbSS) subjects were determined for discrete age groups from childhood through adolescence. Values in sickle cell disease were consistently lower over the entire age range. We further investigated the effect of exogenous sialic acid on the rate of sickling reversion of HbSS erythrocytes and demonstrated that this compound in millimole per liter concentrations could revert pre-sickled erythrocytes to their normal morphology in a concentration-dependent manner. When subjected to partial de-sialation with sialidase (EC 3.2.1.18), the HbSS erythrocytes not only sickled faster upon deoxygenation, they also reverted more slowly on treatment with phenylalanine (a more efficient anti-sickling agent than sialic acid) than did untreated cells. We conclude that, in sickle cell disease, erythrocyte sialic acid content could play a significant role, not only in the control of the sickling rate in vivo, but also, after sickling has occurred, in the rate of recovery from a sickling crisis.
An inexpensive method for sensitive enzymatic determination of oxalate in urine and plasma.
Authors: Berckmans R J , Boer P
Published: January 2020

In this simple, sensitive, and rapid enzymatic method for the determination of oxalate in urine or plasma, oxalate oxidase (EC 1.2.3.4) prepared from barley seedlings is used to convert oxalate to carbon dioxide and hydrogen peroxide, which is determined photometrically at 600 nm, with use of horseradish peroxidase, by oxidative coupling of 3-methyl-2-benzothiazoline hydrazine with N,N-dimethylaniline. Plasma is pre-treated by ultrafiltration and co-precipitation of oxalate with calcium sulfate and ethanol, urine by dilution and reversed-phase chromatography on C18 columns. Analytical recovery for urine is 99 +/- 2%, for plasma 92 +/- 3%. The normal range for urinary excretion is 0.10 to 0.56 mmol/24 h, and for the concentration in plasma 0.4 to 3.7 mumol/L. There were no significant sex-related differences in urinary excretion or plasma concentration. Our within- and between-assay coefficients of variation were, respectively, less than 3.4% and less than 6.0% for urine, and less than 1.5% and less than 4.3% for plasma.
Modified TDx assay for cyclosporine and metabolites, for use with whole-blood samples.
Authors: Vogt W , Welsch I
Published: January 2020

A recently introduced fluorescence polarization immunoassay (FPIA) for determination of cyclosporine A in serum and plasma is discussed with regard to its use for whole-blood samples, with and without hemolysis before the assay. The performance characteristics of the modified method are highly satisfactory (within-run CVs 2.06 to 5.50% and 1.99 to 3.39%, respectively; long-term between-run CVs under routine conditions 5.73 to 8.95%). The limit of detection is 30 micrograms/L. Results agree well with those obtained with the RIAs compared, but the modified FPIA is more convenient and faster.
Improved liquid-chromatographic determination of cyclosporine and its metabolites in blood.
Authors: Shah A K , Sawchuk R J
Published: January 2020

We describe a liquid-chromatographic assay for cyclosporine (CyA) and four of its metabolites in blood. The method is sensitive and specific for this drug and its metabolites. The use of flow programming shortens total chromatographic run time to 30 min, resulting in acceptable separation of metabolites at a lower flow rate and yielding narrower peaks for CyA and the internal standard at the higher flow rate. CyA and metabolites Met 17 and Met 1 were measured in seven heart transplant patients who were undergoing chronic therapy with CyA in oral doses ranging from 3 to 13 mg/kg body wt per day. For the individual patients, the mean trough concentrations in blood of CyA, Met 17, and Met 1 ranged from 111 to 221, 159 to 334, and 36 to 90 micrograms/L, respectively. Met 8 and Met 21 were not observed in any of these patients' blood specimens.
A sensitive bioluminescent immunoinhibition test for CK-B subunit activity and a CK-MB specific ELISA compared: correlation with agarose electrophoresis and influence of CK-isoenzyme profile on results.
Authors: Gorus F , Claessens V , Goubert P , Laureys M
Published: January 2020

Searching for alternatives to the imprecise spectrophotometric tests for low-concentration creatine kinase (EC 2.7.3.2) isoenzyme MB (CK-MB), we investigated the analytical performance of two potentially superior approaches--a bioluminescent immunoinhibition assay (I, LKB-Wallac) and an ELISA (enzyme-labeled immunosorbent assay) technique (II, Hybritech)--in comparison with an electrophoretic method (III, Beckman). Only I showed good between-day precision (CV 8.3%) at the upper reference limit, allowing reproducible assay of CK-B subunit activity down to at least 3 U/L. In conditions where CK isoenzyme assays remained unaffected by CK-MM concentrations, test results were proportional to the amount of CK-MB in the sample up to at least 50 U/L for I, 120 micrograms/L for II, and 100 U/L for III (r greater than 0.998 by linear regression analysis). For CK-MB-positive samples, the data by I correlated more closely with values by III (n = 24; r = 0.994) than did results by II (n = 15; r = 0.909), but both methods were equally effective in discriminating between samples with or without electrophoretically supranormal CK-MB activity (93% sensitivity). II was entirely CK-MB specific, whereas CK-B activity by I was consistently (18/18) increased in CK-MB-negative samples containing CK-BB (n = 6; r = 0.996) or macro CK, types 1 or 2 (n = 12; r = 0.930). I is highly sensitive for screening for increased non-MM CK activity, the nature of which should be subsequently clarified by electrophoresis.
Salivary immunoreactive insulin: a new entry in clinical chemistry?
Authors: Marchetti P , Grossi C , Giannarelli R , Masoni A , Cristofani R , Giannecchini M , Navales R
Published: January 2020

The distribution of salivary immunoreactive insulin (S-IRI) and its relation to plasma insulin (P-IRI) and other clinical and metabolic variables were investigated in 93 nondiabetic subjects (60 males, 33 females, ages 11-70 y). S-IRI was measured by RIA, with monoiodinated insulin as labeled antigen and with standards consisting of serial dilutions in saliva of known amounts of native insulin. The sensitivity of the method was 12.1 pmol/L. The intra- and between-assay CVs were near 10%, and analytical recovery exceeded 93% for various insulin concentrations added to saliva. S-IRI, measured after overnight fast, showed a nonguassian distribution in males (skewness 1.18, kurtosis 1.41) and females (skewness 1.71, kurtosis 1.18); mean and median values were higher in males (14.3 and 12.1 nmol/min) than in females (12.1 and 7.15 nmol/min), but not significantly so. S-IRI was significantly correlated with P-IRI both in males (r = 0.75, P less than 0.0001) and females (r = 0.72, P less than 0.0001). These results, particularly the correlation between S-IRI and P-IRI, indicate the possibility of using S-IRI measurement in clinical practice.
Rapid determination of the hypoxanthine increase in ischemic exercise tests.
Authors: Bolhuis P A , Zwart R , Bär P R , de Visser M , van der Helm H J
Published: January 2020

After ischemic exercise tests, performed to detect glycogenoses or myoadenylate deaminase (EC 3.5.4.6) deficiency, the increases in serum lactate and ammonia usually are measured. Determination of hypoxanthine instead of ammonia can also be used to show myoadenylate deaminase deficiency, but HPLC of hypoxanthine is time-consuming. As a substitute, we developed an indirect enzymatic equilibrium method for hypoxanthine based on coupling the chromogenic system 3,5-dichloro-2-hydroxy-benzenesulfonic acid/4-aminophenazone with formation of hydrogen peroxide by xanthine oxidase (EC 1.1.3.22). The pH optimum is at 7.8 and the absorbance maximum at 510 nm. The calibration curve is linear from 0 to 100 mumol/L and the detection limit is 0.9 mumol/L. Analytical variability (CV) was 1.5% to 3.6% within-run, 4.5% to 8.5% between-run. The assay can be performed with a standard spectrophotometer or a centrifugal analyzer. The coefficient of correlation was 0.68 between hypoxanthine and ammonia increases in plasma from controls who performed the exercise test.
Amino acid concentrations in serum and aqueous humor from subjects with extreme myopia or senile cataract.
Authors: Wuu J A , Wen L Y , Chuang T Y , Chang G G
Published: January 2020

With an amino acid analyzer, we measured amino acids and related compounds in serum and aqueous humor from normal Chinese and some patients with extreme myopia or senile cataracts. Forty peaks were well resolved, and their areas were used to quantify each compound. In the myopic patients, glutamate, alpha-aminoadipate, and methionine concentrations in serum were 10-fold those in the normal subjects. Values for most of the other amino acids and related compounds were also higher in myopic patients' sera. In the cataract patients, concentrations of most of these compounds were lower in serum but higher in aqueous humor than for the normal subjects. Tryptophan was present in significant amounts in sera from the normal subjects, but was not detectable in the senile-cataract patients. The ratio of amino acid concentration in aqueous humor to that in the serum (Ch/Cs) was higher in the cataract patients than in normal subjects for almost all of the compounds we measured.
Age- and sex-specific pediatric reference intervals: study design and methods illustrated by measurement of serum proteins with the Behring LN Nephelometer.
Authors: Lockitch G , Halstead A C , Quigley G , MacCallum C
Published: January 2020

We analyzed blood from 450 healthy children and adolescents, ages one to 19 y, as well as term and preterm infants, to define age- and sex-specific reference intervals for numerous blood constituents. Reference intervals were derived by using nonparametric methods to determine the 0.025 and 0.975 fractiles. Ten serum proteins were measured with the Behring LN Nephelometer. Girls over 10 years of age had higher concentrations of ceruloplasmin and alpha 1-antitrypsin than other children had. There was no sex-related difference in reference intervals for the other proteins tested. Reference intervals are presented for immunoglobulins G, A, and M, complement fractions C3c and C4, ceruloplasmin, transferrin, alpha 1-antitrypsin, retinol-binding protein, and prealbumin (transthyretin).
The Abbott IMx automated benchtop immunochemistry analyzer system.
Authors: Fiore M , Mitchell J , Doan T , Nelson R , Winter G , Grandone C , Zeng K , Haraden R , Smith J , Harris K
Published: January 2020

We describe a new clinical laboratory instrument, the IMx, used to automate immunoassay testing in the clinical laboratory. The IMx incorporates a novel technology called Microparticle capture Enzyme ImmunoAssay (MEIA) for assays of high-molecular-mass analytes, and fluorescence polarization immunoassay (FPIA) for hapten assays. A front-surface fluorometer is used to quantify the enzymatic generation of fluorescent product at a rate proportional to the concentration of the analyte in an MEIA, and a fluorescence polarization optical system is used to quantify results in an FPIA. The microprocessor-based instrument uses a robotic arm with two degrees of freedom and a rotating carousel to process the samples for assay. One assay can be done on each of 24 patients' specimens in 30 to 40 min with "walk-away" automation. Calibration curves are stable for at least two weeks. Instrument control involves software-labeled "command keys," a numeric keypad, and an interactive display. Results are output to a thermal printer or computer interface.
Rapid immunochemical detection of fecal occult blood by use of a latex-agglutination test.
Authors: Väänänen P , Tenhunen R
Published: January 2020

A novel immunochemical test, "Hemolex," is characterized and compared with three guaiac tests: "Hemoccult" (Smith-Kline, U.S.A.), "Hemofec" (Boehringer Mannheim, F.R.G.), and "Fecatwin Sensitive" (Labsystems, Finland). We tested 191 stool specimens from patients, 144 from healthy persons. The sensitivity of Hemolex was 0.6 mL of blood per 100 g of stool. It reacts with neither animal hemoglobin nor human myoglobin, unlike the guaiac tests, nor does it react with the stool of healthy persons under no dietary restrictions, while the guaiac tests react variably. As a rule, Hemolex is insensitive to bleeding in the upper gastrointestinal tract, unlike the guaiac tests. However, Hemolex seems to be more sensitive to bleeding in the lower gastrointestinal canal than are the other tests, and we show it to be suitable for screening for occult blood originating there. It is unaffected by diet. It is simple to perform, requires no special equipment, and stool specimens, stored in the test diluent, are stable for testing for two weeks.
Decreased stability of triglycerides and increased free glycerol in serum from heparin-treated patients.
Authors: Hortin G L , Cole T G , Gibson D W , Kessler G
Published: January 2020

Triglycerides usually are stable in serum. However, for a few patients, we noted marked decreases in measured triglycerides when the same serum specimen was analyzed on successive days. This was found to be ascribable to intravenous administration of heparin. Measured triglyceride in serum of 11 patients being treated with heparin decreased 34% (SD 17%) in samples stored for one day at room temperature. Triglyceride values for sera from control patients remained unchanged. Increases in free glycerol corresponded to the observed decreased in triglycerides. Measurement of free glycerol thus provides a means of recognizing this problem.
Time-dependence of urinary neopterin, a marker of cellular immune activity.
Authors: Auzéby A , Bogdan A , Krosi Z , Touitou Y
Published: January 2020

Neopterin, a marker of cellular immune system activation, is produced by human macrophages after induction by interferon gamma (secreted by T-lymphocytes) and is eliminated mostly in urine. We have documented the circadian rhythm of urinary neopterin in five healthy young men (about 25 years old), using voidings collected during 48 h at fixed 4-h intervals. We repeated the experiment three times, one week apart. Neopterin was measured by high-performance liquid chromatography (HPLC). We clearly show a peak of the excretion of neopterin in the early morning (around 0630 hours +/- 2 h), with total variability (peak-trough difference) reaching 51%. Neopterin is commonly assayed in urinary fractions, so it is imperative to use urine specimens collected at the same time of day--e.g., the first morning urines--to avoid misinterpretation in follow-up of patients.
Determination of the antidepressant fluoxetine and its metabolite norfluoxetine in serum by reversed-phase HPLC with ultraviolet detection.
Authors: Orsulak P J , Kenney J T , Debus J R , Crowley G , Wittman P D
Published: January 2020

A procedure has been developed for measuring fluoxetine and its desmethyl metabolite, norfluoxetine, in serum by reversed-phase high-performance liquid chromatography (HPLC), with ultraviolet detection at 226 nm. Fluoxetine and norfluoxetine are isolated from serum by liquid-liquid extraction. They are then separated by HPLC and quantified, with reduced haloperidol as the internal standard. Fluoxetine, norfluoxetine, and the reduced haloperidol are separated from all interfering peaks in about 15 min. The standard curve is linear (r = 1.000) for both fluoxetine and norfluoxetine concentrations over the range of 25 to 800 micrograms/L. Between-run CVs for 60 and 200 micrograms/L controls (n = 8) were 6.8 and 4.1% for fluoxetine, and 8.8 and 6.2% for norfluoxetine, respectively. In a study of 24 patients with depression who were being treated with 20-60 mg of fluoxetine per day, fluoxetine and norfluoxetine concentrations in serum, measured during the last three weeks of treatment, were 47-469 micrograms/L and 52-446 micrograms/L, respectively.
Production of murine hybrid-hybridomas secreting bispecific monoclonal antibodies for use in urease-based immunoassays.
Authors: Takahashi M , Fuller S A
Published: January 2020

To produce bispecific antibodies (BiAbs) for enzyme immunoassay (EIA) to replace antibody-enzyme conjugates, we developed a panel of 8-azaguanine/ouabain-resistant anti-urease variant hybridoma cell lines for use in hybridoma-hybridoma fusions. These variants represent mouse immunoglobulin subclasses IgM, IgG1, IgG2a, and IgG2b and have growth rates equal to those of the parental hybridomas. We fused an anti-urease-secreting variant hybridoma with an anti-human choriogonadotropin (hCG)-secreting hybridoma (both of IgG1 subclass) and selected the desired product with growth media containing hypoxanthine-aminopterin-thymidine (HAT) and ouabain. Over 95% of the resulting hybrids secreted anti-urease, and 60% of these secreted anti-hCG. The bispecific nature of secreted antibodies was demonstrated in a simultaneous EIA where BiAbs, hCG, and urease (EC 3.5.1.5) were incubated together in anti-hCG-coated microwells. As little as 25 int. units of hCG per liter could be reliably detected, which is equivalent to that for antibody-enzyme conjugates in EIA.
Effect of storage temperature and shaking rate on pH and blood-gas results for two quality-control products.
Authors: Ryder K W , Jay S J , Glick M R , Woods J R
Published: January 2020

Directions for pre-analytical handling of ampules of two commercially available aqueous quality-control products (contrlL and G.A.S.) contain vague instructions such as "store at room temperature" and "shake vigorously" before analysis. We examined the effect of different storage temperatures (25, 31, and 38 degrees C) and shaking rate (one, two, and four shakes per second) on pH and blood-gas results. For both products, increasing the storage temperature significantly decreased pO2 results, the magnitude of the bias being greatest for those solutions with the highest O2 tensions. However, increasing the shaking rate partly offset this bias. Increasing storage temperature also decreased results for pCO2 and increased results for pH for both manufacturers' ampules with the highest CO2 tensions, and this bias was not offset by increasing the shaking rate. We conclude that both storage temperature and shaking rate must be precisely defined and carefully monitored before these products are used in a quality-control program.
Laboratory assessment of poisoning with a carbamate insecticide.
Authors: Remaley A T , Hicks D G , Kane M D , Shaw L M
Published: January 2020

We discuss a case of a 17-year-old white male who intentionally ingested a tick and flea insecticide and was admitted to the emergency room unconscious, with signs and symptoms of cholinergic toxicity. Capillary gas chromatography and electron-impact mass fragmentographic analysis of the patient's urine and serum demonstrated the presence of poly-ethylene glycol and propoxur (o-isopropoxyphenyl N-methyl-carbamate), a carbamate-based cholinesterase inhibitor commonly used in insecticides. The patient fully recovered, but only after a complicated hospital course. We also discuss the laboratory assessment and clinical treatment of poisoning with carbamate and organophosphate insecticides.
Hypophosphatasia: biochemical screening of a Dutch kindred and evidence that urinary excretion of inorganic pyrophosphate is a marker for the disease.
Authors: Macfarlane J D , Poorthuis B J , van de Kamp J J , Russell R G , Caswell A M
Published: January 2020

Hypophosphatasia is an inherited disease in which a deficiency of the bone/liver/kidney or tissue nonspecific isoenzyme of alkaline phosphatase (AP; EC 3.1.3.1) occurs. All forms of the disease are characterized clinically by defective mineralization. Several biochemical abnormalities are associated with the deficiency of AP activity, e.g., increased urinary excretion of inorganic pyrophosphate (PPi) and phosphoethanolamine (PEA). Measurement of these analytes in kindreds of patients with hypophosphatasia may be useful in identifying carriers, and in understanding the inheritance of the disease. We studied biochemically 22 members of the kindred of a 24-year-old woman with hypophosphatasia. We measured activity of AP in serum and leukocytes, and the urinary excretion of PPi and PEA. Within this kindred, urinary excretion of PPi appeared to indicate carrier status, and among the clinically normal adults, values for this analyte were inversely correlated with the activity of AP in serum. These results suggest that urinary excretion of PPi is sensitive to subtle changes in the activity of AP.
One-step protocol for assays of total and direct bilirubin with stable combined reagents.
Authors: Fossati P , Ponti M , Prencipe L , Tarenghi G
Published: January 2020

We describe an improved colorimetric method for assays of total and direct bilirubin in serum. Bilirubin reacts with diazotized sulfanilic acid in an acidic medium to form a blue azopigment. Total bilirubin is assayed in the presence of reaction accelerators (caffeine, urea, and citric acid), direct bilirubin in their absence. The azo compound so formed is read at the same wavelength (570 nm) in both assays. A sample blank is run in parallel. Standard curves are linear for total and direct bilirubin concentrations up to 513.0 and 256.5 mumol/L, respectively. The method is characterized by (a) use of the same protocol for both assays, i.e., a one-step procedure with short reaction time (5 min at room temperature), and (b) use of a single working solution, which, refrigerated, is stable for one month. The method is reliable, yields results that compare closely with those of the classical Jendrassik--Gróf method, is suitable for routine use, and lends itself to automation.
Effect of citrate on the urinary excretion of calcium and oxalate: relevance to calcium oxalate nephrolithiasis.
Authors: Cowley D M , McWhinney B C , Brown J M , Chalmers A H
Published: January 2020

Studies in 24 recurrent oxalate stone-formers have shown that values for urinary calcium excretion for this group on at-home diets vary significantly (P less than 0.001) more than values for creatinine excretions. By placing stone-formers on controlled in-hospital diets and measuring their calcium excretions, we were able to predict probable outpatient hypercalciuria (greater than 7.5 mmol/day) with a sensitivity of 95% and a specificity of 95%. In this study, the renal loss of calcium during low-calcium diets was proportional to the absorptive hypercalciuria during high-calcium diets. Calcium loading experiments in fasted stone-formers and normal subjects indicated that citrate, at citrate:calcium molar ratios ranging from 0.12 to 1, stimulated urinary calcium excretion more than did calcium carbonate loading alone. In addition, citrate also significantly (P less than 0.05) increased the excretion of urinary oxalate by two normal subjects for a given load of calcium oxalate. Malabsorption of citrate and possibly other hydroxycarboxylic acids may thus predispose to oxalate nephrolithiasis by promoting calcium and oxalate absorption.
Extraction of intracellular nucleosides and nucleotides with acetonitrile.
Authors: Au J L , Su M H , Wientjes M G
Published: January 2020

Extraction of intracellular nucleosides and nucleotides with acetonitrile (ACN) and water was compared with extraction with 60 g/L perchloric acid (PCA), followed by neutralization with KOH, 1 mol/L. Freshly isolated rat bone-marrow and intestinal cells were incubated with radiolabeled 5-fluorouracil (FUra) and 5-fluorodeoxyuridine. The ribose and deoxyribose nucleosides and nucleotides of FUra, and ADP and ATP in the soluble extracts were separated by HPLC and measured by scintillation counting or ultraviolet absorbance. The insoluble precipitates were digested in 1 mol/L NaOH and analyzed for the radioactive macromolecule-bound nucleotides. Both extraction methods yielded the same total (i.e., soluble and insoluble) amount of radioactivity. However, the ACN method yielded significantly more FUra nucleosides and triphosphate nucleotides and ATP in the soluble fraction, and more proteins and macromolecule-bound nucleotides in the insoluble fraction. In the PCA method, the soluble fraction contained more monophosphate nucleotides and ADP than in the ACN method. The PCA extraction procedure promoted decomposition of ATP to ADP and interfered with the ion-pairing reversed-phase HPLC assay. The ACN extraction is faster (less than 5 min) than the PCA extraction (greater than 10 min). Moreover, the ACN in the soluble extract fraction can be removed by evaporation and thus does not interfere with the HPLC analysis. Thus the ACN method evidently is suitable for extraction of nucleosides and nucleotides.
Distribution of cyclosporin A metabolites among plasma and cells in whole blood: effect of temperature, hematocrit, and metabolite concentration.
Authors: Lensmeyer G L , Wiebe D A , Carlson I H
Published: January 2020

Drug-free whole-blood samples supplemented with the cyclosporines and samples from 10 transplant patients receiving cyclosporin A (CsA) were equilibrated at 4, 22, and 37 degrees C for 2.5 h; the plasma and cells were separated; and the fractions were assayed by high-performance liquid chromatography (HPLC). Partitioning of CsA and metabolites among plasma and cells was diverse and not always predictable for patients' samples. Overall, although widely variable, greater than 50% of the total concentration of metabolites M1, M8, M9, M10, M16, M17, U1, U8, and U9 in whole blood was associated with the cells, whereas greater than 50% of M13, M18, M21, M25, M26, M203-218, U2, U3, U4, U5, U6, and U7 was associated with plasma. A decrease in hematocrit from 47.8% to 24%, an increase of the sample's temperature (from 4 to 37 degrees C), or an increase in analyte concentration (usually greater than 500 micrograms/L for selected metabolites) increased the relative portion associated with plasma in a nonlinear fashion. Parent CsA was most influenced by these changes; its relative concentrations in plasma varied from 18% to 50%. These data support the preferential use of whole blood for therapeutic monitoring of "cyclosporines." Through additional studies, we suggest possible mechanisms affecting the distribution phenomenon and ascribe chemical structure-distribution relationships.
Immunoradiometric assay for alpha gamma- and gamma gamma-enolase (neuron-specific enolase), with use of monoclonal antibodies and magnetizable polymer particles.
Authors: Paus E , Nustad K
Published: January 2020

Monoclonal antibodies were raised against neuron-specific enolase, gamma gamma-enolase, and used in an immunoradiometric assay (IRMA), with mono-disperse magnetizable particles as the solid phase. The assay's sensitivity was 0.4 microgram/L and the interassay coefficient of variation was less than 5% in the working range from 0.4 to 170 micrograms/L. Compared with our radioimmunoassay based on polyclonal antibodies, the incubation time is shorter, and precision and sensitivity are improved. The IRMA also improved detection of neuron-specific enolase in sera from patients with lung cancer without a concomitant change in measured enolase in the reference population. The better sensitivity of the IRMA results from its ability to measure alpha gamma- and gamma gamma-enolase with equal response. Ninety percent of the small-cell lung carcinoma patients (36 of 40) had increased values before treatment, compared with 7% of non-small-cell lung carcinoma patients (8 of 114).
Stratus automated creatine kinase-MB assay evaluated: identification and elimination of falsely increased results associated with a high-molecular-mass form of alkaline phosphatase.
Authors: Butch A W , Goodnow T T , Brown W S , McClellan A , Kessler G , Scott M G
Published: January 2020

We compared the performance of an automated assay of creatine kinase MB isoenzyme (CK-MB) mass (Stratus) with that of a CK-MB enzymatic assay routinely used at our institutions. Both of these assays use the same CK-MB-specific monoclonal antibody to immunocapture CK-MB, thus providing a direct means of comparing a mass assay with an activity assay. Routine CK-MB measurements for 206 samples within the analytical range of both assays revealed the following relationship: Stratus (micrograms/L) = 0.67 (activity U/L) + 0.18 (r = 0.95, Sx.y = 4.45). The linearity, sensitivity, and precision of the Stratus assay were acceptable for routine clinical use. Icteric, lipemic, and hemolyzed samples do not interfere with the assay. During our evaluation we identified a single, clinically significant false-positive sample. Because this patient had alkaline phosphatase values greater than 1100 U/L, we investigated additional samples with increased activities of alkaline phosphatase and found that samples from 12 of 23 patients selected for alkaline phosphatase values greater than 460 U/L produced falsely increased CK-MB values. We determined that a membrane-associated, high-molecular-mass form of alkaline phosphatase was a cause of these falsely increased values and instituted an approach to identify falsely increased Stratus CK-MB values. Samples from 23 of 1933 patients were falsely increased, the increase being clinically significant in samples from 14 of these patients. Consultation with the manufacturer resulted in the successful reformulation of the substrate/wash solution to minimize interferences from high-molecular-mass forms of alkaline phosphatase.
Solid-phase enzymoimmunoassay for osteocalcin in human serum or plasma, with use of a monoclonal antibody.
Authors: Power M J , Fottrell P F
Published: January 2020

In this solid-phase enzymoimmunoassay on microtiter plates for osteocalcin in serum or plasma, we use an osteocalcin-horseradish-peroxidase conjugate and a monoclonal antibody raised against bovine osteocalcin. We thoroughly standardized the assay for measurement of osteocalcin in both serum and plasma, demonstrating independence of sample volume, and determining the analytical recovery and within-and between-assay CVs. The detection limit was between 0.6 and 1.1 micrograms/L and the ED50 was 16 micrograms/L for a 5-microL sample volume. The intra-assay CV over the range 3 to 74 micrograms/L was less than or equal to 15%. The interassay CV over the range 3.6 to 46 micrograms/L was less than or equal to 16%. Results by this assay and by an in-house radioimmunoassay in which the same monoclonal antibody was used correlated well (r2 = 0.948). Osteocalcin concentrations in serum and plasma as measured with the present assay agreed well with published values.
Fluorometric method for phenylalanine microplate assay adapted for phenylketonuria screening.
Authors: Gerasimova N S , Steklova I V , Tuuminen T
Published: January 2020

We adapted the method of McCaman and Robins for fluorometry of phenylalanine to a microplate assay for routine phenylketonuria screening. Sensitivity is 15 mumol/L for the plasma assay and 30 mumol/L for the dried blood-spot assay, with CV less than 10% for both assays. Results for human plasma by microplate assay correlated well (r = 0.99) with results of amino acid analyzer determination of phenylalanine. When measurements are performed in an automated reader, the microplate assay has considerable advantages over conventional measurements in cuvettes: smaller volumes of reagents and automation enabling high throughput and convenience. Because the described method is a quantitative one, we can postulate that, compared with the semiquantitative Guthrie inhibition bioassay, this microplate assay is more reliable, easier to perform, and about twofold less costly.