Hassan M. Qazzaz

Deglycosylated Products of Endogenous Digoxin-like Immunoreactive Factor in Mammalian Tissue

Digoxin-like immunoreactive factor (DLIF) from adrenal cortex is an endogenous molecule with structural features remarkably similar to those of digoxin, a plant-derived cardiac glycoside (Shaikh, I. M., Lau, B. W. C., Siegfried, B. A., and Valdes, R., Jr.(1991) J. Biol. Chem. 266, 13672-13678). Two characteristic structural and functional features of digoxin are a lactone ring and three digitoxose sugars attached to a steroid nucleus. Digoxin is known to undergo deglycosylation during metabolism in humans. We now demonstrate the existence of several naturally occurring deglycosylated components of DLIF in human serum. The components are identified as DLIF-genin, DLIF-mono, and DLIF-bis, corresponding to the aglycone, and the aglycone with one and two sugars, respectively. Similar components are produced by acid-induced deglycosylation of DLIF isolated from bovine adrenal cortex. The elution pattern and sequence of DLIF-deglycosylation was identical to that of digoxin suggesting identical sugar stoichiometry. However, analysis of these newly discovered congeners by reverse-phase chromatography, spectrophotometry, antibody reactivity, and kinetics of deglycosylation, demonstrates that subtle structural and physical differences do exist when compared to digoxin. DLIF was chromatographically distinct from digoxin, and interestingly, the mobility of the DLIF-genin was shifted toward increased polarity relative to digoxigenin. DLIF and DLIF-bis, -mono, and -genin congeners have absorbance maxima at 216 nm, whereas digoxin and its congeners absorb at 220 nm. Reaction with specific antibodies directed at the lactone portion of these molecules shows DLIF and its deglycosylated congeners to be 103-fold less reactive than digoxin. Kinetics of sugar removal suggests that DLIF is 8-fold more susceptible to deglycosylation than is digoxin. Two less polar DLIF components produced from the DLIF-genin have at 196 nm and are 4-fold less immunoreactive than DLIF. Our data suggest that subtle structural differences exist between DLIF and digoxin at or near the lactone ring as well as in the nature of the sugars. The presence of deglycosylated congeners of DLIF in human serum, including the less polar components, suggests in vivo deglycosylation of these factors. This is the first demonstration of the existence of naturally occurring deglycosylated derivatives of DLIF and establishes the likelihood of active metabolism of DLIF in mammals.

Treatment of human serum with sulfosalicylic acid structurally alters digoxin and endogenous digoxin-like immunoreactive factor.

Pretreatment of human serum with 5-sulfosalicylic acid (SSA) as used in the Abbott TDx digoxin assay produces deglycosylated congeners of digoxin (DIG) and of endogenous digoxin-like immunoreactive factor (DLIF). Using high-performance liquid chromatography analysis, we observed differences in the degree and pattern of DIG breakdown products among five patients. The aglycone digoxigenin was the major product in several samples. Smaller amounts of the bis- and mono-digitoxosides and unidentified products less polar than DIG were sometimes present. Treatment of DLIF-containing plasma with SSA produced similar patterns of DLIF-breakdown products. Incubation of normal plasma containing DIG with SSA for up to 30 min caused little change in measured DIG by TDx and radioimmunoassay (RIA) but decreased to 50% in the ACS DIG assay. These results are consistent with the near 100% cross-reactivities of deglycosylated DIG congeners in the TDx and RIA assays compared to their lower cross-reactivities in the ACS assay. We conclude that the breakdown of DIG and DLIF during treatment of serum with SSA may compromise the accuracy of TDx DIG assays and may explain discrepancies observed in other studies between digoxin immunoassays. This study underscores the importance of understanding the effects of pretreatment strategies used for analytes measured by immunoassay.

Two biologically active isomers of dihydroouabain isolated from a commercial preparation

Ouabain is a plant-derived cardiac glycoside that inhibits the catalytic activity of Na(+),K(+)-ATPase (sodium pump; NKA). Dihydroouabain, a derivative of ouabain with a reduced lactone ring, is commonly used as a sodium pump antagonist. It has been assumed that commercially available dihydroouabain is homogeneous. We now report that preparations of dihydroouabain contain two components each with a different potency for inhibition of sodium pump activity. We used reverse-phase HPLC chromatography, UV spectrophotometry, electrospray ionization-mass spectrometry (ESI-MS), nuclear magnetic resonance (NMR) spectroscopy and two independent bioassays to characterize these compounds. The two dihydroouabain fractions (Dho-A and Dho-B) resolved by 3 min chromatographically, had UV absorbance maxima at 196 nm, and comprised 37% and 63% of the stock dihydroouabain, respectively. The molar potency of each component for inhibition of NKA from porcine cerebral cortex differed by 4. 4-fold (Dho-A, IC(50) = 7.13 +/- 0.8 microM; Dho-B, IC(50) = 1.63 +/- 0.12 microM). The relative potencies were 9% and 40% of those of ouabain, respectively. A similar pattern for phosphorylation of NKA was observed. Mass spectrometry (ESI-MS) and fragmentation patterns are consistent with Dho-A and Dho-B being isomers of identical molecular mass (587 Da) and each with six hydroxyl groups, a deoxyhexose sugar moiety and a lactone ring. Furthermore, NMR spectroscopy revealed structural differences between Dho-A and Dho-B by displaying noticeably different chemical shifts at only two groups of proton resonances assigned to H-21 and H-22. The ESI-MS and NMR results confirm the presence of the isomerism at C20 of the lactone ring. Our results demonstrate the existence of two molecular forms of dihydroouabain, each with a different biological potency. These findings underscore the importance of characterizing the purity of dihydroouabain commercial preparations. It also provides possible molecular models for investigating the metabolism of endogenous ouabain-like factors recently reported in mammals.

P-145 Oral Administration of EGCG, a Green Tea Polyphenol, Both Suppresses and Rescues Mice from DSS-Induced Colitis

Background:The anti-inflammatory effects of green tea polyphenols (GTP) have sparked considerable interest as a potential therapeutic agent for inflammatory bowel disease (IBD). We sought to evaluate the effects of orally administered (−)epigallocatechin-3-gallate (EGCG) on the dextran sulfate sodium (DSS) colitis model to determine whether this agent works as a potential preventive and/or treatment therapy for IBD. Methods:Groups (n = 5) of C57BL/6 mice were exposed to 2.5% DSS +/− EGCG in 2 or 20 &mgr;g/mL concentrations. Control groups were exposed to 2.5% DSS only from days 1 to 9, while EGCG was given as a preventive agent concurrently with DSS from days 1 to 9 or as a treatment agent after DSS administration on days 10 to 16. Animals were weighed daily, and colonoscopies were performed on days 9 and 16 to determine the endoscopic index of severity (EIS). Animals were sacrificed on day 17, and blood and colonic tissue samples were collected. Serum soluble cytokine analyses were performed using Milliplex multiplex assay. Tight junction protein analysis of colonic tissue was performed using Western blot. Results:Control animals developed significant weight loss and colitis by day 9 (EIS = 12). Animals that were given preventive doses of EGCG developed less severe colitis by day 9 (EIS = 5 and 4 for 2 and 20 &mgr;g/mL), while animals given treatment doses developed significant colitis by day 9 (EIS = 7 and 9 for 2 and 20 &mgr;g/mL) that resolved significantly, compared to control animals, by day 16 (EIS = 0.5 and 1 for 2 and 20 &mgr;g/mL). Additionally, preventive doses of EGCG protected animals from significant weight loss, another measure of colitis severity. Serum levels of pro-inflammatory cytokines (IL-6, IFN-&ggr;, and IL-12) were significantly lower in all preventive and treated animals than control animals. Western blot analysis of tight junction protein expressions showed the following modulations by preventive and treatment doses of 20 &mgr;g/mL EGCG, respectively (with 100% equal to protein expression in control animals): occludin, 195.7% and 216.2%; claudin-1, 229.2% and 438.7%; claudin-4, 67.2% and 62.4%; and ZO-1, 163.2% and 181.8%. Conclusions:Oral administration of EGCG protected against weight loss and severe colitis when used in a preventive capacity; moreover, EGCG treatment was effective at rapidly resolving colitis. Furthermore, EGCG administration markedly attenuated pro-inflammatory cytokine response and reinforced tight junction integrity. These data provide further evidence supporting the use of EGCG for treating inflammatory bowel disease and for the anti-inflammatory mechanism that we have seen in IBD patients and models.