Clinical Chemistry / DIGOXIN-LIKE AND DIGITOXIN-LIKE IMMUNOREACTIVE SUBSTANCES IN ELDERLY PEOPLE Digoxin-Like and Digitoxin-Like Immunoreactive Substances in Elderly People Impact on Therapeutic Drug Monitoring of Digoxin and Digitoxin Concentrations Sang Ling Wu, MD, Wei Li, MD, PhD, Alice Wells, MT(ASCP), and Amitava Dasgupta, PhD Key Words: Digoxin-like immunoreactive substances; Digitoxin-like immunoreactive substances; DLIS; DTLIS; Elderly people; Digoxin Abstract We compared digoxin-like (DLIS) and digitoxin-like (DTLIS) immunoreactive substance concentrations for 30 people older than 65 years with those for 25 people younger than 50. None received digoxin or had liver disease, uremia, or volume expansion. We found no DTLIS in any specimen, and only 1 specimen from an elderly person demonstrated a low DLIS concentration. In addition, for 22 non volume expanded patients (8 younger than 50 years and 14 older than 65) receiving digoxin, the fluorescence polarization (FPIA) and the microparticle enzyme (MEIA) immunoassays revealed comparable serum digoxin concentrations, indicating an insufficient DLIS concentration to interfere with digoxin immunoassay results. Therefore, elderly people who are not volume expanded do not have elevated DLIS or DTLIS concentrations. Furthermore, for patients with liver disease or uremia (18 older than 65 years and 20 younger than 50), the DLIS and DTLIS concentrations were elevated. Finally, for 5 patients with liver disease who received digoxin, serum digoxin concentrations were lower by MEIA and higher by FPIA, indicating the patients had elevated DLIS levels that interfered with the assays. Elevated DLIS and DTLIS concentrations are associated with volume expansion and not age. Digoxin-like immunoreactive substances (DLIS) crossreact with antidigoxin antibodies and interfere with the serum digoxin measurement by immunoassays. 1,2 Although the concentration of DLIS in serum samples of healthy people is below the detection limit of a digoxin immunoassay, DLIS concentrations can be elevated substantially in volume expanded conditions, such as uremia, liver disease, essential hypertension, hypertension with water volume expansion, and congestive heart failure. 3-6 DLIS are bound strongly to serum protein, while digoxin is only 25% bound to serum albumin. 7 Interference of DLIS in digoxin measurement by immunoassays has been well documented in the literature. Digoxin has a narrow therapeutic range, and false elevation of serum digoxin concentration due to DLIS is troublesome. 8 Negative interference of DLIS in serum digoxin measurement by the new Abbott nonpretreatment digoxin assay has been reported. 9,10 This is a serious problem because the physician may increase the digoxin dosage based on these falsely lower digoxin levels, resulting in toxic effects of digoxin. Serum from volume-expanded patients also has factors that cross-react with the fluorescence polarization immunoassay (FPIA) of digitoxin. Apparent digitoxin concentrations as high as 21.2 ng/ml (27.8 nmol/l) have been reported in a patient with liver disease but receiving no digitoxin. 11 This is an important cross-reactivity because the therapeutic range of digitoxin is 20.0 to 35.0 ng/ml (26.2-45.9 nmol/l). Ebara et al 12 used an enzyme immunoassay for digitoxin and reported a digitoxin equivalent concentration of 15.0 ng/ml (19.7 nmol/l) in cord blood. Another interesting feature is that a serum specimen from a patient may have a measurable concentration of DLIS but no detected concentration of digitoxin-like immunoreactive substances (DTLIS) and vice versa. The correlation between DLIS and DTLIS concentrations is poor. 11 600 Am J Clin Pathol 2001;115:600-604 American Society of Clinical Pathologists
Clinical Chemistry / ORIGINAL ARTICLE Digoxin is used widely in elderly patients to treat various cardiac disorders. Elderly patients also are sensitive to the toxic effects of digoxin, and their serum digoxin concentrations should be monitored closely. Clinical conditions, such as essential hypertension and renal dysfunction, are more common in elderly people. Such clinical conditions may increase the serum DLIS and DTLIS concentrations. DLIS interference in serum digoxin measurement may be an important factor in elderly patients and should be considered to avoid the toxic effects of digoxin. Digitoxin also is used to treat cardiovascular disease, but this drug is not used commonly in the United States. Digitoxin is used in Europe. DTLIS may similarly affect measurement of serum digitoxin concentrations in elderly people. However, DLIS concentrations in elderly people have been studied inadequately in the past, and DTLIS levels in elderly people have not been studied. Yamamoto et al 13 studied DLIS concentrations in elderly patients not receiving digoxin. They reported a range of DLIS concentrations from 0.00 to 0.74 ng/ml (0.00-0.95 nmol/l) in patients older than 65 years and concluded that DLIS concentrations were significantly higher in patients older than 65 years compared with patients younger than 35 years. 13 In contrast, Lackner et al 14 found no difference in DLIS concentrations between elderly (older than 70 years) and younger (younger than 55 years) people and concluded that serum digoxin concentrations in elderly people were not affected by DLIS. The present study was designed to resolve this conflict. We studied older and younger groups of patients with and without liver disease or uremia. Our hypothesis is that elevated an DLIS concentration is associated with volume expansion and not with age. Materials and Methods Digoxin and digitoxin were purchased from Sigma Chemical (St Louis, MO). High-performance liquid chromatography (HPLC) grade methanol was obtained from Aldrich Chemical (Milwaukee, WI). Standard solutions of digoxin and digitoxin were prepared in HPLC-grade methanol. FPIA kits for measuring digoxin and digitoxin concentrations were purchased from Abbott Laboratories (Abbott Park, IL), and the assays were run using a TDx/FLX analyzer (Abbott Laboratories). The microparticle enzyme immunoassay (MEIA) kits for digoxin also were obtained from the Abbott Laboratories, but the assays were run using an AxSYM analyzer (Abbott Laboratories). Centrifree Micropartition System for ultrafiltration was obtained from Amicon (Danvers, MA). DLIS or serum digoxin concentrations were measured by both FPIA and MEIA. The FPIA for digoxin requires a serum pretreatment step: 200 µl of serum was treated with 200 µl of a precipitating agent containing 5-sulfosalicylic acid in 50% methanol. After mixing, the specimen was centrifuged at a high speed to separate precipitated protein from the supernatant. The clear supernatant was assayed for digoxin. The assay was linear up to a serum digoxin concentration of 5.0 ng/ml (6.4 nmol/l), and the sensitivity of the assay was 0.2 ng/ml (0.3 nmol/l). The MEIA for digoxin does not require serum pretreatment. The assay was linear up to a serum digoxin concentration of 4.0 ng/ml (5.1 nmol/l). The sensitivity of the assay was 0.3 ng/ml (0.4 nmol/l). DTLIS and digitoxin concentrations were measured by the FPIA for digitoxin. The FPIA for digitoxin requires pretreatment. In this step, 300 µl of methanol was added to 100 µl of serum to precipitate proteins. Clear supernatant after centrifugation was used for digitoxin measurement. The assay was linear up to a serum digitoxin concentration of 80 ng/ml (104.8 nmol/l), and the sensitivity of the assay was 2.0 ng/ml (2.6 nmol/l). We measured free digoxin concentrations in protein-free ultrafiltrate prepared by centrifuging serum with a Centrifree Micropartition Ultrafiltrate device for 40 minutes at 1500g. Then 200 µl of protein-free ultrafiltrate was treated with 200 µl of protein precipitating agent. As expected, no protein precipitation was observed. After mixing, the specimen was assayed for digoxin. Serum albumin, creatinine, serum urea nitrogen, and bilirubin concentrations were measured using a Hitachi 747 analyzer and reagent systems supplied by the manufacturer (Boehringer Mannheim, Indianapolis, IN). The manufacturer s recommended protocols were used. Statistical analyses were performed using an independent 2-tailed t test. We considered a difference significant only at a 95% confidence interval (P <.05). We also performed regression analysis to see the correlation between serum digoxin concentrations measured by the MEIA and FPIA in patients receiving digoxin. Results We studied serum samples from 30 people older than 65 years and 25 people younger than 50 years for possible DLIS and DTLIS activities. These subjects had no conditions that might lead to volume expansion. We observed no measurable DTLIS activity in any subject. We also observed no DLIS activity in any subject younger than 50 years and in only 1 subject older than 65 years. This 70-year-old subject showed a measurable DLIS concentration of 0.2 ng/ml (0.3 nmol/l) digoxin equivalent. This value is at the detection limit of the FPIA (detection limit 0.3 nmol/l). Therefore, we consider this DLIS concentration very low. American Society of Clinical Pathologists Am J Clin Pathol 2001;115:600-604 601
Wu et al / DIGOXIN-LIKE AND DIGITOXIN-LIKE IMMUNOREACTIVE SUBSTANCES IN ELDERLY PEOPLE We also studied digoxin concentrations in 14 patients older than 65 years and 8 patients younger than 50 years who were receiving digoxin. These patients had normal creatinine and bilirubin concentrations. We observed excellent agreement between serum digoxin concentrations measured by the MEIA and the FPIA Table 1 in older and younger patients. We observed measurable DLIS and DTLIS concentrations in 11 of 20 subjects with liver disease or uremia who were younger than 50 years. We also observed measurable DLIS and DTLIS concentrations in 9 of 18 subjects who were older than 65 years and had significant liver disease or uremia Table 2. We observed lower serum digoxin concentrations when measured by the MEIA and higher digoxin concentrations when measured by the FPIA in volume-expanded patients receiving digoxin. For example, 1 patient (older than 65 years) receiving digoxin had a total digoxin concentration of 2.9 ng/ml (3.7 nmol/l) when measured by the FPIA and 1.7 ng/ml (2.2 nmol/l) when measured by the MEIA. However, free digoxin concentrations were comparable Table 3. We observed a similar trend in total digoxin concentrations in other volume-expanded subjects who received digoxin. The free fraction of digoxin tended to be elevated in elderly people compared with younger people. The mean free fraction was 78.1% in elderly people and 70.9% in Table 1 Total and Free Digoxin Concentrations in Patients Receiving Digoxin Digoxin Concentration, Total younger people. However, the difference was not statistically significant. Albumin concentrations tended to be lower in elderly people. The mean albumin concentration in elderly people was 3.2 g/dl (32 g/l), while in people younger than 50 years, it was 3.6 g/dl (36 g/l). Again, the difference was not statistically significant. Discussion Our data clearly demonstrated that in elderly people without liver disease or uremia, DLIS and DTLIS are not present in sufficient concentrations to interfere in the serum digoxin assay or the serum digitoxin assay. On the other hand, we observed elevated DLIS and DTLIS concentrations in patients with liver disease or uremia regardless of the age of the patient. Our results resolve the controversy of the association of DLIS with old age. Lackner et al 14 studied patients with normal serum creatinine levels and found no association between DLIS and age. On the other hand, Yamamoto et al 13 reported that higher DLIS concentrations were associated with older age. Some of the elderly patients in the study by Yamamoto et al 13 received drip infusion therapy with a sodium-containing solution, which may have caused volume expansion. The increased DLIS concentrations in these patients may be secondary to volume Patient No. MEIA FPIA Free Free Digoxin, % Albumin, g/dl (g/l) Older than 65 years 1 1.2 (1.5) 1.0 (1.3) 0.7 (0.9) 70.0 3.5 (35) 2 1.3 (1.7) 1.1 (1.4) 0.9 (1.2) 81.8 3.1 (31) 3 1.1 (1.4) 1.2 (1.5) 0.8 (1.0) 66.7 3.9 (39) 4 1.4 (1.8) 1.4 (1.8) 1.2 (1.5) 85.7 2.5 (25) 5 2.2 (2.8) 2.3 (2.9) 2.0 (2.6) 86.9 2.6 (26) 6 0.8 (1.0) 0.8 (1.0) 0.6 (0.8) 75.0 3.2 (32) 7 1.8 (2.3) 1.8 (2.3) 1.5 (1.9) 83.3 3.2 (32) 8 1.1 (1.4) 1.2 (1.5) 1.1 (1.4) 91.6 1.7 (17) 9 1.9 (2.4) 1.9 (2.4) 1.6 (2.0) 84.2 2.9 (29) 10 0.7 (0.9) 0.7 (0.9) 0.5 (0.6) 71.4 3.7 (37) 11 2.0 (2.6) 1.9 (2.4) 1.6 (2.0) 84.2 3.4 (34) 12 0.9 (1.2) 0.9 (1.2) 0.6 (0.8) 66.6 3.8 (38) 13 0.7 (0.9) 0.7 (0.9) 0.5 (0.6) 71.4 3.6 (36) 14 0.9 (1.2) 0.8 (1.0) 0.6 (0.8) 75.0 3.5 (35) Younger than 50 years 1 0.6 (0.8) 0.5 (0.6) 0.3 (0.4) 60.0 4.8 (48) 2 1.9 (2.4) 2.1 (2.7) 1.6 (2.0) 76.1 3.2 (32) 3 0.6 (0.8) 0.5 (0.6) 0.3 (0.4) 60.0 4.1 (41) 4 0.9 (1.2) 0.9 (1.2) 0.7 (0.9) 77.7 3.1 (31) 5 6.9 (8.8) 6.5 (8.3) 4.9 (6.2) 75.3 3.4 (34) 6 0.5 (0.6) 0.5 (0.6) 0.4 (0.5) 80.0 2.8 (28) 7 0.7 (0.9) 0.7 (0.9) 0.5 (0.6) 71.4 3.8 (38) 8 0.3 (0.4) 0.3 (0.4) 0.2 (0.3) 66.6 3.7 (37) FPIA, fluorescence polarization immunoassay; MEIA, microparticle enzyme immunoassay. 602 Am J Clin Pathol 2001;115:600-604 American Society of Clinical Pathologists
Clinical Chemistry / ORIGINAL ARTICLE Table 2 Concentrations of Digoxin-Like and Digitoxin-Like Immunoreactive Substances in Patients With Liver Disease or Uremia Digoxin Equivalent, Digitoxin Equivalent, Patient No. MEIA FPIA FPIA Older than 65 years 1 ND ND 4.7 (6.2) 2 ND 0.2 (0.3) 2.0 (2.6) 3 ND 2.0 (2.6) 9.3 (12.2) 4 ND 0.5 (0.7) 4.1 (5.4) 5 ND 0.9 (1.2) 4.9 (6.4) 6 ND 0.8 (1.0) 6.8 (8.9) 7 ND 0.7 (0.9) 4.3 (5.6) 8 ND 0.7 (0.9) ND 9 ND 0.6 (0.8) 4.5 (5.9) Younger than 50 years 1 ND 0.2 (0.3) 2.7 (3.5) 2 ND 0.5 (0.6) 2.3 (3.0) 3 ND ND 6.1 (8.0) 4 ND 0.5 (0.6) 2.5 (3.3) 5 ND 0.3 (0.4) 2.9 (3.8) 6 ND 0.7 (0.9) 3.3 (4.3) 7 ND 0.6 (0.8) ND 8 ND 0.5 (0.6) 4.0 (5.2) 9 0.4 (0.5) 0.5 (0.6) ND 10 ND 0.4 (0.5) 3.9 (5.1) 11 ND 0.6 (0.8) ND FPIA, fluorescence polarization immunoassay; MEIA, microparticle enzyme immunoassay; ND, not done. Table 3 Total and Free Digoxin Concentrations in Patients With Liver Disease and Receiving Digoxin Total Digoxin, Free Digoxin, Age Group, y/ Patient MEIA FPIA MEIA FPIA Younger than 50 A 0.8 (1.0) 1.0 (1.3) 0.8 (1.0) 0.8 (1.0) B 2.8 (3.6) 4.1 (5.2) 2.6 (3.3) 2.5 (3.2) Older than 65 C 1.7 (2.2) 2.9 (3.7) 2.0 (2.6) 2.0 (2.6) D 1.1 (1.4) 1.5 (1.9) 1.0 (1.3) 1.2 (1.5) E 1.2 (1.5) 1.6 (2.0) 1.1 (1.4) 1.1 (1.4) FPIA, fluorescence polarization immunoassay; MEIA, microparticle enzyme immunoassay. expansion. Our study clearly demonstrated that there is no association between elevated DLIS concentrations and old age. We also demonstrated that elderly people did not have elevated DTLIS concentrations. DTLIS concentrations in elderly people have not been studied previously. Good correlation between serum digoxin concentrations measured by both the MEIA and FPIA for digoxin further indicated that measurement of the serum digoxin concentration in elderly patients receiving digoxin was not affected by DLIS. In the presence of DLIS, there is poor agreement between the digoxin concentrations measured by the MEIA and the FPIA. Usually, the digoxin concentrations measured by the FPIA are falsely elevated. The values are falsely lowered when measured by the MEIA owing to negative interference of DLIS. 10 We showed this poor correlation between serum digoxin concentration in 5 patients with liver disease who also were receiving digoxin. One patient was younger than 50 years, while another patient was older than 65 years (Table 3). Elderly people who have normal creatinine and bilirubin concentrations do not have sufficient concentrations of DLIS or DTLIS to interfere with measurement of the concentration of serum digoxin or digitoxin. However, if an elderly patient has substantial liver disease, uremia, or other pathologic condition that may lead to volume expansion, elevated DLIS concentrations may be present in the serum, and free digoxin should be monitored instead of total digoxin to eliminate such interference. However, we studied a small group of patients. A more detailed study with a larger patient population is needed to confirm our preliminary observations. From the Department of Pathology and Laboratory Medicine, University of Texas Houston Medical School. Address reprint requests to Dr Dasgupta: Dept of Pathology and Laboratory Medicine, University of Texas Houston Medical School, 6431 Fannin, MSB 2.292, Houston, TX 77030. References 1. Nanji AA, Greenway DC. Falsely raised plasma digoxin concentrations in liver disease. Br Med J (Clin Res Ed). 1985;290:432-433. 2. Soldin SJ. Digoxin issues and controversies. Clin Chem. 1986;32:5-12. 3. Graves SW, Brown B, Valdes R Jr. Digoxin-like immunoreactive substances in a patient with renal impairment. Ann Intern Med. 1983;99:604-608. 4. Sault MH, Vasdev SC, Longerich LL. Endogenous digoxinlike substances in patients with combined hepatic and renal failure. Ann Intern Med. 1984;58:748-751. 5. Cloix JF. Endogenous digitalis-like compounds. Hypertension. 1987;10(suppl I):67-70. 6. Shilo L, Adawi A, Solomon G, et al. Endogenous digoxin-like immunoreactivity in congestive heart failure. Br Med J (Clin Res Ed). 1987;295:415-416. 7. Valdes R Jr, Graves SW. Protein binding of endogenous digoxin-immunoactive factors in human serum and its variation with clinical condition. J Clin Endocrinol Metab. 1985;60:1135-1143. 8. Stone JA, Soldin SJ. An update on digoxin. Clin Chem. 1989;35:1326-1331. 9. Jortani S, Miller JJ, Helm RA, et al. Unexpected suppression of digoxin values caused by DLIS [abstract]. Clin Chem. 1996;42:S124. American Society of Clinical Pathologists Am J Clin Pathol 2001;115:600-604 603
Wu et al / DIGOXIN-LIKE AND DIGITOXIN-LIKE IMMUNOREACTIVE SUBSTANCES IN ELDERLY PEOPLE 10. Dasgupta A, Trejo O. Suppression of total digoxin concentrations by digoxin-like immunoreactive substances in the MEIA digoxin assay: elimination of interference by monitoring free digoxin concentrations. Am J Clin Pathol. 1999;111:406-410. 11. Luke M, Dasgupta A. Digitoxinlike and digoxinlike immunoreactivities in sera of patients with uremia and liver disease as measured by fluorescence polarization immunoassays: poor correlation between digitoxinlike and digoxinlike immunoreactivities. Ther Drug Monit. 1997;19:230-235. 12. Ebara H, Suzuki S, Nagashima K, et al. Digoxin and digitoxin-like immunoreactive substances in amniotic fluid, cord blood and serum of neonates. Pediatr Res. 1986;20:28-31. 13. Yamamoto T, Takano K, Sanaka M, et al. Digoxin-like immunoreactive substances in elderly patients and its impact on the TDx digoxin assay. Ther Drug Monit. 1998;20:417-421. 14. Lackner TE, Lau BW, Parvin C, et al. Endogenous digoxinlike immunoreactivity in elderly patients with normal serum creatinine concentrations. Clin Pharm. 1988;7:449-453. 604 Am J Clin Pathol 2001;115:600-604 American Society of Clinical Pathologists