bluuebunny/crossref_metadata_2025 huggingface dataset:
Partial purification of endogenous digitalis-like compound(s) in cord blood
Authors: Balzan Sllvana , Ghione Serglo , Biver Pascal , Gazzetti Psolo , Montali Umberto
Published: January 2020

Increasing evidence indicates the presence of endogenous digitalis-like compound(s) in human body fluids. In this preliminary report, we describe a study of the partial purification by HPLC of these compounds in the plasma of neonates (who have particularly high concentrations of this substance) and adults. Plasma samples from neonates (cord blood) and adults, lyophilized and extracted with methanol, were applied on a 300 x 3.9 mm C18 Nova Pak column and eluted with a mobile phase of acetonitrile/methanol/water (17/17/66 or 14/14/72 by vol) and, after 30 min, with 100% methanol. We assayed eluted fractions for inhibitory activity of 86Rb uptake and for digoxin-like immunoreactivity. The elution profile revealed a first peak of inhibitory activity of 86Rb uptake at the beginning of the chromatography; another peak was eluted with the 100% methanol. The two peaks also cross-reacted with antidigoxin antibodies. Because the second peak could possibly reflect the nonspecific interference of various lipophilic compounds, we focused our attention on the first peak. For these fractions dose-response curves for 86Rb uptake and for displacement of digoxin were parallel, respectively, to those of ouabain and digoxin, suggesting similarities of digoxin-like immunoreactive substance to cardiac glycosides. Similar chromatographic profiles were also obtained for plasma from adults, suggesting that the endogenous glycoside-like compound(s) in the neonate may be the same as those in the adult.
Relationships between the proton nuclear magnetic resonance properties of plasma lipoproteins and cancer
Authors: Otvos J D , Jeyarajah E J , Hayes L W , Freedman D S , Janjan N A , Anderson T
Published: January 2020

We conducted a comprehensive investigation of the origin of nuclear magnetic resonance (NMR) lineshape variability of plasma lipids among healthy individuals and those with cancer. The methyl and methylene resonances of lipid in human plasma, whose linewidths have been reported to correlate with the presence of malignancy, are composed of the overlapping resonances of "mobile" protons from the major lipoproteins (very-low-, low-, and high-density lipoproteins). We tested two hypotheses for the origin of the narrower plasma linewidths observed for cancer patients: (a) malignancy-associated differences in the spectral properties (chemical shift, lineshape) of one or more of the lipoproteins, and (b) differences in the fraction of lipoprotein lipid giving rise to detectable NMR signal. Analysis of the concentrations of lipoprotein lipid and of 500 MHz NMR spectra of the lipoprotein constituents in greater than 100 plasma samples failed to provide support for either hypothesis. Although linewidths were found to be significantly narrower for the cancer group, the difference is entirely attributable to differences in the concentrations of the lipoproteins.
Determination of product shelf life and activation energy for five drugs of abuse
Authors: Anderson Geoffrey , Scott Milda
Published: January 2020

Estimating the shelf life of reagents and controls is a critical step in evaluating new formulations. We describe several stability assessment techniques, giving particular attention to experimental design. Activation energy was experimentally determined for five major drugs of abuse and metabolites: morphine, 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid, amphetamine, phencyclidine, and benzoylecgonine. The use of activation energy in establishing shelf life is illustrated for SENTRY (Hycor Biomedical Inc.) Drugs of Abuse Screen Control, a liquid urine-based product.
Direct measurement of high-density lipoprotein cholesterol by the reflotron assay with no manual precipitation step
Authors: Ng Ronald H , Sparks Katherine M , Statland Bernard E
Published: January 2020

We evaluated the quantification of high-density lipoprotein (HDL) cholesterol in plasma with the Boehringer Mannheim Reflotron reflectance photometric analyzer. The Reflotron is designed for testing in small to medium-size laboratories and physicians' offices. This HDL method does not require a manual precipitation step because the reagent tab contains dextran sulfate (Mr 50,000) and magnesium acetate. It takes 90 s to complete an analysis of 30 microL of plasma. Within-day standard deviations (SDs) were 0.02-0.04 mmol/L (6-16 mg/L). Total SDs over a three-month period were 0.03-0.06 mmol/L (11-23 mg/L). The Reflotron values averaged 0.02 mmol/L (6 mg/L) or 1.3% lower than the Hitachi 737 values; the standard error of the estimate (Sy.x) was 0.07 mmol/L (29 mg/L).
Dry reagent technology for rapid, convenient measurements of blood coagulation and fibrinolysis
Authors: Oberhardt Bruce J , Dermott Sharon C , Taylor Murdock , Alkadi Zaid Y , Abruzzini Anthony F , Gresalfi Nancy J
Published: January 2020

Rapid coagulation and fibrinolysis assays suitable for use with an imprecisely measured sample volume (either whole blood or plasma) have been developed, utilizing a technology based on paramagnetic iron oxide particles (PIOP) that move in response to an oscillating magnetic field. PIOP are combined with appropriate test reagents for clotting and thrombolysis assays and formulated as dry reagents within a capillary test chamber. The minima and maxima of the PIOP oscillations define a two-sided waveform that provides kinetic information on fibrin polymerization and lysis. Subject to the chemistry of the dry reagent formulation, the resulting waveform can be used to define clotting time, lysis onset time, or fibrinogen variables. Applications to one-stage prothrombin time and one-stage activated partial thromboplastin time tests have yielded assays with consistently good correlations with other test methods. Applications to fibrinolysis studies have yielded global assays of thrombolytic activity, in that the assay results reflect the interactions of multiple factors associated with the effectiveness of thrombolytic therapy. Depending on the components utilized in a particular reagent formulation, one can derive information about the activity of such factors as fibrinogen, plasminogen, and related inhibitors, as well as the lytic agent being administered. Use of these assays in a clinical setting should provide a rapid, convenient alternative to conventional testing of coagulation variables and a reliable method for monitoring thrombolytic therapy.
Determination of creatinine in serum and urine by cation-exchange high-pressure liquid chromatography
Authors: Harmoinen Aimo , Sillanaukee Pekka , Jokela Hannu
Published: January 2020

We describe an HPLC method for quantifying creatinine, separating the analyte from other compounds in serum and urine by cation-exchange chromatography and measuring its absorbance at 234 nm. The precision of the method (CV) varied from 2.9% (mean creatinine concentration, 31 mumol/L) to 1.7% (361 mumol/L) within a series of assays and from 3.9% (34 mumol/L) to 2.4% (391 mumol/L) between series. A comparison with the Jaffé method, as performed with a Technicon SMA analyzer, gave the regression line yHPLC = 1.00xJaffé - 12.0 (n = 141, r = 0.998, and Syx = 19). Results also are comparable with those of an enzymatic method, if the enzymatic method is standardized with a serum-based standard when serum samples are measured. An aqueous standard has to be used for enzymatic determination of creatinine in urine.
Neuron-specific enolase concentrations in serum in benign liver diseases
Authors: Collazos Julio , Genollá Jose , Ruibal Alvaro
Published: January 2020

Neuron-specific enolase (EC 4.2.1.11; NSE), a glycolytic enzyme produced by neuronal and neuroendocrine cells, is used as a tumor marker of neuroendocrine cancers, especially small-cell lung cancer. We have evaluated the behavior of NSE in nonmalignant liver diseases in 161 patients who underwent a thorough clinical and biochemical evaluation. Nine of the 161 patients (5.6%)--three of the 86 cirrhotic patients (3.5%), and six of the 75 noncirrhotics (8%)--had abnormal concentrations of NSE. Of the numerous clinical and analytical factors considered, none correlated significantly with NSE. The low false-positive rate, similar to or even lower than for other benign diseases, and the absence of association with the numerous characteristics of liver diseases that we studied, support the lack of any substantial metabolism of NSE by the liver. Consequently, the coexistence of a benign liver disease does not limit the usefulness of this enzyme as a tumor marker.
Influence of monoclonal immunoglobulins in direct determinations of iron in serum
Authors: Bakker A J
Published: January 2020

I compared seven buffers and four chromogens for determining serum iron, to evaluate the frequency of falsely high or low concentrations of iron in 59 sera containing monoclonal immunoglobulins. The results for these direct assays with untreated sera were compared with those obtained by a proposed reference method (Br J Haematol 1978;38:291-4) with protein-free filtrates of the same sera. Sera with monoclonal immunoglobulins sometimes yielded erroneous results; the frequency of errors, which could be as much as 29% (17/59), depended on the composition of buffer and color reagent. Addition of thiourea or a detergent (Triton X-405) to some of the buffers lowered the frequency of errors, but did not abolish them. In only a few of the investigated buffer/chromogen combinations were no errors found. Detection of errors by analyzing the absorbance pattern after mixing sample and buffer was not always successful. Moreover, the presence and magnitude of errors bore no relationship to the type or the concentration of the monoclonal immunoglobulins. Unfortunately, the problem cannot be solved by a simple two- or threefold dilution of the sample.
Single procedure for detection, confirmation, and quantification of benzodiazepines in serum by liquid chromatography with photodiode-array detection
Authors: Puopolo P R , Pothier M E , Volpicelli S A , Flood J G
Published: January 2020

We developed a reversed-phase chromatographic procedure for detecting benzodiazepines and other drugs in serum. A liquid-liquid extraction step with hexane/ethyl acetate isolates the drugs from serum; absolute recoveries are generally greater than 85%. Reconstituted extracts are chromatographed on a 4-microns (particle size) C18 column; 14 drugs and an internal standard (flunitrazepam) are separated in 8 min. Peak detection, purity checking, and identification are performed with a computerized photodiode-array detector. Run-to-run imprecision (CV) for many benzodiazepines is less than 3%. In a study of 126 specimens from Emergency Department patients, the procedure showed excellent agreement with a gas-chromatographic method involving either mass-spectrometric or flame-ionization detection. This single procedure provides rapid and accurate detection, quantification, and confirmation of benzodiazepines in serum.
Quantification of lipoprotein(a) in dried blood spots and screening for above-normal lipoprotein(a) concentrations in newborns
Authors: Van Biervliet J P , Michiels G , Rosseneu M
Published: January 2020

Lipoprotein(a) [Lp(a)] is considered an additional, independent, and largely genetically determined risk factor for the development of premature coronary heart disease. Analogous with increased Lp(a) concentrations that represent an additional risk factor in adults, above-normal concentrations of Lp(a) can be detected in five- to seven-day-old newborns. We describe a simple enzyme-linked immunosorbent assay for measuring Lp(a) in dried blood spots collected by heel-prick in five- to seven-day-old infants. Lp(a) could be quantitatively recovered from blood spots. We chose a cutoff value of 100 mg/L for identifying the newborns at risk, based on the Lp(a) distribution in 180 such infants.
Fibrinogen assays: a collaborative study of six different methods. C.I.S.M.E.L. Comitato Italiano per la Standardizzazione dei Metodi in Ematologia e Laboratorio
Authors: Palareti G , Maccaferri M , Manotti C , Tripodi A , Chantarangkul V , Rodeghiero F , Ruggeri M , Mannucci P M
Published: January 2020

This collaborative study, organized by the Hemostasis Subcommittee of C.I.S.M.E.L., evaluated the accuracy, precision, and comparability of the following six widely used fibrinogen assays: total clottable fibrinogen (Blombäck and Blombäck), clotting time (Von Clauss), turbidimetry (Ellis and Stransky), Chromotime System, prothrombin time (PT)-derived, and radial immunodiffusion (RID). The same frozen samples, with normal and high contents of fibrinogen, were examined in four laboratories. The methods were calibrated with an internal standard whose fibrinogen content was determined gravimetrically. Both the Von Clauss and the RID methods were reliable, accurate, and precise, if adequate calibration was used. The PT-derived method was highly reproducible, but had some problems with accuracy. We demonstrate that an adequate calibration procedure is indispensable for reliable fibrinogen measurements whatever method is used. Because neither the calibration procedures proposed by the manufacturers nor the use of lyophilized commercial plasmas is adequate for this purpose, we urge that an international standard for fibrinogen measurement be promptly established.
Body mass index and liver enzyme activity in serum
Authors: Salvaggio A , Periti M , Miano L , Tavanelli M , Marzorati D
Published: January 2020

The association between body mass index (BMI) and serum liver enzyme activity [gamma-glutamyltransferase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST)] was studied in 3167 subjects, 2373 men and 794 women. The subjects were managers and employees, ages 18-64 years, who were examined during a program of preventive medicine. Analysis of covariance was used to compare the serum liver enzyme activities (expressed as natural logarithms) of the subjects, who were subdivided according to BMI, while also considering age, alcohol and cigarette consumption, and physical activity. In men, the percentage increase in the geometric mean of liver enzyme activity of the obese subjects (BMI greater than 30 kg/m2) compared with that of the normal subjects (BMI less than or equal to 25 kg/m2) was 47.7% (P less than 0.001) for GGT, 55.3% (P less than 0.001) for ALT, and 19.7% (P less than 0.001) for AST; in women, the increase was 63.2% (P less than 0.01) for GGT, 58.4% (P less than 0.001) for ALT, and 7.3% (P greater than 0.05) for AST. Thus, our observations demonstrate a relation between BMI and serum liver enzyme activity.
Factors affecting determinations of manganese in serum by atomic absorption spectrometry
Authors: Nève J , Leclercq N
Published: January 2020

A method was developed for the routine determination of manganese in serum from healthy human subjects by graphite furnace atomic absorption spectrometry. We controlled the experimental conditions rigorously, from sampling to analysis, to minimize contamination and to conserve diluted samples. We optimized the procedure with two serum Reference Materials, one of which had a manganese concentration very close to what is thought to be the physiological concentration in humans. The best analytical performance was obtained by directly injecting into the furnace serum diluted with an equal volume of a solution containing Triton X-100 and sodium EDTA and calibrating by the standard additions procedure or by a calibration graph constructed in a similar matrix. The Zeeman background correction produced better accuracy and precision than did the classical deuterium correction. Within-run CV for a manganese concentration of 12.7 nmol/L in serum was 7.9%, and between-run precision was 16.1%. The mean (SD) serum manganese concentration in 31 healthy adults was 10.8 (SD 3.0) nmol/L. Sex and age of subjects did not affect concentrations.
Detection of toluenediamines in the urine of a patient with polyurethane-covered breast implants
Authors: Chan S C , Birdsell D C , Gradeen C Y
Published: January 2020

Breast prostheses are implanted for augmentation or during reconstructive surgery. One of the more commonly used prostheses is the polyurethane-sponge-covered silicone gel implant. Some clinicians are concerned about the safety of this product because the polyurethane foam disintegrates in vivo, and its subsequent fate is not known. Polyurethane is a polymer formed by reacting diisocyanates and polyols. This study indicates that the polymer sponge breaks down into its reactive monomers, 2,4- and 2,6-toluenediisocyanate, which are converted into their corresponding diamines. We present evidence of the excretion of the diamine metabolites in the urine of a patient implanted with polyurethane-covered prostheses.
Versatile, non-clotting-based heparin assay requiring no instrumentation
Authors: Chen Weiliam , Yang Victor C
Published: January 2020

A non-clotting-based heparin assay requiring no instrumentation has been developed by attaching protamine (a heparin antidote) to a porous filter paper strip and monitoring the subsequent capillary migration of heparin sample through the paper. The area of paper to which heparin adsorbed, which is proportional to the concentration of heparin in the sample, is made visible by spraying Methylene Blue NNX solution onto the paper strip when the sample reservoir has emptied. The dye interacts with heparin to cause a metachromatic shift of the dye's absorption maximum from blue to purple. Heparin concentrations in the samples are estimated according to the length of the purple region on the paper strip. Heparin in plasma (2 to 35 int. units/mL) can be differentiated in approximately 6 min. The plasma heparin values derived by the new method agree reasonably well with those obtained by conventional assays (r greater than 0.99, n = 48).
Relationship of serum estradiol and progesterone concentrations to the excretion profiles of their major urinary metabolites as measured by enzyme immunoassay and radioimmunoassay
Authors: Munro C J , Stabenfeldt G H , Cragun J R , Addiego L A , Overstreet J W , Lasley B L
Published: January 2020

Paired daily blood and urine samples were collected from 10 apparently healthy premenopausal women to compare the hormone profiles of estradiol (E2) and progesterone in serum with those of estrone conjugates (E1Conj) and pregnanediol-3-glucuronide (PdG) in urine. Serum hormones were measured by radioimmunoassay (RIA) kits, whereas the urinary steroid metabolites were assessed by both RIA and enzyme immunoassay (EIA). RIA and EIA values for urinary E1Conj and PdG were not different, and both methods produced urinary profiles that paralleled the profile of the parent steroid in serum. However, the simplicity, flexibility, and economy of EIA will make this method more widely applicable. Mean E1Conj values lagged behind concentrations of serum E2 by one day or less, whereas daily urinary PdG profiles lagged behind serum progesterone by one to two days. Mean urinary profiles of E1Conj were similar whether or not creatinine was used to adjust for urine volume; however, creatinine indexing was beneficial when urinary profiles in individual cycles were compared with changes of serum E2.
Cardiac troponin T in diagnosis of acute myocardial infarction
Authors: Mair Johannes , Artner-Dworzak Erika , Lechleitner Peter , Smidt Jörn , Wagner Ina , Dienstl Franz , Puschendorf Bernd
Published: January 2020

Troponin T is a structurally bound protein found in striated muscle cells. We tested concentrations of its cardiac-specific isotype in peripheral venous blood samples serially drawn from 72 patients with confirmed myocardial infarction. Fifty-nine patients received thrombolytic treatment with intravenous streptokinase, urokinase, or recombinant tissue-type plasminogen activator; because of contraindications, the remaining 13 patients did not. Concentrations of troponin T in plasma, measured by an enzyme-linked immunosorbent assay, started increasing within a few hours after the onset of symptoms (median, 4 h; range, 1-10 h). The sensitivity of troponin T for detecting myocardial infarction was 100% from 10 to 120 h after the onset of symptoms; sensitivity on the seventh day after admission was 84%. Concentrations were increased for up to three weeks in some patients with late or high peak values. Successful reperfusion in Q-wave infarction obviously influences the release of troponin T into plasma, with all such cases showing peak values less than or equal to 26 h (median, 14 h) after the onset of symptoms. Troponin T concentrations in these patients returned to within the reference interval more rapidly than in nonreperfused subjects. In the 13 patients without fibrinolytic therapy, troponin T tended to peak approximately 48 h (median) after the onset of chest pain. Troponin T concentrations in patients for whom thrombolysis was unsuccessful resembled those in patients without fibrinolytic therapy. The specificity of the assay was 96% as tested in samples of 96 emergency-room patients. The reference interval (less than 0.5 micrograms/L) was established from samples of 100 healthy blood donors. Troponin T measurements are a specific and sensitive method for the early and late diagnosis of acute myocardial infarction and could, therefore, provide a new criterion in laboratory diagnosis of its occurrence.
Ligand affinity chromatographic separation of serum IgG on recombinant protein G-silica
Authors: Cassulis Pauline , Magasic Mario V , DeBari Vincent A
Published: January 2020

We purified IgG by an automated chromatographic procedure, using a recombinant DNA-produced Protein G ligand bound to silica as the solid phase. The IgG can be separated from human serum in approximately 20 min with little operator manipulation. Recovery studies indicate that unconcentrated eluates contain 77-100% of the applied IgG. Concentration to volumes approximating that of the applied serum resulted in recovery of 76-98% of the applied IgG. High-resolution protein electrophoresis of the eluates demonstrated retention of the broad gamma band, with virtual absence of other serum proteins. This finding was confirmed by immunoelectrophoresis, which demonstrated homogeneity of IgG in the eluates. IgG subclass distributions in four eluates, compared with the sera from which they were harvested, indicate comparable percentages of IgG1, IgG2, and IgG3 and some enhancement of the IgG4 fraction [serum IgG4: 2.9 +/- 0.4% (SEM); eluate IgG4: 13.8 +/- 1.7%, P = 0.007]. The reactivity of eluates from two sera containing various antibodies to human immunodeficiency virus type 1 was virtually identical to that of the sera from which they were prepared. We conclude that this chromatographic application is an effective purification method for serum IgG and antibodies of the IgG class. The method may be applicable in the specialty clinical laboratory, particularly in those interested in protein and other immunological abnormalities.
Time-integrated measurement of corticosteroids in saliva by oral diffusion sink technology
Authors: Wade Stephen E , Haegele Albert D
Published: January 2020

Saliva, as a medium for assessing adrenocortical function in humans, has many advantages and a few distinct disadvantages. Interpretation of measurements of saliva cortisol is complicated by the contamination of saliva by steroid-binding proteins from blood plasma, enzyme activity in the salivary gland that converts cortisol to cortisone, and the amplification in saliva of the episodic fluctuations in systemic cortisol concentrations. We describe a new measurement technology that rejects artifacts from contamination of saliva by plasma protein, provides for measurement of both cortisol and cortisone, and integrates episodic fluctuations in concentration over a period of hours. This oral diffusion sink technology may greatly enhance the reliable interpretation of corticosteroid concentrations measured in saliva.
Isolation and characterization of zinc coproporphyrin I: a major fluorescent component in meconium
Authors: Horiuchi Kentaro , Adachi Kyoko , Fujise Yutaka , Naruse Hireo , Sumimoto Kazuhiro , Kanayama Naohiro , Terao Toshihiko
Published: January 2020

We isolated a substance from an organic extract of meconium (in neutral pH) that exhibited a porphyrin-like fluorescence peak at 580 nm on excitation at 405 nm and deduced its structure. Spectrometric data suggest that the substance is not free coproporphyrin I or free coproporphyrin III, but is a chelate of coproporphyrin I with Zn2+. We also detected a chelate of coproporphyrin III with Zn2+ by HPLC. These substances may be useful as new indicators of the presence of meconium.
Rapid and specific enzyme immunoassay of serotonin
Authors: Chauveau J , Fert V , Morel A M , Delaage M A
Published: January 2020

A new, highly sensitive enzyme immunoassay (EIA) of serotonin (5HT) is described. The assay is based on the competition between N-succinyl-glycinamide-serotonin (N-SGA-5HT, obtained by acylation of the 5HT in the sample to be assayed) and an enzymic tracer, N-succinyl-5HT-acetylcholinesterase, for binding to rabbit polyclonal antibody coated onto the wells of microtiter plates. The antibody is directed against an immunogen obtained by coupling N-succinyl-5HT to glycyl-bovine serum albumin. The EIA permits the accurate measurement of as little serotonin as 0.5 nmol/L (1.8 pg per well) in blood, plasma, serum, cerebrospinal fluid, urine, platelets, and other tissues, with no significant cross-reactivity with other compounds. The results obtained correlate well with those obtained by HPLC after extraction. The assay has the advantage of permitting the measurement of 5HT in up to 500 samples in as little as 3 h.
Combi scheme: new combined internal/external quality-assessment scheme in The Netherlands
Authors: Steigstra Herman , Jansen Rob T , Baadenhuijsen Henk
Published: January 2020

The Dutch Foundation for Quality Assessment in Clinical Chemistry (SKZL) is the professional organization that conducts external quality-assessment schemes in The Netherlands. However, such schemes in fact assess the performance of the internal quality-control systems of the participating laboratories. In this paper we describe a new concept, relating the data for internal control materials with those for external samples and thereby leading to a combined external/internal scheme (Combi). The statistical principles underlying the Combi scheme are discussed and examples of the graphical presentation of the results are shown. Because the laboratory data are transmitted over the public telephone system to the computers of the SKZL, we also describe the principles of the data communication. At two-month intervals a statistical presentation is sent to all participants. The central database is updated daily with the received results, making possible an on-line consultation regarding the statistics of the accumulated findings of the control materials in use.
Aluminum phthalocyanine-streptavidin: new, sensitive fluorescent tracer for immunoassay
Authors: Kelly T A , Hunter C A , Schindele D C , Pepich B V
Published: January 2020

A new fluorescent reagent based on aluminum phthalocyanine has been developed for general application to immunoassay. This highly sensitive fluorophore, Ultralite 680, may be covalently attached to a variety of biological entities for use as a tracer. We report the use of an Ultralite 680-streptavidin-digoxin conjugate as a tracer in a direct, competitive, heterogeneous, fluorescent immunoassay of digoxin. The assay is performed manually with 50 microL of serum and no sample pre-treatment. Primary anti-digoxin antibody is incubated with a serum sample containing digoxin and Ultralite 680-streptavidin-digoxin. Bound and free tracer are separated, and the bound fluorescence is released and quantified with a commercially available spectrofluorometer. Assay precision was good (CV = 7.05% at a digoxin concentration of 2.00 micrograms/L) and results by our method correlated well with those by a conventional RIA (r = 0.9650, n = 24).
Monoclonal and oligoclonal gammopathies in heart-transplant recipients
Authors: Myara I , Quenum G , Storogenko M , Tenenhaus D , Guillemain R , Moatti N
Published: January 2020

Immunoglobulin abnormalities in serum from 76 heart-transplant recipients were examined by cellulose acetate and agarose gel electrophoresis. Monoclonal components were typed by immunofixation. IgG, IgA, and IgM and total kappa and lambda light chains were quantified by immunonephelometry. We confirm that both monoclonal and oligoclonal immunoglobulin banding are common in serum from these patients. Of the 149 serum samples examined, 21 (15%) had one monoclonal component and 53 (35%) had two or more. These monoclonal immunoglobulins were generally present at a low concentration and were transient. The class of immunoglobulins most commonly involved was IgG (about sevenfold more numerous than IgM); monoclonal IgA components and free light chains were not detected. The nephelometric kappa/lambda and heavy chain/light chain ratios were poor indicators of these abnormalities. Immunoglobulin abnormalities were not correlated with the sex and age of recipients, the pre-existing cardiopathy, the time since transplantation, or plasma concentrations of cyclosporine, but did correlate with plasma immunoglobulin concentration, biopsy findings, and viral infections, especially cytomegalovirus (CMV). A monoclonal IgG purified from a patient with a high titer of anti-CMV antibodies did not react with CMV antigens. The origin of these immunoglobulin abnormalities is unclear. Our data suggest that the presence of monoclonal or oligoclonal banding in heart-transplant recipients is of limited prognostic significance.
Cancer antigen 125 in serum and ascitic fluid of patients with liver diseases
Authors: Molina R , Filella X , Bruix J , Mengual P , Bosch J , Calvet X , Jo J , Ballesta A M
Published: January 2020

Serum concentrations of cancer antigen 125 (CA 125) were determined for 373 patients with various liver diseases: 57 with acute hepatitis, 57 with chronic hepatitis, 244 with liver cirrhosis (86 compensated and 158 decompensated), and 15 with primary liver cancer. The antigen was measured simultaneously in the serum and ascitic fluid of 46 of the patients with liver cirrhosis and sequentially in the serum and ascitic fluid of another 25 cirrhotics treated with paracentesis and (or) diuretics. Abnormal results for CA 125 were detected in sera from 4% of the patients with acute or chronic hepatitis, 60% of the patients with liver cirrhosis, and 67% of the patients with primary liver cancer. The main factor associated with abnormal serum concentrations of this antigen was the presence of ascites, with pathological CA 125 values in 94% of patients with ascites without jaundice (mean 566 +/- 528 arb. units/mL), compared with only 40% of patients with jaundice and without ascites (mean 40.1 +/- 28.5 arb. units/mL) (P less than 0.001). High concentrations of CA 125 were mainly associated with spontaneous bacterial peritonitis. The serum concentration of CA 125 decreased after treatment with paracentesis, but increased in patients treated with diuretics rather than paracentesis. The release of this antigen in liver cirrhosis appears to be independent of the liver disorder and, rather, results from peritoneal synthesis of this antigen.
Free and bound malondialdehyde measured as thiobarbituric acid adduct by HPLC in serum and plasma
Authors: Carbonneau M A , Peuchant E , Sess D , Canioni P , Clerc M
Published: January 2020

Assay of free and total malondialdehyde (MDA) in human serum and plasma from healthy subjects and from patients with high risk of lipoperoxidation was performed as follows: (a) acidic (HClO4, pH 1, at 20 degrees C) or basic (NaOH, pH 13, at 60 degrees C) treatments for 30 min; (b) reaction of the protein-free extract (obtained by acid precipitation) with thiobarbituric acid (TBA); (c) HPLC separation on C18 columns with an eluting solution of methanol/phosphate buffer, 10 mmol/L, pH 5.8 (40/60, by vol), at a flow rate of 1.5 mL/min. Free MDA averaged 0.042 (SEM 0.008) and 0.043 (SEM 0.007) mumol/L, respectively, in serum and plasma from healthy subjects. Free (+/- SEM) MDA increased significantly in the plasma from cancer patients (0.270 +/- 0.047 mumol/L) and from hemodialyzed patients (0.214 +/- 0.035 mumol/L). In serum of hemodialyzed patients, analyses for total MDA were unsuitable because of interfering peaks. MDA bound to NH2 groups constituted 83.2% and 83.5% of total MDA in serum and plasma of healthy subjects, respectively, and only 58% in plasma of hemodialyzed patients.
High-sensitivity laser-induced fluorescence detection for capillary electrophoresis
Authors: Zhang J Z , Chen D Y , Wu S , Harke H R , Dovichi N J
Published: January 2020

Capillary electrophoresis provides very high efficiency separations of biological molecules. Laser-induced fluorescence produces very high sensitivity detection. The combination of the two techniques results in an analytical tool with unprecedented properties: separations with more than a million theoretical plates and detection limits of a few hundred analyte molecules. This paper considers the design of high-sensitivity laser-induced fluorescence detection for capillary zone electrophoresis separation of labeled amino acids and capillary gel electrophoresis separation of DNA sequencing fragments.
Atomic force microscopy: seeing molecules of lipid and immunoglobulin
Authors: Hansma H G , Weisenhorn A L , Edmundson A B , Gaub H E , Hansma P K
Published: January 2020

The atomic force microscope (AFM) can image individual molecules by raster-scanning a sharp tip over a surface. In this paper we present molecular-resolution images of immunoglobulin M (IgM) and of ultraviolet light-polymerized films of the lipid dimethyl-bis(pentacosadiynoyloxyethyl) ammonium bromide ("BRONCO"). The polar head groups of individual lipid molecules can be resolved on the surface of this and other lipid films. These lipid films also provide a good substrate for AFM imaging of DNA and of other molecules such as antibodies. Because the AFM scans surfaces, it is most often successful at imaging either molecules that can form an array on a surface or molecules that are quite firmly attached to a surface. The ability of the AFM to operate under water, buffers, and other liquids makes it possible to study biological molecules under conditions in which they are physiologically active. Imaging of the actual molecular process of fibrin polymerization shows the potential of the AFM for studying biological processes. In the six years since its invention, the AFM has excited much interest and has imaged molecules in a wide range of systems.
Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics
Authors: Blackburn G F , Shah H P , Kenten J H , Leland J , Kamin R A , Link J , Peterman J , Powell M J , Shah A , Talley D B
Published: January 2020

Electrochemiluminescence (ECL) has been developed as a highly sensitive process in which reactive species are generated from stable precursors (i.e., the ECL-active label) at the surface of an electrode. This new technology has many distinct advantages over other detection systems: no radioisotopes are used; detection limits for label are extremely low (200 fmol/L); the dynamic range for label quantification extends over six orders of magnitude; the labels are extremely stable compared with those of most other chemiluminescent systems; the labels, small molecules (approximately 1000 Da), can be used to label haptens or large molecules, and multiple labels can be coupled to proteins or oligonucleotides without affecting immunoreactivity, solubility, or ability to hybridize; because the chemiluminescence is initiated electrochemically, selectivity of bound and unbound fractions can be based on the ability of labeled species to access the electrode surface, so that both separation and nonseparation assays can be set up; and measurement is simple and rapid, requiring only a few seconds. We illustrate ECL in nonseparation immunoassays for digoxin and thyrotropin and in separation immunoassays for carcinoembryonic antigen and alpha-fetoprotein. The application of ECL for detection of polymerase chain reaction products is described and exemplified by quantifying the HIV1 gag gene.
Abbott prism: a multichannel heterogeneous chemiluminescence immunoassay analyzer
Authors: Khalil O S , Zurek T F , Tryba J , Hanna C F , Hollar R , Pepe C , Genger K , Brentz C , Murphy B , Abunimeh N
Published: January 2020

We describe a multichannel heterogeneous immunoassay analyzer in which a sample is split between disposable reaction trays in a group of linear tracks. The system's pipettor uses noninvasive sensing of the sample volume and disposable pipet tips. Each assay track has (a) a conveyor belt for moving reaction trays to predetermined functional stations, (b) temperature-controlled tunnels, (c) noncontact transfer of the reaction mixture between incubation and detection wells, and (d) single-photon counting to detect a chemiluminescence (CL) signal from the captured immunochemical product. A novel disposable reaction tray, with separate reaction and detection wells and self-contained fluid removal, is used in conjunction with the transfer device on the track to produce a carryover-free system. The linear immunoassay track has nine predetermined positions for performing individual assay steps. Assay step sequence and timing is selected by changing the location of the assay modules between these predetermined positions. The assay methodology, a combination of microparticle capture and direct detection of a CL signal on a porous matrix, offers excellent sensitivity, specificity, and ease of automation. Immunoassay configurations have been tested for hepatitis B surface antigen and for antibodies to hepatitis B core antigen, hepatitis C virus, human immunodeficiency virus I and II, and human T-cell leukemia virus I and II.
Total arsenic in urine: palladium-persulfate vs nickel as a matrix modifier for graphite furnace atomic absorption spectrophotometry
Authors: Nixon D E , Mussmann G V , Eckdahl S J , Moyer T P
Published: January 2020

We evaluated the effectiveness of nickel and palladium with or without added potassium persulfate as matrix modifiers for the determination of total arsenic in urine. Complete recovery of pure aqueous solutions of As(III), As(V), cacodylic acid (DMA), monomethylarsinic acid (MMA), and o-arsanilic acid was attained by using both nickel and palladium modifiers. Combined arsenobetaine and arsenocholine (so-called fish arsenic), extracted from a certified control material of dogfish muscle (DORM-1), were completely recovered with Pd-S2O8 matrix modification, but not with nickel. Excellent agreement with target values for arsenic in urines from the Centre de Toxicologie du Quebec, supplied by the Interlaboratory Comparison Program, was attained irrespective of the arsenic source when we used Pd-S2O8 as the matrix modifier.
Prostate-specific antigen in serum occurs predominantly in complex with alpha 1-antichymotrypsin
Authors: Lilja H , Christensson A , Dahlén U , Matikainen M T , Nilsson O , Pettersson K , Lövgren T
Published: January 2020

Immunologic measurements of the serum concentration of prostate-specific antigen (PSA), an abundant prostatic-secreted serine proteinase, are frequently used to monitor patients with prostate cancer, though it has not been ascertained whether this immunoreactivity represents a PSA zymogen, the active proteinase, or PSA complexed to extracellular proteinase inhibitors. To characterize the PSA immunoreactivity in serum, we used monoclonal antibodies produced against PSA and a polyclonal rabbit IgG against alpha 1-antichymotrypsin in the design of three noncompetitive PSA assays: assay T, which detected PSA both when present as the active proteinase and when complexed to alpha 1-antichymotrypsin; assay F, which recognized the active proteinase but most poorly detected PSA complexed to alpha 1-antichymotrypsin; and assay C, which was specific for PSA complexed to alpha 1-antichymotrypsin. We used the three assays to measure PSA immunoreactivity in 64 patients' sera and in the effluent after gel chromatography of sera from four patients. This identified an 80- to 90-kDa complex between PSA and alpha 1-antichymotrypsin as the predominant fraction of the PSA immunoreactivity in blood plasma; an immunoreactive 25- to 40-kDa compound was the minor fraction.
Rapid electrochemiluminescence assays of polymerase chain reaction products
Authors: Kenten J H , Casadei J , Link J , Lupold S , Willey J , Powell M , Rees A , Massey R
Published: January 2020

We demonstrate the first use of an electrochemiluminescent (ECL) label, [4-(N-succimidyloxycarbonylpropyl)-4'-methyl-2,2'- bipyridine]ruthenium(II) dihexafluorophosphate (Origen label; IGEN Inc.), in DNA probe assays. This label allows rapid (less than 25 min) quantification and detection of polymerase chain reaction (PCR)-amplified products from oncogenes, viruses, and cloned genes. For the PCR, we used labeled oligonucleotide primers complementary to human papiloma virus and the Ha-ras oncogene. These samples were followed by ECL analysis or hybridization with specific, Origen-labeled oligonucleotide probes. These studies demonstrate the speed, specificity, and effectiveness of the new ECL labels, compared with 32P, for nucleic acid probe applications. We describe formats involving conventional methodologies and a new format that requires no wash step, allowing simple and rapid sample analysis. These rapid assays also reduce PCR contamination, by requiring less sample handling. Improvements in ECL detectability are currently under investigation for use in DNA probe assays without amplification.
Absorption spectra of human fetal and adult oxyhemoglobin, de-oxyhemoglobin, carboxyhemoglobin, and methemoglobin
Authors: Zijlstra W G , Buursma A , Meeuwsen-van der Roest W P
Published: January 2020

We determined the millimolar absorptivities of the four clinically relevant derivatives of fetal and adult human hemoglobin in the visible and near-infrared spectral range (450-1000 nm). As expected, spectral absorption curves of similar shape were found, but the small differences between fetal and adult hemoglobin absorptivity were important enough that they should be taken into account in multicomponent analysis of hemoglobin derivatives. Common pulse oximeters, however, involving light of 660 and 940 nm, are so insensitive to the presence of fetal hemoglobin that they can be used safely in neonates. The error in pulse oximetry caused by the presence of carboxyhemoglobin is insubstantial, but methemoglobin gives either an understimation or an overestimation at high or low oxygen saturation, respectively, the turning point being near 70% saturation.
Rapid and sensitive chemiluminescent enzyme immunoassay for measuring tumor markers
Authors: Nishizono I , Iida S , Suzuki N , Kawada H , Murakami H , Ashihara Y , Okada M
Published: January 2020

We developed a chemiluminescent enzyme immunoassay (CLEIA) to quantify such tumor markers as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), CA19-9, and CA125. We used a novel chemiluminescent substrate, a derivative of 1,2-dioxetane phosphate (AMPPD), to measure alkaline phosphatase as a labeling enzyme to Fab' fragments of antibody. Regardless of the solid phase, i.e., polystyrene beads (6 mm diameter) or ferrite-coated particles (0.3 microns diameter), the standard curves within the dynamic ranges of the conventional RIA or enzyme immunoassay (EIA) were linear in all cases except for those with AFP. Use of the ferrite particles further shortens the immunoreaction, so the assay can be performed in 30 min. In addition, the relationships between concentrations of the marker and chemiluminescent signals for CA19-9, CA125, and CEA were linear up to concentrations about 10-fold greater than the ordinary dynamic ranges. Intra- and interassay CVs (averages for individual analyte) were 2.2%-4.9% and 2.0%-5.8%, respectively. In an analysis of serum samples, results of the CLEIA correlated reasonably well with those of RIA or EIA. The lower limit of detection by CLEIA with ferrite particles was 390 arb. units/L for CA19-9, 990 arb. units/L for CA125, 0.06 micrograms/L for CEA, and 0.03 micrograms/L for AFP. Thus, the sensitivity increased to between two- and 10-fold that of RIA or colorimetric EIA, depending on the analytes.
Development of an enzyme-linked immunosorbent assay of apolipoprotein E-AII complex in plasma
Authors: Tozuka M , Yoshida Y , Tanigami J , Miyachi M , Katsuyama T , Kanai M
Published: January 2020

Measurement of apolipoprotein (apo) E-AII complex in human plasma is important in determining the role of apoE in lipoprotein metabolism. In this paper, we demonstrate a new and simple method to determine apoE-All complex by using an enzyme-linked immunosorbent assay. Anti-apoE IgG (goat) was used as a capture antibody, and captured apoE-All complexes were detected by an anti-apoAll (rabbit) horseradish peroxidase-conjugated anti-rabbit IgG (goat) system. With this method, apoE-All complex was specifically determined without the interference of apoAll and was not affected by apoE monomer less than 250 mg/L. The content of the complex in reference serum, a normolipidemic serum pooled from five subjects with phenotype E3/E3, was arbitrarily defined as 100%. The coefficients of variation were 3.5%-6.3% within assay and 8.8%-11.6% between assays.
High-Performance Liquid-Chromatographic Measurement of Plasma Creatinine in Newborns
Authors: Scott Paul H
Published: January 2020

This HPLC method for measuring plasma creatinine is based on cation-exchange chromatography and is particularly suitable for use with specimens from babies. A short chromatographic run is performed after simple protein precipitation with zinc sulfate and addition of an internal standard, N-methylnicotinamide. The standard curve for the method is linear up to 200 mumol/L, and analytical recovery of added creatinine is between 101% and 103%. Between-batch precision (CV) is less than 3% for mean creatinine values of 103 and 164 mumol/L. The method is free of interference from other metabolic components and drugs commonly used in neonates in routine clinical practice. Using specimens from neonates, I compared this method with a routinely used automated alkaline picrate method (from Randox Labs., performed on a Cobas MIRA analyzer). Linear-regression analysis yielded a correlation coefficient of 0.90, a slope of 1.00, and an intercept of +0.8 mumol/L. This HPLC method for creatinine should be of use in those circumstances where the alkaline picrate method is known to produce dubious results; however, the latter method is probably more suitable for routine use.
Elimination of Matrix Effects in Electrothermal Atomic Absorption Spectrophotometric Determinations of Bismuth in Serum and Urine
Authors: Dean Shaheb-ud , Tscherwonyi Peter J , Riley William J
Published: January 2020

A sensitive and precise electrothermal atomic absorption spectrophotometric method for determining bismuth concentration is described. Protein precipitation and the use of a palladium modifier reduce the problems of foaming and permit the use of a higher ashing temperature. The detection limit of the assay is 0.9 nmol/L. Total CVs (intra- and interassay) for serum ranged from 3.5% to 15.1% and for urine from 4.8% to 14.5% at concentrations of 60.0 and 6.0 nmol/L, respectively. Analytical recoveries of bismuth added to serum and urine were 102% and 103% over the same range. The method is robust and reproducible and can be accurately calibrated with aqueous standards.