Mahdy KA, Ahmed HH, Mannaa F, Abdel-Shaheed A. Clinical benefits of biochemical markers of bone turnover in Egyptian children with chronic liver diseases. World J Gastroenterol 2007; 13(5): 785-790 [PMID: 17278204 DOI: 10.3748/wjg.v13.i5.785]
Corresponding Author of This Article
Karam A Mahdy, Medical Biochemistry Department, National Research Centre, El Bohouth Street, Dokki 12311, Cairo, Egypt. karammahdy@yahoo.com
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Karam A Mahdy, Medical Biochemistry Department, National Research Centre, Egypt
Hanaa H Ahmed, Hormones Department, National Research Centre, Egypt
Fathia Mannaa, Medical Physiology Department, National Research Centre, Egypt
Azza Abdel-Shaheed, Child Health Department, National Research Centre, Egypt
ORCID number: $[AuthorORCIDs]
Author contributions: All authors contributed equally to the work.
Supported by the National Research Centre during the research plan from 2001-2004 by projects 5/4/5 and 5/5/5 in collaboration with National Liver Institute of Menoufyia University
Correspondence to: Karam A Mahdy, Medical Biochemistry Department, National Research Centre, El Bohouth Street, Dokki 12311, Cairo, Egypt. karammahdy@yahoo.com
Telephone: +2-2-3371499 Fax: +2-2-3370931
Received: August 23, 2006 Revised: October 8, 2006 Accepted: December 20, 2006 Published online: February 7, 2007
Abstract
AIM: To investigate the association between serum insulin-like growth factor 1 (IGF-1), osteocalcin, and parathyroid hormone (PTH) levels with the etiology and clinical condition of patients with chronic liver disease.
METHODS: Eighty children with hepatocellular damage were divided into 3 groups according to the etiology of disease infection: bilharziasis (9 patients), hepatitis B virus (HBV, 12 patients) and hepatitis C virus (HCV, 29 patients). The Child score index was found as A in 24 patients, B in 22 patients, C in 4 patients. Thirty healthy children served as control group. HBsAg, HBcAbIgM, HBcAbIgG, and anti-HCV were detected using ELISA technique. HCV-RNA was measured by reverse transcription polymerase chain reaction (RT-PCR). Anti-bilharzial antibodies were detected by indirect haem- agglutination test. Liver function tests were performed using autoanalyser. Serum IGF-1, osteocalcin and PTH levels were measured by ELISA technique. Abdominal ultrasonography was also conducted.
RESULTS: Serum IGF-1 level was significantly lower in all patient groups with liver diseases, while serum osteocalcin and PTH levels were significantly elevated in patients with HBV and HCV infections compared with the control group. Serum osteocalcin and PTH concentrations were measured with the severity of liver disease from Child A to C. Child A patients unexpectedly showed significantly reduced IGF-1 levels in comparison to patients staged as Child B or C. Serum osteocalcin level was negatively correlated with albumin (14.7 ± 0.54 vs 3.6 ± 0.10, P < 0.05), while that for PTH was positively correlated with total protein (70.1 ± 2.17 vs 6.7 ± 0.10, P < 0.05) in patients with HCV infection.
CONCLUSION: Low serum IGF-1 level seems to play a critical role in the bone loss in patients with chronic liver disease. Elevated biochemical markers of bone remodeling suggest high-turnover in patients with viral infection and reflect severity of the clinical stage.
Citation: Mahdy KA, Ahmed HH, Mannaa F, Abdel-Shaheed A. Clinical benefits of biochemical markers of bone turnover in Egyptian children with chronic liver diseases. World J Gastroenterol 2007; 13(5): 785-790
Bone tissues are metabolically active and undergo continuous remodeling throughout their life. Skeletal remodeling is achieved by two counteracting processes: bone formation which is accomplished through osteoblasts and bone resorption that is attributed to osteoclast activity[1]. Bone manifestations are well-known as extrahepatic complications of chronic liver disease[2]. Patients with chronic liver disease are at increased risk of developing hepatic osteodystrophy manifested as osteomalacia or osteoporosis[3]. Osteoporosis can be described as a disturbance in the bone remodeling process. Bone loss is a result of an imbalance of the bone remodeling process, where bone resorption exceeds bone formation[4].
The prevalence of osteoporosis in patients with chronic liver diseases ranges from 10% to 60%[5,6]. In general, secondary factors such as malabsorption and nutritional deficiencies may cause bone changes in chronic liver disease[7]. Insulin like growth factor (IGF) family is considered as important anabolic hormones, which play a role in anabolic metabolism and stimulating DNA synthesis, cell proliferation and meiotic division throughout life[8]. Since most circulating IGFs are synthesized by hepatocytes, lower levels of these parameters could be found in patients with liver diseases[8].
Regulation of bone metabolism is achieved by various factors such as mechanical motion, minerals, and hormones, all influencing bone turnover[4]. Osteocalcin is a protein produced by osteoblasts. Its level is reduced in the presence of osteodystrophia and can be recommended as a very sensitive and specific marker for bone formation/turnover[9].
Parathyroid hormone has a dual effect on bone cells. It can stimulate osteoblast activity and lead to substantial increase in bone density. In contrast, when secreted continuously at relatively high rates, as in hyperparathyroidism patients it can stimulate oseoclast-mediated bone resorption and suppress osteoblast activity[1]. The anabolic effect of parathyroid hormone (PTH) on osteoblasts is probably both direct and indirect via growth factors such as insulin like growth factor-1 (IGF-1) and transforming growth factor-β (TGF-β)[1].
However, the role of hepatocellular dysfunction in hepatic osteodystrophy is not clear. Therefore, this study aimed to clarify the influence of hepatocellular dysfunction on bone loss, and to outline the clinical benefits of controlling serum IGF-1, osteocalcin and parathyroid hormone levels in children with chronic liver diseases.
MATERIALS AND METHODS
Eighty children were enrolled in this study, from Liver Institute, Menoufyia University Inpatient and Outpatient Clinic and divided into: (1) Bilharziasis group: 9 patients (males whose age ranged from 6 to 15 years). (2) HBV group: 12 patients (10 males and 2 females whose age ranged from 3 to 15 years). (3) HCV group: 29 patients (22 males and 7 females whose age ranged from 2 to 16 years). (4) Control group: 30 apparently healthy children (22 males and 8 females whose age ranged from 3 to 15 years) with no history or clinical evidence of liver disease or any other diseases. (5) History was obtained from all individuals and clinical examination was carried out.
Specimen collection
Under complete aseptic condition, 5 mL of blood was taken from all the patients and controls. Each blood sample was divided into two portions: a small portion (1.8 mL) was taken on 3.8% Na-citrate as anticoagulant (0.02 mL ) to obtain plasma and a larger portion of blood sample was allowed to clot. Sera were separated and stored at -80°C till being tested, while the plasma sample was used immediately to measure prothrombin time and concentration.
Serological test
Hepatitis surface antigen (HBsAg), hepatitis B core antibody IgM (HBcAb IgM) and hepatitis B core antibody IgG (HBcAb IgG) were measured by ELISA technique using kits from Dia-Sorin Biomedica Co., according to the methods of Boniolo et al[10], Tedder and Wilson[11] and Hoofangle et al[12] respectively.
Anti-HCV was detected by third generation of ELISA using kit from the Biochem ImmunoSystem Inc[13], HCV-RNA extraction was carried out by reverse transcription polymerase chain reaction (RT-PCR) according to the method described by Ravaggi et al[14]. Anti-bilharzial antibodies were detected by indirect haem-agglutination test (IHA) according to the method of Hoshino et al[15], using kits of Fumoze-France.
Liver function tests
Serum AST, ALT, alkaline phosphatase (ALP), GGT , total and direct bilirubin, total protein, and albumin levels were measurd using EKTOCHEM 750XRC analyzer. Prothrombin time and concentration were also estimated in plasma according to the method of Poller[16].
Biochemical markers of bone turnover
Serum insulin like growth factor-1 (IGF-1), osteocalcin and PTH levels were measured by ELISA technique using kits of BioSource-Belgium[17-19].
Abdominal ultrasonography
To assess the size and echopattern of the liver, the size of the spleen and the presence of ascitis or any other abnormalities in the abdomen were detected using RT-X200 Prob 3.5 MHZ convex of General Electric Company -USA.
Statistical analysis
The obtained data are presented as mean ± SE. The difference between two groups was calculated using unpaired t-test, while the difference between more than two groups was calculated using one way analysis of variance ANOVA according to Snedecor and Cochran[20]. Least significant difference (LSD) test was used to compare the means of Child classification according to Walter and Duncan[21] at probability 0.05.
RESULTS
Patients with chronic liver diseases were divided into three groups according to the etiology: group 1 (9 patients with bilharziasis whose mean age was 10.8 ± 1 years), group 2 (12 patients with HBV whose mean age was 11.5 ± 0.6 years, and group 3 (29 patients with HCV infection whose mean age was 11.8 ± 1.5 years). Thirty children whose mean age was 10.5 ± 0.5 years and were sex matched with normal liver served as control group.
Table 1 depicts the results of serum IGF-1, osteocalcin and PTH levels in patients with either bilharziasis or HBV and HCV infections as well as in controls. The results revealed that patients with bilharziasis and those with either HBV or HCV infections showed a significant decrease (P < 0.01) in serum IGF-1 level compared to the controls. The most significant decrease in serum IGF-1 was recorded in patients with bilharziasis followed by HBV-infected and HCV-infected patients. Serum osteocalcin level showed a significant increase (P < 0.01) in patients with HBV and HCV infection compared to the controls. The most significant increase in serum osteocalcin level was observed in HBV-infected patients. Patients suffering from bilharziasis had no significant (P > 0.05) change in serum osteocalcin level compared to the controls. Serum PTH level was significantly increased in HBV-infected patients (P < 0.01) and in those with HCV infection (P < 0.05) compared to the controls. No significant change in serum PTH level was detected in patients with bilharziasis compared to the controls (Table 1).
Table 1 Serum IGF-1, osteocalcin and PTH levels in patient groups and control group (mean ± SE).
Liver function tests in patients with bilharziasis or HBV and HCV infection as well as in the control are presented in Table 2. Serum ALT and AST activities showed a significant (P < 0.05) increase in all patient groups compared to the control group. The most significant increase in serum ALT and AST activities was recorded in the patients suffering from HCVinfection. The activities of ALP, GGT and total bilirubin in serum revealed a significant increase (P < 0.05) in patients with HBV and HCV infection compared to the control, while no significant change (P > 0.05) was recorded in patients with bilharziasis as compared to the control group. Serum total protein level showed significant decrease (P < 0.05) in patients with HCV infection, whereas patients with either bilharziasis or HBV had no significant (P > 0.05) change as compared to the control (Table 2). Serum albumin level revealed a significant (P < 0.05) decrease in patients suffering from either HBV or HCV infection compared to the control. No significant change in serum albumin level was observed in patients with bilharziasis compared to the control. Patients suffering from either bilharziasis or HBV and HCV infection had a significant (P < 0.05) decrease in prothrombin concentration compared to the control. The most significant decrease in prothrombin concentration was detected in HCV-infected patients (Table 2).
Table 2 Liver function tests in different patient groups and control group (mean ± SE).
Alterations in serum IGF-1, osteocalcin and PTH level according to the severity of the disease from Child class A to C are depicted in Table 3. Significant (P < 0.05) change was detected in the three serum markers (IGF-1, osteocalcin and PTH) in each grade of the disease. The mean values of the three serum markers were markedly increased with the severity of the disease and the highest value was recorded in Child class C of the disease using LSD at probability 0.05.
Table 3 Alterations in serum IGF-1, osteocalcin and PTH levels according to the severity of the disease from class A to C (mean ± SE).
Noteworthy, our results revealed that serum IGF-1, osteocalcin and PTH levels were not age or sex dependent except for osteocalcin level which was higher in females (Table 4).
Table 4 Effect of age and sex on serum IGF-1, osteocalcin and PTH (mean ± SE).
On correlating the three bone markers with each other and liver function indices using Pearson correlation, there was only significant negative correlation between serum osteocalcin and albumin (r = -0.409, P = 0.027) and significant positive correlation between PTH and total protein (r = 0.451, P = 0.014) in patients with HCV infection (Figure 1A and B), while no significant correlation was recorded between IGF-1, PTH or osteocalcin in our patients.
Figure 1 Correlation coefficient between serum osteocalcin and albumin levels (A) & between serum PTH and total protein levels (B) in patients with HCV infection.
DISCUSSION
Patients with chronic liver disease are prone to develop hepatic osteodystrophy. In the majority of cases it is characterized by development of osteoporosis and/or osteomalacia with possible persistence of minor or major disability[3]. Osteoporosis reflects loss of bone (both matrix and its mineral) and osteomalacia is due to defective mineralization of osteoid[22]. Osteoporosis is a frequent complication of end-stage liver disease irrespective of its etiology. The prevalence varies between 9% and 60%, the highest being observed in cholestatic disorders and alcoholic liver disease[23].
The present study aimed at elucidating the influence of bilharziasis, hepatitis B or C infections on the development of osteoporosis in children. Serum levels of IGF-1, osteocalcin and PTH were measured and correlated with the routine liver functions as well as the child-pough score. The results of the present work showed that serum IGF-1 level was significantly decreased in all children infected with bilharziasis, HBV and HCV, while unexpectedly it showed significant increase with the severity of liver diseases from A to B or C classification but its level was still lower than that in the control.
These results are greatly supported by the finding of Hassan et al[24] who showed a significant reduction in the level of IGF-1 in children with schistosomiasis with or without hepatic fibrosis. Also, our results are in agreement with the findings of Orsini et al[25] who found similar results in hepatosplenic schistosomiasis patients and the results of Mazziotti et al[26] in patients with hepatocellular carcinoma (HCC) and hepatitis C virus-related cirrhosis. The reduction in IGF-1 level also can precede the diagnosis of HCC[26]. Similarly, decreased serum IGF-1 level has been recorded both in patients with viral cirrhosis[9,27] and in patients with hepatitis B infection without cirrhosis[28].
In schistosomiasis patients with hepatic fibrosis, the depressed circulation of IGF-1, free T4 and growth hormone may be responsible for stunted stature[24]. Moreover, low serum IGF-1 level in hepatosplenic schistosomiasis patients has been found to be related to the clinical form of hepatosplenic schistosomiasis[25]. The level of IGF-1 is elevated in patients with schistosomiasis with or without liver fibrosis and relates to its clinical form[25], suggesting that the severity of liver dysfunction can affect the level of IGF-1. As liver function of patients with bilharziasis is usually better than those with cirrhosis due to HBV and HCV infection and usually scored as child A, this may partly explain the relatively unexpected low level of IGF-1 in our patients with less sever liver affection (child A group).
The present study recorded a significant increase in serum osteocalcin level in patients with viral hepatitis infection only. Also a negative correlation was obtained with the level of serum albumin in patients with HCV infection. Moreover, serum osteocalcin level was significantly increased in child B and C patients than in child A patients.
It has been reported that serum osteocalcin level is higher in cirrhotic patients than in control, suggesting that cirrhotic patients have high bone turnover. In addition, osteodystrophy associated with hepatic cirrhosis is due to a defect in alpha-hydroxylation (1, 25(OH)2D) by the kidney due to the decrease in its primary substrate, the liver 25(OH)2D. This could be attributed to the reduced availability of vitamin D, inadequate conversion of vitamin D to 25(OH)2D, accelerated metabolism of 25(OH)2D, and urinary loss of 25(OH)2D with its transport protein[29]. However, some studies reported that serum osteocalcin level is low in cirrhotic patients and osteopenia in these patients is not due to a decrease in bone formation[30,31], but may be a consequence of hepatic osteodystrophy due to low plasma vitamin D and calcium levels[31].
Concerning the changes in serum PTH level, the present study recorded significant increase in patients with Hepatitis B and C infections. As regard a positive correlation was obtained with serum total protein level among the group of HCV infection. Moreover, serum PTH was significantly increased in class B and C patients than patients of class A.
In hepatocellular dysfunction some previous studies reported that serum PTH level is high in patients with primary biliary cirrhosis[32], liver cirrhosis[33], HBV infection[34], as well as in dogs with schistosomiasis[35] and in children with cholestatic and non-cholestatic liver disease[36]. On the other hand, other studies reported that PTH is unchanged in patients with chronic viral liver disease[37] and post hepatic cirrhosis[38]. Kirch and co-workers[33] have found a significant correlation between PTH and the parameters of liver functions such as prothrombin, albumin and bilirubin, suggesting that the increasing PTH level is related to liver dysfunction. This may explain why the elevation of PTH level is due to the impaired liver function rather than secondary hyperparathyroidism[33]. Moreover, elevated PTH-related protein was observed with hyperglobulinemia and hypoalbuminemia in dogs with schistosomiasis[35], and this may explain the obtained positive correlation between PTH and serum total protein levels in patients with HCV infection due to hyperglobulinemia observed in our study.
Hepatic osteodystrophy (HOD) begins at the stage of chronic non-cirrhotic liver injury and bone loss is connected with liver damage, suggesting that the principal pathogenesis of HOD is attributed to intestinal Ca-malabsorption as a result of low serum albumin and villous atrophy[39]. Lower ionized calcium resulting from deficient intestinal absorption due to low 25(OH)2D, leads to increase in PTH level[36].
The significant rise in serum osteocalcin level in our female patients indicated that their bone turnover was accelerated. This result is in agreement with Steinberg et al[40] who found that female sex is a risk factor for bone turnover and low bone density.
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