Effect of glutathione on the cadmium chelation of EDTA in a patient with cadmium intoxication

Hyo-wook Gil, Eun-jung Kang, Kwon-hyun Lee, Jong-oh Yang, Eun-young Lee and Sae-yong Hong

Human and Experimental Toxicology 30(1) 7983 The Author(s) 2011 Reprints and permission: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0960327110369818 het.sagepub.com

Abstract In order to evaluate the efficiency and renal protective effects of glutathione during Ca-EDTA chelation therapy for chronic cadmium intoxication, we measured the renal excretion of cadmium, b2-microglobulin, proteinuria, and hematuria during intravenous administration of glutathione with Ca-EDTA in a 54-yearold patient with chronic cadmium intoxication. We administered 500 mg of Ca-EDTA and 50 mg/kg of glutathione alone or in 1 L of normal saline over the next 24 hours and repeated this over 12 consecutive days. During the first 3 days, the basal levels (only saline administration) were determined; during the second 3 days, Ca-EDTA only was administered, for the third sequence of 3 days, Ca-EDTA with glutathione was provided, and for the last 3 days, glutathione alone was given. One month later, the same protocol was repeated. There were six blood and urine samples to analyze in each group. The blood cadmium level was higher when the EDTA was infused together with glutathione (7.44 + 0.73 mg/L, p < 0.01) compared to the basal level of 4.6 + 0.44 mg/L. Also, the renal cadmium excretion was significantly higher in the EDTA with glutathione group than in the basal group (23.4 + 15.81 mg/g creatinine vs 89.23 + 58.52 mg/g creatinine, p < 0.01). There was no difference in the protein/creatinine and b2-microglobulin/creatinine ratio in the urine (p > 0.05) among the groups. Furthermore, microhematuria and proteinuria did not develop over the observation period of 6 months. These results suggest that glutathione administration with EDTA might be an effective treatment modality for patients with cadmium intoxication. Keywords cadmium, chelation, EDTA, glutathione

Introduction
Cadmium exposure with resultant toxicity usually occurs due to environmental and/or occupational exposure. Welders, solders, and other metal-workers who use cadmium-containing alloys are at risk of developing acute cadmium toxicity by inhalation of the cadmium oxide fumes. Other workers who do not work with metals may develop chronic cadmium toxicity through exposure to cadmium-containing dust.1,2 Upon exposure, cadmium is bound to b2-macroglobulin and albumin in the bloodstream. It is then quickly and preferentially redistributed to the liver and kidneys where it is complexed with metallothionein, an endogenous thiol-rich protein that is produced in both organs.3 The clinical presentation of chronic cadmium toxicity includes renal tubular dysfunction and painful

bone disorders including osteomalacia, osteoporosis, and spontaneous painful bone fractures.4 The management of patients with chronic cadmium toxicity is challenging.5 All agree that reduction of cadmium exposure should be the first line of treatment. However, the use of chelation is controversial; it has not been recommended because attempts at removing the cadmium deposits risks redistributing
Department of Internal Medicine, Soonchunhyang University Cheonan Hospital, Cheonan City, South Korea Corresponding author: Sae-Yong Hong, Department of Internal Medicine, Soonchunhyang University Cheonan Hospital, 23-20, Bongmyung-Dong, Cheonan 330-721, Korea. E-mail: syhong@sch.ac.kr

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the compounds to other organs and exacerbating the toxicity.5 However, many investigators have reported animal studies that demonstrate a possible protective role of antioxidants, especially sulfhydryl amino acids, on cadmium intoxication.6-9 Although the exact mechanism is poorly understood, it is known that cadmium exerts at least some of its toxic effects via the binding of sulfhydryl groups, subsequent denaturing of proteins and/or inactivation of enzymes.10 Sulfhydryl group amino acids have antioxidant affects themselves and their sulfhydryl base may compete to bind cadmium with other tissue proteins.10 Glutathione is a tripeptide of g-glutamylcysteinylglycine, available for intravenous administration. In our previous study,11 we reported that intravenous administration of glutathione increased not only cysteine levels but also the pool of sulfhydryl group amino acids (sum of cysteine, cystine, and methionine). Therefore, we hypothesized that cadmium chelation should be safer and more effective when the chelating agent is administered with glutathione. In order to support this hypothesis, we measured the blood cadmium level, renal excretion of cadmium, a proximal tubule injury marker (b2-microglobulin), and glomerular injury markers (proteinuria, hematuria), during intravenous administration of CaEDTA with and without glutathione in a patient with chronic cadmium intoxication.

mm Hg, with a weak pulse pressure (60/min), and the respiratory rate was 20/min. The results of the routine blood chemistry, CBC, and urinalysis are summarized in Table 1.

Experimental treatment
In an attempt to provide new aggressive treatment, we designed a protocol for Ca-EDTA chelation with glutathione. With the patients consent, we administered 500 mg of Ca-EDTA and 50 mg/kg of glutathione alone or together in 1 L of normal saline over 24 hours using a drop factor and repeated the treatment for 12 consecutive days. The first 3 days were used for the determination of basal levels (only saline administration, basal group); the second 3 days for Ca-EDTA only (EDTA group), the third 3 days for Ca-EDTA with glutathione (EDTA with glutathione group), and the last 3 days for glutathione only (glutathione group). Blood samples and 24-hour urine were obtained everyday to measure creatinine, cadmium, and b2-microglobulin levels. One month later, the same protocol was repeated and six blood and urine samples obtained for each group (Figure 1).

Assay
The cadmium levels were measured by a flameless atomic Absorption Spectrophotometer (AAS 800 Perkin-Elmer, Germany). b2-microglobulin was measured by the radioimmune assay method (SPAC-S b2-microglobulin micro kit, Daiichi Co., Japan).

Material and method Case report

A 54-year-old male presented to the toxicology clinic at Soonchunhyang Cheonan hospital on 4 October 2008. The chief complaint was generalized bone pain, worse at night and relieved during the day time especially by walking. The history was significant for the patient working at a factory producing compressors for air conditioners over the past 24 years (from 1977.3 to 2001.5). In May 2001, abnormally high blood levels of cadmium were noted during a regular screening test by a doctor employed by the industry to monitor workers. The patient reported that, at that time, he had intractable bone pain, insomnia, and general weakness. The patient was then immediately entered into a program for the treatment of cadmium intoxication, and he no longer worked at the factory. During the first visit to the outpatient toxicology clinic, the patient was exhausted, looked chronicallyill and very thin, with a body weight of 49.5 kg and a height of 172 cm. The blood pressure was 100/60

Statistics
The data is presented as mean + standard deviation. The parameters were compared with the KruskalWallis test among groups and a p < 0.05 was considered significant.

Results
The blood cadmium level was higher when EDTA was infused together with glutathione (7.44 + 0.73 mg/L, p < 0.01, Mann-Whitney U) compared to the basal level of 4.6 + 0.44 mg/L (Figure 2). However, there was no significant difference between the basal level and the glutathione alone (5.55 + 0.90 mg/L) or the EDTA alone (5.53 + 1.03 mg/L; Figure 2). The basal renal cadmium excretion was 23.4 + 15.81 mg/g creatinine. It increased to 89.23 + 58.52 mg/g creatinine (p < 0.01) when EDTA was given with glutathione. The renal cadmium excretion was 9.87 +

G Hyo-wook et al. Table 1. Laboratory findings at the beginning and 6 months after chelation therapy Basal CBC WBC (/UL) Hemoglobulin Hematocrit Platelet Blood chemistry Albumin (g/dL) Glucose (mg/dL) Total bilirubin (mg/dL) AST(IU/L) ALT(IU/L) BUN (mg/dL) Creatinine (mg/dL) Calcium (mg/dL) Phosphate (mg/dL) Urine analysis Specific gravity pH Protein RBC (/HPF) WBC (/HPF) Blood cadmium level (mg/L) Urine cadmium level (mg/day) 10,870 13.2 39.5 225,000 4.5 116 0.3 16 26 25 0.9 9.3 3.5 1.026 6.0 1-4 <1 4.4 50.1 Before 2nd session 10,020 13.5 40.1 237,000 4.4 121 0.4 17 22 14.6 0.9 9.2 3.8 1.020 6.0 1-4 <1 4.8 35.5

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After 6 months 10,130 13.8 42 247,000 4.2 132 0.3 17 14 12.3 0.9 9.1 2.9 1.012 7.0 1-4 <1 5.7 10.9

3.02 mg/g creatinine for glutathione alone, and 20.52 + 12.42 mg/g creatinine for EDTA alone (Figure 3). The protein/creatinine ratio in the urine was 109.39 + 17.29 mg/g in the basal state, 117.43 + 14.1 7mg/g with glutathione and calcium EDTA administration, 117.44 + 28.67 mg/g for EDTA administration alone, and 108.81 + 13.95 mg/g for glutathione alone (p > 0.05, Kruskal-Wallis analysis).

The b2-microglobulin/creatinine ratio in the urine was 3378.48 + 1814.20 ng/mg in the basal state, 5674.94 + 2600.93 ng/mg for glutathione with calcium EDTA administration, 4767.45 + 1359.81 ng/ mg for glutathione administration alone, and 7178.45 + 2359.81 ng/mg for EDTA alone (p > 0.05, Kruskal-Wallis analysis). There was no significant change in the serum creatinine levels. In addition,

Figure 1. The time schedule of chelation therapy. Intravenous administration of EDTA 500 mg and/or glutathione 50 mg/ kg in normal saline during 24 hours.

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Figure 2. Comparison of serum cadmium concentration among groups. *p < 0.01 Mann-Whitney test.

microhematuria and proteinuria did not develop over the 6-month observation period (Table 1). The asparate aminotransferase (ALT) and blood urea nitrogen (BUN) decreased after 6months, which meant the safety of this protocol. The results of each session were shown in Table 2.

Discussion
There are many patients with chronic cadmium intoxication worldwide that have renal disease, chronic lung disease, and intractable bone pain. The severity of the bone pain can be extreme. Due to the significant discomfort of patients clinical toxicologists have been investigating new effective treatment modalities that are not only safe, but also reduce the risk for exacerbating end-organ toxicity, in patients with chronic cadmium intoxication. Chelation has not been recommended for a number of reasons. First, there is no evidence that chelation in chronically poisoned animals is associated with longterm improvement.5 Second, most of the cadmium in chronically exposed patients is bound to intracellular metallothionein, which greatly reduces its toxicity. Any attempt to remove cadmium from these deposits risks redistributing the cadmium to other organs.12 Finally, many of the common chelating agents might not be effective for removing significant amounts of cadmium. The results of the present study showed that both the blood cadmium levels (Figure 1) and amount of renal excretion of cadmium (Figure 2) were significantly higher when EDTA was administered with glutathione

compared to EDTA given alone. Therefore, glutathione appears to assist the chelating activity of EDTA for cadmium deposits. Subsequent EDTA and glutathione therapy has not been effective in our study. The preceding glutathione with calcium EDTA application might have mobilized most of the available cadmium. We cannot explain the precise mechanism underlying this synergy. However, the sulfhydryl base of glutathione may compete to bind cadmium with other tissue proteins. Based on our preliminary study11 showing 50 mg/kg of glutathione to be safe and to increase the SH containing amino acid (cysteine, cystine, and methionine) levels for longer than 6 hours, this might be a useful clinical dose. However, further study is necessary to determine the optimum dose of glutathione for cadmium chelation in patients with chronic cadmium intoxication. There was no significant change in the serum creatinine levels. In addition, microhematuria and proteinuria did not develop over the 6-month observation period (Figure 3). Taken together, no difference in the b2-microglobulin/creatinine ratio in urine for this chelation procedure suggests that glutathione could diminish the nephrotoxic effect of EDTA. However, it is not clear whether repeated chelation procedures over long treatment duration would cause renal injury. The final assessment of the clinical outcome should be based on the degree of pain relief provided to the patient; this is the most serious complaint of patients with chronic cadmium intoxication. Although we did not use objective measures of pain, 6 months after our current chelation trial the patient still reported similar levels of pain compared to before the chelation trial.

Figure 3. Comparison of urine cadmium concentration among groups. p < 0.01 Kruskal-Wallis analysis.

G Hyo-wook et al. Table 2. The results of each session (mean) 1st session EDTA with glutathione Serum cadmium (mg/L) Urine cadmium (mg/g creatinine) Urine b2-microglobulin (ng/mg creatinine) Urine protein (mg/g creatinine) 7.45 88.28 6353.94 123.93 Glutathione 5.7 17.11 6514.65 133.18 EDTA 5.65 8.75 5422.95 103.62 2nd session EDTA with glutathione 7.43 90.17 5594.88 110.94 Glutathione 5.40 19.82 6856.28 93.84

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EDTA 5.40 10.98 5681.31 115.99

However, the patient was comforted by the fact that the cadmium was being eliminated from his body. In conclusion, our results suggest that glutathione administration with EDTA might provide a safe and effective chelation treatment modality for patients with cadmium intoxication. References
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6. Sinha M, Manna P, Sil PC. Taurine protects the antioxidant defense system in the erythrocytes of cadmium treated mice. BMB Rep 2008; 41: 657-663. 7. Manna P, Sinha M, Sil PC. Amelioration of cadmiuminduced cardiac impairment by taurine. Chem Biol Interact 2008; 30: 88-97. 8. Koyuturk M, Yanardag R, Bolkent S, Tunali S. The potential role of combined anti-oxidants against cadmium toxicity on liver of rats. Toxicol Ind Health 2007; 23: 393-401. 9. Vicente-Sa nchez C, Egido J, Sa nchez-Gonza lez PD, Pe rez-Barriocanal F, Lo pez-Novoa JM, Morales AI. Effect of the flavonoid quercetin on cadmiuminduced hepatotoxicity. Food Chem Toxicol 2008; 46: 2279-2287. 10. Vestergaard M, Matsumoto S, Nishikori S, Shiraki K, Hirata K, Takagi M. Chelation of cadmium ions by phytochelatin synthase: role of the cysteine-rich C-terminal. Anal Sci 2008; 24: 277-281. 11. Hong SY, Gil HW, Yang JO, Lee EY, Kim HK, Kim SH, et al. Pharmacokinetics of Glutathione and its Meatbolites in Normal Subjects. J Korean Med Sci 2005; 20: 721-726. 12. Dalhamn T, Friberg L. Dimercaprol (2, 3-dimercaptopropanol) in chronic cadmium poisoning. Acta Pharmacol Toxicol 1955; 11: 68-71.

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