Vox Sanguinis (2015) 108, 268–273 © 2014 International Society of Blood Transfusion DOI: 10.1111/vox.12223
ORIGINAL PAPER
Activity of cytochrome P450 1A2 in relation to hepatic iron accumulation in transfusion-dependent b-thalassaemia major patients E. Shteyer,1* I. Nitzan,1* A. Godfarb,2 N. Hemed3 & S. Revel-Vilk4 1
Pediatric Gastroenterology Unit, Hadassah-Hebrew University Medical Center, Jerusalem, Israel Hematology Department, Hadassah-Hebrew University Medical Center, Jerusalem, Israel 3 Liver Unit, Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem, Israel 4 Pediatric Hematology/Oncology Department, Hadassah-Hebrew University Medical Center, Jerusalem, Israel *the first two authors had equal contribution to the manuscript, 2
Background Cytochrome P450 1A2 (CYP1A2) is a cytochrome enzyme with a pivotal role in hepatic drug metabolism. Data from CYP1A2(-/-) mouse suggest that CYP1A2 plays a role in aspects of hepatic iron toxicity. The aim of this study was to assess the activity of CYP1A2 in relation to hepatic iron load in patients with transfusion-dependent b-thalassaemia major. Methods The 13C-methacetin continuous breath test was performed on 30 consecutive patients with transfusion-dependent b-thalassaemia major. CYP1A2 activity was measured by the rate at which the 13C substrate is metabolized and exhaled expressed as percentage dose recovery (PDR) per hour. CYP1A2 activity was correlated with clinical and laboratory parameters and hepatic iron accumulation by T2* magnetic resonance imaging (T2*MRI). Results Cytochrome P450 1A2 activity in patients with transfusion-dependent bthalassaemia major was positivity correlated with plasma ferritin levels. No correlation was found with age, duration and amount of red blood cell transfusion and type of iron chelation therapy. Low CYP1A2 activity was negatively associated with hepatic iron accumulation (T2*MRI ≤ 63 ms); adjusted odds ratio (OR; 95% CI) for hepatic iron accumulation in patients with low CYP1A2 activity was 0047 (0003–072; P = 0021). Of the six patients with decreased activity of CYP1A2, five had no hepatic iron accumulation and one had mild hepatic iron accumulation by T2*MRI.
Received: 1 July 2014, revised 10 October 2014, accepted 21 October 2014, published online 4 December 2014
Conclusion Activity of CYP1A2 is associated with hepatic iron accumulation in patients with transfusion-depended b-thalassaemia major. Further studies are needed to assess the exact role of CYP1A2 in iron metabolism in human. Key words: 13C-methacetin breath test, blood transfusion, cytochrome P450 1A2, iron overload, b-thalassaemia major.
Introduction
Correspondence: Shoshana Revel-Vilk, Pediatric Hematology/Oncology Department, Hadassah –Hebrew University Medical Center, POB 12000, Jerusalem 91200, Israel E-mail: [email protected]
268
Body stores of iron are carefully regulated except in hemochromatosis and disorders such as b-thalassaemia major (b-TM), in which case, patients receive regular red blood cell (RBC) transfusion. Under these conditions, normal iron homoeostasis fails to prevent the harmful accumulation of iron [1]. Patients with b-TM require regular
CYP1A2 and iron overload in thalassaemia 269
RBC transfusion to sustain life that results in progressive iron overload, affecting multiple organs. Untreated cardiac iron accumulation is the leading cause of death in patients with b-TM treated with regular RBC transfusion. In the liver, iron accumulation leads to increased production of reactive oxygen species (ROS), mitochondrial damage and progressive liver disease. Nevertheless, not all patients with iron overload will accumulate iron in the liver and progress to liver damage. Cytochrome P450 1A2 (CYP1A2) is a cytochrome enzyme that plays a pivotal role in the metabolism of many drugs. In relation to hepatic iron metabolism, CYP1A2 was shown to be essential for the development of uroporphyria and liver damage [2]. CYP1A2 (-/-) mice were resistant to injury from iron [3], showing differential damage that correlated with CYP1A2 levels [4]. Still, the direct link between CYP1A2 and iron metabolism in mice is not fully elucidated. Recent work suggests that uroporphyria may be CYP1A2 independent [5]. Although there is a great homology in CYP1A2 gene between human and mouse [6], in human, there is wide genotype variability and no definite genotype–phenotype correlation [7]. CYP1A2 activity in vivo can be measured selectively by various methods [8], including methacetin breath test, which we used in our study. Methacetin is rapidly metabolized into acetaminophen and 13CO2 by cytochrome CYP1A2 with a single O-dealkylation step and is considered a good substrate for evaluating cytochrome P450 enzyme activity [9]. The aim of this study was to assess the hepatic cytochrome CYP1A2 enzyme activity of children and adults with transfusion-depended b-TM using the 13C-methacetin breath test.
Materials and methods Patients The 13C-methacetin breath test was performed in consecutive patients with b-TM from the pediatric and adult Hematology Department at Hadassah Medical Center. The BreathID was done in the morning, prior to the scheduled RBC transfusion. Hospital records were reviewed, and the following data were recorded: age at the time of the study, age when starting regular RBC transfusion, amount of RBCs transfused (ml/kg/year) during the previous year, type of iron chelation therapy at the time of the study, hepatitis C virus (HCV) status and history of splenectomy. All adults or legal guardians gave written informed consent for participation in the study. The study was approved by the Institutional Review Board Committee and the Israel Ministry of Health Committee for Human Clinical Trials. © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 268–273
13
C-methacetin breath test
We used a novel system, the BreathID 13C-methacetin breath test (MBT, Exalenz, Israel), which is based on the measurement of 12CO2 and 13CO2 concentrations by molecular correlation spectroscopy (MCSTM) that can detect variations of 13 s (%) Type of iron chelation Deferoxamine Deferiprone Deferasirox More than one type of chelation in the last year
30 30 (1–60) 29 (08–54) 110 (12–250) 1090 (110–8223) 5 6 4 2 15 7
RBCs, red blood cells; ALT, alanine aminotransferase; PT, prothrombin time. a Median (range).
( 63 ms) in eight patients, mild iron accumulation (T2*MRI 27–63 ms) was found in eight patients, moderate iron accumulation (T2*MRI 14–27 ms) was found in two patients and severe iron accumulation (T2*MRI < 14 ms) was found in four patients. The results of the MBT tests in patients with no, mild and moderate/ severe hepatic iron accumulation is presented in Table 2. Of the six patients with decreased activity of CYP1A2, five had no hepatic iron accumulation and one had mild hepatic iron accumulation. All patients with moderate and severe hepatic iron accumulation had normal CYP1A2 activity. Low CYP1A2 activity was negatively associated with hepatic iron accumulation (T2*MRI ≤ 63 ms; Table 3). Hepatic iron accumulation was not associated with age, time from initiating regular RBC transfusion and amount of RBCs transfused over the previous year (Table 3). The odds ratio (OR; 95% CI) for T2*MRI ≤ 63 ms in patients with low CYP1A2 was 0047 (0003–072) adjusted for age, time from initiating regular RBC transfusion and amount of RBCs transfused over the previous year.
Discussion The aim of the current study was to assess the hepatic cytochrome CYP1A2 enzyme activity of children and © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 268–273
CYP1A2 and iron overload in thalassaemia 271
Table 2
13
C-methacetin breath test results in patients with no, mild, moderate/severe hepatic iron accumulation (measured by MRI T2*)
PDR peak, %/h Low PDR peak,
ORIGINAL PAPER
Activity of cytochrome P450 1A2 in relation to hepatic iron accumulation in transfusion-dependent b-thalassaemia major patients E. Shteyer,1* I. Nitzan,1* A. Godfarb,2 N. Hemed3 & S. Revel-Vilk4 1
Pediatric Gastroenterology Unit, Hadassah-Hebrew University Medical Center, Jerusalem, Israel Hematology Department, Hadassah-Hebrew University Medical Center, Jerusalem, Israel 3 Liver Unit, Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem, Israel 4 Pediatric Hematology/Oncology Department, Hadassah-Hebrew University Medical Center, Jerusalem, Israel *the first two authors had equal contribution to the manuscript, 2
Background Cytochrome P450 1A2 (CYP1A2) is a cytochrome enzyme with a pivotal role in hepatic drug metabolism. Data from CYP1A2(-/-) mouse suggest that CYP1A2 plays a role in aspects of hepatic iron toxicity. The aim of this study was to assess the activity of CYP1A2 in relation to hepatic iron load in patients with transfusion-dependent b-thalassaemia major. Methods The 13C-methacetin continuous breath test was performed on 30 consecutive patients with transfusion-dependent b-thalassaemia major. CYP1A2 activity was measured by the rate at which the 13C substrate is metabolized and exhaled expressed as percentage dose recovery (PDR) per hour. CYP1A2 activity was correlated with clinical and laboratory parameters and hepatic iron accumulation by T2* magnetic resonance imaging (T2*MRI). Results Cytochrome P450 1A2 activity in patients with transfusion-dependent bthalassaemia major was positivity correlated with plasma ferritin levels. No correlation was found with age, duration and amount of red blood cell transfusion and type of iron chelation therapy. Low CYP1A2 activity was negatively associated with hepatic iron accumulation (T2*MRI ≤ 63 ms); adjusted odds ratio (OR; 95% CI) for hepatic iron accumulation in patients with low CYP1A2 activity was 0047 (0003–072; P = 0021). Of the six patients with decreased activity of CYP1A2, five had no hepatic iron accumulation and one had mild hepatic iron accumulation by T2*MRI.
Received: 1 July 2014, revised 10 October 2014, accepted 21 October 2014, published online 4 December 2014
Conclusion Activity of CYP1A2 is associated with hepatic iron accumulation in patients with transfusion-depended b-thalassaemia major. Further studies are needed to assess the exact role of CYP1A2 in iron metabolism in human. Key words: 13C-methacetin breath test, blood transfusion, cytochrome P450 1A2, iron overload, b-thalassaemia major.
Introduction
Correspondence: Shoshana Revel-Vilk, Pediatric Hematology/Oncology Department, Hadassah –Hebrew University Medical Center, POB 12000, Jerusalem 91200, Israel E-mail: [email protected]
268
Body stores of iron are carefully regulated except in hemochromatosis and disorders such as b-thalassaemia major (b-TM), in which case, patients receive regular red blood cell (RBC) transfusion. Under these conditions, normal iron homoeostasis fails to prevent the harmful accumulation of iron [1]. Patients with b-TM require regular
CYP1A2 and iron overload in thalassaemia 269
RBC transfusion to sustain life that results in progressive iron overload, affecting multiple organs. Untreated cardiac iron accumulation is the leading cause of death in patients with b-TM treated with regular RBC transfusion. In the liver, iron accumulation leads to increased production of reactive oxygen species (ROS), mitochondrial damage and progressive liver disease. Nevertheless, not all patients with iron overload will accumulate iron in the liver and progress to liver damage. Cytochrome P450 1A2 (CYP1A2) is a cytochrome enzyme that plays a pivotal role in the metabolism of many drugs. In relation to hepatic iron metabolism, CYP1A2 was shown to be essential for the development of uroporphyria and liver damage [2]. CYP1A2 (-/-) mice were resistant to injury from iron [3], showing differential damage that correlated with CYP1A2 levels [4]. Still, the direct link between CYP1A2 and iron metabolism in mice is not fully elucidated. Recent work suggests that uroporphyria may be CYP1A2 independent [5]. Although there is a great homology in CYP1A2 gene between human and mouse [6], in human, there is wide genotype variability and no definite genotype–phenotype correlation [7]. CYP1A2 activity in vivo can be measured selectively by various methods [8], including methacetin breath test, which we used in our study. Methacetin is rapidly metabolized into acetaminophen and 13CO2 by cytochrome CYP1A2 with a single O-dealkylation step and is considered a good substrate for evaluating cytochrome P450 enzyme activity [9]. The aim of this study was to assess the hepatic cytochrome CYP1A2 enzyme activity of children and adults with transfusion-depended b-TM using the 13C-methacetin breath test.
Materials and methods Patients The 13C-methacetin breath test was performed in consecutive patients with b-TM from the pediatric and adult Hematology Department at Hadassah Medical Center. The BreathID was done in the morning, prior to the scheduled RBC transfusion. Hospital records were reviewed, and the following data were recorded: age at the time of the study, age when starting regular RBC transfusion, amount of RBCs transfused (ml/kg/year) during the previous year, type of iron chelation therapy at the time of the study, hepatitis C virus (HCV) status and history of splenectomy. All adults or legal guardians gave written informed consent for participation in the study. The study was approved by the Institutional Review Board Committee and the Israel Ministry of Health Committee for Human Clinical Trials. © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 268–273
13
C-methacetin breath test
We used a novel system, the BreathID 13C-methacetin breath test (MBT, Exalenz, Israel), which is based on the measurement of 12CO2 and 13CO2 concentrations by molecular correlation spectroscopy (MCSTM) that can detect variations of 13 s (%) Type of iron chelation Deferoxamine Deferiprone Deferasirox More than one type of chelation in the last year
30 30 (1–60) 29 (08–54) 110 (12–250) 1090 (110–8223) 5 6 4 2 15 7
RBCs, red blood cells; ALT, alanine aminotransferase; PT, prothrombin time. a Median (range).
( 63 ms) in eight patients, mild iron accumulation (T2*MRI 27–63 ms) was found in eight patients, moderate iron accumulation (T2*MRI 14–27 ms) was found in two patients and severe iron accumulation (T2*MRI < 14 ms) was found in four patients. The results of the MBT tests in patients with no, mild and moderate/ severe hepatic iron accumulation is presented in Table 2. Of the six patients with decreased activity of CYP1A2, five had no hepatic iron accumulation and one had mild hepatic iron accumulation. All patients with moderate and severe hepatic iron accumulation had normal CYP1A2 activity. Low CYP1A2 activity was negatively associated with hepatic iron accumulation (T2*MRI ≤ 63 ms; Table 3). Hepatic iron accumulation was not associated with age, time from initiating regular RBC transfusion and amount of RBCs transfused over the previous year (Table 3). The odds ratio (OR; 95% CI) for T2*MRI ≤ 63 ms in patients with low CYP1A2 was 0047 (0003–072) adjusted for age, time from initiating regular RBC transfusion and amount of RBCs transfused over the previous year.
Discussion The aim of the current study was to assess the hepatic cytochrome CYP1A2 enzyme activity of children and © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 268–273
CYP1A2 and iron overload in thalassaemia 271
Table 2
13
C-methacetin breath test results in patients with no, mild, moderate/severe hepatic iron accumulation (measured by MRI T2*)
PDR peak, %/h Low PDR peak,
