bluuebunny/crossref_metadata_2025 huggingface dataset:
Optimized conditions for determining activity concentration of alpha-amylase in serum, with 1,4-alpha-D-4-nitrophenylmaltoheptaoside as substrate.
Authors: Rauscher E , Neumann U , Schaich E , von Bülow S , Wahlefeld A W
Published: January 2020

We describe a method for measuring the catalytic activity of alpha-amylase (EC 3.2.1.1) in serum and urine, by use of a defined substrate: 1,4-alpha, D-4-nitrophenyl maltoheptaoside. We use a phosphate buffer of pH 7.10, containing chloride as activator and alpha-glucosidase (EC 3.2.1.20) as the auxiliary enzyme. After a lag phase of 4 min at 25 degrees C or 30 degrees C, or 3 min at 37 degrees C, the increase of absorption of 4-nitrophenol is measured at 410 nm or 405 nm. The pH value of the assay mixture is a compromise between optimum pH for the alpha-amylase reaction, shortest possible lag phase, and an acceptable absorptivity of 4-nitrophenol. Because the dissociation of 4-nitrophenol depends strongly on pH and temperature, we determined its absorptivity with various combinations of these variables in the assay. Heparin-treated plasma can be used, but not EDTA, fluoride, or citrate. Lipemia, hemoglobin less than or equal to mumol/L, bilirubin less than or equal to 170 mumol/L, glucose less than or equal to 100 mmol/L, and ascorbic acid less than or equal to 1 mmol/L of sample do not interfere in the assay.
Direct measurement of antigens in serum by time-resolved fluoroimmunoassay.
Authors: Kuo J E , Milby K H , Hinsberg W D , Poole P R , McGuffin V L , Zare R N
Published: January 2020

A long-lived fluorescence label (Tb3+) has been attached to the antigen of interest by using a bifunctional chelating agent 1-(p-benzenediazonium)-EDTA. A nonequilibrium competitive-binding immunoassay protocol, in conjunction with time-resolved detection of the long-lived fluorescence label, allows the antigen to be analyzed directly in samples containing diluted human serum. Results obtained for immunoglobulin G with this simple and rapid procedure correlated well (r = 0.93) with those by a commercially available fluorescence immunoassay method.
Efficiency in the diagnosis of acute pancreatitis increased by improved electrophoresis of amylase isoenzyme P3 on cellulose acetate.
Authors: Massey T H
Published: January 2020

Serum from patients who have suffered acute pancreatitis contains P3, an isoenzyme of pancreatic-derived amylase (EC 3.2.1.1). Heretofore, complete resolution of P3 from the major salivary isoenzyme in serum, S1, has not been possible, thus compromising the diagnostic potential of P3 for pancreatitis. I describe an electrophoretic method for the essentially complete resolution of P3 from S1 by including CaCl2, 1 mmol/L, in the Tris barbiturate electrophoresis buffer (25 mmol/L, pH 8.8). I evaluated the clinical utility of the method for 129 consecutive patients suspected of having pancreatitis, by using receiver operating characteristic curve analysis for results for total amylase, P2, and P3 activity. For a true-positive rate of 90% with a prevalence of pancreatitis of 7.8%, the diagnostic efficiency was increased from 82% (total amylase) to 91% (P2) to 98% (P3). Thus, including P3 activity in the diagnostic criteria will eliminate most false-positive results for pancreatitis based on total amylase activity alone, and should decrease the need for expensive radiologic procedures currently required to confirm the presence of pancreatitis. I conclude that P3 can be of significant value in the differential diagnosis of pancreatitis from other syndromes with hyperamylasemia.
New test for urinary glucose (BM33071) evaluated.
Authors: Smalley D L , Bradley M E
Published: January 2020

Results for urinary glucose by the Boehringer Mannheim BM33071 test pad and a hexokinase-based method agree well. The new test, which involves the glucose oxidase/peroxidase reaction, measures as little as 260 mg of glucose per liter. Acetoacetate, beta-hydroxybutyrate, and human hemoglobin do not interfere.
Four methods for determining total protein compared by using purified protein fractions from human serum.
Authors: Nishi H H , Kestner J , Elin R J
Published: January 2020

The proportional bias of four methods frequently used for determining low concentrations of protein was evaluated with human serum protein fractions (Cohn Fractions II, III, IV, and V). Each fraction was assigned a protein concentration value as determined by the biuret method, calibrated with purified human serum albumin monomer. All four methods (Coomassie Brilliant Blue dye-binding, the method of Lowry et al., ultraviolet absorption, and immunonephelometry gave acceptable results for Fraction V (albumin). The ultraviolet absorption and the Lowry methods overestimated the three globulin fractions (II, III, and IV), whereas the other two methods underestimated these fractions. The method of Lowry et al. gave the least proportional bias for the globulin fractions.
Determination of thyrotropin in serum by time-resolved fluoroimmunoassay evaluated.
Authors: Kaihola H L , Irjala K , Viikari J , Näntö V
Published: January 2020

We evaluated a new, highly sensitive time-resolved fluoroimmunoassay for thyrotropin (TSH) in serum. This direct immunometric "sandwich"-type assay involves two monoclonal antibodies against TSH, one immobilized, the other labeled with europium. Extremely high specific activity of the label and the use of labeled antibody in large excess make the method sensitive enough to measure TSH values falling below the normal reference interval. The standard curve is nearly linear over a wide range of TSH concentrations (standard concentrations range from 0.25 to 324 milli-int. units/L). The lowest concentration detectable was 25 micro-int. units/L. The CV for the assay was less than 6% at 0.5 milli-int. unit/L or higher, 11.3% at 0.1 milli-int. unit/L. For a CV of 10% the lower limit of the working range would be around 0.1 milli-int. unit/L. The interassay CV was 6.7 to 11.8% for TSH concentrations of 0.31 to 19.6 milli-int. units/L. The 95% confidence interval for sera from 111 healthy persons was 0.6-3.8 (range 0.3-3.8) milli-int. units/L. For hyperthyroid patients and thyroid cancer patients treated with thyroxin after thyroidectomy, serum TSH values were all below the reference interval (most were less than 25 micro-int. units/L).
Oligoclonal banding in cerebrospinal fluid assessed by electrophoresis on agarose after centrifugal sample concentration through a microconcentrator membrane.
Authors: Christenson R H , Russell M E , Gubar K T , Silverman L M , Ebers G C
Published: January 2020

We describe use of a microconcentrator membrane with a 30 000-Da cutoff for treatment of cerebrospinal fluid (CSF) specimens before detection of oligoclonal bands by electrophoresis on agarose. After centrifugation at 2000 X g for 25 min, 0.5-, 1.0-, and 2.0-mL aliquots of CSF were concentrated 15-, 25-, and 40-fold. Analytical recovery of immunoglobulins G and A from the microconcentrators was about 90% (CV 5-8%). We found good correlation between results by this method and by a silver-stain procedure in a study comparing oligoclonal banding in CSF from multiple sclerosis and control patients. After 40-fold concentration of 2 mL of CSF, 2 mg of immunoglobulin G per liter can be detected, because the analytical sensitivity of the electrophoresis is 80 mg/L.
Determination of dehydroepiandrosterone sulfate in plasma by a one-step enzyme immunoassay with a microtitre plate.
Authors: Dhar T K , Müller C , Schöneshöfer M
Published: January 2020

We have developed a rapid and cost-effective enzyme immunoassay for dehydroepiandrosterone sulfate (DHEA-S) in plasma, performed with samples on a microtitre plate within 2.5 h. No extraction or centrifugation steps are involved. The 3-hemisuccinate of dehydroepiandrosterone is labeled with horseradish peroxidase, then mixed with hydrogen peroxide substrate in the presence of the chromogen, tetramethylbenzidine. The detection limit of the assay is 12.5 pg of DHEA-S per well. Intra- and interassay CVs at three steroid concentrations (12.8, 1.28, and 0.16 mumol/L) ranged from 2.3 to 5.4% and 6.1 to 8.4%, respectively. Results correlated well (r = 0.95) with those of a radioimmunoassay with iodinated DHEA-S. The turnaround time for 41 samples (in duplicate) is 2.5 h, which includes 2 h of incubation time. The sensitivity of this one-step version and the linearity of its standard curve are equivalent to those of a less practicable two-step version. This technique may replace coated-tube enzyme immunoassays for routine use.
Fluorescence energy transfer immunoassay of digoxin in serum.
Authors: Plebani M , Burlina A
Published: January 2020

We have evaluated the reliability of the new fluorescence energy transfer immunoassay (FETI) for determining concentrations of digoxin in serum. The method demonstrated good stability of the standard curve and satisfactory precision in both within-and between-run assay. We have also found a significant correlation between FETI and two RIA methods (r=0.97). Furthermore, the association between results by FETI and by the EMIT method (a conventional enzyme immunoassay) was a linear one. Finally, the assay encountered no significant interference from endogenous substances (hemoglobin, bilirubin , and triglycerides).
Comparison of creatinine as determined with the Ames Seralyzer and by three Jaffé-based methods.
Authors: Kroll M H , Olson D , Rawe M , King C , Hagengruber C , Elin R J
Published: January 2020

We compared results for urinary creatinine, serum creatinine, and creatinine clearance, as determined with the Ames Seralyzer, with results determined with the Beckman ASTRA, the DuPont aca, and Technicon's AutoAnalyzer and SMAC. Results for urinary creatinine from the Seralyzer differed significantly (p less than 0.05) from those obtained with the ASTRA and AutoAnalyzer, but not with the aca. The Seralyzer results for serum creatinine were at least 1.0 mg/L higher (p less than 0.05) than by the other three methods. Results for creatinine clearance from the Seralyzer were 8 to 11 mL/min lower (p less than 0.05) than results by the other three methods. These differences are related to the positive interference by bilirubin in the Seralyzer creatinine method. We also evaluated 23 other compounds for interference with these methods for creatinine.
Analytical patterns and biochemical properties of macro creatine kinase type 2.
Authors: Stein W , Bohner J , Bahlinger M
Published: January 2020

Here we describe our findings for 105 patients' sera containing macro creatine kinase (CK) type 2, as confirmed by exclusion chromatography. Depending on the technique used for determining isoenzyme CKs (electrophoresis, ion-exchange chromatography, immunoinhibition), this variant CK shows characteristic patterns and interferes in CK-MB assays by different mechanisms and to various degrees, thus complicating test interpretation. Macro CK type 2 evidently is not of cytoplasmic origin; rather it is a separate CK activity of human serum, characterized by its heat stability and, especially, by its increased molecular mass and high energy of activation. These latter characteristics have never been associated with the normal-size, dimeric cytoplasmic CK isoenzymes, but are typical for mitochondrial CK isolated from human tissues. We conclude that mitochondrial CK released after severe cell damage usually appears in blood in macromolecular forms (macro CK type 2), not in a dimeric form.
Enzymatic determination of serum ethanol with membrane-bound dehydrogenase.
Authors: Takei H , Nakashima K , Adachi O , Shinagawa E , Ameyama M
Published: January 2020

In this enzymatic method for detecting ethanol in blood by use of membrane-bound microbial alcohol dehydrogenase (no EC no. assigned), the enzyme catalyzes the reaction irreversibly and the rate of oxidation can be monitored by spectrophotometry of the reduction of the indicator dye. No pyridine nucleotides such as NAD+ or NADP+ are used. The calibration curve is linear in the range of 0.1 to 4.0 g of ethanol per liter. Assays of 45 samples of serum having ethanol values ranging from 0.4 to 3.2 g/L by the described technique and a gas-chromatographic method gave respective means of 1.734 and 1.732 g/L (r = 0.954).
Fluorescent substances in uremic and normal serum.
Authors: Shaykh M , Bazilinski N , McCaul D S , Ahmed S , Dubin A , Musiala T , Dunea G
Published: January 2020

We measured the fluorescence, at various excitation (Ex) and emission (Em) wavelengths, of serum ultrafiltrates and fractions of serum resolved by chromatography on Sephadex G15, studying both normal subjects and patients in chronic renal failure requiring hemodialysis. We found hitherto undescribed fluorescence at Ex 380 nm/Em 440 nm and Ex 400 nm/Em 460 nm, the intensity being greatly increased in patients with chronic renal failure in comparison with normal subjects (p less than 0.005). This fluorescence persisted unaltered when serum was filtered through membranes having cutoffs ranging from 10 000 to 500 Da. Each serum fraction resolved by gel chromatography demonstrated a characteristic fluorescence, which was generally much more intense in uremics. The most intense fluorescence (Ex 380 nm/Em 440 nm and Ex 400 nm/Em 460 nm) was emitted in the higher-Mr fractions.
Elimination of the "chromogen oxidase" activity of bilirubin oxidase added to obviate bilirubin interference in hydrogen peroxide/peroxidase detecting systems.
Authors: Maguire G A
Published: January 2020

The use of bilirubin oxidase to remove interference by bilirubin in hydrogen peroxide/peroxidase detecting systems is hampered by its inherent "chromogen oxidase" activity (its ability to oxidize the chromogens used in the systems). This unwanted activity is greater than 99% inhibited by 0.5 mmol/L cyanide, 97% inhibited by 20 mmol/L azide. At these same concentrations, they inhibit bilirubin oxidase activity by 95% and 73%, respectively. Sequential addition of reagents allows the use of bilirubin oxidase without interference by the chromogen oxidase activity.
Direct-measurement ion-selective electrodes: analytical error in hyponatremia.
Authors: Cowell D C , McGrady P M
Published: January 2020

Using two commercial direct ion-selective-electrode sodium and potassium analyzers, we found lower sodium values in cases of hyponatremia as compared with those by flame emission spectrophotometry or indirect ion-selective-electrode analyzers. It is shown that these errors can be eliminated by modifying the calibrant, and that there is a requirement for internationally agreed-upon reference standards for use by manufacturers and laboratory personnel to assess analytical performance of these analyzers.
Injurious effect of EDTA contamination on colorimetry of serum iron.
Authors: Koopman B J , Hindriks F R , Lokerse Y G , Wolthers B G , Orverdijk J F
Published: January 2020

Colorimetry of iron in serum with Ferrozine (as used in the Technicon SMAC) or with bathophenanthroline (as used in the Du Pont aca) is influenced by EDTA, in contrast to such measurements with atomic absorption spectroscopy. Therefore EDTA contamination should be avoided with these colorimetric methods. If, however, contamination with EDTA is suspected, addition of zinc sulfate to serum or to the SMAC "ascorbic acid reagent" will cancel the influence of EDTA on measurements of iron in the SMAC.
Measurement of glycosylated whole-blood protein for assessing glucose control in diabetes: collection and storage of capillary blood on filter paper.
Authors: Little R R , Wiedmeyer H M , England J D , Knowler W C , Goldstein D E
Published: January 2020

We present data on the use of filter-paper blood collection for measurement of glycosylated whole-blood proteins (gWB) (hemoglobin and plasma proteins). A capillary blood sample, obtained by fingerprick, is spotted directly onto filter paper (Schleicher & Schuell 903). The blood spot is washed briefly with alcohol (ethanol or isopropanol) to remove free glucose and dried before shipment to the laboratory. In the laboratory, the blood is eluted from the paper and analyzed for gWB by a colorimetric method. The gWB is primarily a measure of glycosylated hemoglobin (gHb) with a small contribution from glycosylated plasma protein. Concentrations of gWB and gHb are highly correlated (r = 0.91). The filter-paper method offers advantages over currently available methods for quantifying gHb and may be particularly useful in screening for diabetes and for assessing glycemic control in patients from remote areas.
International survey of apolipoproteins A1 and B measurements (1983-1984).
Authors: Cooper G R , Smith S J , Wiebe D A , Kuchmak M , Hannon W H
Published: January 2020

We report here results of a worldwide survey of apolipoprotein (apo) A1 and B measurements. A lyophilized pool of human serum was developed for the survey to test its suitability as a candidate Reference Material. Seventy-five laboratories from 18 countries responded to invitations to participate. Using several methods, they evaluated the mass concentration of apo A1 and B in the proposed Reference Material. The bias between laboratories and between analytical methods was then characterized. Fifty-five laboratories reported results before the deadline on measurement of at least one apolipoprotein by one method. Variation in apo A1 and B among collaborators constituted the largest source of error in the study (68% of the total variation for apo A1, 73% for apo B). Variation among method means was not significant for apo A1, but for apo B it constituted 20% of the total variation. Differences due to vials or in replicate measurements within vials were found to constitute 6% or less of the total variability. Within-laboratory, among-vial, and among-replicate CV values were less than 5% for the study. These results suggest that future standardization efforts can be successful.
Quality control of lipid measurements in epidemiological studies: the U.S. Air Force HEART program.
Authors: Hainline A , Hill P , Garbaczewski L , Winn C
Published: January 2020

A special standardization and quality assurance program, similar to that created for the Lipid Research Clinics Program (LRC), was developed for the American Health Foundation Laboratory by the Centers for Disease Control (CDC) to assure the quality of lipid measurements in the U.S. Air Force Health Evaluation and Risk Tabulation (HEART) Program. This study was designed to test the feasibility of reducing the incidence of heart disease in active-duty U.S. Air Force personnel through life-study intervention. During the 18-month study, CDC provided serum calibrators and reference materials for internal control and an external surveillance program for measurements of total cholesterol (TC) and high-density-lipoprotein cholesterol (HDLC). The Laboratory, using an automated enzymic method to measure cholesterol, achieved an overall goal for accuracy of less than 2% error (av systematic error, -30.6 mg/L) for TC, as measured on nine reference pools for which values were assigned by CDC. The average bias of measurements of HDLC with heparin-manganese to separate the lipoproteins in five CDC reference pools was -4.6 mg/L. Bias was estimated relative to the values assigned to the reference materials by the CDC reference methods for TC and HDLC. The average CV for TC was 0.89%, for HDLC 2.66%. Accuracy of cholesterol measurements can be assured over time with a standardization and quality-assurance program that incorporates accurately labeled reference materials for calibration, internal quality control, and external surveillance.
Facilitated determination of ionized calcium.
Authors: Urban P , Buchmann B , Scheidegger D
Published: January 2020

Using a calcium-containing heparin preparation for anticoagulation, we determined [Ca2+], the mean concentration of ionized calcium, in whole blood of 120 healthy blood-donors to be 1.23 (SD 0.04) mmol/L. Similarly, for 50 intensive-care patients selected without conscious bias, the correlation between [Ca2+] in serum (mean 1.15, SD 0.10 mmol/L) and in whole-blood samples anticoagulated with the same heparin preparation (mean 1.15, SD 0.09 mmol/L) was very good (r = 0.95). Storing samples anaerobically on ice for as long as 2 h did not alter whole-blood [Ca2+]. On the other hand, various concentrations of calcium-free heparin preparations all induced a significant decrease in measured [Ca2+]. By using whole-blood samples, rather than plasma or serum, for [Ca2+] determination with a calcium-selective electrode, repetitive measurements can be made with simple handling procedures, facilitating rapid implementation of appropriate therapeutic measures for critically ill patients.
Carboxyhemoglobin determination by second-derivative spectroscopy.
Authors: Parks J , Worth H G
Published: January 2020

In this procedure hemoglobin is converted to its reduced form and the magnitude of the zero-order spectral shift of the reduced hemoglobin peak at 430 nm to the carboxyhemoglobin peak at 418 nm is determined by second-derivative spectrum analysis. The method is simple, straightforward to set up, and rapid. A result may be obtained within 15 min of receiving the sample. It is sufficiently sensitive to differentiate carboxyhemoglobin concentration in the blood of smokers and nonsmokers.
Hyperlipoproteinemia with albumin-lipid complex: a novel type of hyperlipoproteinemia associated with insulin-resistant diabetes mellitus.
Authors: Habbal Z M , Apelian A Z
Published: January 2020

We describe a new phenotype of hyperlipoproteinemia in two members of a family with a high degree of consanguinity. Both have a history of uncontrolled diabetes mellitus without ketoacidosis, and a family history of coronary artery disease at a relatively early age. A high degree of insulin resistance was found. The abnormal lipoprotein(s) has alpha-lipoprotein mobility on cellulose acetate electrophoresis and has a relative density of less than 1.006 as determined by ultracentrifugation of serum collected after a short fast. The fraction isolated by ultracentrifugation contains about half of the serum cholesterol and triglycerides and most of the phospholipids; the major protein component is albumin. Immunoelectrophoresis showed low concentrations of beta-lipoproteins in both sera, and two abnormal precipitin bands against monospecific antiserum to antilipoprotein A; a third member of the family showed only one abnormal precipitin band against the same antibody. We tentatively propose an abnormal gene(s) as the underlying mechanism. The insulin-resistant diabetes mellitus, probably inherited separately, may aggravate the hyperlipidemia.
Particle-counting immunoassay (PACIA) of pregnancy-specific beta 1-glycoprotein, a possible marker of various malignancies and Crohn's ileitis.
Authors: Fagnart O C , Mareschal J C , Cambiaso C L , Masson P L
Published: January 2020

Pregnancy-specific beta 1-glycoprotein (SP1) was assayed by particle-counting immunoassay (PACIA) with a sensitivity of 1 microgram/L. In serum from 50 men, the SP1 concentration was less than 1 microgram/L, whereas three of the specimens from 46 nonpregnant women had values exceeding 1 microgram/L. In 29% of 950 consecutive patients' sera, SP1 concentrations exceeded 1 microgram/L--in sarcoma (six of six), in malignant hemopathies (101/127, 80%) such as myeloma (20/26, 92%) and acute myeloblastic leukemia (23/27, 90%), and in various other types of cancer (11/19, 58%) except for bronchial epithelioma, which did not lead to any significant increase of SP1 in the five patients examined. The concentration of SP1 was also frequently increased in patients with Crohn's ileitis (28/43, 65%) but not in patients with other inflammatory disorders.
Estimation of thyroxin, triiodothyronine, thyrotropin, free thyroxin, and triiodothyronine uptake by use of magnetic-particle solid phases.
Authors: Witherspoon L R , Shuler S E , Gilbert S
Published: January 2020

We evaluated solid-phase radioassays involving small, uniform magnetic particles (MAGIC, Corning Medical), obtaining data on total serum thyroxin (T4), triiodothyronine (T3), thyrotropin (TSH), free thyroxin (FT4), and T3 uptake for a total of 301 serum samples from euthyroid patients; patients receiving replacement thyroxin; patients receiving estrogen, or who were pregnant; hyperthyroid, hypothyroid, and nonthyroidally ill patients; and patients receiving salicylates, phenytoin, or heparin. We found each procedure to be technically simple and precise (between-assay CVs for T4 2.8-8.2%, T3 9-7.4%, TSH 2.9-4.5%, FT4 3.6-11%, T3 uptake 2.2-3.7%). The magnetic separations are rapid (2 to 5 min), reproducible, and complete. Each MAGIC assay produced clinically appropriate results. These assay systems offer the convenience associated with noncentrifugation assays and excellent analytical performance.
Radiochemical microassay of delta-aminolevulinic acid dehydratase activity in whole blood and bone marrow.
Authors: Kondo M , Urata G
Published: January 2020

We describe an assay for delta-aminolevulinic acid dehydratase (EC 4.2.1.24) activity. Radioactive [14C]porphobilinogen, formed by action of this enzyme on [14C]delta-aminolevulinic acid, is purified by passage through an ion-exchange chromatographic column before measurement with a liquid scintillation counter. The radioactive substance in the final solution was identified as solely [14C]porphobilinogen by paper-chromatographic analysis. The present assay procedure requires only a 0.1-microL sample of blood and is about 100-fold more sensitive than the conventional colorimetric methods involving Ehrlich's reagent. Using this method, we found that activity of this enzyme in the bone marrow of rats decreases abruptly and sharply two weeks after birth.
Chemiluminescence enzyme immunoassay for thyroxin with use of glucose oxidase and a bis(2,4,6-trichlorophenyl)oxalate-fluorescent dye system.
Authors: Arakawa H , Maeda M , Tsuji A
Published: January 2020

In this chemiluminescence enzyme immunoassay for thyroxin, glucose oxidase (EC 1.1.3.4) is the enzyme label and the bound and free fractions are separated by the double-antibody solid-phase technique. In the assay of enzyme activity, hydrogen peroxide generated from glucose substrate is measured by its chemiluminescence reaction with bis(2,4,6-trichlorophenyl)oxalate and 8-anilino-1-naphthalene-sulfonic acid. The measurable range of thyroxin is 2.5 to 200 micrograms/L. The coefficients of variation for thyroxin concentrations of 43.2 and 12.8 micrograms/L were 7.0% and 8.6% (intra-assay), and 6.4% and 12.3% (interassay), respectively. The proposed method is applicable to large-scale preliminary screening of neonates for congenital hypothyroidism, with samples consisting of dried blood spotted on filter paper.
Headspace measurement of ethanol in blood by gas chromatography with a modified autosampler.
Authors: Penton Z
Published: January 2020

An autosampler intended for liquid injection into a gas chromatograph was modified to serve as an automatic headspace sampler by replacing the syringe with a 100-microL gas-tight syringe and shortening the needle that normally enters the vials for sampling. Instead of the usual 1.0 to 1.5 mL of liquid sample, the vials contained 200 microL of liquid; the vapor above the liquid was injected into the gas chromatograph. Blood samples from California drivers were analyzed for ethanol by using the modified autosampler and the values were compared with those obtained by two other methods: the Smith modified version of the Widmark diffusion desiccation oxidation method (J Lab Clin Med 38: 762, 1951) and direct injection of diluted blood into the gas chromatograph. The correlation coefficient between the headspace method described in this paper and the titration method was 0.984; with direct injection, 0.997.
Steroid receptor analysis by size-exclusion liquid chromatography: considerations for the clinical laboratory.
Authors: Pavlik E J , Nelson K , van Nagell J R , Robinson J C , Hanson M B , Donaldson E S , Flanigan R C , Kenady D E
Published: January 2020

Steroid receptor activity can be more quickly estimated by size-exclusion chromatography than by conventional analysis on sucrose gradients, and profiles of receptor activity are better resolved. Here we discuss several factors affecting this form of analysis in clinical laboratories. The composition of the elution buffer influences steroid receptor elution and recovery: high ionic strength, neutral pH, and the presence of dimethylformamide are important. Of the TSK-SW (Beckman) size-exclusion chromatographic columns we considered, the TSK-G2000SW column appeared to be the most appropriate. "Reference" elution profiles are presented for several marker proteins and estrogen receptor forms generated under different sample-treatment conditions. In examining the sensitivity of receptor analyses by this method, we used fresh rodent preparations, a commercial receptor reference (Estrocept), and human tumor material obtained by needle biopsy. We also compared frozen and lyophilized receptor preparations with fresh ones.
Multiple forms of gamma-glutamyltransferase: a clinical study.
Authors: Wenham P R , Horn D B , Smith A F
Published: January 2020

We have measured the amounts of different molecular forms of gamma-glutamyltransferase (EC 2.3.2.2), leucine aminopeptidase (EC 3.4.11.2), and alkaline phosphatase (EC 3.1.3.1) in serum of patients with different types of liver disease. A high-molecular-mass (greater than 1 000 000 Da) form of gamma-glutamyltransferase and of each of the other enzymes is present in greatest amounts in patients with jaundice from extrahepatic obstruction. A gamma-glutamyltransferase form of intermediate molecular mass (250 000 to 500 000 Da) is present in the serum from most patients with liver disease and can be separated by electrophoresis into several bands. We found that one of these bands predominated in patients with extrahepatic obstructive jaundice, whereas the others predominated in patients with other liver diseases. Electrophoresis of serum gamma-glutamyltransferase may be of clinical value in distinguishing extrahepatic from intrahepatic causes of jaundice.
Amniotic fluid acetylcholinesterase activity and alpha-fetoprotein in chromosomal anomalies and neural tube defects.
Authors: Buamah P K , Skillen A W , Harrison J , Davison V
Published: January 2020

Measurement of alpha-fetoprotein concentration and acetylcholinesterase activity in amniotic fluid can be used to identify chromosomal defects as well as neural tube defects. In seven cases of trisomy 21 and one case of partial trisomy 3, alpha-fetoprotein concentrations were below the reference range but values for acetylcholinesterase activity were normal for the appropriate gestational age. One case of trisomy 13 had an increase in acetylcholinesterase activity and normal alpha-fetoprotein concentration.
Determination of serum ferritin by a one-step immunoenzymoassay, and comparison of four liver-ferritin standards.
Authors: Revenant M C , Goudable J , Beaudonnet A , Mailliavin A , Pichot J , Monnet L
Published: January 2020

We describe a one step "sandwich"-type immunoenzymoassay for ferritin in human serum. The solid-phase consists of glutaraldehyde-treated polypropylene tubes coated with rabbit antibody to human ferritin. Liver ferritin is the standard. Peroxidase-conjugated antiserum to ferritin and a sensitive chromogen, o-phenylenediamine, are used. The assay requires 90 min. The standard curve is linear up to 400 micrograms of ferritin per liter of serum. Within- and between-run CVs are less than 6% for low, high, and medium concentrations and are about 13.0% at the decision level for iron deficiency. Results by a two-step "sandwich" procedure (New England Immunology Associates kit) correlated well, r = 0.98. We assessed four liver ferritin standards from different manufacturers with the described method. The mean absorbance for the 40 micrograms/L ferritin standard was 1.5 for that from Diagnostics Biochem and National Institute for Biological Standards and Controls, 1.0 for that from Dako, and 0.4 for that from Sigma. Consequently, to standardize results, all liver ferritin standards should be calibrated vs the National Institute for Biological Standards and Controls reference standard.
Placental alkaline phosphatase and cancer antigen 125 in sera of patients with benign and malignant diseases.
Authors: Eerdekens M W , Nouwen E J , Pollet D E , Briers T W , De Broe M E
Published: January 2020

Human placental alkaline phosphatase (hPLAP; EC 3.1.3.1), cancer antigen 125 (CA 125), and carcinoembryonic antigen (CEA) were determined in sera of patients with malignant and nonmalignant disorders. For CA 125 we used two different commercial assay systems, based on the same monoclonal antibody. hPLAP had the same sensitivity (20%) as CA 125 for detecting non-ovarian neoplasia, whereas that of CEA was 45%. For detecting ovarian cancer CA 125 (Cis kit) was slightly more sensitive (50%) than hPLAP (45%), much more than CEA (10%). hPLAP was increased in sera of 2% of patients with nonmalignant disorders, CA 125 in 23%, and CEA in 18%. hPLAP was increased in only one of 10 diabetic patients and two of 50 patients on chronic renal dialysis. CA 125 and CEA were respectively increased in 45% and 23% of all liver pathologies studied and in 12% and 17% of patients with renal insufficiency. The sensitivity of hPLAP for detecting ovarian cancer is slightly inferior to that of CA 125, but its specificity is much higher. We found the Abbott system for CA 125 to be more sensitive than the Cis system.
Direct spectrophotometry of magnesium in serum after reaction with hexokinase and glucose-6-phosphate dehydrogenase.
Authors: Tabata M , Kido T , Totani M , Murachi T
Published: January 2020

We describe a simple method for determining magnesium in serum by using hexokinase (EC 2.7.1.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). The method is based on determination of the reaction rate of hexokinase activated by Mg2+, which participates in the hexokinase reaction as the substrate in the form of a Mg X ATP2- complex. The reaction rate is determined from the change in absorbance at 340 nm as NADPH is produced by the action of glucose-6-phosphate dehydrogenase. This simple and rapid spectrophotometric method does not require expensive instrumentation, but results correlate satisfactorily with those obtained by atomic absorption spectroscopy. Thus, the present method gives a "true" value for magnesium in serum, a value appreciably lower than that obtained by an earlier colorimetric method, the Xylidyl Blue II method (Biochem Med 7: 208-217, 1973), which lacks specificity.
Studies on vitamin D binding protein in the nephrotic syndrome.
Authors: Colston K , Williams N J , Cleeve H J
Published: January 2020

We studied the properties of vitamin D binding protein in plasma and urine from nine patients with nephrotic syndrome. Samples were incubated with 25-[3H]hydroxyvitamin D3, after which we determined binding capacity and apparent dissociation constants. Binding capacity was markedly less in plasma from patients with nephrotic syndrome than that from normal subjects, but binding affinity was unchanged. Specific binding of 25-hydroxy[3H]vitamin D3 could be demonstrated in urine from all the nephrotic patients, and sucrose density-gradient analysis of these urines revealed a single binding peak with sedimentation characteristics similar to those of vitamin D binding protein in plasma.
Dry-reagent strips used for determination of theophylline in serum.
Authors: Rupchock P , Sommer R , Greenquist A , Tyhach R , Walter B , Zipp A
Published: January 2020

A reagent strip for quantifying theophylline in serum or plasma has been developed for use with the Apoenzyme Reactivation Immunoassay System (ARIS) with the Ames Seralyzer reflectance photometer. The test takes 80 s and involves comparison with a two-point calibration line, which can validly be stored in the instrument for two weeks. Results for theophylline in clinical serum samples correlate well (r greater than 0.98) with results by liquid chromatography, fluoroimmunoassay, and enzyme immunoassay procedures. The within-run CV for four concentrations of controls ranged from 3.5 to 6%; the between-run CVs ranged from 3 to 5%. This assay for use in therapeutic drug monitoring is convenient, rapid, and simple, and thus is appropriate for use in emergency rooms, physician's offices, and small laboratories.
Gamma-glutamyltransferase and its isoenzymes: progress and problems.
Authors: Nemesánszky E , Lott J A
Published: January 2020

Because of its sensitivity to disturbances of excretory liver function, gamma-glutamyltransferase (GGT; EC 2.3.2.2) assay has become one of the most important diagnostic tests for hepatobiliary disorders. However, its value in the differential diagnosis of liver diseases is limited. It is present in kidney, liver, pancreas, and intestine in electrophoretically distinct but not necessarily organ-specific forms. Much effort has been expended to relate diseases of these organs to isoenzyme findings in serum. Although the activities of GGT isoenzymes in disease differ from those in health, the existence of disease-specific patterns is controversial. Much is known about the biochemistry, structure, and immunochemistry of GGT, but more work is needed on methods, standardization of terminology, and correlation of isoenzyme findings with diseases. Further progress in the last area should improve the clinical applicability of the isoenzyme activities.
Fluorometric assay of alpha-L-iduronidase in serum for detection of affected and carrier animals in a canine model of mucopolysaccharidosis I.
Authors: Shull R M , Hastings N E
Published: January 2020

We investigated measuring serum alpha-L-iduronidase (EC 3.2.1.76) by a sensitive fluorometric assay in 28 members of a canine family with mucopolysaccharidosis I. If assayed on the day of collection, serum was an acceptable specimen for identifying affected, carrier, and normal dogs. The overall correlation (r) between iduronidase activity in serum and mononuclear leukocytes was 0.966. However, iduronidase was extremely labile in serum refrigerated or frozen for 48 h.
Stability of vitamin A in frozen sera.
Authors: Driskell W J , Lackey A D , Hewett J S , Bashor M M
Published: January 2020

We compared values for vitamin A measured in fresh sera with values obtained after storage at -20 degrees C for five to eight years. Loss of vitamin A from long-stored sera during the extraction step was eliminated by adding ascorbic acid to the extraction solvent, ethanol. Retinol-binding protein was also measured in the stored sera. Not only did vitamin A values remain stable during the years of storage, but also the correlation between concentrations of vitamin A and retinol-binding protein in the stored sera was typical of that observed with fresh sera.
Acid phosphatase and zinc in semen of subjects with no clinical evidence of prostatic disease.
Authors: Vaubourdolle M , Clavel J P , Cynober L , Piton A , Galli A
Published: January 2020

We evaluated the distribution of values for prostatic acid phosphatase (EC 3.1.3.2) activity and zinc concentration in semen of 80 men without clinical evidence of prostatic disease. Both substances have log normal distributions. Reference intervals were defined by parametric tests after logarithmic transformation of data and by nonparametric tests. There is a correlation between zinc concentration and prostatic acid phosphatase activity in semen.
Determination of vitamin E in microsamples of serum by liquid chromatography with electrochemical detection.
Authors: Chou P P , Jaynes P K , Bailey J L
Published: January 2020

In this procedure for determination of vitamin E by "high-performance" liquid chromatography with electrochemical detection, 25-microL serum specimens are deproteinized with ethanol. Vitamin E (alpha-tocopherol), its derivatives (beta- and gamma-tocopherols), and the internal standard (delta-tocopherol) are extracted into heptane and the extract is evaporated and the residue reconstituted with methanol before injection into the chromatograph. Within- and between-run CVs for an alpha-tocopherol concentration of 13.6 mg/L were 5.1% (n = 28) and 6.0% (n = 5), respectively. The standard curve is linear to 100 mg/L; the minimum concentration detectable is 0.1 mg/L. Analytical recovery ranged from 99.8% to 104.8%. In 36 specimens collected from apparently healthy subjects who were not taking vitamin supplements, alpha-tocopherol as determined by this method ranged from 4.3 to 9.7 mg/L, from 1.8 to 3.9 mg/L for beta- and gamma-tocopherols. Results by this method (y) and an HPLC-ultraviolet method (x) correlate reasonably (r = 0.81): y = 0.88x - 0.55 mg/L (n = 45). This procedure is adaptable to automated analysis, and the small sample requirement facilitates its applicability to neonates.
Immunoreactive phospholipase A2 in serum in acute pancreatitis and pancreatic cancer.
Authors: Nevalainen T J , Eskola J U , Aho A J , Havia V T , Lövgren T N , Näntö V
Published: January 2020

Immunoreactive phospholipase A2 (EC 3.1.1.4) was measured by a new sensitive time-resolved fluoroimmunoassay in the serum of 58 healthy subjects and 103 patients with acute pancreatitis. Patients with acute pancreatitis were grouped according to the etiology and clinical severity of the disease. The mean phospholipase A2 concentration in the reference (healthy) group was 5.5 (SD 1.9) micrograms/L. In acute pancreatitis the mean phospholipase A2 concentration was increased on the first day after hospital admission in all groups, and returned to normal somewhat more slowly than did serum amylase, especially in the patients with severe alcoholic pancreatitis. In this latter group the mean concentration of serum phospholipase A2 on the first day was 42.6 (SD 29.5) micrograms/L. In patients with pancreatic cancer, serum phospholipase A2 was 29.2 (SD 21.3) micrograms/L. The phospholipase A2 and amylase values were closely associated in all groups. The clinical sensitivities were 90.9% for severe alcoholic pancreatitis and 87.5% for pancreatic cancer. Immunochemical determination of phospholipase A2 in serum provides fast and specific detection of injury to pancreatic acinar cells. In addition to the early diagnosis of acute pancreatitis, follow-up determinations of phospholipase A2 seem to be useful in differentiating between mild and severe forms of pancreatitis.
Variability of plasma lipids and lipoproteins: the Jerusalem Lipid Research Clinic Study.
Authors: Friedlander Y , Kark J D , Stein Y
Published: January 2020

We examined the variability of lipid and lipoprotein concentrations in plasma from a population sample from the Jerusalem Lipid Research Clinic study. Coefficients of variation of about 8% for plasma cholesterol, 11% to 15% for low- and high-density-lipoprotein cholesterol, and about 30% for triglyceride were reported, both for 17-year-olds and adults examined twice, with a median period of two months between measurements. Stability was similar in a subsample of adults who had an additional measurement a median of 28 months later. Within-assay analytical variation (CV) was 1.9-2.0% for cholesterol, 1.5-2.3% for triglyceride, and 4.5% for high-density-lipoprotein cholesterol. Between-assay variation was 3-5% for cholesterol and triglyceride and 10% for high-density-lipoprotein cholesterol. The lower stability of the lipoprotein fractions of cholesterol than of total cholesterol emphasizes the need for repeated measurements of these fractions for more accurate characterization of subjects, especially those with extreme values, both for clinical use and for predicting outcome in follow-up studies.
Direct solid-phase radioimmunoassay for screening 17 alpha-hydroxyprogesterone in whole-blood samples from newborns.
Authors: Hofman L F , Klaniecki J E , Smith E K
Published: January 2020

We describe a direct, solid-phase RIA for 17 alpha-hydroxyprogesterone (17-OH-P) that we are using to screen neonates for congenital adrenal hyperplasia. Phosphate buffer containing danazol and anti-17-OH-P is placed in tubes coated with antibody to IgG. The tubes also contain standards, controls, or blood samples on filter paper discs 3 mm in diameter. 125I-labeled 17-OH-P is added to each tube. The mixture is vortex-mixed and incubated overnight. The fluid and disc are removed, the radioactivity remaining in the tubes is counted, and the amount of 17-OH-P per disc is calculated by using a log-logit transformation of the standard curve. Results compare favorably with those by two extraction assays. Inter- and intra-assay CVs were less than 11% and less than 9%, respectively. Sensitivity was 2 pg per assay tube. There is no significant cross reactivity with structurally related steroids at their physiological concentrations. Analytical recovery of added 17-OH-P averaged 104%. 17-OH-P in whole blood spotted on filter paper is stable for at least six months.
Measuring bile-salt concentrations lacks clinical value for detecting hepatic dysfunction in infants receiving parenteral nutrition.
Authors: Beckett G J , Glass E J , Callaghan M O , Elton R A , Hume R
Published: January 2020

Concentrations of conjugated cholate, chenodeoxycholate, direct bilirubin, and alanine aminotransferase (ALT, EC 2.6.1.2) were measured in plasma of 122 low-birthweight infants receiving parenteral nutrition. Eighteen (15%) of them developed hepatic dysfunction. We observed two distinct biochemical patterns in these infants. In the Type A pattern (12 infants), concentrations of direct-reading bilirubin and bile salts increased with no change in ALT activity. In the Type B pattern (six infants), increases in the concentrations of bile salt and direct bilirubin were followed by increases in ALT activity. Hepatic dysfunction persisted significantly longer in infants who developed the Type B pattern. The two patterns did not differ significantly in the times at which values for bile salts or direct bilirubin in plasma became abnormal or became normal at resolution, nor did maximal concentrations of bile salts in plasma differ significantly. Maximal concentrations of direct bilirubin were higher in the Type B infants. We conclude that, in such infants, measurement of bile-salt concentrations in plasma offers no advantages for detecting hepatic dysfunction over the more conventional measurement of direct bilirubin in plasma.
Immunochemical properties of immunoglobulin G conjugated with lactate dehydrogenase.
Authors: Sudo K , Maekawa M , Kanno T
Published: January 2020

Quantitative binding affinities of immunoglobulin G (IgG) for the five isoenzymes of lactate dehydrogenase (LD; EC 1.1.1.27) were determined for IgG isolated from three patients' sera that contained LD-IgG complexes. These three IgGs formed soluble complexes with four (LD 1, 2, 3, 5) of the five LD isoenzymes in two of the patients and in the third they bound with three (LD 2, 3, 4) of the five LD isoenzymes without inhibiting the enzyme activity or precipitating with the enzyme molecules. When rabbit antibody to human IgG was added to these sera, the complexes between the LD isoenzymes and the patients' isolated IgG were completely precipitated. The equilibrium constants for the respective isolated IgG complexes with LD-3 were 0.102, 2.58, and 7.49 X 10(8) L/mol. In these three cases, LD-3 evoked the strongest response from the prepared IgG, demonstrating that the site of antigen recognition of LD-linked IgG was not associated with the structure of individual H and M subunits.