bluuebunny/crossref_metadata_2025 huggingface dataset:
Urinary p-aminobenzoic acid determined in the pancreatic function test by liquid chromatography, with electrochemical detection.
Authors: Ito S , Maruta K , Imai Y , Kato T , Ito M , Nakajima S , Fujita K , Kurahashi T
Published: January 2020

We describe a liquid-chromatographic procedure for determining urinary p-aminobenzoic acid (PABA) in the exocrine pancreatic function test. The urine specimen, collected for 6 h after oral administration of N-benzoyl-L-tyrosyl-p-aminobenzoic acid, is hydrolyzed in 4 mol/L NaOH containing m-hydroxybenzoic acid as internal standard, to free PABA metabolites, then diluted and injected into the chromatograph. PABA and the internal standard are eluted from a reversed-phase C18 column with phosphate buffer (0.2 mol/L, pH 3.5)/acetonitrile, 87.5/12.5 (by vol), and detected with an electrochemical detector at +1100 mV. Urinary PABA concentration as measured by this method agreed well with that by colorimetry involving p-dimethylaminocinnamaldehyde. The present procedure is specific for PABA, precise, and fairly rapid. We hope that the present method will minimize instances of interference from drugs, which cannot be avoided in the colorimetric method.
Measurement of alpha-fetoprotein in maternal serum: three commercial radioimmunoassay kits and two non-commercial radioimmunoassays compared.
Authors: Forest J C , Verreault F , Pouliot M
Published: January 2020

We evaluated three commercial radioimmunoassay kits (Amersham, Dainabot, Clinical Assays) and two non-commercial methods for determining alpha-fetoprotein in maternal serum during pregnancy. All five procedures were found to be acceptable with respect to practicability, sensitivity, linearity, and precision. Similar results were obtained with Dainabot, Clinical Assays, and the two non-commercial methods, but the Amersham method revealed a proportional error, results being about 20% lower than those by the other methods. Use of the international unit system is suggested for reporting results for AFP, to facilitate comparison between methods and laboratories.
Incidence and mechanism of spurious increase in serum thyrotropin.
Authors: Howanitz P J , Howanitz J H , Lamberson H V , Ennis K M
Published: January 2020

We previously reported spuriously high values for thyrotropin (TSH), presumably owing to an antibody in human serum that reacts with both reagent rabbit antibodies in an immunoradiometric assay (IRMA). We used this IRMA to measure TSH. Five of 20 sera from laboratory animal handlers showed spuriously high values. When we added 2 mL of nonimmune rabbit serum per liter to the labeled IRMA rabbit antibody reagent and reassayed the five affected specimens, the results were within the reference interval. Smaller additions partly corrected the TSH values, but nonimmune sera of eight other species had no effect. Substitution of goat solid-phase antibody decreased, but did not eliminate, the increases in TSH in three of the five affected sera. Chromatographic properties, results of rheumatoid factor testing, and measurement of human anti-rabbit immunoglobulin suggest that the interference is ascribable to an antibody of the IgG class that reacts with rabbit antibody. Evidently, antibody interference with IRMA procedures may be common in certain populations. It can be avoided by including nonimmune serum corresponding to the species used to produce reagent antibody.
Total estriol in serum and plasma as determined by radioimmunoassay.
Authors: Brooks C T , Copas J B , Oliver R W
Published: January 2020

We present and discuss the results of a statistical study of a set of radioimmunoassay data obtained from parallel determinations of the total estriol content of samples of serum and plasma prepared from 98 specimens of whole blood, taken from pregnant woman. Estriol concentrations in the serum exceed by an average of 7% the values found for the plasma prepared from the same whole-blood specimen. The clinical significance of this analytical finding is discussed, and we conclude with the recommendation that serum is the fluid of choice for this assay.
Radioimmunoassay of "free thyroxin" in dried blood spots on filter paper - preliminary observations on the effective differentiation of subjects with congenital hypothyroidism from those with subnormal thyroxin-binding globulin and normal subjects.
Authors: Mizuta H , Miyai K , Ichihara K , Amino N , Harada T , Nose O , Tanizawa O
Published: January 2020

In this sensitive, simple method for measuring "free thyroxin" (FT4) in eluates of dried blood spots on filter paper by use of a radioimmunoassay kit (Amerlex Free T4 RIA), the measurable range of FT4 is 1.8 to 57 ng/L (equivalent to the concentration in serum), or 7 to 237 fg/tube. The mean coefficients of variation for within assay-within spots, within assay-between spots, and between assays were 5.3%, 5.0%, and 6.2%, respectively. FT4 in blood spotted on filter paper is stable for at least a month when dried and kept at either -20 degrees C, 4 degrees C, room temperature (about 25 degrees C), or 37 degrees C. The results for FT4 in dried blood spots correlated closely with the free-T4 concentration in serum (r = 0.99). The method can be used to differentiate cases of primary and secondary hypothyroidism from normal subjects and those with subnormal thyroxin-binding globulin. This method may be useful in screening for congenital hypothyroidism, because sample-retesting is not necessary.
Micellar improvement of the calmagite compleximetric measurement of magnesium in plasma.
Authors: Abernethy M H , Fowler R T
Published: January 2020

Magnesium in plasma is determined by diluting a 50-microL sample with 5.0 mL of stable calmagite reagent containing the amphoteric detergent Empigen BB, and buffered with 2-amino-2-methyl-1-propanol at pH 11.5. The calcium response is masked with strontium-buffered [ethylene-bis(oxyethylenenitrilo)]tetraacetate (EGTA), while iron is masked with triethanolamine. The detergent causes an increased separation of the test and blank spectral absorbance bands. The test absorbance peaks, which may be measured immediately, are at 520 nm, while the blank peak shifts from 610 to 655 nm on addition of detergent. The blank absorbance at 520 nm is relatively decreased by the detergent. This allows the use of a more concentrated calmagite reagent, which in turn extends the linear relation of absorbance to magnesium concentration to 5 mmol/L in plasma. This method (y) has been compared with atomic absorption procedure (x) of McDonald and Watson (Clin, Chim. Acta 14: 233, 1966) and gives favorable regression statistics (y = 1.001X + 0.057 mmol/L).
Rapid assay for amino acids in serum or urine by pre-column derivatization and reversed-phase liquid chromatography.
Authors: Turnell D C , Cooper J D
Published: January 2020

This method for estimating clinically important amino acids in serum or urine within 40 min involves o-phthalaldehyde/2-mercaptoethanol derivatization and reversed-phase "high-pressure" liquid chromatography. Homocysteic acid is an internal standard, and homoserine and norvaline are reference peaks. For all the amino acids estimated, the between-run coefficients of variation ranged from 2.0 to 13.5%, and the mean analytical recoveries from both serum and urine samples was 101%. Peak areas vary linearly with concentration up to 1500 mumol/L for all the amino acids assayed. The limit of detection for each amino acid was estimated to be 38 fmol.
Two-dimensional electrophoretic analysis of muscle contractile proteins in patients with idiopathic scoliosis.
Authors: Whalen R G , Ecob M S
Published: January 2020

Muscle biopsies from patients with idiopathic scoliosis were analyzed by two-dimensional gel electrophoresis. Both isoelectric focusing and nonequilibrium pH-gradient electrophoresis were used in the first dimension of the separations, to obtain maximum information from each biopsy specimen. The identification of the various contractile proteins and their isoforms was considered, based in part by analogy with studies on animal models. The myosin light chains and the TN-C subunit of troponin were the most useful qualitative markers indicative of the muscle phenotype. Convex and concave paraspinal muscles were analyzed from 13 patients with idiopathic scoliosis. Eight of the patients had light chain and TN-C subunits characteristic of both fast and slow contracting muscle. However, five patients had mixtures of fast and slow proteins only in the concave muscle, the convex muscle being composed exclusively of slow light chains and slow TN-C. Possible mechanisms leading to such a situation and the implications of these results concerning the muscle pathology are discussed.
Lymphocyte, monocyte, and granulocyte proteins compared by use of two-dimensional electrophoresis.
Authors: Gemmell M A , Anderson N L
Published: January 2020

We compared cellular proteins from normal human blood lymphocytes, monocytes and granulocytes, using high-resolution two-dimensional electrophoresis. The leukocytes were isolated from peripheral blood by centrifugation on density step gradients (yielding a fraction of purified granulocytes and a lymphocyte/monocyte mixture) and monocytes were subsequently separated from lymphocytes by virtue of their adherence to plastic. Wright-stained smears indicated that each of the three resulting fractions was 90 to 95% pure. The cells were labeled with [35S]methionine after various intervals in culture, then solubilized and analyzed by two-dimensional electrophoresis. Although most proteins in each cell type are common to all three, there are nevertheless several specific marker proteins that distinguish one cell type from another. We also examined the appearance of these markers in three lines of cultured cells from humans (GM607, a B-lymphoblastoid line; HL-60, a promyelocytic leukemic line; and 1494, a normal skin fibroblast).
Interference by ascorbic acid in test systems involving peroxidase. I. Reversible indicators and the effects of copper, iron, and mercury.
Authors: White-Stevens R H
Published: January 2020

I describe the mechanism whereby ascorbic acid can hamper test systems involving a peroxide-generating system, peroxidase, and a benzidine-type indicator. In test media such as urines, abnormally high concentrations of ascorbic acid may give rise to false negatives in the determination of analytes such as glucose. Absorbance measurements in solution or reflectance measurements on commercially available paper reagent strips demonstrate either inhibition of visible activity by ascorbic acid or a lag time in the development of oxidized indicator color. The duration of the lag time is proportional to the ascorbic acid concentration, is inversely proportional to the enzyme concentration, and is also affected by concentrations of hydrogen peroxide and o-tolidine indicator. The same results were seen in both citrate buffer pH 5 and phosphate buffer pH 7. Because the complete system (o-tolidine indicator, hydrogen peroxide, and peroxidase) must be present if the ascorbate is to be oxidized rapidly, this indicates that ascorbic acid inhibits color development by re-reducing oxidized indicator as fast as it is formed; the o-tolidine then acts catalytically in oxidizing ascorbic acid. Added Cu2+ and Fe3+, both known to react with ascorbic acid, had measurable but small effects on the system. In contrast, Hg2+ abolished the ascorbic acid-elicited lag time, even when present in near-stoichiometric concentration. Hg2+ showed little inhibitory effect on the activity of either glucose oxidase or peroxidase. Presumably it rapidly oxidizes ascorbic acid to dehydroascorbate. The reaction of mercuric ion with ascorbate was measured by reflectance measurements of paper reagent strips in addition to absorbance measurements of solution assays; equivalent results were obtained. Incorporation of Hg2+ into reagent strips can thus render both strips and solution diagnostic tests insensitive to interfering substances such as ascorbic acid.
Interference by ascorbic acid in test systems involving peroxidase. II. Redox-coupled indicator systems.
Authors: White-Stevens R H , Stover L R
Published: January 2020

Ascorbic acid hampers some test systems based on use of peroxidase (EC 1.11.1.7) and redox indicators, by producing a lag time in color development. With reversible indicators, no color development occurs during the ascorbic acid lag time. With oxidatively coupled indicator systems, such as 3-methyl-2-benzothiazolinone hydrazone (MBTH) and a suitable coupler such as chromotropic acid (CTA; 4,5-dihydroxynaphthalene-2,7-disulfonic acid), ascorbic acid diminishes the rate of color development, but does not abolish it. The effect of ascorbic acid strongly depends on the reaction pH as well as the nature of the coupler used. The ascorbate-elicited reduction (or lag) in color development was inversely proportional to the concentrations of MBTH and virtually unaffected by changes in CTA coupler concentration. The rate of color development following the lag was directly proportional to the concentration of MBTH but unaffected by the CTA. These observations suggest that peroxidase with H2O2 catalyzes the oxidation and activation of MBTH to an oxidized species (MBTHox). This species is reduced by ascorbic acid and at the same time couples oxidatively with CTA. Thus, the activity during the ascorbate-induced lag time reflects this competition of ascorbic acid and coupler for MBTHox. This study of peroxidase/ascorbate lag time with the redox coupled indicator system has led to the selection of fast couplers that are highly resistant to interference by ascorbic acid. Suitable resistant couplers (e.g., chromotropic acid, Chicago acid, and H acid) appear to be aromatic ring systems with highly activating substituents and directing toward electrophilic aromatic substitution at the ortho and para positions.
Monitoring octanoic and decanoic acids in plasma from children with intractable epilepsy treated with medium-chain triglyceride diet.
Authors: Haidukewych D , Forsythe W I , Sills M
Published: January 2020

We describe a procedure for gas-chromatographic determination of n-octanoic and n-decanoic acids in 100 microL of plasma from children with intractable epilepsy treated with medium-chain triglyceride (MCT) diet. With n-nonanoic acid as the internal standard, the extraction efficiencies for octanoic and decanoic acids were 98 and 105%, respectively. Within-run CVs for octanoic acid at 0.5, 1.0, 2.5, 5.0, 7.5, and 10.0 micrograms/0.1 mL were 8.8, 7.9, 8.5, 6.5, 4.3, and 5.7%, respectively. For decanoic acid at identical concentrations, the CVs were 10.0, 7.4, 4.9, 4.0, 2.6, and 3.5%, respectively. For 10 children on MCT diet (45.9% of calories supplied as MCT) the mean concentrations of octanoic and decanoic acids were 44.2 and 27.0 mg/L, respectively. Presence of relatively "high" quantities of these acids in plasma may contribute to seizure control when MCT diet is prescribed for intractable childhood epilepsy.
Computer analysis of two-dimensional gels: semi-automatic matching.
Authors: Miller M J , Vo P K , Nielsen C , Geiduschek E P , Xuong N H
Published: January 2020

We describe a computer program system for finding, quantitating, and matching the protein spots resolved on a two-dimensional electropherogram. The programs that locate and quantitate the incorporation of radioactivity into individual spots are totally automatic, as are the programs for matching protein spots between two exposures of the same gel. A semi-automatic method is used to match protein spots between different gels. This procedure is quite fast with the use of a computer-graphic display, which is also helpful in the editing process. A data base is set up and programs have been written to correlate matched protein spots from multi-gel experiments and to efficiently plot out quantitative data from sequences of equivalent spots from many gels or even many multi-gel experiments. The practical use of this system is discussed.
Two-dimensional gel electrophoresis of serum specimens from a normal population.
Authors: Tracy R P , Currie R M , Young D S
Published: January 2020

We examined sera from a normal population by two-dimensional gel electrophoresis, to establish the normal pattern of serum proteins and to investigate genetic polymorphisms. With such information in hand, specimens from patients with certain diseases may be readily evaluated. Towards this goal, we optimized the ISO-DALT system (Proc. Natl, Acad, Sci, USA 74: 5421--5425, 1977) for routine phenotyping of alpha 1-antitrypsin, haptoglobin, GC-globulin, alpha 2-HS-glycoprotein, and transferrin, as well as a previously unknown polymorphic protein. We examined the effects of aging the specimens for 2 h at room temperature (no changes) or at -20 degrees C for several months (small changes), as well as serum/plasma differences and the effect of protease inhibitors. Silver-stain methods were modified to allow simultaneous staining of 10 gels, with reasonably good reproducibility of stain intensity. We quantitated silver-stained gels by densitometry of photographic transparencies. Very small samples suffice with this stain (0.5 microL of serum or plasma), allowing the use of "finger-stick" methods instead of venipuncture, yet the patterns are better resolved and easier to read than those for 10-microL specimens processed on gels stained with Coomassie Blue. Our techniques for rapidly removing albumin and IgG allow the investigator to examine areas on the gel that ordinarily are obscured. The region of haptoglobin has been examined by using serum from an ahaptoglobinemic donor. Finally, we present an expanded "normal" map illustrating the composite protein pattern.
Reproducibility and quality assurance of two-dimensional gel electrophoresis of serum specimens.
Authors: Tracy R P , Currie R M , Young D S
Published: January 2020

Currently we are using two different ISO-DALT two-dimensional gel electrophoresis systems, designated MC-Iso 1 and MC-Iso 2, for the analysis of serum and plasma samples. Here we report quality-assurance data for both of these systems. CV values for the slopes of the pH gradient (ISO dimension) are 5.6% of less; CV values for the slopes of the molecular-mass curves (log Mr vs relative mobility in the DALT dimension) are 3.4% or less. We examined the various steps of the analysis in detail for reproducibility and protein loss, using radiolabeled albumin, alpha 2-macroglobulin, and beta 2-microglobulin. Generally, in the first dimension, less protein enters the MC-Iso 2 gels (our routine system in which silver stain is used) than enters the MC-Iso 1 gels (our wide-range system for myeloma serum samples, in which the gel is stained with Coomassie Blue), on the average, 87% as much. The CV at this stage for both systems is 5--8%. During equilibration, considerable amounts of protein are lost (approximately 30% in 10 min) from the ISO gel, and the reproducibility is also decreased. Resolution in the DALT dimension has, in most cases, little or no effect on either recovery or reproducibility. Overall, for most proteins expected to appear in an ISO gel of a given pH range, approximately 50--60% of the starting material may be expected to reside in the sodium dodecyl sulfate slab gel, under our conditions. The two most important variables affecting recovery are the concentration of the NaOH (used as catholyte) and the pH of the starting sample. The overall CV for the process is between 8 and 12%.
Proteins of human amniotic fluid. II. Mapping by two-dimensional electrophoresis.
Authors: Burdett P , Lizana J , Eneroth P , Bremme K
Published: January 2020

In an earlier study we used various electrophoretic techniques to investigate the proteins in amniotic fluid, including two-dimensional electrophoresis. However, the high proportion of albumin dominated the pattern and tended to distort the pH gradient. Improved methodology, based upon the removal of albumin with Blue Sepharose CL6B and the silver-staining technique, has been used in the present work. These modifications have minimized problems of distortion. We have extended our work in a attempt to complete the two-dimensional map of the proteins in amniotic fluid. Over 200 proteins were seen, ranging in molecular mass from 10 000 to 100 000, including a relatively high proportion of polypeptides of low-molecular mass. The spots seen in the map are discussed in relation to some of the proteins, peptides, and hormones that have been reported in amniotic fluid. Eventually, it is hoped that polypeptides will be found that will provide better indicators of the condition of the fetus.
Proteins of human urine. III. Identification and two-dimensional electrophoretic map positions of some major urinary proteins.
Authors: Edwards J J , Tollaksen S L , Anderson N G
Published: January 2020

We mapped the proteins of human urine by high-resolution two-dimensional electrophoresis, utilizing the ISO-DALT system. Wide-range pH gradients and narrow-range acid gradients were both used in the first-dimension separations. The patterns revealed proteins ranging in relative molecular mass from 10 000 to 90 000. Proteins identified in the map included transferrin, albumin, hemopexin, alpha 2-HS glycoprotein, alpha 1-antitrypsin. Gc globulin, alpha 1-acid glycoprotein, Zn alpha 2-glycoprotein, retinol binding protein, beta 2-microglobulin, the immunoglobulin light chains, and MAUP (most acid urinary protein). The use and utility of internal-charge and molecular-mass standards are described. We used electrophoretic transfer of proteins to nitrocellulose sheets and subsequent detection by immunological methods to identify some proteins.
Familial porphyria cutanea tarda: the pattern of porphyrins formed from porphobilinogen by hemolysates.
Authors: Alleman M A , Wilson J H , van den Berg J W , Edixhoven-Bosdijk A , van Gastel-Quist L M
Published: January 2020

Porphyria cutanea tarda is thought to result from an inherited deficiency of uroporphyrinogen decarboxylase (EC 4.1.1.37) in some patients. Present methods for determining uroporphyrinogen decarboxylase activity are time consuming, so we examined the pattern of porphyrins formed from porphobilinogen by hemolysates as a possible marker for hereditary porphyria cutanea tarda. After the hemolysates are incubated with porphobilinogen, the porphyrins are converted to their methyl esters and examined by liquid chromatography, with fluorometric detection. The porphyrinic patients examined, and some of their relatives, showed a characteristic pattern of porphyrin production, with high uroporphyrin/coproporphyrin and (uroporphyrin + heptacarboxylic porphyrins)/coproporphyrin ratios, at least partly ascribable to increased uroporphyrinogen I synthetase (EC 4.2.1.8) activity in patients' hemolysates, and also to a relative deficiency of uroporphyrinogen decarboxylase. Examination of the pattern of porphyrins produced from porphobilinogen by hemolysates is a suitable technique for detecting asymptomatic individuals with porphyria cutanea tarda.
Quantitation of beta-lipoprotein in cord serum by rate nephelometric immunoassay: a potential screening test for familial hypercholesterolemia.
Authors: Brewster T G , Waite D J , Hudson G A
Published: January 2020

We measured beta-lipoprotein in 232 cord sera by rate nephelometry and compared the results with those obtained by electroimmunoassay. The mean (mean +/- two standard deviations) was 544 (293-1009) mg/L by rate nephelometry and 503 (265-955) mg/L by electroimmunoassay. The between-assay correlation coefficient was 0.891. Between-run coefficients of variation were 3.5-4.4% for nephelometry and 11-14% for electroimmunoassay. Nephelometry was faster, more precise, and less labor intensive than electroimmunoassay. For these reasons, rate nephelometry appears to be a more suitable assay system for mass screening, should detection of familial hypercholesterolemia prove feasible and desirable.
Simplified fluorometry of total metanephrines in urine.
Authors: Christenson R H , McGlothlin C D , Hedrick R , Cate J C
Published: January 2020

We describe a simplified fluorometric method for quantitatively determining metanephrines in urine. The method is based on the elution of metanephrines from an ion-exchange column followed by production of a fluorescent derivative that has excitation and emission maxima of 405 nm and 505 nm, respectively. Metanephrine concentrations as great as 7.0 mg/L were within the linear range of the assay. Analytical recovery of metanephrines was 82-87%. Cross contamination by catecholamines was insignificant. The upper range of normal was 520 micrograms/24 h. Within-run and between-run CV's were 5% and about 9%, respectively. Twenty-four-hour urine specimens from five patients with pheochromocytoma showed above-normal amounts of the metanephrines by this method.
Immune complexes: characteristics, clinical correlations, and interpretive approaches in the clinical laboratory.
Authors: Ritzmann S E , Daniels J C
Published: January 2020

Immune-complex-mediated injury is thought to play a role in diseases such as rheumatoid arthritis, systemic lupus erythematosus, serum sickness, various infectious diseases, and malignancies. With increased appreciation of the biological and pathological significance of circulating immune complexes has come efforts to develop appropriate techniques for identifying and measuring them. Common approaches exploit such phenomena as the attachment of complement components to antigen-antibody complexes, the presence of specialized receptors for immune complexes at the surface of cells, and the ability of rheumatoid factor to bind with immune complexes. This variety of assay systems for immune complexes has yielded abstruse results in numerous human pathological conditions. Unfortunately, these results seldom correlate with one another in a given disease. Thus, use of a panel of immune complex assays has been recommended. Indirect consequences of immune complex disease may still be appraised and evaluated with some confidence in clinical medicine: measurements of C3 and C4, cryoglobulins, serum viscosity, and turbidity of serum samples. Measurement of immune complexes may be useful in diagnosis, prognosis, and therapeutic monitoring, but it is the characterization of immune complexes that holds the greatest potential for better understanding of disease mechanisms.
Oxygen dissociation curves for whole blood, recorded with an instrument that continuously measures pO2 and sO2 independently at constant t, pCO2, and pH.
Authors: Zwart A , Kwant G , Oeseburg B , Zijlstra W G
Published: January 2020

We describe a method for recording oxygen dissociation curves for whole-blood specimens. The blood sample is placed in a thermostated measuring chamber, and pO2 and SO2 are measured continuously by polarography and by reflectometry, respectively. During the recording of an oxygen dissociation curve, the pO2 and SO2 signals are stored in a data-acquisition system, while pH, pCO2, and temperature are kept constant. Determination of precision and error discussion indicated that the coefficient of variation (CV) of the determination of the oxygen dissociation curve is mainly determined by the error in the measurement of SO2. The overall CV of pO2 values belonging to the lower, mid-, and upper parts of the SO2 range is estimated to be about 2.6, 3.1, and 2.1%, respectively. In practice the measurements are about 30% more precise than estimated. With our method, the fixed-acid-induced Bohr effect (H+ factor) can be determined over the entire SO2 range with much greater precision than hitherto.
Dexamethasone suppression of adrenocortical function.
Authors: Sharp A M , Handelsman D J , Ristuccia R M , Turtle J R
Published: January 2020

To investigate the potency of dexamethasone in suppressing adrenocortical function, we gave various evening doses to laboratory staff members. Plasma corticotropin, cortisol, and dehydroepiandrosterone sulfate concentrations were measured the following day, to quantitate the degree of suppression after both short- and long-term treatments. We found that the minimum amount for effective suppression was 1 mg for a single evening dose, 0.5 mg per day for a long-term evening dose. On these regimens plasma corticotropin and cortisol were consistently suppressed, but plasma dehydroepiandrosterone sulfate was suppressed only with long-term treatment.
The Hycel-M multichannel analyzer--a model for evaluation.
Authors: Arntsen K W , Deacon A C , Worth H G
Published: January 2020

We evaluated the Hycel-M Multichannel Analyzer on behalf of the British Department of Health and Social Security. The purpose of the evaluation was to determine the precision of the instrument over a prolonged period of time, to compare its results with those of well-tried laboratory procedures, and to determine how the instrument would perform under the rigors of routine analysis. In addition, experiments were designed and carried out to test specifically certain new features of the instrument. Some new approaches to the analysis of the data generated have been examined and are discussed. The findings are presented as a demonstration of an evaluation procedure.
Hydrolysis by human alpha-amylase of p-nitrophenyloligosaccharides containing four to seven glucose units.
Authors: David H
Published: January 2020

Kinetic and "high-pressure" liquid-chromatographic studies were performed on the reaction of human pancreatic and salivary alpha-amylase (EC 3.2.1.1) with p-nitrophenyloligosaccharides containing four to seven glucose units. The kinetic studies indicate that, as the temperature increases from 25 to 37 degrees C, the apparent Km of the enzyme for a given substrate increases slightly. However, as the number of glucose units in the substrate increases, the apparent Km decreases. The apparent Vmax increases as the temperature increases, and decreases as the number of glucose units in the substrate increases. In contrast, chromatographic data indicate that the relative rate of hydrolysis by alpha-amylase is pG7 greater than pG6 greater than pG5 greater than pG4. The differences between the kinetic and the chromatographic results are discussed. p-Nitrophenyl-alpha-maltopentaoside and p-nitrophenyl-alpha-maltohexaoside are superior to the other p-nitrophenyloligosaccharides as substrates for determining alpha-amylase activity in an alpha-glucosidase-coupled assay system.
A multi-stage Gaussian transformation algorithm for clinical laboratory data.
Authors: Boyd J C , Lacher D A
Published: January 2020

We have developed a multi-stage computer algorithm to transform non-normally distributed data to a normal distribution. This transformation is of value for calculation of laboratory reference intervals and for normalization of clinical laboratory variates before applying statistical procedures in which underlying data normality is assumed. The algorithm is able to normalize most laboratory data distributions with either negative or positive coefficients of skewness or kurtosis. Stepwise, a logarithmic transform removes asymmetry (skewness), then a Z-score transform and power function transform remove residual peakedness or flatness (kurtosis). Powerful statistical tests of data normality in the procedure help the user evaluate both the necessity for and the success of the data transformation. Erroneous assessments of data normality caused by rounded laboratory test values have been minimized by introducing computer-generated random noise into the data values. Reference interval endpoints that were estimated parametrically (mean +/- 2 SD) by using successfully transformed data were found to have a smaller root-mean-squared error than those estimated by the non-parametric percentile technique.
Use of alumina, sephadex G10, and ion-exchange columns to purify samples for determination of epinephrine, norepinephrine, dopamine, homovanillic acid, and 5-hydroxyindoleacetic acid in urine.
Authors: Westerink B H , Bosker F J , O'Hanlon J F
Published: January 2020

We investigated the usefulness of small Sephadex columns, prepared in Pasteur pipettes, in purifying samples to be analyzed for catecholamines, homovanillic acid, and 5-hydroxyindoleacetic acid. After free catecholamines in urine samples were purified on alumina followed by Sephadex G10, a reliable and simultaneous quantification of epinephrine, norepinephrine, and dopamine was achieved by using high-performance liquid-chromatography with electrochemical detection. Purification of urine on Bio-Rad prepacked ion-exchange columns followed by Sephadex G10 resulted in a reliable, fast (200 samples processed per week) method for determination of free catecholamines in urine. Analytical recoveries of both methods were between 80 and 95%, with a CV of about 3%. Single purification on Sephadex G10 sufficed to allow simultaneous determination of homovanillic acid and 5-hydroxyindoleacetic acid in urine. Analytical recovery for this method was about 90%, with a CV of about 5%. Sephadex G10 columns, which can be re-used without regeneration for at least one year, appear to have a great potential in clinical chemistry.
Free amino acid content of lymphocytes nd granulocytes compared.
Authors: Fukuda K , Hirai Y , Yoshida H , Nakajima T , Usui T
Published: January 2020

For extracting free amino acids from human leukocytes, we find that disruption of the cells by ultrasonication is more reliable than freezing and thawing. The amount of free amino acids extracted by the latter method depends on the temperature and duration of thawing. We extracted free amino acids from leukocytes of healthy men by ultrasonication and compared their concentrations in lymphocytes and granulocytes. The amino acid content of granulocytes significantly (p less than 0.001) exceeded that of lymphocytes. Of the amino acids extracted from granulocytes, 76% was taurine; for lymphocytes taurine, aspartic acid, and glutamic acid were predominant, respectively composing 44%, 17%, and 26% of the total. Taurine is proposed as an index of cell disruption.
Solid-phase colorimetric determination of potassium.
Authors: Charlton S C , Fleming R L , Zipp A
Published: January 2020

A nonpolar organic film (plasticized polyvinyl chloride) containing the ionophore valinomycin was incubated with an aqueous solution containing potassium ion and a detectable anion (erythrosin B). The amount of erythrosin B retained by the film after washing could be measured by absorbance or reflectance, and was directly related to the potassium concentration. This dye-binding method is quantitative for potassium and is suitable for both aqueous and serum-based solutions. There was no interference by sodium in the range found in serum. Several polyvinyl chloride plasticizers and anionic dyes and some other ionophores were found to be useful. The anion binding is thought to be restricted to the surface.
An innovative technology for "random-access" sampling.
Authors: Smith J , Svenjak D , Turrell J , Vlastelica D
Published: January 2020

A major instrumental limitation to rapid, "random-access" clinical analysis has been cross-contamination at sampling and reagent probes. Use of an immiscible, nonreactive fluid as a positive barrier between the liquid sample and reagent and the interior and exterior surfaces of their respective probes provides an inert, deformable surface that both prevents carryover and ensures accurate delivery. Being totally nonreactive under clinical chemistry conditions, the fluid does not affect the ensuing analysis. Application of this technology to the Technicon RA-1000 (trademark of Technicon Instruments Corp.) system allows transfer of a liquid sample and any of 14 liquid reagents to the reaction vessel with a carryover of less than 1/10(6) with a precision (CV) of 0.5%. Its use provides for a rapid analysis rate of 240 tests per hour, and for transfer of microliter quantities of serum, with carryover of less than 1/8000, again at an average CV of 0.5%.
On-line direct liquid introduction interface for micro-liquid chromatography/mass spectrometry: application to drug analysis.
Authors: Eckers C , Skrabalak D S , Henion J
Published: January 2020

We describe an integrated micro-liquid chromatograph/mass spectrometer (micro-LC/MS) system capable of performing routine determinations for 1--10 ng of drugs and their metabolites extracted from biological fluids. The micro-LC is constructed from conventional "high-performance" liquid-chromatographic instrumentation by using commercially available components. The mass spectrometer is operated in the chemical ionization mode. The direct liquid introduction micro-LC/MS interface can be constructed from commercially available materials. Chromatographic and mass spectral results demonstrate the ability of the micro-LC and micro-LC/MS system to separate and determine multiple components in standards of trace concentrations and in equine urinary extracts. The stability and sensitivity of this micro-LC/MS system are demonstrated through determinations of trichlormethiazide.
Relationship between direct-potentiometric and flame-photometric measurement of sodium in blood.
Authors: Apple F S , Koch D D , Graves S , Ladenson J H
Published: January 2020

The relationship between direct-potentiometric and flame-photometric measurements of sodium has been studied in human sera and other, simpler systems. When water content was varied by using an inert substance (silica), de-ionized sera, or de-ionized individual proteins, the percentage that the flame photometric values were of the direct potentiometric Na+ values was identical to the measured water content. However, in 35 patients the percentage of Na+ values was 99.1% and the water content was 92.0%, a discrepancy of 7.1%. De-ionization of sera removed this discrepancy, apparently because of the lower pH of the de-ionized sera. The percentage of flame-photometric to direct-potentiometric Na+ values varied as a function of pH in pooled sera; in lyophilized and reconstituted sera; and in de-ionized, lyophilized, and reconstituted sera. Four explanations for the discrepancy between the percentage of Na+ values and the water content are discussed: a calibration problem, a measurement artifact, Na+ binding, and water binding. At this time there are no definitive data concerning which explanation is correct. We expect that our finding of a pH dependency for the percentage of measured Na+ values can be used to develop model systems to elucidate the mechanism producing the discrepancy between the percentage of Na+ values and the water content.
Further studies on danazol interference in testosterone radioimmunoassays.
Authors: Sharp A M , Fraser I S , Caterson I D
Published: January 2020

Investigating the interference of danazol in testosterone radioimmunoassays, we measured serum testosterone concentrations in the serum of women receiving danazol, with three testosterone radioimmunoassay kits. All of these showed positive interference by danazol or its metabolites (or both). Values for testosterone obtained with two of the methods (Diagnostic Products Corp. and Farmos Diagnostica) depended on the volume of serum extracted per assay tube. A detailed column-chromatographic method is presented in which testosterone is resolved from danazol and its metabolites.
Multi-enzyme membrane electrodes for determination of creatinine and creatine in serum.
Authors: Tsuchida T , Yoda K
Published: January 2020

An enzyme electrode system for the determination of creatinine and creatine was developed by utilizing three enzymes: creatinine amidohydrolase (CA), creatine amidinohydrolase (Cl), and sarcosine oxidase (SO). These enzymes were co-immobilized onto the porous side of a cellulose acetate membrane with asymmetric structure, which has selective permeability to hydrogen peroxide. Two kinds of multi-enzyme electrodes were constructed by combining a polarographic electrode for sensing hydrogen peroxide and an immobilized CA/Cl/SO membrane or Cl/SO membrane for creatinine plus creatine or creatine, respectively. The multi-enzyme electrodes responded linearly up to 100 mg of creatinine and creatine per liter in human serum. Response time was 20 s in the rate method and the detection limit was 1 mg/L. Only 25 microL of serum sample is required. Analytical recoveries, precisions, and correlations with the Jaffé method were excellent, and the multi-enzyme electrodes were sufficiently stable to perform more than 500 assays. No loss of activity of immobilized enzymes was observed after nine months of storage at 4 degrees C in air.
Time-resolved fluoroimmunoassay of human choriogonadotropin.
Authors: Pettersson K , Siitari H , Hemmilä I , Soini E , Lövgren T , Hänninen V , Tanner P , Stenman U H
Published: January 2020

We describe time-resolved fluoroimmunoassay for human choriogonadotropin involving monoclonal antibodies directed against the beta- and alpha-subunits. The latter antibody was labeled with europium, which was measured by counting for 1 s after the immunoreaction was completed. In the solid-phase sandwich assay, both a one-step and two-step procedure were used; the respective measuring ranges were 0.7-135 and 0.7-350 int. units/L, the latter covering a 500-fold dynamic range. The CV within the assay range was between 4 and 8%, depending on the dose. Cross reactivity with lutropin in the one- and two-step procedures was 1.6% and 1.0%, respectively.
Measurement of glucose in plasma by a differential pH technique.
Authors: Luzzana M , Dossi G , Mosca A , Granelli A , Berger D , Rovida E , Ripamonti M , Musetti A , Rossi-Bernardi L
Published: January 2020

A new automatic apparatus based on the differential measurement of pH between two solutions has been developed. Two 25-microL (internal volume) glass capillary electrodes are used to measure the results of automated (under microcomputer control) chemical reactions that lead to the liberation or the uptake of hydrogen ions. The sensitivity of the differential pH measurements is better than +/- 0.0001 pH unit, and the change in H+ concentration that can be detected by such an apparatus is 1 mumol/L for plasma and 3 mumol/L for whole blood. The technique has been applied to the measurement of glucose in plasma, giving results in agreement with the specifications of the Food and Drug Administration reference method for quantitative determination of glucose (hexokinase/glucose-6-phosphate dehydrogenase method).
Simple, rapid spectrophotometry of urinary N-acetyl-beta-D-glucosaminidase, with use of a new chromogenic substrate.
Authors: Noto A , Ogawa Y , Mori S , Yoshioka M , Kitakaze T , Hori T , Nakamura M , Miyake T
Published: January 2020

We have developed a new spectrophotometric assay for urinary N-acetyl-beta-D-glucosaminidase (NAGase) with use of sodio m-cresolsulfonphthaleinyl N-acetyl-beta-D-glucosaminide (MCP-NAG). MCP-NAG was synthesized from acetochloro-glucosamine and m-cresolsulfonphthalein (MCP) in four steps. MCP-NAG reacts well with NAGase (Km = 0.41 mmol/L) and is highly water soluble. The absorption maximum and molar absorptivity of the aglycone MCP are 580 nm and 40 670, respectively. Spectral overlap of interfering substances at 580 nm is almost negligible, so that the urine blank can be omitted from the assay procedure. The high molar absorptivity of MCP gives sufficient analytical sensitivity at a reaction time of 15 min. The correlation between the MCP-NAG method (y) and the fluorimetric method (x) involving 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide is represented by the equation y = 0.995x - 0.669 (r = 0.991). Thus, the present method provides practical advantages over conventional methods, for use in the routine laboratory.
Comparison of a logistic and a mass-action curve for radioimmunoassay data.
Authors: Raab G M
Published: January 2020

This paper compares a logistic curve and a mass-action curve in their ability to represent radioimmunoassay standard curves. A data base from 10 different assays is used in this comparison. Six of the 10 assays could be modeled equally well by either the logistic or the mass-action curve. In one case the mass-action curve gave a slightly better fit, though the practical distinction was negligible. In the remaining three assays the mass-action curve performed very much worse than the logistic curve. It is shown that the mass-action curve can assume only a very limited range of shapes, which explains its difficulty with these three assays. In particular, the mass-action curve cannot represent assays where the standard curve slope is less than a specified value. The paper also discusses the extension of each of these curves to more complicated equations, and their application to immunoassay data.
Kinetic enzymic method for automated determination of total cholesterol in serum.
Authors: Deeg R , Ziegenhorn J
Published: January 2020

We describe a rapid, kinetic, fixed-time method for determining serum total cholesterol by use of cholesterol esterase, cholesterol oxidase, and the indicator reaction with peroxidase, 4-aminophenazone, and phenol. On addition of the competitive inhibitor 3,4-dichlorophenol the Michaelis constant of cholesterol oxidase is apparently increased, which extends the linear relation between absorbance change and cholesterol concentration to 20.7 to 25.9 mmol/L, depending on the analyzer being used. For calibration, a single standard is used. Total analysis time is in the range of 80 to 210 s. Incubation temperature is 25 degrees C or 37 degrees C. The single-reagent procedure has been adapted to three different centrifugal analyzers and to the Eppendorf ACP 5040 analyzer. It yields precise and accurate results and is insensitive to potential interferences.
Kinetic enzymic assay for D(-)-lactate, with use of a centrifugal analyzer.
Authors: Ludvigsen C W , Thurn J R , Pierpont G L , Eckfeldt J H
Published: January 2020

The D(-) isomer of lactic acid appears to cause a form of metabolic encephalopathy experienced by patients who have had jejunoileal bypass for morbid obesity. However, analysis for D(-)-lactate is not routinely available in clinical or reference laboratories. We describe an enzymic centrifugal-analyzer assay for D(-)-lactate in plasma or serum, with use of D(-)-lactate dehydrogenase. The method involves two-point kinetic calibration and preincubation of specimen and NAD+, thus eliminating the need for specimen-blanking or protein-precipitating pretreatment. This rapid, accurate, and precise assay should be helpful in evaluating patients with "short-bowel syndrome" who display confusion, lethargy, ataxia, or other central nervous-system disturbances that may be ascribable to D(-)-lactic acidosis.
Evaluation of a radioimmunoassay for estradiol in unextracted serum.
Authors: Mertens R , Liedtke R J , Batjer J D
Published: January 2020

We describe the performance of a commercial (Steranti/EIR) RIA reagent kit for measuring 17 beta-estradiol directly in serum. Day-to-day precision data for control sera were as follows: mean = 102.8 ng/L, CV = 6.8%, n = 20; mean = 231.1 ng/L, CV = 5.3%, n = 21; mean = 747.7 ng/L, CV = 9.4%, n = 21. Analytical recovery of added estradiol from seven different serum pools from men, to which three different concentrations of estradiol had been added, was (mean +/- SD): 98.6 +/- 7.0% at 107.5 ng/L added; 98.8 +/- 4.7% at 322.5 ng/L added; 108.2 +/- 4.8% at 645 ng/L added. Overall recovery of estradiol in these experiments (mean +/- SD for 21 determinations) averaged 101.9 +/- 7.0%. Assay of 32 serum specimens from women by both the direct (y) and an extraction method (x) gave the following linear regression statistics: y = 1.12x - 1.3, r = 0.998, Sy/x = 30.2 ng/L, mean y = 438.2 ng/L, mean x = 391.4 ng/L. Hemoglobin, bilirubin, and moderate lipemia do not interfere. Sensitivity of the direct assay was 2.6 ng/L. Compared with the extraction assay, the direct estradiol assay has advantages of speed and simplicity.
The chemical basis and specificity of the nitrosonaphthol reaction.
Authors: Knight J A , Robertson G , Wu J T
Published: January 2020

1-Nitroso-2-naphthol reacts with various 5-hydroxyindoles as well as para-substituted phenols and guaiacols. Consequently, this reaction has long been used to measure tyrosine in tyrosinosis and tyrosinemia, homovanillic acid in neuroblastoma, and 5-hydroxy-3-indoleacetic acid in the carcinoid tumor. We report here on the specificity of this reaction, as well as our early observations with respect to the chemical basis of the reaction.