bluuebunny/crossref_metadata_2025 huggingface dataset:
Exposure to mixtures and congeners of polychlorinated biphenyls
Authors: Skerfving S , Svensson B G , Asplund L , Hagmar L
Published: January 2020

There are 209 congeners of polychlorinated biphenyls (PCBs), the metabolism and toxicity of which vary by congeners. Use of PCBs is now restricted, but environmental contamination and human exposure persist. Analysis for "total PCBs" in biological samples gives limited information; congener-specific analysis is far more informative, but more complicated. Concentrations of congeners in serum/plasma, adipose tissue, or milk are useful biomarkers of exposure. Lipids may contain similar concentrations and congener patterns, but these vary between exposures and are different from those of the corresponding exposure mixtures; hence, analysis of lipids cannot be used to identify the original exposure. Some non- and mono-ortho congeners may attain a coplanar conformation, which renders them capable of a dioxin-like action. Toxic equivalency factors (TEFs) have been used to sum that risk as toxic equivalents (TEQs), which are considerably different from congener concentrations. No reliable data have been developed on the relationship between concentrations of "total PCBs" or congeners in biological samples and effects of PCBs on human health, mainly because of the various analytical procedures involved and confounding exposures.
Quality assurance, accreditation, and certification: needs and possibilities
Authors: Dybkaer R
Published: January 2020

Demonstrable quality of laboratory services entails two parts. First, one needs a quality policy statement, identification of user needs, choice of measurement procedures, reference measurement system to provide traceability, control materials, and proficiency testing with materials having reference-measurement-assigned values. Second, it may be useful to obtain recognition of competence in addition to the director's certificate, such as Good Laboratory Practice (when studying toxicity of chemicals), ISO 9000 certification of a self-defined quality system, nongovernmental professional accreditation, or, most demanding, governmental accreditation according to European Standard EN 45,001 with some modifications. These require external quality audit; ISO and EN also require a quality manual, which is valuable documentation in any case.
Monitoring the worker and the community for chemical exposure and disease: legal and ethical considerations in the US
Authors: Ashford N A
Published: January 2020

Biomonitoring of workers and communities raises important legal and ethical concerns, but the two contexts are different. Monitoring workers is usually done by, or at the instigation of, the employer who, in law, is responsible for their health and safety. Whenever worker monitoring leads to the removal of workers, difficult issues emerge affecting labor-management relations, labor law, and discrimination law. Resulting legal and ethical questions are usually framed with the context of the employment contract or labor relationship. In contrast, public health or environmental officials may be the driving force behind biomonitoring of the community. No employer-employee relationship exists, and the doctor-patient relationship may be tenuous. The community may often request biomonitoring, but the situation is no less contentious. On the basis of an historical view of monitoring events within the US, mechanisms are suggested to promote positive interactions between employers and workers and among agencies, individuals, and groups in the monitoring of chemically contaminated communities.
Determination of lead in urine and whole blood by stable isotope dilution gas chromatography-mass spectrometry
Authors: Aggarwal S K , Kinter M , Herold D A
Published: January 2020

A stable isotope dilution gas chromatography-mass spectrometry (GC-MS) method is described for the determination of lead (Pb) in urine and whole blood. The use of lithium bis(trifluoroethyl)dithiocarbamate Pb(FDEDTC) as a chelating agent showed strong memory effect, restricting the range of Pb isotope ratios that can be measured in unknown samples. To overcome this carryover problem, we further derivatized the Pb(FDEDTC)2 chelate with 4-fluorophenyl magnesium bromide to form Pb(FC6H4)4. The sequential analyses of solutions of natural Pb and enriched 204Pb with Pb(FC6H4)4 chelate by GC-MS demonstrated no observable memory effect. Precision and accuracy of Pb isotope ratio measurements with Pb(FC6H4)4 were established, and the isotope dilution GC-MS method was validated by determining Pb concentrations in urine standards from the National Institute of Standards and Technology, urine and blood reference materials from the New York State Department of Health, and blood Pb survey samples from the College of American Pathologists.
Mechanism and elimination of aspirin-induced interference in Emit II d.a.u. assays
Authors: Linder M W , Valdes R
Published: January 2020

The presence of salicylates in urine reduces the signal in Emit assays (Syva), potentially yielding false-negative drugs-of-abuse screening results. We demonstrate that the principal urinary metabolite of salicylate, salicyluric acid (SUA; 2-hydroxybenzoylaminoacetic acid), interferes with the measurement of NADH formed in the assay by reducing the molar absorptivity of NADH at 340 nm. Thus, for a given concentration of d.a.u. analyte the change in absorbance over the assay time interval is less in the presence of SUA. With the Emit cocaine assay on the Hitachi 704 analyzer, the rate of absorbance change (delta AR) monitored at 340 nm for a specimen containing approximately 270 micrograms/L benzoylecgonine (BE) was 57 +/- 1.9 mA/min without SUA and 29 +/- 2.7 mA/min with 5 g/L SUA (n = 20). In contrast, delta AR determined at 376 nm was 18.6 +/- 0.5 mA/min with and 17.9 +/- 0.8 mA/min without 5 g/L SUA (n = 20). Measuring the Emit assay signal at wavelengths where SUA has no absorbance (376 nm) eliminates the interference due to SUA while maintaining the precision of the assay near the cutoff concentration for BE (300 micrograms/L).
Familial high serum concentrations of the carboxyl-terminal propeptide of type I procollagen
Authors: Sorva A , Tähtelä R , Risteli J , Risteli L , Laitinen K , Juntunen-Backman K , Sorva R
Published: January 2020

We describe a family with an apparently autosomal-dominant trait that caused extremely high circulating concentrations of the carboxyl-terminal propeptide of type I procollagen (PICP). All family members examined had normal values for other biochemical markers of bone formation and degradation and no related clinical abnormalities. Furthermore, their serum concentrations of the amino-terminal propeptide of type I procollagen (PINP) were normal. Although PINP and PICP are released from the same precursor molecule, PINP is cleared from the circulation via the scavenger receptor in liver endothelial cells, whereas PICP is cleared via the mannose receptor of these cells. We thus hypothesize that the clearance of circulating PICP is compromised in the affected subjects of this family, the result of either a defective mannose receptor function or an abnormal molecular structure of their PICP.
Conceptual framework for evaluating laboratory tests: case-finding in ambulatory patients
Authors: Silverstein M D , Boland B J
Published: January 2020

In response to concerns about inappropriate testing and healthcare costs, clinicians and laboratorians should work together to evaluate laboratory tests. Studies of routine laboratory tests ordered for screening and case-finding in the preoperative setting, at hospital admission, and in the ambulatory setting have not provided sufficient evidence of the benefits of case-finding when evaluated against commonly used criteria for screening procedures. We adapt a conceptual framework developed from radiology for evaluating imaging technologies to evaluate laboratory tests. The framework addresses diagnostic efficacy (whether the test correctly identifies abnormalities), diagnostic effectiveness (whether the test changes the physician's diagnoses), therapeutic efficacy (whether the test changes patient management), and therapeutic effectiveness (whether the test changes patients' outcomes). We propose that clinicians and laboratorians use this framework to evaluate the diagnostic efficacy, diagnostic effectiveness, therapeutic efficacy, and therapeutic effectiveness of laboratory tests in the ambulatory setting and in other settings.
Components of biological variation in prolyl endopeptidase and dipeptidyl-peptidase IV activity in plasma of healthy subjects
Authors: Maes M , Scharpé S , De Meester I , Goossens P , Wauters A , Neels H , Verkerk R , De Meyer F , D'Hondt P , Peeters D
Published: January 2020

We investigated the components of biological variation in plasma prolyl endopeptidase (PEP; EC 3.4.21.26) and dipeptidyl-peptidase IV (DPP IV; EC 3.4..14.5) activity in healthy individuals. We took monthly blood samples from 26 healthy volunteers for determination of plasma PEP and DPP IV activity during one calendar year. The estimated CVs for PEP activity were: total (CVt) = 25.0%, interindividual (CVg) = 13.9%, and intraindividual (CVi) = 16.8%. There was a statistically significant (P < 0.0001) seasonal pattern in plasma PEP activity, with significantly higher values in the fall than in the other seasons. The peak-trough difference in the yearly variation in PEP activity, expressed as a percentage of the mean, was as high as 56.8%. The estimated CVs for DPP IV activity were: CVt = 17.1%, CVg = 14.5%, and CVi = 8.2%. DPP IV activity was significantly (P < 0.0001) higher in summer than in the other seasons but the amplitude of the yearly variation was small.
Microwave-induced rapid preparation of fluoro-derivatives of amphetamine, methamphetamine, and 3,4-methylenedioxymethamphetamine for GC-MS confirmation assays
Authors: Thompson W C , Dasgupta A
Published: January 2020

We prepared trifluoroacetyl, pentafluoropropyl, and heptafluorobutyl derivatives of amphetamine, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA) in 45 s, 1 min, and 6 min, respectively, by using microwave irradiation. Conventional techniques require heating the reaction mixture for 15 min at 40 degrees C for trifluoroacetyl derivatives, 15 min at 75 degrees C for pentafluoropropyl derivatives, and 40 min at 60 degrees C for heptafluorobutyl derivatives. The mass-spectral fragmentation patterns and the gas-chromatographic retention times of the derivatives obtained by both microwave irradiation and conventional heating were similar. Perfluorooctanoyl derivatives of amphetamine can be prepared quantitatively by either heating the reaction mixture for 30 min at 60 degrees C or by 1 min of microwave irradiation. Conversion of methamphetamine and MDMA to the corresponding perfluorooctanoyl derivatives was not quantitative by either technique, although the yield of the derivative in the conventional technique was much higher.
Fatty acids in erythrocytes measured by isocratic HPLC
Authors: Abushufa R , Reed P , Weinkove C
Published: January 2020

We developed an isocratic reversed-phase HPLC method to measure arachidonic, palmitoleic, linoleic, eicosatrienoic, oleic, palmitic, and stearic acids from hydrolyzed erythrocytes. Washed erythrocytes were heated in methanol:HCl and the fatty acids extracted into hexane:amyl alcohol. After derivatization with 4-bromomethyl-7-methoxycoumarin, samples diluted in mobile phase (acetonitrile:water, 85:15 by vol) were injected onto a 250 x 4.6 mm C18 column, and the eluted fatty acids were detected fluorometrically. For all analytes, the mean within-batch CV was 8.2% (5.5-10.8%), the mean limit of detection was 7.0 mumol/L, a linear response was maintained up to 400 mumol/L, and results agreed well with those by gas chromatography. The addition of antioxidant (butylated hydroxytoluene) was essential for sample stability. We discuss hydrolysis and extraction times, derivatization temperature, critical steps in chromatography, and concentration units.
High-density lipoprotein subfractions measured in stored serum
Authors: Bausserman L L , Saritelli A L , Milosavljevic D
Published: January 2020

We compared the effects of freezing serum on the determination of high-density lipoprotein (HDL) subfractions by two dual-precipitation methods, heparin and manganese chloride/dextran sulfate (HM/DS) (Gidez et al., J Lipid Res 1982;23:1206-23) and DS/DS (Warnick et al., Clin Chem 1982;28:1574), and by ultracentrifugation. Storing serum for 1 month at -70 degrees C resulted in reduced HDL3-cholesterol by ultracentrifugation and reduced total and HDL3-cholesterol by the DS/DS method. There was no change in either total HDL-cholesterol or HDL3-cholesterol with the HM/DS method. Additional studies involving only HM/DS indicated that total HDL-cholesterol in serum stored at 4 degrees C begins to decline after 3 days (-3.1 +/- 3.5%, P < 0.1). HDL was stable at -20 degrees C for 2 weeks but both total and HDL3-cholesterol decreased significantly after 1 month. Storage of serum at -70 degrees C resulted in no changes for 1 year; however, at 18 months, HDL3-cholesterol was reduced 13% (P = 0.002). We conclude that HDL subfractions can be determined accurately in serum as well as in plasma after storage at -70 degrees C for up to 1 year.
Phospholipase A2, C-reactive protein, and white blood cell count in the diagnosis of acute appendicitis
Authors: Grönroos J M , Forsström J J , Irjala K , Nevalainen T J
Published: January 2020

We compared the predictive value of determining group II phospholipase A2 (PLA2) in serum for diagnosing acute appendicitis with the predictive values of white blood cell count (WBC) and measurement of C-reactive protein (CRP). In this prospective study, we included 186 patients who were undergoing appendectomy after clinical diagnoses of acute appendicitis. The performance of each test was measured by receiver-operating characteristic curves. WBC was the test of choice in diagnosing uncomplicated acute appendicitis. However, in contrast to CRP and PLA2, which increased in patients with protracted inflammation, there was not a concomitant increase in WBC. Therefore, especially CRP, but also PLA2, were better indicators of appendiceal perforation or abscess formation than was WBC. Increased WBC, CRP, and PLA2 values did not unequivocally corroborate the clinical suspicion of appendicitis, but if all three values were within normal limits, acute appendicitis could be excluded with a 100% predictive value. PLA2 values showed a highly significant correlation with CRP but not with WBC values, which supports the view that PLA2 represents an acute-phase reactant.
ELISA of pentosidine, an advanced glycation end product, in biological specimens
Authors: Taneda S , Monnier V M
Published: January 2020

Pentosidine is a fluorescent protein cross-link and glycoxidation marker for the advanced glycation reaction in diabetes, aging, and uremia. We raised polyclonal antibodies in New Zealand White rabbits against this hapten coupled to keyhole limpet hemocyanin. The antibodies detected by ELISA reacted strongly with free pentosidine but not with pentosidine-like compounds. The working range of the competitive ELISA for standard pentosidine was 0.1-100 pmol. Pentosidine was detectable in bovine serum albumin incubated with ribose as a function of incubation time. Immunoblotting studies showed that pentosidine specifically stained in oligomers of lysozyme incubated with ribose. Digestion with protease (Pronase E, 20 g/kg) as well as acid hydrolysis enhanced the immunoreactivity of samples, the pentosidine values in digested human plasma correlating with those measured by HPLC (r = 0.98). Pentosidine in diabetic and uremic plasma digested with Pronase E was significantly higher than normal (P < 0.01; mean +/- SD): 1620 +/- 1940 and 2630 +/- 1320 [corrected] nmol/L, respectively, vs 151 +/- 55 nmol/L (normal). Amounts of pentosidine in hydrolyzed skin collagen increased with age and were increased in diabetes and uremia. This ELISA provides a new tool for assessing the role of the advanced Maillard reaction in aging and age-related diseases.
Nanotechnology: basic concepts and definitions
Authors: Kaehler T
Published: January 2020

Molecular nanotechnology is an engineering discipline in which the goal is to build devices and structures that have every atom in the proper place. By means of this general purpose material-processing technology it will be possible to build almost any rigid, covalently bonded structure. Identical parts will be truly identical, enabling energy conversion and computation systems to have extremely high performance and reliability. Assembly will be done by mechanosynthesis, the process of holding two reactive molecules in contact with each other in a controlled orientation. Synthesis will be done on nanoscale assembly lines called molecular mills, where systems of moving belts will press individual molecules together and catalyze 10(6) reactions/s per station. High-performance mechanical computers will use moving rods to block the motion of other rods. The most important physical limit will be radiation damage. Without redundancy, a subsystem lasting 100 years will be limited to 10(6) nm3.
Kromoscopic analysis: a possible alternative to spectroscopic analysis for noninvasive measurement of analytes in vivo
Authors: Sodickson L A , Block M J
Published: January 2020

Light that penetrates scattering media shows nonlinearities that mask the broad and shallow perturbations made by trace analytes on the background illuminant spectrum. Narrow-band spectroscopic decomposition and deconvolution of such weak bands is a formidable analytical task that pushes the fundamentally linear spectroscopic method beyond practical limits. Kromoscopy is a high-dimensional analog of human color perception; it has broad-band spectrally overlapping detectors similar to those of the visual system, but in the infrared. Analyte bands are integrated fully in two or more detectors with different relative weightings. As in color vision, the analyte information is coded in the direct correlations between detector signals, which individually have higher signal-to-noise ratios than their spectroscopic counterparts. Our Kromoscopic instrument responds directly to glucose in aqueous solution, is not affected by temperature disturbances, and is fast enough to measure physiologically induced Kromoscopic changes in the arterial pulse waveform with high precision.
The white IRIS leukocyte differential analyzer for rapid high-precision differentials based on images of cytoprobe-reacted cells
Authors: Kasdan H L , Pelmulder J P , Spolter L , Levitt G B , Lincir M R , Coward G N , Haiby S I , Lives J , Sun N C , Deindoerfer F H
Published: January 2020

Combining unique cytoprobe, rapid hemacyte fractionation, and novel color image analysis, The White IRIS (TWI) extends automated intelligent microscopy to leukocyte differentiation. TWI provides flow cytometry precision and microscopical resolution to review specimens flagged by hematology analyzers with differential capabilities or to complement other analyzers without these capabilities. The system includes compartments for closed sampling, rapid leukocyte-rich plasma preparation, cytoprobe-induced metachromasia, and collection and color analysis of leukocyte images, and presents the results as a single-view 500-cell differential on a 20-in. (50-cm) touch-screen monitor. Method correlations for the five mature cell types averaging r > 0.90 were obtained with a prototype system. Classification of normal and abnormal specimens showed 95% agreement with a reference method without any undetected significant morphologic abnormality. False-positive and false-negative rates of 7.27% and 3.53%, respectively, exceeded performance of current commercial systems. Case studies demonstrate the ease and speed with which unusual pathologies and leukemias can be observed and interpreted.
D-lactic acidemia and aciduria in pediatric and adult patients with short bowel syndrome
Authors: Bongaerts G , Tolboom J , Naber T , Bakkeren J , Severijnen R , Willems H
Published: January 2020

D-Lactate produced by abundant intestinal lactobacilli during acidotic episodes in short bowel (SB) patients is commonly regarded as a main factor in the pathogenesis of SB syndrome-associated (D-lactic) acidosis. Since we had observed that gram-positive bacteria, mainly lactobacilli, were abundant even in the absence of acidosis, we studied serum concentrations and urinary excretions of D- and L-lactate in young and adult SB patients, especially during nonacidotic periods. Serum L-lactate and urinary L-lactate excretion were similar in adults and children. Serum D-lactate and urinary D-lactate excretion were higher in SB children than in SB adults. Food consumption affects D-lactate production and alters D-lactic acidemia and aciduria. We conclude that D-lactate is frequently present in serum of SB patients even in the absence of acidosis. High serum concentrations and urinary excretions may reflect dietary factors in these patients.
Increased hepatic secretion of very-low-density lipoprotein apolipoprotein B-100 in cholesteryl ester storage disease
Authors: Cummings M H , Watts G F
Published: January 2020

Using a stable isotope method, we measured the hepatic secretion rate of very-low-density lipoprotein apolipoprotein B-100 (VLDL apoB) in a 26-year-old women who had dyslipidemia due to cholesteryl ester storage disease (CESD) and in five normolipidemic subjects. [1-13C]Leucine was administered by a primed constant intravenous infusion and the enrichment of VLDL apoB was determined by gas chromatography-mass spectrometry. The absolute secretion rate (ASR) of VLDL apoB in the patient was more than twice the mean ASR of the normolipidemic group (17.1 vs 8.0 +/- 0.8 mg/kg body wt. per day). The plasma mevalonic acid concentration, a measure of intrahepatic cholesterol synthesis, was also greater in the patient than in the normolipidemic subjects (8.3 vs 4.4 +/- 1.8 micrograms/L). The findings are consistent with the hypothesis that in CESD increased intrahepatic synthesis of cholesterol stimulates hepatic secretion of VLDL apoB and this may partly account for the dyslipidemia.
Enhanced chemiluminescence labeled-antibody immunoassay (Amerlite-MAB) for free thyroxine: design, development, and technical validation
Authors: Christofides N D , Sheehan C P
Published: January 2020

We describe the development and validation of a one-step nonradioactive immunoassay for free thyroxine (Amerlite-MAB FT4) in serum or plasma, in the dedicated Amerlite enhanced luminescence assay system. A monoclonal antibody to thyroxine (T4), conjugated with horseradish peroxidase (HRP; EC 1.11.1.7), competes for binding with FT4 or with a conjugate of a protein and L-triiodothyronine immobilized onto the surface of the assay microwells. The signal is generated by antibody-HRP bound to the protein-T3 conjugate, with luminol as substrate. The assay design exploits the high sensitivity of luminescence signal detection, permitting minimal sample dilution and T4 sampling by the antibody. It withstands progressive dilution of serum and is unaffected by T4-binding proteins in serum. The disclosure and validation of this FT4 assay follows guidelines recommended by the American Thyroid Association.
Structural and functional polymorphism of lipoprotein(a): biological and clinical implications
Authors: Scanu A M
Published: January 2020

Lipoprotein(a) [Lp(a)], a variant of low-density lipoprotein, is heterogeneous in density because of variability in the content and composition of its core lipids and size polymorphism of its specific glycoprotein component, apolipoprotein(a) [apo(a)]. In some individuals, density polymorphism may also derive from the fact that Lp(a) contains 2 mol of apo(a) per mole of apoB100, contrary to the more common 1:1 molar stoichiometry. Moreover, the size of apo(a) is polymorphic because of variations in the number of kringle 4 type 2 repeats. Another type of apo(a) polymorphism is related to sequence mutations at the kringle level. Two mutations can occur in kringle 4 type 10: one, Trp72-->Arg, is affiliated with an Lp(a) that is lysine-binding defective; the other, Met66-->Thr, with a normal lysine-binding function. Thus, Lp(a) is structurally and functionally polymorphic, a notion that must be considered in assessing the cardiovascular pathogenicity of this lipoprotein variant and in immunoquantification assays.
Plasma homocyst(e)ine and arterial occlusive diseases: a mini-review
Authors: Malinow M R
Published: January 2020

Homocysteine (HCY), which is derived from the intracellular metabolism of methionine, is exported into plasma, where it circulates mostly in oxidized forms (i.e., homocystine and cysteine-HCY disulfide) and mainly bound to proteins. Concentrations of total HCY, or homocyst(e)ine [H(e)], are increased in 15-40% of patients with coronary, cerebral, or peripheral arterial diseases. Such association of H(e) with arterial occlusive diseases has been documented in retrospective, cross-sectional, and prospective studies. Concentrations of H(e) are also increased in subjects having thickened carotid arteries, as determined by ultrasonography, and who are asymptomatic for atherosclerosis. Statistical analyses of data from several series of patients demonstrate that H(e) concentrations are associated with coronary artery disease, independently from most other risk factors for atherosclerosis. The increased concentrations of H(e) are readily corrected by folic acid, occasionally supplemented with pyridoxine, vitamin B12, choline, or betaine. Whether these supplements affect the evolution of atherosclerotic disease needs to be established by prospective, placebo-controlled clinical trials.
Estrogen and progesterone receptors in breast cancer microsamples simultaneously quantified by enzyme-ligand immunoassay
Authors: Krambovitis E , Hatzidakis G , Hatzoglou A , Romain S , Durand A , Stefanakis A , Castanas E
Published: January 2020

A new method (enzyme-ligand immunoassay, ELIA) is described for the estimation of estrogen (ER) and progesterone (PR) receptors in microsamples of human breast cancer tissue. The technique, based on the nonisotopic measurement of receptor-bound estradiol and progesterone, involves three steps: (a) simultaneous saturation of active receptors with their respective authentic ligands, (b) heat treatment of the cytosol to release the steroids from their cognate receptors before or after absorption with dextran-coated charcoal, and (c) measurement of both steroids present in the cytosol by a modified competitive-inhibition enzyme immunoassay. The useful range of the method was 10-4000 pmol/L for ER and 6.5-1000 pmol/L for PR. The correlation coefficient (r) between the one-point and Scatchard plot analysis was 0.95 for ER and 0.99 for PR. Comparison of the one-point ELIA and expected values with the radioligand binding assay (RLBA) results for EORTC samples gave r = 0.88 and 0.99 for ER and PR, respectively. Further comparison of the one-point ELIA with RLBA and with a commercial enzyme immunoassay, in blind testing of cancer tissue microsamples from 70 patients, gave good agreement for ER with r = 0.95-0.97 and concordance of 92.9-94.4% (cutoff, 15 pmol/g protein) against the other two methods. The results were more disperse in all three methods for PR estimation, the assay correlating perhaps better with the enzyme immunoassay (r = 0.90) at a concordance of 89.4% (same cutoff value).
Finnish-type aspartylglucosaminuria detected by oligonucleotide ligation assay
Authors: Delahunty C M , Ankener W , Brainerd S , Nickerson D A , Mononen I T
Published: January 2020

Aspartylglycosaminuria (AGU) is a recessively inherited lysosomal storage disease that occurs with much higher frequency in Finland than elsewhere. AGU is caused by a deficiency in glycosylasparaginase (GA), which results in the accumulation of glycoasparagines in lysosomes. In the Finnish population, a single nucleotide change in the gene encoding GA is responsible for the disease. We have used the oligonucleotide ligation assay (OLA) to detect the mutation in polymerase chain reaction (PCR)-amplified DNA samples from normal, carrier, and affected individuals. Screening for AGU among 415 random Finnish DNA samples with PCR/OLA revealed five carriers of the mutant allele and demonstrated the potential of the method for use in carrier screening. PCR/OLA provides a rapid, reliable, nonisotopic method to detect the mutation responsible for AGU that can readily be applied to large population screening.
Standardization of glycohemoglobin results and reference values in whole blood studied in 103 laboratories using 20 methods
Authors: Weykamp C W , Penders T J , Miedema K , Muskiet F A , van der Slik W
Published: January 2020

We investigated the effect of calibration with lyophilized calibrators on whole-blood glycohemoglobin (glyHb) results. One hundred three laboratories, using 20 different methods, determined glyHb in two lyophilized calibrators and two whole-blood samples. For whole-blood samples with low (5%) and high (9%) glyHb percentages, respectively, calibration decreased overall interlaboratory variation (CV) from 16% to 9% and from 11% to 6% and decreased intermethod variation from 14% to 6% and from 12% to 5%. Forty-seven laboratories, using 14 different methods, determined mean glyHb percentages in self-selected groups of 10 nondiabetic volunteers each. With calibration their overall mean (2SD) was 5.0% (0.5%), very close to the 5.0% (0.3%) derived from the reference method used in the Diabetes Control and Complications Trial. In both experiments the Abbott IMx and Vision showed deviating results. We conclude that, irrespective of the analytical method used, calibration enables standardization of glyHb results, reference values, and interpretation criteria.
Cloned enzyme donor immunoassay (CEDIA) for drugs-of-abuse screening
Authors: Armbruster D A , Hubster E C , Kaufman M S , Ramon M K
Published: January 2020

Large numbers of specimens (5000-18,000) were screened for amphetamines, barbiturates, cocaine, marijuana, opiates, and phencyclidine by RIA (Roche), Emit II (Syva), and a new immunoassay, CEDIA (cloned enzyme donor immunoassay, Microgenics). All immunoassays performed equivalently for cocaine, opiates, and phencyclidine. All immunoassays detected the same amphetamine/methamphetamine-positive specimens, but all also detected numerous specimens containing cross-reacting sympathomimetic amines. CEDIA detected 100%, Emit II 93%, and RIA 82% of the barbiturate-positive specimens. Emit II and CEDIA detected 86-88% of the specimens found by RIA to be marijuana positive. A subset of specimens was additionally screened by OnLine (Roche) and TDx (Abbott) for amphetamines, cocaine, and marijuana. OnLine and TDx also detected all of the amphetamine-positive specimens and numerous specimens containing cross-reacting sympathomimetic amines. All immunoassays performed equivalently for cocaine, and the four nonisotopic tests detected 86-89% of the marijuana positives found by RIA. Interfering sympathomimetic amine drug compounds can be eliminated by using an oxidizing agent, thus decreasing the number of unconfirmable amphetamine presumptive positives. The CEDIAs for all of the major drugs of abuse are reliable and effective for large-volume urine screening programs.
Are reference intervals for carboxyhemoglobin appropriate? A survey of Boston area laboratories
Authors: Marshall M D , Kales S N , Christiani D C , Goldman R H
Published: January 2020

CO is a leading cause of poisoning deaths in the US today. Treating physicians use the carboxyhemoglobin (COHb) % saturation to guide the diagnosis and treatment of CO intoxication. We conducted a telephone survey of hospitals and laboratories in the Boston area, focusing on methodology for COHb determination and accompanying COHb reference intervals. Among 130 facilities, 23 (18%) provide COHb analysis. All facilities that perform the COHb test utilize dedicated multiwave-length photometry. Reference intervals for COHb varied widely among facilities. Eight of 21 (38%) facilities give unacceptably high "normal intervals" for nonsmokers when compared with values available in the literature. Thirteen of 20 (65%) use reference intervals for smokers that are too low, and 3 of 20 (15%) use values that are too high. These reference values provided by the testing facilities may be misleading to the ordering physicians unfamiliar with background COHb saturations. This may lead to misdiagnoses, false reassurances, and perhaps less aggressive treatment than might be warranted. The results of this study argue for wider adoption of COHb reference intervals supported by the current literature.
Measurement of a more stable region of osteocalcin in serum by ELISA with two monoclonal antibodies
Authors: Rosenquist C , Qvist P , Bjarnason N , Christiansen C
Published: January 2020

Intact human osteocalcin purified from femoral bones as well as tryptic fragments of the intact molecule [amino acids (aa) 1-19, 20-43, and 45-49] were used to raise and screen monoclonal antibodies (MAbs). A two-site ELISA for measurement of human osteocalcin in serum was developed with use of these MAbs. A MAb recognizing midregion human osteocalcin (aa20-43) was used as capture antibody, and an NH2 terminus (aa7-19)-specific peroxidase-conjugated MAb was used for detection. Human osteocalcin obtained from bone was used for calibration, and parallelism was observed for osteocalcin from serum samples, NH2-terminal midfragments (aa1-43), and synthetic human osteocalcin. Both inter- and intraassay variations were < 7%. Serum osteocalcin in healthy premenopausal women (n = 49) was 18.3 +/- 4.2 micrograms/L (mean +/- SD) and 28.6 +/- 9.7 micrograms/L in early postmenopausal women (n = 114). The mean serum concentration (n = 10) decreased by 10% after 7 days of storage at 4 degrees C, whereas the concentration of intact human osteocalcin was reduced 63%. The N-MID ELISA and an IRMA measuring intact human osteocalcin were used to monitor the effect of hormone replacement therapy in a retrospective study. A significant decrease to the premenopausal concentration was detected only in the N-MID ELISA.
Transferability of blood lead determinations by furnace atomic absorption spectrophotometry and continuum background correction
Authors: Qiao H , Parsons P J , Slavin W
Published: January 2020

We have examined and proved feasible the transfer of a method for blood lead determination, developed and optimized for a Zeeman-corrected instrument, to a continuum-corrected furnace atomic absorption spectrophotometer. Numerous reference materials analyzed with the continuum-corrected instrument gave results within 10 micrograms/L (0.05 mumol/L) at low values and varied by < 6% at values > 200 micrograms/L (0.97 mumol/L). Forty-four routine human blood specimens were analyzed by the same method with both continuum- and Zeeman-corrected instrumentation, and gave results that agreed within about the same limits as found with reference materials. The day-to-day precision was about 1/5 the accuracy results. The detection limit was approximately 5 micrograms/L (0.025 mumol/L).
Identification and quantification of intermediates of unsaturated fatty acid metabolism in plasma of patients with fatty acid oxidation disorders
Authors: Onkenhout W , Venizelos V , van der Poel P F , van den Heuvel M P , Poorthuis B J
Published: January 2020

The free fatty acid and total fatty acid profiles in plasma of nine patients with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, two with very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency and two with mild-type multiple acyl-CoA dehydrogenase (MAD-m) deficiency, were analyzed by gas chromatography-mass spectrometry. In the plasma of patients with MCAD deficiency we found increases of octanoic acid (8:0), decanoic acid (10:0), 4-decenoic acid (10:1 omega 6), and 4,7-decadienoic acid (10:2 omega 3), all present almost exclusively in free form. The patients with VLCAD deficiency showed increases of mainly 5-tetradecenoic acid (14:1 omega 9) and to a minor extent 5-dodecenoic acid (12:1 omega 7), 5,8-tetradecadienoic acid (14:2 omega 6), and 7,10-hexadecadienoic acid (16:2 omega 6), in both the free and esterified fatty acid fraction. The MAD-m patients showed variable increases of all the unusual fatty acids present in MCAD- and VLCAD-deficient plasma. The 14:1 omega 9, 14:2 omega 6, and 16:2 omega 6 fatty acids were present mainly in the esterified form. Measurement of these fatty acids in plasma by the relatively simple method presented here provides a sensitive and specific aid in the diagnosis of acyl-CoA dehydrogenase deficiency disorders.
Biochemical markers of bone turnover in patients with metastatic bone disease
Authors: Demers L M , Costa L , Chinchilli V M , Gaydos L , Curley E , Lipton A
Published: January 2020

Several biochemical markers of bone formation and bone resorption have recently been developed. These markers have been evaluated for clinical utility in patients with metabolic bone disease, including Paget disease and osteoporosis, and for their potential use in cancer patients whose disease has metastasized to bone. We have evaluated seven markers of bone turnover in the plasma and urine of 94 patients with newly diagnosed or progressive malignancy with and without clinical evidence of bone metastases. As determined by a positive bone scan and (or) bone survey, 30 patients had metastases to bone; 51 patients had metastatic cancer without overt bony involvement; and 13 patients had local disease without bone metastases. To evaluate the predictive value of these markers in the metastatic population, we utilized a "Z-score" and logistic regression analysis to distinguish patients with documented bone metastatic disease from those patients without clinical evidence of bone metastases. The higher the Z-score, the better the marker predicts the presence of bone metastases. With this statistical approach, urine N-telopeptide measurements had the highest Z-score and the most significant association with the probability of bone metastases. Urine deoxypyridinoline was the second most predictive marker of bone metastases. Thus, biochemical markers of bone resorption might be of use to predict the presence of bone metastases in cancer patients and to monitor the efficacy of antiresorptive therapy in patients treated for metastatic bone disease.
Blood and plasma glutathione measured in healthy subjects by HPLC: relation to sex, aging, biological variables, and life habits
Authors: Michelet F , Gueguen R , Leroy P , Wellman M , Nicolas A , Siest G
Published: January 2020

We report an HPLC method for measuring the concentrations of reduced (GSH) and total (GSHt) free glutathione in human plasma and whole blood. The chromatographic step was coupled with a postcolumn derivatization reaction and fluorometric detection. The linear range was 0.81-13.02 mumol/L, and the detection limit was 0.13 mumol/L. In healthy adults (ages 18-73 years), mean concentrations were 941 +/- 155 mumol/L for GSHt and 849 +/- 63 mumol/L for GSH in blood (107 men, 94 women), and 3.39 +/- 1.04 mumol/L for GSH in plasma (66 men, 58 women). Blood GSHt but not GSH was significantly lower in children (32 boys, 32 girls: 872 +/- 157 mumol/L) than in adults. Blood GSHt and GSH appeared to be correlated positively with the number of cigarettes smoked per day and the regular practice of physical exercise, and negatively with alcohol abstinence. We observed positive correlations between blood GSHt and cholesterol and calcium concentrations, and between blood GSH and cholesterol concentration.
Fluorescence in situ hybridization: powerful molecular tool for cancer prognosis
Authors: Fox J L , Hsu P H , Legator M S , Morrison L E , Seelig S A
Published: January 2020

We review several aspects of fluorescence in situ hybridization (FISH) technology that demonstrate its breadth and power in detecting and monitoring genetic abnormalities associated with cancers. The clinical utility of FISH in disease management is demonstrated in several examples, including trisomy 8 detection with high specificity and sensitivity in patients with myeloid leukemias; trisomy 12 detection with higher efficiency than conventional cytogenetics in patients with chronic lymphocytic leukemia; assessment of engraftment success, chimerism, and relapse in opposite sex bone marrow transplantation; and correlation of trisomy 7 with survival time in patients with prostate tumors. Advances in FISH technology include multicolor analyses, which permit the simultaneous detection of several genetic abnormalities by using cohybridization of probes labeled with several fluorescent labels or label combinations, and comparative genomic hybridization, a relatively new method whereby a single hybridization can reveal aberrations across the entire genome.
Purification and characterization of different molecular forms of prostate-specific antigen in human seminal fluid
Authors: Zhang W M , Leinonen J , Kalkkinen N , Dowell B , Stenman U H
Published: January 2020

We have developed a new procedure for purifying prostate-specific antigen (PSA) from human seminal fluid. The method is based on ammonium sulfate precipitation, hydrophobic interaction chromatography, gel filtration, and anion-exchange chromatography. It can be completed within 2 days with a recovery of intact PSA of 30%. By anion-exchange chromatography, five isoforms of PSA (A, B, C, D, and E) can be separated. The major form (PSA-B) consists of the intact enzyme, as shown by the occurrence of only one band of 33 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or nonreducing conditions, and by amino acid sequencing, which reveals only one amino-terminal sequence corresponding to the reported amino-terminal sequence of intact PSA. The specific absorbance of 1 g/L PSA-B at 280 nm was 1.61, and 80% of the PSA-B formed a complex with alpha 1-antichymotrypsin, indicating that it is enzymatically active. Three cleaved forms of PSA with different nicking sites and low enzymatic activity were separated from intact PSA by ion-exchange chromatography. In addition, we isolated a glycosylation variant, PSA-A, which showed a higher isoelectric point (pI = 7.2) than PSA-B (pI = 6.9) but similar enzymatic activity; this form accounts for 5-10% of total PSA. After treatment with sialidase, PSA-A and B had the same isoelectric point value (pI = 7.7).
Serum catalase as marker of graft-vs-host disease in allogeneic bone marrow transplant recipients: pilot study
Authors: Yasmineh W G , Kaur T P , Blazar B R , Theologides A
Published: January 2020

We evaluated the efficacies of serum catalase (CAT), 5'-nucleotidase (5'NT), and tumor necrosis factor-alpha (TNF) as diagnostic markers of acute graft-vs-host disease (GVHD) in 28 allogeneic bone marrow transplant recipients by comparing their abilities to discriminate between GVHD-related and non-GVHD-related complications. Mean peak serum CAT concentrations for patients with GVHD-related complications (n = 17) were about fivefold higher than concentrations in patients with non-GVHD-related complications (n = 25; P = 0.003), whereas the mean peak concentrations of serum 5'NT and TNF were not substantially different. Similarly, the sensitivity and specificity of serum CAT (100% and 88%, respectively) for use as a diagnostic marker of GVHD were much better than those of serum 5'NT (88% and 24%, respectively) or serum TNF (65% and 4%, respectively). Receiver-operating characteristic plots of all possible sensitivity-specificity pairs obtained over the whole range of results also showed that serum CAT has the best diagnostic accuracy. Low specificities of serum TNF and 5'NT were caused mainly by their increase in septicemia, fungal infection, and veno-occlusive disease and after the use of granulocyte-macrophage colony-stimulating factor to stimulate donor cell engraftment. Serum CAT may prove to be a rapid and relatively noninvasive test for the diagnosis of acute GVHD.
Relation between cathepsin D expression and other prognostic factors in breast carcinomas
Authors: Shaheen R M , Miseljic S , Wiehle R D , Wittliff J L
Published: January 2020

We evaluated cathepsin D concentrations in 318 breast carcinoma specimens with a standardized IRMA and established distribution values of 5.9-217.8 nmol/g (median 51.8). Concentrations of cathepsin D did not correlate with age or with concentrations of HER-2/neu oncoprotein, estrogen receptor, or epidermal growth factor receptors. A significant correlation was observed between cathepsin D and progestin receptor (P = 0.009), but only in postmenopausal patients. In our role as a National Reference Laboratory for conducting interlaboratory comparisons of tumor markers, we evaluated cathepsin D assay proficiency by using control samples with intra- and interassay CVs of 2-8% and 10-13%, respectively. Human reference specimens containing known quantities of cathepsin D were developed to facilitate standardized testing. The IRMA procedure and the use of quality-assurance samples permits evaluation of the clinical significance of cathepsin D in human breast cancer trials.
Different responses of biochemical markers of bone resorption to bisphosphonate therapy in Paget disease
Authors: Blumsohn A , Naylor K E , Assiri A M , Eastell R
Published: January 2020

We examined the response of different biochemical markers of bone resorption to bisphosphonate therapy (400 mg of etidronate daily for 6 months) in mild Paget disease (n = 14). Urinary markers included hydroxyproline (OHP), total (T) and free (F) pyridinolines (Pyds) determined by HPLC, immunoreactive FPyds, immunoreactive TPyds, and the N- and C-terminal telopeptides of type I collage (NTx, CL). Serum measurements included tartrate-resistant acid phosphatase (TRAcP) and the C-terminal telopeptide of type I collagen (ICTP). ICTP and TRAcP showed a minimal response to therapy (% change at 6 months, -13.1 +/- 6.8 and -6.7 +/- 3.4, respectively). The response was greatest for urinary telopeptides (NTx and CL; % change -75.7 +/- 7.5 and -73.4 +/- 8.9, respectively). The response was somewhat greater for TPyds than for FPyds. We conclude that: (a) ICTP and TRAcP are unreliable indicators of changes in bone turnover; (b) oligopeptide-bound Pyds and telopeptide fragments of type I collagen in urine show a somewhat greater response to therapy than do FPyds and may be more sensitive indicators of bone resorption; and (c) as yet no evidence suggests that these markers are substantially better predictors of the clinical response to therapy than serum total alkaline phosphatase or urinary OHP. There are several problems with the interpretation of these measurements in Paget disease, and the clinical utility of these measurements remains uncertain.
High-throughput method for determination of apolipoprotein E genotypes with use of restriction digestion analysis by microplate array diagonal gel electrophoresis
Authors: Bolla M K , Haddad L , Humphries S E , Winder A F , Day I N
Published: January 2020

Molecular epidemiological research has identified the association of a common apolipoprotein E (apo E) isoform (E4 as opposed to E3), with risk both of coronary artery disease and of Alzheimer dementia. In addition, the role of apo E genotype (usually E2/E2) in Type III hyperlipidemia is well known. However, both for diagnostic and research purposes, apo E genotyping is cumbersome. The preferred approach is electrophoretic sizing of restriction digestion fragments, enabling simultaneous analysis of the two codons (112 and 158) that represent the six common genotypes (E2/E2; E2/E3; E2/E4; E3/E3; E3/E4; E4/E4). However, the consequent demands of high-yield PCR, high-resolution, high-throughput electrophoresis, and sufficient detection sensitivity have left shortfalls in published protocols. In conjunction with a high-throughput electrophoresis system we described recently, microplate array diagonal gel electrophoresis (MADGE), we have constructed extensively optimized, simplified protocols for DNA isolation from mouthwash samples for PCR setup and high-yield PCR, for restriction digestion, and for subsequent MADGE gel image analysis. The integral system enables one worker to readily undertake apo E genotyping of as many as hundreds of DNA samples per day, without special equipment.