Comparison of four digoxin immunoassays with respect to interference from digoxin-like immunoreactive factors

OBJECTIVES: Comparison of a new monoclonal digoxin assay with three polyclonal digoxin assays for their cross-reactivity to digoxin-like immunoreactive factors (DLIF) and digoxin metabolites. DESIGN AND METHODS: Sixty-six nondigitalized patient samples from 5 different groups: neonates, women in 3rd trimester pregnancy, and patients with liver or renal diseases, or undergoing organ transplants, and 139 samples from digoxin-treated patients of 4 categories (hospital sick, liver, renal, and outpatients) were compared in 4 different digoxin assays: (a) ACS Digoxin (ACS) developed for the automated chemiluminescent Ciba Corning ACS 180 system, (b) Baxter Stratus (Stratus, a fluoroimmunoassay), (c) Ciba-Corning Magic (Magic, a radioimmunoassay), and (d) an in-house radioimmunoassay (RIA). The ACS and RIA were also compared for their cross-reactivity to four principal digoxin metabolites. RESULTS AND CONCLUSION: Among the nondigitalized specimens, no significant DLIF interference was found for all 4 assays among the pregnant women or liver and transplant patients. However, the neonates registered high DLIF interference with Magic and RIA, but none for ACS or Stratus. DLIF interference in renal samples was highest in the Magic assay and lowest in RIA. Among the specimens with digoxin, a higher number of discrepant samples were found from the sick patients than from outpatients. In 75% of such discrepant samples, the ACS result was less than other assay results, suggesting DLIF as the probable cause. The two assays differed most in their cross-reactivity to the deglycated metabolites, digoxigenin and its mono-digitoxoside.     

Disorders of the ability to distinguish colors during digitalis therapy (author's transl)

31 hospitalized patients treated with beta-methyl digoxin and 34 treated with beta-acetyl digoxin were examined for color vision disturbances and compared with a control group (n = 17). The serum digoxin concentration was determined radioimmunologically. A significant correlation (p less than 0,01) was found between the severeness of the disturbances in color vision and the concentration of digoxin in the serum. The disturbances concerned different wave length regions. The lapse of time for disturbances in the distinguishing colors is being described in a case of suicidal intoxication.     

Digoxin-quinidine interaction in patients with renal failure

Investigations were performed in order to study whether or not quinidine would exert similar effects on the serum digoxin concentration in patients with renal failure as in normal subjects. Fourteen out of fifteen patients showed a significant increase of the serum digoxin level after four days of quinidine application. This indicates, that the quinidine effect is not solely caused by a decrease of the renal digoxin clearance, although nine patients, not being hemodialysed, revealed a correlation between their creatinine clearance and the rise of the serum digoxin concentration after quinidine. As however, the patients on hemodialysis did not show higher digoxin levels than those treated conservatively, it is suggested that the degree of the uremic intoxication might be responsible for the observed correlation.     

Serum digoxin concentration: dependence on body weight and age

In patients of a cardiological practice, 121 digoxin serum concentrations were determined by radioimmunoassay (RIA). Some drugs were suspected of interfering with the RIA or with the pharmacokinetics of digoxin. Patients having such additional drugs or patients with elevated serum creatinine were not included. The daily maintenance dose of digoxin was roughly adjusted to body weight. Patients with 0.5 mg digoxin daily showed unexpectedly low serum digoxin levels not fully explained by the relatively high body weight. This dose group was not included in the following correlations. At a maintenance dose of 0.25 and 0.375 mg digoxin and in the age groups 40-69 years (n = 66) there was an approximately inverse proportionality between serum digoxin concentration (per 0.25 mg digoxin daily) and body weight. When all age classes from 20 to 89 years were included (n = 96), a week positive correlation between serum digoxin concentration (per 0.25 mg digoxin daily and per 69.28 kg body weight) and age was found. A similar positive correlation resulted between serum digoxin concentration (per 0.25 mg digoxin daily) and the reciprocal of the nomographically determined creatinine clearance, always within the normal serum creatinine range. Based on these correlations, two simplified formulas are presented to predict the serum concentration and therapeutic maintenance dose of digoxin. The formulas are valid for the normal serum creatinine range and for digoxin tablets of optimal bioavailability.     

Total simultaneous repair of coarctation and intracardiac pathology in adult patients

OBJECTIVE: To determine whether neutralizing antibodies (NABs) to interferon beta (IFNbeta)-1a (Avonex) and IFNbeta-1b (Betaseron) cross-react. BACKGROUND: A total of 38% of MS patients treated with IFNbeta-1b and 22% of those treated with IFNbeta-1a were reported to develop NABs, which could reduce the clinical efficacy of the drug. METHODS: Blood from 10 MS patients was collected before and at 3 and 6 months after initiating treatment with IFNbeta-1a. ELISA was performed to detect binding antibodies to IFNbeta-1a. Sera from patients who tested positive for binding antibodies to IFNbeta-1a were then screened for NABs to IFNbeta-1a in a biologic assay based on neutralization of antiviral activity. These serum samples were subsequently tested for cross-reactivity with IFNbeta-1b both in the ELISA and the biologic assay. In the second part of the study, sera from patients who participated in the phase III IFNbeta-1b trial at the University of Maryland were examined for cross-reactivity with IFNbeta-1a in the ELISA and the biologic assay. RESULTS: Of the 10 patients treated with IFNbeta-1a, three developed binding as well as NABs to IFNbeta-1a 6 months after treatment, and these antibodies cross-reacted with IFNbeta-1b both in the binding and the biologic assay. Similarly, sera from six patients with NABs to IFNbeta-1b showed cross-reactivity with IFNbeta-1a in the binding assay. Three of these six serum samples tested for neutralizing activity against IFNbeta-1a demonstrated the presence of NABs to IFNbeta-1a. CONCLUSIONS: NABs to IFNbeta-1a (Avonex) and IFNbeta-1b (Betaseron) cross-react, both in the binding and the biologic assays. This suggests that switching to alternate IFNbeta preparation in patients who develop NABs may not be clinically beneficial. Studies examining cross-reactivity between NABs to IFNbeta-1a and IFNbeta-1b in a large number of patients are indicated.     

Immunoassay of digoxin in hair

Digoxin analysis in blood is an essential tool for therapeutic drug monitoring in cardiology because compliance with the treatment is a critical issue for the patient. Unfortunately, in postmortem cases blood digoxin concentration is of poor quality because there is a possible drug redistribution in the corpse and because of digoxin-like factors present in some people's blood. On the other hand, no biological fluid can be obtained at the autopsy. The aim of the present study was to evaluate the ability of an immunological method to determine digoxin in hair, in order to confirm that hair analysis can provide information on digoxin use before death. We studied 35 elderly patients who had been taking digoxin (60-250 micrograms/day) for 1-5 years. Two decontamination procedures were tested: washing by dichloromethane or by water and methanol. Three extraction procedures were compared: crushing in a ball mill and chloroform/acetone: crushing and methanol; enzymatic digestion. Immunoassays were performed by a microparticulate enzyme immunoassay. Serum digoxin levels were also assayed when sampling hair. The best results were obtained after decontamination with water and methanol followed by enzymatic digestion. Hair digoxin concentrations range from 3.6 to 11.4 pg/mg. Those very low concentrations are probably due to low and narrow range serum digoxin levels (0.3-1.4 ng/ml). No correlation was found between hair and blood digoxin. A forensic case is presented with 5 pg/mg digoxin in hair.     

Sensitive radioimmunoassay for digoxin in plasma and urine

A reproducible and sensitive radioimmunoassay for digoxin in either serum, plasma or urine is described. Using 0.5 ml of serum or plasma, the assay sensitivity is 0.05 ng of digoxin/ml. The antiserum and tracer solutions employed are available in a kit sold in the United States. All other reagents were prepared in the laboratory. The assay allows measurement of digoxin in plasma or serum for 96 hours after single 0.5 mg doses of digoxin; this is necessary in human bioavailability studies to accurately estimate the total area under the digoxin concentration, time curve from zero to infinite time. In contrast, with the kit assay, employing 0.2 ml of plasma or serum, it has been reported that the 12 hr serum digoxin levels, after single 0.5 mg doses, are, in most subjects, below the sensitivity limit (about 0.5 ng/ml) of the assay.     

Biochemical evaluation of digoxin dosage in patients with heart failure

The investigations were carried out in 56 patients aged 54 to 84 years, treated with a supporting dosage 0.25 mg of digoxin because of chronical insufficiency of the heart, according to the NYHA classification II and III degree, in whom the functions of liver and kidneys have not been ascertained. A fourfold determination of digoxin concentrations in the blood was established in the time of distribution balance. From among the examined patients three groups were separated: receiving the drug chronically at 8.00 a.m. (group A), receiving it at 8.00 p.m. (group B) and group C, for which the sacral method was used. Depending on medical indications the patients received during the examination other drugs. In group C the therapy was limited to diuretic drugs. In no clinical symptoms of digitalism could be observed. Subtherapeutic levels of digoxin (< 0.8 ng/ml) were found in the three groups on an average in 50% of the patients. The high percentage of patients with nontherapeutic concentration in blood serum confirms once more, that treatment with digoxin without checking their concentration in the serum does not give the certainty of suitable dosage. The results of the studies show that the optimalization of digoxin therapy from the point of pharmacological view should be based on a penetrating estimation of the whole of the clinical image, the checking of the image with the help of the concentration determinations of the drug.     

Evaluation of a radioimmunoassay for serum unconjugated estriol using commercial reagents

A radioimmunoassay using a commercially available antiserum was evaluated for measurement of serum unconjugated estriol in pregnancy. The evaluation showed an inter-assay variance of 12.1%, intra-assay variance of 6.8%, sensitivity of 0.2-0.6 ng/ml (0.7-2.1 nmol/l), and average recovery of 85.3%. The assay is specific for unconjugated estriol, showing less than 1% cross-reactivity with estriol-3-sulfate and estriol-16-glucuronide. Normal limits were established from 7 to 40 weeks' gestation using 175 serum samples. No diurnal variation could be demonstrated at 8 a.m. and 3 p.m. Eighty-nine serum specimens and 82 urine specimens obtained from 18 high-risk pregnancies were within normal limits except in cases of intruterine fetal death, pre-eclampsia, and suspected placental sulfatase deficiency. Serial urinary estriol levels fluctuated as much as 75%, while serial serum samples varied by only 30%.     

An in vitro assay for detection of glomerular binding IgG autoantibodies in patients with systemic lupus erythematosus

The deposition of anti-dsDNA antibodies in the glomerulus is believed to play a critical role in the pathogenesis of nephritis in SLE. However, an absolute correlation between serum levels of anti-dsDNA antibodies and renal disease has not been found. Recently a glomerular binding assay (GBA) has been developed to detect IgG binding to isolated rat glomeruli. We have used the GBA to study sera from four groups of SLE patients: (A) + anti-dsDNA antibodies, active nephritis; (B) - anti-dsDNA antibodies, active nephritis; (C) + anti-dsDNA antibodies, no nephritis; and (D) - anti-dsDNA antibodies, no nephritis. The serum anti-dsDNA antibodies in group A and group C patients could not be distinguished on the basis of isotype, charge, or cross-reactivity with histones. Nevertheless, the mean intensity of glomerular immunofluorescence was significantly higher in group A than in the three other patient groups and distinguished between patients with serum anti-dsDNA antibodies who had nephritis and those without clinically apparent nephritis. GBA reactivity was unaffected by DNase treatment of sera, but was partially inhibited by preincubation with dsDNA. These findings are consistent with the hypothesis that some anti-dsDNA antibodies cross-react with glomerular components and that the presence of this cross-reactivity is associated with, and may be responsible for, the development of nephritis. In addition, we have identified a group of SLE patients with renal disease and typical renal histopathology and immune deposits who do not have serum anti-dsDNA antibodies or antibodies that directly bind to glomeruli in the GBA. The mechanism of renal immune deposition in these patients remains to be determined.     

Serum digoxin in the presence of digibind: determination of digoxin by the Abbott AxSYM and Baxter Stratus II immunoassays by direct analysis without pretreatment of serum samples

We have reevaluated the feasibility of using direct immunochemical methods to track free digoxin in patients receiving Digibind. We report here that results obtained by the Stratus II and AxSYM immunoassays on patients receiving digoxin (without Digibind), digoxin-fortified serum samples supplemented with Digibind, and a digitoxic patient treated with Digibind, show no clinically significant biases. We conclude that useful free digoxin concentrations may be obtained for Digibind-treated patients using either the AxSYM or Stratus immunoassays without subjecting samples to ultrafiltration before analysis.     

Clinical value of the determination of serum digoxin levels (author's transl)

The authors refer to the technique of the serum digoxin enzyme immunoassay, and they report the results of 297 dosages concerning 111 hospitalized patients. The normal plasmatic rates are of 1,4 +/- 0,6 microgram/l in patients who present no sign of digitalic overdosage. The rates are of 5,2 +/- 1,6 microgram/l in cases of intoxication. The difference between these rates is greatly significant (p less than 0.001). The limit between therapeutic and toxic rates is situated around 3 microgram/l with an overlapping from 2 to 3 microgram/l. Authors then examine the individual factors that intervene in digoxin metabolism and especially study the influence of age, myocardic factors and renal insufficiency. On the basis of these results and review of the literature, they emphasize the interest of serum digoxin determination in the diagnosis of digitalis toxicity, as well as in the management of high risk patients, and of cardiopathies difficult to stabilize.     

A specific antidote for the treatment of digitalis poisoning in uremic patients

Two chronic haemodialyzed patients with digitalis intoxication are reported. One of them took digoxin 0.25 mg three times daily for an unknown period and the other took digitoxin 0.1 mg twice daily for two weeks. The symptoms of intoxication were mainly concealed by uremic syndrome. The diagnosis was established by noticed sinus bradycardia, first- and second-degree atrioventricular block in ECG and the determination of sera levels of glycosides (serum digoxin concentration was 7.36 ng/ml, serum digitoxin concentration was 46.5 ng/ml) in both cases. Considering the probable long elimination period of digitalis and the potentially life-threatening situation the patients were given digoxin-specific antibody (Fab) fragments with potassium replacement therapy. The symptoms disappeared within a few hours after therapy, side effects and rebound toxicity did not develop. In connection with these cases the aim of this report is to publish a method which can reverse the life-threatening digitalis intoxication in patients suffering from renal failure as well. As to the above method, the authors have not found any similar case reports in the Hungarian medical literature.     

Plasma-digoxin concentration in patients at time of hospital admission (author's transl)

Plasma-digoxin and serum creatinine concentrations were determined on admission in 145 unselected patients previously digitalized as outpatients. Adequate digitalization was found in 62.7%, inadequate doses in 15.9% of patients. In the latter group the daily dosage reported by the patients failed to correlate with the plasma-digoxin concentration by radioimmunoassay. One-fifth of all patients had clinical evidence of digitalis intoxication. Of these, 69% had plasma-digoxin concentrations of more than 2.0 ng/ml and 31% less than 2.0 ng/ml. Mean digoxin concentration for all patients with signs of digitalis intoxication was 2.5+/-0.9 ng/ml. In patients simultaneously receiving spironolactone or canrenoate-K+ there was danger of falsely high values for digoxin because of interference of those drugs with the radioimmunoassay.     

Evaluation of bone resorption: a common problem during impaired mobility

Osteoporosis, the result of an imbalance between bone resorption and bone formation, is a potential problem for the individual with a spinal cord injury because of the immobility commonly associated with this impairment. This study was performed to determine the diagnostic value of a new assay for urinary Pyridinium crosslink (UPyr). Assays were performed on 62 first morning voided and 50 24-hour urine specimens from clients in a bone health clinic. Higher than normal levels of UPyr were observed in females with osteoporosis. UPyr correlated well with urinary hydroxyproline (r = 0.429, p = 0.005; conversely, there was an inverse relationship between bone density and UPyr (r = -0.489, p = 0.01), positive correlation (r = 0.43, p = 0.011) between the 24-hour UPyr and a serum marker of bone resorption. The study confirms that UPyr has the ability to identify states of high bone resorption. This assay should be a welcome addition to the bone health assessment of individuals with risk factors such as impaired physical mobility.     

Specific insulin assays, insulin sensitivity and blood pressure

Serum insulin concentrations have been used as markers of insulin resistance in population studies examining the relationship between insulin resistance and blood pressure, but the relationship is variable among studies. We hypothesized that differences in cross-reactivity of insulin assays with proinsulin and its split/des-amino products might account for the variation. We therefore examined fasting and post-glucose load serum insulin concentrations (determined by both specific and conventional assays), insulin sensitivity (measured by the euglycaemic clamp technique), and blood pressure, in a group of 56 diabetic (NIDDM) and non-diabetic subjects. Insulin concentrations as measured by the two methods were highly correlated (r = 0.97, p < 0.0001), and the relationships among serum insulin concentrations, insulin sensitivity and blood pressure were independent of assay method; for example, in non-diabetic subjects the univariate correlation between log10AUC insulin and insulin sensitivity index was similar with both methods [r = -0.81 vs. r = -0.82, p < 0.0001 (specific vs. conventional assay)]. Discrepancies between studies in the relationship between serum insulin concentrations and blood pressure are unlikely to be due to cross-reactivity of conventional insulin assays with proinsulin-like molecules.     

Serum concentration of digitalis glycosides as a therapeutic guide

The concentration of digitalis glycosides in serum may serve as a useful guide in adjusting digitalis glycoside dosage to individual needs. Radio-immunological determination of digoxin is mainly indicated when patients with renal failure, hypothyroidism or hyperthyroidism or elderly subjects are treated with digoxin, when signs of digitalis intoxication are present or when patient compliance has to be assessed. However, factors which alter myocardial sensitivity to digitalis glycosides must be considered in the interpretation of serum concentrations of digitalis glycosides.     

The serum digoxin concentration: ten questions to ask

Although the role of digoxin therapy has been the subject of debate, the drug is generally accepted as effective in the treatment of heart failure due to systolic dysfunction and as therapy for atrial fibrillation and supraventricular tachyarrhythmias. Serum digoxin concentrations are commonly used to gauge patient response to digoxin. Digoxin pharmacokinetics are complex, and many factors can confound the interpretation of digoxin concentrations. The exact therapeutic range of serum digoxin varies in the literature but should be considered to be from 0.8 to 2.0 ng per mL, on the basis of population data regarding therapeutic response and toxicity. Renal function plays a major role in digoxin pharmacokinetics and is an important factor in determining digoxin doses. Many medications, including quinidine, amiodarone and verapamil, alter digoxin pharmacokinetics and can result in two- to three-fold increases in the serum digoxin concentration. Effective interpretation of the digoxin concentration requires consideration of the patient's renal function and clinical status, possible drug interactions, time of the assay and other variables.     

Resistance to tomudex (ZD1694): multifactorial in human breast and colon carcinoma cell lines

ZD1694 (Tomudex; TDX) is a quinazoline antifolate that, when polyglutamated, is a potent inhibitor of thymidylate synthase (TS), the enzyme that converts dUMP to dTMP. Continuous exposure of MCF-7 breast and NCI H630 colon cells to TDX, with stepwise increases in TDX up to 2.0 microM, resulted in stably resistant cell lines (MCFTDX and H630TDX) that were highly resistant to TDX. Initial studies revealed 34-fold increase in TS protein levels in MCFTDX and a 52-fold increase in TS levels in H630TDX cell lines. Despite continued exposure of these cells to 2.0 microM TDX, TS protein and TS mRNA expression decreased to parental levels in H630TDX cells, whereas in MCFTDX cells TS mRNA expression and TS protein levels remained elevated. Southern blot analysis revealed a 20-fold TS gene amplification in the MCFTDX cell line. TDX uptake was 2-fold higher in resistant MCFTDX cells than in parental MCF-7 cells, whereas in H630TDX cells TDX uptake was 50-fold less than that observed in parental H630 cells. In contrast, no change in the transport of either leucovorin or methotrexate into H630TDX cells was noted when compared with the H630 parental cells. In H630TDX cells, folylpolyglutamate synthetase (FPGS) activity was 48-fold less compared to parent H630 cells; however, FPGS mRNA expression was similar in both lines. H630TDX cells were also highly resistant to ZD9331, a novel quinazoline TS inhibitor that does not require polyglutamation, suggesting that defective transport by the reduced folate carrier was also an important mechanism of resistance in these cells. In MCFTDX and H630TDX resistant cells, several mechanisms of resistance are apparent: one increased TS expression; the others evolved over time from increased TS expression to decreased FPGS levels and decreased TDX transport.