RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS
FOR DISSERTATION
1. / NAME OF THE CANDIDATEAND ADDRESS
(in block letters) / Dr. SHRIKANT CHANDRAKAR
POST GRADUATE STUDENT,
DEPARTMENT OF BIOCHEMISTRY,
J.J.M. MEDICAL COLLEGE,
DAVANGERE – 577004,
KARNATAKA.
2. / NAME OF THE INSTITUTION / J.J.M. MEDICAL COLLEGE,
DAVANGERE- 577004.
3. / COURSE OF STUDY AND SUBJECT / M.D. BIOCHEMISTRY
4. / DATE OF ADMISSION TO COURSE / 31-05-2011
5. / TITLE OF THE TOPIC / “CYSTATIN- C AS AN EARLY BIOMARKER OF RENAL IMPAIREMENT IN PATIENTS WITH TYPE -2 DIABETES”
6. / BRIEF RESUME OF THE INTENDED WORK :
6.1 Need for the study :
India has the largest number of diabetic patients in the world, estimated to be approximately 40.9 million in the year 2007 and expected to increase to approximately 69.9 million by year 2025.1 Diabetic Nephropathy is the single most common cause of chronic renal failure and is a rapidly growing problem worldwide.2
Diabetic nephropathy is an acquired sclerotic injury associated with thickening of the glomerular basement membrane secondary to long standing effects of hyperglycemia, advanced glycation end products and reactive oxygen species.2 Diabetic nephropathy clinically characterized by increasing rates of urinary albumin excretion starting from normoalbuminuria, which progress to microalbuminuria, macroalbuminuria and eventually to end stage renal disease.1 Microalbuminuria is a potent risk factor for cardiovascular events and deaths in patients with type-2 diabetes.2 So it is important to assess renal function as accurately as possible, because renal disease has different clinical presentations and patients are often asymptomatic.3
GFR is a useful index to asses kidney function.3 GFR can be measured directly by clearance studies of exogenous markers such as Iohexol, Iothalamate and Cr51- EDTA but these are costly, time consuming, labor – intensive and require administration of substances that make them in compatible with routine monitoring.4
Endogenous marker plasma creatinine and urea concentration despite their limitations are commonly used because they are cheap and easily available.5
Urea is freely filtered by the glomerulous and not secreted by the tubules.6 However a large portion (40-70%) is passively reabsorbed from renal tubules so underestimate GFR and also its concentration in plasma may vary depending on diet, hepatic function and state of numerous diseases.5
Serum creatinine is an insensitive indicator of diminished GFR because its concentration is affected by meat intake, gender, muscle mass, malnutrition and ageing.6 Serum creatinine is freely filtered by glomerulous, not reabsorbed by the proximal tubules but is secreted in small amounts leading to over estimation of GFR.5
Recently Cystatin-C as a surrogate endogenous marker for estimating early decline in GFR in diabeties.7 Cystatin-C is a low molecular weight of 13 kDa protein belonging to cystatin super family consists of 120 amino acids and its gene located on chromosome 20.7 Its concentration is not significantly affected by age, gender, race, muscle mass, diet, infection, liver function, malignancies, myopathies and body fat content and has a desirable traits as a marker because filtered solely by the glomerulous, not secreted by renal tubules, completely reabsorbed by the tubules and then completely catabolized and generated at a constant rate by all cells in body.7
The present study will be undertaken to evaluate the levels of cystatin-C as a marker of early renal impairment in type-2 diabetic patients in turn helping in the early intervention and management of diabetic nephropathy cases.
6.2 Review of literature :
In one of the study cystatin-C levels of serum and urine increased with increasing degree of albuminuria, reaching higher levels in macroalbuminuric patients and could be a useful marker for renal dysfunction in type-2 diabetic patients with normoalbuminuria.8
It is also showed in this study that cystatin-C and cystatin-C based estimation of GFR may be useful and more sensitive than creatinine in detecting mild acute renal insufficiency in diabetic patients.5
It is found out in the study that measurement of serum cystatin-C concentration provides a simple and accurate method for detecting early renal impairment in subjects with diabetes.9
In one of the study it is showed that serum cystatin-C and creatinine were significantly higher in macroalbuminuric type-2 diabetic patients with renal dysfunction than macroalbuminuric type-2 diabetic patients with normal renal function, the microalbuminuric group as well as normoalbuminuric group.10
In the study it is found out that serum cystatin-C is a reliable marker of GFR in patients with mild to moderately impaired kidney function and has a higher diagnostic accuracy than serum creatinine and calculated creatinine clearance from the Cockcroft and Gault formula in female patients.11
It is showed that use of cystatin-C to measure renal function will optimize early detection, prevention and treatment strategies for diabetic nephropathy.12
It is found out in one of the study that there is good correlation of serum cystatin-C levels with GFR typically better than that of serum creatinine levels.13
It is showed that serum cystatin-C may be more sensitive and specific than serum creatinine for detecting early changes in isotopically determined Glomerular Filtration Rate.14
6.3 Objectives of the study :
· To study the role of serum cystatin-C as an early biomarker of renal impairment in type-2 diabetic patients.
· To compare the serum levels of cystatin-C and creatinine in normoalbuminuric, microalbuminuric and macroalbuminuric in type-2 diabetic patients and controls and to detect early decline of GFR using the staging of chronic kidney disease defined by the National Kidney Foundation in type-2 diabetic patients.
7. / MATERIALS AND METHODS
7.1. Source of data
A study will be carried out for a period of one year from 2012 -2013. The patients will be selected from Chigateri General Hospital and Bapuji Hospital, Davangere (both hospitals are attached to the teaching institute J.J.M. Medical College, Davangere).
The study will be carried out in type-2 diabetic patients and age and sex matched healthy controls. Both cases and controls will be interviewed to obtain relevant data.
Inclusion Criteria:
Cases: 60 proven cases of type-2 diabetic patients in age group of 30 – 80 years.
Controls: 30 cases of age and sex matched healthy controls will be compared.
All patients suffering from type-2 diabetes diagnosed and confirmed by physician with FBS and PPBS according to American Diabetes Association criteria (FBS ≥126 mg/dl & 2 hour PPBS ≥ 200 mg/dl).
Exclusion criteria:
Patients with thyroid disorders, under thyroid medications, under steroid therapy, uncontrolled hypertensive patients and cardiovascular disease patients.
7.2. Method of data collection:
After obtaining informed consent, about 5ml of fasting venous blood samples will be drawn under aseptic precautions in to a sterile bulb from selected subjects. Serum will be separated by centrifugation. Parameters will be estimated by the following methods :
1) Estimation of serum cystatin-C by Immunoturbidimetry:15
Principle: Turbidimetry measures the reduction in light transmission caused by particle formation, and it quantifies the residual light transmitted. It is based on the principle of agglutition reaction. The test specimen is mixed with the activation buffer and the anti human antibody solution and allowed to react.
Presence of cystatin-C in the test specimen forms an insoluble complex producing a turbidity, which is measured at wavelength 550nm. The resulting turbidity corresponds to the concentration of the cystatin-C in the test specimen.
2) Estimation of serum creatinine by Jaffe’s Method:16
Principle: Creatinine reacts with picric acid in alkaline medium to form a reddish yellow complex, intensity of which is directly proportional to the concentration of creatinine in the specimen and can be measured at 520nm (green filter).
3) Estimation of urinary microalbumin by Immunoturbidometry:15
Principle: Turbidimetry measures the reduction in light transmission caused by particle formation, and it quantifies the residual light transmitted. It is based on the principle of agglutition reaction. The test specimen is mixed with the activation buffer and the anti human antibody solution and allowed to react.
Presence of albumin in the test specimen forms an insoluble complex producing a turbidity, which is measured at wavelength 340nm. The resulting turbidity corresponds to the concentration of the albumin in the test specimen.
4) Estimation of Albumin by Bromocresol Green method:17
Principle: When bound with bromocresol green, albumin exhibits a change in absorbance at 628 nm, which related to concentration of albumin.
5) Estimation of serum Glucose by Hexokinase method:18
Principle: Hexokinase is an enzyme that catalyzes the phosphorylation of glucose by adenosine triphosphate (ATP) to form glucose-6-phosphate and adenosine diphosphate (ADP). A second enzyme, glucose-6-phosphate dehydrogenase catalyzes the reaction of the glucose-6-phosphogluconic acid and reduced NADPH. The NADPH is formed in direct proportion to the amount of glucose originally present. NADPH has a high absorbance at 340nm that is proportional to the original amount of glucose in the sample.
Statistical analysis:
Results will be subjected for appropriate statistical analysis.
1) ANOVA will be used for multiple comparisons followed by Post Hoc Tukui Test (student T test) for group wise comparison.
2) Co- relation analysis will be used to measure the relationship between the biomarkers
3) Diagnostic validity test such as specificity, sensitivity, positive predictive value, negative predictive value will be used to assess the utility of these parameters as biomarkers in diabetic nephropathy and to differentiate type-2 diabetic cases from healthy controls.
7.3 Does the study require any investigations or interventions to be conducted on patients or other humans or animals? If so, please describe briefly.
Yes.
Estimation of serum cystatin-C, creatinine, Glucose using human venous blood sample and albumin level in urine of type-2 diabetic patients and controls.7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Yes
8. / LIST OF REFERENCES :
1) Ranjit Unni Krishnan, Mohan Rema et al. Prevalence of Risk Factors of Diabetic Nephropathy in an Urban South Indian Population, Diabetes Care, 2007 August Volume 30, Number 8: 2019 – 2024.
2) Hugh R. Brady, Yvonne M. O’Meara, Barry M. Brenner, Glomenular Disease In: Fausi, Braunwald, Kasper, Hauser, Longo Jameson Loscalzo. Harrison’s Principles of Internal Medicine 17th ed. New York: McGraw-Hills; 2008: p 5573-5598.
3) M.Kannapiran, D.Nisha, A.Madhusudhana Rao. Underestimation of Impaired Kidney Function with Serum Creatinine. Ind j. Clin Biochem 2010 Oct- Dec 25(4): 380-354.
4) Omar F. Laterza, Christopher P. Price, and Mitchell G.Scott. Cystatin-C: An Improved Estimator of Glomerular Filtration Rate. Clinical Chemistry 2002; 48:5: 699-707.
5) Kati Jarvela, Pasi Maaranen, Aimo Harmoinen, Heini Huhtala and Terosisto. Cystatin-C in diabetics as a marker of mild renal insufficiency after CABG. Ann Thorac Cardiovasc Surg 2011; Vol 17: 277- 282.
6) Raymond L. Heilman and Marec J. Mazur. Cystatin-C as a More Sensitive Indicator of Diminished Glomerulor Filtration Rate. Liver Transplantation 2005 Mar Vol- 11, No.-3; p: 264- 266.
7) Jan Mares, David Stejskal, Jitta Vavroskova, Karel Urbanek, Roman Herzig, Peter Hlustik. Use of Cystatin C Determination in Clinical Diagnostics. Bio Med Papers 2003; 147 (2), 177- 180.
8) Yun Kyung Jeon , Mi Ra Kim , Jung Eun Huh , Ji Young Mok, Sang Heon Song , Sang Soo Kim , Bo Hyun Kim , Soo Hyong Lee , Yong Ki Kim , and In Joo Kim. Cystatin-C as an early biomarker of nephropathy in patients with type-2 diabetes. J Korean Med Sci 2011: 258- 263
9) Richard J Mac Isaac, Erosha Premaratne, George Jerums . Estimating Glomerular Filtration Rate in diabetics using serum cystatin-C. Clin Biochem Rev 2011 May; Vol 32: 61-67.
10) El- Shafey E M, El-Nagar G F, Selim M F, El-Sorogy H A , Sabry A A. Is Serum Cystatin-C an Accurate Endogenous Marker of Glomerular Filtration Rate for Detection of Early Renal Impairment in Patients with type-2 Diabetes Patients? Ren Fail 2009; 31 (5): 355-9.
11) Radovan Hojos, Sebastjan Bevc, Robert Ekart, Mak Similjan Gorenjak and Ludvik Puklavec. Serum Cystatin-C as an Endogenous Marker of Renal Function in Patients with Mild to Moderately Impairment of Kidney Function. Nephro Dial Transplant 2006; 21: 1855-1862.
12) Laura Pucci, Stefano Triscornia et al. Cystain-C and Estimates of Renal Function: Searching for Better Measure of Kidney Function in Diabetic patients. Clinical chemistry 2007; 53:3:480- 488.
13) Richard A. Lafayette, Ronald D. Perrone, and Andrew S. Levey. Laboratory Evaluation of Renal Function. In: Robert W. Schrier Diseases of the Kidney and Urinary Tract 7thed Philadelphia, USA; Lipincott Williams Wilkins; 2001; Vol.1: p. 346- 347.
14) Bertram L. Kasiske, William F. Keane. Laboratory Assessment of Renal Disease: Clearance, Urinanalysis and Renal Biopsy. In: Brenner and Rector’s The Kidney 6thed., United States of America; Saunders Company, 2000; Vol.1: p.1136.
15) Pesce AJ, Fring C S, Gauldie J. Spectral Techniques. In: Kaplan L A, Pesce A J, Kazmlerczak S Cedts, Clinical Chemistry. 4th ed; Mosby 2008: p. 100- 103.
16) Clinical tests in kidney disease. In: Godkar P B Edt, Clinical Biochemistry – Principles and Practice. Bombay, India: Bhalani Publishing House; 1994: p.122-125.
17) Dorris L. Ross. Proteins. In: Michael L. Bishop, Janet L. Duben – Von Lauten, Edward P. Fody. Clinical chemistry principles, procedures, correlations. Philadelphia; J.B Lippincott Company; 1985: p.184.
18) Kathleen Becan Mc Bride. Carbohydrates. In: Michael L. Bishop, Janet L. Duben –Von Lauten, Edward P. Fody. Clinical chemistry principles, procedures, correlations. Philadelphia; J.B Lippincott Company; 1985: p 300.
9. / SIGNATURE OF THE CANDIDATE
10 / REMARKS OF THE GUIDE / Study of serum cystatin-C help us to detect early renal impairment in type-2 diabetes mellitus in turn it helps in management of diabetic nephropathy cases.
11 / NAME & DESIGNATION.
11.1 GUIDE
11.2 SIGNATURE / Dr. BHARATHI B.K M.D
READER,
Department Of Biochemistry ,
JJM Medical College,
Davangere.
11.3 CO-GUIDE
(if any)
11.4 SIGNATURE / Dr. RAJEEV AGRAWAL
M.D DNB(Nephrology)
PROFESSOR,
Department Of Medicine,
JJM Medical College,
Davangere.
11.5 HEAD OF THE DEPARTMENT
11.6 SIGNATURE / Dr. D.S. JAYAPRAKASH MURTHY
B.Sc.,M.B.B.S.,M.D.,
PROFESSOR & HOD,
DEPARTMENT OF BIOCHEMISTRY,
JJM MEDICAL COLLEGE,
DAVANGERE.
12 / REMARKS OF THE CHAIRMAN &
PRINCIPAL
12.1 SIGNATURE