Persistence of hapten-antibody complexes in the circulation of immunized animals after a single intravenous injection of hapten.

To study the fate of a low molecular weight antigen (hapten) in the circulation of animals whose sera contain antibodies specific for that low molecular weight antigen, a single injection of digoxin-(3)H (0.4 mg/kg) was administered intravenously to 18 rabbits. Thirteen animals (nine nonimmunized and four immunized with bovine serum albumin) served as control animals. In five rabbits which had been immunized with a digoxin-bovine serum albumin conjugate and whose sera contained digoxin-specific antibodies, the mean 12-h serum digoxin concentration was 8,300 ng/ml (control: 92 ng/ml) and the mean serum concentration 12 mo after the single injection of digoxin-(3)H was 85 ng/ml. In digoxin-immunized rabbits, less than 10% of the digoxin-(3)H was excreted in the first 10 days (control: 77% recovered in urine and feces) and the mean biological half-life of digoxin, as calculated from serum digoxin-(3)H disappearance curves, was 72 days (control: 3.4 days). In sera of digoxin-immunized rabbits, more than 90% of the circulating digoxin-(3)H was immunoglobulin bound, as determined by the double-antibody and dextran-coated charcoal methods. The serum disappearance rate of (125)I-antidigoxin antibodies was similar in nonimmunized and in immunized animals and in the presence or absence of digoxin. It is concluded that the biological half-life of a hapten may be markedly prolonged when the hapten is bound to specific antibody. The persistence of antibody-hapten complexes in the circulation suggests that these complexes may not be deposited in tissues and raises the possibility that low molecular weight determinants may be capable of preventing or reversing the deposition of immune complexes, containing macromolecular antigens, in the tissues of experimental animals and man.

Digoxin-inactivating bacteria: identification in human gut flora.

Digoxin, the most widely used cardiac glycoside, undergoes significant metabolic conversion in many patients to cardioinactive metabolites in which the lactone ring is reduced. This appears to occur within the gastrointestinal tract. An attempt was made to isolate and identify the organisms capable of reducing digoxin from stool cultures obtained from human volunteers. Of hundreds of isolates studied, only Eubacterium lentum, a common anaerobe of the human colonic flora, converted digoxin to reduced derivatives. Such organisms were also isolated in high concentrations from the stools of individuals who did not excrete these metabolites when given digoxin in vivo. When the growth of E. lentum was stimulated by arginine, inactivation of digoxin was inhibited. Neither the presence of these organisms alone nor their concentration within the gut flora appeared to determine whether digoxin would be inactivated by this pathway in vivo.

Reversal of digoxin toxicity with specific antibodies.

To determine whether digoxin-specific antibodies can reverse established digoxin toxicity in the dog, digoxin intoxication was produced by the intramuscular administration of digoxin, 0.09 mg/kg, on each of 3 consecutive days. All animals developed toxic arrhythmias (atrioventricular block, ventricular premature contractions and/or ventricular tachycardia). In control animals not receiving antidigoxin antibodies, the arrhythmias persisted throughout a 6 hr study period. Seven of the nine control dogs were dead within 24 hr and one moribund animal was sacrificed at that time; the last animal died within 48 hr.In contrast, in six of eight dogs given digoxin-specific antibodies in canine plasma and/or rabbit serum, the arrhythmias reverted to a sinus mechanism within 30-90 min after the start of the infusion. At the end of a 6 hr period of study, these six dogs were in normal sinus rhythm and all eight were alive and in normal sinus rhythm at the end of 72 hr. This study provides evidence that digoxin-specific antibodies can reverse severe established digoxin toxicity in the dog.

Biologic activity of digoxin-specific antisera.

Digoxin-specific antibodies are capable of removing essentially all intracellular digoxin from rat renal cortical slices or from human erythrocytes. In removing digoxin from erythrocytes, these antibodies are capable of reversing an effect of the drug on cellular potassium transport. This study provides direct evidence that antibodies are capable of removing, and thereby reversing the biological effect of, physiologically active low molecular weight substances after they have been taken up by mammalian cells. This biologic property of digoxin-specific antibodies suggests that autidigoxin sera may prove useful in the reversal of digoxin toxicity. Rapid and essentially quantitative removal of digoxin from red cells by antibody is not accompanied by an immediate restoration of patassium influx to normal levels. Identification of the mechanism of this phenomenon may provide useful information concerning the mode of action not only of digoxin, but also of the cation transport system of human erythrocytes.

Immunological protecion against dixin toxicity.

The lethal dose of digoxin was determined by administering 10 ml of a digoxin-saline solution to 26 nonimmunized rabbits through an ear vein over a 10 min period. Rabbits receiving less than 0.45 mg/kg digoxin showed no toxic effect, whereas all 15 rabbits that received 0.5 mg/kg developed an early arrhythmia and died within 1 hr. Moreover, eight rabbits which had been immunized with antigens unrelated to digoxin or injected with Freund's adjuvant mixture all died after receiving 0.6 mg/kg digoxin. Thus, it was concluded that 0.6 mg/kg digoxin was uniformly lethal in rabbits that had not been immunized or had received antigens unrelated to digoxin. By way of contrast, 17 rabbits immunized with a digoxin-albumin conjugate in complete Freund's adjuvant formed digoxin-specific antibodies and survived doses of digoxin varying between 0.6 and 0.9 mg/kg. In 10 rabbits immunized with digoxin-albumin conjugates, digoxin-specific antibody titers were determined following the administration of digoxin. There was a significant fall in antibody titer. This study indicates that rabbits protected by digoxin-specific antibodies suffer no acute adverse effects from an amount of digoxin which is uniformly lethal in nonimmunized rabbits and in rabbits immunized with other antigens.

The electrophysiologic effects of low and high digoxin concentrations on isolated mammalian cardiac tissue: reversal by digoxin-specific antibody.

The effects of digoxin on electrophysiologic properties were evaluated in isolated perfused cardiac tissue. In canine Purkinje fiber (PF)-ventricular muscle (VM) preparations, control measurements, using microelectrode technique, were made of: resting potential (RP), action potential (AP) amplitude, rate of rise, overshoot, duration (APD), membrane responsiveness, conduction velocity (CV), and refractory period. The preparation was then exposed to 1 x 10(-7) M digoxin and repeat measurements were carried out every 15 min. At slow (30/min) rates of stimulation APD initially prolonged then markedly shortened. With more rapid stimulation (75 and 120/min) no initial APD prolongation was observed. When stimulated at 75/min, RP and AP rate of rise, amplitude, and CV remained near control values for 60-75 min then rapidly decreased until electrical inexcitability (110+/-15 min). At that time fibers were perfused with serum containing digoxin-specific antibody (DSA) or one of a group of test solutions. In the preparations exposed to DSA, membrane characteristics improved by 15 min, and by 60 min approximated control values. No beneficial effect was seen with the various test solutions. DSA also reversed digoxin-induced enhanced phase 4 depolarization in PF. Effective (ERP) and Functional (FRP) refractory periods of rabbit atrioventricular (AV) node preparations were measured in the control state. The tissue was then exposed to 1 x 10(-7) M digoxin and refractory period measurements repeated. At a time when AV conduction prolonged by 20%, associated with marked prolongation of ERP and FRP, DSA or various test solutions were perfused. The prolongation in ERP, FRP, and AV conduction time rapidly returned to normal only in the DSA perfused tissue. It is concluded that DSA has the ability to reverse pronounced toxic electrophysiological effects of digoxin in in vitro cardiac tissue.

Radioimmunoassay of human fibrinopeptide B and kinetics of fibrinopeptide cleavage by different enzymes.

Thrombin converts fibrinogen to fibrin monomer by cleaving fibrinopeptides A and B (FPA and FPB) from the amino terminal ends of the A (alpha) and B (beta) chains. A radioimmunoassay capable of measuring the A peptide in human blood as an index of thrombin action in vivo has been described previously. This paper describes the development of a radioimmunoassay for FPB and the use of both assays in the demonstration of distinctive patterns of cleavage of the amino terminal ends of the A (alha) and B (beta) chains of fibrinogen by various enzymes. Antisera were raised in rabbits to a synthetic analogue of FPB coupled to bovine serum albumin. FPB analogue was couple to desaminotyrosine and radiolabeled with 125I by the chloramine-T technique. The radiolabeled peptide was bound by the antiserum, and binding was inhibited by synthetic or native FPB. Unbound tracer was separated from bound tracer by charcoal adsorption. The senistivity of the assay was such that 50% inhibition of binding of the tracer was caused by 1.25 ng of the native FPB. Fibrinogen was treated with thrombin, plasmin, trypsin, Reptilase, and an extract of the venom from Ancistrodon contortrix contortrix (ACC). After ethanol precipitation and centrifugation, dialysates of enzymatically altered fibrinogen were assayed for FPA and FPB. The action of thrombin on fibrinogen resulted in a rapid release of FPA and a slower release of FPB. Plasmin cleaved a segment(s) of the B (beta) chain which included FPB but cleaved no detectable FPA-containing material for the first 2 h of incubation. In the case of plasmin-treated fibrinogen, the dialysates had been further treated with thrombin before being assayed for FPA and FPB. Trypsin rapidly cleaved both peptides, the B before the A. Reptilase cleaved only FPA in 24 h. ACC cleaved FPB at a rapid rate, with a slowere cleavage of FPA. The distinctive cleavage patterns produced by the serine proteases may be useful in interpreting the levels of FPA and FPB measured in human blood and in studying the generation of FPA and FPB in clinical blood samples.

Effects of digoxin-specific antibodies on accumulation and binding of digoxin by human erythrocytes.

THE PRESENT STUDIES INDICATE THAT ACCUMULATION OF DIGOXIN BY INTACT HUMAN ERYTHROCYTES IS THE RESULT OF TWO PROCESSES: binding of digoxin to the erythrocyte membrane and uptake of digoxin across the membrane into the cell. In contrast, accumulation of ouabain by human erythrocytes is entirely attributable to binding of this glycoside to the plasma membrane. Digoxin binding to the erythrocyte membrane involves a single class of binding sites, is a saturable function of the extracellular digoxin concentration, reversible, temperature-sensitive, dependent on the cation composition of the incubation medium, inhibited by other cardioactive steroids, and correlates with the inhibition of erythrocyte potassium influx. Digoxin uptake across the membrane into the cell is also temperature-sensitive and reversible but is a linear function of the extracellular digoxin concentration, not altered by changes in the cation composition of the incubation medium, not inhibited by other cardioactive steroids, and does not correlate with inhibition of erythrocyte potassium influx. Digoxinspecific antibodies can both prevent and reverse effects of digoxin on potassium influx in human erythrocytes by virtue of the capacity of the antibodies to decrease the amount of digoxin that is bound to the erythrocyte membrane. These antibodies also reduce uptake of digoxin across the plasma membrane into the erythrocyte; however, this portion of cellular digoxin is not responsible for the observed inhibition of potassium influx. In the presence of digoxin-specific antibodies, the changes in digoxin binding to the erythrocyte membrane and in digoxin uptake across the membrane into the cell reflect the ability of the antibodies to form complexes with "free" digoxin molecules in the incubation medium and thereby decrease the effective concentration of digoxin.

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.

Measurement of fibrinopeptide A in human blood.

Since thrombin cleaves fibrinopeptides A (FPA) and B from the NH(2)-terminal end of the fibrinogen molecule, measurement of fibrinopeptide levels in plasma may provide a direct index of thrombin action. Recently a radioimmunoassay for FPA has been developed, and in the present paper, we describe the application of this assay to the measurement of FPA levels in clinical blood samples. Since fibrinogen cross-reacts with antibodies to FPA, dialysis was used to extract the peptide from plasma. In vitro generation of FPA was prevented by removing the fibrinogen from the plasma by precipitation with ethanol before dialysis. The processing technique permitted recovery of 75% of FPA added to blood in vitro. Evidence that the immunoreactivity measured in plasma is due to FPA was provided by the results of experiments in which two antisera to FPA with different specificities showed comparable results and addition of thrombin caused no change in immunoreactivity. In contrast, extracts of streptokinasetreated plasma showed a five-fold increase in activity when treated with thrombin and markedly different immunoreactivity with the two antisera. Plasma FPA levels in 30 normal men were below 2 ng/ml, with a mean of 0.5 ng/ml. FPA levels in 12 patients with reduced fibrinogen levels or reduced platelet counts or both ranged between 4 and 289 ng/ml. FPA levels in 13 patients with normal or elevated fibrinogen levels, including 6 patients with clinical evidence of venous thrombosis or pulmonary embolism or both, ranged between 5 and 23 ng/ml. FPA and fibrinogen degradation product levels did not correlate, and in several patients, elevated FPA levels were found in the presence of normal fibrinogen degradation product levels. After infusion of FPA-containing solutions in four normal individuals, FPA showed a disappearance rate from the plasma consistent with a t((1/2)) of 3-5 min. Heparin infusions in six patients with venous thrombosis or pulmonary embolism or both and elevated FPA levels were followed by a prompt decline in FPA level at a mean rate equivalent to a 3-5 min t((1/2)).

The generation of fibrinopeptide A in clinical blood samples: evidence for thrombin activity.

Plasma fibrinopeptide A (FPA) concentrations were measured in clinical blood samples incubated in the collecting syringe for different time periods before addition to heparin and Trasylol, and the rate of in vitro generation of FPA was calculated as the mean increment in FPA concentration per minute over the linear portion of the generation curve. 36 normal individuals had a mean plasma FPA level of 0.64 +/- 0.56 pmol/ml and an FPA generation rate of less than 0.5 pmol/ml per min. Clinical samples with elevated plasma FPA levels manifested slow (less than 1 pmol/ml per min) (28 patients) or rapid FPA generation (greater than 1 pmol/ml per min) (33 patients). Slow FPA generation was found in 10/10 patients with venous thrombosis, in 4/4 with aortic aneurysm, and in several patients with acquired hypofibrinogenemia. In one such patient, addition of fibrinogen resulted in rapid FPA generation whereas thrombin addition was without effect. Rapid FPA generation was generally linear, was usually associated with slower fibrinopeptide B generation and was inhibited by parenteral or in vitro heparin. It is thought to reflect increased thrombin activity and was seen in patients with pulmonary embolism, active systemic lupus erythematosus, renal transplant rejection, and after infusion of prothrombin concentrates. The initial rate of FPA cleavage by thrombin at fibrinogen concentrations from 0.05 to 4 mg/ml showed little change between 2 and 4 mg/ml with a Km of 2.99 muM. At a fibrinogen concentration of 2.5 mg/ml the FPA cleavage rate was 49.2 +/- 1.6 nmol/ml per min per U of thrombin. Exogenous thrombin added to normal blood generated 21.7 nmol/ml per U of thrombin FPA in the first minute with a nonlinear pattern reflecting inactivation of thrombin and the presence of alternative substrates. Hence, the thrombin concentration in the blood cannot be calculated from the FPA generation rate. The FPA generation rates in clinical samples with rapid generation (1-28 pmol/ml per min) could be produced by 2 X 10(-5) to 5.6 X 10(-4) thrombin U/ml acting on purified fibrinogen at physiological conditions of pH, ionic strength, and temperature.

Effects of sheep digoxin-specific antibodies and their Fab fragments on digoxin pharmacokinetics in dogs.

Intact sheep antidigoxin antibodies and their Fab fragments have both been found to exert profound effects on digoxin pharmacokinetics in [3H] digoxin-treated dogs. Both classes of molecule remove digoxin from the extravascular space and sequester it in the circulation in protein-bound form, a form in which the digoxin is presumably inactive. These two classes of molecule differ, however, in that the intact antibody molecules interfere with digoxin excretion, thereby promoting the retention of the glycoside; this retained digoxin is eventually released in free, active form when the administered antibody is metabolically degraded. In contrast, urinary excretion of digoxin continues in Fab-treated dogs, with significant quantities of digoxin being excreted promptly in the urine in complex with Fab fragments. These differences in urinary excretion, together with the probable decreased immunogenicity of sheep antidigoxin Fab fragments, suggest that such fragments possess potential advantages over intact antibody molecules for use in the therapy of life-threatening digoxin intoxication in man.

A potential krimpsiekte vaccine.

Krimpsiekte, a chronic form of cardiac glycoside poisoning, is an important plant-induced intoxication of small stock in South Africa. It is caused by cumulative, neurotoxic bufadienolides, such as cotyledoside. A cotyledoside-bovine serum albumin conjugate was synthesized to immunize animals. The efficacy of the cotyledoside-conjugate in inducing an immunological response was ascertained in rabbits (n = 4) and sheep (n = 4) by determining cotyledoside antibody titres with an ELISA using cotyledoside-hen ovalbumin as antigen. The formation of anticotyledoside antibodies was induced in both rabbits and sheep following immunization with the cotyledoside-protein conjugate. Protection provided by the vaccine was demonstrated by challenging sheep (n = 4) with repeated, daily doses of cotyledoside (0.015 mg/kg) administered intravenously, commencing 45 days after the initial vaccination. One control animal died on Day 3 of the challenge period and the other was severely affected after administration of the third cotyledoside dose. The immunized ewes (n = 2) remained clinically unaffected and the challenge was suspended following six daily injections. Vaccination as a means of preventing krimpsiekte seems to be quite feasible and deserves further investigation.

Treatment of 63 severely digitalis-toxic patients with digoxin-specific antibody fragments.

Sixty-three patients with life-threatening digitalis intoxication were treated with purified fragments of digoxin-specific antibodies (Fab) obtained from sheep. Twenty-eight patients developed toxicity as the result of digitalis ingestion in a suicide attempt, 5 ingested a large amount of digoxin accidentally and 30 developed toxicity in the course of treatment for underlying heart disease. The dosage of digoxin-specific Fab was calculated to be equimolar to the amount of cardiac glycoside in the patient's body. Digitalis toxicity was completely reversed in most cases, with onset of effect usually within 30 minutes of administration of Fab fragments. Unbound and, therefore, active digoxin serum concentrations decreased to undetectable levels within minutes after administration of the fragments. In all patients who had elevated serum potassium concentrations caused by massive digitalis toxicity, treatment with the Fab fragments reversed the hyperkalemia. There were no obvious adverse reactions to treatment. Potentially life-threatening digitalis intoxication can be rapidly and safely reversed by treatment with purified digoxin-specific Fab fragments.

Cellular electrophysiologic effects of vertebrate digitalis-like substances.

Substances structurally and functionally similar to digitalis glycosides are produced by several vertebrate species. There also is evidence for a digitalis-like substance of human origin. Standard microelectrode techniques were used to study the direct effects on the cellular electrophysiology of canine Purkinje fibers of 1) bufalin, an unconjugated cardiotonic steroid molecule that is produced by the toad Bufo marinus, and 2) an extract of human bile that showed digitalis-like immunoreactivity on radioimmunoassay. The goal of this study was to determine whether these substances have arrhythmogenic effects comparable with those seen with toxic doses of digitalis glycosides. Bufalin, 2 x 10(-8) M, significantly (p less than 0.05) reduced maximal diastolic potential, action potential amplitude and duration and maximal rate of rise of phase 0 (Vmax) within 40 min of onset of exposure. All six fibers developed delayed afterdepolarizations and two developed triggered rhythms. Ouabain was less potent, in that a 2 x 10(-7) M concentration was required to comparably reduce maximal diastolic potential, action potential amplitude and duration and Vmax within 30 min. These Purkinje fibers also developed delayed afterdepolarizations and triggered rhythms. A sample of an extract of human bile that showed digitalis-like immunoreactivity with an antibufalin serum also reduced maximal diastolic potential, action potential amplitude and duration and Vmax, and produced delayed afterdepolarizations and triggered activity. In contrast, immunologically unreactive bile extracts had no appreciable effect on the action potential. In summary, the cardiac toxicity of digitalis substances produced by lower vertebrates is comparable with that induced by the glycosides. Moreover, it appears that humans may produce digitalis-like substances that may be cardiotoxic.

Decreased digoxin cardioinactive-reduced metabolites after administration as an encapsulated liquid concentrate.

The generation by intestinal bacteria of large amounts of cardioinactive metabolites of digoxin with a reduced lactone ring (digoxin reduction products, or DRP) may be associated with increased dosage requirements. Since DRP excretion varies inversely with bioavailability, we compared the 6-day urinary excretion (CUE) of digoxin and DRP after 0.4-mg doses of an encapsulated liquid concentrate and a standard tablet in 22 normal subjects known to form substantial amounts of DRP. Mean (+/- SE) CUE of digoxin was greater with the capsules than the tablets (195.9 +/- 8.6 and 137.5 +/- 6.3 micrograms). CUE of DRP was less after the capsules (60.8 +/- 5.5 and 102.7 +/- 9.5 micrograms). Percent DRP was greater after the tablets in every subject (mean for tablets, 41.2 +/- 2.7%; capsules, 23.5 +/- 1.8%). Patterns of DRP excretion differed with the two preparations, probably reflecting differences in the routes whereby digoxin reached the colon. The use of highly bioavailable capsules in subjects with heavy DRP production should minimize metabolic inactivation during digoxin therapy.

Serum digitalis measurements in the assessment of digitalis resistance and sensitivity.

Antibodies to digitalis glycosides have been elicited in experimental animals and have been utilized in the development of rapid, sensitive, specific and convenient radioimmunoassay methods for the clinical measurement of digoxin and other cardiac glycosides in man. The use of these assay methods has supplemented earlier studies with radiolabeled digitalis preparations and has made it possible to obtain much new information concerning factors which may contribute to the well known patient to patient variability in digitalis dosage requirements and in sensitivity to the toxic effects of cardiac glycosides. In some patients with a poor clinical response to digitalis, the finding of a serum concentration which is relatively low for the dose prescribed may suggest that true digitalis resistance is not present and may raise questions of poor patient compliance, tablet inadequacies, intestinal malabsorption, increased metabolic degradation or hyperthyroidism; if the cause of the low serum level cannot be identified or corrected, serial serum measurements should enable safe and rational upward adjustment of dosage. In some patients with digitalis toxicity, the finding of a serum level which is relativity high for the dose prescribed may suggest that the patient is not sensitive to digitalis but rather is excreting it slowly; in such instances in elderly patients (with decreased glomerular filtration rates) and in patients with renal disease, serial digitalis measurements are useful adjuncts to clinical observation in determining optimal digitalis dosage schedules. A knowledge of serum digitalis concentrations should enable us to develop sound principles for a more rational approach to the clinical administration of cardiac glycosides, especially in patients with unusually high dosage requirements or with unusual sensitivity to relatively small doses of digitalis.

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.

Immunoelectron microscopic localization of platelet factor 4 and fibrinogen in the granules of human platelets.

To determine the storage site of platelet fibrinogen and of platelet factor 4 (PF4) in human platelets by immunoelectron microscopic techniques, washed human platelets were briefly exposed to Karnovsky's fixative and embedded in water-soluble Durcupan. Thin sections of platelets were exposed to Fab fragments of rabbit anti-human fibrinogen or of goat anti-human PF4, followed by a peroxidase conjugate of Fab fragments of antibodies to rabbit immunoglobulin (Ig) G or to goat IgG. The technique enabled preservation of the antigenic determinants of the platelet proteins, accessibility of Fab fragments to the platelet proteins, and maintenance of the ultrastructural integrity of the platelets. Using this approach, it was directly demonstrated that platelet fibrinogen and PF4 are stored in the alpha-granules of human platelets.

Digoxin inactivation by the gut flora in infancy and childhood.

Inactivation of digoxin by reduction of the lactone ring has recently been shown to occur in one third of adults and to be mediated by anaerobic intestinal bacteria. Children from birth through adolescence were studied to determine the pattern of development of this gut flora-mediated process. None of 36 digitalized infants 8 months of age or less excreted reduced digoxin metabolites in the urine. The adult pattern of digoxin reduction product excretion by one third of patients was observed after 16 months of age; however, high levels of digoxin reduction products such as are found in 10% of adults were not encountered in children less than 9 years of age. Even though reduced metabolites were not formed in vivo early in life, stool cultures of 20 of 73 infants younger than 8 months of age contained digoxin reduction product-forming bacteria at high concentrations, in some instances as early as the second week of life. Maturation of the gut flora with respect to digoxin metabolism appears to be a protracted process. The relative digoxin resistance of infants and children is not due to bacterial inactivation.