IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 The Effect of [2-(9-anthryl) -3- (1,3,4- Triazole -1- yl) -2,3- Dihydro -5,6- ene – 1,3- Oxazepine- 4,7-Dione] on Serum AST and ALT Activities H. S. Jasim Departme nt of chemistry, College of Education Ibn – Al- Haitham, Unive rsity of Baghdad Abstract In this study , the effect of an organic comp ound p rep ared as derivative of o xazep ine tested on the activities of asp artate amino trasferase (AST) and alanin amino transferase (ALT). The kinetic study of such enzy mes is in the p resence of oxazepine derivative. The results revealed that the organic comp ound is a non comp etitive inhibitor for both enzy mes. The Km value for A ST is 1.3 × 10 -3 M and Vmax for the uninhibited is 200 U/mL and for the inhibited is 111.1 U/mL while Km valu e for ALT is 2.5 × 10 -3 M and Vmax are 89.66 U/mL and 56.77 U/mL for the uninhib ited and inhibited enzy me resp ectively. Introduction Aspartate amino transferase (AST) is an enzy me belonging to the class of transferases. It is commonly reffered to as a transaminases and is involved in the transfer of an amino group between asp artate and α- keto acids. The older terminolo gy , serum glutamic o xalo acetic transaminases ( SGOT or GOT), may also be used. The transamination reaction is important in intermediary metabolism because of its function in the sy nthesis and degredation of amino acids. The Keto acids formed by the reaction are altimatly oxidized by the tricarboxy lic acid cycle to p rovide a source of ener gy [1,2]. Asp artate amino transferase is widely distributed in human tissue. The highest concentrations are found in card iac tissue, liv er and ske letal muscle, with smaller amounts found in the kidney , p ancrease and erythrocytes.The clinical use of A ST is limited mainly to the evaluation of hepato cellular disorders and skeletal muscle involvement[3]. Alanin amino transferase (ALT) is a transferase with enzy matic activity similar to AST. Sp ecifically, it c atalyzes the transfer of an amino group from alanin to α- Keto glutarate with the formation of glutamate and py ruvate. The older terminology was serum glutamic p y ruvic transaminase (SGPT or GPT).It is distributed in many tissues , with comp aratively high concentrations in the liver. It is consid ered more liver sp ecific enzy me of the transferases.Clinical applications of ALT assays are confined mainly in evaluation of hepatic disorders. Hi gher elevations are found in hepato cellular disorder, then in extra hep atic or intrahepatic obstructions of the liver.The elevation of ALT activity are frequently higher than those of AST and tend to remain elevated longer as a r esult of t he longer half-life of ALT in serum[1,2,4]. Derivative of oxazep ine organic comp ound was used to study its effect on liver enzy me's function (i.e A ST and ALT). T he compound [2- (9-anthry l)-3-(1,3,4- triazole -1-yl) -2,3-dihy dro – 5,6-ene -1,3-o xazep ine -4,7- dione] is amon g tricy clic antidepressant comp ounds. M any of these comp ounds are adminstrated orally for the treatment of dep ression as well as anxiety or agitation associated with depression , through blocking p ost sy naptic dop amine receptors in the central nervous sy stem. M any of the metabolic products formed have therap eutic actions. T he IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 rate of metabolism of these agents is variable and influenced by a wide variety of factors. As a result, the half-life of tricarboxy lic acid v aries considerably among p atients. The rate of elimination can also be influenced by the Co adminstration of other drugs t hat are eliminated by hepatic metabolism. The toxicity of tricarboxy lic acids are dose dep endant[5,6] . Experime ntal Preparation of organic solutions :- Thirty seven mg of the organic comp omd was dissolved in 10 mL absolute ethanol to p rep are 10 -2 M stock solution . Series of dilutions were mad e to p rep are [10 -3 , 10 -4 , 10 -5 and 10 -6 M ] solutions. The enzyme activities were measured according to Biomghreb Kit No. 20039 France. Principle of GOT (AS T) activity measurment :- Colorimetric d etermination of GOT activity according to the following reaction :- L- Asp artate + α - Ketoglutarate oxalo acetate + L- Glutamate The oxalo acetate formed is measured from derivative with 2,4 Dinitrop heny l hydrazone at 505nm [7]. Principle ALT (GPT) acti vity measurment :- Colorimetric d etermination of GPT activity according to the following reaction L- Alanine + α- Keto glutarate p y ruvate + L- Glutamate. The py ruvate formed was measured from derivative with 2,4 – Dinitrop heny l hy drazone at 505 nm [6]. The effect of ethanol used as a solvent and diluent was determined by adding a quantity equivalent to the samp le and all st eps completed as in the p rocedure used for the determination of GOT and GPT activities [6]. De termination of the percentage of inhi bition :- Using the series of dilutions p rep ared (10 -6 -10 -2 M) of the organic comp ound, while the concentration of the subst rate was kept fixed to get the percentage of inhibition according to the equation :- (The [S] according to the kit is ) Activity withinhibitor % Inhibition = 100 - ( 100) Activity without inhibitor  The inhibitor concentration which is closer to the Km value obtained from the M ichaelis – M enten plot of the uninhibited enzy me is used for determination of the type of inhibition which was p erformed by using different concentrations of substrate with t he fixed concentration of the organic compound. The same method of GOT and GPT,activities used by utilizing the same concentrations of substrate without the addition of 10 -3 M organic compound as inhibitor. Results and Discussion The Conc. of substrate was obtained by multiply ing the volume p ipetted times the total Conc. (202 mmole/L)divided by total volume (24.4).The f actor of conversion from ml p ipeted to Conc.is 8.28(202/24.4) For obtaining the activities of GOT and GPT in U/mL , a st andard curve was dr awn accordin g to the instruction in the Kit. The y - axis is the absorbance and the x- axis for the activity in U/mL. Figures 1A and 1B represented the GOT and GPT calibration curves resp ectively. To obtain U/mL from calibration curve the followin g equation was app lied G PT IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 Slope =Abs/(U/mL) Slope=434.78 for GOT ; Slope=170.36 for GPT Figures 2 and 3 showed M ichaelis-M enten p olt for GOT and GPT resp ectively. from Fig.2 the Km value was found to be 1.32 × 10 -3 M , and Vmax is 195.7 U/mL for the reaction cataly zed by GOT. From Fig.3 the km value is found to be 1.16 × 10 -3 M and Vmax 68.1 U/mL for GPT. Table 1 and 2 showed the effect of differ ent concentrations of t he organic comp ound on GOT and GPT activities resp ectively. Figures 4 and 5 were the lineweaver-burk p lot for the effect of organic comp ound on GOT and GPT activities. It is clear that the organic comp ound has a non-comp etitive inhibitory effect on bot h enzy mes. The Km value for GOT is 1.3 × 10 -3 M and the Vmax for the uninhib ited GOT is 200 u/mL.Vmaxapp. for the inhibited GOT is 111.1 U/mL in a non-comp etitive inhibitors. The Km value for GPT is 2.5 × 10 -3 M for both uninhibited and inhibited enzy me and the Vmax is 89.66 U/mL for the uninhibited enzy me. Vmaxapp. for the inhibited GPT is 56.77 U/mL in a non – comp etitive inhib itors, the Vmax for a reaction catalyzed by the enzy me is reduced in the presence of inhibitor even if subst rate were saturating ( S >> Km),the observed Vmax will be lower than it would be in the absence of the inhibitor because the non-comp etitative inhibition, the inhibitor binds to Enzy me E and (ES) enzy me substrate complex[8]. Non- competitive inhibition occurs when the inh ibitor and subst rate bind at different sites on the enzy me. Non –competitive inhibition can not be overcome by incr easin g the con centration of substrate thus non-comp etitive inhibitors decrease the app arent Vmax of the reaction , Since non-comp etitive inhibitors donot interfere with the binding of substrate to enzy me. Thus t he enzy me shows the same Km in the p resence or absence of the non-comp etitive inhibitor[9,10]. There are no studies in the literature about the effect of oxazep ine derivatives on AST and/or ALT. The variable inhib itory effect of the sy nthesized derivative under st udy on serum AST and ALT may be due to t he change in the st ereost ructure of the enzy me in the presence of such derivative or the binding of this derivative to some side chains of the amino acids p resent in the active site. Re ferences 1- Bishop ,M .L.; Fody , E.P. and Schoeff, L (2005)″ Clin ical chemistry ″ 5 th ed . Lip p incott Williams and wi lkins, Philadelp hia. 2- M urray ,R.K.; Granner, D.K. and Rod well, V.W.(2006)″ Harp er ◌ُs Illust rated Biochemist ry ″ 27 th ed. The M c Graw Hill . U.S.A. 3- M ahe shwari , N. (2008)″ Clinical Biochemistry ″ Jayp ee Brothers medical publishers (P) LTD New Delhi. 4- Berg, J.M .; Ty moczko, J.L. and Stry er, L. (2007)″Bioch emistry ″, 5 th ed . W.H. Free man and company New York. 5- Linder, M .W.; KeCK P.E.; Standards of laboratory p ractice : antidep ressant drug monitoring . National Ac ademy of clinical Biochemistry (1998)Clin. chem. 44:1073- 1084. 6- Schatzbery ,A.F.; (2002),p harmacological p rinciples of antidepressant efficacy . Human p sy cop harmacol (supp l 2 ) : 17-22. 7- Britman, S. ;Frankel, S. (1957)Am.J. clin. Path. 28,56-63. 8- Voet, D .and Voet, J. G. (2004)″ Biochemistry ″ , 3 rd ed. Wiely International cdition U.S.A. IHJPAS 9- Devlin ,T.M . (2006)″ Text book of Biochemistry with clinical correlations ″ 6 th ed. A.John wiley and Sons INC ., p ublication , U.S.A. 10- Champ e, P.C.; Hawey, R.A and Ferrier, D.R. (2008)″ Illustrated Reviews Biochemist ry ″ 4 th ed. Lipp incott Williams and wilk ins , Philadelphia. Table: (1) Effect of different concentrations of the organic compound on GOT activity Conc. 1*10 -5 1*10 -4 1*10 -3 1*10 -2 %GOT 22 17.1 9.8 26.8 Table: (2) Effect of different concentrations of the organic compound on GPT acti vity Conc. 1*10 -5 1*10 -4 1*10 -3 1*10 -2 %GPT 9.05 6.91 10.6 16.4 Table: (3) kinetic paramete rs of AS T and ALT before and after the addition of oxazepine derivative. Before Aft er Vmax U/ mL Km Vmaxapp. U/mL Kmapp. AST 200 1.3×10 -3 M 111.1 1.3×10 -3 M ALT 89.66 2.5×10 -3 M 56.77 2.5×10 -3 M enzyme s Kinetic paramet ers IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 Fig.(1A)Calibration curve of GOT Fig. (1B)Calibration curve of GPT Fig.(2) Michaelis-Menten polt of GOT Fig.(3) Michaelis-Menten polt of GPT 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0 20 40 60 80 100 120 140 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0 0.5 1 1.5 2 2.5 10 - a b s. 5 0 5 n m U/m 0 0. 05 0. 1 0. 15 0. 2 0. 25 0. 3 0. 35 0 20 40 60 80 100 120 140 160 U/m a b s. 5 0 5 n m V u /m l (a b s.× 4 3 4 .7 8 ) 0 0. 05 0.1 0. 15 0.2 0. 25 0.3 0. 35 0.4 0. 45 0.5 0 0. 5 1 1.5 2 2.5 10 - V u /m l (a b s.× 1 7 0 .3 6 ) [S ]=mL×8.28 [S ]=mL×8.28 IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 Fig.(4) Line weaver-burk plot for the effect of organi c compound on GOT activity Fig.(5) Line weaver-burk plot for the effect of organi c compound on GPT activity 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 (1 /V u /m l )× 1 7 0 .3 6 1/[S ] 10 -3 With out inhibitor With inhibitor 0 2 4 6 8 10 12 14 -0.8 -0.6 -0.4 -0.2 0 0 .2 0.4 0.6 0.8 1/[S ] 10 -3 1/V 10 -3 With out inhibitor With inhibitor IHJPAS 2010) 2( 32المجلد مجلة ابن الهیثم للعلوم الصرفة والتطبیقیة اي د– 3، 2 –) یل – 1 –ترایازول - 4، 3، 1( -3-)انثریل -9(-2 [تأثیر فعالیة في ]دایون – 7، 4 –بین اوكساز – 3، 1 –ین ی – 6، 5 –ھایدرو في مصل دم االنسان AST ،ALTانزیمات حسام سلمان جاسم ، جامعة بغدادابن الھیثم –كلیة التربیة ، قسم الكیمیاء الخالصة فعالیة انزیمات اسبارتیت امینو ترانسفریز في االوكسازبین مشتق مثل الدراسة دراسة تأثیر مركب عضويتم في هذه (AST)، ینو ترانسفریز واالنین ام(ALT) . خاصة ان الدراسة الحركیة لهذه االنزیمات بوجود مشتقات االوكسازبین لها اهمیة ملیة ازالة هذه العقاقیر یتم بصورة واسعة تستعملالن بعض هذه المشتقات عملیات أیض من خالل مضادات للكآبة وبما ان ع .عمل الكبد تحتل اهتمام خاص یةكفا الكبد لذا فأن تأثیر هذه العقاقیر على Kmوان قیمة ثابت مایكلس . النزیمین الى اضوي مثبطًا ال تنافسیًا بالنسبة ن المركب العاأظهرت نتائج الدراسة 10هي ASTالنزیم -3 ×1.3 M وان السرعة القصوىVmax 200لالنزیم غیر المثبط هي U/mL واالنزیم المثبط هي 111.1 U/mL قیمة بینما كانتKm النزیمALT 10هي -3 U/mL 56.77و U/mL Vmax 89.66 و، 2.5× . لالنزیم غیر المثبط والمثبط على التوالي IHJPAS