The Significance of Hepatobiliary Enzymes for Differentiating Liver and Bone Diseases : A Case Control Study from Manipal Teaching Hospital of Pokhara

Serum alkaline phosphatase (ALP) is a member of a family of zinc metalloprotein enzymes and is secreted mainly by the liver, bone, mucosal epithelia of small intestine, proximal convoluted tubule of kidney and placenta. High mitochondrial aspartate transaminase (AST) is seen in extensive tissue necrosis during myocardial infarction and also in chronic liver diseases like liver tissue deterioration and necrosis. Marked elevations of alanine transaminase (ALT) levels are observed with diseases that involve primarily hepatocytes such as viral hepatitis, ischemic liver injury (shock liver) and toxin-induced liver damage. Serum gamma –glutamyl transferase (γ-GT) activity is mainly attributed to the hepatobiliary system and most commonly raised in alcoholic liver disease. The objective of this study is to diagnose hepotobiliary and bone diseases with the facilitation of various biochemical markers as single enzyme lacks the specificity.


Background
The hepatobiliary enzymes varies substantially in viral hepatitis, extrahepatic cholestasis, Paget's disease and osteomalacia.Hepatitis types A, B, and C are the highest critical forms of viral hepatitis in the United States.Epidemics of hepatitis A and endemic transmission of both hepatitis B and hepatitis C is usually due to high-risk sexual behaviour and drug use.HCV infection was responsible for 1.3% of acute viral hepatitis in Nepal.The existence of numerous drug abusers in the country may result in the emergence of HCV as a vital cause of chronic liver disease in Nepal in future 1 .
Extrahepatic cholestasis occurs outside the liver.It can be caused by bile duct tumors, cysts, narrowing of the bile duct (strictures), stones in the common bile duct, pancreatitis, pancreatic tumor or pseudocyst, pressure on an organ due to a nearby mass or tumor and primary sclerosing cholangitis.In Asia, there is a much higher incidence of extrahepatic cholestasis than in Western countries 2 .Paget's disease of bone is a chronic disorder, characterized by focal areas of excessive osteoclastic bone resorption accompanied by a secondary increase in osteoblastic bone formation.Paget's disease of bone shows an increasing frequency of occurrence with age and also varies with genetic, cultural, environmental, social, racial differences.The prevalence of Paget's disease of bone is about 3% of people aged over 40; 10% by age 80 and prevalence rate for Paget's disease of bone is approximately 1 in 33 or 3.00%in the USA 3 .Osteomalacia is the softening of the bones caused by inadequate or delayed mineralization of osteoid in mature cortical and spongy bone secondary to inadequate amounts of available phosphorus and calcium.The causes of osteomalacia are lack of calcium and vitamin D in the diet, not enough exposure to sunlight, malabsorption of vitamin D by the intestines, use of very strong sunscreen, cancer, hereditary or acquired disorders of vitamin D metabolism, kidney failure and acidosis, liver disease, phosphate depletion associated with not enough phosphates in the diet and as side effects of medications used to treat seizures 4 .Serum alkaline phosphatase (ALP) is a member of a family of zinc metalloprotein enzymes and is expressed mainly from the liver, bone, mucosal epithelia of small intestine, proximal convoluted tubule of kidney, and placenta .Though ALT is found in kidney, heart and muscle, its greater concentration is in liver when compared to other tissues of the body.ALT is purely a cytoplasmic enzyme and it catalyses the transamination reaction.Marked elevations of ALT levels are observed with diseases that involve primarily hepatocytes such as viral hepatitis, ischemic liver injury (shock liver) and toxininduced liver damage 9 .γ-GT is a membrane bound enzyme present in hepatocytes and biliary epithelial cells, renal tubules, pancreas and intestine.It also helps in transport of peptides into the cell across the cell membrane and involves in glutathione metabolism 10 .Serum γ-GT activity is mainly attributed to hepatobiliary system and is most commonly seen in alcoholic liver disease.The objective of this study is to diagnose the hepotobiliary and bone diseases with the help of various biochemical markers as a single enzyme lacks specificity.

Study design and the participants
A hospital based case control study was carried out in the Department of Biochemistry of Manipal Teaching Hospital, Pokhara, Nepal.It was chosen for the study because Manipal teaching hospital is a tertiary care 825 bedded hospital and it was expected that most of the patients will come to this hospital from Western Nepal.

Data collection
The present study was undertaken between 1  11 .Estimation of serum alkaline phosphatases was done by standard enzymatic method 12 .
Assay of serum gamma-glutamyl transferase activity was based on the hydrolysis of the substrate L-gamma-glutamyl-3-carboxy-4-nitranilide 13 . All these laboratory parameters were analyzed using Human reagent kits and with the help of semi autoanalyser (Human, Germany).

Inclusion criteria
Patients with increased aminotransferase levels (greater than 1.5 times normal) for at least six months, presence of anti-HCV and anti-HBV in serum, minimal to nil alcohol consumption (less than 30 g/day for men and less than 20 g/day for women) with chief complaints related to vomiting, hepatomegaly, jaundice or ascites were included in our study.Extrahepatic cholestasis group comprised of 300 subjects with intraluminal, intramural or extraluminally obstruction.Patients have a diagnosis of Paget's disease, bone deformities and radiological signs of Osteomalacia were also included.Control group comprised of 300 healthy subjects with age and sex distribution similar to the clinical groups.

Exclusion criteria
Exclusion criteria were patients with history of an alcohol intake of more than 40 g per day, severe cardiac or renal disease, and active intravenous drug abuse.Patients receiving previous treatment with interferon or immunosuppressive agents or were taking medication that could cause steatosis (i.e.salicylates, nonsteroidal antiinflammatory drugs, corticosteroids, valproic acid, amiodarone, perhexiline maleate), treated with zoledronic acid who relapsed and was retreated with anti-resorptive bisphosphonate or calcitonin therapy within the last 12 months, Bisphosphonate hypersensitivity, patients with suspected/ proven metastases at retreatment, Active primary hyperparathyroidism, hypoparathyroidism, presence of tubulopathy, hypercalciuria were also excluded from the study.

Sample size calculation
For the sample size calculation with 95% confidence interval and significance level α = 5%.We conducted a pilot study of 100 cases each of all the diseases included in this study.In extra hepatic cholestasis, σ= SD of the ALP = 285, allowable error = 35, and required sample size was 255.In Paget's disease, σ= SD of the ALP = 220, allowable error = 25, and required sample size was 298.In Osteomalacia, σ= SD of the ALP = 200, allowable error = 24, and required sample size was 267.σ= SD of the ALP in Osteomalacia cases.In Viral hepatitis, σ=500, allowable error = 57, and required sample size was 296.σ= SD of the ALT in cases of viral hepatitis

Outcome variables
The levels of AST, ALT, ALP, γ-gt were assessed in cases of viral hepatits, extra hepatic cholestasis, Paget's disease osteomalacia and controls.

Explanatory variables
Factor at individual level were age, gender (male and female).

Ethical committee approval
Preceding the study, approval for the study was obtained from the institutional research ethical committee.

Data management and statistical analysis
Analysis was done using descriptive statistics and testing of hypothesis.The data was analyzed using Excel 2003, R 2.8.0, Statistical Package for the Social Sciences (SPSS) for Windows Version 16.0 (SPSS Inc; Chicago, IL, USA) and the EPI Info 3.5.1 Windows Version.The One way ANOVA was used to examine the statistical significant difference between groups.Post Hoc test LSD used for the comparison of means of control versus case groups.A p-value of <0.05 (two-tailed) was used to establish statistical significance.

Results
Of 1500 subjects enrolled in our study, 1200 were cases and rest 300 were controls.Cases taken were of viral hepatitis, extraheaptic cholestasis, Paget's disease and osteomalacia.For all subjects, mean values and confidence interval was calculated with all variables taken into our study.
Table 1 illustrates that values of AST and ALT were markedly raised in cases of viral hepatitis when compared to controls, Paget's disease, osteomalacia.The difference observed was found to be statistically significant (p Value).ALP was maximally raised in cases of Paget's disease 1434.8±219.5(CI1409.8,1459.8),extrahepatic cholestasis 907.5±282.7(CI875.5,939.7),osteomalacia 511.7±198.9(CI 489.2,534.4).ALP values were within normal limits in controls 140.2 ±28.4 (CI 137.0, 143.5).The difference in between cases and controls for ALP was found to be statistically significant.γ-GT values was found to be significantly raised in cases of extrahepatic cholestasis 205.6 ±44.0(CI 200.6,210.6)when compared to controls 18.4±3.9(CI17.9,18.9).The difference was found to be statistically significant.γ-GT values for viral when compared to controls.There was a statistically significant difference for γ-GT in cases of extrahepatic cholestasis when compared to controls. .The incidence of cholangiocarcinoma in US which remains the most frequent cause of extrahepatic cholestasis is approximately 0.8 per 100000 people per year.The annual incidence per 100000 population is 7.3 in Israel, 6.5 in American Indians and 5.5 amongst the Japanese 21 .Paget's disease is not known to exhibit a proclivity for any race.Nonetheless, remarkable patterns of prevalence have been illustrated.The prevalence of Paget disease is maximum in Europe (predominantly England, France, and Germany).The disease is infrequent in Asian countries, particularly China, India, and Malaysia, and in the Middle East and Africa [22][23] .The overall incidence of osteomalacia is 1 in every 1,000 individuals.Pregnancy and breastfeeding increase a woman's need for vitamin D and therefore increase the risk of deficiency.Women are affected slightly more often than men 24 .Isolated alterations of biochemical markers of liver damage can present a challenge for the clinician.Individual markers may correctly identify a cause but appear to have insufficient specificity and sensitivity .The mean values of ALP in extrahepatic cholestasis (907.6±SD282.8)was less than that of Paget's disease(1434.8±SD219.5).In Paget's disease, increased ALP was due to osteoblastic activity 33 .Likewise, in extrahepatic cholestasis, accumulating bile salts increase ALP synthesis and liberation from surface of bile duct epithelia 34 .Further, we found that values of ALP in cases of osteomalacia (511.8±SD198.9) was lesser than both Paget's disease and extrahepatic cholestasis.The level of ALP was within normal range in controls and cases of viral hepatitis.

γ-GT
γ-GT is a membrane bound enzyme present in hepatocytes and biliary epithelial cells.γ-GT was significantly raised in cases of extrahepatic cholestasis(205.6±SD44.1) when compared to viral hepatitis 19.5±SD3.8),Paget's disease(20.5±SD4.2),osteomalacia (19.0±SD3.7)and controls(18.4±SD3.9).Our results concurred with the findings of Castro-e-Silva et al 35 .An increase in γ-GT levels in patients with obstructive liver disease is associated with bile duct damage and fibrosis.Thus, because of its lack of specificity but high sensitivity for liver disease, γ-GT can be valuable for identifying causes of altered ALP levels, in concert with other biochemical abnormalities.An elevated alkaline phosphatase with normal γ-GT in cases of Paget's disease and osteomalacia stalwartly imply the bone origin.

Clinical relevance
Our study signifies that AST and ALT are the foremost specific markers of viral hepatitis.ALP is the main marker of Paget's disease and osteomalacia.γ-GT and ALP together are most important markers of extrahepatic cholestasis.Thus, deviation in levels of hepatobiliary enzymes precisely distinguishes between liver and bone diseases.

What this study adds
In regular clinical practices, there is an inclination of diagnosing bone and liver diseases with single hepatobiliary enzymes.The present study emphasizes that comparing the values of various hepatobiliary enzymes increase the sensitivity and specificity of accurately differentiating and obtaining the diagnosis of disease.

Future scope of study
Isoenzymes of different enzymes can be studied for the differentiation of liver and bone diseases.Moreover, by observing the serum levels of different enzymes, causes and diagnosis of hepatobiliary disorders and different types of liver diseases can be established.

Conclusion
It is not easy for clinicians to differentiate and diagnose liver and bone diseases correctly with single hepatobiliary enzyme.If a systematic approach is adopted, based on additional non-invasive serological tests and imaging procedures covering the most frequent liver diseases, the cause is often apparent.Correlation of variation in several biochemical markers increases the sensitivity and specificity for segregate and identification of liver and bone diseases for proper treatment and prognosis.

Table 2 : Detailed evaluation of mean values of variables in cases and controls of males
† p Value <0.001 statistical significance between groups Table2illustrates that in cases of males, variation in values of all variables were somewhat similar to those of total cases.Out of total 300 cases, 197 cases of viral hepatitis were males.In cases of Paget's disease, the number of males (169) suffering were more than females (131).The In cases of osteomalacia, ALP was moderately raised (491.6±199.0)andvalues were less than that of Paget's disease (1431.6±233.5)andextrahepatic cholestasis (910.9 ±281.7).γ-GT values was maximally increased in cases of extrahepatic cholestasis (210.4±45.1).The mean values of γ-GT was found to be in normal range in cases of viral hepatitis (20.1± 3.8) Paget's disease (21.1± 4.1) and osteomalacia(18.2±2.8).

Table 3 : Detailed evaluation of mean values of variables in cases and controls of females
† p Value <0.001 statistical significance between groupsTable 4 depicts that there was statistically significant difference for AST in cases of viral hepatitis when compared to controls.There was statistically significant difference for ALT in cases of viral hepatitis and extraheaptic cholestasis when compared to controls.There was statistically significant difference in ALP in cases of viral hepatitis, extrahepatic cholestasis, Paget's disease and osteomalacia † Nepal Journal of Epidemiology 2011;1(5): 153-59 Copyright © 2011 INEA Published online by NepJOL-INASP www.nepjol.info/index.php/NJE157

Discussion Prevalence and Diagnosis of viral hepatitis, extrahepatic cholestasis, Paget's disease, and osteomalacia
The mean age of patients of extrahepatic cholestasis 63.9±SD0.1 (63.5, 64.5) was little higher than those of viral hepatitis.Paget's disease is noticeably unusual in persons younger than 25 years and augments in occurrence with increasing age.Paget's disease mostly develops in persons in the fifth decade of life and is most frequently diagnosed in the sixth decade.The incidence of Paget's disease in persons older than 80 years is approximately 10%.The mean age of the patients in our study was 64.5±SD2.9(64.2,64.8)and it concurred with the selection of patients of Poor G et al 32PALP was highly elevated in cases of Paget's disease32