Medical Journal of Babylon-Vol. 11- No. 2 -2014 مجلة بابل الطبية- المجلد الحادي عشر-العدد

مجلة بابل الطبية- المجلد ال حادي عشر -العدد ال ثاني - Medical Journal of Babylon-Vol. 11 - No. 2 -201 4 201 4

Correlation of New Vitamin C Derivatives with Alanine Amino Transferase and Aspartate Amino Transferase Activities

Mufeed J.Ewadha Seenaa Badra Ibtesam H. Al Musawib Hamid G. Hasanc Hiba Resheed Behayaaa a College of Medicine, University of Babylon, Hilla, Iraq b Nahrain Forensic DNA Training Center, Nahrain University, Baghdad, Iraq. c College of Education, University of Baghdad, Baghdad Iraq,

Received 8 January 2014 Accepted 10 February 2014 Abstract Background: Liver enzymes are commonly found in liver cells and when the liver is damaged, liver cells release their enzymes into the blood stream. Vitamin C is a low-weight molecular antioxidant and is effective in the aqueous phase in protecting different parts of cells against free radicals. An enzyme inhibitor is a molecule, which binds to enzymes and affect their activity. Objective: This study was carried out to investigate the effect of new vitamin C derivative on serum liver enzymes. Research design and methods: Eight samples were used. Liver enzymes activity measured with and without vitamin C derivative. Results: The result showed a reduced activity of aspartate aminotransferase (AST), and alanine transferase (ALT) after addition of vitamin C derivative. Conclusion: It is concluded that the decrease in ALT, and AST activities were due to noncompetitive .inhibition of enzymes by vitamin C derivative Key Words: Alanine aminotransferase, aspartate aminotransferase, vitamin C derivative, enzyme inhibition. الخلصه أنزيمات الكبد موجودة عادة في خليا الكبد وعند تلف الكبد يتم الفراج عن النزيمات الخاصة في مجرى الدم. فيتامين C هو منخفض الوزن الجزيئي للكسدة وفعال في المرحلة المائية في حماية أجزاء مختلفة من الخليا ضد الجذور الحرة. مثبط النزيم هو جزيء، الذي يرتبط بالنزيمات ويؤثر على نشاطهم. أجريت هذه الدراسة لمعرفة تأثيرمشتق فيتامين C الجديد على انزيمات الكبد في الدم. استخدمت ثمانية عينات. النشاط قياس انزيمات الكبد بدون ومع فيتامين C المشتق. أظهرت النتيجة انخفاض نشاط ASTو ALT بعد إضافة فيتامين C المشتق. ويستنتج من ذلك أن النخفاض في ALT، AST نتيجة التثبيط غير التنافسي على النزيمات الذي اظهره مشتق فيتامين C.

29 مجلة بابل الطبية- المجلد ال حادي عشر -العدد ال ثاني - Medical Journal of Babylon-Vol. 11 - No. 2 -201 4 201 4 transaminase (SGPT) and glutamate- may present in acute (recent infection, oxaloacetate transaminase (SGOT) relatively rapid onset) or chronic respectively [10]. forms. The most common causes of Oxidative stress, to some viral hepatitis are the five unrelated extent, is seen in most diseases and hepatotropic viruses: Hepatitis A [2], certainly all inflammatory diseases. Hepatitis B, Hepatitis C, Hepatitis D, Liver disease is without exception in and Hepatitis E. In addition to the this case [11]. Many liver diseases nominal hepatitis viruses, other viruses have high levels of reactive oxygen that can also cause liver inflammation species (ROS) and reactive nitrogen include Herpes simplex, species (RNS) with substantial Cytomegalovirus, Epstein-Barr virus, evidence that the magnitude of or Yellow fever [3] oxidative protein and lipid In developing countries, and in modifications correlates with disease regions with poor hygiene standards, severity and is also linked to disease the incidence of infection with virus is progression [11, 12]. This has led to an high and the illness is usually enthusiasm for the possibility of contracted in early childhood. As antioxidant therapy in liver diseases. incomes rise and access to clean water Vitamin C is a low-weight molecular increases, the incidence of viral antioxidant and is effective in the hepatitis decreases [5]. Worldwide, aqueous phase in protecting different HAV is responsible for an estimated parts of cells against free radicals.The 1.4 million infections annually [6]. biological role of ascorbate is to act as HBV causes more than 4 million cases a reducing agent, donating electrons to of acute hepatitis per year throughout various enzymatic and a few non- the world, and it is estimated that enzymatic reactions. The one- and approximately 350 million people are two-electron oxidized forms of vitamin chronically infected with the virus [7]. C, semidehydroascorbic acid and HBV leads to 1 million deaths dehydroascorbic acid, respectively, can annually as a result of viral hepatitis– be reduced in the body by glutathione induced liver disease. The worldwide and NADPH-dependent enzymatic annual incidence of acute HCV mechanisms [13,14]. The presence of infection is not easily estimated, glutathione in cells and extracellular because patients are often fluids helps maintain ascorbate in a asymptomatic. An estimated 170 reduced state [15]. million people are chronically infected An enzyme inhibitor is a with HCV worldwide [8]. molecule, which binds to enzymes and Liver enzymes are commonly decreases their activity. Since blocking found in liver cells and when the liver an enzyme's activity can kill a is damaged, liver cells release their pathogen or correct a metabolic enzymes into the blood stream; thus, imbalance, many drugs are enzyme increased levels of these enzymes are a inhibitors. The binding of an inhibitor symptom of liver damage. The first can stop a substrate from entering the step in the diagnosis of liver damage is enzyme's active site and/or hinder the a simple blood test that shows the enzyme from catalyzing its reaction. presence of certain liver enzymes in Inhibitor binding is either reversible or the blood. Aminotransferases are the irreversible [16]. most sensitive liver enzyme. The two Types of reversible inhibitors transaminases commonly measured are There are four kinds of reversible ALT and AST [9]. Which previously enzyme inhibitors. They are classified were called glutamate-pyruvate

30 مجلة بابل الطبية- المجلد ال حادي عشر -العدد ال ثاني - Medical Journal of Babylon-Vol. 11 - No. 2 -201 4 201 4

ALT and AST measured for all according to the effect of varying the participants using enzymatic concentration of the enzyme's substrate colorimetric method-Randox company on the inhibitor [16]. (United Kingdom) [17]  Competitive inhibition Different concentration of substrate  Uncompetitive inhibition (2,4 dinitrophenylhydrazine) were used  Mixed inhibition alone first then procedure repeated  Non-competitive inhibition using vitamin C derivative (2,3,5,6 tetra acetic acid L-ascorbic acid) in Aims of the Study same concentration as substrate ,the Assessment of the kinetic effect of activity was measured in presence and vitamin C derivatives on ALT and absence of vitamin C derivative to AST. know the effect of vitamin C derivative Subjects and Methods on enzyme activity in vitro. The study was conducted in the Different concentration of 2,3,5,6 tetra city of Hilla, from August 2013 to acetic acid L-ascorbic acid were October 2013; this study enrolled 4 prepared by serial dilution with diluted patients with viral hepatitis which mineral acid (sulphoric acid). attended Marjan medical city and 4 apparently healthy subjects. Informed Results consent was obtained from all The activity of ALT and AST enzyme participants; the practical side of the with and without vitamin C derivative study was performed at Biochemistry for selected samples were mentioned in Department / College of medicine / table -1and 2. University of Babylon.

Table 1 The activity of ALT enzyme with and without vitamin C derivative for selective patient and healthy subject. Subjects Concentration Activity of Activity of enzyme in presence of substrate enzyme of vitamin C derivative

(mmol/l) (U/l) (U/l) 0.5 0.13 0.07 1 0.24 0.10 1.5 0.27 0.19 2 0.44 0.24 Patient 2.5 0.44 0.24 3 0.44 0.24 3.5 0.44 0.24 4 0.44 0.24 0.5 0.12 0.06 1 0.26 0.14 1.5 0.3 0.22 2 0.35 0.26 Healthy 2.5 0.35 0.26 3 0.35 0.26 3.5 0.35 0.26 enzymes measured4 by using 0.35Maximum velocity0.26 (Vmax), Lineweaver–Burk plots by applying Micheleas–Menten constant (Km) for

31 مجلة بابل الطبية- المجلد ال حادي عشر -العدد ال ثاني - Medical Journal of Babylon-Vol. 11 - No. 2 -201 4 201 4 each samples as figures 1,2,3and 4 the different concentrations of revealed. substrate and the obtained activities for

Figure 1 Lineweaver–Burk plots of ALT in healthy subject with and without vitamin C derivative.

Figure 2 Lineweaver–Burk plots of ALT in hepatic patient with and without vitamin C derivative.

Table 2 The activity of AST enzyme with and without vitamin C derivative for selective patient and healthy subject. subjects Concentration Activity of enzyme Activity of enzyme in of substrate presence of vitamin C (U/l) derivative (mmol/l) (U/l) 0.5 0.12 0.07 1 0.29 0.16 1.5 0.35 0.23 2 0.41 0.29 Patient 2.5 0.41 0.29 3 0.41 0.29 3.5 0.41 0.29 4 0.41 0.29 0.5 0.44 0.17 1 0.63 0.27 1.5 0.83 0.5 2 0.91 0.59 Healthy 2.5 0.91 0.59 3 0.91 0.59 3.5 0.91 0.59 4 0.91 0.59

Figure 3 Lineweaver–Burk plots of AST in healthy subject with and without vitamin C derivative.

Figure 4 Lineweaver–Burk plots of AST in hepatic patients with and without vitamin C derivative. Conclusion Discussion Vitamin C derivative (2,3,5,6 This study found that Vmax of tetra acetic acid L-ascorbic acid) act as ALT and AST decrease by presence of non-competitive inhibitor for ALT and vitamin C derivative while Km remain AST enzymes. constant, which indicate that this compound act as non-competitive References inhibitor because competitive 1.Burkitt HG, Young B, Heath JW. inhibitors bind to enzyme (E), but not Wheater’s functional histology: a text to enzyme substrate complex (ES). and colour atlas. 3rd ed. Edinburgh: Competitive inhibition increases Km Churchill Livingston, 1993. (i.e., the inhibitor interferes with 2. Ryan KJ, Ray CG. Sherris Medical substrate binding), but does not affect th Microbiology ,4 ed. McGraw Vmax (the inhibitor does not hamper Hill. 2004; 541–4 catalysis in ES because it cannot bind 3.Miguet JP, Coaquette A, Bresson- to ES) while non-competitive Hadni S, Lab M. The other types of inhibitors have identical affinities for E viral hepatitis. 1990;40 (18): 1656–9 and ES (Ki = affect substrate binding) 4. Steffen R. "Changing travel-related but decreases Vmax (i.e., inhibitor global epidemiology of hepatitis A". binding hampers catalysis. Mixed-type Am. J. Med. 2005;118: 46S–49S. inhibitors bind to both E and ES, but 5. Jacobsen KH, Koopman JS. "The their affinities for these two forms of effects of socioeconomic development the enzyme are different (Ki ≠ Ki'). on worldwide hepatitis A virus Thus, mixed-type inhibitors interfere seroprevalence patterns". Int J with substrate binding (increase Km) Epidemiol . 2005;34 (3): 600–9. and hamper catalysis in the ES 6.Schmilovitz-Weiss H, Ben-Ari Z, complex (decrease Vmax).So this Sikuler E, Zuckerman E, Sbeit W, compound may use in pharmacological Ackerman Z, et al. Lamivudine aspect and follow up study needed to treatment for acute severe hepatitis B: prove its effect in vivo rather than in a pilot study. Liver Int. 004; 24(6):547- vitro. 51.

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