Pharmacological Research 48 (2003) 515–518
Increased excretion of urine coproporphyrins during daunorubicin administration in patients affected by acute myelogenous leukemia Arnaldo Pinelli a,∗ , Cirillo Mussini b , Boris Bertolini a , Marina Buratti c , Silvio Trivulzio a a
Department of Pharmacology, Chemotherapy and Medical Toxicology, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy b Department of Internal Medicine, University of Modena, Modena, Italy c Laboratorio di Tossicologia Professionale, Istituti Clinici di Perfezionamento, Milan, Italy Accepted 15 May 2003
Abstract Increased erythrocyte porphyrin values and high urine porphyrin levels have been reported in leukemic patients, but it is not clear whether the alteration in porphyrin metabolism is due to the leukemia or its treatment. The aim of this study was to compare porphyrin levels in leukemic patients undergoing chemotherapy or not. We analysed porphyrin values in patients with acute emyelogenous leukemia, who had or had not received chemotherapy according to Gale. Erythrocyte and urine porphyrin levels were increased as a result of the leukemic process, but urine coproporphyrins were further increased by daunorubicin treatment. These higher urine coproporphyrin levels were attributed to the activity of daunorubicin, which is known to interfere with the coproporphyrinogen decarboxylation process leading to the accumulation and high excretion of coproporphyrins in urine. © 2003 Elsevier Ltd. All rights reserved. Keywords: Acute myelogenous leukemia; Daunorubicin; Urine coproporphyrins; Erythrocyte protoporphyrins
1. Introduction There is considerable interest in the possible connection between modified porphyrin metabolism and neoplastic diseases . Altered porphyrin metabolism has been reported in leukemia patients in whom porphyrins may accumulate in erythrocytes or be excreted in urine. Increased urine excretion of coproporphyrins has been described in patients affected by various types of acute or chronic leukemias , and high levels of free protoporphyrins or coproporphyrins have been found in patients with acute myeloblastic, chronic myeloid or chronic lymphoid leukemias . These increased porphyrin levels have been attributed to reduced heme synthetase activity or deficient porphyrinogen decarboxylase [2,3]. However, as the porphyrin measurements were made in patients undergoing chemotherapy, it is not clear whether the metabolic alteration is due to the disease, its treatment or both. ∗ Corresponding author. Tel.: +39-02-5031-7054; fax: +39-02-5031-6949. E-mail address: [email protected]
1043-6618/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1043-6618(03)00190-7
The aim of this study was to investigate whether porphyrin levels are altered in patients with acute myelogenous leukemia, and further modified by antineoplastic treatment.
2. Materials and methods 2.1. Patients All of the patients and controls gave their informed consent to participate in the study, which was conducted in accordance with the recommendations of the Declaration of Helsinki and the local Ethics Committee. The patients were affected by acute myelogenous leukemia, which was diagnosed on the basis of a clinical examination and peripheral blood and bone marrow cytology and cytochemistry . Four patients were analysed as controls before the administration of antineoplastic treatment; a further six patients were treated with daunorubicin and ara-C according to Gale’s protocol . Daunorubicin intravenously administered at a dose of 45 mg/m2 of body surface area every day for 3 days, with the cycle being repeated every 21 days.
A. Pinelli et al. / Pharmacological Research 48 (2003) 515–518
2.2. Methods Blood and 24-h urine samples were collected from the controls and patients (on the third day of the first daunorubicin cycle). Urine coproporphyrins were detected as methyl esters using the method described by Pinelli and Gaspari . Erythrocyte protoporphyrins were extracted using a mixture of methanol and sulphuric acid (10:1) , and then also evaluated as methyl esters . The protoporphyrin and coproporphyrin data are expressed as mean values ± S.E., and the differences between the controls and leukemic patients, and between the treated and untreated patients, were evaluated using Tukey’s test . Statistical significance was assumed at ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
3. Results In the control subjects, average urine coproporphyrin excretion was 58.92 ± 12.64 g/24 h and the levels of erythrocyte protoporphyrins were 54.00 ± 12.19 g/100 ml RBC. The porphyrin values in the untreated leukemic patients appeared to be higher than in the control subjects: urine coproporphyrin levels were 98.70 ± 4.99 g/24 h and erythrocyte protoporphyrins were 105.25 ± 9.40 g/100 ml RBC.
Antineoplastic treatment increased coproporphyrin amount to 294.10 ± 33.74 g/24 h, significantly higher than the values observed in the untreated leukemic patients or controls. The treatment did not affect the levels of erythrocyte protoporphyrins: 92.92±11.39 g/100 ml RCB versus 105.25± 9.40 g/100 ml RCB in the untreated patients (Table 1). No signs of myocardial damage were observed in the patients when porphyrins were detected.
4. Discussion In comparison with the controls, erythrocyte protoporphyrin and urine coproporphyrin levels were clearly higher in all of the subjects with acute myelogenous leukemia, and paralleled the severity of the disease. Other authors have reported similar data in patients with leukemia. Altered erythrocyte porphyrin levels have been described in some leukemic patients , and increased urine coproporphyrin excretion was reported in subjects with various types of acute and chronic leukemias . The cause of these elevated porphyrin levels is not clear. Some authors have attributed them to an increase in porphyrin biosynthesis due to a marked decrease in heme content, thus leading to the stimulation of the enzymatic
Table 1 Levels of erythrocyte and urine porphyrins in controls and in patients affected by acute myelogenuos leukemia in absence or presence of daunorubicin administration
Controls 1 2 3 4 5
23 46 44 27 21
M M M F M
− − − − −
Leukemic patients 1 26 2 37 3 66 4 35 5 6 7 8 9 10
12 29 56 25 11 15
Erythrocyte protoporphyrins (g/100 ml RBC) 28 68 24 88 64
37 95 32.2 47.6 82.8
54.00 ± 12.19b
58.92 ± 12.64
M M M F
− − − −
118.5 117 107.5 78
105 88.2 92.4 109.2
M M M F M M
+ + + + + +
105.25 ± 9.40 75.5 78 127.5 123 57.5 96
98.70 ± 4.99 269.4 235.2 433.2 220.8 252 354
92.92 ± 11.39 Significance
Controls vs. leukemic untreated Controls vs. leukemic treated Leukemic untreated vs. leukemic treated a
294.10 ± 33.74
** * N.S.
* *** ***
The antineoplastic therapeutic scheme was performed according to the Gale’s protocol. Mean ± S.E. ∗ Significance levels: (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. b
Urine coproporphyrins (g/24 h)
A. Pinelli et al. / Pharmacological Research 48 (2003) 515–518
activities of the heme biosynthetic chain  as it appears from some reports [9,10]. Increased uroporphyrinogen synthetase activity and high levels of free protoporphyrins have been detected in the erythrocytes of patients with lymphoproliferative diseases , as well as augmented ALA-dehydrase and coproporphyrinogen synthetase activity have been described in children with acute lymphoblastic leukemia in whom high levels of urine coproporphyrins, uroporphyrins and their precursors ALA and PBG have also been found . It is important to note that the urine coproporphyrin levels observed in our untreated leukemic patients were significantly increased by daunorubicin-based antineoplastic therapy, thus indicating a cause/effect relationship. The elevated coproporphyrin levels may be due to daunorubicin, which has been reported to induce heme oxygenase  and to interfere with coproporphyrinogen decarboxylation, thus leading to an accumulation of non-metabolised coproporphyrins that are subsequently excreted in the urine . Furthermore, daunorubicin may affect the transfer of coproporphyrinogen to the mitochondria because it damages mitochondrial membranes and affects the coproporphyrinogen decarboxylation process associated with mitochondrial structures [13,14]. Anthracycline antibiotics react with cytochrome P-450 reductase in the presence of NADH to form semiquinone radical intermediates that interact with oxygen to produce superoxide anion radicals. These can generate hydrogen peroxide and hydroxyl radicals, both of which are highly destructive of cells [15–17]. Particularly, daunorubicin may also damage the heart, and may lead to the appearance of the toxic cardiac effects reported in varying percentages of patients [18,19] and in several experimental conditions [14,15,20–22]. Zanon and Lambertenghi-Deliliers have described severe degenerative alterations in the myocardial mitochondria of mice given a single high dose of daunorubicin . The redox cycling of anthracyclines by cardiac mitochondria leads to the formation of radicals by NADH dehydrogenase . Furthermore, superoxide anions are produced by doxorubicin analogues in the heart sarcosomes and mitochondria as a result of NADH dehydrogenase involvement ; mitochondrial cytochrome C oxidase has been shown to be a target site for daunorubicin in heart tissue . In our daunorubicin-treated subjects, we found higher urine coproporphyrin levels without any clinical signs of cardiac toxicity. As high urine coproporphyrin levels have been detected in patients with acute myocardial infarction [23,24], it can be hypothesised that an increase in urine coproporphyrin excretion may occur in patients treated with daunorubicin before the appearance of the biochemical signs (such as plasma cardiac necrotis markers)  and clinical symptoms of heart failure. As coproporphyrins have been demonstrated to be excreted in the urine during myocardial infarction [23,24] and as c-troponin I is released from damaged heart into blood stream , it would be interesting to perform an extensive
longitudinal study of daunorubicin administration in order to evaluate whether coproporphyrins may increase in plasma or urine either before the appearance of c-troponin I or in presence of this cardiac necrosis marker and may thus act as early indicator of heart damage.
References  McColl KE, Goldberg A. Abnormal porphyrin metabolism in diseases other than prophyria. Clin Haematol 1980;9:427–44.  Palma-Carlos AG, Palma-Carlos ML. Free erythrocyte porphyrins in leukaemia. Sangre 1975;20:127–33.  Lottsfeldt FI, Schwartz S, Krivit W. Hyperbaric oxygen, whole-body X irradiation, and cyclophosphamide combination therapy in mouse leukemia L1210. J Nat Cancer Inst 1966;36:37–43.  Hayhoe FGJ, Quaglino D. Haematological cytochemistry. London: Churchill Livingstone; 1980.  Gale RP. Advances in the treatment of acute myelogenous leukemia. New Engl J Med 1979;300:1189–99.  Pinelli A, Gaspari R. A new precise and sensitive method for the assay of urine porphyrins using combined thin-layer chromatography and spectrophotofluorimetric analysis. Clin Chim Acta 1972;39:135– 42.  Schwartz S, Berg MH, Bossenmayer I, Dinsmore H. Determination of porphyrins in biological materials. Glick D Methods Biochem Anal 1960;8:221–92.  Armitage P. Statistica medica, 10th ed. Milano: GG Feltrinelli; 1991.  el-Sharabasy MM, el-Waseef AM, Hafez MM, Salim SA. Porphyrin metabolism in some malignant diseases. Br J Cancer 1992;65:409– 12.  Epstein O, Lahav M, Schoenfeld N, Nemesh L, Shaklai M, Atsmon A. Erythrocyte uroporphyrinogen synthase activity as a possible diagnostic aid in the diagnosis of lymphoproliferative diseases. Cancer 1983;52:828–32.  Wissel PS, Drummond GS, Kappas A. Protective effect of Sn-protoporphyrin against doxorubicin-induced perturbations of heme metabolism. Life Sci 1990;47:1595–9.  Gajdos A, Gajdos-Ttorok M. Porphyrines et porphynes. Paris: Masson; 1969, p. 139.  Praet M, Pollakis G, Goormaghtigh E, Ruysschaert JM. Damages of the mitochondrial membrane in Adriamycin treated mice. Cancer Lett 1984;25:89–96.  Zanon PL, Lambertenghi-Deliliers G, Pozzoli EF, Nava M, Soligo DA, Praga C, et al. Selective mitochondrial alterations induced by a single dose of daunorubicin or 4-demethoxydaunorubicin in mouse ventricular myocardium. Tumori 1980;66:27–34.  Chabner BA, Ryan PP, Paz-Arez L, Garcia-Carbonero R, Calabresi P. Antineoblastic agents. In: Goodman Gilman’s Harman JG, Limbirds LL, editors. The pharmacological basis of therapeutics, 10th ed. McGraw-Hill, New York; 2001. p. 1427.  Pollakis G, Goormaghtigh E, Delmelle M, Lion Y, Ruysschaert JM. Adriamycin and derivatives interaction with the mitochondrial membrane: O2 consumption and free radicals formation. Res Commun Chem Pathol Pharmacol 1984;44:445–59.  Doroshow JH. Anthracycline antibiotic-stimulated superoxide, hydrogen peroxide, and hydroxyl radical production by NADH dehydrogenase. Cancer Res 1983;43:4543–51.  Bonadonna G, Monfardini S. Cardiac toxicity of daunorubicin. Lancet 1969;1:837.  Cowan JC, Baig MW, Tan LB. Disorders of the heart. In: Davies DM, editor. Textbook of adverse drug reactions, 4th ed. Oxford: Oxford University Press; 1991. p. 99–147.
A. Pinelli et al. / Pharmacological Research 48 (2003) 515–518
 Davies KJ, Doroshow JH. Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracycline radical formation by NADH dehydrogenase. J Biol Chem 1986;261:3060–7.  Gervasi PG, Agrillo MR, Lippi A, Bernardini N, Danesi R, Del Tacca M. Superoxide anion production by doxorubicin analogs in heart sarcosomes and by mitochondrial NADH dehydrogenase. Res Commun Chem Pathol Pharmacol 1990;67:101– 15.  Papadopoulou LC, Tsiftsoglou AS. Mitochondrial cytochrome c oxidase as a target site for daunomycin in K-562 cells and heart tissue. Cancer Res 1993;53:1072–8.  Koskelo P, Toivonen I. Separation of urinary coproporphyrin isomers 1 and 3 by thin-layer chromatography. Studies in healthy subjects
and patients with myocardial infarction. Scand J Clin Lab Invest 1966;18:543–9.  Koskelo P, Heikkila J. Urinary excretion of porphyrin precursors in myocardial infarction. Acta Med Scand 1965;178:681–6.  Adamcova M, Gersl V, Hrdina R, Melka M, Mazurova Y, Vavrova J, et al. Cardiac troponin T as a marker of myocardial damage caused by antineoplastic drugs in rabbits. J Cancer Res Clin Oncol 1999;125:268–74.  Bertinchant JP, Robert E, Polge A, Marty-Double C, Fabbro-Peray P, Poirey S, et al. Comparison of the diagnostic value of cardiac troponin I and T determination for detecting early myocardial damage and the relationship with histological findings after isoprenaline-induced cardiac injury in rats. Clin Chim Acta 2000;298:13–28.