Studies on human low serum IgD phenotype and Gm markers.

The human "low serum IgD phenotype" was studied by simultaneous Gm typing and IgD immunoassay of several populations. An association between Gm (f+b+) haplotype and low human IgD was confirmed and extended to the "low serum IgD phenotype"--as defined from population distribution and genetic studies by Dunnette et al. 1978. Further, it was shown that Black American sera determined by Gm haplotype, had a similar percentage of "low serum IgD phenotype" samples (16%) although they lacked the "associated" Gm(f+b+) haplotype of White American samples. Sardinian sera showed a low incidence of the "low serum IgD phenotype" which was not correlated with Gm haplotype distribution. Familial aggregation of the "low serum IgD phenotype" was observed. No association was found between "low serum IgD phenotype" and serum IgE values. Age related abiotrophy of IgD could not be attributed to selective survival of "low serum IgD phenotype" persons.

Immunochemical studies of antisera to human fibrinopeptide-B.

The immunochemical specificity of rabbit antisera to human fibrinopeptide-B (FPB) has been studied by comparing the relative abilities of FPB and of various proteins and peptides containing the NH2-terminal segment of the B beta-chain of human fibrinogen to inhibit the binding of a radioiodinated FPB derivative by each of seven anti-FPB sera. Anti-FBP sera varied in the extent to which they cross-reacted with fibrinogen, the NH2-terminal disulfide knot of fibrinogen (N-DSK), B beta 1(Pyr)-118(Met), B beta 1(Pyr)-42(Arg), and desarginyl-FPB. Anti-FPB sera have been identified that discriminate effectively between FPB and larger FBP-containing peptides; such antisera can be used to measure FPB in the absence of the larger peptides or to demonstrate the presence of larger peptides such as B beta 1(Pyr)-42(Arg) in extracts of clinical plasma samples by means of an increase in FPB immunoreactivity following thrombin treatment. One anti-FPB serum has been identified that is capable of detecting desarginyl-FPB, and this antiserum has been used in the development of a radioimmunoassay for desarginyl-FPB. Thus, by precisely defining the specificity of anti-FPB sera, it has been possible to identify antisera that are useful, not only in the measurement of FPB, but also in the detection of other important related molecules, such as B beta 1(Pyr)-42(Arg) and desarginyl-FPB. The immunochemical detection of these FPB-related peptides should provide useful information concerning the action of proteolytic enzymes, such as plasmin on the NH2-terminal segment of the B beta-chain of fibrinogen, and of carboxypeptidase-B on free FPB, in human plasma.

The development and application of a radioimmunoassay for dihydrodigoxin, a digoxin metabolite.

The cardioinactive digoxin metabolite, dihydrodigoxin, has been conjugated to bovine serum albumin and to bovine pancreatic ribonuclease by the periodate oxidation method. Rabbits immunized with the dihydrodigoxin-bovine serum albumin conjugate formed antibodies which bound a radioiodinated dihydrodigoxin-ribonuclease conjugate. This binding was inhibited by dihydrodigoxin. After affinity chromatography on a digoxin-ribonuclease-Sephacryl immunoadsorbent to remove antibodies which cross-reacted with digoxin, dihydrodigoxin was 300 times more effective than digoxin in inhibiting the binding of tracer by antibody. Digoxin-absorbed antidihydrodigoxin antibodies were coupled to Sephacryl and were used to develop a solid-phase radioimmunoassay capable of detecting 250 to 500 pg of dihydrodigoxin in 1 ml of human serum or urine. This radioimmunoassay has been used to define the pharmacokinetics of the metabolite in four normal human volunteers who ingested 125 to 500 micrograms of dihydrodigoxin by mouth. Dihydrodigoxin was quickly absorbed, with maximal serum concentrations achieved within 45 to 105 min, followed by a rapid fall in serum immunoreactivity over 2 to 4 hr and then by a slower, more gradual decline. The terminal half-life (beta) in serum varied from 4.24 to 11.9 hr (mean +/- S.E. = 8.1 +/- 1.3 hr). Most of the administered dose was excreted in the urine, with cumulative urinary recovery varying inversely with the dose. Urinary half-lives averaged 13.8 +/- 2.1 hr, and renal clearance rates were similar to those of creatinine. Dihydrodigoxin is rapidly absorbed and excreted in man and appears to be eliminated from the body at a faster rate than digoxin.

Inactivation of digoxin by the gut flora: reversal by antibiotic therapy.

In approximately 10 per cent of patients given digoxin, substantial conversion of the drug to cardioinactive, reduced metabolites (digoxin reduction products, or DRPs) occurs. The site and clinical importance of this conversion is unknown. In four normal volunteers taking digoxin daily for four weeks, urinary excretion of DRPs was greatest after a poorly absorbed tablet was ingested, and least after intravenous administration, Stool cultures from subjects known to make DRPs in vivo ("excretors") converted digoxin to DRPs; cultures from nonexcretors did not. Three excretors were given tablets for 22 to 29 days. A five-day course of erythromycin or tetracycline, administered after a base-line period of 10 to 17 days, markedly reduced or eliminated DRP excretion in urine and stool. Serum digoxin concentrations rose as much as twofold after antibiotics were given. We conclude that in some persons digoxin is inactivated by gastrointestinal bacteria. Changes in the enteric flora may markedly alter the state of digitalization.

Effect of quinidine on the digoxin receptor in vitro.

To investigate the basis for a clinically important digitalis-quinidine interaction that is characterized by increases in serums digoxin concentrations when quinidine is administered to digoxin-treated patients, we have studied in vitro the interaction of quinidine with the digoxin receptor. Evidence has been obtained that quinidine is capable of decreasing the affinity for digoxin of cardiac glycoside receptor sites on purified Na,K-ATPase and on intact human erythrocyte membranes. As others have shown, quinidine is capable of inhibiting Na,K-ATPase activity, and evidence has been obtained in the current study that, while quinidine can reduce the affinity of the enzyme for digoxin, it is also capable of acting together with digoxin in inhibiting enzyme activity to a degree greater than the inhibitory effect of digoxin alone. The concentrations of digoxin and quinidine used in this study were considerably greater than their therapeutic serum concentrations. Nevertheless, these observations are consistent with the hypothesis that the increases in serum digoxin concentrations and the decreases in volumes of digoxin distribution observed clinically when quinidine is administered to digoxin-treated patients may reflect, at least in part, a decrease in the affinity of tissue receptors for digoxin. The possibility must also be considered that enhanced cardiac effects of digoxin may occur clinically as the result of an augmentation, by quinidine, of digoxin effects, which more than compensates for the modest reduction in digoxin binding.

Lung injury induced by antibody fragments to angiotensin-converting enzyme.

Rabbits given goat anti-rabbit angiotensin-converting enzyme antibodies or derived antibody fragments develop rapidly fatal pulmonary edema. Endothelial cell injury is manifested by bleb formation and the disintegration of cell membranes. Platelets are found along the injured endothelium and leukocytes block capillary lumens. The pathologic features are similar when immune IgG, F(ab')2, or Fab are given. In vitro studies of complement activation show that solubilized, purified angiotensin-converting enzyme alone activates C1, with consumption of C4 and C3. Addition of immune IgG plus converting enzyme enhances this activation. F(ab')2 plus enzyme enhances only C3 consumption, while Fab with enzyme produces no additional complement utilization. Thus, while complement activation may be involved in the pathogenesis of injury induced by IgG or F(ab')2, the mechanism of Fab-induced endothelial injury remains unclear.

Urinary excretion of reduced metabolites of digoxin.

The urinary excretion of the relatively cardioinactive reduced metabolites of digoxin, dihydrodigoxin and related compounds was measured by radioimmunoassay in 131 normal subjects during studies of the bioavailability of digoxin preparations. Digoxin reduction products (DRP) constitute more than 5 percent of the excretion of digoxin and its metabolites in one-third of the volunteers after the administration of single or multiple doses of digoxin. There was little or no output of DRP during the first 8 hours after a single dose, with maximal excretion usually occurring on the second day. Most subjects who excreted more than 5 percent DRP on one occasion did so with each subsequent exposure to digoxin. Six volunteers, however, in whom substantial amounts of DRP had previously been found, failed to excrete detectable quantities after subsequent doses. In two, this change occurred shortly after they took erythromycin. Urinary DRP were less after the intravenous administration compared to the oral administration of digoxin. After oral doses, DRP excretion tended to vary inversely with the bioavailability of the preparation. The findings are consistent with the hypothesis that DRP are formed as the result of the activity of a variable component of the intestinal flora. Prospective studies will be necessary to prove this hypothesis.