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PubMed1992

Leukotriene uptake by hepatocytes and hepatoma cells.

The uptake of tritiated cysteinyl leukotrienes (LTC4, LTD4, LTE4) and LTB4 was investigated in freshly isolated rat hepatocytes and different hepatoma cell lines under initial-rate conditions. Leukotriene uptake by hepatocytes was independent of an Na+ gradient and a K+ diffusion potential across the hepatocyte membranes as established in experiments with isolated hepatocytes and plasma membrane vesicles. Kinetic experiments with isolated hepatocytes indicated a low-Km system and a non-saturable system for the uptake of cysteinyl leukotrienes as well as LTB4 under the conditions used. AS-30D hepatoma cells and human Hep G2 hepatoma cells were deficient in the uptake of cysteinyl leukotrienes, but showed significant accumulation of LTB4. Moreover, only LTB4 was metabolized in Hep G2 hepatoma cells. Competition studies on the uptake of LTE4 and LTB4 (10 nM each) indicated inhibition by the organic anions bromosulfophthalein, S-decyl glutathione, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate, probenecid, docosanedioate, and hexadecanedioate (100 microM each), but not by taurocholate, the amphiphilic cations verapamil and N-propyl ajmaline, and the neutral glycoside ouabain. Cholate and the glycoside digitoxin were inhibitors of LTB4 uptake only. Bromosulfophthalein, the strongest inhibitor of leukotriene uptake by hepatocytes, did not inhibit LTB4 uptake by Hep G2 hepatoma cells under the same experimental conditions. Leukotriene-binding proteins were analyzed by comparative photoaffinity labeling of human hepatocytes and Hep G2 hepatoma cells using [3H]LTE4 and [3H]LTB4 as the photolabile ligands. Predominant leukotriene-binding proteins with apparent molecular masses in the ranges of 48-58 kDa and 38-40 kDa were labeled by both leukotrienes in the particulate and in the cytosolic fraction of hepatocytes, respectively. In contrast, no labeling was obtained with [3H]LTE4 in Hep G2 cells. With [3H]LTB4 a protein with a molecular mass of about 48 kDa was predominantly labeled in the particulate fraction of the hepatoma cells, whereas in the cytosolic fraction a labeled protein in the range of 40 kDa was detected. Our results provide evidence for the existence of distinct uptake systems for cysteinyl leukotrienes and LTB4 at the sinusoidal membrane of hepatocytes; however, some of the inhibitors tested interfere with both transport systems. Only LTB4, but not cysteinyl leukotrienes, is taken up and metabolized by the transformed hepatoma cells.

Affinity Labels↗
PubMed1978

Sodium-dependent cardiac glycoside binding: experimental evidence and hypothesis.

1 The influence of increasing Na+ concentrations on the binding of digitoxin, digoxin and ouabain was examined in a Na+-K+-ATPase preparation of guinea-pig hearts. 2 Two distinct processes seem to be involved in this interaction: one binding process was activated at low Na+ concentrations. The maximum binding capacities were different and the K0.5 values were nearly identical for the cardiac glycosides studied. 3 In contrast, the second binding process was activated at appreciably higher Na+ concentrations, the maximum binding capacities were almost identical and the K0.5 values were different for the cardiac glycosides studied. 4 On the basis of these results attempts are made to explain the well known differences in the myocardial accumulation of cardiac glycosides.

Adenosine Triphosphatases↗
PubMed1988

High sensitivity of the Na+, K+-pump of human red blood cells to genins of cardiac glycosides.

1. Four different cardiac glycosides (ouabain, digitoxin, digoxin and gitoxin) and their corresponding genins were tested on Na+, K+-pump fluxes measured under steady-state and initial rate conditions (non equilibrium conditions) in human and rat erythrocytes and in mouse macrophages. 2. In human red cells, Na+, K+-pump fluxes exhibited up to 8 fold higher sensitivity to genins than to glycosides. In addition genins, but not the corresponding glycosides, exhibited double reactivity with regard to the erythrocyte Na+, K+-pump (with the exception of gitoxigenin). A weak reactivity component was similar to the one of the corresponding glycosides (IC50 of about 10(-6) M) and a high reactivity component exhibited IC50 values varying from 0.1 to 0.5 X 10(-6) M for digitoxigenin and ouabagenin respectively. 3. In contrast with human red cells, the initial rate of Na+, K+-pump fluxes in rat erythrocytes and mouse macrophages was less sensitive to genins than to the corresponding cardiac glycosides. 4. Dihydroouabain was 3, 10 and 75 times less active than ouabain in inhibiting the initial rate of Na+, K+-pump fluxes in human and rat erythrocytes and in mouse macrophages respectively. 5. In conclusion, Na+, K+-pump fluxes measured under initial rate conditions in human erythrocytes exhibit an unusually high sensitivity to genins of cardiac glycosides. This property probably results from the fast binding rate constants of genins and the slow association rates of glycosides to human red cells.

Animals↗
PubMed1993

Digoxin activates sarcoplasmic reticulum Ca(2+)-release channels: a possible role in cardiac inotropy.

1. The effect of digoxin on rapid 45Ca2+ efflux from cardiac and skeletal sarcoplasmic reticulum (SR) vesicles was investigated. Additionally the interaction of digoxin with single cardiac and skeletal muscle SR Ca(2+)-release channels incorporated into planar phospholipid bilayers and held under voltage clamp was determined. 2. Digoxin (1 nM) increased the initial rate and amount of Ca(2+)-induced release of 45Ca2+ from cardiac SR vesicles, passively loaded with 45CaCl2, at an extravesicular [Ca2+] of 0.1 microM. The efflux in the presence and absence of digoxin was inhibited at pM extravesicular Ca2+ and blocked by 5 mM Mg2+. 3. To elucidate the mechanism of action of digoxin, single-channel recording was used. Digoxin (1-20 nM) increased single-channel open probability (Po) when added to the cytosolic but not the luminal face of the cardiac channel in the presence of sub-maximally activating Ca2+ (0.1 microM-10 microM) with an EC50 of 0.91 nM at 10 microM Ca2+. The mechanisms underlying the action of digoxin appear to be concentration-dependent. The activation observed at 1 nM digoxin appears to be consistent with the sensitization of the channel to the effects of Ca2+. At higher concentrations the drug appears to interact synergistically with Ca2+ to produce values of Po considerably greater than those seen with Ca2+ as the sole activating ligand. 4. Digoxin had no effect on single-channel conductance or the Ca2+/Tris permeability ratio. In channels activated by digoxin the Po was decreased by Mg2+. Single-channels were characteristically modified to along lasting open, but reduced, conductance state when 100 nM ryanodine was added to the cytosolic side of the channel.5. Activation of the cardiac SR Ca2+-release channel was observed with similar concentrations of digitoxin, however, higher concentrations of ouabain were required to increase PO. In contrast, a steroid which is not positively inotropic, chlormadinone acetate, had no effect on either cardiac or skeletal SR Ca2+-release channel activity.6. At concentrations up to 1 microM, digoxin had no effect on Ca2+-induced 45Ca2+ efflux from skeletal muscle SR vesicles nor did it affect skeletal SR Ca2+-release channel Po, reflecting a difference between the cardiac and skeletal isoforms of the Ca2+-release channel.7. Since activation of the cardiac SR Ca2+-release channel occurs within the range of concentrations of digoxin encountered therapeutically, it is possible that activation of this channel contributes to the positive inotropic effect observed with this drug. Further, activation of the channel by higher concentrations of digoxin may contribute to the toxic effects seen clinically.

Animals↗
PubMed2003

Nongastric H,K-ATPase: structure and functional properties.

Nongastric H,K-ATPases whose catalytic subunits (AL1) encoded by human ATP1AL1 and homologous animal genes comprise the third distinct group within the X,K-ATPase family. No unique nongastric beta has been identified. Precise in situ colocalization and strong association of AL1 with beta1 of Na,K-ATPase was detected in apical membranes of rodent prostate epithelium. In this tissue, beta1NK serves as an authentic subunit of both the Na,K- and nongastric H,K-pumps. Upon expression in Xenopus oocytes the human AL1 can assemble with beta1NK, and more efficiently with gastric betaHK, into functional H,K-pumps. Both AL1/beta complexes exhibit a similar K-affinity, and their K-transport depends on intra- and extracellular Na. These data provide new evidence that nongastric H,K-ATPase can perform Na/K-exchange, and indicate that beta does not significantly affect this ion-pump function. Analysis of human nongastric H,K-ATPase expressed in Sf-21 insect cells revealed that AL1/betaHK exhibits substantial enzymatic activities in K-free medium and K stimulates, but Na has inhibitory effect on ATP hydrolysis. Thus, although the nongastric H,K-ATPase can function as Na/K exchanger, its reaction mechanism is different from that of the Na,K-ATPase. Human nongastric H,K-ATPase is highly sensitive to bufalin, digoxin, and digitoxin, but almost resistant to digoxigenin and ouabagenin.

Animals↗
PubMed2004

Digitalis-induced signaling by Na+/K+-ATPase in human breast cancer cells.

Because beneficial effects of digitalis treatment in breast cancer patients have been suggested by epidemiological studies, we explored the mechanism of the growth inhibitory effects of these drugs on the estrogen receptor-negative human breast cancer cell line MDA-MB-435 s. Ouabain concentrations (100 nM or lower) that caused less than 25% inhibition of the pumping function of Na+/K+-ATPase had no effect on cell viability but inhibited proliferation. At the same concentrations, ouabain 1) activated Src kinase and stimulated the interaction of Src and Na+/K+-ATPase with epidermal growth factor receptor (EGFR); 2) caused a transient and then a sustained activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2); 3) increased the expression of p21Cip1 but decreased that of p53; and 4) activated c-Jun NH2-terminal kinase (JNK) but not p38 kinase. These data, in conjunction with our previous findings on the signaling role of Na+/K+-ATPase in other cells, suggest that ouabain-induced activation/transactivation of Src/EGFR by Na+/K+-ATPase leads to activation of ERK1/2, the resulting increase in the level of cell cycle inhibitor p21Cip1, and growth arrest. Cooperation of JNK with ERK1/2 in this process is also suggested. Digoxin and digitoxin concentrations close to or at the therapeutic plasma levels had effects on proliferation and ERK1/2 similar to those of ouabain, supporting the proposed potential value of digitalis drugs for the treatment of breast cancer.

Breast Neoplasms↗
PubMed1977

Antagonism of biogenic amine-induced depression of cerebral cortical neurones by Na+, K+-ATPase in inhibitors.

The effects of iontophoretically applied Na+-, K+-dependent adenosinetriphosphatase (Na+,K+-ATPase) (EC 3.6.1.3) inhibitors (ouabain, digitoxin, digitoxigenin, strophanthin K, strophanthidin, thevetin A and B, ethacrynate, and harmaline) on the depression of rat cerebral cortical neurones by noradrenaline, 5-hydroxytryptamine, and histamine have been studied. The inhibitors antagonized depressions of spontaneously active neurones evoked by these amines, but not those evoked by gamma-aminobutyric acid, adenosine, adenosine 5'-monophosphate, or calcium. The antagonistic potencies of the various inhibitors appeared to be proportional to their known potencies as inhibitors of Na+, K+-ATPase. The data therefore support the hypothesis that amines depress central neurones by activating an electrogenic sodium pump.

Adenosine Triphosphatases↗
PubMed1987

Mechanisms of stimulated 45Ca efflux in skinned skeletal muscle fibers.

Excitation-contraction (E-C) coupling in skeletal muscle can be studied in skinned fibers by direct assay of 45Ca efflux and simultaneous isometric force, under controlled conditions. Recent work provides evidence that such studies can address major current questions about the mechanisms of signal transmission between transverse tubules and sarcoplasmic reticulum and sarcoplasmic reticulum calcium release, as well as operation of the sarcoplasmic reticulum active Ca transport system in situ. Stimulation by imposed ion gradients at constant [K+][Cl-] product results in 45Ca release with two components: a large Ca2+-dependent efflux, responsible for contractile activation, and a small Ca2+-insensitive efflux. The Ca2+-insensitive stimulation is sustained, consistent with sustained depolarization, and appears to gradate the Ca2+-dependent stimulation; this component is likely to reflect intermediate steps in E-C coupling. Several lines of evidence suggest that the depolarizing stimulus acts on the transverse tubules. It is inhibited by the impermeant glycoside ouabain applied before skinning, which should specifically inhibit polarization of subsequently sealed transverse tubules. Sealed polarized transverse tubules also are the only plausible target for stimulation of 45Ca release by monensin and gramicidin D, which can rapidly dissipate Na+ and K+ gradients; a protonophore and the K+-specific ionophore valinomycin are ineffective. Ionophore stimulation is prevented by the permeant glycoside digitoxin; it is also highly Ca2+ dependent. Stimulation of 45Ca release by imposed ion gradients is potentiated by perchlorate, which potentiates charge movements and activation in intact fibers, and is inhibited selectively in highly stretched fibers, presumably by transverse tubule-sarcoplasmic reticulum uncoupling. These results relate the Ca2+-dependent sarcoplasmic reticulum efflux channel to the physiological transverse tubule-sarcoplasmic reticulum coupling pathway, which also could involve Ca2+.

Adenosine Triphosphate↗
PubMed1989

Uptake of bumetanide into isolated rat hepatocytes and primary liver cell cultures.

Uptake of bumetanide into rat liver cells was investigated using isolated hepatocytes and primary cell cultures. The kinetics of [3H]-bumetanide uptake revealed two saturable components in addition to an unsaturable component. Saturable bumetanide uptake consists of a high-affinity, sodium-dependent uptake and a low-affinity transport system. Bumetanide uptake into isolated rat hepatocytes is energy dependent and temperature sensitive. At low temperatures, bumetanide uptake is due to diffusion with a permeability coefficient of 1.16 x 10(-6) cm/s. In primary liver cell cultures, uptake of bumetanide decreases rapidly over 3 days. AS-30D ascites hepatoma cells do not take up bumetanide but bind small amounts of the loop diuretic. Hepatocytes metabolized bumetanide extensively. The metabolites were secreted into the surrounding incubation buffer. Two hydroxylated and at least one conjugated biotransformation product could be separated by thin-layer chromatography. Isolated rat hepatocytes possess carrier proteins for uptake of bumetanide and very likely also for uptake of other loop diuretics like furosemide, piretanide, and torasemide. Several inhibitors of multispecific transport systems in the kidney and liver were tested as potential inhibitors of hepatocellular bumetanide or furosemide uptake. Probenecid, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, iodipamide, digitoxin, bile acids, and bromosulfophthalein inhibited uptake of loop diuretics. Inhibition by taurocholic acid was competitive with a Ki of 24 microM. Taurocholic acid inhibited [3H]bumetanide uptake in the presence but not in the absence of Na+. Deoxycholic acid and bromosulfophthalein were noncompetitive inhibitors of hepatocellular bumetanide uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1987

Ammonium ion substitutes for K+ in ATP-dependent Na+ transport by basolateral membrane vesicles.

Ion-transporting cells from posterior gills of blue crabs (Callinectes sapidus) acclimated to low salinity were used as starting material for the preparation of microsomal membrane vesicles by density gradient centrifugation. The Na+-K+-adenosinetriphosphatase (ATPase)-enriched basolateral vesicles were loaded with KCl- or NH+4-containing medium by dilution and centrifugation, and initial rates of 22Na+ uptake into the vesicles were measured by a rapid filtration procedure. Varying the extravesicular sucrose concentration altered equilibrium uptake of 22Na+, indicating the existence of osmotically sensitive vesicles. Monensin, a sodium-specific ionophore, enhanced passive uptake of 22Na+ across the vesicle membrane in the absence of ATP. With 100 mM KCl in the intravesicular medium, addition of ATP to the extravesicular medium increased initial rates of 22Na+ uptake 10- to 20-fold over levels measured without ATP. A nonhydrolyzable ATP analog failed to stimulate 22Na+ uptake. Intravesicular K+ could be replaced by NH+4 but not by choline. With NH+4 as counterion, Na+ transport was inhibited by digitoxin, but valinomycin had no effect. A study of the kinetic effects of intravesicular K+ and NH+4 on initial rates of 22Na+ uptake indicated the existence of two classes of binding sites, one responding to counterion concentrations in the millimolar range and a second class responding to counterion concentrations over 50 mM. Our results indicate that ATP-dependent 22Na+ uptake by membrane vesicles from Callinectes sapidus gill, mediated by Na+-K+-ATPase, can utilize either K+ or NH+4 as counterion.

Adenosine Triphosphate↗
PubMed2006

The cardenolide UNBS1450 is able to deactivate nuclear factor kappaB-mediated cytoprotective effects in human non-small cell lung cancer cells.

Non-small cell lung cancers (NSCLC) are associated with very dismal prognoses, and adjuvant chemotherapy, including irinotecan, taxanes, platin, and Vinca alkaloid derivatives, offers patients only slight clinical benefits. Part of the chemoresistance of NSCLCs results from the constitutive or anticancer drug-induced activation of the nuclear factor-kappaB (NF-kappaB) signaling pathways. The present study shows that human A549 NSCLC cells display highly activated cytoprotective NF-kappaB signaling pathways. UNBS1450, which is a cardenolide belonging to the same class of chemicals as ouabain and digitoxin, affected the expression and activation status of different constituents of the NF-kappaB pathways in these A549 tumor cells. The modifications brought about by UNBS1450 led to a decrease in both the DNA-binding capacity of the p65 subunit and the NF-kappaB transcriptional activity. Using the 3-(4,5-dimethylthiazol-2yl)-dephenyltetrazolium bromide colorimetric assay, we observed in vitro that UNBS1450 was as potent as taxol and SN38 (the active metabolite of irinotecan) in reducing the overall growth levels of the human A549 NSCLC cell line, and was more efficient than platin derivatives, including cisplatin, carboplatin, and oxaliplatin. The chronic in vivo i.p. and p.o. UNBS1450 treatments of human A549 orthotopic xenografts metastasizing to the brains and the livers of immunodeficient mice had a number of significant therapeutic effects on this very aggressive model.

Animals↗
PubMed1979

Reversal of digitalis effects by specific antibodies.

Highly digoxin-specific or ouabain-specific antibodies can readily be obtained by immunizing rabbits or sheep with repeated injections of the glycoside coupled to protein carriers. By virtue of their binding capacity digoxin-specific antibodies are capable of removing digoxin concentrations from red blood cells and renal tissue specimens. As evidenced by various experiments with human erythrocytes and isolated cardiac preparations in vitro, digoxin effects are rapidly reversible in the presence of digoxin-specific antibodies. In vivo antidigoxin antibodies can protect animals from digoxin effects and promptly abolish established toxic effects, associated with marked alterations of digoxin pharmacokinetics. However, due to the large molecular weight, complete antibodies cannot be eliminated via the renal route. The use of antigen-binding (Fab) fragments of digoxin-specific antibodies offer the advantage of rapid renal elimination of bound and inactivated digoxin. So far, due to potential immune reactions, the clinical use of purified digoxin-specific antibodies of Fab fragments is restricted to life-threatening accidental or suicidal digoxin or digitoxin poisoning.

Animals↗
PubMed1976

Neural basis for the genesis and control of digitalis arrhythmias.

The pharmacokinetics of ouabain associated with toxicity were studied in the cat and the guinea pig both in vivo and in vitro using ouabain-H3. After spinal cord transection a higher dose of ouabain was required to reach the lethal endpoint. This intervention also increased the myocardial and serum levels associated with toxicity were studied in the cat and the guinea pig both in vivo and in vitro using ouabain-H3. After spinal cord transection a higher dose of ouabain was required to reach the lethal endpoint. This intervention also increased the myocardial and serum levels associated with death. These findings were corroborated in experiments using digitoxin H3. In vitro, substantially higher ouabain tissue contents were associated with a lethal event. In addition, in cats and guinea pigs, the lethal myocardial ouabain content did not change when the infusion rate of ouabain was varied in vivo or the perfusate ouabain concentration was changed in vitro. In vivo, propranolol increases the myocardial ouabain content associated with death to in vitro levels. In vitro, the drugs prolongs the time to death by retarding the myocardial uptake of ouabain. These data suggest that the toxic effects of ouabain in the whole animal are largely neural and in the isolated heart, substantially myocardial.

Animals↗
PubMed1972

Ouabain-specific antibodies: immunochemical properties and reversal of Na + , K + -activated adenosine triphosphatase inhibition.

Antibodies with high affinity and specificity for the cardiac glycoside ouabain were raised in rabbits. The antigen used was a conjugate of ouabain linked through its rhamnose moiety to terminal alpha-amino groups of poly D,L alanyl-human serum albumin. Ouabain-specific antibodies were present as early as 3 wk, and rose steadily in titer over the initial 20-33 wk of immunization. Levels as high as 6.5 mg specific immunoglobulin per ml antiserum were reached in one rabbit at the end of 45 wk. The average intrinsic association constants for ouabain were 1.3 x 10(9) M(-1) and 1.6 x 10(9) M(-1) in antisera studied in detail, and there was evidence of restricted heterogeneity of binding site affinities. A high degree of specificity was demonstrated. Significant cross-reactivity occurred only with other cardioactive steroid compounds such as acetyl strophanthidin, digoxin, and digitoxin, while endogenous steroids did not cross-react even when present in 1000-fold excess. A rapid and convenient radioimmunoassay procedure for plasma or urine ouabain concentrations was developed using these antibodies. Competition between ouabain-(3)H tracer and unlabeled ouabain for specific antibody binding sites allowed the measurement of ouabain concentrations as low as 0.1 ng/ml or less without need for extraction procedures. The high association constants observed in these studies permit antibody reversal of established myocardial effects of ouabain. Both blockade and reversal of ouabain inhibition of canine myocardial microsomal Na(+), K(+)-activated ATPase by antibody were documented, suggesting a possible mechanism for reversal of cellular effects.

Adenosine Triphosphatases↗
PubMed1984

Anaphylactoid reaction caused by intravenous doxycycline during general anesthesia and beta-blockade treatment.

A 71-year-old woman with icterus was treated with doxycycline orally for one week. She then was admitted for an exploratory laparotomy under general anesthesia with barbiturate, pancuronium, fentanyl, and nitrous oxide. She developed rapid atrial fibrillation and received digitoxin and beta-blockade. Thirty minutes after her last medication she received doxycycline 100 mg in 200 ml sodium chloride 0.9% infused over five to ten minutes. Soon after the completed infusion, she developed a severe anaphylactoid reaction with bronchospasm, hypotension, and generalized urticaria, which was treated successfully with ephedrine, aminophylline, hydrocortisone, furosemide, metaraminol, ketamine, and epinephrine. The possibility of beta-blockade treatment worsening the reaction is discussed and five other reports of anaphylactoid reactions to intravenous doxycycline are mentioned.

Adrenergic beta-Antagonists↗
PubMed1979

Cimetidine, a histamine H2 receptor antagonist, occupies androgen receptors.

The histamine H2 receptor antagonist cimetidine is in increasing usage in the medical management of peptic ulcer. In clinical trials, its most frequent side effect is gynecomastia. Such estrogenic/antiandrogenic manifestations are well known side effects of treatment with digitoxin or spirolactones. Both of these drugs share a common skeleton with the steroid hormones and have been shown to occupy estrogen and/or androgen receptors. Cimetidine has no measurable affinity for rat uterine estradiol receptors, but competes for tritiated dihydrotestosterone-binding sites in mouse kidney preparations with a displacement curve parallel to that for unlabeled dihydrotestosterone. Steroid receptor-mediated side effects, therefore, may not be confined to molecules with a common skeleton, such as steroids, spirolactones, and cardiac glycosides, but may extend to such apparently unrelated molecules as histamine antagonists and androgens.

Animals↗
PubMed2001

Stereoselectivity and species difference in plasma protein binding of KE-298 and its metabolites.

In vitro protein binding of KE-298 and its plasma metabolites, deacetyl-KE-298 (M-1) and S-methyl-KE-298 (M-2), was high in rat (>97%), dog (>89%) and human plasma (>99%), respectively. Human serum albumin (>93%) was the main protein involved in the binding to plasma proteins, while the binding to human serum globulins was low (16-33%). The binding of KE-298 and its metabolites in all species of plasma was stereoselective. The (+)-(S)-enantiomers of these compounds bound rat, dog and human plasma proteins to a greater extent than did the (-)-(R)-enantiomers, except that the case of KE-298 was opposite in rat plasma. The stereoselective plasma levels of these compounds in rats, dogs, or humans would likely be due to stereoselective differences in binding to plasma albumin. The protein binding of M-1 in adjuvant-induced arthritis rat plasma was >97%, and the stereoselectivity was similar to the case of normal rat plasma. KE-298 and its metabolites remarkably displaced [14C]warfarin, which bound on albumin in a solution of diluted rat serum albumin. Similarly, there was a displacement of [14C]warfarin in solutions of dog and human serum albumin, and concomitantly the displacement of [14C]diazepam. [3H]Digitoxin was not displaced by any of the enantiomers in each albumin solution. No stereoselectivity was found in displacement by enantiomers of the three compounds. These results suggest that stereoselective protein binding can be attributed to quantitative differences in binding to albumin rather than to the different binding sites.

Animals↗
PubMed2002

Establishment of enzyme immunoassay for measuring beta-methyldigoxin levels in human serum by specific antiserum.

We investigated the specificity of obtained antisera to beta-methyldigoxin by the enzyme immunoassay. Three types of hapten-bovine serum albumin (BSA) conjugates were synthesized to obtain high specific antisera to beta-methyldigoxin. The haptens were linked to the carrier protein through hemisuccinate at C-3' and C-3'' positions in the digitoxose chain and at C-12 position in the aglycone. Anti-beta-methyldigoxin 3'-hemisuccinate-BSA antiserum showed a low detection limit (0.2 ng/ml) and possessed high specificity for beta-methyldigoxin, exhibiting low cross-reactions with digoxigenin bisdigitoxoside (8.3%), dihydrodigoxin (4.8%), digitoxin (1.5%), and digoxigenin monodigitoxoside (0.95%), except for cross-reaction with digoxin (43%). Compared with commercial antidigoxin antiserum, clinically used to monitor beta-methyldigoxin concentration in human serum, cross-reaction data of anti-beta-methyldigoxin 3'-hemisuccinate-BSA antiserum showed higher specificity for beta-methyldigoxin. The intra-assay and inter-assay variations using this antiserum were less than 6.9% and 8.1%, respectively. The recovery tests were good, within the range of 96.2-104.3%. Phenyl boric acid (PBA) column treatment was effective to rapidly and selectively separate beta-methyldigoxin from the mixture of beta-methyldigoxin and its metabolites in human serum. The recovery tests of beta-methyldigoxin with PBA column in human serum were about 110% and interference of metabolites of beta-methyldigoxin was negligible. These results suggest that anti-beta-methyldigoxin 3'-hemisuccinate-BSA antiserum and PBA column treatment are useful to more precisely monitor the unchanged type of beta-methyldigoxin concentration in human serum.

Animals↗