AN IMMUNOMETRIC ELISA FOR 15-F,, ISOPROSTANE, AN URINARY BIOMARKER FOR OXIDANT STRESS Dime Sasnh. Ying Yuan, Kath Gikas, Douglass Roberts, JJ, Denis Callewaert. t: xford Biomedical MJ, USA 48371
Taber, Jason Morrow, L. Jackson Research, P.O. Box 522, Oxford
Several isoprostanes found in human tissues are molecular biomarkers for oxidant stress. In human urine the major metabolite of 15-F,,-Isoprostane (8-iso-PGF,,,,,,,,,,,) is 2,3-dinor-5,6-dihydro-15-F,,-Isoprostane (FZ-IsoP-M). Thus, an ELISA assay specific for F2-IsoP-M in urine should provide a non-invasive quantitative index of oxidative status. We have (1) generated a polyclonal antibody specific for FZ-IsoP-M, using synthetic isoprostane conjugated to canine thyroglobulin and (2) developed a competitive immunometric F2-IsoP-M ELISA,using synthetic FZ-IsoP-M conjugated to bovine serum albumin (BSA). In this assay, F2-IsoP-M-BSA was immobilized on microtiter plates, to which anti-FZ-IsoP-M and either standard or unknowns were added. Anti-FZ-IsoP-M bound to the immobilized F2-IsoP-M ligand was subsequently measured using horseradish peroxidase-conjugated to a secondary antibody. The ELISA exhibited a detection limit of 0.1 ng/ml and negligible crossreactivity with prostaglandin F,, or 15-F,iIsoprostane. ELISA measurements of F2-IsoP-M in unextracted normal human urine samples (pH 6-7) ranged from lo-200 ng/ml, which is much greater than the reported range (0.3-3.0 ng/ml) obtained by GC/MS, and may reflect interference due to other compounds in urine, crossreactivity of anti-F2-IsoP-M with other isoprostane metabolites in urine or conjugated forms of isoprostanes that are removed by purification procedures required by GC/MS. [Supported in part by National Institute of Environmental Health Services contract No. N43-ES-854291
UK, AB24 2TZ
Aerobic organisms continually face exposure to free radicals and have evolved sophisticated antioxidant systems to effectively remove them. any process that increases the production of free radicals or weakens the defence system may ultimately lead to oxidative stress and tissue damage, unless compensatory changes occur. we examined whether long-term cold exposure enhanced catalase (cat) and selenium-dependant glutathione peroxidase (gpx) activities in various tissues of short-tailed field voles microtus agrestis. two groups of 7 male and 7 female voles were born at and maintained at 8&3-C or 22s~C. resting oxygen consumption (measured at 30-C). body mass, lean and fat mass were recorded at age 51M.9 day-l. cat and gpx activities were measured (61f1.9 day-l) in liver, heart, kidney, skeletal muscle (hindlimb) and duodenum. voles at &3-C or 22?3mC did not differ in body mass, lean or fat mass. resting oxygen consumption was over 30% higher in voles kept at 8fl-C, with cat and gpx activities also elevated in all tissues compared to voles at 22&-X, particularly skeletal muscle (71% and 25% higher, cat and gpx respectively), heart (40% and 43% respectively) and kidney (20% cat). protein content did not differ in any tissue between the two groups. our results show that long-term cold exposure and increased oxygen consumption leads to an enhancement of the enzymatic antioxidant defence system, especially in skeletal muscle and heart.
I 106 LIVING
EFFECT OF LONG-TERM COLD EXPOSURE ON ANTIOXIDANT ENZYME ACTIVITY IN FIELD VOLES M. AGRESTIS s;splmea J McLnm, M Himanka, W Peacock, J Speakman Dept. of Zmlogy,
FAST AND DYING
a SELMAX SAM SNART, JANE McLAREN, PAULA REDMAN, ELA KROL, DJANE JACKSON, MARJA JOHNSON t? JOHN SPEAKMAN. DEPT. OF ZOOLOGY, UNIVERSITY OF ABERDEEN, ABERDEEN, UK, AB24 2TZ
it is almost axiomatic that increases in energy metabolism lead to increased rates of mortality and thus shortened lifespan. this link is consistent with the free-radical theory of ageing and has important implications for our understanding of the physiological basis and evolution of life history trade-offs in animal ecology. no previous studies have examined the consequences for lifespan of intraspecific variations in energy expenditure of animals living in a constant environment. here we report such a study in a group of 42 female mfl mice. between 6 and 13 months of age we monitored food intake, daily energy expenditure, body mass, fat mass, lean mass, assimilation efficiency and body temperature. after 13 months of age the mice were monitored daily until they died. we sought relationships between lifespan and traits measured when the mice were 6-13 months old. contrary to expectations there were significant positive relationships between lifespan and daily energy expenditure, residual energy expenditure and metabolic intensity (energy expenditure per gram body mass). lifespan was not related to body mass, fat mass, lean mass or body temperature. in this cohort of mice, living fast was associated with dying more slowly, calling into question the physiological inevitability of the linkage previously described.
TISSUE GLUTATHIONE AND THIOREDOXIN FOLLOWING LIPOIC ACID SUPPLEMENTATION Chandnn Mustafa A&Jay, Snvita Khannn,Leena Vider, Sashwatz Roy, Osmo Haatkinen. Lawrence Berkeley National Laboratory and University of Kuopio,
Thiol antioxidants play a central role in bolstering antioxidant defenses as well as in regulating redox-sensitive signal transduction processes. alpha-Lipoic acid (LA) is a nutritional supplement and clinical drug. In vitro studies have shown that supplementation of cells in culture by LA increase cellular GSH levels by improving availability of cysteine inside the cell. In vivo data are limited. At present there is no published data documenting the possible influence of LA supplementation on thioredoxin expression. In this study we examined the effect of intragastric LA supplementation (150 mg/kg, 8 weeks) on GSH and thioredoxin levels in various rat tissues. LA supplementation increased the level of free LA in the red gastrocnemius muscle, and increased total glutathione (TGSH) levels in the liver and blood. Classically LA is known for its role as an essential cofactor in oxidative metabolism. Although there is no evidence that orally supplemented LA is incorporated into mitochondrial proteins it is frequently assumed that LA supplementation facilitates oxidative metabolism. In vertebrates, endogenous lipoate is found in five mitochondrial proteins as lipoyllysine. We observed that tightly protein-bound lipoyllysine pool in tissues is independent of the loosely-bound or free LA status in the tissue and that tissue lipoyllysine status is not influence by LA supplementation. LA supplementation increased thioredoxin expression in certain rat tissues. Consistent results related to the regulation of thioredoxin expression by LA were obtained when Jurkat T cells were treated with 0.1 mM or 0.25 mM LA. Supported by Finnish Ministry of Education and Academy Grants to CKS.