1.1. Overview of Hepatitis
Hepatitis generally means inflammation of liver. Hepatitis is caused due to use of certain drugs, toxins, heavy use of alcohol, bacterial and viral infections. Hepatic diseases are a major fear for severe health related problems throughout the world. Viral hepatitis is the name of family which is caused by different viral infections. Viral Hepatitis is classified into different types such as Hepatitis A, Hepatitis B, Hepatitis C, etc. In various researches data predicted that East Asia is the most affected region of the world with hepatitis due to certain economic and social factors. Hepatitis C is one of the primary causes of illness and mortality in Pakistan and other developing countries of world. Among them Hepatitis B and Hepatitis C are blood borne. Most common causes to become infected with HCV virus are sharing of shaving blades, needles, syringes, or other surgical equipment to inject drugs in various health care settings or hospitals or baby born by an infected mother who has already infected with Hepatitis C .
1.2. Pathophysiology of Hepatitis C
Researchers collect data worldwide as Hepatitis C virus infects about 150 - 170 million people and among them chronic infection occurs in almost 40% - 80% of cases and then it often centrals to cirrhosis, chronic liver inflammation, liver necrosis or hepatocellular carcinoma. Existence cycle of HCV is not unstated properly due to lack of productive cell culture system. Some external factors increase risk of cirrhosis, such as chronic alcohol depletion and viral infections. Moreover participative role of HCV proteins is also not clearly understood in hepatic cell carcinogenesis. Further progress by the help of research work and experimental studies in understanding of HCV infection waits for the advent of new technologies and health model system .
1.3. Physiology of Hepatitis C and Liver Functions
The physiology of the liver contains basic contents such as its metabolism, excretion, and body defense. In terms of cellular functioning, the liver is the elementary place of multiple biochemical responses which are very essential to the human organism including synthesis, degradation, transformation and biotransformation of certain substances or other biomolecules. The relationship between structure and functions of specific hepatic processes which control normal liver activities under normal conditions is essential for understanding of liver immune responses to observe clinical diseases distressing the liver normal physiology. The overview of hepatic physiology underline some of common facts of regular hepatic anatomy and functions of liver in relation to the physician challenging liver abnormalities and its diverse extents necessary for treating liver abnormalities for patient’s benefit in future .
1.4. Significance of Liver Function Tests in Hepatitis
Many people with hepatitis C feel physically well and healthy and have no significant findings on routine physical examination that would predicts a health care provider to suspect and diagnose liver disease. Majority of people with hepatic disease that leads to cirrhosis have a normal physical examination and have a healthy appearance but the diagnosis, evaluation, findings, clinical laboratory reports and treatment of liver disease particularly in hepatitis C places a large necessity on laboratory tests results (especially liver function tests) to diagnose, predict and evaluate response to therapy.
The liver performs several functions and collectively it is called the body's manufacturing center and filtering plant. Different blood tests used to evaluate abnormal liver conditions can be divided into those which represent liver cell damage or cholestasis. In various cases, the damage to the hepatocytes or liver cell causes elevation in these enzymes as compared to their normal values and the degree of elevation is very important in diagnosis of liver damage in acute disease but is not much important in chronic diseased condition. The reasons of elevated levels of amino transferases are basically fatty liver, autoimmune hepatitis, medication induced hepatitis, viral hepatitis, liver necrosis and alcoholic liver disease so an abnormality or modification in these liver function tests does not mean that liver is not functioning properly .
In fact mostly patients with elevated levels of amino transaminases, have normal liver function tests and have no alterations seen in them as compared to normal values.In most cases abnormality seen in chronic hepatitis C infection is often an elevated level of enzyme termed as alanine aminotransferase (ALT) but it was also observed in 60% of patients infected with hepatitis C have a normal transaminase level in them in any stage of hepatitis. The elevation in level of serum ALT does not associate with specified disease condition and it might be normal in any stage of chronic hepatitis C. Innovative researches on hepatitis C indicates that an increase in ALP and total bilirubin as well as thrombocytopenia (low platelets) are also observed along with other abnormalities seen due to elevated levels of liver function enzymes and other parameters .
1.5. Effect of Ascorbic Acid (Vitamin C) on Human Health
Vitamin C has a vital role in metabolism of protein and correlated with collagen synthesis which is necessary element of connective tissues which plays an important role in wound healing. In addition to its metabolic functions it was suggested that vitamin C plays a major role in advancement of immune functions and improves the absorption of ferrous form of iron in RBC’s.
Inadequate intake of vitamin C causes vitamin C deficiency disease known as scurvy(scorbutus), which is characterized by mouth and gum lesions, bleeding through lips, lethargy or lassitude, gums inflammation, severe connective tissue weakness, deferment in wound healing and capillary fragility .
2. Sample Groupings
Experimental Patients are divided into 2 groups;
1) Group A: 50 hepatitis C patients selected randomly who were not treated with ascorbic acid supplementation but they were receiving their anti HCV treatment. Their liver function test parameters such as serum alanine aminotransferase, serum aspartate aminotransferase, serum alkaline phosphatase, serum total bilirubin, direct bilirubin, indirect bilirubin, serum proteins (total, albumin, globulin, A/G ratio) and serum ascorbic acid was estimated by using appropriate method. Then these values were again estimated after one month and compare pre values with their post values.
2) Group B: 50 HCV positive patients were selected randomly for vitamin C supplementation and then their liver function test parameters such as serum alanine aminotransferase, serum aspartate aminotransferase, serum alkaline phosphatase, serum total bilirubin, direct bilirubin, indirect bilirubin, serum total protein, albumin, globulin, A/G and serum ascorbic acid were estimated by using appropriate methods before and one month after intake of vitamin C supplementation.
3. Research Methodology
To perform liver function tests, blood sample of normal individuals and HCV patients were taken randomly from local private sector hospitals who followed inclusion criteria. Patients were recruited, by convenience purposive sampling, to the study with polymerase chain reaction (PCR) confirm Hepatitis C finding. Inclusion criteria included male and female patients of age 25 to 65 years with detectable HCV RNA in serum by PCR, elevated ALT (>80 IU/L), and who were put on anti-viral treatment. Exclusion criteria included the patients with any history of Peg-interferon treatment, presence of any other form of liver disease (including viral hepatitis A and B), decompensated cirrhosis or concomitant disease such as Diabetes and other hormonal diseases. It also included the use of hepato-tonic drugs, silymarin, garlic oil, usage within 2 weeks period, pregnancy or lactation and refusal to participate in the study. The recruited patients were divided in two groups and then evaluate liver function parameters such as serum alanine aminotransferase, serum aspartate aminotransferase, serum alkaline phosphatase, serum total bilirubin, direct bilirubin, indirect bilirubin, serum total protein, albumin, globulin and A/G ratio were evaluated by using appropriate methods as described below. Appropriate techniques of handling the samples, chemicals and laboratory instruments were used in whole research work.
4. Statistical Analysis
The results of this research study were expressed as mean ± S.D, standard error of the mean (SEM) and percentage where applicable statistical analysis was performed by using IBM SPSS software (statistical package of social sciences).The ‘independent sample t test’ was used for analysis of independent variables and ‘paired t test’ were used to compare means of two groups (normal individuals vs. HCV patients group).To prove study hypothesis Values p < 0.01 considered highly significant, p < 0.05 were considered significant and p > 0.05 were considered insignificant .
5. Results and Discussion
Data composed from different study groups such as normal individuals and hepatitis C patients (with and without intake of supplementation) were collected before intake of supplementation and observed parameters were again accessed after one month of supplementation intake to evaluate consequences of ascorbic acid supplementation on different LFT parameters. Data was collected after permission granted by local ethical committee of University of Sargodha because ascorbic acid is vitamin supplement and has not any serious side effect on health and do not interfere with hepatitis medication treatment. The concentrations of serum ALT, serum AST, serum ALP, serum bilirubin levels and serum proteins (total protein, albumin, globulin, A/G) were assessed and difference in LFT levels were evaluated before ascorbic acid supplementation intake and after supplementation intake (Appendix 1 and Appendix 2).
5.1. Consequence of Ascorbic Acid Supplementation on LFT Parameters
Due to this fact that ascorbic acid reduces hepatocytes inflammation and increase antioxidant capacity, present study was based on evaluation of protective effects of ascorbic acid supplementation in hepatitis C patients. According to various clinical data it was observed that in normal individuals ascorbic acid supplementation produced certain healthy effects as well as use of these supplementation improve serum ascorbic acid level which was decreased markedly in some individuals due to lower consumption of vitamin C or deficiency of serum ascorbic acid in them in normal daily routine. Moreover, different serum liver function parameters were also improved by use of ascorbic acid supplementation especially bilirubin and serum ALT level in HCV patients as shown in their clinical values compared to pretest values before intake of supplementation.
5.2. Effect of Ascorbic Acid Supplementation on Serum Alt
Mean ± SD of serum ALT calculated as (195 ± 65.715) before intake of supplementation and Mean ± SD of serum ALT (155 ± 40.854) after intake of supplementation and p value observed as 0.042 which indicate that p value is significant because p < 0.05. As it was shown that means of the two levels lies in appropriate range and from direction of t-value, we can determine that there was a significant improvement in ascorbic acid level following the intake of ascorbic acid supplementation.
It was observed that ALT level was much elevated in HCV patients 195 ± 65.715 before use of ascorbic acid supplementation due to certain reasons which cause liver toxicity and in association to hepatotoxicity liver enzymes become elevated than their normal levels. After use of ascorbic acid supplementation by hepatitis C patients, it was observed that ALT enzymatic level markedly decreased and shifted towards lower levels which indicate that ascorbic acid has a potential beneficial effect on elevated level of ALT in hepatitis C patients and improves abnormal transaminase levels in HCV patients as shown in Figure 1.
In another study ALT levels were observed in patients having hepatic complaints. 45 patients were observed for 6 months of supplementation therapy with antioxidant vitamin without any substantial side effects. Vitamins supplementation treatment resulted in a statistically significant progress in elevated aminotransferase level because p = 0.002. But there was no perfection in necrotic inflammatory activity or ALT activity in chronic cases with this combination therapy . These observations support our results due to significant observed values.
5.3. Effect of Ascorbic Acid Supplementation on Serum AST
Mean ± S.D was 114.12 ± 31.18 before supplementation and then after one month of supplementation intake, the observed value of AST was maintained as
Figure 1. Effect of ascorbic acid supplementation on serum ALT.
102.15 ± 24.42 and p values were p < 0.048. 50 HCV patients take vitamin C supplementation for one month. Their Mean ± SD of serum AST calculated as (105 ± 43.217) before intake of supplementation and Mean ± SD of serum AST (85 ± 38.054) after intake as shown in Figure 2.
Studies recommend that anti-oxidative supplements may obstruct in advancement of HCV, to improve LFT levels and to reduce interferon antiviral therapy more effective. Some other studies also suggested that intake of antioxidants reduce LFT levels in HCV patients. In the existing research, vitamin C was linked with decline in serum liver enzymes levels of in patients who revealed increased levels before the supplementation involvement. Administration of vitamin C induced a significant reduction also in other liver enzymes such as serum AST, ALT, ALP (p < 0.04) levels along with reducing oxidative stress induced by hepatotoxicity and signifying that this antioxidant vitamin protect against liver damage and elevated LFT levels induced by hepatotoxicity .
5.4. Influence of Ascorbic Acid Supplementation on Serum ALP
Mean ± SD of serum ALP calculated before use of supplementation in HCV patients group without any intake of vitamin C supplementation was (212 ± 36.76) and after supplementation it was observed as (209 ± 32.408) and p values is (0.054).These results shown that only a little substantial change was observed on serum ALP level in these individuals without intake of ascorbic acid.
Serum ALP level of 50 HCV patients was observed before and after intake of vitamin C supplementation for one month. Their Mean ± SD of serum ALP calculated as (215 ± 35.213) before intake of supplementation and Mean ± SD of serum ALP enzyme was (185 ± 29.39) after intake of supplementation as shown in Figure 3 and p values observed as 0.051 which indicate that p values are nonsignificant for ALP because p < 0.05.
5.5. Effect of Ascorbic Acid Supplementation on Serum Bilirubin Levels
It was observed in present study that increased values of total bilirubin, direct
Figure 2. Effect of ascorbic acid supplementation on serum AST level.
bilirubin and indirect bilirubin can be transferred towards normal reference range after intake of vitamin C supplementation for prescribed time period and when comparison made between observed values of bilirubin before administration of vitamin C and after one month use of vitamin C it indicated slight difference between these two values. Because mean ± SD of total bilirubin was 2.409 ± 0.365 before supplementation and it became 2.207 ± 0.654 after one month of supplementation and p < 0.046. The consumption of vitamin C supplementation has a mild affective benefit on bilirubin level in HCV infected individuals because p values were not much clearly significant.
The results for Mean ± SD of serum total bilirubin calculated as (2.409 ± 0.365) before intake of supplementation and Mean ± SD of serum total bilirubin was (2.207 ± 0.654) after intake of supplementation and p values observed as 0.046 which indicate that p values are significant. Similarly, calculated values of Mean ± SD of serum direct bilirubin observed as (0.986 ± 0.356) before intake of supplementation and Mean ± SD of serum direct bilirubin was (0.812 ± 0.20) after intake of supplementation and p values observed as 0.048. Mean ± SD of serum indirect bilirubin observed as (1.423 ± 0.432) before intake of supplementation and Mean ± SD of serum indirect bilirubin was (1.395 ± 0.134) after intake of supplementation and p values observed as 0.053 and these observations are mentioned in Figure 4. These observations showed that there was no clearly significant change observed in serum direct bilirubin and indirect bilirubin.
5.6. Effect of Ascorbic Acid Supplementation on Serum Protein Levels
Serum total protein, albumin and globulin values were observed in 50 HCV patients before and after intake of vitamin C supplementation for one month. Mean ± SD of serum total protein shown in Figure 5, calculated as (6.561 ± 0.321) before intake of supplementation and Mean ± SD of serum ascorbic acid is (6.741 ± 0.426).
Mean ± SD of serum albumin shown in Figure 6 calculated as (3.510 ± 0.4633) before intake of supplementation and Mean ± SD of serum albumin is
Figure 3. Effect of ascorbic acid on serum ALP level.
Figure 4. Effect of ascorbic acid on serum bilirubin.
Figure 5. Effect of ascorbic acid on serum total protein.
Figure 6. Effect of ascorbic acid on serum albumin level
(3.710 ± 0.4987) after intake of supplementation and p values observed as 0.053. Mean ± SD of serum total globulin is calculated as (2.951 ± 0.3853) before intake of supplementation and Mean ± SD of serum globulin after ascorbic acid supplementation was calculated as(2.752 ± 0.4987) and p values observed as 0.055 which indicate that p values are not significant for serum globulin because p < 0.05. Mean ± SD of A/G observed as (1.189 ± 0.275) before intake of supplementation and Mean ± SD of A/G after supplementation was (1.348 ± 0.364) and p values observed as 0.056 (shown in Figure 7) which indicate that p values are not significant and finally it was observed that t-test values for serum total
Figure 7. Effect of ascorbic acid on serum globulin level.
proteins proves to be insignificant and intake of vitamin C supplementation produce a slight improvement in levels of serum protein in HCV infected patients.
Dietary vitamin C supplements markedly decline endogenous level of protein related oxidative damage related to liver. Assessments presented that administration of monosodium glutamate at various measure levels of 0.6 mg and then 6, 12, 30 and 60 mg/kg for 2 weeks increased serum ALT and AST .
Another study indicated that antioxidants levels of vitamins A, C and E were decreased (P < 0.001) paralleled to controls. The indicators of lipid peroxidation, antioxidant status, antioxidant activities and liver function were observed in blood and urine chronic hepatitis C patients. Oxidative stress shown in blood and urine due to antioxidant markers and it is a momentous feature of hepatitis C infection. Clear evidence of oxidative strain in non-cirrhotic patients was observed more. Therefore, it may be suggested that antioxidant remedy have a role in reducing disease advancement to hepatic and cirrhotic patients .
7. Comparison between Different LFT Parameters
Serum ascorbic acid supplementation was given in Hepatitis C patients for one month and selected parameters were observed in both groups classified as hepatitis C patients with supplementation and without supplementation. Sampling techniques, dose of supplementation, duration of supplementation, sample size, the observed parameters, observations before supplementation and after supplementation remained same throughout the study. The results of observed parameters showed that ascorbic acid supplementation improve serum ascorbic acid level in group named as hepatitis C patients treated by vitamin C supplements but significantly serum ascorbic acid level was predominantly increased in normal individuals as compared to HCV affected patients after intake of vitamin C but on the other hand vitamin C supplementation markedly improved some specific parameters of liver function tests especially it decrease ALT and serum direct bilirubin level in HCV effected patients as compared to HCV patients without intake of vitamin C supplementation. A very comparable change (p < 0.0527) was observed in serum ascorbic acid level in HCV patients without intake of vitamin C supplementation and (p < 0.038) in hepatitis C patients after a suggestive intake of ascorbic acid supplementation. These results indicate that vitamin C might be helpful to decrease the oxidative strain linked with abnormal liver functions and also help to recover the antioxidant defense system. There is therefore a strong outcome of this study was to accomplish that antioxidants are useful beneficial agents for hepatitis C to normalize elevated liver function tests. Moreover significant difference was observed between levels of serum ALT, AST, serum bilirubin in hepatitis C patients by intake of vitamin C which indicate that use of vitamin C supplementation is effective for HCV patients to improve their LFT values and help to minimize elevation in certain LFT parameters which otherwise proven to be fatal if abnormally raised for long duration.
In the present study, the use of ascorbic acid supplementation has a beneficial effect on serum ascorbic acid level in HCV patients because ascorbic acid level improved in 2nd group in which HCV patients were treated by ascorbic acid supplementation. Studies reported that the patients of HCV with elevated levels of LFT values, when take vitamin C, have a substantial decrease in serum aminotransferase levels, in addition to reducing oxidative stress induced by hepatotoxicity. Thus it was shown in present study that intake of vitamin C might defend against liver damage due to its antioxidant status because antioxidant property of vitamin C also prevent cirrhosis induced by HCV and also be helpful to normalize the elevated LFT parameters and related liver enzymes.
Vitamin C regulated intensities of hepatic function parameters along with blood hydro-peroxide value in hepatic cells. It was helpful to conserve cell integrity and potentiates actions of alanine aminotransferase. In that study, treatment with vitamin C (1000 g/day) helps to normalize the above-mentioned parameters or shifted these parameters from abnormal elevated levels towards the normal reference range .
Antioxidants are basic and most commonly used supplements by healthy individuals as well as patients with hepatitis viral infection. The use of ascorbic acid supplements causes beneficial effects on biochemical parameters because oxidative stress causing damage to host inflammatory processes and stimulation in different diseases especially by viral proteins. Successfully increasing antioxidants level in body, it is able to decrease the possible risk of hepatocellular toxicity and so decrease liver injury and other inflammatory responses. Presence of oxidative stress in hepatitis is well recognized because of fact that various oxidized protein cause damage to hepatic cells causing necrosis and inflammation. Different biochemical studies on vitamins had shown that ascorbic acid (vitamin C) is effective antioxidant which can produce its effect by searching free reactive oxygen species (ROS). Another study has equally shown protection from hepatic oxidative damage due to anti-oxidant effect of ascorbic acid and other vitamins. .
The results of previous studies related to effect of vitamin C against liver injury. Although this study was done on male rats but results were encouraging and characterized with increased activities of serum enzymatic levels of AST and ALP. Abnormal hepatic functions and liver injuries also decreased the concentrations of various other parameters such as serum protein, albumin and globulin. The result of that study shown, that treatment of individuals having liver injury due to oxidative damage when treated with vitamins C resulted in significant reduction in levels of serum ALT, AST and ALP as well as serum vitamin C level. It was helpful in substantial increase in serum total protein levels. Vitamin C also have an effect on reduction of liver cell necrosis and the safe administration of vitamin C make it capable to normalize histological damage associated.In addition to this, vitamin C is known to show activity by blocking the antioxidant chain that averts cell membrane damage averts by ROS in hepatic cells. It was signifying that, the flavanone containing antioxidant vitamin might protect against liver damage produced due to various pathological conditions. Such types of results were observed after supplementation with other antioxidant nutrients alone or combined with vitamin A and E as detected in subjects submitted to cadmium induced hepatotoxicity .
The present study indicates that use of vitamin C by HCV patients helps to decrease the elevated abnormal level of different enzymes, which considered to be abnormally raised in hepatitis C participants. A significant difference (p < 0.042) was observed in serum ALT values observed in patients of HCV after intake of vitamin supplementation. The significant p values showed that, elevated serum ALT levels can be decreased after regular intake of vitamin C that may be due to its effective role in body chemistry. Similarly significant difference (p < 0.048) observed in serum direct bilirubin level after intake of vitamin C which indicate that LFT values can be changed in HCV patients after supplementation with vitamin C.
In another similar study again on rats, vitamin C was described to minimize the liver damage caused by various chemical mediators. In a study it was observed that it helped to normalize the abnormal levels of ALT, AST and ALP. Ascorbic acid was capable to conserved cellular integrity and restrained activity of alanine aminotransferase and aspartate aminotransferase. Such observation was reported in Wister rats for estimation of hepatic functions and enzymes related to significant liver activities. SGOT (AST), SGPT (ALT), alkaline phosphatase and gamma-GT also significantly improved. Studies results also shown that pretreatment with ascorbic acid 200 mg/kg efficiently regulated liver related parameters and help to minimize elevated liver enzymes values .
In another study it was to investigate the effect of different doses of vitamin C on the biochemical parameters of normal and streptozotocin (STZ)-induced hepatic subjects. Liver and kidney enzymes were elevated after the onset of diseases. Moderate doses of vitamin C significantly (P < 0.0008) reduced plasma gamma-glutamyl level. The plasma level of electrolytes, such as calcium and sodium, also changed significantly (P < 0.00001) after oral administration of vitamin C. Antioxidants, such as vitamin C, may ameliorate the biochemical parameters of diseased patients .
Various etiologic factors in Hepatitis may lead to oxidative stress and thereby inflammation of liver. Abnormal hepatic functions and liver injuries also decreased the concentrations of various other parameters such as serum protein, albumin and globulin. The result of this study shown, that treatment of individuals having liver injury due to oxidative damage when treated with vitamins C resulted in significant reduction in levels of serum ALT, AST and ALP as well as serum vitamin C level. It was helpful in substantial increase in serum total protein levels. Vitamin C also have an effect on reduction of liver cell necrosis and the safe administration of vitamin C make it capable to normalize histological damage associated. In addition to this, vitamin C is known to show activity by blocking the antioxidant chain that averts cell membrane damage averts by ROS in hepatic cells. Hence, it may be proposed that decreased blood ascorbic acid level may leads to signs or severity of magnitude of pathologic event. A powerful defense system may be compromised in ascorbic acid deficiency and possibly considered to be improved after this vitamin intake. It is proposed that the consumption of vitamin C may lessen the oxidative pressure associated with abnormal liver functions, and its addition in diet may be beneficial to restore the antioxidant defense system. There ascorbic acid may be a useful adjuvant therapeutic agent in hepatitis C along with anti-viral treatment to normalize abnormal elevated liver function tests and to cure the disease.
Appendix 1. Demographic Data of Individuals of Serum Ascorbic Acid and LFTS in HCV Patients with no Ascorbic Acid Supplementation
^(A.A = ascorbic acid, ALT1, ALT2 = serum alanine enzyme before and after, ALP1, ALP2 = ALP enzyme before and after, TB = total bilirubin, DB = direct bilirubin, IB = indirect bilirubin, TP = total protein, Alb = albumin, Glb = globulin,).
Appendix 2. Demographic Data of Individuals of Serum Ascorbic Acid and LFTS in HCV Patients with Ascorbic Acid Supplementation
^(A.A = ascorbic acid, ALT1, ALT2 = serum alanine enzyme before and after, ALP1, ALP2 = ALP enzyme before and after, TB = total bilirubin, DB = direct bilirubin, IB = indirect bilirubin, TP = total protein, Alb = albumin, Glb = globulin).
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