bluuebunny/crossref_metadata_2025 huggingface dataset:
Solid-phase extraction with strong anion-exchange columns for selective isolation and concentration of urinary organic acids.
Authors: Verhaeghe B J , Lefevere M F , De Leenheer A P
Published: January 2020

This is a new method for isolating and concentrating urinary organic acids before gas chromatography--mass spectrometry. Sulfate and phosphate anions are removed by precipitation with Ba(OH)2, and the urine pH is adjusted to 8-8.5. The sample is then applied onto small, disposable, strong-anion-exchange columns. Neutral and basic compounds are washed out with water, which is then removed by centrifugation and by rinsing the columns with methanol and diethyl ether. The organic acids are eluted with a 4/1 (by vol) mixture of an organic solvent--either n-butanol or ethyl acetate--and formic acid containing HSO4- (0.1 mol/L) as a highly selective counter-ion, and finally with methanol alone. Sulfate ions are retained, and the eluate is evaporated before trimethylsilyl derivatization. Analytical recoveries (about 100%) of organic acids compare favorably with those obtained with solvent extraction. This convenient procedure is selective and reproducible and is a suitable alternative to the more cumbersome diethylaminoethyl-Sephadex extraction method.
Effect of glucose loading on concentrations of chromium in plasma and urine of healthy adults.
Authors: Morris B W , Griffiths H , Kemp G J
Published: January 2020

We report here a small study designed to identify the effect of a 75-g oral glucose load on concentrations of chromium in plasma and urine of apparently healthy volunteers. We detected a consistent and significant (P less than 0.01) decline in plasma chromium after glucose administration, the nadir of the chromium response coinciding with the zenith of the glucose concentration.
An analysis for blood manganese used to assess environmental exposure.
Authors: Hams G A , Fabri J K
Published: January 2020

In this graphite-furnace atomic-absorption spectrometric method for measuring manganese in whole blood, we use a pyrolytic platform to minimize interference by sample matrix. For optimal sample ashing we denature the sample within the furnace with nitric acid and use oxygen as the purge gas at low temperatures. The mean manganese concentration found in blood from 15 unexposed city dwellers was 215 (2 SD 135) nmol/L. By comparison, the range of manganese concentrations in blood sampled from a group of Australian aborigines living near a surface manganese ore deposit on Groote Eylandt, Northern Territory, was much higher (median 405 nmol/L, range 175 to 990 nmol/L).
Anion transport as related to hemoglobin A1c in erythrocytes of diabetic children.
Authors: Brahm J , Mortensen H B
Published: January 2020

We determined chloride and bicarbonate transport [Jcl and Jbic, nmol/(cm2.s)] under physiological conditions (Cl- 110 and HCO3- 25 mmol per liter, respectively, pH 7.4, 38 degrees C) across the erythrocyte membrane in blood samples from 12 diabetic (Jcl 26.1, SD +/- 3.7, n = 24; Jbic 7.6, SD +/- 0.9, n = 19) and 10 non-diabetic children (Jcl 30.6, SD +/- 4.6, n = 16; Jbic 7.3, SD +/- 1.0, n = 20) with mean hemoglobin A1c values of 11.08% (SD +/- 2.45%) and 5.36% (SD +/- 0.25%), respectively. The concentration of HbA1c, which also reflects the degree of glycation of the membrane proteins, differed significantly (P greater than 0.001) between the two groups, whereas there was no significant variation (P greater than 0.1) in Jcl and Jbic. We conclude that glycation of the integral transport protein in the erythrocyte membrane, capnophorin (also called "band 3"), which mediates a tightly coupled anion exchange, does not change the capacity of the transport system under physiological conditions. Thus the rate-limiting step of the exploitation of the CO2 transport capacity of the blood is not impaired in diabetics and consequently does not endanger the compensatory hyperventilation after ketoacidosis.
Fiber optic immunochemical sensor for continuous, reversible measurement of phenytoin.
Authors: Anderson F P , Miller W G
Published: January 2020

This fiber optic sensor, based on a homogeneous fluorescence energy-transfer immunoassay, operates in a continuous, reversible manner to quantify the anticonvulsant drug phenytoin. B-Phycoerythrin-phenytoin and Texas Red-labeled antibody to phenytoin were sealed inside a short length of cellulose dialysis tubing, which was cemented to the distal end of an optical fiber. When the sensor was alternately placed into solutions with various concentrations of free phenytoin, the drug crossed the dialysis membrane and displaced a fraction of the B-phycoerythrin-phenytoin from the antibody. The resulting change in fluorescence signal was measured with a fiber optic fluorometer. A typical competitive-binding calibration curve was seen between 5 and 500 mumol of phenytoin per liter. Equilibrium response time ranged from 5 to 30 min for different sensors. Replicate equilibrium measurements with one sensor, alternated eight times between two solutions of phenytoin, gave a CV of 2.1% (n = 16) at 0 mumol/L and 2.4% (n = 13) at 100 mumol/L. A reversible immunochemical sensor can be made that has a response time suitable for continuous concentration measurements.
In vivo 31P nuclear magnetic resonance spectroscopy of bone mineral for evaluation of osteoporosis.
Authors: Brown C E , Battocletti J H , Srinivasan R , Allaway J R , Moore J , Sigmann P
Published: January 2020

The mineral content of stationary bone samples can be quantified by 31P nuclear magnetic resonance (NMR) spectroscopy. The assay can be performed in regions of the anatomy that pose problems for absorptiometric techniques, because the mineral content is measured within a selected volume without concern for the geometry of the bone. In vivo 31P NMR spectra of the bones in human fingers and wrist are reported. Soft tissue such as marrow and skeletal muscle contributes little to the 31P NMR spectra of human fingers and wrist and thus should not seriously affect the accuracy of the mineral assay. 31P NMR spectrometry should prove helpful for confirming rapid bone mineral loss in those at risk and for monitoring response to treatment.
Radioimmunoassay of cholecystokinin in plasma.
Authors: Ohgo S , Takemura J , Oki Y , Nishizono F , Ishikawa E , Yoshimi T , Yamamura T , Seino Y , Matsukura S
Published: January 2020

A sensitive and specific radioimmunoassay for cholecystokinin (CCK) has been developed. Synthetic unsulfated carboxy-terminal fragment, CCK-8, was radioiodinated by the conventional Chloramine-T method. Antibodies were raised against sulfated CCK-8 covalently coupled to bovine thyroglobulin via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. By purification, highly immunoreactive 125I-labeled CCK-8 was obtained. The antiserum was highly avid, and plasma could be assayed directly. The detection limit of the assay was 5 pmol of sulfated CCK-8 per liter. The assay measured fragments CCK-8, CCK-33, and CCK-39 with equimolar potency. CCK-4, gastrin, and vasoactive intestinal polypeptide were not detected, even at higher concentrations. The concentration of CCK, as the sum of these CCK peptides, in plasma during fasting was low (10.5 +/- 2.1 pmol/L, mean +/- SEM) but still detectable in all normal subjects examined (range, 6.4-20.1 pmol/L). After ingestion of a test meal, CCK in plasma increased rapidly, peaking at 41.3 (SEM 5.7) pmol/L at 40 min and remaining high for 3 h after the meal. This supports the concept that CCK has important roles in digestion and absorption.
A computerized classification technique for screening for the presence of breath biomarkers in lung cancer.
Authors: O'Neill H J , Gordon S M , O'Neill M H , Gibbons R D , Szidon J P
Published: January 2020

A simple computer-based screening technique has been developed for classifying human expired air components into 16 chemical classes, based on empirical formulas. The sort procedure was developed to simplify the screening of the composition of expired air samples by sorting all components into chemical classes and classifying components at the greater than 75% and greater than 90% occurrence levels. Both occurrence-rate components are then evaluated as diagnostic markers in a discriminant function model for their ability to detect lung cancer. Of the 386 components detected in the gas chromatography/mass spectrometry (GC/MS) data files, 45 components were present at the greater than 75% occurrence level and 28 components at the greater than 90% occurrence level. Thus, this preliminary sort routine, performed by using a simple macro program installed into a standard personal-computer spread-sheet, greatly reduces the amount of data required for statistical treatment. Such a sort routine can also be applied as easily to other complex GC/MS data files for the purpose of data reduction.
Age- and sex-specific pediatric reference intervals and correlations for zinc, copper, selenium, iron, vitamins A and E, and related proteins.
Authors: Lockitch G , Halstead A C , Wadsworth L , Quigley G , Reston L , Jacobson B
Published: January 2020

Age- and sex-specific reference intervals based on the 0.025 and 0.975 fractiles of data derived from a healthy pediatric population are presented for zinc, copper, selenium, iron, ferritin, retinol, alpha-tocopherol, and related analytes in serum. Age was an important covariate for copper, selenium, retinol, and tocopherol, and ferritin in boys. Strong correlations were found between retinol and retinol-binding protein, prealbumin (transthyretin), alpha-tocopherol, and selenium. Tocopherol was highly correlated with both cholesterol and triglycerides. We found no relationship between serum zinc and either retinol or retinol-binding protein. Despite exclusion of children in whom anemia, microcytosis, or variant hemoglobins were found, the 0.025 fractile for iron in several age groups was even less than the concentration considered to indicate poor iron nutritional status.
Sensitivity of immunofixation electrophoresis for detecting IgM paraproteins in serum.
Authors: Kahn S N , Bina M
Published: January 2020

Detection and characterization of small IgM paraproteins by immunoelectrophoresis are often difficult because of (a) slow diffusion of the macromolecular immunoglobulin, and (b) the obscuring of the light chain, especially kappa, by the light chains of background polyclonal immunoglobulin (the "umbrella effect"). Various methods of sample pretreatment have been tried in an attempt to overcome this problem. Using immunofixation electrophoresis (IFE), we were able to detect and characterize IgM paraproteins in serum at concentrations as low as 0.25 g/L, even when the polyclonal immunoglobulin background was increased. With routine IFE, our laboratory identified 58 patients with IgM paraproteinemia in a four-year period. In 36, the paraproteins were present at less than 5 g/L; 10 of these were detectable only by IFE.
Production and characterization of genetically engineered antibody molecules.
Authors: Morrison S L , Canfield S , Porter S , Tan L K , Tao M H , Wims L A
Published: January 2020

Expression of antibody heavy- and light-chain genes by transfection permits the production of monoclonal antibodies with improved biological and antigen-binding properties. The immunoglobulin genes are placed in vectors containing a gene for encoding a protein that provides a biochemically selectable function in eukaryotic cells; these vectors are transfected into myeloma and hybridoma cells. Selection of drug-resistant cells permits the efficient isolation of the rare cells that express the transfected DNA. By placing heavy and light chains on plasmids with different selectable markers, one can deliver heavy- and light-chain genes simultaneously to the same cell. The transfected immunoglobulin genes are efficiently expressed and the proteins produced are a faithful mirror of the genes that were introduced. Using the standard techniques of genetic engineering and gene transfection, we can now produce antibodies of widely varying structures, including chimeric antibodies with segments derived from different species. These antibodies provide useful reagents to study structure-function relationships within the antibody molecule. Ultimately it will be possible to produce a new generation of antibody molecules with improved antigen-binding properties and effector functions.
Analysis of idiotypic and anti-idiotypic antibodies as models of receptor and ligand.
Authors: Linthicum D S , Bolger M B , Kussie P H , Albright G M , Linton T A , Combs S , Marchetti D
Published: January 2020

Antibodies to small bioactive ligands and peptides may mimic the binding characteristics of the natural receptor; in turn, the anti-idiotypic antibodies generated against the binding sites of such anti-ligand antibodies may mimic some aspects of small bioactive ligands and peptides. Among the several levels of investigation of such antibody-receptor networks are (a) the quantitative structure-activity relationships of ligand binding to antibody as compared with natural receptor; (b) the molecular modeling of antibody-receptor binding sites and the genomic basis for such structures; and (c) the characteristics of the molecular mimicry exhibited by "mimetopes" on anti-idiotypic antibodies. To illustrate the analysis encountered at each of these levels, we discuss here antibody and anti-idiotypic systems that are directed to small neuroactive ligands and their receptors.
High-performance immunoaffinity chromatographic detection of immunoregulatory anti-idiotypic antibodies in cancer patients receiving immunotherapy.
Authors: Phillips T M
Published: January 2020

Anti-idiotypic antibodies are regulatory antibodies responsible for the shutdown of active immune responses against growing tumor cells. In an attempt to study these antibodies, a technique for isolating specific anti-idiotypic antibodies by immunoaffinity chromatography was devised. Human anti-tumor antibodies were isolated by affinity absorption to fixed autologous tumor cells. These antibodies were biotinylated, immobilized on streptavidin-coated beads, and used as a ligand to isolate reactive anti-idiotypes from the plasma of patients during periods when immune reactivity against their tumors could not be detected. The isolated anti-idiotypes demonstrated the ability to react with the original antibodies and to inhibit their binding to autologous tumor cells. Thus functional anti-idiotypic antibodies can be isolated by immunoaffinity chromatography with the original idiotype as the ligand. This technique can be used to monitor regulatory antibodies in cancer patients receiving immunotherapy.
Inhibition of mannosidase in hybridomas yields monoclonal antibodies with greater capacity for carbohydrate labeling.
Authors: Simonson R B , Ultee M E , Long C G , Gillette R W , McKearn T J , Rodwell J D
Published: January 2020

Labeling an antibody site specifically through its carbohydrate residues preserves more of its antigen-binding activity than does labeling through protein moieties. To boost the amount of immunoglobulin G carbohydrate capable of being labeled, we treated hybridoma cells with a mannosidase inhibitor, deoxymannojirimycin (dMM). Polyacrylamide gel electrophoresis showed formation of a glycoprotein with high mannose content, in that endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96) could digest the antibody from the dMM-treated cells, but not from control cultures. Carbohydrate analysis confirmed this conclusion, indicating that the antibody from the dMM-treated cells had twice as much mannose as did the control antibody. The glucosamine content of the treated-cells' antibodies was half that of the control, and no additional carbohydrate residues were detectable in the antibodies secreted by the dMM-treated cells. We conjugated both the dMM and control antibodies through their carbohydrate to a chelator. In labeling, the dMM antibody conjugate incorporated approximately threefold as much 111In isotope as the control conjugate. The two labeled antibodies were injected into mice and showed similar organ distributions.
Desirable performance characteristics and clinical utility of immunoglobulin and light-chain assays derived from data on biological variation.
Authors: Ford R P , Mitchell P E , Fraser C G
Published: January 2020

Using automated assays of IgG, IgA, and IgM, and kappa (kappa) and lambda (lambda) light-chains, we assessed the analytical, intra-individual, and interindividual components of variation in a cohort of 12 apparently healthy subjects to examine the utility of the Kallestad Immunochemical Evaluation system. The immunoglobulins and light-chains had small intra-individual variation, and, in consequence, good analytical precisions (CV) of 2.3%, 2.5%, 3.0%, 2.4%, and 2.4% are required for IgG, IgA, IgM, kappa, and lambda assays, respectively. All of these analytes, and the derived kappa/lambda and heavy-/light-chain ratios, have marked individuality. Conventional population-based reference values are of limited utility; therefore, the assays are unlikely to have high diagnostic sensitivity but will be very useful for monitoring individuals. The kappa/lambda ratio may be of particular value in monitoring because a change exceeding 4% does imply a significant (P less than or equal to 0.05) difference in serial results.
High sensitivity, homogeneous particle-based immunoassay for thyrotropin (Multipact).
Authors: Wilkins T A , Brouwers G , Mareschal J C , Cambiaso C L
Published: January 2020

We describe the first homogeneous, nonradioactive, high-sensitivity assay for human thyrotropin (TSH). The assay is based on particle immunoassay techniques, wherein 800-nm particles form the basis for the immunochemistry, delivery, and the detection technologies, respectively. Our assay also is the first to involve the use of fragmented monoclonal antibodies (to eliminate serum interferences) covalently coupled to particles without loss of their binding properties. Assays are performed in a semiautomated mode with use of a new modular system (Multipact). Equilibrium is reached in less than 2 h. Precision profile, sensitivity, and clinical studies indicate that the assay is accurate, has good precision at low concentrations, and that detection-limit characteristics compare well with those of a leading commercial high-sensitivity immunoradiometric assay (IRMA) for TSH. Dilution characteristics were satisfactory down to the assay's detection limit for a range of clinical samples. Correlation studies vs a reference IRMA method yielded the regression equation, present method = 0.976 (IRMA) + 0.002 milli-int. unit/L (r = 0.98), for 223 samples with TSH concentrations in the range 0 to 30 milli-int. units/L. For 40 samples with TSH less than or equal to 1.0 milli-int. unit/L it was: present method = 0.94 (IRMA) + 0.005 milli-int. unit/L (r = 0.96).
Immunoblotting with (sub)class-specific antibodies reveals a high frequency of monoclonal gammopathies in persons thought to be immunodeficient.
Authors: Radl J , Wels J , Hoogeveen C M
Published: January 2020

Immunoblotting, in combination with high-resolution electrophoresis and the use of mouse monoclonal antibodies to human (sub)class immunoglobulin (Ig) isotypes, substantially increased the sensitivity with which homogeneous Ig components (H-Ig) could be detected. Using this technique, we reinvestigated 40 selected sera, previously found to be negative for H-Ig by agar electrophoresis and immunofixation, from two groups of individuals thought to have an age-related immunodeficiency, i.e., persons older than 95 years and recipients of kidney grafts who were undergoing immunosuppressive treatment. In both groups, small single or multiple H-Ig components were found, in frequencies of 76% and 79%, respectively. For comparison, the Ig spectrum of 10 sera from patients on dialysis treatment and of 33 sera from young adult blood donors was ordinarily heterogeneous, except for one elderly patient and one blood donor with a previously unknown IgG2 deficiency. These results are complementary to the observations in some immunodeficiencies in children and indicate that the appearance of single or multiple H-Ig components in low concentration can be considered a very sensitive indicator of certain immune system disorders forming a separate category of monoclonal gammopathies.
Skin-puncture and blood-collecting technique for infants: update and problems.
Authors: Meites S
Published: January 2020

This is updated information on acceptable practice in skin puncture and blood collection in infants, as well as on the devices used, with the additional aim of emphasizing major problem areas and some tentative solutions. Consensus standards for skin puncture have little experimental support, and evade the hard fact that studies are needed to clarify optimum sites for puncture and depth and width of lancets, and to assess the effects of compression and skin resistance in the puncturing process. Preliminary data revealed that the puncturing depth of 2.4 mm recommended for the newborn is excessive. In four of 14 newborns at necropsy, the distance from posterior planar skin surface to underlying bone ranged between 2.0 and 2.2 mm. An experimental lancet, with a 1.8-mm tip length and a diameter of 0.79 mm yielded customary blood volumes from newborns in three of the four pediatric centers where it was tested. Lack of success with the lancet was attributed to inexperienced phlebotomists, not to the lancet's decreased size. Also reviewed are problems with common devices used, and the need for examining the "economy" of blood collection.
A multi-factor experimental design for evaluating random-access analyzers.
Authors: Krouwer J S , Stewart W N , Schlain B
Published: January 2020

A multi-factor experimental design for evaluating random-access analyzers has been developed and tested for the Ciba Corning "550 Express" random-access analyzer. The 12-sample design estimates imprecision, slope, nonlinearity, linear drift, and reagent carryover to the next assay. The design was constructed so that estimates of the factors' effects are almost entirely uninfluenced by each other. Use of the design is illustrated by an example in which reagent-to-assay carryover was pinpointed as an apparent cause of high imprecision. This led to a modification of the analyzer such that carryover was insignificant. The appendix contains a 27-sample design that provides additional estimates. Software to perform such calculations is available on request.
Laboratory evaluation of the Du Pont "Dimension Clinical Chemistry System".
Authors: Knight D , Singer R , White J M , Fraser C G
Published: January 2020

We evaluated the analytical performance of the Du Pont Dimension Clinical Chemistry System to assess its suitability as a replacement for the Du Pont aca III, currently routinely used for analysis of pediatric, high-risk, and emergency specimens. Twenty-six analyses were studied. These generally fulfilled pre-set analytical goals for precision based on biological variation or "state of the art." Significant economies were achieved and we found the Dimension to be reliable, easy to operate, and simple to maintain. It also fulfilled safety requirements for analysis of specimens of high risk to laboratory staff.
Evaluation of a radioimmunoassay of urinary cortisol without extraction.
Authors: Huang C M , Zweig M
Published: January 2020

We evaluated the Kallestad "Quanticoat" cortisol RIA for direct (no extraction) measurement of urinary free cortisol, which requires no solvent extraction. An analytical-recovery study showed a linear regression of y (measured) = 0.65x (added) + 37.5 micrograms/L (Sy.x = 21.4 micrograms/L, r = 0.978, n = 48). Recovery appeared to vary with the urine used and with the concentrations of cortisol added. Within- and between-run CVs were less than or equal to 4.1% and less than or equal to 3.8%, respectively. Cross reactivities were low, except for prednisolone (20.5%). This no-extraction method gave higher values for urinary free cortisol than did either an RIA method involving extraction or an HPLC method. A comparison study with the HPLC (x) and with the method involving extraction (x') gave the following Deming-debiased regression equations: y = 1.60x + 68.8 (Sy.x = 34.4, n = 29) and y = 1.33x' + 0.69 (Sy.x = 40.3, n = 66), respectively. We conclude that the no-extraction method may give misleading results for patients' diagnosis or management if this cross reactivity is not taken into account.
Coloscreen VPI test kit evaluated for detection of fecal occult blood.
Authors: Scriven A J , Tapley E M
Published: January 2020

The usefulness of the Coloscreen VPI test kit in occult blood detection has been examined and compared with our current method. The results show the Coloscreen procedure to be a simple, sensitive method that is much less subject to vegetable peroxidase interference than are other available screening procedures.
A rapid sample-preparation technique for thin-layer chromatographic analysis for 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid in human urine.
Authors: King D L , Gabor M J , Martel P A , O'Donnell C M
Published: January 2020

In this rapid TLC method of analysis for the urinary cannabinoid metabolite, 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), hydrolyzed urine specimens are aspirated through a porous, alkyl-silica extraction layer located along the lower edge of a biphasic thin-layer chromatogram; THC-COOH is simultaneously extracted from the sample and concentrated on the TLC plate for subsequent migration and detection. The method, evaluated in a blind study, identified all samples containing THC-COOH greater than or equal to 20 micrograms/L. The technique increases samples throughput, sensitivity, and specificity as compared with conventional TLC methods and is applicable to a wide variety of compounds.
Effects of estrogen on mineral metabolism in postmenopausal women as evaluated by multiple assays measuring parathyrin bioactivity.
Authors: Stock J L , Coderre J A , Posillico J T
Published: January 2020

Data on the effect of estrogen on immunoreactive parathyrin (iPTH) in postmenopausal women are conflicting. We administered estrogen or placebo to 21 postmenopausal women for 12 weeks and measured PTH bioactivity (bioPTH), using the renal cytochemical bioassay. Before treatment, there was a negative correlation between nephrogenous cAMP and the tubular maximum for urinary phosphate excretion and a positive correlation between values measured by a mid-region-specific PTH RIA and those measured in an immunoradiometric assay for intact PTH. Values measured by the midregion-specific RIA were also positively correlated with nephrogenous cAMP. BioPTH values were not correlated with other indices of PTH activity but were increased compared with values for younger subjects. After estrogen treatment there was no change in bioPTH activity despite an early decrease in serum osteocalcin and a later increase in nephrogenous cAMP. PTH concentrations measured by mid-region-specific or intact RIAs were unchanged, but sample size may have been insufficient to exclude the possibility of significant changes in these values. The effects of estrogen on mineral metabolism in postmenopausal women are time-dependent. Early effects are independent of PTH, and later effects are variably associated with increased PTH activity.
Liquid-chromatographic determination of acetylated hemoglobin.
Authors: Turpeinen U , Stenman U H , Roine R
Published: January 2020

In this liquid-chromatographic assay for acetylated hemoglobin in human blood, glycated hemoglobins in hemolysates are first removed by affinity chromatography on boronate-agarose columns. Acetylated hemoglobin in the nonretained fraction is determined by cation-exchange chromatography. The absorbance of the effluent is monitored at 415 nm. The mean within-assay CV was 8.5%, the between-assay CV 17%. The mean proportion of acetylated hemoglobin in 20 pregnant, nondiabetic women was 1.9%, and in 17 alcoholics it was 2.7%. Rapid and reproducible, this method is suitable for use in routine determination of acetylated hemoglobin.
An improved radioimmunoassay of C-peptide and its application in a multiyear study.
Authors: Myrick J E , Gunter E W , Maggio V L , Miller D T , Hannon W H
Published: January 2020

A commercial radioimmunoassay (RIA) for human proinsulin C-peptide was modified to improve its ruggedness and specificity, to decrease the influence of specimen matrix, and to shorten "hands-on" time. In the new protocol, we prepare calibrators in a C-peptide-free serum pool, prepared by treatment with activated charcoal (biological matrix), instead of in a defined matrix. This yielded essentially 100% analytical recoveries for C-peptide concentrations up to 300 pmol/L, a broader analytical range. We also corrected calibrators and unknown samples for nonspecific binding (NSB). Decreasing the concentration of ethanol (from 950 to 880 mL/L) for differential precipitation of the antigen-antibody complex resulted in an NSB of less than 10%, while maintaining high bound/total count percentages for samples and calibrators. C-peptide is thermally unstable without aprotinin at -20 degrees C and with or without aprotinin at 4 degrees C or above, but multiple freeze-thaw cycles do not affect C-peptide in serum. The modified C-peptide assay was applied to plasma from a multiyear study (fasting and post-carbohydrate-challenge subjects). During the four years of the study CVs ranged from 1.9% to 8.6% for replicate analyses of C-peptide in samples with concentrations less than or equal to 500 pmol/L. Between-run CVs were 3.8% to 8.2%, total CVs 3.8% to 10.7%.
Enzyme immunoassay of hemoglobin A1c: analytical characteristics and clinical performance for patients with diabetes mellitus, with and without uremia.
Authors: Engbaek F , Christensen S E , Jespersen B
Published: January 2020

We evaluated an enzyme immunoassay (Novo BioLabs) for determination of hemoglobin A1c and measured the contribution of carbamylated hemoglobin to the hemoglobin A1c results obtained by HPLC for patients with chronic renal failure. The reference interval, determined for blood donors, was 0.035-0.050 (i.e., 3.5% to 5% of the total hemoglobin). For non-diabetic patients with chronic renal failure, on hemodialysis, the mean proportion of hemoglobin A1c was 0.038 (SD 0.0045); for umbilical cord blood it was 0.006-0.009. For diabetic patients with normal or near-normal renal function, results by enzyme immunoassay (y) and HPLC (x) correlated well (r = 0.83): y = 0.99x - 0.005. Added fetal hemoglobin or carbamylated hemoglobin did not interfere with enzyme immunoassay, but added carbamylated hemoglobin co-chromatographed with hemoglobin A1c. Measurements for patients with chronic renal failure, with or without diabetes, by enzyme immunoassay and by HPLC, were compared with measurements for patients with diabetes mellitus but no renal failure. Carbamylated hemoglobin contributed 0.019 to the hemoglobin A1c results obtained by HPLC for patients with chronic renal failure.
Direct solid-phase enzymoimmunoassay of testosterone in saliva.
Authors: Howard K , Kane M , Madden A , Gosling J P , Fottrell P F
Published: January 2020

This competitive, solid-phase enzymoimmunoassay for testosterone in saliva is carried out on microtiter plates and involves no chromatographic or extraction steps. With an overnight incubation the detection limit of the assay is 230 fg per well (16.1 pmol/L). There was a good correlation (correlation coefficient 0.95) between testosterone concentrations measured with and without prior extraction of the saliva samples. Repeated assay of three control saliva samples containing a range of testosterone concentrations (200-1000 pmol/L) gave within- and between-assay coefficients of variation of 5.5-13.2%. The analytical procedure is simple and closely resembles already published procedures for the determination of progesterone and estrone (with extraction) in saliva. One person can assay 200 samples in 24 h and the assay is suitable for reproductive and sports medical applications, particularly for projects involving serial sampling and yielding large numbers of samples.
Monoclonal antibody to the gamma chain of human fetal hemoglobin used to develop an enzyme immunoassay.
Authors: Moscoso H , Shyamala M , Kiefer C R , Garver F A
Published: January 2020

A monoclonal antibody (mAb) that recognizes the gamma chain of human fetal hemoglobin (Hb F) has been produced by cell hybridization techniques. The mAb reacts with Hb F (alpha 2 gamma 2), Hb Bart's (gamma 4), and Hb Kenya (gamma-beta hybrid), but does not cross-react with Hb A (alpha 2 beta 2) or Hb A2 (alpha 2 delta 2). We describe a direct enzyme-linked immunoassay (ELISA) for measurement of Hb F, in which hemoglobins from standards or from unknown hemolysates are covalently bound to the wells of microtiter plates. The antigen is quantified by addition of the gamma-specific mAb, followed by anti-mouse IgG conjugated with horseradish peroxidase, and incubation with the substrate, tetramethylbenzidine. Absorbances at 630 nm are directly proportional to the amount of Hb F present in the standards or samples. Results for Hb F in 53 hemolysates agreed well with values obtained by "high-performance" liquid chromatography, RIA, alkali denaturation, and magnetic affinity immunoassay. This ELISA can detect a 0.5% proportion of Hb F in 1 h and offers distinct advantages over other techniques currently in use.
Mechanisms responsible for increasing immunoreactivity of apolipoprotein A-I with storage: the role of oxidation.
Authors: Wang X L , Dudman N P , Wang J , Wilcken D E
Published: January 2020

Atherogenesis has been linked to low concentrations of high-density lipoprotein and its principal carrier protein, apolipoprotein (apo) A-I, in serum. We measured apo A-I by enzyme-linked immunosorbent assay, using polyclonal antiserum raised against purified apo A-I and conjugated to peroxidase. During storage at 4 degrees C and above, the immunoreactivity of apo A-I increased so that measured values in stored serum were difficult to interpret. The immunoreactivity of serum apo A-I also increased substantially (up to 50-fold) during exposure to NaIO4. This increase was apo A-I concentration dependent, was linearly dependent on the concentration of NaIO4 up to 200 mmol/L at pH 7.4 and 25 degrees C, and was not ascribable to nonspecific binding of antibody-peroxidase conjugate to the reaction wells. Treatment with NaIO4 caused apo A-I immunoreactivity to increase to a peak or plateau, then gradually decline. In 100 mmol/L NaIO4 (pH 7.4, 25 degrees C), this plateau occurred 30-60 min after oxidation commenced. When frozen serum and stored nonfrozen serum were both oxidized with NaIO4 under the same conditions, the apo A-I from the frozen serum had a significantly (P = 0.0004) greater increase in immunoreactivity. Sera from different patients with different apo A-I concentrations did not have proportionately increased immunoreactivity after treatment with NaIO4. Our findings suggest that the increase in apo A-I immunoreactivity on storage at 4 degrees C or higher involves atmospheric oxidation, and they raise the possibility of in vivo oxidized and nonoxidized forms of apo A-I.
Antipyrine, indocyanine green, and lorazepam determined in plasma by high-pressure liquid chromatography.
Authors: Awni W M , Bakker L J
Published: January 2020

This HPLC method for measuring antipyrine, lorazepam, and indocyanine green in 0.5 mL of plasma can be used in studies of liver function in which these "model" compounds are used. After a fast, simple, one-step extraction procedure with acetonitrile, an isocratic HPLC system is used, with a single detection wavelength (214 nm) and a single internal standard (1-acetamidopyrene). The mobile phase is a 47/53 (by vol) mixture of acetonitrile and 50 mmol/L phosphate buffer, pH 6. The three compounds are separated on an LC-18 reversed-phase column. The low cost of the HPLC method makes feasible the routine clinical measurement of all three compounds.
Interaction between pyridine nucleotide coenzymes and heme proteins as a possible source of error in assay of activities of coenzyme-linked enzyme.
Authors: Jeyasingham M D , Pratt O E , Roopral H K
Published: January 2020

The ultraviolet absorbance spectra of pyridine nucleotide coenzymes change in the presence of heme-containing proteins. The positions of each of the two main absorbance peaks of NADH are shifted progressively towards shorter wavelengths in the presence of increasing concentrations of hemoglobin, and the third peak, at 220 nm, disappears altogether. Similar changes are seen in the spectra of NAD+ and NADPH, and similar effects on these spectra are produced by myoglobin and cytochrome c, but not by comparable concentrations of albumin. The spectral shifts are generally accompanied by a decreased peak height. This finding may help explain problems reported by previous workers in the measurement of the activity of enzymes such as transketolase or lactate dehydrogenase in erythrocyte hemolysates. Errors may be considerable if allowance is not made for this effect, especially if the concentration of heme protein in the spectrophotometer cuvette much exceeds 1 g/L. The interaction appears to indicate some form of bonding, occurring generally between pyridine nucleotide coenzymes and the heme group in proteins. We relate the findings to measurement of activities of pyridine nucleotide-linked enzymes in erythrocyte lysates and in plasma containing myoglobin after muscle breakdown.
Changes in concanavalin A-reactive proteins in inflammatory disorders.
Authors: Silvestrini B , Guglielmotti A , Saso L , Cheng C Y
Published: January 2020

Quantitative changes of concanavalin A (Con A)-reactive proteins in serum samples obtained from rats with induced inflammation and from patients with inflammatory and autoimmune diseases were examined by use of lectin blots. Treatment of rats with a single dose of fermented yeast to induce inflammation caused an extensive increase in Con A-reactivity. These changes were time dependent and were similar in both sexes of the animals. When we examined serum samples obtained from patients with various inflammatory disorders for their Con A-reactive proteins as compared with normal donors, we noted that the Con A-reactivity increased in patients with rheumatoid arthritis and systemic lupus erythematosus. Among all the glycoproteins examined by lectin blots with use of Con A, a set of five proteins was selected for detailed analysis by densitometric scanning. These included alpha 2-macroglobulin, P-150, P-95, P-40, and P-35, of Mr 180,000, 150,000, 95,000, 40,000, and 35,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Densitometric scanning analysis of the lectin blots revealed that the Con A-reactivity of these proteins increased during inflammation. Because alpha 2-macroglobulin is not an acute-phase protein in humans, an increase in Con A staining of this protein suggested that altered glycation is associated with autoimmune diseases. Thus, study of changes in Con A-reactive proteins in human sera may facilitate our understanding of the etiology and pathophysiology of autoimmune diseases.
A rapid, automated micromethod for measuring free fatty acids in plasma/serum.
Authors: Jeevanandam M , Hsu Y C , Ramias L , Schiller W R
Published: January 2020

The investigation of mobilization and utilization of fat as fuel in human subjects requires the quantification of free fatty acids (FFA) in the circulating plasma. Methods in current use involve tedious extraction and titration, or enzymatic reaction with colorimetric or fluorometric detections. A rapid and reliable micro-technique is needed. We have adapted the manual enzymatic method of the Wako NEFAC kit to an automated rapid assay performed with a micro-centrifugal analyzer. This method depends upon the specific acylation of CoA by FFA, followed by oxidation and condensation to form a purple adduct measurable at 550 nm. The acylation step requires incubation at 37 degrees C for 10 min, a critical step for serum/plasma analysis. Only 4 microL of plasma is needed, and 20 tests can be performed in 20 min. The precision (CV) of sample analysis is within 2%. The results for the samples analyzed by this technique are within 4% (SEM 1%) of results by the manual method. Thus accurate results are achieved at reduced cost, time, and sample size.
An improved pyrogallol red-molybdate method for determining total urinary protein.
Authors: Orsonneau J L , Douet P , Massoubre C , Lustenberger P , Bernard S
Published: January 2020

We adapted the pyrogallol red-molybdate method for total urinary protein to the Cobas Bio centrifugal analyzer. The method is simple, rapid, sensitive, and inexpensive. Addition of 25 mg of sodium dodecyl sulfate per liter to the reagent modifies protein reactivities so that the chromogenicity of human gamma globulins is the same as that of albumin. Results by this method and a comparison method that included gel filtration and a modified biuret reaction correlated well (r = 0.951).
Comparative results with five cannabinoid immunoassay systems at the screening threshold of 100 micrograms/L.
Authors: Wells D J , Barnhill M T
Published: January 2020

Five commercially available immunoassay techniques were compared for the detection of cannabinoid metabolites in urine at a positive cutoff of 100 micrograms/L. Numerical values, available for four of these techniques, were used to evaluate inter-assay equivalence at this threshold. The total number of positive results differed for each assay, with some individual samples exhibiting wide variations in numerical values. As a group, the radioimmunoassay systems gave a greater proportion of positive specimens than did enzyme-linked or fluorescence-polarization-based techniques. The single threshold of detection suggested by federal guidelines does not provide for equivalent screening of cannabinoids with these immunoassay techniques.
Time-resolved fluoroimmunoassay of aflatoxins.
Authors: Degan P , Montagnoli G , Wild C P
Published: January 2020

Preparation of purified and Eu-labeled antibodies specific for aflatoxins is described. Their use is illustrated by a solid-phase competitive time-resolved fluoroimmunoassay, results of which were correlated with those of an enzyme-linked immunosorbent assay based on use of the unmodified antibody to aflatoxin. This procedure is discussed as a quick, sensitive, and reliable immunoassay for use in mycotoxin screening in foodstuffs and body fluids.
Influence of O.C.T. embedding compound on determinations of estrogen and progestin receptors in breast cancer.
Authors: Pasic R , Djulbegovic B , Wittliff J L
Published: January 2020

Estrogen receptor (ER) and progestin receptor (PR) concentrations in tumor biopsies are important predictive indicators of a clinical response to endocrine therapy of breast cancer. To assess interference of O.C.T. (optimum cutting temperature) embedding compound in assays of ER and PR by radioligand binding, we determined specific binding capacities and affinities of ER and PR in cytosols by a multipoint titration method, using split samples of 14 breast-tumor biopsies, one portion serving as untreated control, the other treated with O.C.T. There was no statistically significant difference between these two groups. We then compared these data with those of historical controls analyzed both in the presence and absence of sodium molybdate (10 mmol/L). Eighty breast-tumor specimens (mean +/- SD patients' ages, 59 +/- 14 y) embedded in O.C.T. compound and analyzed without molybdate gave ER and PR values that differed insignificantly from those for 306 samples (patients' ages, 61 +/- 14 y) untreated with O.C.T. Thirty-nine specimens (patients' ages, 58 +/- 15 y) embedded in O.C.T. compound were analyzed in the presence of molybdate and compared with the results for 288 specimens (patients' ages, 61 +/- 14 y) untreated with O.C.T. Again, there was an insignificant difference in the concentrations and affinities of receptors in the two groups. Evidently O.C.T. compound does not alter the receptor status of tumor biopsies.
Lactate dehydrogenase isoenzyme composition of human platelets.
Authors: Miyada D S , Fagin A , Pirkle H , Ocariz J , Vaziri N D
Published: January 2020

The LDH isoenzyme composition of 12 platelet preparations was determined by electrophoresis. The mean (+/- SD) percentages of LDH-1, LDH-2, LDH-3, LDH-4, and LDH-5 were 16.6 +/- 1.7, 30.1 +/- 1.0, 34.2 +/- 1.3, 18.2 +/- 1.3, and 0.9 +/- 1.1, respectively. In comparison with previous data, these data show identical ranking of the prevalence of each isoenzyme but significantly different percentages, particularly of LDH-1 and LDH-4. The release of platelet LDH by freezing and thawing differed little from that by homogenization.
Usefulness of data on albumin and prealbumin concentrations in determining effectiveness of nutritional support.
Authors: Bernstein L H , Leukhardt-Fairfield C J , Pleban W , Rudolph R
Published: January 2020

In this ongoing study, albumin and prealbumin (transthyretin) changes were compared in 40 patients managed with enteral and (or) parental support with attainment of caloric/protein goals. The concentration of prealbumin in serum changed rapidly and more accurately reflected current nutritional status of these patients than did that of albumin. We determined concentrations of albumin and prealbumin that reflected significant improvement in nutritional status, using Rudolph's approach based on Shannon information measures. Reference values for albumin and prealbumin in the treatment populations were 25 g/L and 107 mg/L, respectively. A prealbumin concentration of 135 mg/L or greater reflected a return to stable status.
Detection of poisoning by plant-origin cardiac glycoside with the Abbott TDx analyzer.
Authors: Cheung K , Hinds J A , Duffy P
Published: January 2020

Cardiac glycoside poisoning caused by ingestion of plant material is common in tropical and sub-tropical areas. In evaluating the use of the Abbott TDx Digoxin II assay to detect such cases of poisoning, we found it a rapid and convenient method for confirming the ingestion of glycosides from the plants Nerium oleander, Thevetia peruviana, and Adonis microcarpa, and from the toad Bufo marinus. Here we report some clinical cases illustrating our experience with the use of this assay, and describe results of cross-reactivity studies with compounds structurally similar to digoxin. Because of the competitive nature of the immunoassay as well as the complexity of the mixture of cross-reacting cardiac glycosides present in the plant material, the measured apparent digoxin concentration is not linearly related to the cardiac glycoside concentration.
Patterns of lactate dehydrogenase isoenzymes 1 and 2 in serum of patients performing an exercise test.
Authors: Rotenberg Z , Weinberger I , Sagie A , Fuchs J , Davidson E , Sperling O , Agmon J
Published: January 2020

Values for total lactate dehydrogenase (LD, EC 1.1.1.27) activity and LD isoenzymes were determined in serum from 56 patients and 40 healthy subjects before and 24, 48, and 72 h after they performed an exercise test. The mean (for all four times) total LD activity concentration and proportion of LD-2 were within the normal range for all 96 subjects. Mean LD-1 values for serum, although within the normal range in all subjects, were significantly higher in patients with positive exercise test results than in subjects with negative results: 75 (SD 12) U/L in 35 patients with ST depression greater than 2 mm; 63 (SD 14) U/L in 16 patients with ST depression of 1-2 mm; 43 (SD 11) U/L in subjects with negative test results, by 48 h after the test. The LD 1:2 ratio was also markedly higher in the group of patients with positive test exercise results, especially in those with ST depression greater than 2 mm (1.02, SD 0.06), compared with those subjects with negative results (0.60, SD 0.04). A similar trend was also found 24 and 72 h after the exercise test. We conclude that exercise-myocardial ischemia may lead to an increased LD 1:2 ratio in serum, and demonstrate a correlation between the degree of ischemia and the LD 1:2 ratio. Determination of the LD 1:2 ratio, even in the presence of normal total LD activity, may assist in the clinical evaluation of patients performing an exercise test.