bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrophoretic fractionation of 5'-nucleotidase
Authors: Panteghini M
Published: January 2020

Human isonucleotidases were separated by electrophoresis on a cellulose acetate membrane. Three 5'-nucleotidase forms, NTP1, NTP2, and NTP3, were resolved with this method and quantified by densitometry. The procedure was not only simple and rapid but also sufficiently precise (between-run CV < 20%) and sensitive (detected nucleotidase fractions of > 0.5 U/L). The effects of various treatments (heat, neuraminidase, glycosidases, proteases, lectins, and detergents) on the electrophoretic pattern of 5'-nucleotidase were studied. NTP1 (mean 12% of total 5'-nucleotidase, SD 5%), NTP2 (mean 30%, SD 8%), and NTP3 (mean 58%, SD 8%) were found in all normal persons studied. The increase in total 5'-nucleotidase in patients with hepatobiliary disease was mainly due to the NTP1 isoform.
Combined polymerase chain reaction-hybridization microplate assay used to detect bovine leukemia virus and Salmonella
Authors: Rasmussen S R , Rasmussen H B , Larsen M R , Hoff-Jørgensen R , Cano R J
Published: January 2020

Here we describe the use of an assay that integrates the polymerase chain reaction (PCR) with hybridization of the amplified product for detection in the same microwell. Traditional PCR requires transportation of the amplified product to another system for characterization of samples. Transportation means time-consuming manipulation and risk of contaminating the laboratory with amplified product. Integration of amplification and specific product detection greatly reduces sample manipulations and the risk of contamination. We used the assay for detection of bovine leukemia virus and Salmonella. The results were identical with those produced by two traditional PCR methods. This assay could easily be adapted for other organisms, simply by using other primers and probes.
Effects of specimen turbidity and glycerol concentration on nine enzymatic methods for triglyceride determination
Authors: Sampson M , Ruddel M , Elin R J
Published: January 2020

We compared the effects of specimen turbidity and glycerol concentration on nine enzymatic methods for triglyceride measurement. We assayed 51 specimens with triglyceride concentrations of 0.85-8.21 mmol/L (75-727 mg/dL) and turbidity at 420 nm equivalent to > or = 0.1 mmol/L (8.8 mg/dL) triglyceride (measured as part of our comparison method). The data were analyzed by multiple regression, which gave coefficients for the effects of glycerol concentration and the change in turbidity during the reaction. The effects of specimen turbidity and glycerol concentration were method-dependent and ranged from 6.20% to -15.67% of the measured result. The magnitude of the turbidity effect (in assays with a significant turbidity interference) was similar to that for glycerol (in assays with a significant glycerol interference). A triglyceride assay with a bichromatic measurement was less subject to interference from turbidity.
Neuropeptides and anxiety: focus on cholecystokinin
Authors: Lydiard R B
Published: January 2020

Cholecystokinin (CCK), a gastrin-like neuropeptide, exists in the central nervous system in several forms. The octapeptide (CCK-8) occurs in predominantly sulfated form (CCK-8S), and the tetrapeptide (CCK-4) occurs in smaller but significant quantities. This review highlights recent developments in preclinical and clinical research into the potential role for CCK in mediating anxiety states. Relevant animal and human studies of administration of CCK agonists are discussed, as well as recent data regarding the concentration of CCK-8S in cerebrospinal fluid from patients with panic disorder, bulimia nervosa, and obsessive compulsive disorder. Finally, the development of agents that specifically antagonize CCK receptors will be described, as will potential therapeutic uses for these new compounds.
Selected strategies for improving sensitivity and reliability of immunoassays
Authors: Kricka L J
Published: January 2020

Selected recent advances in immunoassay are reviewed. Development has continued on new labels (beta-lactamase, pyrophosphatase, luciferases, photoproteins, pyridopyridazines, europium cryptates, metal carbonyl complexes, porphines, phosphors) and label-detection methods (e.g., chemiluminescence assays, thermometric assays, NADP(+)- and FADP-based coupled assays). Various methods have been explored to increase assay sensitivity, including label amplification via catalyzed reporter deposition (peroxidase label) and immuno-polymerase chain reaction (DNA label). The focus of new immunoassay strategies has been on improved reliability (bispecific antibodies), assay simplification (piezoelectric and surface plasmon resonance immunosensors, phase-modulation fluorescence spectroscopy, polymerized bilayer assemblies), and simultaneous multianalyte testing (e.g., quadruple labeling with lanthanides, one-step test devices for drugs of abuse).
Commercial assays for serum osteocalcin give clinically discordant results
Authors: Masters P W , Jones R G , Purves D A , Cooper E H , Cooney J M
Published: January 2020

Serum samples from 9 healthy controls and from subjects with primary hyperparathyroidism (n = 5), Paget disease (n = 3), pregnancy (n = 5), glucocorticoid therapy (n = 5), postmenopausal osteoporosis (n = 10), and renal failure (n = 10) were used to assess the clinical agreement among eight commercially available assay kits for osteocalcin (OC). These kits differ in their assay configurations (six radioimmunoassays, two immunoradiometric assays), standards (five bovine, three human), and antibodies (six polyclonal, two monoclonal). Individual results were divided by the mean OC of the control subjects for each assay and expressed as percentage deviations. The expected wide variation in absolute OC concentrations between kits was only partially reduced by this transformation. Agreement was equally poor when absolute OC concentrations were compared with the reference ranges quoted by the manufacturers. The discordance was particularly marked in renal failure, presumably because of immunoreactive fragments, and in osteoporosis. Systematic differences could not be attributed to assay format, species source of standard, or antibody specificity. We conclude that results cannot be compared between assays even when normalized against healthy subjects, and that standardization is needed.
Hematofluorometric determination of erythrocyte zinc protoporphyrin: oxygenation and derivatization of hemoglobin compared
Authors: Louro M O , Tutor J C
Published: January 2020

We compared oxygenation and derivatization of hemoglobin for the hematofluorometric determination of erythrocyte zinc protoporphyrin (ZPP). No statistically significant differences were found when the volume ratio of sample to ProtoFluor reagent (which converts hemoglobin to cyanohemoglobin) was changed from 1:1 to 1:4. With the derivatizing reagent, results were significantly higher than those obtained after thorough aeration of the blood sample (P < 0.001). The differences between the results obtained by the two procedures were greater for ZPP values in the reference range. Although the correlation between methods was high (r = 0.997), interconversion of the results by means of the regression equation was not acceptable because the standard error of the estimate from the regression (Syix = 0.36 micrograms/g hemoglobin) was greater than the error acceptable medically (criterion of Harris: Arch Pathol Lab Med 1988; 112:416-20).
Detecting benzodiazepines: immunoassays compared with negative chemical ionization gas chromatography/mass spectrometry
Authors: Fitzgerald R L , Rexin D A , Herold D A
Published: January 2020

We tested 231 urine samples by six immunoassay methods--EMIT d.a.u., EMIT II, Roche Abuscreen Online, Abbott TDx, Diagnostic Products Corp. (DPC) double-antibody radioimmunoassay (RIA), and Biosite Triage--and by negative chemical ionization gas chromatography/mass spectrometry to determine how the immunoassays performed on samples selected for suspected benzodiazepine use (n = 100) and in random urine drug screening (n = 131). In general, all of the assays were successful in detecting oxazepam and related metabolites, even at concentrations below the stated cutoffs. However, the negative predictive value of benzodiazepine immunoassays for samples selected for suspected benzodiazepine use ranged from 86% to 96%. A primary difference between the test kits was the ability of DPC RIA and Triage to detect lorazepam when other assays did not. Contrary to previous reports, pretreatment of specimens with glucuronidase was not necessary to detect oxazepam-related metabolites with these immunoassays.
Enzymatic diagnosis of aspartylglycosaminuria by fluorometric assay of glycosylasparaginase in serum, plasma, or lymphocytes
Authors: Mononen I , Mononen T , Ylikangas P , Kaartinen V , Savolainen K
Published: January 2020

Serum, plasma, and lymphocytes from aspartylglycosaminuria (AGU) patients and carriers and from normal controls were incubated with a fluorescent glycosylasparaginase substrate, L-aspartic acid beta-(7-amido-4-methylcoumarin), and the release of 7-amino-4-methylcoumarin was measured fluorometrically after incubation for 1-4 h. The mean glycosylasparaginase (EC 3.5.1.26) activity in normal serum, plasma, and lymphocytes was 20.2 (SD 5.0) mU/L (n = 24), 17.5 (SD 5.0) mU/L (n = 24), and 242 (SD 108) mU/g protein (n = 17), respectively. The corresponding values in the Finnish AGU patients were 0.7 (SD 0.4) mU/L (n = 10), 0.3 (SD 0.3) mU/L (n = 10), and 6.0 (SD 4.6) mU/g protein (n = 7). No overlapping values were obtained between the AGU patients and the carriers in any of the samples, but the values between the carriers and controls were overlapping in 28 of 29 serum, 22 of 29 plasma, and 4 of 21 lymphocyte samples. Thus, the fluorometric glycosylasparaginase assay in various blood samples allows specific detection of the enzyme defect in AGU, but cannot be used for reliable detection of carriers of the disease.
Limitations of the Friedewald formula for estimating low-density lipoprotein cholesterol in alcoholics with liver disease
Authors: Matas C , Cabré M , La Ville A , Prats E , Joven J , Turner P R , Masana L , Camps J
Published: January 2020

The accuracy of the Friedewald formula in estimating low-density lipoprotein (LDL) cholesterol was investigated in 47 alcoholic patients with liver disease (21 minimal-change, 26 cirrhotic) by comparing the results with those obtained by sequential preparative ultracentrifugation. In 14% of subjects with minimal-change disease, the error in the estimated LDL cholesterol was 50% +/- 9% (mean +/- SD; range 40-59%) and was related to the degree of attendant hypertriglyceridemia (r = 0.98; P < 0.001). A similar degree of error was observed in patients with cirrhosis, despite the absence of hypertriglyceridemia; an abnormal VLDL cholesterol: triglyceride ratio was the contributory factor in the discrepancy. We conclude that, as is the case in other clinical pathologies in which abnormalities of lipoprotein composition have been described (e.g., diabetes), the Friedewald formula to estimate LDL cholesterol may be inappropriate in chronic alcoholics, particularly those in whom a degree of hepatic dysfunction may be suspected.
Retinol, alpha-tocopherol, lutein/zeaxanthin, beta-cryptoxanthin, lycopene, alpha-carotene, trans-beta-carotene, and four retinyl esters in serum determined simultaneously by reversed-phase HPLC with multiwavelength detection
Authors: Sowell A L , Huff D L , Yeager P R , Caudill S P , Gunter E W
Published: January 2020

We describe the use of HPLC with multiwavelength detection to measure retinol, alpha-tocopherol, lutein/zeaxanthin, beta-cryptoxanthin, lycopene, alpha-carotene, trans-beta-carotene, beta-carotene, and the linoleate, oleate, palmitate, and stearate esters of retinol in a single 200-microL serum sample. The method is sensitive enough to detect individual retinyl esters in fasting serum from a nonhyperlipidemic population and requires only 12 min for each sample. Serum concentration ranges and means are reported for retinol, alpha-tocopherol, lutein/zeaxanthin, beta-cryptoxanthin, lycopene, alpha-carotene, trans-beta-carotene, and the sum of the retinyl esters from serum analyses of 3480 participants from several different studies.
Measurement of gonadotropins in dried blood spots
Authors: Worthman C M , Stallings J F
Published: January 2020

We describe direct immunofluorometric assays for luteinizing hormone (hLH) and follicle-stimulating hormone (hFSH) in fingerstick blood spots dried on filter paper, based on modifications of commercially available kits. Determinations are made from 2.5-mm-diameter discs (3 microL of dried blood) punched out from blood spot standards and samples. Sample dose detection limits of the assays (IU/L) are 0.26 for LH and 0.13 for FSH, with mean interassay CVs of 11.6% (LH) and 7.8% (FSH) at low concentrations. Analytical recoveries of added hormone averaged 100% for LH and 95% for FSH. Clinical studies showed that values for blood spots (x) and directly assayed plasma (y) are highly correlated, so that results from blood spots can be converted directly to plasma equivalents, as follows: yLH = 0.07 + 1.90 xLH, and yFSH = 0.424 + 2.207 xFSH. These gonadotropins are stable in blood spots for at least a year under refrigeration; LH for at least 8 weeks and FSH 6 weeks at 22 degrees C; and both hormones for a week at 37 degrees C. These methods thus allow self-sampling, serial sampling, and mailing of specimens.
Early detection of metabolic abnormalities in preterm infants impaired by disorders of blood glucose concentrations
Authors: Lyall H , Burchell A , Howie P W , Ogston S , Hume R
Published: January 2020

We recently reported (Acta Paediatr Scand 1992;8: 580-4) three preterm infants with severe respiratory distress syndrome and abnormal glucose profiles for the first 5 days of life who subsequently died in infancy; only at autopsy were they shown to have abnormal glucose-6-phosphatase activity. We have therefore studied retrospectively in a matched cohort of 109 infants the blood glucose profiles correlated with the severity of respiratory distress syndrome (expressed as the fraction of inspired oxygen, FiO2): group A, mild, FiO2 < 0.25; group B, moderate, FiO2 0.26-0.50; group C, severe, FiO2 > 0.51. All groups had a similar frequency of low blood glucose values (15% < or = 2.2 mmol/L; 29% < or = 2.6 mmol/L), but high blood glucose values and greater variability in glucose values were more common in groups B and C despite lower caloric intakes (A, 4.3%; B, 9.3%; C, 9.6% > or = 7 mmol/L). We conclude that the early blood glucose patterns in those three previously described preterm infants with abnormal hepatic glucose-6-phosphatase activity at autopsy cannot be viewed as abnormal when considered against a matched cohort of infants. Preterm infants at risk of genetic or developmental delays in blood glucose homeostasis should be reassessed after recovery from their acute illnesses.
Different forms of insulin-like growth factor-binding protein-3 detected in serum and seminal plasma by immunofluorometric assay with monoclonal antibodies
Authors: Koistinen H , Seppälä M , Koistinen R
Published: January 2020

Monoclonal antibodies against recombinant insulin-like growth factor-binding protein-3 (IGFBP-3) were produced to study the various forms of IGFBP-3 and to develop a specific immunofluorometric assay. We tested seven antibodies that showed no cross-reactions with the other five human IGFBPs. By immunoblotting, the main bands in normal serum were seen at > 90, 45, 41, 29, and 25 kDa. In pregnancy serum, the 29-kDa was stronger, and the double band at 41-45 kDa was weaker or totally absent. We characterized two immunofluorometric assays. IGF-I had no effect on either assay. Added IGF-II lowered the amount of recombinant IGFBP-3 measured by the 5C11/7F8 assay, but not by the 1B6/5C11 assay. In normal serum and follicular fluid, IGFBP-3 measurements were lower by the 5C11/7F8 assay, but in most pregnancy sera and amniotic fluids the assays gave similar results. The 5C11/7F8 assay also detected IGFBP-3 in seminal plasma, whereas the 1B6/5C11 assay did not. We conclude that the results of IGFBP-3 measurement depend on the antibodies used, and that different antibodies are suitable for the IGFBP-3 measurement in different body fluids.
When is a serum iron really a serum iron? The status of serum iron measurements
Authors: Tietz N W , Rinker A D , Morrison S R
Published: January 2020

Although serum iron determinations play an important role in the diagnostic process, the accuracy of routine methods is suspect. The poor performance of currently used methods is well documented in the quarterly College of American Pathologists survey reports. Compared with the 1990 method of the International Committee for Standardization in Haematology (ICSH), the 1978 ICSH Reference Method shows a +3.4% bias and the standard method of the National Committee for Clinical Laboratory Standards shows a -17.8% bias for iron results. All routine methods checked--DuPont aca, Abbott TDx, Kodak Ektachem (except one lot), BM/Hitachi 717, and Synermed--show a negative bias over the entire analytical range, a significant negative intercept, and extremely poor correlation with the 1990 ICSH method for iron values < 750 micrograms/L. Individual discrepancies of several hundred percent were observed. Imprecision of the methods is not the reason for these discrepancies. We question whether most routine methods measuring low iron concentrations provide results sufficiently reliable for confirmation of iron deficiency.
Replacing creatinine measurements with specific gravity values to adjust urine cotinine concentrations
Authors: Haddow J E , Knight G J , Palomaki G E , Neveux L M , Chilmonczyk B A
Published: January 2020

Creatinine and specific gravity (relative density) measurements both allow differences in urine concentration to be taken into account in determining urine cotinine concentrations. In this study we demonstrate that the variance of urine cotinine measurements is reduced comparably when either creatinine or specific gravity measurements are used for correction. This reduction in variability improves the correlation between urine cotinine measurements and clinical endpoints. In this study, the clinical endpoints were pulmonary function in a population of nonsmoking children with asthma, 42% of whom were reported to have been exposed to environmental tobacco smoke. When corrected by either creatinine measurements or specific gravity values, the urine cotinine measurements performed as well or better than reported exposure (and comparably with each other) in assessments of lung function. A dose-response relationship was also more consistently apparent. Specific gravity values can be used reliably in place of creatinine values to adjust urine cotinine measurements for both research and clinical purposes.
Citrate as the main small molecule binding Al3+ in serum
Authors: Ohman L O , Martin R B
Published: January 2020

A recent proposal (Clin Chem 1992;38:1809-18) that inorganic phosphate is the predominant small molecule binding Al3+ in blood serum is shown to be incorrect. Experiments in which Al3+ is added to solutions containing phosphate and citrate in the same proportions as in serum clearly show that citrate prevents formation of any aluminum phosphate precipitate, and analysis of these data indicates that the assumed binding constants for soluble phosphate are two orders of magnitude too strong. Consideration of the effect of competitive binding of alkaline earth metal ions to citrate on Al3+ binding and of a set of transferrin-Al3+ stability constants leads to the conclusion that the proportions are in close agreement with previously published results. We conclude on the basis of stability constants that, of the Al3+ in blood serum, approximately 89% (+/- 5%) binds to transferrin and approximately 11% (+/- 5%) to citrate.
Simultaneous determination of mercury speciation in biological materials by GC/CVAFS after ethylation and room-temperature precollection
Authors: Liang L , Bloom N S , Horvat M
Published: January 2020

We developed a method for the simultaneous determination of monomethyl mercury (MMHg), inorganic mercury [Hg(II)], and total mercury (THg) in biological materials. A variety of biological materials can be digested in methanolic KOH solution. The MMHg and Hg(II) present are converted to volatile ethyl derivatives, methylethyl mercury and diethyl mercury, by an aqueous-phase ethylation reaction with sodium tetraethylborate. The ethyl derivatives are precollected onto a trapping column at room temperature, in case of disconnection with the separation/detection system, and then thermally desorbed into a packed isothermal gas chromatography (GC) column. Eluted organo-Hg compounds from the GC column are decomposed into Hg0, and detection is completed by cold vapor atomic fluorescence spectrometry (CVAFS). Pure standard solutions can be used for calibration. The sum of MMHg and Hg(II) obtained by this method equals the THg value obtained by digestion with HNO3 and H2SO4, reduction with SnCl2, and single-stage amalgamation/CVAFS for all biological materials studied. Absolute detection limits are 0.6 pg and 1.3 pg of Hg as MMHg and Hg(II), respectively, corresponding to 0.3 ng and 0.6 ng/g (wet) of sample.
Steady-state concentration of cyclosporin G (OG37-325) and its metabolites in renal transplant recipients
Authors: Langman L J , Leichtman A B , Weitzel W F , Yatscoff R W
Published: January 2020

The steady-state concentrations of cyclosporin G (OG37-325) (CsG) and six of its metabolites (GM1, GM9, GM4N, GM1c, GM1c9, GM19) were measured throughout the 12-h dosing interval in six renal transplant recipients receiving CsG as prophylaxis against acute cellular rejection. The mean 12-h whole-blood trough concentrations (micrograms/L) were CsG, 131 +/- 26; GM1, 79 +/- 55; GM9, 110 +/- 114; GM4N, 28 +/- 18; GM1c, 31 +/- 18; GM1c9, 216 +/- 145; and GM19, 303 +/- 217. The relative concentration of the primary metabolites (GM1, GM9, GM4N) remained stable with respect to CsG throughout the dosing interval, whereas that of the secondary metabolites increased. The secondary metabolites GM19 and GM1c9 exhibited extensive between-patient variation. We investigated the effect of these metabolites on commercially available monoclonal antibody-based fluorescence polarization immunoassays (FPIA) and RIAs adapted for measurement of CsG. The 12-h whole-blood trough concentrations measured by FPIA and RIA exceed those measured by HPLC by 19% and 36%, respectively. These measured biases corresponded closely with the calculated biases (FPIA 19%, RIA 28%) based on the known cross-reactivities of CsG metabolites and their concentrations. These results suggest that cross-reactivity with metabolites account for a large part of the bias observed in immunoassays of CsG.
Human urinary arsenic excretion after one-time ingestion of seaweed, crab, and shrimp
Authors: Le X C , Cullen W R , Reimer K J
Published: January 2020

We studied chemical speciation of arsenic compounds in urine samples by using HPLC with inductively coupled plasma mass spectrometry detection. We examined urinary arsenic excretion patterns and the arsenic species excreted from nine human subjects who ingested seaweed products and crab (or shrimp). Fast urinary excretion of unchanged arsenobetaine was seen after ingestion of crab and shrimp, which contain arsenobetaine as the major arsenic species. In contrast, the arsenosugars, which comprise the major arsenic species in seaweed, are metabolized and have a longer retention time in the human body. When nine volunteers ingested the commercial seaweed product nori, both the urinary arsenic excretion pattern and the excreted arsenic species varied from individual to individual, and as many as six metabolites could be detected. It seems that arsenosugars are not decomposed by stomach acid and that reactions involving enzymatic and (or) microbial activity in the human body may be responsible for the metabolism of arsenosugars.
Two-site immunoassay of recombinant hirudin based on two monoclonal antibodies
Authors: Mille B , Condamines O , Herbert J M , Maftouh M , Picard C , Dussossoy D , Pau B
Published: January 2020

Two monoclonal antibodies (mAbs), 10-2 and 10-5, both directed against recombinant hirudin variant 2-Lys47 (rHV2), were selected for their high affinity and epitopic specificities to develop a two-site immunoassay of rHV2. The mAb concentrations, incubation time, and temperature were optimized. The immunoassay has a detection limit for rHV2 of 45 ng/L in plasma and 30 ng/L in urine. The reactivity of the mAbs was tested against rHV2 and several forms of this protein truncated in the carboxyl terminus. The capture mAb 10-2 was found to be mainly directed against rHV2, whereas tracer mAb 10-5 was independent of the carboxyl-terminal region of the protein. This explains the high specificity of the immunoassay for the 65-amino acid form of hirudin.
Measurement of felbamate by wide-bore capillary gas chromatography and flame ionization detection
Authors: Rifai N , Fuller D , Law T , Mikati M
Published: January 2020

Felbamate, a newly developed antiepileptic agent, has been demonstrated to control partial and generalized seizures effectively. We have developed a gas-chromatographic method for the determination of felbamate, using a wide-bore capillary column, a flame ionization detector, and a simple extraction procedure. The assay day-to-day precision (n = 20) was 5.2% and 3.6% for drug concentrations of 50 and 150 mg/L, respectively; average recovery over a wide range of felbamate concentrations was 95%; the detection limit was 5 mg/L; and assay linearity extended to 300 mg/L. Although 9 of the 27 drugs tested were coextracted with felbamate, they exhibited significantly different retention times and showed no interference. A short-term stability study showed that plasma felbamate is stable at 4, -20, or -78 degrees C for at least 1 month. Plasma felbamate concentrations in 66 pediatric patients ranged from 7 to 154 mg/L (mean +/- SD 44 +/- 24.7). We consider the method ideally suited for therapeutic monitoring of plasma felbamate concentration.
New enzymatic assay of iron in serum
Authors: Fujita T , Hamasaki H , Furukata C , Nonobe M
Published: January 2020

A new enzymatic method for assaying iron in serum samples, suitable for automated analyzers, is reported. Three reagent mixtures are used: dilution buffer (pH 3.0; ascorbate), reagent 1 (pH 6.7; apoaconitase), and reagent 2 (pH 7.7; citrate, magnesium, and isocitrate dehydrogenase). Sera are diluted with dilution buffer. Fe3+ is liberated from transferrin in sera under acidic conditions, and then reduced by ascorbate. Reagent 1 is added to diluted specimens, and apoaconitase is reactivated by Fe2+ at neutral pH. The resulting solutions are mixed with reagent 2, so that holoaconitase hydrolyzes citrate to isocitrate and the isocitrate and NADP+ are converted to 2-oxoglutarate, NADPH, and CO2. Serum iron is determined linearly up to 70 mumol/L, with within-run CVs < or = 2.4% and day-to-day CVs < or = 2.9%. This method (y) gives results correlating with those of a Reference Method (x) proposed by the International Committee for Standardization in Haematology: y = 0.98x + 0.38 mumol/L (n = 72, r = 0.996, Sylx = 0.63 mumol/L). The mean (+/- SD) serum iron concentrations measured by our method were 18.5 +/- 5.4 and 15.2 +/- 6.0 mumol/L for 63 males and 166 females, respectively.
Clinical utility of liquid-chromatographic analysis of effusions for hyaluronate content
Authors: Nurminen M , Dejmek A , Mårtensson G , Thylen A , Hjerpe A
Published: January 2020

A previously described HPLC method for determining hyaluronate in effusions was used to analyze a consecutive series of effusions from 1039 patients with pleural fluids and from 571 patients with peritoneal fluids. A mesothelioma was verified histologically in 50 of the cases. The results were used to estimate the clinical utility of the analysis. With a cutoff of 75 mg/L for hyaluronate-derived uronic acid, assay specificity for a malignant mesothelioma was 100% and the sensitivity 56%. Only 20% of the effusions from the mesothelioma patients showed no evidence of increased production of hyaluronate. Cytological smears from the associated cell pellets were evaluated as malignant or suspicious for malignancy in only 28% or in a further 46% of the mesothelioma cases, respectively, leaving 30% of the pellets as cytologically false-negative. We also analyzed effusions from selected cases submitted from other hospitals, 154 of which had been diagnosed histologically as mesotheliomas. Concentrations of hyaluronate were increased in these cases too, but a considerable proportion of the samples showed evidence of losses of hyaluronate; consequently, the sensitivity of the assay in these samples was lower.
Enzymatic method for assaying calcium in serum and urine with porcine pancreatic alpha-amylase
Authors: Kayamori Y , Katayama Y
Published: January 2020

We developed a kinetic assay for calcium in serum and urine, based on the activation of porcine pancreatic alpha-amylase (EC 3.2.1.1) with 2-chloro-4-nitrophenyl-alpha-maltotrioside as substrate. The kinetic generation of 2-chloro-4-nitrophenol, monitored at 405 nm, was proportional to the concentration of calcium in serum and urine. The assay was developed and evaluated with the Cobas Bio centrifugal analyzer. The average within-run and between-day imprecision (CV) was 0.96/1.26% and 1.07/1.63%, respectively, for serum calcium and 1.50/2.54% and 1.70/2.64%, respectively, for urine calcium. Results of the proposed method (y) correlated well with those determined by atomic absorption spectrophotometry (x): y(serum) = 1.005x + 0.028 mmol/L (Sy/x = 0.058, r = 0.974, n = 50), and y(urine) = 1.017x-0.115 mmol/L Sy/x = 0.30, r = 0.981, n = 25). We also present data showing that the method is highly sensitive, rapid, relatively free of interference, and amenable to automation.
Changes in plasma amino acid concentrations in response to HIV-1 infection
Authors: Hortin G L , Landt M , Powderly W G
Published: January 2020

Plasma concentrations of 21 amino acids were determined for 20 control subjects and 20 subjects infected with human immunodeficiency virus type 1 (HIV). Compared with the control subjects, the HIV-infected group had lower cystine, tryptophan, and methionine (decreased 67%, 52%, and 32%, respectively, P < 0.001 for each) and increased taurine (230%, P < 0.001) and lysine concentrations (30%, P < 0.001). Other amino acid concentrations changed modestly. Amounts of cystine, tryptophan, methionine, taurine, and lysine did not differ significantly between subgroups of HIV-infected subjects with > 200 (n = 6) or < 200 (n = 14) CD4+ lymphocytes per microliter, suggesting that the concentrations decrease soon after infection and change little thereafter. Activation of metabolism of cystine to taurine may explain reciprocal changes in these amino acids and known depletion of cystine and glutathione. The selective changes in amino acid profiles observed during HIV infection differ from those recognized for malnutrition or other pathological processes.
Purification of human procollagen type I carboxyl-terminal propeptide cleaved as in vivo from procollagen and used to calibrate a radioimmunoassay of the propeptide
Authors: Pedersen B J , Bonde M
Published: January 2020

We purified human procollagen type I carboxyl-terminal propeptide (PICP) that had been cleaved as in vivo from procollagen. PICP in serum-free medium from cultured human fetal fibroblasts was purified by thiophilic adsorption chromatography, low-pressure gel filtration, and HPLC gel filtration. The purity and homogeneity of the protein was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal amino acid sequencing showed that the sequences of the alpha 1 and alpha 2 chains of this PICP were identical to those of the PICP produced in vivo. The monocomponent PICP thus purified was used as calibrator in a simple equilibrium-type RIA of PICP with polyclonal antibodies raised in rabbits. The measuring range is 0.15-3.75 nmol/L, and the assay detection limit is 0.03 nmol/L. The within-run and total CVs are 2% and 4%, respectively. The reference interval for the plasma concentration of PICP in healthy women of ages > 30 years is 0.36-1.44 nmol/L (geometric mean 0.72 nmol/L, n = 154).
Carnitine determination by an enzymatic cycling method with carnitine dehydrogenase
Authors: Takahashi M , Ueda S , Misaki H , Sugiyama N , Matsumoto K , Matsuo N , Murao S
Published: January 2020

We describe a highly sensitive and specific method for determining L-carnitine in serum by use of carnitine dehydrogenase (EC 1.1.1.108). The method involves a new enzymatic cycling technique with NADH, thio-NAD+, and carnitine dehydrogenase, and measures the increase of absorbance at 415 nm of thio-NADH produced at 37 degrees C during the reaction: [formula: see text] The calibration curve for L-carnitine in serum was linear between 5 and 250 mumol/L. Analytical recovery was 96.5-106%, and within-run and between-run imprecisions (CV) were 0.66-4.33% and 1.02-2.56%, respectively. This method was free from interference by bilirubin, hemoglobin, various acyl-DL-carnitines, and ascorbate. The procedure is simple, rapid, accurate, and automatable. The amount of free L-carnitine in serum (53.6 +/- 11.7 mumol/L, n = 200) was greater in men than in women (45.1 +/- 14.2 mumol/L, n = 200) (mean +/- SD).
Characteristics of a two-site immunoradiometric assay for human skeletal alkaline phosphatase in serum
Authors: Panigrahi K , Delmas P D , Singer F , Ryan W , Reiss O , Fisher R , Miller P D , Mizrahi I , Darte C , Kress B C
Published: January 2020

This two-site IRMA includes specific monoclonal antibodies for measuring skeletal alkaline phosphatase (B-ALP) in human serum. Assay calibration is based on mass units (micrograms per liter) and was established with purified B-ALP from a human osteosarcoma cell line, SAOS-2. Precision studies demonstrated intra- and interassay CVs of 3-5% and 5-7%, respectively. Relative reactivity studies showed that the assay has a sevenfold preference for detecting B-ALP compared with the liver isoenzyme in serum. The normal reference interval for 478 healthy adults was 5-22 micrograms/L. Method comparison studies showed good correlation between this B-ALP assay (y) and commercially available electrophoretic methods (x) (y = 0.3540x + 20.5, R2 = 0.929) in a pagetic population. Temporal profiles for total ALP, this IRMA B-ALP assay, and B-ALP by electrophoresis in three pagetic patients were parallel. We conclude that this assay demonstrates good analytical performance and would be useful for the clinical assessment of metabolic bone disorders.
Changes in manganese content of mononuclear blood cells in patients receiving total parenteral nutrition
Authors: Matsuda A , Kimura M , Takeda T , Kataoka M , Sato M , Itokawa Y
Published: January 2020

We measured the manganese content of mononuclear blood cells and the manganese concentrations in whole blood and plasma from 31 healthy volunteers and 25 patients receiving total parenteral nutrition (TPN) (i.e., no dietary manganese). The manganese content (mean +/- 2 SD) of mononuclear blood cells and the manganese concentration in whole blood and plasma in the healthy volunteers were 8.84 +/- 4.18 ng/10(8) cells, 11.21 +/- 4.68 micrograms/L, and 1.21 +/- 0.72 micrograms/L, respectively. The manganese content of mononuclear blood cells from patients receiving TPN gradually decreased, falling below the lowest limit of the normal range by 21 weeks (95% confidence limits, 13-30 weeks) after starting TPN. Manganese concentrations in whole blood and plasma were within normal limits during our study period. These results suggest that the manganese content of mononuclear blood cells may better indicate manganese nutritional status than whole-blood or plasma concentrations.
Age-related reference values for urinary organic acids in a healthy Turkish pediatric population
Authors: Guneral F , Bachmann C
Published: January 2020

Organic acid concentrations were quantified by gas chromatography and the individual acids identified by mass spectrometry in urine specimens from a healthy Turkish pediatric population of ages 2 days to 16 years, subdivided into five age groups. We quantified 69 organic acids (32 major and 37 minor components) and report here the median values and percentiles for each compound, adjusted for creatinine content, for the five groups. Concentrations of most of the organic acids tend to decrease with age but display substantial differences between age groups. This emphasizes the importance of comparing patients' data with age-matched reference data. Correlations between the excretion of organic acids and protein or caloric intake were significant for several compounds.
Rapid HPLC determination of total homocysteine and other thiols in serum and plasma: sex differences and correlation with cobalamin and folate concentrations in healthy subjects
Authors: Jacobsen D W , Gatautis V J , Green R , Robinson K , Savon S R , Secic M , Ji J , Otto J M , Taylor L M
Published: January 2020

High-performance liquid chromatography with fluorescence detection has been utilized for the rapid determination of total homocysteine, cysteine, and cysteinylglycine in human serum and plasma. Our earlier procedure (Anal Biochem 1989;178:208), which used monobromobimane to specifically derivatize thiols, has been extensively modified to allow for rapid processing of samples. As a result, > 80 samples a day can be assayed for total homocysteine, cysteine, and cysteinylglycine. The method is sensitive (lower limit of detection < or = 4 pmol in the assay) and precise (intra- and interassay CV for homocysteine, 3.31% and 4.85%, respectively). Mean total homocysteine concentrations in plasma and serum were significantly different, both from healthy male donors (9.26 and 12.30 mumol/L, respectively; P < 0.001) and healthy female donors (7.85 and 10.34 mumol/L, respectively; P < 0.001). The differences in total homocysteine between sexes were also significant (P = 0.002 for both plasma and serum). Similar differences were found for cysteine and cysteinylglycine. We found a significant inverse correlation between serum cobalamin and total homocysteine in men (P = 0.0102) and women (P = 0.0174). Serum folate also inversely correlated with total homocysteine in both sexes.
Limit of detection (LQD)/limit of quantitation (LOQ): comparison of the empirical and the statistical methods exemplified with GC-MS assays of abused drugs
Authors: Armbruster D A , Tillman M D , Hubbs L M
Published: January 2020

The limit of detection (LOD) for any analytical procedure, the point at which analysis is just feasible, may be determined by a statistical approach based on measuring replicate blank (negative) samples or by an empirical approach, consisting of measuring progressively more dilute concentrations of analyte. The limit of quantitation (LOQ), or concentration at which quantitative results can be reported with a high degree of confidence, may likewise be determined by either approach. We used both methods to determine LOD and LOQ for forensic gas chromatographic-mass spectrometric (GC-MS) analyses of abused drugs. The statistically determined LOD and LOQ values for these assays underestimated the LOD because of the large imprecision associated with blank measurements and the inability of blank samples to meet typical GC-MS acceptance criteria. The empirical method provided much more realistic LOD values, supported by reasonable experimental data, and are 0.5-0.03 the magnitude of the corresponding statistical LODs. The empirical LODs and LOQs are identical for these GC-MS assays. The observations made here about the LOD/LOQ for specific forensic GC-MS procedures are generally applicable to any type of analysis.
Simultaneous separation and quantification of free and metal-chelated protoporphyrins in blood by three-dimensional HPLC
Authors: Sato H , Ido K , Kimura K
Published: January 2020

In this new method for simultaneous separation and quantification of free and metal-chelated porphyrins in blood, the porphyrins are extracted from blood samples with a mixture of diisopropylamine:water:methanol (25:100:900, by vol) and separated by HPLC elution. The data are collected in three-dimensional form with a microcomputer. This method permits a high recovery of protoporphyrin (PP) and Zn-chelated PP as well as heme from blood. By this method, the amounts of these analytes in erythrocytes from normal or abnormal subjects can be determined more accurately than by conventional methods.
Enzymatic quantification of 1,5-anhydro-D-glucitol: evaluation and clinical application
Authors: Phillipou G , James S K , Frith R G , Farrant R K , Phillips P J
Published: January 2020

We investigated an enzymatic colorimetric procedure for quantification of plasma 1,5-anhydro-D-glucitol (AG) with respect to its reproducibility and application for evaluation of diabetes mellitus. Assay specificity is critically dependent on the two-layer ion-exchange column chromatography procedure to remove glucose from the sample. For female nondiabetic subjects (n = 110, ages 22-36 years) the median plasma AG concentration was 143 mumol/L (range 70-209 mumol/L); there was no relation to body mass index (BMI), fasting plasma glucose, or insulin, but an inverse association was noted for age. For a group of older subjects (n = 69, 70-85 years), no association between AG concentrations and sex, age, BMI, or various medical conditions was found. In diabetic subjects (n = 170) a significant inverse nonlinear relation existed between plasma AG and glycohemoglobin (GHb) such that at GHb > 8.5%, AG concentrations were typically < 50 mumol/L. The findings confirm that plasma AG, in the presence of normal renal function, is a reliable marker for hyperglycemia.
Role of biomarkers in identifying and understanding environmentally induced disease
Authors: Silbergeld E K , Davis D L
Published: January 2020

Establishing associations between environmental agents and disease presents challenges to both epidemiologists and toxicologists, particularly in cases of complex gene-environment interactions and when there is a long latency between exposure and disease. Biologic markers, physiological signals that reflect exposure, early cellular response, or inherent or acquired susceptibilities, provide a new strategy for resolving some of these problems. Biomarker research assumes that toxicant-induced diseases are progressive and that injury proceeds from entry of the toxicant into target cells, which induces subcellular biochemical events, to cell- and organ-level events that eventually induce irreversible or persistent organism dysfunction. The epidemiologic value of a biomarker lies in its ability to predict backward toward exposure and forward toward risk of clinical outcome, which is largely unknown. Research in mechanistic toxicology will advance the range of useful biomarkers in epidemiology and clinical medicine.
Reference limits in occupational toxicology
Authors: Aitio A
Published: January 2020

Two categories of reference limits can be discerned in biological monitoring: The first category identifies individuals who have been exposed to a toxic agent at work, and is based on the distribution of the concentration of the agent or its metabolite in the population that has not been exposed to the agent at work. The second category, for which the term biological action level (BAL) is proposed, provides a guideline on the level of exposure that is acceptable. These levels may be either directly health-based or derived from good working practices. Thus BAL is a biological equivalent for the generic term occupational exposure limit. BAL should be independent of legal overtones, and implies that workers' exposure should be reduced.
Cadmium: exposure markers as predictors of nephrotoxic effects
Authors: Lauwerys R R , Bernard A M , Roels H A , Buchet J P
Published: January 2020

Cadmium (Cd) is a cumulative element with a biological half-life of > 10 years in humans. The total amount of Cd accumulated in the liver and in the kidney can be measured in vivo by neutron activation (or x-ray fluorescence), but this technique does not necessarily measure the fraction that is biologically active. At low exposure (i.e., general environmental exposure or moderate occupational exposure), blood Cd is mainly influenced by the last 2 to 3 months of exposure. Under such conditions, the Cd concentration in urine mainly reflects the amount of Cd stored in the body, particularly in the kidney. In Europe and the US, the Cd reference values are usually < 2 nmol/mmol creatinine. Because most of the Cd in urine is probably bound to metallothionein, the changes in the urinary metallothionein concentration parallel those of Cd. The determination of Cd concentration in hair is of limited value because in humans it is difficult to distinguish between externally deposited and endogenous Cd. Fecal Cd is a good indicator of the oral daily intake. The results of several cross-sectional epidemiologic studies of the relation between the prevalence of renal dysfunction and Cd concentration in urine led us to propose a biological limit value for Cd of 5 and 2 nmol/mmol creatine for adult male workers and the general population, respectively.
Methylmercury: significance of intrauterine and postnatal exposures
Authors: Grandjean P , Weihe P , Nielsen J B
Published: January 2020

Outbreaks of methylmercury poisoning in Japan and Iraq have demonstrated the sensitivity of the fetus to neurotoxic effects. Based on toxicokinetics and considerations of practicability, the optimal biomarker of methylmercury exposure is the hair concentration, but whole-blood measurements of mercury are also useful. Dose-response relations are still incompletely known, especially concentrating developmental neurotoxicity under conditions of chronic exposure. Available evidence indicates that neurobehavioral dysfunction in children may occur if the maternal mercury concentration in hair is > 6 micrograms/g (30 nmol/g). This value corresponds to a blood mercury concentration of approximately 24 micrograms/L (120 nmol/L). The period of maximum sensitivity of the nervous system to methylmercury toxicity is unknown, but the transfer of mercury to the newborn through human milk may represent an additional risk. In view of the wide occurrence of mercury contamination in developing countries, increased use of the exposure biomarkers is encouraged.
Chlorinated dibenzo-p-dioxins and -furans: problems in analysis of biomarkers
Authors: Schlatter C
Published: January 2020

Although dioxin concentrations in adipose tissue or blood lipids can suitably be used for toxicological evaluation, such data usually are not yet asked for in the risk assessment process, which is still based mainly on theoretical calculations of possible environmental exposure instead of measured concentrations in humans. The latter is necessary because the substantial kinetic differences between experimental animals and humans observed for dioxins lead to different organ concentrations and distribution patterns. Hence the classical extrapolation method for assessing toxicity on the basis of the administered oral dose is inappropriate. Also, the applied models and theoretical assumptions that often predicted a considerable human exposure by the oral, dermal, or inhalatory route in the case of contaminated soil and dust have not proved to be pertinent, since the actual burdens determined by biomonitoring in people living at heavily contaminated sites did not show markedly increased concentrations.