IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Biochemical Study on Diabetic Nephropathy Patients H. Sh. Ahmed, E. Abd-Ali, M. R. Abdullah Department ofChemistry,College of Education Ibn Al-Haitham.University of Baghdad Abstract This study deals with thirty non-insulin dependent diabetes mellitus patients suffering from diabetic nephropathy in addition to twenty five healthy control.Some biochemical parameters were determined in the serum of all subjects enrolled in the study.These parameters are serum glucose,serum urea,serum creatinine,total serum protein and serum albumin.The aim of the present study was to estimate these parameters in diabetic nephropathy patients. The results of the present study revealed a significant increase in glucose,urea and creatinine in patients as compared to controls . Also a significant decrease was found in total serum protein, serum albumin and albumin to globulin ratio (A/G) in patients compared to controls,while a significant increase in serum globulin in patients compared to control was found (P<0.001).In conclusion no statistical differences were found in the study parameters between gender and between groups with different ages. Key words:Diabetes mellitus,total serum protein,albumin,chronic renal failure,end stage renal disease. Introduction Diabetes mellitus is a group of metabolic disorders of carbohydrate metabolism in which glucose is underutilized by the body tissues,producing hyperglycemia.The diabetic patients are at increasing risk of developing specific complications such as renal failure nephropathy [1].Diabetic nephropathy is the most common cause of end-stage renal failure in patients starting dialysis in the developed 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 prognosis from cardiovascular disease.This includes increased mortality after an acute coronary syndrome and after precutaneous coronary intervention (PCI) with or without stenting [4],[5].Chronic renal failure (CRF) is a pathophysiologic process 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) represents a clinical state or a condition in which there has been irreversible loss of endogenous renal function,a degree sufficient to render the patient permanently dependent upon renal replacement therapy (dialysis or transplantation) in order to avoid life-threatening uremia.The main causes of CRF are glomerulonephritis, polycystic kidney, accelerated hypertension, calculi disease,analgesic nephropathy , diabetes mellitus, amyloid, hereditary nephritis and interstitial nephritis[5].Hypoalbuminemia although not synonymous of malnutrition, is highly related to it.Poor nutrient, frequently observed in uremia ,may cause malnutrition and subsequently hypoalbuminemia. In addition ,it has been recently reported that a systemic inflammatory response may participate in developing hypoalbuminemia in CRF.Other conditions non- associated to inflammatory response,such as the protein losses through dialysis,may cause and increase malnutrition. Overhydration ,frequently present in patient 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].Many symptoms 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 parameters,serum albumin level has been identified as the most powerful laboratory predicator of mortality and as an independent risk factor for death [6]. The aim of the present work is to evaluate urea, creatinine,total serum protein,serum albumin and globulin in diabetic patients suffering from renal failure according to genders. Experimental Part: -Selection of subjects and blood sampling: This study was conducted during the period from December 2008 until the end of March 2009.The blood samples were taken from patients whose ages ranged from (38-72) year admitted to Medical City in Baghdad.Ten ml of venous blood wasl obtained after a 12 hour fast from thirty patients with non insulin dependent diabetes mellitus and non-hepatitus, these patients 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 plain tube, allowed to stand for 15 minutes at room temperature, centrifuged at 3500 rpm for 10 minutes.The resulting serum was separated and used for the estimation of glucose, urea, creatinine, total serum protein, serum albumin, and globulin. Methods blood – -Determination of serum glucose: o o Glucose was determined by using the enzymatic method (GOD-POD) according to the Triender P.method [7]. Glucose in the fasting serum is oxidized to gluconic acid by glucose oxidase. Hydrogen peroxide, which is also formed in the reaction, reacts with the indicator reagents 4-aminoantipyrine in the presence of peroxides enzyme to form a stable red quinine compound which absorbs at 500 nm. -Determination of serum urea:o was d Urea was determined by using coloremetric method (Urease-Berthelot Modified) according to the Fawcet and Scot method [8],which urease catalyses the convertion of urea to ammonia ion and bicarbonate ,then ammonia hydrolysed to form ammonium ion,which reacts with salicylate and hypochlorite to give colored complex absorbance at 580 nm - Determination of serum creatinine: - Creatinine was measured by using kinetic method [9].Creatinine reacts with alkaline picrate yielding an orange yellow complex which absorbs at 462nm.It is measured within one minute thereby avoiding interferences of other compounds which also react with picrate,but more slowly than creatinine. aaaAAAaaa( by using of s --Determination of total serum protein: that recognizes a specific Total serum protein was measured by (Coloremetric Biuret Method).Copper salts in an alkaline medium reacts with the peptide bonds of the protein producing a violet colour which is proportional to the amount of protein present [10].Absorbance at 546nm substance called an are -Determination of serum albumin::: 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 sulphonphthalein).The albumin-BCG-complex absorbs maximamlly at 578nm. percent of the bone marrow.It i -Globulin was calculated using the relation: (Total Serum Protein=Albumin+Globulin). IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Statistical Analysis: All values were expressed as mean ± standard deviation (M±SD). Statistical analysis were performed using student ’s T-Test (p ≤ 0.01) the lowest limit of significance difference between the studied groups [12]. Rsults and Discussion Table (1) shows a marked significant increase in blood glucose,urea,and creatinine in diabetic nephropathy patients as compared to controls. econd most separated and The mean (±SD) of serum glucose in the control group was (85.36±10.22) mg/dl,while in patient group was (218.60±59.21) mg/dl which exhibited a significant elevation (P<10 -5 ) from the control level.Fasting glucose increases per decade throught life.Renal threshold (the point at which glucose sp ills in the urine) also increases with age; there is also an altered insulin response to glucose [13]. Table (1) also showed elevation in urea and creatinine.Increases in urea in renal failure are caused by impaired ability to excrete protein aceous catabolites because of marked reduction in glomerular filtration rate(GFR).Urea is directly related to the protein content of the diet.Urea can also be increased by gastrointestinal hemorrhage,enhanced protein 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 patients with renal disease [14]. Table (2) showed a marked significant decrease in total serum protein,serum albumin and A/G ratio in patients group,while a significant increase was found in serum globulin as compared with their controls.Indicate that those patients have a sever malnutrition state nephropathies and controls. Patients with CRF develop hypoalbuminemia to a complex setting of conditions,with systemic inflammatory response as a major cause, not with standing ,other factors such as malnutrition and overhydration can also p lay a relevant role [15]. It has been postulated 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 nephropathy patients and control group are shown in table (3) ,and table(4). It has been concluded from the results of this study that no statistical differences were found in parameters( serum glucose, serum urea, serum creatinine, total serum protein and serum albumin) between gender and between groups with different ages.develop n-associated to inflammatory a 444444444444444444444444444444444455 References 1- Shivananda,N.B.(2008):"Manipal manual of clinical biochemistry".,3 rd ed.,P 70. 2- Joss,N.;Paterson,C. ;Deighan Simps- on ,K. and Jones ,B.M.(2002).Diabetic nephropathy.How effective is treatment in clinical practice ?.Q.J.M ed.,95(1):41-49. 3- Emil,A.and Jack,W.(2000).″A lang medical book″.Smith general neurology.Chronic renal failure and dialysis.,15th ed.,Ch.36 ,P 610. 4- United State Renal Data System.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. ; Fauci ,A.S. ;Kasper, 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- Weichselbaum,T.E.and Amer,J.(1974):"Clinical Chemistry, principles 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)."Medical biostatistics & epidemiology:Examination & board review". Norwalk ,Connecticut, Appleton & Lange",1 st ed.,47-88. 13- Michael ,L.;Bishop ;Edward,P.; Fody ,and Lary –Schoeff(2005)."Clinical Biochemistry".,5 th ed., 648-649. 14- Burits and Ashwood,E.(1999)."Tietz text-book of clinical chemistry".,2nd ed.,Ch 22, 2: 15- CuetoManzano,A.B.(2001).Hypoalbuminemia in dialysis.Is it a marker for malnutrition or inflammatory?.Rev.Clin.,53(2):152-158. 16- Radbill,B.(2008)."Rationale 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 approach to acute renal failure and protein-losing nephropathy."Diabetic nephropathy",4298(1):8. "Diabetic nephropathy " 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): Serum glucose,urea and creatinine in patients group and their 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 urea 0.98±0.51 8.03±1.34*** Serum creatinine ***P<10-5 Table (2): Total serum protein,serum albumin & globulin in (g/l) and (A/G) ratio in patients group and their controls (mean±SD). 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 gender. 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% 100.0% N % N % N % Male Female Gender Total Healthy control Patients Studied groups Total IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Table (4):The (mean±SD) in diabetic nephropathies patients and healthy control groups according to gender Healthy control Patients Clinical data 47.20±9.25 54.37±11.35 Age/years (mean±SD) 31 38 Minimum 65 72 Maximum 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 د ،ابن الهیثم- كلیة التربیة،قسم الكیمیاء جامعة بغدا 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 ــاهمین فـــ ــــ ــــخاص المســـ ــــ ــــــع األشــ ــــ ـــة لجمی ــــ ـــــدوال الكیموحیویـــ ــــ ــــض الـ ــــ بعــ ـدیر .البـروتین الكلــي و األلبـومین فــي أمصـال المسـاهمین فــي الدراسـة،الكریــاتنین،الیوریـا،الكلوكـوز ة تقـ الهــدف مـن هــذه الدراسـ .هذه المتغیرات لدى مرضى اإلعتالل الكلوي ـا و الكریـاتنین فــ الـى الحالیـة تشــیر الدراســة نتـائج ة فـي تركیــز الكلوكـوز و الیوریـ ـاك زیـادة معنویــ ي أمصـال المرضــى أن هنـ P< 10)(كانـت اذ ،مقارنة مـع األصـحاء -5 معنـوي ملحـوظ فـي البـروتین الكلـي و األلبـومین و نسـبة نقصـان كـذلك هنـاك . ة مـع فـي معنوي إرتفاع بینما وجد ، األلبومین الى الكلوبیولین في مصل الدم تركیـز الكلوبیـولین فـي أمصـال المرضـى مقارنـ .P< 0.001)(كانت ،اذ األصحاء ـــتنتاج ة ب یمكـــــن اإلســ ــــ ــــن هـــــذه الدراســ ــــمـ ـــتهاأنـ ــم دراســ ـــي تـــ ـــي الـــــدوال التــ ـــد أخـــــتالف معنـــــوي فـــ ـا (ه ال یوجــ ــــ 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