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A Pt electrode modified by polyaniline-poly(3-methylthiophene)-poly(3,3'-diaminobenzidine) was used for amperometric determination of epinephrine in a solution of NaHS[O.sub.4]/[Na.sub.2]S[O.sub.4] (pH 2.0). For these studies, potentials between 0.40 and 0.50 V were applied, and the best response was obtained at 0.45 V. The limit of detection, limit of quantification, and the linear dynamic range were 1.23 x [10.sup.-4], 4.10 x [10.sup.-4], and 4.10 x [10.sup.-4] to 100.0 mmol [L.sup.-1], respectively. These results were compared with determinations using Pt electrodes that were coated and uncoated with homopolymers. To check the accuracy of the developed method and the matrices interference, determination of epinephrine was performed in human serum samples using this modified electrode. The epinephrine concentrations were adjusted to 1.0 and 5.0 mmol [L.sup.-1], and recovery values were calculated as 100.4% and 96.8%, respectively. It is significant that the determination of epinephrine was carried out at a lower potential (0.45 V) than the oxidation potential of epinephrine (0.65 V) without any matrix effect. Keywords: epinephrine, polyaniline, poly(3-methylthiophene), poly(3,3'-diaminobenzidine), amperometric determination. Nous avons utilise une electrode de platine modifiee par des polymeres constitues de polyaniline, de poly(3-methylthiophene) et de poly(3,3'-diaminobenzidine) pour effectuer le dosage amperometrique de l'epinephrine dans une solution de NaHS[O.sub.4] et de [Na.sub.2]S[O.sub.4] (pH 2,0). Aux fins d'etudes amperometriques, nous avons applique des potentiels allant de 0,40 a 0,50 V et avons obtenu la meilleure reponse a 0,45 V. Le seuil de detection, le seuil de quantification et le domaine de linearite ont ete respectivement de 1,23 x [10.sup.-4], 4,10 x [10.sup.-4] et de 4,10 x [10.sup.-4] a 100,0 mmol [L.sup.-1]. Nous avons compare ces resultats pour les electrodes de platine recouvertes d'homopolymeres et l'electrode nue. En vue de verifier l'exactitude de la methode mise au point et l'interference des supports, nous avons effectue le dosage de l'epinephrine dans des echantillons de serum humain a l'aide de cette electrode modifiee. Nous avons ajuste les concentrations d'epinephrine a 1,0 et 5,0 mmol [L.sup.-1], et avons calcule des valeurs du rendement de 100,4% et de 96,8%, respectivement. Il est important de noter que le dosage de l'epinephrine a ete effectue a potentiel inferieur (0,45 V) au potentiel d'oxydation de l'epinephrine (0,65 V), sans effet du support. [Traduit par la Redaction] Mots-cles: epinephrine, polyaniline, poly(3-methylthiophene), poly(3,3'-diaminobenzidine), dosage amperometrique., Introduction Catecholamines are water-soluble organic compounds that are produced in the body from tyrosine, and they are the main neurotransmitters that mediate central nervous system functions such as motor control, [...]

The 130-kDa smooth muscle myosin light chain kinase (smMLCK) is a [Ca.sup.2+]/CaM-regulated enzyme that plays a pivotal role in the initiation of smooth muscle contraction and regulation of cellular migration and division. Despite the critical importance of smMLCK in these processes, little is known about the mechanisms regulating its expression. In this study, we have identified the proximal promoter of smMLCK within an intron of the mouse mylk gene. The mylk gene encodes at least two isoforms of MLCK (130 and 220 kDa) and telokin. Luciferase reporter gene assays demonstrated that a 282-bp fragment (- 167 to + 115) of the smMLCK promoter was sufficient for maximum activity in A10 smooth muscle cells and 10T1/2 fibroblasts. Deletion of the 16 bp between-167 and-151, which included a CArG box, resulted in a nearly complete loss of promoter activity. Gel mobility shift assays and chromatin immunoprecipitation assays demonstrated that serum response factor (SRF) binds to this CArG box both in vitro and in vivo. SRF knockdown by short hairpin RNA decreased endogenous smMLCK expression in A 10 cells. Although the SRF coactivator myocardin induced smMLCK expression in 10T1/2 cells, myocardin activated the promoter only two-to fourfold in reporter gene assays. Addition of either intron 1 or 6 kb of the 5' upstream sequence did not lead to any further activation of the promoter by myocardin. The proximal smMLCK promoter also contains a consensus GATA-binding site that bound GATA-6. GATA-6 binding to this site decreased endogenous smMLCK expression, inhibited promoter activity in smooth muscle cells, and blocked the ability of myocardin to induce smMLCK expression. Altogether, these data suggest that SRF and SRF-associated factors play a key role in regulating the expression of smMLCK. myocardin; serum response factor; GATA; transcriptional regulation; gene expression; telokin

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.jada.2005.01.004 Byline: Mary Dean Coleman, Sharon M. Nickols-Richardson Abstract: This study examined the effect of a low-carbohydrate/high-protein diet on serum and urine ketone body concentrations. Thirteen overweight premenopausal women aged 32 to 45 years consumed [less than or equal to]20 g carbohydrate/day with liberal intakes of protein and fat for 2 weeks; thereafter, carbohydrate intake increased 5 g/week for 10 weeks. Women were weighed and provided fasting urine and blood samples to detect urinary ketones and quantify serum ketone concentrations, respectively, at baseline and weeks 1 to 4, 6, and 12. Women lost 8.3%[+ or -]2.8% of initial body weight by week 12. Serum [beta]-hydroxybutyrate production was highest at week 1 and declined weekly, with all values higher than baseline (P<.05). Each week, serum [beta]-hydroxybutyrate was correlated with presence of urinary ketones (P<.05), but no relationship was found between weekly weight change and serum ketone production. Urinary ketones are detected in premenopausal women complying with a low-carbohydrate/high-protein diet and are associated with serum ketone concentration. However, serum ketones do not reflect weight loss.

Herpesvirus B (Cercopithecine herpesvirus 1) has been implicated as the cause of approximately 40 cases of meningoencephalitis affecting persons in direct or indirect contact with laboratory macaques. However, the threat [...]

Our research seeks to identify a serum profile, or serotype, that reflects substantial changes in food intake. Earlier studies demonstrated that a number of low-molecular-weight, redox-active compounds of metabolome were sufficiently stable analytically and biologically to identify biomarkers of dietary restriction (DR, restriction of total food intake) in rats. A second initial requirement is to demonstrate feasibility, i.e., that concentration changes in selected serum metabolites can contain sufficient information to classify rats by diet. The current study distinguished 101 (female) and 112 (male) chromatographically identifiable compounds that differ between ad libitum (AL) consumption and DR 6-mo-old rats. In a cohort of female rats, both hierarchical cluster analysis (HCA) and principal component analyses (PCA) could distinguish dietary groups with 100% efficiency (101 metabolites). Repeating the classification studies using the 63 biologically and analytically most robust metabolites decreased noise without affecting categorical separation. In a cohort of male rats, PCA, but not HCA, distinguished the original dietary groups with 100% accuracy (112 metabolites). A subset of 52 of the 112 metabolites enabled both HCA and PCA to group the male rats with 100% accuracy. These data demonstrate that quantitative analysis of selected serum metabolites can yield sufficient information by which to classify the dietary intake of a group of rats, identify such markers chromatographically and set the stage for validation of these metabolic serotypes in independent datasets. J. Nutr..132: 1031-1038, 2002. KEY WORDS: * dietary restriction * serum metabolite * HPLC * multivariate analysis * biomarker * rats

A sensitive and simple one-step immunoassay was developed and validated for quantitative determination of Cd(II) in human serum. In this method, a monoclonal antibody that recognizes Cd(II)-EDTA complexes was directly immobilized onto microwell plates. The serum sample containing metallothionein(MT)-bound and non-MT-bound Cd(II) was acidified to displace the Cd(II) from MT. The sample was then treated with metal-free EDTA to convert Cd(II) to Cd(II)-EDTA complexes. A mixture of Cd(II)-EDTA complexes derived from serum samples and Cd(II)-EDTA conjugated with peroxidase enzyme was incubated in the wells to compete for binding sites of the immobilized antibody. After addition of peroxidase substrate, the bound fraction of the enzyme conjugate was measured by a microplate reader, and the signal was inversely proportional to the concentration of the Cd(II) in the sample. The assay limit of detection was 0.24 [micro] g/ L, and the effective working range at coefficient of variation of [less than or equal to] 10% was 0.24-100 [micro] g/L. Analytical recovery of spiked Cd(II), in the concentration range between 0.8 and 50 [micro] g/L, was 97.8 [+ or -] 4.0%. The assay was selective for Cd(II); other metal ions (Mn, Co, Cu, Zn, Mg, Hg, Ca, Ni, Fe, and Pb), tested at concentrations considerably higher than those present in human serum, did not significantly interfere with the assay. The assay results correlated well with those obtained by graphite furnace atomic absorption spectrometry (r = 0.984).

The type III cytotoxin ExoS of Pseudomonas aeruginosa exhibits ADP-ribosyltransferase activity at its C-terminal domain. Data reveal that ExoS ADP ribosylates the G-protein Rap at arginine-41 residue site leading to the inhibition of Rap interaction with its guanine nucleotide exchange factor.

Isolation and structure analysis of a new toxin from the bivalve mussels, the chlorosulfolipid, are discussed. Data indicate that the new toxin is structurally very different from the known polyether diarrhetic shellfish poisoning toxins as revealed by nuclear magnetic resonance spectroscopy.

A procedure for the direct determination of arsenic in diluted serum by electrothermal atomization laser-excited atomic fluorescence spectrometry (ETA-LEAFS) is reported. Laser radiation needed to excite As at 193.696 and 197.197 nm is generated as the second anti-Stokes stimulated Raman scattering output of a frequency-doubled dye laser operating near 230.5 and 235.5 nm, respectively. Two different LEAFS schemes have been utilized and provide limits of detection of 200-300 fg for As in aqueous standards. When measurements of serum samples diluted 1:10 with deionized water are performed, a stable background signal is observed that can be accounted for by taking measurements with the laser tuned off-wavelength. No As is detected in any of the bovine or human sermn samples analyzed. Measurements of 100 pg/mL standard additions of As to a diluted bovine serum sample utilizing either inorganic or organic As species demonstrate a linear relationship of the fluorescence signal to As spike concentration, but exhibit a sensitivity of approximately haft that observed in pure aqueous standards. The limit of detection for As in 1:10 diluted serum samples is 65 pg/mL or 650 fg absolute mass, which corresponds to 0.65 ng/mL As in undiluted serum. To our knowledge, the ETA-LEAFS procedure is currently the only one capable of directly measuring As in diluted serum at these levels.

Byline: Arend Bokenkamp (1), Michael Domanetzki (1), Raymund Zinck (2), Gerhard Schumann (2), Johannes Brodehl (1) Keywords: Key words: Renal function tests; Reference values; Cystatin C; Creatinine Abstract: Cystatin C, a low molecular weight protein, is a new endogenous marker of renal function whose serum concentration correlates better with glomerular filtration rate than creatinine. The aim of the present study was to define a reference interval for cystatin C concentrations in children. Cystatin C was measured by an immunoturbidimetric assay in sera obtained from 258 children (93 girls, 165 boys, median age 6.29 years, range 1 day to 18 years) without evidence of kidney disease. The reference interval was calculated non-parametrically using the 2.5th and 97.5th percentiles. For comparison, creatinine was measured in the same samples. The cystatin C concentration was highest on the first days of life (range 1.64--2.59 mg/l) with a rapid decrease during the first 4 months. Beyond the 1st year, the cystatin C concentration was constant, with a reference interval of 0.7--1.38 mg/l. In contrast, serum creatinine concentrations steadily increased with age until adulthood. Compared with creatinine, cystatin C facilitates the recognition of abnormal renal function in children as its reference range is constant beyond the 1st year of life. The higher levels of cystatin C in the 1st year of life probably reflect the low glomerular filtration rate of neonates and infants. Author Affiliation: (1) Children&apos;s Hospital, Hannover Medical School, Hannover, Germany, DE (2) Department of Clinical Chemistry I, Hannover Medical School, Hannover, Germany, DE Article note: Received June 23, 1997 received in revised form August 27, 1997 accepted August 28, 1997

We have developed a recombinant human serum albumin (rHSA) from Pichia pastoris which expresses high levels of heterologous proteins. rHSA is used clinically in high concentration (approximately 250 mg/ml in a 50 mL vial). We had to consider not only proteins from host cells as impurities but also mannan, which exhibits harmful effects on humans. However, the analysis of mannan in biopharmaceuticals produced from yeast has not been reported. Contaminating mannans in the final product were one important index to assess the clinical safety of rHSA. We have developed a highly sensitive enzyme immunoassay (EIA), utilizing an avidin-biotin system, for the detection of either the protein or mannan polysaccharide components from P. pastoris components (PPC) in rHSA. In addition, we used anion exchange chromatography with pulsed amperometric detection (AE-PAD) for monosaccharide analysis of glycoconjugates for the detection of mannan from PPC in rHSA. The detection limits of the EIA for PPC (PPC EIA) and the AE-PAD were 1 ng of protein/250 mg of rHSA and 180 ng of mannose/mg of rHSA, respectively. The mannan content in partially purified rHSA as determined by the AE-PAD was about same as the PPC content as determined by the PPC EIA. We showed that the PPC EIA and the AE-PAD are useful methods for the purity analysis of biopharmaceuticals produced from yeast.

The transcription of Egr-1 protein and an increase in messenger ribonucleic acid (nRNA) supply was demonstrated in cells of the renal inner medullary collecting duct cell in rats. The improved mRNA supply was found to be related to the increased Egr-1 transcription. The results further revealed that epidermal growth factor, urea and peptide mitogen contributed to the activation of Egr-1 transcription without water treatment, with the 50% medium dilution water causing an additive effect.

Byline: T. Oyama (1), N. Kohno (2), A. Yokoyama (2), Y. Hirasawa (2), K. Hiwada (2), H. Oyama (1), Y. Okuda (1), K. Takasugi (1) Keywords: Key words: Interstitial pneumonitis--Rheumatoid arthritis--KL-6. Abstract: In rheumatoid arthritis (RA), interstitial pneumonitis is one of the major extraarticular complications that worsens a patient&apos;s prognosis. KL-6, a human MUC1 mucin, has been reported to be a sensitive serum marker for activity of interstitial pneumonitis. We investigated the clinical significance of serum KL-6 level in patients with RA. Serum levels of KL-6 and RA-associated inflammatory markers were evaluated in 177 RA patients. The diagnosis of active interstitial pneumonitis was made by clinical symptoms, pulmonary function tests, chest X-ray film, and high resolution CT. Serum KL-6 was increased in 8 of 9 (88.9%) RA patients with active interstitial pneumonitis but in only 1 of 168 (0.6%) RA patients without active interstitial pneumonitis. No significant correlation was found between KL-6 level and conventional clinical parameters. In RA, abnormal elevation of serum KL-6 strongly indicates the complication of active interstitial pneumonitis. Author Affiliation: (1) Department of Internal Medicine, Dohgo Spa Hospital, Onsen-gun, Ehime 791-02, Japan, JP (2) Second Department of Internal Medicine, Ehime University School of Medicine, Onsen-gun, Ehime 791-02, Japan, JP Article note: Accepted for publication: 11 February 1997

A cross-sectional survey was made in 56 exceptionally healthy males, ranging in age from 20 to 84 years. Measurements were made of selected steroidal components and peptidic hormones in blood serum, and cognitive and physical tests were performed. Of those blood serum variables that gave highly significant negative correlations with age (r > -0.6), bioavailable testosterone (BT), dehydroepiandrosterone sulfate (DHEAS), and the ratio of insulin-like growth factor 1 (IGF-1) to growth hormone (GH) showed a stepwise pattern of age-related changes most closely resembling those of the age steps themselves. Of these, BT correlated best with significantly age-correlated cognitive and physical measures. Because DHEAS correlated well with BT and considerably less well than BT with the cognitive and physical measures, it seems likely that BT and/or substances to which BT gives rise in tissues play a more direct role in whatever processes are rate-limiting in the functions measured and that DHEAS relates more indirectly to these functions. The high correlation of IGF-1/GH with age, its relatively low correlation with BT, and the patterns of correlations of IGF-1/GH and BT with significantly age-correlated cognitive and physical measures suggest that the GH-IGF-1 axis and BT play independent roles in affecting these functions. Serial determinations made after oral ingestion of pregnenolone and data from the literature suggest there is interdependence of steroid metabolic systems with those operational in control of interrelations in the GH-IGF-1 axis. Longitudinal concurrent measurements of serum levels of BT, DHEAS, and IGF-1/GH together with detailed studies of their correlations with age-correlated functional measures may be useful in detecting early age-related dysregulations and may be helpful in devising ameliorative approaches.

Human serum albumin (HSA) in fresh frozen and freeze-dried serum reference materials was examined by mass spectrometry and a variety of affinity chromatography techniques. The relative molecular mass distribution of HSA in fresh frozen serum was found to be identical to that of an HSA standard. However, the HSA in the freeze-dried reference serum exhibited a relative molecular mass distribution that was shifted to higher mass, broader, and substantially more heterogeneous than that of HSA in fresh frozen serum. A proteolytic cyanogen bromide digestion of the HSA from freeze-dried serum contained adducts [approximately]162 u higher in mass than digest fragments 124-298 and 447-548, suggesting glycation. The presence of glycation on fragments 124-298 and 447-548 correlates with the known sites of HSA glycation. Glycation was further confirmed by the mass spectral analysis of the retained and unretained fractions from glycoaffinity chromatography of HSA from freeze-dried serum. The relative molecular weight of the HSA in the retained fraction indicated the presence of a doubly glycated species. The chemical heterogeneity of Cys-34, the site of the only free thiol in HSA, was examined and found not to be a substantial source of molecular mass heterogeneity for HSA from either fresh frozen or freeze-dried serum.

Three novel cytotoxic compounds were isolated from deep-water Lamellomorpha strongylata. The extraction of the cytotoxic compounds such as swinholide H, calyculinamide A and B involved repeated extractions from frozen specimens by utilizing methanol followed by the bioassay-guided purification of the extract. Swinholide H was detected in the extract as a cytotoxic metabolite with a molecular formula of C80H136O20. Furthermore, Apehadex LH-20 chromatography produced a mixture of calyculins A, B and E including calcyculins A, B, E and F from the extract.

The NIST/NCI Micronutrient Measurement Quality Assurance Program has conducted 33 interlaboratory comparison exercises for fat-soluble vitamin-related compounds in human sera over the past 12 years. Periodic reanalysis of lyophilized serum samples prepared from more than 70 different sera has enabled estimation of the short- and long-term measurement characteristics. Median- and interquartile-range-based statistics adequately estimate the distribution of results from laboratories that are in analytical control from total distributions that include a significant minority of outlier data. Short-term interlaboratory reproducibility standard deviations (SDs) are predictable functions of analyte concentration, with an asymptotic limit at low analyte concentration and a linear relationship at high concentrations. Long-term trends in the interlaboratory reproducibility can be estimated by standardizing the short-term SD at the observed analyte concentration to an expected SD at a given physiologically significant analyte concentration. The 'average' laboratory's same-day analytical repeatability SD is about one-third of the estimated interlaboratory reproducibility; repeatability for longer periods between analyses is, on average, no better than the reproducibility. While a few exceptional laboratories have maintained excellent repeatability over the entire decade, long-term study measurements generated within a single laboratory are not generally more internally consistent than results from multiple laboratories. Enhanced and more consistently implemented intralaboratory quality control and quality assurance methods are required to further improve and maintain interlaboratory measurement comparability.

The serum of healthy elderly and adult men were analyzed after intravenous infusion of calcium gluconate to determine the effects of aging on the levels of blood ionized calcium and serum parathyroid (PTH) hormone. The morning fasting serum PTH levels of 74-year-old males were significantly higher than 39-year-old males. Elderly males with glomerular filtration rates that are greater than 70 milliliters per minute also exhibited sustained decrease in blood ionized calcium levels.

Serum electrolyte abnormalities due to vomiting in patients of bulimia nervosa, may serve as markers of vomiting behavior in young adult women. Abnormally low levels of potassium are found in patients with bulimia nervosa and detection of such low potassium levels in a healthy women may imply that they are bulimic. Detection of potassium levels below the normal, can help in the diagnosis and treatment of bulimic patients.

The nutrient density estimates, derived from a combination of food frequency and food records, is a useful predictor of the serum concentrations of vitamins A and E, and carotenoids in the dietary intake of free-living adults. It is a better predictor than the dietary estimates from food frequency and food records alone. Nutrient density estimates correlate with the serum concentrations more than the nutrient intake estimates. The time or season fails to affect the serum nutrient concentrations significantly.

Quantification of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated dibenzo-p-dioxins (PCDDs) usually requires preconcentration and cleanup prior to analysis. These procedures often involve using large amounts of toxic organic solvents. The sample preparation from serum is even more complex because of the coextraction of lipids and other nonpolar serum components. We describe the unprecedented use of cloud-point extraction to preconcentrate, extract, and clean up PAHs and PCDDs from human serum using the nonionic surfactant Triton X-100. The samples were analyzed by high-performance liquid chromatography with ultraviolet detection. The phase separation was induced by the addition of salt to the micellar serum solutions. The surfactant-rich phase was treated with acetonitrile and water to precipitate and remove some of the unwanted substances in the serum sample extract without significantly affecting the recoveries of the analytes. The favorable characteristics of cloud-point extraction discussed here strengthen its potential use as an alternative to other techniques of separation.

A direct stereoselective reversed-phase HPLC method is described for the determination of the enantiomers of a proton pump inhibitor, pantoprazole (PAN), in human serum. The enantiomers were separated with high resolution on a cellulose-based chiral stationary phase (Chiral-cel OJ-R) following on-line solid phase sample cleanup with a column-switching device. A mixture of acetonitrile and 50 mM sodium perchlorate was used as the mobile phase at a flow rate of 0.5 mL/min. Pantoprazole enantiomers were detected by monitoring the column effluent with UV light at a wavelength of 290 nm. The calibration curve for each enantiomer was linear from 0.1 to 5.0 [[micro]gram]/mL. Under these conditions, the determination of pantoprazole enantiomers in human serum can be achieved with satisfactory selectivity, sensitivity, precision, and accuracy. The described procedure is very simple and rapid since labor-intensive sample preparation is not required. The method was applied to the analyses of the serum samples obtained from a volunteer who received an 80-mg oral dose of racemic PAN. The samples showed the (+)/(-) isomer ratios ranging from 0.74 to 1.03 up to 6 h after dosing, indicating that there is only a small difference in the concentrations of (+)- and (-)-PAN.

A new silicone rubber matrix carbonate-selective membrane and a high-pH buffer diluent are used to enhance the performance of the electrode measurements for serum carbon dioxide. The proposed membrane employs one-component silicone rubber as the matrix and trifluoro-acetyl-p-decylbenzene as the neutral ionophore. The optimized membrane formulation incorporates as high as 21.9 wt% plasticizer (e.g., bis(2-ethylhexyl) adipate). The highly plasticized silicone rubber membranes not only function equivalently, in terms of the carbonate response, to the conventional PVC matrix membranes, but they also exhibit substantially reduced interfering response toward salicylate. Furthermore, the silicone rubber membrane exhibits better adhesion to the solid surface than do PVC or PU membranes. The use of higher pH buffers (e.g., 2-amino-2-methyl-1-propanol (AMP)-[H.sub.2]S[O.sub.4], pH 9.510.5) further enhances the selectivity of the carbonate electrode measurement system for total C[O.sub.2] species over other anions. It is shown that the combined use of the silicone rubber matrix membrane and the high-pH AMP buffer provides a carbonate sensor system that is substantially less subject to interference from salicylate and chloride than is the conventional measurement system employing the PVC-based electrode with the lower pH (8.4-8.8) buffer diluent.