bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences between concentrations of salivary cortisol and cortisone and of free cortisol and cortisone in plasma during pregnancy and postpartum
Authors: Meulenberg P M , Hofman J A
Published: January 2020

We measured cortisol and cortisone in plasma--both total and free--and in saliva during the course of pregnancy and postpartum. Antepartum and postpartum concentrations and morning and afternoon concentrations of both steroids were compared. The mean concentrations of cortisol and cortisone increased towards term and were significantly greater at the end of pregnancy than postpartum, except for free cortisol in plasma in the afternoon. The daily rhythm of both steroids was maintained throughout pregnancy and postpartum. The correlations between salivary and plasma free concentrations of cortisol and cortisone as well as of the sum of cortisol + cortisone were highly significant. The mean concentrations of cortisone in saliva accurately reflected both total and free concentrations in plasma. For cortisol, however, the change of the concentrations in saliva, was somewhat different from that in plasma. Moreover, the mass ratio of plasma free cortisol to salivary cortisol was about 2, whereas for cortisone the ratio was only about 0.5, probably owing to the conversion of cortisol to cortisone by 11 beta-hydroxysteroid dehydrogenase in the salivary gland. Furthermore, the passage of cortisol and cortisone from plasma to saliva should not be regarded as simple diffusion, because in the first half of pregnancy the sum of the concentrations of cortisol + cortisone in saliva significantly exceeded the sum of their free concentrations in plasma.
Quantification of chloride in sweat with the Cystic Fibrosis Indicator System
Authors: Warwick W J , Hansen L G , Werness M E
Published: January 2020

We examined the relation between chloride concentration and the area of complexed chloride of Medtronic's Cystic Fibrosis Indicator System, using a high-resolution x-y coordinated digitizer to measure the circumference of the chloride precipitation ring. These digitized points were entered directly into an IBM PC computer, where the area of the chloride precipitation was calculated with use of a repetitive rectangular estimation program. Using these data, we determined the relationship between the area of chloride precipitation and the chloride concentration of the standard NaCl solutions. When the area of the ring of chloride precipitation in the system's patch is measured immediately after the sweat test is completed, the concentration of chloride in the sweat can be calculated with a reproducibility equal to that of the Gibson-Cooke sweat test.
Automated quantification of creatine kinase MB isoenzyme in serum by radial partition immunoassay, with use of the Stratus analyzer
Authors: Chapelle J P , el Allaf M
Published: January 2020

We evaluated the analytical and clinical performances of a new radial partition immunoassay for measuring the mass concentration of creatine kinase (CK)-MB in serum. All pipetting, washes, incubations and data reduction were performed in 8 min by the Stratus (Dade) fluorometric analyzer. Within-assay and between-assay CVs were respectively 5.5% and 8.4% at 21 micrograms/L, and 4.2% and 3.4% at 48 micrograms/L. Assaying serial dilutions of serum samples with high CK-MB concentrations demonstrated excellent linearity. Results of the Stratus technique correlated well (n = 115, r = 0.98) with those of the Tandem-E CKMB II assay. There was no interference from hemolysis, bilirubin, rheumatoid factor, or added CK-MM (up to 3500 U/L); consequently, CK-MB can be determined in undiluted serum, even in the presence of high total CK activity. The mean CK-MB concentration in 105 blood donors was 1.9 (SD 1.3) micrograms/L. For seven myocardial infarction patients who received prompt fibrinolytic therapy, the mean CK-MB concentration was 4.5 (SD 1.8) micrograms/L at admission, and maximum concentrations, 119 (SD 94) micrograms/L, were recorded 16 h later. CK-MB returned to concentrations less than 10 micrograms/L within 72 h.
Radioimmunoassay of erythropoietin: analytical performance and clinical use in hematology
Authors: Schlageter M H , Toubert M E , Podgorniak M P , Najean Y
Published: January 2020

We report here the performance of a recently commercialized radioimmunoassay kit for determining erythropoietin (EPO) in serum or plasma. The lower detection limit of the method was 3 U/L. Precision, analyzed by the variation coefficients between different assay runs and in the same experiment, was always less than 10%; accuracy was assessed by recovery and dilution tests. In anemic patients (hematocrit 18-39%), the concentration of EPO was logarithmically related to hematocrit. A relatively large dispersion of the results was noted, as reported by others with various RIAs. Patients with severe renal failure demonstrated a very low EPO value, whatever the degree of their anemia. In some chronic anemias resulting from malignancy, EPO concentrations were also relatively low. In the polycythemia vera group, the EPO mean was below normal for greater than 95% of the patients, whatever their clinical stage (first evaluation, relapse, or remission). In contrast, 91% of the patients with pure erythrocytosis had a normal or increased EPO value, even when the etiology was unknown. Measurement of EPO concentration may be useful for the clinical differentiation of myeloproliferative disorders and, subsequently, for their prognosis and choice of treatment.
Colorimetric liposome lysis for assay of anti-streptolysin O antibody
Authors: Kubotsu K , Ushio I , Yoshikawa K , Kida M , Ishikawa K , Matsuura S , Sakurabayashi I
Published: January 2020

We developed an automated colorimetric assay based on liposome lysis for measurement of the titer of anti-streptolysin O antibodies (ASO) in human sera by using liposomes in which alkaline phosphatase (EC 3.1.3.1) was entrapped. The assay involved the inhibition by ASO of liposome lysis caused by streptolysin O. The procedure was fully automated for use with a routine clinical automated analyzer. The inter- and intra-assay precision showed that results were reproducible. Potential interfering substances such as bilirubin, hemoglobin, and triglycerides did not affect the results. With this assay, results correlated well with those by the Rantz-Randall method and the latex agglutination method (r = 0.900 and 0.890, respectively). This automated colorimetric assay for ASO should be of use for diagnosing streptococcal infections.
A model for molecular screening of newborns: simultaneous detection of Duchenne/Becker muscular dystrophies and cystic fibrosis
Authors: Prior T W , Highsmith W E , Friedman K J , Perry T R , Scheuerbrandt G , Silverman L M
Published: January 2020

Gene mutations responsible for the majority of Duchenne/Becker muscular dystrophy (DMD/BMD) and cystic fibrosis (CF) chromosomes have been identified. We describe a DNA-based strategy, rather than the traditional biochemical assays, for screening newborns. DNA sequences spanning the CF mutation and several DMD/BMD deletion-prone exons are amplified simultaneously via a multiplex polymerase chain reaction. The gel is visually inspected for DMD/BMD deletions and then blotted and hybridized with allele-specific oligonucleotides to determine the presence or absence of the CF mutation. We determined that blood spots provide sufficient DNA for the molecular analysis, so the procedure can be used in screening programs of newborns.
Predicting effects of quality-control practices on the cost-effective operation of a stable, multitest analytical system
Authors: Westgard J O , Oryall J J , Koch D D
Published: January 2020

The cost-effective operation of an analytical system depends on quality-control (QC) practices such as the QC procedure itself (control rules, number of control measurements); the batch size or the run length; and the use of bracketed, nonbracketed, or pre-control modes of operation. Predictive value models that predict the defect rate and test yield of each test, as well as of the system as a whole, have been used to study these practices and to develop strategies for improving the quality and productivity of a multitest analyzer. Quality was optimized for most tests by achieving high error detection and low false rejection by the QC procedures. For a few tests where ideal QC performance could not be achieved, predictive models indicate that high quality is achieved, predictive models indicate that high quality is achieved as long as the observed stabilities (low frequencies of errors) of the measurement procedures are maintained. In our laboratories, productivity gains of 2.9% ($17,400/year) were achieved by changing QC procedures. Predictive models indicate that further gains are possible by increasing batch size and changing from bracketed to nonbracketed control operation. In general, the common practice of bracketed control on stable analytical systems may need to be re-examined owing to its effect on the cost of operation.
Time-resolved fluoroimmunoassay for unconjugated estrogen in urine: comparison with a fluorometric assay for total estrogen and application in an in vitro fertilization program
Authors: Thomas C M , van den Berg R J , Segers M F , Geelen L M , Hollanders J M , Doesburg W H , Houx P C
Published: January 2020

A time-resolved fluoroimmunoassay (TR-FIA) for unconjugated estrogens in human urine is described. 6-Keto-17 beta-estradiol-6-(O-carboxymethyl)oxime:bovine serum albumin is immobilized onto microtiter strip wells and the coated wells are incubated with 17 beta-estradiol standard preparations or unknowns with a polyclonal antiserum to 17 beta-estradiol-16,17-monosuccinyl:albumin. The antiserum-bound estrogen is detected by incubation with a europium-labeled anti-rabbit IgG that serves as both second antibody and tracer. After the immunoreactions, the bound portion of the labeled antiserum is quantified by dissociating the Eu3+ in a fluorescence-enhancement solution and measuring its fluorescence with a time-resolved fluorometer. The detection limit of the TR-FIA is 24 pmol of 17 beta-estradiol per liter; the analytical range extends to 1.8 nmol/L. This assay is a convenient alternative to radioimmunoassay and to the automated Kober-Ittrich fluorometry of total estrogen. Its advantages include short counting times; use of nonradioactive, stable reagents, all of which are commercially available; and more nearly complete automation. We conclude that this TR-FIA, compared with the Kober-Ittrich fluorometric assay (J Endocrinol 1957; 16:49-56), provides the clinician with equivalent information during follicular development therapy as part of an in vitro fertilization program.
Erythrocyte porphobilinogen deaminase activity in porphyria cutanea tarda
Authors: Siersema P D , de Rooij F W , Edixhoven-Bosdijk A , Wilson J H
Published: January 2020

Porphyria cutanea tarda (PCT) results from a metabolic block in heme synthesis at the level of uroporphyrinogen decarboxylase. We measured the activity of one of the enzymes preceding it in the heme biosynthetic pathway, porphobilinogen deaminase (PBGD; EC 4.3.1.8), in erythrocytes of 47 patients with symptomatic or asymptomatic familial or sporadic PCT. PBGD activity was significantly increased in all four PCT groups, compared with controls. To study the mechanism of this increased PBGD activity, we determined, using polyclonal antibodies, the amount of immuno-detectable PBGD per 100 units of PBGD activity (Ig PBGD/100 U) and the total amount of immuno-detectable PBGD (Ig PBGD) in erythrocytes from all 47 patients and from controls. In both familial and sporadic PCT, Ig PBGD/100 U was decreased compared with that in controls (P less than 0.05). Especially in asymptomatic patients of the familial PCT group there was an inverse correlation between increasing PBGD activity and Ig PBGD/100 U (r = -0.90). In familial PCT, and to a minor degree in sporadic PCT, an increase in PBGD activity was accompanied by an increased Ig PBGD, compared with controls (familial PCT: P less than 0.001, sporadic PCT: P less than 0.05). In familial and sporadic PCT an increase in erythrocyte PBGD activity can, at least partly, be explained by a diminished degradation of PBGD. In familial PCT, in the symptomatic group more than in the asymptomatic group, and to a minor degree in sporadic PCT, there is in addition an increase in the absolute amount of PBGD.
Proposed reference method for iron in serum used to evaluate two automated iron methods
Authors: Valcour A A , Krzymowski G , Onoroski M , Bowers G N , McComb R B
Published: January 2020

The manual Reference Method of the Centers for Disease Control for serum iron (CDC/RM/Fe) and a semiautomated adaptation of it were used to evaluate two working methods: one, a detergent solubilization procedure for the Roche Cobas-Bio analyzer, the other, the Kodak Ektachem 700 procedure, based on dry-film technology. The CDC/RM/Fe and its semiautomated version gave essentially the same results for 40 sera from hospital patients. This semiautomated version was in turn compared with the two working procedures in a study involving 200 patients. Each of the working methods correlated well with the semiautomated CDC/RM/Fe method. Separate recovery and interference studies indicated satisfactory analytical recovery of iron in all cases, but the detergent solubilization method was found to be susceptible to interference by hemoglobin, lipemia, and bilirubin.
Colorimetry and constant-potential coulometry determinations of transferrin-bound iron, total iron-binding capacity, and total iron in serum containing iron-dextran, with use of sodium dithionite and alumina columns
Authors: Jacobs J C , Alexander N M
Published: January 2020

After the parenteral administration of iron-dextran (imferon), the increased total iron concentrations in serum can be determined by atomic absorption spectroscopy and by colorimetric methods involving sodium dithionite, which reductively dissociates iron from the dextran complex. We report that constant-potential coulometry detects only about 55-70% of dextran-bound iron before dithionite reduction and variable amounts after reaction with the reducing agent. In addition, we have developed a procedure for determining transferrin-bound iron, total iron-binding capacity (TIBC), total iron, and dextran-bound iron with the Kodak Ektachem colorimetric system. In determining total serum iron, the sample is first mixed with sodium dithionite, which rapidly dissociates all dextran-bound iron, but does not remove iron from either transferrin or hemoglobin. After the mixture is applied to an Ektachem slide, transferrin-bound iron is released at pH 4 and is detected together with the iron previously bound to dextran. TIBC is determined by mixing serum with ferric citrate in moderate excess and filtering through a small alumina (Al2O3) column, which binds excess free iron and iron-dextran; the iron in the column eluate represents the TIBC. Transferrin-bound iron is determined by applying diluted serum without added ferric citrate to an alumina column and measuring the iron in the column eluate. Dextran-bound iron is equivalent to the difference between total and transferrin-bound iron. Using this method, we found that transferrin iron-binding sites are saturated in vitro by excess iron-dextran less efficiently than by ferric citrate.
Improved method for identifying alpha 1-antitrypsin Pi M subtypes by isoelectric focusing in agarose
Authors: Nowicki M L , Freier E F
Published: January 2020

We describe an improved method for the classification of alpha 1-antitrypsin variants by isoelectric focusing in agarose. Identification of the three Pi M subtypes can now be made by using a narrow-range carrier ampholyte (pH 4.2-4.9) and pretreating serum with dithioerythritol-iodoacetic acid to enhance band resolution. Phenotype results for two groups of Pi M homo- and heterozygotes are compared to illustrate the improved accuracy of the new method.
Deuterium enrichment of plasma determined by nuclear magnetic resonance spectroscopy: dilution kinetics of 2H2O and estimation of total body water
Authors: Brans Y W , Schwartz C A , Hood R J , Ksebati M B , Konduri G G
Published: January 2020

We demonstrate the feasibility of quantifying the abundance of 2H in plasma by nuclear magnetic resonance (NMR) spectroscopy. After adding internal standard (tert-butyl-d9 alcohol) to deproteinized plasma samples containing 2H2O, we determined the ratio of NMR peak areas for 2H2O and tert-butyl-d9 alcohol. This peak-area ratio was directly proportional to the exogenous 2H enrichment of plasma (difference between measured and naturally occurring 2H) between 0 and 0.272 atom % (r = 0.999). The coefficient of variation was 1.34% at an exogenous enrichment of 0.136 atom %. We applied this method to a study of the dilution kinetics of 2H2O to determine the optimal time and method of blood sampling for estimation of total body water content. The 2H enrichment of plasma stabilized by 4 h after intravenous injection of 2H2O, 1 g/kg of body weight, and fluctuated within 2-4% of the 4- to 8-h mean thereafter.
Simultaneous determination of acidic 3,4-dihydroxyphenylalanine metabolites and 5-hydroxyindole-3-acetic acid in urine by high-performance liquid chromatography
Authors: Stroomer A E , Overmars H , Abeling N G , van Gennip A H
Published: January 2020

We describe a simple and rapid quantitative method for the simultaneous determination of 3,4-dihydroxyphenylalanine acid metabolites and 5-hydroxyindole-3-acetic acid. After solvent extraction from acidified urine, the acids are analyzed by reversed-phase high-performance liquid chromatography. For detection and quantification, we used a fluorescence detector in combination with an amperometric detector to obtain a high degree of specificity. Sample preparation and chromatographic analysis can be completed within an hour. Results by the method correlate well with those by a previously used gas-chromatographic method, but the new method is faster and avoids the need for derivatization.
Improved phenotyping of apolipoprotein E: application to population frequency distribution
Authors: Hill J S , Pritchard P H
Published: January 2020

A simple procedure for phenotyping apolipoprotein (apo) E directly from plasma has been developed for use in the lipid clinic laboratory. In this new method, 10 microL of serum or plasma is pretreated with neuraminidase (EC 3.2.1.18), which removes the sialic acid residues from apo E and eliminates additional bands, thereby ensuring correct phenotype assignment. After a rapid delipidation step, the samples are focused in vertical polyacrylamide mini-slab gels and immunoblotted with a polyclonal goat anti-apo E antibody, followed by a Protein G-peroxidase conjugate. The accuracy of this method was confirmed by comparison with the established procedure of phenotyping by isoelectric focusing of delipidated very-low-density lipoprotein. In addition, sera from 203 subjects from Vancouver, selected without conscious bias, were used to determine the local distribution of the apo E alleles. We estimate that the relative frequencies of apo E alleles epsilon 2, epsilon 3, and epsilon 4 in this population are 0.086, 0.761, and 0.153, respectively. The speed and convenience of using minigels make this procedure ideal for clinical laboratory applications and large population studies.
Study of lipoprotein particles LpAI and LpAI:AII in patients before coronary bypass surgery
Authors: Coste-Burel M , Mainard F , Chivot L , Auget J L , Madec Y
Published: January 2020

Lipids, apolipoproteins, and LpAI and LpAI:AII particles were studied in 43 men (mean age 51, SD 7, years) 24 h before their coronary bypass surgery and in 54 control men (mean age 46 SD 9, years). LpAI and LpAI:AII were analyzed by electroimmunodiffusion and by a noncompetitive enzyme-linked immunoassay, respectively. Concentrations of LpAI and LpAI:AII in the bypass patients were significantly lower (P less than 0.001) than those in the controls. Apolipoprotein AI was significantly correlated with LpAI (P less than 0.001) and LpAI:AII (P less than 0.01) in controls, but only with LpAI:AII (P less than 0.001) in bypass patients. Discriminant analysis between controls and patients showed apolipoprotein AI to be the most powerful discriminant factor; the addition of LpAI and LpAI:AII did not improve discriminant power. We conclude that the determination of LpAI and LpAI:AII particles reflects metabolic disorders in patients but does not significantly influence the discrimination of such patients into risk groups.
Flexibility of noncompetitive avidin-biotin immunoassay: immunoassay of lutropin applied to different signal detection systems
Authors: Vilja P
Published: January 2020

I adapted the noncompetitive avidin-biotin immunoassay (NABA) of lutropin (LH) for use with radiometric, fluorometric, time-resolved fluorometric, and luminometric detection systems by changing the enzyme substrate or conjugating avidin with different labels. The corresponding methods were used to determine LH in 40 urine samples, and the results were compared with those obtained by an immunoenzymometric assay (IEMA), also based on the NABA principle. Scatter plots of the results showed a linear relation with high correlation coefficients (r = 0.972-0.983), and bias plots showed good agreement between the four comparison methods and the IEMA. The results indicate that the effectiveness of the NABA of LH is independent of the signal detection system. This means good flexibility for the user in choice of instrument and signal detection system.
Gradient centrifugation to isolate platelets for determination of trace elements by neutron-activation analysis
Authors: Bibow K , Mundal H H
Published: January 2020

A nonionic iodinated compound, Nycodenz, was used for density-gradient isolation of platelets before analysis for trace elements by instrumental neutron activation. The platelet samples were almost free from plasma and erythrocytes, and the trace element contribution from additives was insignificant for the elements investigated. The reproducibility of the trace element analysis in platelets attests to the usefulness of density-gradient separation. Platelet samples were obtained twice, three weeks apart, from 10 healthy young women (ages 24 +/- 2 years). The mean (SD) concentrations were as follows: Se, 0.62 (0.16) mg/L and 4.7 (1.4) ng/10(9) platelets; Zn, 44 (10) mg/L and 333 (80) ng/10(9) platelets; Rb, 3.0 (0.5) mg/L and 23 (5) ng/10(9) platelets; and Cs, 24 (5) micrograms/L and 0.18 (0.04) ng/10(9) platelets.
Impact of protein measurements on standardization of assays of apolipoproteins A-I and B1
Authors: Henderson L O , Powell M K , Smith S J , Hannon W H , Cooper G R , Marcovina S M
Published: January 2020

In 1989 the Committee on Apolipoproteins of the International Federation of Clinical Chemistry and the Centers for Disease Control conducted an international survey of total-protein measurements of isolated low-density lipoproteins (LDL) and delipidated high-density lipoproteins (HDL), and of their relationships to the National Institute of Standards and Technology (NIST) bovine serum albumin (BSA) Standard Reference Material (SRM). Most of the 93 apolipoprotein laboratories surveyed use the Lowry total-protein method. Results reported with the LDL preparations demonstrated a large bias and variation among methods; those with delipidated HDL were not as great, but were similar to those for BSA. Performance improved appreciably with use of the Lowry-sodium dodecyl sulfate method and the NIST BSA SRM for protein measurement. The total CVs, including among-laboratory and within-laboratory errors, averaged approximately 50% and 23% for LDL, 17% and 11% for HDL, and 18% and 11% for BSA solutions by all methods and by the Lowry methods, respectively. Regardless of the methods used, greater variability of the protein measurements was seen with the normally occurring LDL than with the nonlipoprotein BSA or delipidated HDL. The mean CV values for all samples among laboratories averaged between 10% and 15% with the modified Lowry methods; the biuret method gave the highest among-laboratory CV, 34%; the Kjeldahl had the lowest, 7.7%. Use of the same methodology and primary nonapolipoprotein standard is essential for comparability of protein results for apolipoprotein primary standard solutions. This is especially true for apolipoprotein B, because its inherent properties and lability make protein analysis difficult. This study supports the use of a standardized selected Lowry-sodium dodecyl sulfate method traceable to quantitative amino acid analysis as a point of reference for determining the protein concentration of primary calibration reference materials for apolipoproteins.
Intestinal variant alkaline phosphatase in plasma in disease
Authors: Kuwana T , Rosalki S B
Published: January 2020

We investigated by enzyme electrophoresis after prolonged neuraminidase treatment the activity of "intestinal variant" (alpha 2-globulin mobility) alkaline phosphatase (EC 3.1.3.1; ALP) in the plasma of 189 patients selected for disorders (diabetes mellitus, liver cirrhosis, and chronic renal failure) with a known high frequency of increased plasma intestinal (beta-globulin mobility) ALP activity. The overall frequency of the variant ALP was 23.8%, whereas in the samples showing intestinal ALP it was 45.0%. The variant ALP was not observed in the absence of intestinal ALP, nor in patients of blood group A. Its frequency did not differ significantly between the different patient groups. Quantification of the variant ALP by densitometry was unsatisfactory but the quantity could be estimated by subtracting the intestinal ALP activity measured by electrophoresis from the activity determined by immunoassay with monoclonal antibody that reacts with both the intestinal and the variant forms. This indicated median activity of 12 U/L for the variant, approximately equal to that of the concomitant intestinal ALP. From the effects of papain and bromelain treatments, we suggest that "intestinal variant" represents intestinal ALP with attached membrane-binding domain.
Analysis of 8-methoxypsoralen by high-performance liquid chromatography
Authors: Ketchum C H , Robinson C A , Huang S T
Published: January 2020

We report a simple and rapid procedure for assaying 8-methoxypsoralen (8-MOP) in plasma by high-performance liquid chromatography (HPLC). The standard curve for the assay is linear for 8-MOP from 15 to 500 micrograms/L (y = 0.002x-0.01, r = 0.99) with a lower limit of detection of 1.5 micrograms/L. Intra-assay precision (CV) was 6.0% at the 100 micrograms/L concentration and 10.0% at 50 micrograms/L (n = 30 each). Interassay precision was 6.4% at 100 micrograms/L and 7.0% at 50 micrograms/L (n = 50 each). Extraction recovery of 8-MOP was 98%. Common antiarrhythmics, sedatives, and hypnotics were found not to interfere.
Concentrations of total and free dehydroepiandrosterone in plasma and dehydroepiandrosterone in saliva of normal and hirsute women under basal conditions and during administration of dexamethasone/synthetic corticotropin
Authors: Swinkels L M , Ross H A , Smals A G , Benraad T J
Published: January 2020

Using a specific and sensitive radioimmunoassay involving extraction with diethyl ether and chromatographic separation of steroids, we measured concentrations of salivary and plasma dehydroepiandrosterone (DHEA) in 22 women with normal ovulatory cycles (ages 18-45 years). Salivary DHEA values closely correlated with total and free DHEA in plasma. In the follicular phase the mean concentrations of salivary and plasma free DHEA were virtually equal [mean (SD): 0.61 (0.32) and 0.56 (0.34) nmol/L, respectively]. In the luteal phase, salivary DHEA slightly exceeded the plasma free DHEA [0.68 (0.40) vs 0.56 (0.38) nmol/L, P less than 0.01]. Also, during combined dexamethasone/synthetic corticotropin administration in 25 patients with androgenizing disorders and in 10 normal subjects (each in the follicular and luteal phases), the concentration of DHEA in saliva strongly correlated with total and free DHEA in plasma. During these dynamic tests, the mean concentrations of free DHEA in plasma and salivary DHEA in the hirsute women were significantly higher than the mean concentrations in the control women at all times before and after corticotropin infusion (P less than 0.05- less than 0.0001). In contrast, plasma total DHEA in patients exceeded nonhirsute values only at 15 min after corticotropin administration. In six of 25 patients total DHEA during combined administration of dexamethasone/synthetic corticotropin exceeded normal values by at least 2 SD. The response of salivary and free DHEA to synthetic corticotropin in this subgroup was also excessive.
Fluorometric determination of carnitine in serum with immobilized carnitine dehydrogenase and diaphorase
Authors: Matsumoto K , Yamada Y , Takahashi M , Todoroki T , Mizoguchi K , Misaki H , Yuki H
Published: January 2020

A fluorometric flow-injection method for determining carnitine with use of immobilized enzymes carnitine dehydrogenase (EC 1.1.1.108) and diaphorase (EC 1.8.1.4) was developed and applied to the assay of carnitine in serum of patients treated with valproic acid. After fractionation and hydrolysis of carnitines in serum samples by perchloric acid and potassium hydroxide, liberated carnitine was converted to resorufin by immobilized carnitine dehydrogenase and diaphorase in the presence of beta-NAD+ (1.0 mmol/L), resazurin (12.5 mumol/L), and Tris acetate (0.6 mol/L, pH 9.0) at 37 degrees C. The fluorescence intensity of resorufin was monitored at lambda Ex 560 nm and lambda Em 580 nm. The calibration curve was linear for carnitine amounts from 0.1 to 1.0 nmol. Quantitative analytical recovery and satisfactory within- and between-run imprecision of carnitine in each carnitine fraction were obtained. Interference by bilirubin, serum albumin, and hemoglobin was negligible. Carnitine deficiencies were detected in about 20% of the valproic acid-treated patients (n = 198). The present method should be useful for monitoring carnitine deficiencies in clinical laboratories.
Diagnosis of ectopic pregnancy by the rate of increase of choriogonadotropin in serum: diagnostic criteria compared
Authors: Emancipator K , Bock J L , Burke M D
Published: January 2020

We compare three methods for using the rate of change of human choriogonadotropin (hCG) concentration in serum to diagnose ectopic pregnancy. With Method I, the lower limit for the rate of increase of serum hCG in normal pregnancy is 66% per 48 h. With Method II, a different lower limit of normal is specified for each of four discrete sampling intervals of hCG. With Method III, the lower limit of normal is determined by a continuous discriminant function of the initial hCG concentration. If the initial hCG concentration is less than or equal to 2000 int. units/L (Second International Standard), all three methods have acceptable diagnostic efficiencies, and there are no statistically significant differences among conclusions from the methods. None of the three methods performs satisfactorily if the initial hCG concentration is greater than 2000 int. units/L. We recommend Method I because it is simpler than the other two.
Proficiency testing for creatine kinase isoenzyme CK-2 (CK-MB) in Ontario
Authors: McQueen M J , Patten R L , Henderson A R , Krishnan S , Wood D E , Webb S
Published: January 2020

Three surveys of the measurement and interpretation of creatine kinase (CK; EC 2.7.3.2) isoenzyme 2 (CK-MB) were conducted in Ontario, Canada, in 1989. Of the clinical laboratories participating, 66% used immunological methods and 24% used electrophoretic methods. Although reference ranges and interpretative routines varied widely, 95% of the laboratories reported correct interpretations for 10 of the 15 vials tested. The only major problems occurred with samples with very low total CK activity. Within-survey duplicate results compared well, and 89% of the laboratories had consistent between-survey results, even for specimens with low total CK activity. Errors were proportional to the frequency of use of the different analytical methods. The lyophilized testing material gave higher results with methods for measuring the mass of CK-2, suggesting that the material contained inactive but immunologically intact CK-2. The surveys indicate that laboratories should review their protocols for measuring CK-2 when only a single sample from the patient is available.
Simplified fluorometric assay of total selenium in plasma and urine
Authors: Sheehan T M , Gao M
Published: January 2020

We describe here a single-tube assay that may be applied to the whole range of selenium status in adult and pediatric patients, including depletion during parenteral or other nutrition. A specimen or aqueous standard, 100 microL, is digested with 0.5 mL of HNO3/HCIO4 (4/1 by vol, at 190 degrees C for 90 min), reduced with 0.5 mL of concentrated HCI (150 degrees C, 30 min), and complexed with 0.5 mL of 6.3 mmol/L 2,3-diaminonaphthalene (DAN) reagent in the presence of EDTA (60 degrees C, 30 min). The resulting fluorophore is extracted into cyclohexane and its fluorescence measured (excitation at 366 nm; emission at 544 nm). It is not necessary to control pH during the complexing step or to protect the DAN from light. The limit of detection of selenium is 10 micrograms/L (0.126 mumol/L); linearity of results extends to 2000 micrograms/L (25.3 mumol/L). Between-batch precision is 5%, analytical recovery 90%-96%. Performance is good as tested against Reference Materials and by participation in a National Quality Assurance Scheme.
Centrifugal ion-selective electrode system for potassium in whole blood
Authors: Brackett J , Durley B , Janczak R , Kazlauskas V , Kmiec J , Norlie J , Rosencranz R , Schultz S , Spring T , Theusch W
Published: January 2020

We describe a novel ion-selective electrode (ISE) system that can be used in the Abbott Vision analyzer. A reusable sensor pack and a disposable test pack have been miniaturized to approximately the size and weight of a conventional Vision test pack, thus eliminating the need for a separate ISE module. The sensor pack contains a fluid path, battery-powered electronics, and screw-in electrodes. The test pack contains separate chambers and fluid channels for a blood specimen and two aqueous calibrators. During a run, plasma is separated from blood cells and is moved sequentially, along with the two calibrators, into and out of the sensor pack by centrifugal force. Each run includes checks for electromechanical integrity, fluid contamination, calibration slope and intercept, and specimen hemolysis. The system operates with CVs of less than 2% and gives results that correlate well with those by flame photometry and direct ISE methods. Either skin-puncture or venipuncture whole blood can be assayed, as well as serum or plasma.
Rhabdomyolysis secondary to lovastatin therapy
Authors: Manoukian A A , Bhagavan N V , Hayashi T , Nestor T A , Rios C , Scottolini A G
Published: January 2020

We report a case of lovastatin-induced rhabdomyolysis and resulting life-threatening renal failure. Lovastatin, a hypocholesterolemic agent, decreases endogenous cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.88). This agent has been implicated in causing rare serious side effects in various clinical settings; however, the mechanism of these adverse reactions is not understood. The clinical course of our patient was characterized by profound muscle weakness with marked increases in serum creatine kinase and myoglobin. Light- and electron-microscopic studies of skeletal muscle of our patient demonstrated a noninflammatory myopathy suggestive of ongoing rhabdomyolysis with vacuolization and focal degeneration of myocytes. The patient's symptoms and the laboratory values referable to rhabdomyolysis resolved after discontinuation of the drug. We speculate that the rhabdomyolysis was due to mitochondrial damage secondary to inadequate synthesis of coenzyme Q and heme A, members of the electron-transport system of the inner mitochondrial membrane.
Testicular cancer secretes intact human choriogonadotropin (hCG) and its free beta-subunit: evidence that hCG (+hCG-beta) assays are the most reliable in diagnosis and follow-up
Authors: Saller B , Clara R , Spöttl G , Siddle K , Mann K
Published: January 2020

Human choriogonadotropin (hCG) and free hCG-beta values for 934 serum samples from patients with seminomatous or nonseminomatous testicular cancer were measured by highly specific immunoradiometric assays (IRMAS). In non-seminoma samples, hCG and hCG-beta were highly correlated (r = 0.82, P less than 0.001). Of 112 "marker-positive" seminoma samples, only 46 (41.1%) showed both increased hCG and hCG-beta. In 39 cases (34.8%) only hCG-beta and in 27 cases (24.1%) only dimer-hCG was increased. This makes the determination of hCG and hCG-beta, either by two assays or by a single hCG (+hCG-beta) assay, most reliable in these patients. For all samples, hCG (+hCG-beta) was measured by a polyclonal RIA and a monoclonal IRMA, which differed in their cross-reactivities with hCG-beta (234% and 720%, respectively). The hCG (+hCG-beta) IRMA, as a result of its higher hCG-beta cross-reactivity, was superior to the hCG (+hCG-beta) RIA in detecting slightly increased hCG-beta. Additionally, 11 widely used commercial hCG kits were tested for their hCG-beta cross-reactivities and showed values between less than 3% and 264%.
SCC antigen measured in malignant and nonmalignant diseases
Authors: Molina R , Filella X , Torres M D , Ballesta A M , Mengual P , Cases A , Balaque A
Published: January 2020

SCC antigen was measured in the serum of 214 patients with benign diseases and in 251 patients with various cancers. With 2.5 micrograms/L as the upper normal limit for serum, values were positive in 2.9% of 69 healthy subjects (I), 29.0% of 214 patients with benign pathologies (II), and 41% of 217 patients with active cancer (III). The highest values in group II were for patients in renal failure (64%) or with lung diseases (40%) or head-and-neck diseases (21.2%). Specificity of SCC increased (91.1%) when we excluded patients in renal failure or with creatinine values greater than 133 mumol/L. In group III, SCC values were abnormal in 57.7% of patients with squamous cell carcinoma, but in only 9.3% of those with other histological types (P less than 0.001). In squamous cell carcinoma of the lung, cervix, or head and neck, SCC values were related to tumor stage, values being highest in patients with metastases.
3-Hydroxy-3-methylglutaryl-CoA lyase deficiency as detected by radiochemical assay in cell extracts by thin-layer chromatography, and identification of three new cases
Authors: Gibson K M , Lee C F , Kamali V , Johnston K , Beaudet A L , Craigen W J , Powell B R , Schwartz R , Tsai M Y , Tuchman M
Published: January 2020

In this rapid radiochemical assay for 3-hydroxy-3-methylglutaryl-coenzyme A lyase (I) activity in cell extracts, DL-3[glutaryl-3-14C]hydroxy-3-methylglutaryl-coenzyme A is used as substrate and the radiochemical product, [3-14C]acetoacetic acid, is converted to the more stable [3-14C]-3-hydroxybutyric acid in the presence of added NADH and 3-hydroxybutyrate dehydrogenase. Substrate and product are separated and quantified by thin-layer chromatography on cellulose (solvent system: butanol/water/formic acid, 77:13:10 by vol). All reagents for the assay are commercially available. No detailed column chromatography or spectrophotometry is required. Thus the assay is suited for any clinical laboratory. Using this procedure, we studied cultured fibroblasts or lymphocytes isolated from whole blood from five patients in whom the urinary organic acid profile was suggestive of deficiency of I. Three patients had less than or equal to 18% of control I activity in fibroblast or lymphocyte extracts. The other two had activity within the normal range. In one of the latter cases, urinary excretion of three of the characteristic acids disappeared with age, and 3-hydroxyisovaleric acid excretion was within normal limits. The other case presented with urinary excretion of moderate amounts of all four metabolites and the characteristic absence of urinary ketone bodies. Evidently, confirmatory enzyme studies should be undertaken, even when the profile of urinary organic acids appears definitive for this deficiency.
Increased activities of cytosol aminopeptidase and lactate dehydrogenase in serum originate from lymphocytes in necrotizing lymphadenitis
Authors: Sugaya N , Kanno T , Nirasawa M , Mitamura K , Takeuchi Y , Osano M
Published: January 2020

In three pediatric patients with necrotizing lymphadenitis, cytosol aminopeptidase activity (c-AP; EC 3.4.11.1) in serum was markedly increased to 509, 417, and 191 U/L, respectively (normal range 25-60 U/L). Lactate dehydrogenase (LD; EC 1.1.1.27) was also increased, with LD-3 predominating. The increased concentrations of c-AP and LD presumably originated from the destruction of infected, activated lymphocytes, especially T lymphocytes. Necrotizing lymphadenitis is probably caused by a lymphocytotropic virus.
Calibration in three dimensions: optimizing a two-parameter calibration technique to extend the range of an immunoturbidimetric urinary albumin assay into antigen excess
Authors: Tillyer C
Published: January 2020

I describe a general technique, called "two-parameter calibration," which allows precise determination of analyte from non-monotonic calibration plots and the calibration of immunoturbidimetric assays in antigen excess. Using three-dimensional calibration plots and relative-sensitivity curves, two optimal parameters may be selected from a number of possible options by using criteria presented here. Choosing two different values of delta At1-t2, the change in absorbance from time t1 to t2--as the reaction parameters in an immunoturbidimetric assay for albumin, I have optimized the choice of time interval for two-parameter calibration and extended the working range of the assay by three- to fourfold. The albumin assay shows excellent agreement of observed and expected values (r = 0.996) and also with results of a routine kinetic dye-binding method used on diluted plasma samples (r = 0.970).
"Microgel diffusion blotting" for sensitive detection of antibodies to extractable nuclear antigens
Authors: De Keyser F , Verbruggen G , Veys E M , Nimmegeers J , Schatteman L , Goethals K , Vandenbossche M
Published: January 2020

A fast immunoblotting procedure, termed "microgel diffusion blotting," is used to detect and identify antibodies to extractable nuclear antigens (i.e., to Sm, RNP, and SSB) in patients with rheumatic diseases. The method differs from the standard immunoblotting techniques by the use of ultra-thin microgels for polyacrylamide gel electrophoresis: the very thin gel layer allows transfer of proteins to a nitrocellulose membrane by simple diffusion. Principal advantages of this variant technique are its simplicity, rapidity, and reproducibility--characteristics that make the test suitable for routine application. We compared the sensitivity of the test with that of double immunodiffusion in agarose for the evaluation of humoral antinuclear immunity. Microgel diffusion blotting detected antibodies in serum at concentrations less than 0.001 of those detectable by immunodiffusion.
Mitochondrial aspartate aminotransferase determined by "Fast Protein Liquid Chromatography"
Authors: Grøstad M , Rej R , Huseby N E
Published: January 2020

We describe an improved separation of the isoenzymes of aspartate aminotransferase (EC 2.6.1.1), based on ion-exchange chromatography. Involving the "Fast Protein Liquid Chromatography" system (Pharmacia) with a MonoQ column, this rapid, reproducible method for quantifying the mitochondrial enzyme shows good resolution and sensitivity, and results correlate well with those by an established immunochemical method.
Evaluation of determination of lactate dehydrogenase isoenzyme 1 by chemical inhibition with perchlorate or with 1,6-hexanediol
Authors: Paz J M , Garcia A , Gonzales M , Treviño M , Tutor J C , Jaquet M , Rodriguez-Segade S
Published: January 2020

We have evaluated the determination of lactate dehydrogenase (EC 1.1.1.27) isoenzyme 1 activity by chemical inhibition of the other isoenzymes with perchlorate and with 1,6-hexanediol. In the hexanediol method, we studied the effect of the duration of incubation with the inhibitor; a 5-min incubation yielded results closest to those of an immunochemical technique (Isomune-LD). The perchlorate method was the most precise, and the hexanediol method the least, although for none of the techniques did the coefficient of variation exceed the medically acceptable limit prescribed by the College of American Pathologists. Pairwise correlation among the immunoprecipitation, electrophoretic, and chemical inhibition methods was good (r greater than 0.991), although the differences between means were statistically significant (except for the comparison of the two chemical inhibition methods). Because of its ease, low cost, and precision, we recommend the perchlorate method for routine use.
Effects of lyophilization of serum on the measurement of apolipoproteins A-I and B
Authors: Marcovina S M , Adolphson J L , Parlavecchia M , Albers J J
Published: January 2020

A common accuracy-based standardization program is indispensable for establishing reference intervals for the clinical use of apolipoproteins. The development and distribution of reference materials and quality-control materials that do not exhibit matrix effects between methods is essential to the standardization process. We examined the suitability of lyophilized material as a common reference material for the measurement of apolipoproteins A-I and B. We determined values for apolipoproteins A-I and B in frozen and lyophilized serum pools, using different immunochemical approaches. We found little or no differences in apolipoprotein A-I values between frozen and lyophilized pools as determined by the different methods. In contrast, values for apolipoprotein B in lyophilized samples were consistently lower than those obtained for frozen samples. After adjusting for the effect of dilution due to reconstitution, the difference in the apolipoprotein B values for lyophilized as compared with frozen samples ranged from -26% to 4%, depending upon the assay method. Evidently, serum pools in lyophilized from are not a suitable matrix for reference materials for apolipoprotein B measurements but can be used for apolipoprotein A-I measurements.
Radioimmunoassay of type I collagen that mainly detects degradation products in serum: application to patients with liver diseases
Authors: Hartmann D J , Trinchet J C , Ricard-Blum S , Beaugrand M , Callard P , Ville G
Published: January 2020

This radioimmunoassay for type I collagen mainly detects degradation products of the molecule in human serum samples. Type I collagen antigenicity in serum can be separated into two peaks by gel-filtration chromatography. The larger form represents collagen molecules (as shown by immunoblotting experiments), and (or) type I collagen with aminoterminal propeptide or intact procollagen molecules. The smaller form, the exact nature of which is not known, is quantitatively the principal antigenic form and is derived from degradation of type I collagen. The concentration of type I collagen in serum is increased mainly in cirrhotic patients, with or without active liver disease, but also somewhat in alcoholic patients without cirrhosis.