Biomedical subjects

A N Butt

Publications and source records attributed to A N Butt.

5 recordsLinked to original sources

PubMed2000

Binding of ouabain and human ouabainlike substance to different Na+, K+-ATPase isoforms.

There is very little on the affinity of the human immunoreactive ouabainlike substance (OLS) to individual alpha-isoforms of Na+,K+-ATPase. The present study addresses this issue by comparing ouabain and OLS binding to dog kidney alpha1, rabbit kidney alpha1 and porcine cerebral cortex alpha3 Na+,K+-ATPase. OLS was initially isolated by solid phase extraction from human serum using C18 columns. The extract was further purified by reverse phase HPLC in an acetonitrile/water (containing 0.1% TFA) step-up gradient (16-80%). In this system, two distinct ouabain immunoreactive peaks were resolved. Peak I demonstrated a polarity identical with that of authentic ouabain. In contrast, peak II was relatively non-polar and eluted later in the run. The final step in the purification of OLS involved immuno-affinity chromatography of peak I using a specific sepharose immobilized mouse monoclonal anti-ouabain antiserum. Dose response curves (range 0-100 nmol/l) for ouabain with canine alpha1 and porcine alpha3 Na+,K+-ATPase showed similar inhibitory profiles (IC50=15 nmol/l), whilst rabbit alpha1 Na+,K+-ATPase was relatively insensitive to ouabain and purified peak I OLS. Two fold serial dilution of Peak I OLS, with subsequent analysis by canine and porcine Na+,K+-ATPase inhibition assays and RIA, demonstrated strong positive correlations between OLS determined by RIA and both canine (y=0.945x-2.532, r2=0.977) and porcine (y=0.428x-1.685; r2=0.993) Na+,K+-ATPase assays. The difference in the respective slopes suggests, however, that peak I OLS has a greater affinity for the canine derived enzyme compared to the porcine. In conclusion, these data suggest that like authentic ouabain, peak I OLS is a-isoform and species selective.

Animals↗

PubMed1998

Endogenous ouabain secretion in man is not regulated by ACTH.

It has been suggested that endogenous ouabain-like substance (OLS) is of adrenal origin and the secretion of OLS may be ACTH dependent. To determine if OLS is influenced by the pituitary-adrenal axis, we studied the effect of adrenal stimulation (0.25 mg Synacthen) and suppression (1 mg Dexamethasone) on two separate groups of nine subjects. Serum OLS was measured by a radioimmunoassay (RIA) developed in our lab, and cortisol and ACTH were measured by commercial assay kits. Dexamethasone significantly (P< 0.001) suppressed serum cortisol and ACTH concentrations, without effecting endogenous OLS concentration (0.64+/-0.17 vs 0.85+/-0.18nmol/l). Synacthen increased the concentration of cortisol in serum (p < 0.001) over the test period; OLS concentration, again, remained unchanged (0.45+/-0.04 vs 0.43+/-0.05 nmol/l). In further studies, serum concentrations of cortisol and OLS were compared between left (LAV) and right (RAV) adrenal veins with that from the inferior vena cava (IVC). Concentration of cortisol in the LAV and RAV was five-fold greater than that in IVC. However, there was no difference in OLS concentration at the corresponding sites. In addition, serum OLS concentrations in patients having undergone bilateral adrenalectomy or diagnosed with Addison's disease (0.62+/-0.19 nmol/l) were similar to concentrations in healthy subjects (0.67+/-0.21 nmol/l). Examination of bovine adrenal, liver, kidney, heart and human placenta demonstrated that OLS content of bovine adrenal was comparable with other tissues analysed. HPLC studies of human serum and bovine adrenal gland produced identical elution profiles, resolving a single peak which coincided with the retention time observed for standard ouabain. We conclude that the adrenal is unlikely to be the source of endogenous OLS, the secretion of which appears to be independent of ACTH.

Addison Disease↗

PubMed1997

Effect of high salt intake on plasma and tissue concentration of endogenous ouabain-like substance in the rat.

The effect of high salt intake on serum concentration and tissue distribution of ouabain-like substance (OLS) was examined in rats. Sprague-Dawley rats (n=8) were placed on a high salt diet by the inclusion of 1.8% sodium chloride in drinking water for 7 days and a 'control' group (n=8) was maintained on normal drinking water during the study period. Serum and tissue OLS was measured by radioimmunoassay after solid phase extraction. High salt intake significantly increased serum OLS concentration (1.43 +/- 0.06 vs 1.14 +/- 0.05 nmol/L; mean +/- SEM, P=0.002). In both groups, the adrenal showed significantly (p < 0.001) higher OLS content compared to liver, kidney, heart and brain. HPLC of rat serum extract resolved a major peak with a retention time identical to that of standard ouabain, further confirming the nature of OLS. We conclude that high salt intake increases endogenous production of OLS, which appears to originate from the adrenal gland in the rat.

Animals↗

PubMed1996

Effect of salt intake on excretion of endogenous ouabain-like substance, measured by RIA.

Recent studies suggest that ouabain or a ouabain-like substance (OLS) may be present endogenously in humans. We developed a RIA for ouabain with antisera raised in goat against ouabain conjugated to keyhole limpet hemocyanin and ovalbumin. The antiserum was of high antibody titer (200,000) and was specific for ouabain, with little cross-reactivity with common steroids and structurally related compounds such as ouabagenin (4%), strophanthidin (4%), and dihydroouabain (2%). The RIA had a working range of 0.06-2.0 nmol/L, and the intra- and interassay CV was 6.5% at a concentration of 1.7 nmol/L. With this assay the effect of salt loading on urinary excretion of OLS was examined in 10 healthy volunteers (ages 18-22 years) who increased their salt intake (sodium) for 5 days and reduced it for the next 5 days. Urine was collected and OLS concentration was measured by RIA after solid-phase extraction with a Bond Elut C18 column. Excretion of OLS and sodium were maximal on day 5 and lowest on days 9 and 10. Urine excretion of OLS on day 5 (2.66 +/- 1.22 nmol/24 h) was significantly higher (P < 0.0001) than on day 10 (1.47 +/- 0.69 nmol/24 h). We conclude that (a) the assay developed has sufficient sensitivity and specificity to detect endogenous OLS present in biological fluids, and (b) salt intake increases the excretion of OLS.

Adolescent↗

PubMed1990

Cholesterol absorption by the gall bladder.

Model and real biles were used to investigate factors influencing cholesterol and dextran (70,000 molecular weight) absorption by the gall bladder. Cholesterol absorption was proportional to cholesterol concentration when real bile was used, but model biles showed maximal absorption at cholesterol saturation. Reduction of temperature reduced cholesterol absorption and serosal secretion, but had little effect on dextran absorption. This indicates differences in uptake where cholesterol undergoes passive diffusion but dextran is taken up by fluid-phase endocytosis. Model bile prepared with a single bile salt showed lowest cholesterol uptake from cholate bile, but there was no difference in serosal secretion. Dextran uptake was also lowest from cholate bile, although serosal secretion was highest. These results show that an increase in the biliary content of dihydroxy bile salts increases gall bladder permeability to both hydrophobic and hydrophilic molecules and may lead to the accumulation of lipids in the mucosa, as seen in cholesterolosis.

Absorption↗