- Email: [email protected]
Amiodarone is a pharmacologically safe drug for porphyrias
General Pharmacology 32 (1999) 259–263
Amiodarone is a pharmacologically safe drug for porphyrias Manuel Me´ndez a, Victoria Parera a, Rafael Enrı´quez de Salamanca b, Alcira Batlle a,* a
Centro de Investigaciones sobre Porfirinas y Porfirias-CIPYP (CONICET, Department Biochemistry, School of Sciences, University of Buenos Aires), Argentine b Unidad de Porfirias, Departamento de Medicina Interna, Hospital Universitario Doce de Octubre, Madrid, Spain Manuscript received May 19, 1998; accepted manuscript June 15, 1998
Abstract Amiodarone (AD) is an effective antidysrythmic drug, however, there can be serious side effects, such as hepatic and neurological alterations, as well as skin photosensitization, as seen in porphyrias. Clinical signs in porphyrias might be triggered by the socalled porphyrinogenic drugs. Without sound basis, Amiodarone has been classified as an unsafe drug for porphyric patients. The aim of this work has been to study the effect of AD, both in vivo and in vitro, on heme metabolism. In the in vivo assays, the activities of 5-aminolevulinate synthetase (ALA-S), ALA dehydratase (ALA-D), porphobilinogenase (PBGase) and PBG-deaminase (PBG-D) in blood, liver, and kidney; hepatic and fecal porphyrins, urinary ALA, PBG and porphyrins in male mice strain CF1 treated with AD (100 mg i.p. daily) for 1 week and 1 month, were measured. No significanat differences were found for any of these parameters in the AD treated animals as compared to controls. In the in vitro experiments human bood, and mice blood, liver, and kidney, were used to measure the activities of ALA-S, ALA-D, PBGase, PBG-D and uroporphyrinogen decarboxylase, in the presence of varying concentrations of AD (0.0172–4.304 mM). AD did not modify any of the enzyme activities. All of the above biochemical parameters were studied in 17 cardiac patients under AD treatment for 3 to 20 years. Neither the activieis of the heme enzymes, nor the levels of precursors and porphyrins in urine and plasma were altered. These findings clearly demonstrate that AD is a pharmacologically safe drug and can be used for the treatment of associated pathologies in porphyrias. 1999 Elsevier Science Inc. All rights reserved. Keywords: Porphyrin metabolism; Amiodarone; Porphyrinogenic; Safe drug; Porphyrias
Amiodarone (AD), a benzofurane derivative, was originally introduced in Europe for the treatment of cardiopathies and its potent antidysrhythmic properties are well recognized. Since 1970 it has been used for treating ventricular and supraventricular arrhythmias (Anson and Colin, 1987). AD is an efficient drug, however, it is considered the drug of last resort because of its many side effects, including hepatic, neurologic, thyroidal, pulmonary, ophthalmologic, dermatologic, and cardiovascular alterations. Nevertheless, in most patients AD is well-tolerated, although in some, its adverse actions can become very serious (Harris et al., 1983). From the dermatologic point of view, AD induces skin photosensitivity in 75% of patients, both light and dose dependent (Rappersberger et al., 1989). In 82% of pa-
* Corresponding author. Present address: Viamonte 1881 10A, 1056 Buenos Aires, Argentine. Tel.: 154 1 812 3357; Fax: 154 1 811 7447; E-mail: [email protected].
tients receiving AD, an increase in the activity of hepatic enzymes has been found. However, in most patients these anomalies are transitory and often return to basal levels in time; only 3% of patients develop severe and persistent clinical signs (Lewis et al., 1989; Kerin et al., 1989). In experimental animals, it has been shown that administration of high doses of AD leads to the formation of an inactive complex, Cyt P450-Fe(II)-AD, in liver microsomes, which can not bind CO. As a result, Cyt P450 levels are reduced and so is the activity of some monooxygenases (Larrey et al., 1986). The neurotoxic effects of AD depend on both the accumulated and the maintenance dose of the drug. High doses provoke neurological damage in 35–75% of patients, while low doses are much safer (Kerin et al., 1989). Porphyrias are known metabolic disorders of the heme pathway, each due to a partial primary deficiency of one of the seven enzymes of porphyrin biosynthesis, after 5-aminolevulinate synthetase (ALA-S). They are characterized by a specific pattern of accumulation and
0306-3623/99/$–see front matter 1999 Elsevier Science Inc. All rights reserved. PII: S0306-3623(98)00202-X
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M. Mendez et al./General Pharmacology 32 (1999) 259–263
Fig. 1. Control (h) and AD treated ( ) mice, for 1 week. In vivo measurements. (A) ALA and PBG concentration in urine; (B) Porphyrin concentration in urine, feces, and liver. Other experimental conditions as indicated in the text.
excretion of precursors, ALA, and porphobilinogen (PBG) and/or porphyrins. The excess of these metabolites is responsible for the clinical signs of these diseases, which can be either cutaneous or neurologic. Genetic or acquired porphyrias can become clinically symptomatic because of many factors, including certain drugs, toxic substances, fasting, and stress. This is why lists exist to classify drugs as either safe or unsafe for use in the treatment of associated pathologies in porphyric patients. Although AD has been identified for a long time as an unsafe porphyrinogenic drug, there is very little information about its action on heme metabolism, either in humans or animals. Because of the resemblance between some of its side effects and the clinical signs seen in porphyrias, the aim of the present work has been to study the action of AD on heme biosynthesis, both in vivo, by administrating AD to animals or in patients receiving the drug, and in vitro, by measuring different
heme parameters, in several tissues, in the presence of varying amounts of AD.
1. Materials and methods Reagents used were obtained from Merck, Mallinckroft, or Sigma Companies at Buenos Aires dealers. PBG was prepared according to Sancovich et al., 1970. AD was kindly provided by Dr. J.C. Pico from Roemmers Laboratories, Buenos Aires. For in vivo experiments AD (1.5 gm/100 ml) was dissolved in 7% Tween 80. For in vitro studies AD (30 mg/ml or 50 mg/ml) was dissolved in 10% methanol. 1.1. Animals, tissues, samples preparation For both in vivo and in vitro studies animals were obtained from the Animal House of the School of Sciences, University of Buenos Aires. They were kept on
Fig. 2. In vivo measurements of ALA-S, ALA-D, PBGase and PBG-D activities in liver of CF1 male mice. Control (h), AD treated ( ) during, (A) 1 week; and (B) 1 month. ALA-S and ALA-D specific activities expressed 3 105. Other experimental conditions as indicated in the text.
M. Mendez et al./General Pharmacology 32 (1999) 259–263
261
Fig. 3. ALA-D in vitro activity in blood (h), liver ( ), and kidney ( ) of CF1 mice, measured in the presence of different concentrations of AD in the incubation system. Other experimental conditions as indicated in the text.
a Purina-3 diet (Molinos Rı´o de la Plata, Buenos Aires) and drank water ad libitum. For in vivo experiments, groups of CF1 male mice, each between 30 and 36 g body weight, were used. One group received daily 100 mg AD/kg, i.p. and the control group received the solvent only, for 1 week, 1 and 2 months. On the last day of treatment, animals were housed in metabolic cages to collect 24 h urine and feces and were fasted until killed. Studies were also performed with blood obtained from 17 cardiac patients under AD therapy for periods ranging from 3 months to 20 years and healthy volunteers. For in vitro experiments, blood from healthy volunteers and normal animals was used. One ml of heparinized blood was hemolized by adding 0.2 ml of 5% Triton-X-100 and 3.8 ml of 0.05 M Tris-HCl buffer pH 7.4. After collecting the blood, to measure ALA-S activity, a portion of liver was immediately excised, then the animal was perfused with saline solution; liver and kidney were extracted and frozen until processing; then part of the liver was separated for measuring porphyrin content and the remaining tissue and kidney were used to measure ALA dehydratase (ALA-D), porphobilinogenase (PBGase), and PBG-deaminase (PBG-D). To determine ALA-S, liver without perfusion was homogenized in a mixture of 9% NaCl (25 ml), 0.1 M EDTA (0.125 ml), and 1 M Tris-HCl buffer pH 7.4 (0.25 ml) at a ratio of 1:3 (wt:v). The resulting homogenate was used as enzyme source. To determine ALA-D, PBGase, and PBG-D, perfused liver and kidneys were homogenized in 0.25 M su-
Fig. 4. (A) ALA-D, (B) PBG-D, and (C) URO-D activities in vitro, in human blood, measured in the presence of different concentrations of AD in the incubation system. Other experimental conditions as indicated in the text.
crose (1:10, wt:v), centrifuged 15 min at 15,000 3 g and supernatant employed as enzyme source. 1.2. Determination of ALA, PBG, porphyrins and enzyme activities ALA and PBG were measured according to Mauzerall and Granick (1956). Urinary porphyrins were measured following methods described in Batlle (1998) and liver and fecal porphyrins were measured according to With (1975). Plasma porphyrins were estimated as described by Schoua and Batlle (1987) and total blood porphyrins following Polo et al. (1988). To quantify ALA and PBG in plasma, heparinized blood was centrifuged 15 min at 2,000 rpm at room temperature, then to 1 ml of plasma, 0.2 ml of 50% TCA was added, the mixture centrifuged for 15 min at 2,000 rpm. One ml of the resulting supernatant was taken to
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Fig. 5. Biochemical parameters in plasma and blood from cardiac patients. ALA (h); PBG ( ); Porphyrin Plasma Index (IPP) ( ); ALA-D ( ); PBGase ( ); PBG-D ( ); URO-D ( ); Total Porphyrins in Blood (PTS) (j). RBC: Red Blood Cells. Normal values were: ALA-D (nmole PBG/h. ml RBC): Female 0.794 6 0.247: Male: 0.688 6 0.232; PBGase (nmoles porphyrins/h. ml RBC) Female: 38.74 6 6.05; Male: 34.63 6 8.22; PBG-D (nmoles porphyrins/h. ml RBC): Female: 81.51 6 11.96; Male: 73.13 6 13.62; URO-D (nmoles Hepta 1 Hexa 1 Penta 1 Copro)/h. ml RBC: 4.00 6 0.25 (Substrate Uroporphyrinogen I). ALA (mg/ml plasma): 0.130 6 0.02; PBG (mg/ml plasma): 0.9 6 0.087; IPP: #1.3 (lem 5 615 nm) PTS (mg/100 RBC): 150 6 40.
pH 6–7 with 5 M NaOH, and then ALA and PBG were measured as indicated. ALA-S activity was estimated following Marver et al. (1966); ALA-D activity measured according to Batlle et al. (1967); PBGase and PBG-D activities as described by Batlle et al. (1978); uroporphyrinogen decarboxylase (URO-D) activity was studied following Batlle et al. (1985). One enzyme unit (EU) is defined as the amount of enzyme catalyzing the formation of 1 nmole of product under the standard conditions of incubation: Specific activity (Sp. Act.) as the number of EU per mg of protein. Protein concentration was estimated as described by Lowry et al. (1951). 1.3. Statistical analysis Data were statistically analyzed using nonpaired Student’s t-test, with Sigma Plot 2.0 statistical software for IBM. 2. Results and discussion 2.1. In vivo studies 2.1.1. Precursors and porphyrins in urine, liver and feces Neither the levels of urinary precursors (Fig. 1A), nor porphyrins (Fig. 1B), nor the levels of porphyrins in
feces and liver (Fig. 1B), were altered after one week of AD treatment. After one or two months of AD administration no changes were observed either (data not shown). 2.1.2. Enzyme activities ALA-S, ALA-D, PBGase and PBG-D activities were measured in animals receiving AD after 1 week and 1 month of treatment, in liver, blood, and kidney. Data obtained in liver are illustrated in Fig. 2, where it can be seen that no significant differences were found for any of the enzymes at either period. Similar findings were obtained in blood and kidney, even when treatment with AD was prolonged for 2 months (data not shown). 2.2. In vitro studies 2.2.1. ALA-D in tissues from mice Fig. 3 shows that there were not significant changes in the activity of ALA-D in either blood, liver, or kidney tissues from mice, when different concentrations of AD were added to the incubation system. 2.2.2. Enzyme activities from human blood Fig. 4 shows the activity levels of ALA-D (Fig. 4A), PBG-D (Fig. 4B) and URO-D (Fig. 4C) measured in normal human blood in the presence of varying concentrations of the drug. Once again, no significant modifi-
M. Mendez et al./General Pharmacology 32 (1999) 259–263
cations were found under any condition for any of the three enzymes. 2.2.3. Biochemical parameters in blood from cardiac patients Fig. 5 shows the levels of ALA, PBG, plasma, and total blood porphyrins, as well as erythrocyte ALA-D, PBGase, PBG-D, and URO-D activities in samples from 17 cardiac patients, including a true case of porphyria cutanea tarda (diagnosed 8 years before, now in remission); all of them were under AD treatment for different periods of time. In none of these patients were there any pathological alterations of any of the heme metabolism parameters tested. So far, only two other studies related to the use of AD and its effects on porphyrin metabolism have been published. Waitzer et al. (1987) and Parodi et al. (1988) found normal concentrations of porphyrins in patients who receive AD and developed photosensitization. They proposed that AD includes clinical pseudoporphyria without actually producing any biochemical disturbances in the heme pathway. Our results are in agreement with these authors and we clearly demonstrate that AD does not exhibit porphyrinogenic properties, in spite of having been classified as such. Therefore, it is important to emphasize that AD should be included among the safe drugs wich can be used for the treatment of other associated pathologies in the case of porphyric patients.
Acknowledgments AB and VEP are Superior and Associate Researcher at the Argentine National Research Council (CONICET). MM is a fellow from the same Council. The technical assistance of Mrs. V. Castillo, Mrs. B. Riccillo, and Lic. L. Dato is acknowledged. This work was supported by grants from the CONICET and the University of Buenos Aires. AB and RES are grateful to the Ministerio de Educacio´n y Ciencia (MEC) of Spain for special help.
References Anson, S.W., Colin, F., 1987. Spectroscopic studies of cutaneous photosensitizing agents IX-A spin trapping study of the photolysis of
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amiodarone and desethylamiodarone. Photochem Photobiol 45, 191–197. Batlle, A.M.C., Ferramola, A.M., Grinstein, M., 1967. Purification and general properties of d-Aminolaevulate dehydratase from cow liver. Biochem J 104, 244–249. Batlle, A.M.C., Wider, E., Stella, A.M., 1978. A simple method for measuring erythrocyte porphobilinogenase and its use in the diagnosis of acute intermittent porphyria. Int J Biochem 9, 871–876. Batlle, A.M.C., Enrı´quez de Salamanca, R., Chinarro, S., Afonso, S.G., Stella, A.M., Berges, L., Wider, E., 1985. An Med Intern 2, 264–268. Batlle, A.M.C., 1998. Porfirias y Porfirinas - Aspectos clı´nicos, bioquı´micos y biologı´a molecular. Actualizaciones Me´dico Bioquı´micas. Acta Bioquı´mica Clin Latinoamer (suppl. 3), 145–171. Harris, L., McKenna, W., Rowland, E., Holt, D., Storey, G., Krikler, D., 1983. Side effects of long-term amiodarone therapy. Circulation 67, 45–51. Kerin, N., Aragon, E., Faitel, K., Frumin, H., Rubenfire, M., 1989. Long-term efficacy and toxicity of high and low dose amiodarone regimens. J Clin Pharmacol 29, 418–423. Larrey, D., Tinel, M., Letteron, P., Geneve, J., Descatoire, V., Pessayre, D., 1986. Formation of an inactive cytochrome P450-Fe(II)Metabolite complex after administration of amiodarone in rats, mice and hamsters. J Hepatol 3, 14b. Lewis, J., Ranard, R., Caruso, A., Jackson, L., Mullick, F., Ishak, K., Seeff, L., Zimmerman, J., 1989. Amiodarone hepatotoxicity: prevalence and clinicopathologic correlations amoung 104 patients. Hepatology 9, 679–685. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265–275. Marver, H.S., Tschudy, D.P., Pelroth, M.G., Collins, A., 1966. a-Aminolevulinic acid synthetase I. Studies in liver homogenates. J Biol Chem 241, 2803–2809. Mauzerall, D., Granick, S., 1956. The occurrence and determination of a-Aminolaevulic acid and porphobilinogen in urine. J Biol Chem 219, 435–446. Parodi, A., Guarrera, M., Rebora, A., 1988. Amiodarone-induced pseudoporphyria. Photodermatol 5, 146–147. Polo, C.F., Schoua, E., Batlle, A.M.C., 1988. Determinacio´n de porfirinas totales en sangre (PTS). Acta Bioq Clin Lat XXII, 437–438. Rappersberger, K., Honigsmann, H., Ortel, B., Tanew, A., Konrad, K., Wolff, K., 1989. Photosensitivity and hyperpigmentation in amiodarone-treated patients: incidence, time course and recovery. J Invest Dermatol 93, 201–209. Sancovich, H., Batlle, A.M.C., Grinstein, M., 1970. Preparation of Porphobilinogen. In: Collowick, S., Kaplan, N. (Eds.), Methods in Enzymology. Academic Press, New York, pp. 220–222. Schoua, E., Batlle, A.M.C., 1987. Indice de porfirinas plasma´ticasIPP. Un me´todo ra´pido para la caracterizacio´n de las porfirias. Rev Arg Dermatol 68, 79–85. Waitzer, S., Butany, J., From, L., Hanna, W., Ramsay, C., Downar, E., 1987. Cultaneous ultrastructural changes and photosensitivity associated with amiodarone therapy. J Am Acad Dermatol 16, 779–787. With, T.K., 1975. Clinical use of porphyrin ester chromatography of urine and feces. Dan Med Bull 22, 80–86.
Amiodarone is a pharmacologically safe drug for porphyrias Manuel Me´ndez a, Victoria Parera a, Rafael Enrı´quez de Salamanca b, Alcira Batlle a,* a
Centro de Investigaciones sobre Porfirinas y Porfirias-CIPYP (CONICET, Department Biochemistry, School of Sciences, University of Buenos Aires), Argentine b Unidad de Porfirias, Departamento de Medicina Interna, Hospital Universitario Doce de Octubre, Madrid, Spain Manuscript received May 19, 1998; accepted manuscript June 15, 1998
Abstract Amiodarone (AD) is an effective antidysrythmic drug, however, there can be serious side effects, such as hepatic and neurological alterations, as well as skin photosensitization, as seen in porphyrias. Clinical signs in porphyrias might be triggered by the socalled porphyrinogenic drugs. Without sound basis, Amiodarone has been classified as an unsafe drug for porphyric patients. The aim of this work has been to study the effect of AD, both in vivo and in vitro, on heme metabolism. In the in vivo assays, the activities of 5-aminolevulinate synthetase (ALA-S), ALA dehydratase (ALA-D), porphobilinogenase (PBGase) and PBG-deaminase (PBG-D) in blood, liver, and kidney; hepatic and fecal porphyrins, urinary ALA, PBG and porphyrins in male mice strain CF1 treated with AD (100 mg i.p. daily) for 1 week and 1 month, were measured. No significanat differences were found for any of these parameters in the AD treated animals as compared to controls. In the in vitro experiments human bood, and mice blood, liver, and kidney, were used to measure the activities of ALA-S, ALA-D, PBGase, PBG-D and uroporphyrinogen decarboxylase, in the presence of varying concentrations of AD (0.0172–4.304 mM). AD did not modify any of the enzyme activities. All of the above biochemical parameters were studied in 17 cardiac patients under AD treatment for 3 to 20 years. Neither the activieis of the heme enzymes, nor the levels of precursors and porphyrins in urine and plasma were altered. These findings clearly demonstrate that AD is a pharmacologically safe drug and can be used for the treatment of associated pathologies in porphyrias. 1999 Elsevier Science Inc. All rights reserved. Keywords: Porphyrin metabolism; Amiodarone; Porphyrinogenic; Safe drug; Porphyrias
Amiodarone (AD), a benzofurane derivative, was originally introduced in Europe for the treatment of cardiopathies and its potent antidysrhythmic properties are well recognized. Since 1970 it has been used for treating ventricular and supraventricular arrhythmias (Anson and Colin, 1987). AD is an efficient drug, however, it is considered the drug of last resort because of its many side effects, including hepatic, neurologic, thyroidal, pulmonary, ophthalmologic, dermatologic, and cardiovascular alterations. Nevertheless, in most patients AD is well-tolerated, although in some, its adverse actions can become very serious (Harris et al., 1983). From the dermatologic point of view, AD induces skin photosensitivity in 75% of patients, both light and dose dependent (Rappersberger et al., 1989). In 82% of pa-
* Corresponding author. Present address: Viamonte 1881 10A, 1056 Buenos Aires, Argentine. Tel.: 154 1 812 3357; Fax: 154 1 811 7447; E-mail: [email protected].
tients receiving AD, an increase in the activity of hepatic enzymes has been found. However, in most patients these anomalies are transitory and often return to basal levels in time; only 3% of patients develop severe and persistent clinical signs (Lewis et al., 1989; Kerin et al., 1989). In experimental animals, it has been shown that administration of high doses of AD leads to the formation of an inactive complex, Cyt P450-Fe(II)-AD, in liver microsomes, which can not bind CO. As a result, Cyt P450 levels are reduced and so is the activity of some monooxygenases (Larrey et al., 1986). The neurotoxic effects of AD depend on both the accumulated and the maintenance dose of the drug. High doses provoke neurological damage in 35–75% of patients, while low doses are much safer (Kerin et al., 1989). Porphyrias are known metabolic disorders of the heme pathway, each due to a partial primary deficiency of one of the seven enzymes of porphyrin biosynthesis, after 5-aminolevulinate synthetase (ALA-S). They are characterized by a specific pattern of accumulation and
0306-3623/99/$–see front matter 1999 Elsevier Science Inc. All rights reserved. PII: S0306-3623(98)00202-X
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Fig. 1. Control (h) and AD treated ( ) mice, for 1 week. In vivo measurements. (A) ALA and PBG concentration in urine; (B) Porphyrin concentration in urine, feces, and liver. Other experimental conditions as indicated in the text.
excretion of precursors, ALA, and porphobilinogen (PBG) and/or porphyrins. The excess of these metabolites is responsible for the clinical signs of these diseases, which can be either cutaneous or neurologic. Genetic or acquired porphyrias can become clinically symptomatic because of many factors, including certain drugs, toxic substances, fasting, and stress. This is why lists exist to classify drugs as either safe or unsafe for use in the treatment of associated pathologies in porphyric patients. Although AD has been identified for a long time as an unsafe porphyrinogenic drug, there is very little information about its action on heme metabolism, either in humans or animals. Because of the resemblance between some of its side effects and the clinical signs seen in porphyrias, the aim of the present work has been to study the action of AD on heme biosynthesis, both in vivo, by administrating AD to animals or in patients receiving the drug, and in vitro, by measuring different
heme parameters, in several tissues, in the presence of varying amounts of AD.
1. Materials and methods Reagents used were obtained from Merck, Mallinckroft, or Sigma Companies at Buenos Aires dealers. PBG was prepared according to Sancovich et al., 1970. AD was kindly provided by Dr. J.C. Pico from Roemmers Laboratories, Buenos Aires. For in vivo experiments AD (1.5 gm/100 ml) was dissolved in 7% Tween 80. For in vitro studies AD (30 mg/ml or 50 mg/ml) was dissolved in 10% methanol. 1.1. Animals, tissues, samples preparation For both in vivo and in vitro studies animals were obtained from the Animal House of the School of Sciences, University of Buenos Aires. They were kept on
Fig. 2. In vivo measurements of ALA-S, ALA-D, PBGase and PBG-D activities in liver of CF1 male mice. Control (h), AD treated ( ) during, (A) 1 week; and (B) 1 month. ALA-S and ALA-D specific activities expressed 3 105. Other experimental conditions as indicated in the text.
M. Mendez et al./General Pharmacology 32 (1999) 259–263
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Fig. 3. ALA-D in vitro activity in blood (h), liver ( ), and kidney ( ) of CF1 mice, measured in the presence of different concentrations of AD in the incubation system. Other experimental conditions as indicated in the text.
a Purina-3 diet (Molinos Rı´o de la Plata, Buenos Aires) and drank water ad libitum. For in vivo experiments, groups of CF1 male mice, each between 30 and 36 g body weight, were used. One group received daily 100 mg AD/kg, i.p. and the control group received the solvent only, for 1 week, 1 and 2 months. On the last day of treatment, animals were housed in metabolic cages to collect 24 h urine and feces and were fasted until killed. Studies were also performed with blood obtained from 17 cardiac patients under AD therapy for periods ranging from 3 months to 20 years and healthy volunteers. For in vitro experiments, blood from healthy volunteers and normal animals was used. One ml of heparinized blood was hemolized by adding 0.2 ml of 5% Triton-X-100 and 3.8 ml of 0.05 M Tris-HCl buffer pH 7.4. After collecting the blood, to measure ALA-S activity, a portion of liver was immediately excised, then the animal was perfused with saline solution; liver and kidney were extracted and frozen until processing; then part of the liver was separated for measuring porphyrin content and the remaining tissue and kidney were used to measure ALA dehydratase (ALA-D), porphobilinogenase (PBGase), and PBG-deaminase (PBG-D). To determine ALA-S, liver without perfusion was homogenized in a mixture of 9% NaCl (25 ml), 0.1 M EDTA (0.125 ml), and 1 M Tris-HCl buffer pH 7.4 (0.25 ml) at a ratio of 1:3 (wt:v). The resulting homogenate was used as enzyme source. To determine ALA-D, PBGase, and PBG-D, perfused liver and kidneys were homogenized in 0.25 M su-
Fig. 4. (A) ALA-D, (B) PBG-D, and (C) URO-D activities in vitro, in human blood, measured in the presence of different concentrations of AD in the incubation system. Other experimental conditions as indicated in the text.
crose (1:10, wt:v), centrifuged 15 min at 15,000 3 g and supernatant employed as enzyme source. 1.2. Determination of ALA, PBG, porphyrins and enzyme activities ALA and PBG were measured according to Mauzerall and Granick (1956). Urinary porphyrins were measured following methods described in Batlle (1998) and liver and fecal porphyrins were measured according to With (1975). Plasma porphyrins were estimated as described by Schoua and Batlle (1987) and total blood porphyrins following Polo et al. (1988). To quantify ALA and PBG in plasma, heparinized blood was centrifuged 15 min at 2,000 rpm at room temperature, then to 1 ml of plasma, 0.2 ml of 50% TCA was added, the mixture centrifuged for 15 min at 2,000 rpm. One ml of the resulting supernatant was taken to
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Fig. 5. Biochemical parameters in plasma and blood from cardiac patients. ALA (h); PBG ( ); Porphyrin Plasma Index (IPP) ( ); ALA-D ( ); PBGase ( ); PBG-D ( ); URO-D ( ); Total Porphyrins in Blood (PTS) (j). RBC: Red Blood Cells. Normal values were: ALA-D (nmole PBG/h. ml RBC): Female 0.794 6 0.247: Male: 0.688 6 0.232; PBGase (nmoles porphyrins/h. ml RBC) Female: 38.74 6 6.05; Male: 34.63 6 8.22; PBG-D (nmoles porphyrins/h. ml RBC): Female: 81.51 6 11.96; Male: 73.13 6 13.62; URO-D (nmoles Hepta 1 Hexa 1 Penta 1 Copro)/h. ml RBC: 4.00 6 0.25 (Substrate Uroporphyrinogen I). ALA (mg/ml plasma): 0.130 6 0.02; PBG (mg/ml plasma): 0.9 6 0.087; IPP: #1.3 (lem 5 615 nm) PTS (mg/100 RBC): 150 6 40.
pH 6–7 with 5 M NaOH, and then ALA and PBG were measured as indicated. ALA-S activity was estimated following Marver et al. (1966); ALA-D activity measured according to Batlle et al. (1967); PBGase and PBG-D activities as described by Batlle et al. (1978); uroporphyrinogen decarboxylase (URO-D) activity was studied following Batlle et al. (1985). One enzyme unit (EU) is defined as the amount of enzyme catalyzing the formation of 1 nmole of product under the standard conditions of incubation: Specific activity (Sp. Act.) as the number of EU per mg of protein. Protein concentration was estimated as described by Lowry et al. (1951). 1.3. Statistical analysis Data were statistically analyzed using nonpaired Student’s t-test, with Sigma Plot 2.0 statistical software for IBM. 2. Results and discussion 2.1. In vivo studies 2.1.1. Precursors and porphyrins in urine, liver and feces Neither the levels of urinary precursors (Fig. 1A), nor porphyrins (Fig. 1B), nor the levels of porphyrins in
feces and liver (Fig. 1B), were altered after one week of AD treatment. After one or two months of AD administration no changes were observed either (data not shown). 2.1.2. Enzyme activities ALA-S, ALA-D, PBGase and PBG-D activities were measured in animals receiving AD after 1 week and 1 month of treatment, in liver, blood, and kidney. Data obtained in liver are illustrated in Fig. 2, where it can be seen that no significant differences were found for any of the enzymes at either period. Similar findings were obtained in blood and kidney, even when treatment with AD was prolonged for 2 months (data not shown). 2.2. In vitro studies 2.2.1. ALA-D in tissues from mice Fig. 3 shows that there were not significant changes in the activity of ALA-D in either blood, liver, or kidney tissues from mice, when different concentrations of AD were added to the incubation system. 2.2.2. Enzyme activities from human blood Fig. 4 shows the activity levels of ALA-D (Fig. 4A), PBG-D (Fig. 4B) and URO-D (Fig. 4C) measured in normal human blood in the presence of varying concentrations of the drug. Once again, no significant modifi-
M. Mendez et al./General Pharmacology 32 (1999) 259–263
cations were found under any condition for any of the three enzymes. 2.2.3. Biochemical parameters in blood from cardiac patients Fig. 5 shows the levels of ALA, PBG, plasma, and total blood porphyrins, as well as erythrocyte ALA-D, PBGase, PBG-D, and URO-D activities in samples from 17 cardiac patients, including a true case of porphyria cutanea tarda (diagnosed 8 years before, now in remission); all of them were under AD treatment for different periods of time. In none of these patients were there any pathological alterations of any of the heme metabolism parameters tested. So far, only two other studies related to the use of AD and its effects on porphyrin metabolism have been published. Waitzer et al. (1987) and Parodi et al. (1988) found normal concentrations of porphyrins in patients who receive AD and developed photosensitization. They proposed that AD includes clinical pseudoporphyria without actually producing any biochemical disturbances in the heme pathway. Our results are in agreement with these authors and we clearly demonstrate that AD does not exhibit porphyrinogenic properties, in spite of having been classified as such. Therefore, it is important to emphasize that AD should be included among the safe drugs wich can be used for the treatment of other associated pathologies in the case of porphyric patients.
Acknowledgments AB and VEP are Superior and Associate Researcher at the Argentine National Research Council (CONICET). MM is a fellow from the same Council. The technical assistance of Mrs. V. Castillo, Mrs. B. Riccillo, and Lic. L. Dato is acknowledged. This work was supported by grants from the CONICET and the University of Buenos Aires. AB and RES are grateful to the Ministerio de Educacio´n y Ciencia (MEC) of Spain for special help.
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