Introduction

Chronic hepatitis due to persistent hepatitis C virus (HCV) infection may lead to liver cirrhosis (LC) and eventually hepatocellular carcinoma (HCC) [1]. To prevent these complications, maintaining serum alanine aminotransferase (ALT) levels as low as possible is considered very important [2].

Interferon therapy has been demonstrated to reduce the risk of development of LC and HCC and improve the survival of HCV-infected patients. However, some patients are unwilling to continue interferon therapy because of its unpleasant side effects and cost. Furthermore, interferon therapy has the potential to cause serious adverse effects, especially in elderly patients and in patients having arteriosclerosis, thrombocytopenia, or neuropsychological disorders. For such patients, hepatoprotective therapies, including regular intake of ursodeoxycholic acid (UDCA) and/or glycyrrhizin injection (GI), are considered, but these are sometimes insufficient for maintaining serum ALT levels within the normal range.

Recently, the usefulness of iron reduction therapy for HCV-infected patients has been established [3–7]. We hypothesized that the addition of minor bloodletting, named petit phlebotomy (PP), prior to each GI could adequately reduce serum ALT concentrations, and we planned a 12-month randomized prospective study to evaluate this.

Patients, material, and methods

Patients

From October 2004 to September 2007, 76 HCV-infected patients treated with regular GI at Showa Inan General Hospital and Iida Municipal Hospital were enrolled in this study after informed consent was obtained. All patients were positive for serum HCV-RNA and had demonstrated persistent elevation of serum ALT levels (>45 U/l) for more than 6 months. The exclusion criteria at entry were: (1) previous interferon therapy within 6 months; (2) serum ferritin levels of less than 20 ng/ml; (3) hemoglobin values of less than 11 g/dl; (4) serum albumin concentrations of less than 3.6 g/dl7; (5) decompensated LC; (6) malignancy complications or cardiac, pulmonary, renal, or hematological disease; and (7) pregnancy. All patients were treated with UDCA in addition to regular GI for more than 6 months before entry. These agents were not changed after entry. None of the patients had received regular administration of branched-chain amino acids, diuretics, or albumin infusion. The enrolled patients were assigned randomly to GI + PP or GI groups using sealed opaque envelopes.

All patients were advised to reduce their intake of iron-rich foods and were counseled by a registered dietitian. To aid with compliance, each patient was given a comprehensive list of iron-rich foods, as well as instructions on how to complete dietary records, which required a listing of all food and drink consumed over a 3-day period once every 4 months throughout treatment. Iron intake was assessed based on dietary records using the nutrition-analysis software BASIC-4 for Windows version 2.0 (Kagawa Nutrition University Publishing Division, Tokyo, Japan).

Body mass index (BMI) was calculated at entry. Patients were considered to have hypertension if their systolic/diastolic pressure was greater than 140/90 mmHg, or if they were taking anti-hypertensive drugs. Patients were considered to have diabetes if they had a fasting glucose level equal to or higher than 126 mg/dl, or if they were taking insulin or oral hypoglycemic drugs [8–10].

Diagnosis of LC

The initial diagnosis of LC was made from the histological findings of a percutaneous liver biopsy. In patients who refused liver biopsy, the diagnosis was made using a formula for estimating LC proposed by Ikeda et al. [11]. Diagnoses were then further corroborated by imaging findings, such as the presence of liver surface irregularities, swelling of the left or caudal lobes, the presence of splenomegaly, and the development of esophageal and/or gastric varices.

Procedure of PP

PP was performed before each regular GI for 12 months, using a 24- or 26-gauge needle. The volume of blood drawn was set at a total 1-week volume of 60 ml, divided equally by the number of GIs given; for example, in patients receiving GI twice a week, 30 ml of blood was removed prior to each GI. The primary PP endpoint was a serum ferritin level of less than 20 ng/ml and the secondary PP endpoint was a hemoglobin value of less than 11 g/dl. Upon reaching these endpoints, PP was suspended and conventional hepatoprotective therapies only were continued. If serum ferritin levels began to increase, PP was resumed to again lower them to 20 ng/ml or less. Other discontinuance criteria were the appearance of peripheral edema, ascites, HCC or other serious adverse events, or the patient’s refusal to continue PP.

Laboratory examination

Complete blood counts, including hemoglobin values and platelet counts, and biochemical parameters were measured monthly using standard automated analyzers. The amount of serum HCV-RNA was measured by the Amplicor monitoring method (Roche Diagnostic Systems, Basel, Switzerland).

Assessment of adverse effects

The presence of any adverse effects from PP was verified prior to every GI by a member of the nursing staff and monitored once a month by a medical interview and physical and blood examinations by a doctor.

Ethics

This study was carried out in accordance with the World Medical Association Helsinki Declaration, and was approved by the ethics committees of the applicable hospitals.

Statistics

Statistical analyses were performed using SPSS software 11.0J for Windows (SPSS, Chicago, IL, USA). Qualitative variables were expressed as percentages and were compared using the χ2 test. Quantitative data were expressed as mean ± SD and were compared using a paired or unpaired two-tailed Student’s t test. A P value of less than 0.05 was considered to be statistically significant.

Results

Effects of addition of PP

Of the 76 patients enrolled in this study, 2 patients in each group withdrew because of the appearance of HCC. The remaining 72 patients (36 in each group) completed the 12-month treatment. There were no differences in the baseline characteristics between the groups (Table 1). Twenty patients (56%) in the GI + PP group reached serum ferritin levels of less than 20 ng/ml at 7.2 months on average after starting PP. At the end of the study period, significant differences in serum aspartate aminotransferase (AST) and ALT levels, hemoglobin values, and iron profiles, were found between the groups (Table 2). Serum ALT levels were significantly decreased in the GI + PP group after the treatment period (from 67 ± 34 to 44 ± 14 U/l, P < 0.001; Fig. 1), but remained unchanged in the GI group (from 75 ± 33 to 71 ± 28 U/l). Due to the addition of the PP regime, hemoglobin values and serum ferritin levels were also decreased (from 14.0 ± 1.6 to 12.3 ± 1.7 g/dl and from 163 ± 127 to 25 ± 21 ng/ml, respectively, both P < 0.001). Serum albumin, cholinesterase (ChE), cholesterol, and α-fetoprotein levels did not differ between the two groups at the end of the treatment course (Table 2).

Table 1 Baseline characteristics of the patients
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Table 2 Characteristics of the patients at the end of the study
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Fig. 1
figure 1

Changes in serum alanine aminotransferase (ALT) levels. Data are expressed as mean ± SD. Statistical analysis was conducted using the two-tailed Student’s t test. Filled squares, Glycyrrhizin injection (GI) + petit phlebotomy (PP) group (n = 36); filled circles, GI group (n = 36); *P < 0.05 compared with the GI group at the same time point (unpaired t test); #P < 0.05 compared with the baseline in the GI + PP group (paired t test)

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In chronic hepatitis C patients receiving regular GI, the addition of PP significantly lowered serum ALT and ferritin levels (from 79 ± 46 to 43 ± 14 U/l and from 186 ± 172 to 26 ± 21 ng/ml, respectively, both P = 0.002; Table 3).

Table 3 Changes in the laboratory data of patients with chronic hepatitis C
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Effects of the addition of PP in patients with compensated LC

The additive effects of PP were also investigated in patients with HCV-related compensated LC. Serum ALT levels, as well as serum AST and ferritin levels and hemoglobin values, were markedly decreased in the GI + PP group after the treatment period (Table 4). These decreases were not seen in the 26 patients with LC in the GI group (Table 4). Significant reductions in serum albumin, ChE, or cholesterol concentrations were not observed by the addition of PP (Table 4).

Table 4 Changes in the laboratory data of patients with HCV-related cirrhosis
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Adverse effects of PP

PP was well tolerated. One patient complained of transient faintness just after PP, but immediately recovered without any treatment. No patients in the GI + PP group showed new signs of peripheral edema or ascites due to decreased serum albumin concentrations throughout the treatment.

Discussion

This study clearly demonstrates the usefulness of the addition of PP in HCV-infected patients with persistently abnormal ALT levels, regardless of repeated GI. Long-term GI is believed to be a very effective hepatoprotective therapy, but may sometimes be insufficient to normalize serum ALT levels. Our results show that the addition of PP to regular GI can further lower serum AST and ALT levels and reinforce hepatoprotection, with few adverse effects and at relatively little cost. Thus, PP can be performed in most HCV-infected patients, including elderly patients and those with arteriosclerosis, thrombocytopenia, or neuropsychological disorders.

From the standpoint of iron reduction therapy, this procedure is already considered to be quite useful. In a previous report [6], regular biweekly phlebotomies of 200 or 400 ml were successfully performed in chronic hepatitis C patients until serum ferritin levels reached 10 ng/ml. However, unpleasant symptoms associated with sudden blood loss, such as general fatigue, dizziness, and orthostatic hypotension, may sometimes appear, especially in elderly or female patients. Moreover, the thick needles (16–18 gauge) used in regular phlebotomies often cause stress from puncture pain. In contrast, a thin needle (24–26 gauge) was used for PP in our study, and the volume of blood removed was too small to noticeably affect systemic circulation (20–60 ml). Therefore, we presume that this method will be easily accepted and adhered to by many patients, even elderly ones and those with delicate superficial veins. We are now planning a future trial to compare the efficacy and adherence rates between PP and conventional phlebotomy.

It is noteworthy that PP was continued safely for 1 year in patients with compensated LC. In cirrhotic patients, conventional repeated phlebotomies are sometimes discontinued because of decreases in serum albumin concentrations; some adjustments, such as a reduction of the removed blood volume or extension of intervals between extractions, are required [7]. On the other hand, the extent of decreases in serum albumin, ChE, and cholesterol levels was minimal in PP. Thus, PP is presumed to be a safe and effective iron reduction therapy for patients with compensated LC as well.

Lastly, it has been reported that long-term intermittent GI successfully reduces the incidence of HCC in patients with HCV-related chronic liver disease [12]. Similar results have been found for long-term iron reduction therapy [13, 14]. Based on these findings, it is plausible that the combination of GI and PP could reduce the risk of HCC development more than GI alone. A long-term follow-up of the patients enrolled in the present study is needed to address this issue.

In conclusion, this study confirmed that addition of PP to regular GI successfully reduced serum aminotransferase and ferritin levels in patients with HCV-related chronic liver disease. Serious adverse events did not appear from PP, even in patients with compensated LC. For HCV-infected patients with persistently abnormal serum aminotransferase levels in spite of regular GI, the addition of PP just prior to every GI could become a promising therapeutic option.