IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 Biochemical Study on Diabetic Nephropathy Patients H. S h. Ahmed, E. Abd-Ali, M. R. Abdul lah De partment ofChe mistry,Colle ge of Education Ibn Al-Haitham.University of Baghdad Abstract This st udy deals with thirty non-insulin dependent diabetes mellitus p atients suffering from diabetic nep hrop athy in addition to twenty five healthy control.Some biochemical p arameters were determined in the serum of all subjects enrolled in the st udy .These p arameters are serum glucose,serum urea,serum creatinine,total serum p rotein and serum albumin.The aim of the p resent study was t o estimate these parameters in diabetic nephrop athy p atients. The results of the p resent st udy revealed a significant increase in glucose,urea and creatinine in p atients as comp ared to controls . Also a significant decrease was found in total serum p rotein, serum albumin and albumin to globulin ratio (A/G) in p atients comp ared to controls,while a significant increase in serum globulin in p atients comp ared to control was found (P<0.001).In conclusion no st atist ical differences were found in the st udy p arameters between gender and between group s with different ages. Key words:Diabetes mellitus,total serum protein,albumin, chronic renal failure,end st age renal disease. Introduction Diabetes mellitus is a group of metabolic disorders of carbohy drate metabolism in which glucose is underutilized by the body tissues,p roducing hy p erglycemia.The diabetic p atients are at increasing risk of developing sp ecific comp lications such as renal failure nephrop athy [1].Diabetic nephrop athy is the most common cause of end-stage renal failure in p atients st arting dialysis in the develop ed world [2].In individual cases,it is often difficult to establish the duration of renal failure[3].Chronic kidney disease (CKD) is associated with an advers effect on p rognosis from cardiovascular disease.This includes increased mortality after an acute coronary sy ndrome and after precutaneous coronary intervention (PCI) with or without st enting [4],[5].Chronic renal failure (CRF) is a p athop hy siologic p rocess with multiple etiologies, resulting in the alteration of nephrone number and function,and frequently leading to end-stage renal disease.In turn,end stage renal disease (ESRD) rep resents a clinical st ate or a condition in which there has been irreversible loss of endogenous renal function,a degree sufficient to render the p atient p ermanently dependent up on renal replacement therap y (dialysis or transp lantation) in order to avoid life-threatening uremia.The main causes of CRF are glomerulonephritis, p olycyst ic kidney , accelerated hy p ertension, calculi disease,analgesic nephrop athy , diabetes mellitus, amy loid, hereditary nephritis and interst itial nephritis[5].Hy p oalbuminemia although not sy nony mous of malnutrition, is highly related to it.Poor nutrient, frequently observed in uremia ,may cause malnutrition and subsequently hy p oalbuminemia. In addition ,it has been recently reported that a sy st emic inflammatory resp onse may p articip ate in developing hy p oalbuminemia in CRF.Other conditions non- associated to inflammatory resp onse,such as the protein losses t hrough dialysis,may cause and increase malnutrition. Overhy dration ,frequently p resent in p atient with renal failure, on the one hand causes dilution of serum albumin concentration,and on the other hand ,is a cause of onset and/or enhancement of congestive cardiac failure,which in turn may be associated with malnutrition[6].M any sy mptoms of uremia result from accumulation of urea,creatinine,and other nitrogenous end products of amino acid and protein metabolism in blood.Among the IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 nutritional p arameters,serum albumin level has been identified as the most p owerful laboratory p redicator of mortality and as an indep endent risk factor for death [6]. The aim of the p resent work is to evaluate urea, creatinine,total serum p rotein,serum albumin and globulin in diabetic p atients suffering from renal failure according to genders. Experimental Part: -S election of subjects and blood sampli ng: This st udy was conducted during the p eriod from December 2008 until the end of M arch 2009.The blood samples were taken from p atients whose ages ranged from (38-72) y ear admitt ed to M edical City in Baghdad.Ten ml of venous blood wasl obtained after a 12 hour fast from thirty p atients with non insulin dependent diabetes mellitus and non-hepatitus, t hese p atients were on hemodialysis treatment for different durations, and twenty five healthy individuals as control group and their ages ranged from (31-65) Year. Blood samples were transferred into p lain tube, allowed to st and for 15 minutes at room temp erature, centrifuged at 3500 rp m for 10 minutes.The resulting serum was sep arated and used for the est imation of glucose, urea, creatinine, total serum protein, serum albumin, and globulin. Methods blood – -De termination of se rum glucose : o o Glucose was determined by using the enzy matic method (GOD-POD) according to the Triender P.method [7]. Glucose in the fasting serum is oxidized to gluconic acid by glucose oxidase. Hy drogen p eroxide, which is also formed in the reaction, reacts with the indicator reagents 4-aminoantipy rine in the p resence of p eroxides enzy me to form a st able red quinine comp ound which absorbs at 500 nm. -De termination of serum urea:o was d Urea was determined by using coloremetric method (Urease-Berthelot M odified) according to the Fawcet and Scot method [8],which urease cataly ses t he convertion of urea to ammonia ion and bicarbonate ,then ammonia hy drolysed to form ammonium ion,which reacts with salicylate and hy p ochlorite to give colored complex absorbance at 580 nm - Determination of serum creatinine: - Creatinine was measured by using kinetic method [9].Creatinine reacts with alkaline p icrate y ielding an orange y ellow comp lex which absorbs at 462nm.It is measured within one minute thereby avoiding interferences of other comp ounds which also react with p icrate,but more slowly than creatinine. aaaAAAaaa( by using of s --De termination of total se rum protei n: that recognizes a sp ecific Total serum p rotein was measured by (Coloremetric Biuret M ethod).Cop p er salts in an alkaline medium reacts with the peptide bonds of the p rotein p roducing a violet colour which is p rop ortional to the amount of p rotein p resent [10].Absorbance at 546nm substance called an are -De termination of serum albumi n::: 1Serum albumin was measured by bromo cresol green (BCG) [11].The measurement of serum albumin is based on its quantitative binding to the indicator (3,3',5,5'-tetra bromo-m- cresol sulp honp hthalein).The albumin-BCG-complex absorbs maximamlly at 578nm. p ercent of the bone marrow.It i -Globulin was calcul ated using the rel ation: (Total Serum Prot ein=Albumin+Globulin). IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 S tatisti cal Analysi s: All values were exp ressed as mean ± st andard deviation (M ±SD). Statistical analysis were performed using st udent ’s T-Test (p ≤ 0.01) t he lowest limit of significance differ ence between the studied group s [12]. Rs ults and Discussion Table (1) shows a marked si gnificant increase in blood glucose,urea,and creatinine in diabetic nep hrop athy p atients as compared to controls. econd most sep arated and The mean (±SD) of serum glucose in the control group was (85.36±10.22) mg/dl,while in p atient group was (218.60±59.21) mg/dl which exhibited a significant elevation (P<10 -5 ) from the control level.Fast ing glucose increases p er decade throught life.Renal threshold (the p oint at which glucose sp ills in the urine) also increases with age; there is also an altered insulin resp onse to glucose [13]. Table (1) also showed elevation in urea and creatinine.Increases in urea in renal failure are caused by imp aired ability to excrete p rotein aceous catabolites because of marked reduction in glomerular filtration rate(GFR).Urea is directly related to the p rotein content of the diet.Urea can also be increased by gastrointest inal hemorrhage,enhanced p rotein catabolism.Increases in creatinine are a result of decreased renal excretion.Because many external factors may influence urea concentration ,creatinine is often used as a more reliable indicator of glomerular filtration rate in p atients with renal disease [14]. Table (2) showed a marked significant decrease in total serum p rotein,serum albumin and A/G ratio in p atients group ,while a significant increase was found in serum globulin as comp ared with their controls.Indicate that those p atients have a sever malnutrition st ate nephrop athies and controls. Patients with CRF develop hy p oalbuminemia to a comp lex setting of conditions,with sy st emic inflammatory resp onse as a major cause, not with st anding ,other factors such as malnutrition and overhydration can also p lay a relevant role [15]. It has been p ost ulated that increased incidence of renal disease and a reduced ability to handle the excretion of drugs.Problems affecting renal function are related to damage from infections or drugs disorders such as diabetes mellitus [16],[17].The sex dependent and gender of diabetic nephrop athy p atients and control group are shown in table (3) ,and table(4). It has been concluded from the results of this st udy that no st atist ical differences were found in p arameters( serum glucose, serum urea, serum creatinine, total serum p rotein and serum albumin) between gender and between group s with different ages.develop n-associated to inflammatory a 444444444444444444444444444444444455 Re ferences 1- Shivan anda,N.B.(2008):"M anip al manual of clinical b iochemist ry ".,3 rd ed.,P 70. 2- Joss,N.;Paterson,C. ;Deighan Simps- on ,K. and Jones ,B.M .(2002).Diabetic nephrop athy .How effective is treatment in clinical practice ?.Q.J.M ed.,95(1):41-49. 3- Emil,A.and Ja ck,W.(2000).″A lang medical book″.Smith general neurolo gy .Chronic renal failure and dialysis.,15 th ed.,Ch.36 ,P 610. 4- United State Renal Data Sy st em.National Institute of Diabetes and Digestive and Kidney Disease(2008).Am.J.Kidney Dis. Suppl (1):S1. 5- Post,T.W.;Gibson,C.M .and Henrich,W.L(2008).Chronic kidney disease. Am.J.Kidney .,65:2380. IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 6- Braunwald, E. ; Fau ci ,A.S. ;Kasp er, D.L.,Hauser,S.L.;Longo,D.L. and Jameson,J.(2001)."Harrison´s p rinciple of internal medicine".Chronic renal failure.,15 th ed.,P 1551. 7- Trinder,P.(1969).Ann.Clin.Biochem. 6 :24 8-Patton ,J.K.and Crouch,S.R(1977).Anal. Chem., 46:464-469. 9-Bartles,H.and Bohmer ,M .(1978): Clin.Chem. Acta. ,37:193 10- Weichse lbaum,T.E.and Amer,J.(1974):"Clinical Ch emistry , p rinciples and techniques".Harper and Row.,2 nd ed. 11- Doumas,B.T.;Watson,W.A.and Bigg,H.G.(1971):Clin.Chem.Acta.,31:87 12- Sorlie,D.E.(1995)."M edical biost atist ics & epidemiology :Examination & board review". Norwalk ,Connecticut, App leton & Lange",1 st ed.,47-88. 13- M ichael ,L.;Bishop ;Edward,P.; Fody ,and Lary –Schoeff(2005)."Clin ical Biochemist ry ".,5 th ed., 648-649. 14- Burits and Ashwood,E.(1999)."Tietz text-book of clinical chemistry ".,2 nd ed.,Ch 22, 2: 15- CuetoM anzano,A.B.(2001).Hypoalbuminemia in dialysis.Is it a marker for malnutrition or inflammatory ?.Rev.Clin.,53(2):152-158. 16- Radbill,B.(2008)."R ationale and strategies for early detection and management of diabetic kidney disease.Rev.Clin.,83(12) :1373-1381. 17- Robertson,J.and Sequin,M .A.(2006).Renal diaease- case-based app roach to acute renal failure and protein-losing nephrop athy ."Diabetic nephrop athy ",4298(1):8. "Diabetic nephrop athy " 7 455 44 4444444 354444324444444 444444444 IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 444444 4444 Table (1): Se rum glucose,urea and creatinine in patients group and the ir controls (mean±SD) in mg/dl. Healthy control Patients Clinical data 25 30 Number 47.20±9.25 54.37±11.35 Age 85.36±10.22 218.60±59.21*** Serum glucose 23.36±6.58 174.83±38.81*** Serum ur ea 0.98±0.51 8.03±1.34*** Serum creatinine ***P<10 -5 Table (2): Total se rum protein,serum albumi n & globulin in (g/l) and (A/G) ratio i n patients group and their controls (mean±S D). Healthy control Patients Clinical data 69.04±0.71 63.23±0.70*** Total serum protein (g/l) 41.36±0.44 30.83±46.8*** Serum albumin (g/l) 27.68±0.80 32.40±0.80** Serum globulin (g/l) 1.64±0.56 1.04±0.38*** (A/G) ratio ***P<10 -5 , **P < 0.001 Table (3):Distribution of diabetic nephropathies patients and healthy control groups according to ge nder. 12 16 28 48.0% 53.3% 50.9 % 13 14 27 52.0% 46.7% 49.1 % 25 30 55 100.0% 100.0% 1 00.0 % N % N % N % Male Fem ale Gender Total Healthy control Patients Studie d groups Total IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 Table (4):The (mean±SD) in diabeti c nephropathi es patients and healthy control groups according to gender Healthy control Patients Clinical data 47.20±9.25 54.37±11.35 Age/y ears (mean±SD) 31 38 M inimum 65 72 M aximum 0.014 t-test (P-value) IHJPAS 2010) 3( 23 مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد كلوي السك ري عتالل ال دراسة كیموحیویة لمرضى اإلعتالل الكلوي السكري حمد رعد عبد اهللام، ایمان عبد علي ،هند شاكر أحمد جامعة بغداد ،ابن الهیثم- كلیة التربیة،قسم الكیمیاء الخالصة بداء السكري غیر المعتمد على اإلنسولین ممـن یعـانون مـن اإلعـتالل ن مریضًا مصاباً یعامل هذه الدراسة مع ثالثتت .عشرین شخصأ من األصحاءخمسة و عن الضفالكلوي السكري ــــ ــــ ـــــدوال تقیسـ ــــ ـــــذه الـ ــــ ـــــت هـ ــــ ــــة و كانـ ــــ ـــي الدراســ ــــ ــــــاهمین فـــ ــــــــخاص المســـ ــــــع األشــ ـــة لجمیــــ ــــ ـــــدوال الكیموحیویـــ ــــ ــــض الـ ــــ بعــ الهــدف مـن هــذه الدراسـة تقــدیر .البـروتین الكلــي و األلبـومین فــي أمصـال المسـاهمین فــي الدراسـة،الكریــاتنین،الیوریـا،الكلوكـوز .هذه المتغیرات لدى مرضى اإلعتالل الكلوي ي أمصـال المرضــى أن هنــاك زیـادة معنویــة فـي تركیــز الكلوكـوز و الیوریــا و الكریـاتنین فــ الـى الحالیـة تشــیر الدراســة نتـائج P< 10)(كانـت اذ ،مقارنة مـع األصـحاء -5 معنـوي ملحـوظ فـي البـروتین الكلـي و األلبـومین و نسـبة نقصـان كـذلك هنـاك . تركیـز الكلوبیـولین فـي أمصـال المرضـى مقارنـة مـع فـي معنوي إرتفاع بینما وجد ، األلبومین الى الكلوبیولین في مصل الدم .P< 0.001)(كانت ،اذ األصحاء ـــتنتاج ــــة ب یمكـــــن اإلســ ــــن هـــــذه الدراســ ــــمـ ـــتهاأنـ ـــي تـــــم دراســ ـــي الـــــدوال التــ ـــد أخـــــتالف معنـــــوي فـــ ـــــا (ه ال یوجــ الكلكوز،الیوریـ .حیث العمربین المرضى من حیث الجنس ،وكذلك بین المرضى من ) الكلي،وااللبومین،الكریاتینین،البروتین .المراحل النهائیة لعجز الكلیة،العجز الكلوي،األلبومین،البروتین الكلي،مرض السكري: كلمات مفتاحیة IHJPAS IHJPAS IHJPAS IHJPAS IHJPAS