Between-run Coefficients Of Variation Research Articles

Neutralizing antibodies to interferon beta in multiple sclerosis: Analytical evaluation for validation of a cytopathic effect assay

BackgroundRecent guidelines have recommended the use of validated assays for the measurement of neutralizing antibodies (NABs) to interferon beta (IFNβ) in patients with multiple sclerosis (MS). In an attempt of validation, we studied the analytical performance of a bioassay based on antiviral cytopathic effect (CPE) using WISH cells and the vesicular stomatitis virus (WISH/VSV CPE). MethodsNAB titres measured with the WISH/VSV CPE assay in 63 sera from IFNβ-treated MS patients were compared to those obtained with the reference CPE method using A549 cells and the encephalomyocarditis virus. Binding antibodies (BABs) were measured using a capture ELISA as a screening test for NABs. ResultsNo false-negative BAB was obtained in our patients. The between-run coefficients of variation (CVs) determined with log10 titres of the NIH anti-IFNβ (G038-501-572) yielded good results (≤10.4%) and within-run variability was excellent (CV≤2%). The log10 titres obtained with both CPE assays were highly correlated (r=0.969 and r=0.884 for anti-IFNβ-1a and anti-IFNβ-1b, respectively). The same patients were found NAB-positive with both CPE assays. ConclusionBecause of its good precision, sensitivity and excellent correlation with the reference CPE method, the WISH/VSV CPE bioassay can be used in the follow-up of IFNβ-treated MS patients.

Determination of everolimus in whole blood using the Abbott IMx® sirolimus microparticle enzyme immunoassay

Objectives: Everolimus (Certican®) is a new immunosuppressant derived from sirolimus (Rapamune®) with a 2-hydroxyethyl chain at position 40 of the macrolide ring. The aim of our study was to evaluate the possible determination of everolimus in whole blood using a commercialized microparticle enzyme immunoassay for sirolimus determination. Design and methods: Everolimus concentrations were determined in blood samples from 11 kidney transplant patients ( n = 51) and different control materials ( n = 35) using the Seradyn Innofluor® Certican® fluorescence polarization immunoassay (FPIA) and the Abbott IMx® sirolimus microparticle enzyme immunoassay (MEIA). Results: The MEIA gave a concentration-dependent cross-reactivity with the everolimus, with a linear regression between the assigned values ( y) for the Innofluor® Certican® calibrators and those obtained ( x) using this immunoassay: y = 0.96 x + 0.67 (ma68 = 0.21 μg/L, r = 0.974, p < 0.001). The within- and between-run coefficients of variation using the MEIA were ≤ 7.3%. In analyzing the different blood samples from patients and control materials using the MEIA and FPIA, a linear regression was found: MEIA = 0.99FPIA − 0.46 (ma68 = 0.32 μg/L, r = 0.967, p < 0.001). Correcting the MEIA results by means of the linear regression equation found between the assigned values for the Innofluor® Certican® calibrators and those obtained using this immunoassay led to a reduction in the deviation with respect to the FPIA values. The possible effect of the hematocrit on the results is analogous for both immunoassays. Conclusions: The Abbott IMx® sirolimus MEIA permits the simple and precise determination of everolimus in whole blood, making it a valid alternative for the therapeutic monitoring of this immunosuppressant agent.

High performance liquid chromatographic analysis and preclinical pharmacokinetics of the heteroarotinoid antitumor agent, SHetA2

SHetA2 {[(4-nitrophenyl)amino][2,2,4,4-tetramethylthiochroman-6-yl)amino]methane-thione], NSC 726189} is a sulfur-containing heteroarotinoid, which selectively inhibits cancer cell growth and induces apoptosis without activation of nuclear retinoic acid receptors (RARs). The objective was to develop and validate a HPLC/UV method for the determination of SHetA2, and study the pharmacokinetics of SHetA2 in the mouse. SHetA2 and the internal standard, methylated XK469 (MeXK469) were isolated from 0.2 ml of mouse plasma by solid phase extraction. The analytes were separated on a narrow-bore C18 column, with the mobile phase consisting of 60% acetonitrile in water at a flow rate of 0.2 ml/min. UV detection was set at 341 nm. Pharmacokinetic studies of SHetA2 were carried out in mice following i.v. bolus dose at 20 mg/kg and oral administrations at 20 and 60 mg/kg. The standard curves were linear between 25 and 2,500 nM and the lower limit of quantification (LLOQ) was 25 nM. The within-run coefficients of variation (CVs) were 11.1% at 10, 9.4% at 100, and 5.2% at 2,500 nM and the respective between-run CVs were 10.9, 3.1, and 1.5% (all n=5). The recovery was 85.8% for SHetA2 and 80.6% for MeXK469. Following i.v. bolus dose, plasma concentrations of approximately 10 microM were achieved at 5 min in mice and declined biexponentially with detectable levels at 60 h. The data were fitted with a two-compartment model, which gave a mean initial t1/2 of 40 min and terminal t1/2 of 11.4 h (n=6). The total body clearance was approximately 1.81 l/h/kg. The volume of distribution at steady state (Vdss) was 20.8 l/kg. Plasma protein binding was found to be 99.3-99.5% at low micromolar concentrations. Plasma concentration data for the i.v. and p.o. doses were also fitted interactively to a two-compartment deconvolution model. From this result, oral bioavailability values of 15% at 20 mg/kg and 19% at 60 mg/kg were obtained. A highly sensitive HPLC/UV method for the quantification of SHetA2 in plasma has been developed to support pharmacokinetics of SHetA2 in the mouse. Pharmacokinetic behaviors of this drug appear to be favorable for future development.

Analytical performance of a new two-step ADVIA Centaur® estradiol immunoassay during ovarian stimulation

Measurement of estradiol is useful in women undergoing ovarian stimulation for in vitro fertilization and embryo transfer (IVF-ET). The analytical performance of a new two-step estradiol assay (ADVIA Centaur) estradiol-6 III from Bayer Diagnostics) was evaluated in 41 sera from 11 women undergoing ovarian stimulation. The results were compared to those obtained with two radioimmunoassays (RIAs; RIA Estradiol Immunotech IM 1663 from Beckmann Coulter and Coat-A-Count Estradiol from Diagnostic Products Corporation) and with one chemiluminescent immunoassay (CLIA; ADVIA Centaur estradiol-6). The ADVIA Centaur) estradiol-6 III assay was the most sensitive assay, with a functional sensitivity of 55 pmol/L. Within- and between-run coefficients of variation calculated for the new ADVIA Centaur assay ranged from 3.3% to 9%, which was better than the precision obtained for the other assays. A dilution test showed serum interferences when estradiol was measured in non-diluted samples. No statistical difference was observed between the estradiol results obtained in diluted sera with the new two-step ADVIA Centaur assay and those measured with the Immunotech RIA and the other CLIA. In conclusion, this new, two-step estradiol assay performed on the ADVIA Centaur system displays suitable sensitivity, precision and intermethod agreement with the Immunotech RIA for the measurement of serum estradiol concentrations in women undergoing ovarian stimulation and IVF-ET. For correct linearity, estradiol measurement should be performed on diluted samples.

Can an Immunoassay Become a Standard Technique in Detecting Oxycodone and Its Metabolites?

Opiate toxicology testing is routinely performed in the hospital setting to identify abusers and/or to determine those patients who are not taking prescribed opiate analgesics such as oxycodone. Commercially available assays for opiate detection in urine have decreased sensitivity for oxycodone, which contributes to a high false-negative rate. Functioning as a beta site, our Veterans Affairs hospital evaluated a new enzyme immunoassay, DRI Oxycodone Assay, for its use in the qualitative and semiquantitative detection of oxycodone in urine. We hypothesize that an immunoassay for oxycodone with superior sensitivity and specificity, when compared to the traditional opiate assays, would reduce the need for more expensive and time-consuming confirmatory testing. We used the new liquid homogenous enzyme immunoassay to determine oxycodone results in a total of 148 urine samples from 4 different sample groups. Gas chromatography-mass spectroscopy was subsequently used to confirm the presence or absence of oxycodone (or its primary metabolite, noroxycodone). We also evaluated within-run, between-run, and linearity studies and conducted a crossover study to establish a cutoff value for oxycodone. In our patient population, we used the new DRI immunoassay to evaluate 17,069 urine samples to estimate oxycodone misuse profiles (patients not taking prescribed oxycodone or taking oxycodone without a prescription) during a 4-month period. The sensitivity and specificity of the new oxycodone immunoassay were 97.7% and 100%, respectively, at the cutoff concentration of 300 ng/mL. The assay linearity was 1,250 ng/mL, and the sensitivity was 10 ng/mL. Within-run precision and between-run coefficient of variation were 2.3% and 1.8%, respectively. None of the 15 compounds that we evaluated for interference had crossover significant enough to produce a positive oxycodone result when using 300 ng/mL as the cutoff value. None of the 17,069 oxycodone immunoassays was followed with a request for confirmation. Among patients with positive results (n = 224), 93 (41.5%) were not prescribed oxycodone. The new DRI Oxycodone Assay is a sensitive and specific screening test for the determination of oxycodone. The improved opiate screening results may lead to better patient and prescription management, to decreased laboratory spending, and to the identification of oxycodone abusers, which could result in decreased oxycodone-related mortality.

Analytic Performance of a Point-of Care Instrument for Measurement of Human Chorionic Gonadotropin Hormone

The aim of the study was to examine and validate the feasibility of the point-of care instrument the Stratus® CS STAT Fluorimetric Analyzer (Dade Behring, Inc, Deerfield, IL) for measurement of hCG. The Stratus® βHCG procedure is a 2-site sandwich fluorimetric immunoassay that measures the intact hCG dimer and the free β form in plasma from heparinized blood (on-board centrifugation) or in previously separated plasma or serum in less than 15 minutes. The analytic performance was compared with the Vidas® assay (bioMérieux, Marcy l'Étoile, France), according to the Valtec protocol designed by the French Society of Clinical Biology. Within-run and between-run coefficients of variation were less than 3%. The linearity extended to 1250 IU/L, and the detection limit was under 1 IU/L. Comparative methods gave: The differences observed between SCS and Vidas are explained by the different specificities of antibodies used and by the influence of heparin on βhCG SCS (−10%). We verified that there was no hook effect with high concentrations of hCG (2 × 106 IU/L) and no interference with FSH or LH. This assay is a good tool in the diagnosis of early detection of pregnancy.

Quantification of Serum 25-Hydroxyvitamin D 2 and D 3 Using HPLC-Tandem Mass Spectrometry and Examination of Reference Intervals for Diagnosis of Vitamin D Deficiency

Serum levels of 25-hydroxyvitamin D are important in establishing true vitamin D levels in humans. The purposes of this study were to develop a sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection of 25-hydroxyvitamin D(2) and D(3) and establish reference intervals for these analytes. Chromatographic separation of 25(OH)D(2) and 25(OH)D(3) was achieved after adding deuterated Delta(9)-tetrahydrocannabinol (D(9)-THC-D(3)) and organic extraction. The 3 ions were ionized using positive electrospray ionization and detected in the multiple-reaction monitoring mode using mass (m)/charge (z) transitions of 318.15 > 196.20 (Delta(9)-THC-D(3)), 401.15 > 365.2 (25(OH)D(3)), and 413.15 > 355.20 (25(OH)D(2)). Reference interval study results were compared with current 25(OH)D recommendations. Elution of 25(OH)D(2), 25(OH)D(3), and Delta(9)-THC-D(3) was achieved after 3.0 minutes (total run time, 6.0 minutes). Within- and between-run coefficients of variation were less than 11%. Deming regression of radioimmunoassay and LC-MS/MS methods for total 25(OH)D levels yielded a slope of 0.97 (95% confidence interval, 0.88-1.05) and y-intercept of -1.74 ng/mL. Reference intervals were less than recommended levels (D(2), 0.0-12.1; D(3), 5.5-41.4; total vitamin D, 6.0-43.5 ng/mL [0-30, 14-103, 15-109 nmol/L, respectively]) with no statistically significant differences in race, age, or sex. This LC-MS/MS method provides a rapid, accurate, sensitive, and cost-effective alternative to other methods for detection of 25(OH)D(2) and 25(OH)D(3) at nanomolar concentrations.

Intracellular Levels of Oblimersen Sodium and Target Downregulation Correlates to Clinical Activity of bcl-2 Antisense in Acute Myeoid Leukemia (AML).

Oblimersen sodium (Ob, Genasense™) is an 18 mer phosphorothioate antisense directed against bcl-2, an antiapoptotic protein that mediates chemoresistance in malignant cells. In order to assess whether detectable intracellular concentrations (ICs) of Ob are achievable in vivo, we have developed a sensitive and specific ELISA-based assay. This assay involves Ob hybridization to a 5′-end overhang of a 3′-biotinylated capture probe ligated to a digoxigenin (Dig)-labeled probe, and detection by an anti-Dig-alkaline phosphatase system. In K562 cell extract, the assay was linear within 50–2000 pM range with a limit of quantification (LOQ) of 50 pM (equivalent to 5.0 fmol/100 μL). The withtin-run coefficients of variation (CVs) in 4 spiked concentrations were between 3–7% in 6 replicates with accuracy values between 93–109%. The between-run CVs were between 6–12% and accuracy values between 97–102%. The specificity of the assay was demonstrated by low cross reactivity with mismatched oligonucleotides and putative 3′-end metabolites shortened by 1, 2 or 3 nucleotides. Validation of IC measurement was performed in vitro in K562 cells treated with fluorescent labeled Ob conjugated to oligofectamine. At Ob concentrations between 0.1 – 10 μM, Bcl-2 mRNA downregulation measured by real-time RT-PCR occurred efficiently. Nonlinear regression analysis of a dose-response curve showed that 50% Bcl-2 downregulation (IC50) occurred at approximately 0.29 μM, corresponding to an Ob IC concentration of 37 pmole/mg protein. Cellular uptake was confirmed by microscopy and flow cytometry. To validate these results in vivo, we measured Ob IC in bone marrow samples from untreated AML pts aged > 60 yrs enrolled on the phase I study OSU 0164. These pts were induced with Ob 7 mg/kg/d CIVI on days 1–10, cytarabine 100 mg/m2/d CIVI on days 4–10 and daunorubicin administered iv at two dose levels (45 mg/m2/d IV and 60 mg/m2/d) on days 4–6. Among 21 pts assessable for clinical response and Bcl-2 levels, at pretreatment, Bcl-2 copy numbers (normalized to ABL) were higher among 12 pts who achieved a CR (median 85,325; range 19,120–149,100) than among 9 non-responsive (NR) pts (32,100 bcl-2 /abl copies; range 1,488–163,500) (P=.04; Mann-Whitney test). Following 72 hr Ob infusion, a decrease (−38%) in median Bcl-2/ABL mRNA copies in CR patients and an increase (+115%) in Bcl-2/ABL copies in NR pts (P=.002; Mann-Whitney test) were observed by real time RT-PCR. A trend in higher median IC of Ob was observed in CR pts (17.0 pmole/mg protein; range 1.5–30.0) as compared to NR pts (4.4 pmole/mg protein; range 0.33–28.0) (P=.06; Mann-Whitney test). Six of 7 pts with IC above the median obtained a CR. No differences were observed in the Ob plasma PKs between the CR pts [median steady state concentration (Css) 2.8 μg/mL, area-under-the-curve (AUC) 772 μg*hr/mL and clearance (Cl) 9.6 L/hr) and the NR pts (median Css 3.4 μg/mL, AUC 752 μg*hr/mL, Cl 6.4 L/hr). Although the number of samples analyzed was small, our data suggest that, despite interpatient variability of both Bcl-2 mRNA expression and Ob uptake, this antisense can be successfully delivered to pts and result in clinically relevant target downregulation. A Cancer and Leukemia Group B phase III AML study to characterize prospectively the interplay of IC levels of the Ob and Bcl-2 downregulation is in progress.

Optimization of a liquid chromatography-tandem mass spectrometry method for quantification of the plant lignans secoisolariciresinol, matairesinol, lariciresinol, and pinoresinol in foods.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of the four major enterolignan precursors [secoisolariciresinol, matairesinol, lariciresinol, and pinoresinol] in foods. The method consists of alkaline methanolic extraction, followed by enzymatic hydrolysis using Helix pomatia (H. pomatia) beta-glucuronidase/sulfatase. H. pomatia was selected from several enzymes based on its ability to hydrolyze isolated lignan glucosides. After ether extraction samples were analyzed and quantified against secoisolariciresinol-d8 and matairesinol-d6. The method was optimized using model products: broccoli, bread, flaxseed, and tea. The yield of methanolic extraction increased up to 81%, when it was combined with alkaline hydrolysis. Detection limits were 4-10 microg/(100 g dry weight) for solid foods and 0.2-0.4 microg/(100 mL) for beverages. Within- and between-run coefficients of variation were 6-21 and 6-33%, respectively. Recovery of lignans added to model products was satisfactory (73-123%), except for matairesinol added to bread (51-55%).

A microplate-based enzymatic assay for the simultaneous determination of phenylalanine and tyrosine in serum

Background: Several methods have been reported for the quantitation of phenylalanine (Phe) or tyrosine (Tyr) in blood, but the simultaneous determination of Phe and Tyr usually requires sophisticated equipment. Here we describe a simple and reliable microplate assay for measurement of Phe and Tyr in serum of patients with phenylketonuria (PKU). Methods: Serum was deproteinised and incubated with phenylalanine ammonia-lyase at 37 °C for 2 h. Phe and Tyr were converted to trans-cinnamate and trans-coumarate, respectively, which were determined UV spectrophotometrically. Results: The assay demonstrated linearity up to 3200 μmol/l Phe and 640 μmol/l Tyr. The sensitivity of the assay was 15 μmol/l for Phe and 5 μmol/l for Tyr. Analytical recovery was 105% and 97% for Phe and Tyr, respectively. The between-run coefficient of variation (CV) varied with concentration but was 4.7–18.8% for Phe and 4.2–8.8% for Tyr. The within-run CV was 2.7% for Phe and 6.5% for Tyr. Results obtained with the assay correlated well with those of an ion exchange chromatographic method. Conclusions: This novel assay is sensitive, accurate, precise and suitable for routine monitoring of PKU patients.

Cortisol and cortisone analysis in serum and plasma by atmospheric pressure photoionization tandem mass spectrometry

Objectives: Cortisol metabolism is controlled by 11β-hydroxysteroid dehydrogenase (11β-HSD) isoenzymes, which interconvert cortisol and cortisone. Accurate measurement of the cortisol and cortisone concentrations and their ratio provide useful information about 11β-HSD activity. Methods: Cortisol and cortisone were extracted with methyl- tert-butyl ether from 100 μl of serum or plasma. The extract was evaporated, reconstituted with mobile phase, and analyzed by tandem mass spectrometry using a photoionization interface. The transitions monitored were: m/ z 363 to 121 and 363 to 97 for cortisol, 361 to 163 and 361 to 105 for cortisone. Results: Within-run and between-run coefficients of variation were less than 6% and 12%; 14% and 22%; 11% and 21% for cortisol, cortisone, and their ratio, respectively. The limit of detection was 1 μg/l for cortisol and 5 μg/l for cortisone. Normal ranges for cortisol and cortisone concentration and for their ratio in plasma ( n = 120) determined as the central 95% were 33–246 μg/l for cortisol, 8–27 μg/l for cortisone, and 0.081–0.301 for the cortisone/cortisol ratio. Conclusions: We developed a simple sensitive method for cortisol and cortisone analysis in plasma and serum that uses a small sample volume. The method is very specific, fast, does not have any known interference, and is useful for diagnosis of variety of disease and pathologic conditions.

Measurement of faecal immunoglobulin a levels in young children.

A nephelometric immunoassay was developed to quantify immunoglobulin A (IgA) in children's stools. This method enables IgA in faecal protein extracts to be measured over a large range of concentrations (1.61-51.50 mg/L) with good accuracy (linear recovery in dilution-overloading assay) and precision (within- and between-run coefficients of variation (CVs) of 1-6%). An excellent recovery (105%) was obtained in stool samples overloaded by purified colostral IgA, demonstrating that the method used for faecal IgA extraction is adapted, not to induce significant IgA degradation, and probably allow a complete extraction of IgA. The amount of faecal IgA, as determined in stool samples from 125 children (6-24 months old), was an average of 14 mg per 100 g of stools (about 10% of the total protein stool content), with large individual variation (3-30 mg per 100 g of stools). No correlation was observed between faecal IgA amounts and the children's age or sex. Such an immunoassay could enable exhaustive noninvasive investigations of the maturation of the intestinal immune system, as well as accurate studies of the effect of oral dietary supplementation on IgA regulation.

A simple method for quantifying fentanyl, sufentanil, or morphine in discard syringes from anesthesia procedures.

Fentanyl, sufentanil, and morphine are commonly used in the conduct of anesthesia. Medical staff working with these drugs are at high risk of addiction. To detect and prevent diversion, a method was developed to quantify these drugs in discard syringes using the BioRad REMEDi HS Drug Profiling System. For fentanyl, the lowest concentration detected is 0.1 microg/mL, and the assay is linear to 5.0 microg/mL; the within-run coefficient of variation (CV) is 0.9% (n = 5), and between-run CV is 2.5% (n = 20). For sufentanil, the lowest concentration detected is 0.5 microg/mL, and the assay is linear to 11.0 microg/mL; the within-run CV is 2.0% (n = 5), and the between-run CV is 2.4% (n = 20). For morphine, the lowest concentration detected is 0.5 microg/mL, and the assay is linear to 10.0 microg/mL; the within-run CV is 11.6% (n = 5), and between-run CV is 11.3% (n = 20). Other drugs commonly used in the operating room were checked for cross-reactivity on the REMEDi HS; none cross-reacted. The REMEDi HS can be used for rapid, accurate quantification of fentanyl, sufentanil, and morphine in discard syringes from anesthesia procedures or related medical applications.

An enzymatic assay for lysophosphatidylcholine concentration in human serum and plasma

Objectives: Several methods for measuring lysophosphatidylcholine (LPC) concentrations have been reported. However, these methods are not practical because they are either too complicated and/or too time-consuming for LPC determinations in human serum and plasma. Design and Methods: We have developed a new enzymatic LPC assay, which uses lysophospholipase, glycerophosphorylcholine phosphodiesterase and choline oxidase, and which determines the quantities of hydrogen peroxide generated in the presence of peroxidase using an oxidative chromogenic reagent and 4-aminoantipyrine. Results: Various samples were mixed with LPC assay reagents, and their changes in absorbance were measured. The present method produced a linear calibration line between LPC concentration and absorbance change. It also measured only LPC, and not other phospholipids such as phosphatidylcholine, sphingomyelin and lysophosphatidic acid. The within-run and between-run coefficients of variation were 0.3–0.7% and 0.7%, respectively. The recovery of exogenous LPC added to control serum was 99.5–102.1%. The correlation coefficient obtained in a comparison with a method for analyzing fatty acids was 0.9122. Conclusions: The present method is simple, specific for LPC, and can be applied with an automatic analyzer. It may also be useful for further studies of the biological functions of LPC as well as clinical applications in various disorders.

Chiral high-performance liquid chromatographic analysis of the enantiomers of XK469, a new antitumor agent, in plasma and urine

XK469 (NSC697887), (±)-2-[4-(7-Chloro-2-quinoxaliny)oxy]-phenoxy propionic acid, an analog of the herbicide Assure®, which possesses antitumor activity, especially against murine solid tumors and human xenografts, has recently been found to be the first topoisomerase IIβ poison. Both R(+) and S(−) isomers are cytotoxic, although the R-isomer is more potent. A chiral high-performance liquid chromatography (HPLC) assay that utilizes Chirobiotic T column for the measurement of enantiomers of XK469 in plasma has been developed with a limit of quantitation (LOQ) of 0.2 μg/ml using a 0.2 ml plasma sample. Chloroqinoxaline sulfonamide (CQS) was used as the internal standard and the assay has been validated in rat plasma. The within-run coefficient of variations (CVs) were 5.9, 5.0, and 3.1% for the S-isomer and 8.1, 4.2, 6.4% for R(+)-XK469 at 0.2, 1, and 2 μg/ml, respectively. The between-run CVs were 10.5, 5.3, and 1.9% for S(−)- and 10.9, 6.3, and 3.6% for R(+)-XK469. Using this chiral assay, a plasma concentration time data of R(+)-,S(−)-XK469 in a Fischer 344 rat receiving i.v. dosing of S(−)XK469 at 10 mg/kg was monitored. S(−)XK469 was found to be significantly converted to the R-enantiomer in circulation even when the S-enantiomer was administered. The predominant inversion from S(−)- to R(+)-XK469 was also observed in the mouse and dog plasma. In the rat, the plasma concentration–time profiles for both isomers follow two compartmental pharmacokinetics with the t 1/2β for the R-enantiomer slightly longer and the clearance of the S-enantiomer higher than the R-enantiomer.

Determination of lead in whole blood: Comparison of the LeadCare blood lead testing system with Zeeman longitudinal electrothermal atomic absorption spectrometry

This study compares the efficiency of blood lead level analysis by graphite furnace atomic absorption spectrometry (GFAAS) and the portable LeadCare Blood Lead Testing System (LCS). Recoveries of two added lead concentrations of 22 and 42 micrograms/dL ranged from 102.4 to 105.5% for LCS and from 96.3 to 97.2% for GFAAS. Measurement of a certified sample (Certified Danish Whole Blood) at a blood lead concentration of 26.2 micrograms/dL gave within- and between-run coefficients of variation which were both approximately 8% by LCS and 2% by GFAAS. Comparison of the tested method (LCS) versus GFAAS from analysis of 76 samples of blood lead collected from workers in different industrial sectors showed imperfect overall correlation (r = 0.95). The LCS is quite suitable for screening purposes, but requires the use of non-frozen blood collected less than 24 h before. Conservative threshold values should be applied when using the LCS for initial screening in the field.

Development and evaluation of a sandwich ELISA for quantification of the 20S proteasome in human plasma

Because quantification of the 20S proteasome by functional activity measurements is difficult and inaccurate, we have developed an indirect sandwich enzyme-linked immunosorbent assays (ELISA) for quantification of the 20S proteasome in human plasma. This sandwich ELISA uses a combination of a monoclonal antibody (mcp 20) recognizing the C2-beta subunit of human 20S proteasome (Mr≈30,000) and a polyclonal rabbit anti-20S antibody which labels different subunits of the complex. The detection limit of the assay was established as 10 ng/ml ( n=10, mean of zero standard+2 S.D.) and the recovery rate ranged from 96% to 104%. The within-run and between-run coefficients of variation (CV) ranges were 2.8–3.3 and 3.0–3.4, respectively. Using serial dilutions of plasma to which various amounts of purified 20S proteasome were added, a linear dose–response was observed between 102 and 2050 ng/ml with a slope of 1.004 and a coefficient of determination r 2 of 0.99. In a preliminary experiment performed on a limited number of patients, the present assay was used to quantify the 20S proteasome in plasma from healthy subjects ( n=11) and from a limited number of patients with various diseases (two patients with each of the following diagnoses: acute myeloid leukaemia, chronic myeloproliferative syndromes, Hodgkin's disease and solid tumors). The average concentration of 20S proteasome in plasma from normal subjects was found to be 2319±237 ng/ml ( n=11). With reference to this normal range, the plasma proteasome concentration was found to be increased in most of these pathological state and as high as 1200% when solid tumors had been detected. For patients with Hodgkin's disease, the changes were more variable whereas in patients with chronic lymphocytic leukaemia, the proteasome concentration was raised during the acute phase of disease and decreased during therapy. We suggest that this robust, accurate and highly reproducible assay could be used to quantify proteasome in human plasma and investigate its value as a biological marker for various malignant and nonmalignant diseases.

An aid to the early detection and management of diabetic nephropathy: assessment of a new point of care microalbuminuria system in the diabetic clinic.

To compare the performance of the DCA2000 microalbuminuria system for albumin and creatinine concentrations and the albumin:creatinine ratio (ACR) with laboratory measurements in the hospital diabetes clinic and to assess the ease of use and applicability by standard clinic personnel. Urine albumin and creatinine concentration and ACR were measured in 154 diabetic patient samples and in 77 normal subjects. Both albumin assays are based on immunoturbidimetry. The DCA2000 system utilizes reagent cartridges processed automatically. Control material within-run precision (coefficient of variation (CV)) for albumin and creatinine ranged up to 7.1% and 3.3% respectively. Between-run CVs ranged from 2.1% to 4.3%. Method comparisons yielded correlation coefficients > 0.99 for albumin, creatinine and ACR, only a small negative bias of 3.2 mg/l for albumin and 0.10 mg/mmol for ACR, no concentration-related bias for ACR and no between-method difference for either albumin (P = 0.195) or ACR (P = 0.341). At a laboratory albumin concentration cut-off of 20 mg/l the sensitivity, specificity, negative and positive predictive values were 92.4% 100% 92.7% and 100%. Normal reference range mean albumin, creatinine and ACR values for the DCA2000 and the laboratory were 7.7 mg/l vs. 9.0 mg/l 13.0 mmol/l vs. 12.6 mmol/l and 0.66 mg/mmol vs. 0.81 mg/mmol respectively. Clinic personnel found that the DCA2000 system was easy to use suited the clinic environment and generated confidence in the results. This point of care system safely substitutes laboratory-based measurements. Ease of use and low cost make it suitable for screening and monitoring diabetes treatment. It facilitates the use of random urines, and may obviate the need for timed samples. This approach has a clear place in the battle to reduce the diabetic vascular disease burden.

An enzymatic cycling method for the measurement of myo-inositol in biological samples

Introduction: A sensitive and simple enzymatic cycling method is described for the quantitation of myo-inositol in biological samples. Methods: The method involves the use of a sensitive and simple enzymatic cycling method is described for the quantitation of myo-inositol in biological samples. The method involves use of thio-NAD +, NADH and thermostable myo-inositol dehydrogenase (IDH; EC. 1.1.1.18) and measurement of the increase in absorbance at 405 nm of thio-NADH at 37 °C. Results: The calibration curve for myo-inositol was linear ( r=1.00) between 10 and 400 μmol/l. Analytical recoveries of exogenous myo-inositol added to serum and urine were 100–105% and 98–103%, respectively. Within-run and between-run coefficient of variation (CV) were 0.6–2.1% and 1.1–3.0%, respectively. This method was free from interference by hemoglobin, bilirubin, ascorbate, chyle, various sugars, sugar alcohol and myo-inositol phosphates. With the use of myo-inositol as a standard solution, the serum myo-inositol concentration (mean±SD) was significantly greater in patients with diabetes mellitus (DM) without nephropathy (73.0±13.8 μmol/l, n=7) than in healthy individuals without DM (61.0±12.4 μmol/l, n=20). The urinary myo-inositol concentration was also significantly greater in patients with DM without nephropathy (793.3±870.3 μmol/l, n=7) than in healthy individuals without DM (76.0±63.0 μmol/l, n=13). Conclusions: This new method is simple, sensitive and enables quantitative analysis of myo-inositol.

Quantitative determination of parathyroid hypertensive factor by enzyme-linked immunosorbent assay

A new competitive enzyme immunoassay for the detection parathyroid hypertensive factor (PHF) in human plasma using a PHF–horseradish peroxidase conjugate and IgM antibody adsorbed on the microtiter plate was established. The antibodies raised against rat PHF could recognize human PHF. Cross-reactivity of anti-PHF antibodies with other serum haptens and proteins was negligible. Conjugation of PHF with horseradish peroxidase did not neutralize the antigen activity. The limit of detection of PHF was 0.02 U/mL in reference units and PHF levels between 0.02 and 1 U/mL could be detected. Within-run coefficient of variation (CV) was less than 10%, and between-run CV was less than 15% for over the dynamic range of the assay. Preliminary clinical studies were performed with plasma samples from hypertensive patients with confirmed diagnosis. Parathyroid hypertensive factor levels, as detected with this immunoassay, were positively correlated with PHF levels detected with the semiquantitative blood pressure (BP) bioassay previously used. Parathyroid hypertensive factor levels detected with the enzyme-linked immunosorbent assay (ELISA) were also correlated with BP in patients. The PHF ELISA provides a selective, simple, and rapid method that can be used for routine determination of PHF in human plasma, and provides useful clinical information.