IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Comparative Study of Serum lactic Acid, Lactate Dehydrogenase and Lipid Profile in Ischemic Heart Disease Patients and Healthy Control M. R. Abdullah Departme nt of Chemistry, College of Education Ibn AL – haithem, Unive rsity of Baghdad Abstract The term ischemic heart disease (IHD) defines a disease sp ectrum of diverse etiology , with the common factor being on imbalance between myocardial oxy gen sup p ly and demand . Fifty p atients (30 male and 20 female) att ending Ibn- Al- betar cardic center, the mean age of male was 65 y ears and 58 y ears for female were included in the p resent st udy , Thirty healthy subjects ( 15 male and 15 female ) of matched age were used as control group s. Some biochemical p arameters including lip id and lip op rotein, total cholesterol (TC) , triglycerides (T G), high density lip op rotein (HDL) and low density lip op rotein (LDL), in addition to lactic acid and lactate dehydrogenase (LDH) activities , were evaluated in the sera of IHD p atient group s and control group . The results indicate a significant increase in all p arameters excep t HDL which showed a significant decrease in the sera of male and female p atient group s comp ared with matched sex and age control group . The results indicate the imp ortance of using the above p arameters as risk factors in addition to new biomarkers for differential dignosis and evaluation of the severity of IHD. Introduction Ischemic heart disease involves a p rogression of p athologic conditions that include erosion and rup ture of coronary artery p laqes, activation of p latelets and thrombi. This p rogression is termed acute coronary sy ndrome and ranges from unst able angina to extensive tissue necrosis in acute myocardial infarction[1]. Coronary heart disease is caused by a lack of nutrients and oxy gen reaching the heart muscle and resulting in myocardial ischemia. Ischemia is a reduced blood sup p ly to one erea of the heart and is often a result of atherosclerosis, t hrombosis, sp asms, or embolisms but may also be a result of anemia, carboxy hemoglobinmia, or hy p otension, which IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 causes reduced blood flow to the heart. M ost frequently, ischemia is the result of abnormal coronary arteries, usually caused by an obst raction in one or more of these arteries. At herosclerosis is a thickening and hardening of the artery walls caused by deposits of cholestrol- lip id-calcium p laque in the lining of the arteries [2]. Lactic acid is a by -p roduct of an emergency mechanism that p roduces a small amount of AT P when oxy gen delivery is severely limited. Py ruvate is the normal end p roduct of glucose metabolism (glycoly sis). The conversion of p y ruvate to lactate is activated when a deficiecy of oxy gen leads t o an accumulation of excess NADH. As aresult, only 2 AT P are p roduced for each mole of glucose metabolized to lactate, with the excess lactate released into the blood. This case has a clinical imp ortance because the accumulation of excess lactate in blood is an early, sensitive, and quantitative indicator of the severity of oxy gen dep rivation. As oxy gen delivery decreases below a critical level, blood lactate concentration rises rapidly and indicates t issue hy p oxia earlier than PH. M easurements of blood lactate are useful for metabolic monitoring in critically p atients, for indicating the severity of the illness, and for objectivly determining p atient p rognosis. There are two ty p es of lactic acidosis. Ty p e A is associated with hy p oxic condition, such as shook, myocardial infarction, severe congestive heart failure, p ulmonary edema, or severe blood loss. Ty p e B is of metablic origin, such as with diabetes mellitus, severe infection, leukemia, liver or renal disease, and toxins (ethanol, methanol, or salicylate poisoning)[3]. Lactate dehydrogenase (LDH) is an enzy me that cataly zes the interconversion of lactic and p y ruvic acids. It is a hy drogen-transfer enzy me that uses the coenezy me NAD + . Lactate dehydrogenase is widely distributed in the body . High activities are found in the heart, liver, skeletal muscle, kidney , and erythrocytes; lesser amounts are found in the lung, smooth muscle, and brain. Because of its widesp read activity in numerous body tissues, LDH is elevated in a variety of disorders. Increased levels are found in cardiac, hep atic , skeletal muscle, and renal diseases, as well in several hematologic and neop last ic disorders[4]. An increased serum cholesterol concentration has been shown to have a st rong association with atherosclerosis. Lowering the serum cholesterol, esp ecially the low-density lip op rotein (LDL) cholesterol fraction, has been shown to decrease the incidence of coronary artery disease and slow the progression of coronary atherosclerosis[5]. Experimental part The chosen p atients were affected by IHD and were referred to the intensive care unit (ICU) in Ibn- Albetar cardiac center according to their sp ecialist surgeon diagnosis which was confirmed by (ECG) electro cardio graphs, X– ray and echo st udy , and were subjected to angio – catheterization and found to be angio – p ositive. This st udy includes 50 p atients 30 male and 20 female .The mean age of male was 65 y ear and 58 year for female. Blood samples were collected from 30 healthy subjects to be used as control group (15 male and 15 female), the mean age of the control group was 63 y ears for male and 55 y ears for female. Both group s (study and control) have no other medical diseases, which may interfere IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 with the tests of our st udy , like viral hepatitis, renal failure, diabetes mellitus, and endocrine disorders. 8ml of blood samples were collected from all subjects by venip uncture, and left for 45 minutes for clotting, centrifuged to get the serum, which was refrigerated unless worked immediately . The method used is modificed from that of classical method by David.(1963). Lactate concentration was determined by using a couple enzy matic reaction, and was monitored colorimetrically .First lactate oxidized to p y ruvate and H2O2 Lactate + O2 Lactate oxidase Py ruvate + H2O2 Secondly p eroxide in the p resence of p-chlorop henol and 4-aminoantipy rine y ield quinoneimine, a red comp lex absorbing light at 500 nm. H2O2 + p - chlorop henol + 4- aminoantip y rine p eroxidase quinoneimine + 2 H2O Totale serum LDH activity was measured by utilizing a colorimetric method (Wroblewski and Due,1955), where a readymade kit from Randox laboratories England is used, this method is based on the reduction of py ruvate to lactate in p resence on NADH by the action of LDH. Py ruvate + NADH + H LDH Lactate + NAD+ The p y ruvate that remains unchanged reacted with 2,4- dinitrop henyl hy drazine to give the corresp oding p henylhydrazone, which is determined colorimetrically in alkaline medium by measuring maximum absorbance at 520 nm. Enzy me activity was exp ressed in U/L. Determination of total serum cholesterol [12] involves the use of three enzy mes , cholesterol esterase , cholesterol oxidase and peroxidase . In the p resence of the former mixture ( N- ethy l prop y l - m - anisidine ) and 4 - amino - antip y rine are condensed by hy drogen p eroxide to form quinoneimine dye prop ortional to the concentration of cholesterol , when the absorbance of the samp les was measured against the reagent blank within 60 minutes at 500 nm. The Triglycerides were determined after enzy matic hy drolysis with lipases . The indicator is aquinoneimine formed from hy drogen p eroxide , 4-aminophenazone , and 4 – chlorop henol under the cataly tic influence of perioxidase . The absorbance was measured for test and st andard against the reagent blank within 6 minutes at 500 nm [13]. In determination of high density lip op rotein – cholesterol HDLc (14), the method uses a selective p recipitations of chylomicrones and the apolip op rotein containing lip op rotein VLDLc and LDLc by addition of 4% p hosp hotungst ic acid solution , which contains 10 % magnesium chloride PH 6.2 . Sedimentation of the p recipitant is by centrifugation, and subsequent enzy matic analysis of HDLc as residual cholesterol remaining in the clear sup ernatant, from which the cholesterol can be determined as described above according to [12]. Low density lip op rotein IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 cholesterol LDLc was determined by using emp irical Friedwald formula which was based on the assump tion that VLDLc is p resent in serum at a concentration equals to one fifth of the TG concentration. This formula is as follow [15]: when all concentration are given in milligrams p er deciliter:- LDLc(mg / dl) = Total cholesterol - ( HDLc +VLDLc ) VLDLc = 1/5 TG LDLc (mmo l/l) = Total cholesterol – (HDLc + TG/2.2) S tatistical Analysis of Data To comp are the significance of the differences in the mean values of any two group s student’s t-test was app lied and P value less t han 0.05(p<0.05) was considered st atistically significant. Results and Discussion Lactic acid concentration and lactate dehydrogenase activity in all group s p articip ated in this st udy are shown in Table (1). A significant increase in lactic acid in serum of p atient group s (male and female) comp ared to control group s (male and female) was noticed, while no significant differences between male control group comp ared to female control group was found. On the other hand, a significant increase in serum lactic acid for female p atient group comp ared to that for male p atient group was found (252.7±18.7 vs. 227.3±29.3 mg/dl, <0.05). Ischemia (not enough oxy genated blood gett ing to a certain area), sever oxy gen deprivation of tissues results in a switch from aerobic to anaerobic metabolism, Since lactate is the main p roduct of anaerobic metabolism, it accumulates and leads to lactic acidosis. As most of lactate is metabolized p redominantly in the liver (60%) and kidney (30%), so any liver disease and renal diseases will lead to disturbance of p roduction of H + (or of the lactate anion) and imp airment of its excretion from the body . Also the fact that only the liver and kidney that have the enzy mes that can convert lactate to glucose.Orchard,1990(8) reported that the effects of lactic acidosis on the cardiovascular sy st em are p articularly p ernicious and can include decreased cardiac outp ut, decreased arterial blood p ressure, deceased hepatic and renal blood flow, and centralization of blood volume[ 6]. Yong et al, 1997 reported that ischemia resulted in a significant elevation in lactate levels in blood[ 7]. The significant differences found between lactic acid in the sera of p atient group s (male and female), could be due to excessive p roduction or reduced utilization associated with st age, duration or the severity of the disease. A significant increase in the activity of LDH in the sera of male p atient group comp ared to male control group , also in female patient group to that in female control group was found. Lactate dehy drogenase is among other diagnost ic aid of biomarkers for acute my ocardial ischemia [8]. IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Also Adams and M iracle(1998) reported that measurement of cardiac enzy mes do not alway s p rovide accurate clinical diagnosis, p articularly in p atient with other concomitant diseases , and alternative biomarkers of cardic disease should be used such as , cardiac trop onins and myoglobin[9]. It has been reported that LDH activity is inhibited in the p ostischemic my ocardium, which is associated with p oor glucose oxidation and impaired my ocardial p erformance [10]. A st udy conducted on rat reported that a three fold increase in LDH content in rat heart comp ared with liver, the authors also demost rated that greater basol content of LDH kinase, which inhibits LDH activity , in rat liver compared with heart[11]. Serum levels of total cholesterol and triglycerides in all st udied group s are shown in table (2). A significant increase in male p atient group comp ared to male control, also a significant increase in female p atient group comp ared to female control group was found. Our results are comp atable with many st udies anticip ated that p atients with both hy p ercholest erolemia and hy p ertriglyceridemia , show imp aried endothelium – dependent vasodilation even before hemogy namically significant arterial st enosis develops, and the association between TG and ischemic heart disease events may be related to the p resseuss of atherogenic triglyceride rich p articles in the p lasma , also elevated levels of cholesterol found to be related to t he evolution of ischemic heart diseases which may lead to cardic death [12,13]. While elevated p lasma cholesterol levels are believed to be the major factor in p romoting atherosclerosis , it is now recognized that t riglycerides are also an indep endent risk factor [14]. Serum levels of high density lip op rotein and low density lip op rotein in all of the st udied group s are shown in Table (3). A significant increase in LDL levels and a significant decrease in HDL levels in the serum of both p atient group s (male and female) compared to that in control group s was found. The drastic decrease in serum HDL and elevation of LDL level were common features among p atient group s comp ared to control group s in this st udy which agree with other studies reported that t he excess TC p resent in the form of LDL p articles to that t he form of HDL can be used to evaluate susp tibility to the development of heart diseases , where LDL/HDL ratio may give a p romising evidence when evaluated carefully with sufficient samp les , and could be conclusive in this resp ect , due to p rotective effect of HDL , which transp ort circulating cholesterol to the liver for clearance , exerts anti _ atherogenic effect , and the significant decrease in it ’ s value in both p atient group s , and the high levels of LDL which is more susp table to oxidation (ox-LDL) , forming foam cells which become trapp ed in the walls of blood vessels and contribute to the formation of atherosclerosis p laques that cause arterial narrowing and lead to heart att ack[15] Studies conducted on p atients with different lip id p rofile abnormalities , reported that the higher (ox-LDL) and the lower HDL levels showed the severety of clinical sy mptoms of IBN AL- HAITHAM J. FO R PURE & APPL. SC I VOL. 23 (1 ) 2010 endothelial dy sfunction indicating that ox-LDL and HDL p lay the crucial role even in the early st ages of atherosclerosis [13][16]. In conclusion, the abnormal lip id p rofile detected by elevation in TC , TG and LDL could be among other crucial factors in the cascade leading to ischemic damage in the cardium , and p rolonged ischemic caused accumulation of non-esterified fatt y acid intra- and extra cellularly which may change the p ermeability of p lasma membrane of heart, which may lead to the leakage of cellular substance and enzy mes out side the cells , therefore more work is required, considering careful selection of IHD p atients, with st rict control of the variables which affect the st udied p arameters , and the time course of the onset and duration of the acute IHD att ach. Re ferences 1.Christenson, RH, Duh SH.(1999).Scand J Clin lab invest .59(supp l 230):90-102. 2.Thomas, CL,. (1997). Tabe , s Cy clop edic M edical Dictionary ,18 th ed. Philadelp hia: FA Davis;168. 3.Burtis, Ashwood ER, (2001). Titz Fundamentals of clinical chemist ry . Philadelp hia: WBsaunders, 496:451. 4.Lott , JA and Stang JM . (1980). Serum enzy mes and isoenzy mes in the diagnosis and differential d iagnosis of my ocardial ischemia and necrosis. Clin Chem ,475. 5.Gazes, PC. (1997). Clinical cardiolo gy : A cost-effective App roach. New York: Chapman and Hall, 535. 6.Juarez, U.; Trejo, W.; Whente, M ., Contreras, G.; Cardenas, M . and Rey es,(1986). Arch.Inst. Cardiol.M ex. 68:214. 7.Yon g, M .Y.; Yan, Y.; Ye, W.; lian , R.L. and Zhi, Y. S. , (1997). China n atl. J. New Gast roenterol, 3(4): 225-227. 8.Orchard, C.H.and Kentish, J.C., (1990). Amer.J. Phy siol., 258:967-981. 9.Adams, J.E. and M iracle,V.A. (1998). Am. J. Care 7 :418. 10.M c Veigh, J.J. and lop aschuk, G.D., Amer.J.Physiol.(1990). Heart cric physiol.28:1079-1085. 11.Priestman, D.A.; M istry , S.C. Halsall, A. and Randle,P.J. (1994). Bioch em.300:659-664. 12.Jahn , S. and Schmied, R.E., (2003), Curr Hy p ertension , 41 : 1301-1307. 13.Kraml, P.; Sy rovatka , P.; Stipek , S.; Flalova , L. and Andel , M .,(2004), Phy siol, Res 53: 471-480. IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 14.M urry , R.K., Granner, D.K. and Rodwell, V.W. (2006) harp er , s Illustrated Biochemist ry ,27 th ed. M c Graw Hill , M edical Publishin g division , U SA. 15.Berg, J>M ., Tymoczko , J.L. and Stry er , L. , (2007). Biochemistry . 5 th ed. W.H., Freeman and company, newy ork. 16.Dave, G.S. and Kalia , K. (2007) , Cell . M ol. Biol. 53: 68-78. Table (1) :Se rum Lactic acid and lactate dehydrogenase in all studied groups *represent P value between male and f emale for control group ** represent P value between male and f emale for p atient group S = sign ificant NS = non _ significant N = number of subject Group p arameters Control n=30 Patients n=50 p -value Lactic acid (mg/dL) M ean  SD M ale n=15 M ale n=30 P < 0.05 (s) P * > 0.05 (NS) 131.2  19.7 227.3  29.3 Female n=15 Female n=20 P < 0.05 (s) P ** > 0.05 (s) 128.8  27.1 252.7  18.7 Lactate Dehy drogenase (LDH) (U/L) M ean  SD M ale n=15 M ale n=30 P < 0.05 (s) P * < 0.05 (s) 182.8  22.3 430.4  30.3 Female n=15 Female n=20 P < 0.05 (s) P **< 0.05 (s) 136.7 28.6 415.6  34.2 IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Table (2): Se rum triglyceride and total cholesterol in patient and control groups. Group p arameters Sex Control p atients P-value TC (mmole /L) M ean  SD M ale 3.46  1.21 5.8  1.36 P < 0.05 (s) Female 4.2  0.3 5.07  1.26 P < 0.05 (s) TG (mmole /L) M ean  SD M ale 1.5  0.47 2.25  0.4 P < 0.05 (s) Female 1.4  0.38 2.31  0.48 P < 0.05 (s) Table (3): Se rum lipoprotei n levels in patients and control groups. Group p arameters Sex Control Patients P-value HDL mmole /L M ean  SD M ale 1.2  0.23 0.98  0.07 P < 0.05 (s) Female 1.1  0.28 0.96  0.23 P < 0.05 (s) LDL mmole /L M ean  SD M ale 2.3  0.87 3.1  1.001 P < 0.05 (s) Female 2.46  0.59 3.73  1.009 P < 0.05 (s) LDL/HDL M ean  SD M ale 1.9  0.21 3.16  0.2 P < 0.05 (s) Female 2.23  0.17 3.88  0.21 P < 0.05 (s) 2010) 1( 23مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد دراسة مقارنة لحامض اللبنیك وفعالیة أنزیم الالكتیك دي ھایدروجینیز و صورة الدھون في امصال مرضى القلب الزاویة و األصحاء محمد رعد عبدهللا ة بغدادابن الھیثم ، جامع -ة یقسم الكیماء ، كلیة الترب الخالصة علـى المـرض الناشـئ مـن عـدم تـوزان كمیـة االوكسـجین المطلوبـة لعضـلة (IHD)یطلق مصطلح امراض القلـب الزاویـة . القلب وكمیة االوكسجین المجهزة یراجعون مستشـفى ابـن البیطـار وكانـت معـدل اعمـار الـذكور ) اناث 20و اذكر 30(تضمنت الدراسة خمسین مریضًا متقـاربین فــي معــدالت اعمــارهم لمجموعــة ) انــاث 15و اذكــر 15( ســنة وكــذلك ثالثـین مــن االصــحاء 58االنــاث سـنة و 65 .المرضى مجامیع سیطرة ،ریدات الثالثیـــةســـیلوالك ،وشـــملت الـــدهون والبروتینــات الدهنیـــة وهـــي الكولســـترول الكلـــي الحیویـــةبعـــض الـــدوال تقیســ حامض اللبنیك وفعالیـة الالكتیـك دي هیـدروجینیز فضال عنوالبروتینات الدهنیة واطئة الكثافة ،والبروتینات الدهنیة عالیة الكثافة .في امصال مجامیع المرضى ومجامیع السیطرة عـدا البروتینـات الدهنیـة عالیـة الكثافـة التــي ةالحیویـة المدروسـ دلـت النتـائج علـى وجـود زیـادة معنویـة فـي جمیـع الــدوال .نوي في امصال مجامیع المرضى مقارنة مع مجموعة السیطرة المتماثلة في الجنس والعمر ظهر فیها انخفاض مع امها دالدراسـةً اهمیـة اسـتخدام الـدوال المـذكورة اعـاله عامـل خطـورة مقترنـًا مـع مؤشـرات جدیـدة یمكـن اسـتخ لنـا اظهـرت .حیویابعة المرضى معیار كیمو حیوي للتشخیص التمییزي وتقییم شدة امراض القلب الزاویة ومتا