A CORRELATIVE STUDY OF COPPER, CERULOPLASMIN, IRON, TOTAL IRON BINDING CAPACITY AND TOTAL ANTIOXIDANT CAPACITY IN DIABETIC NEPHROPATHY

BRIEF RESUME OF THE INTENDED WORK:

6.1 Need for the study :

Diabetic mellitus is one of the leading causes of mortality and morbidity in this modern world. This clinical condition characterized by chronic hyperglycemia, presents with various metabolic and clinical complications. Nephropathy is a common microvascular complication of diabetes mellitus. It was estimated that in the year 2007, there were 40.9 million diabetic individuals in India; the prevalence of overt nephropathy was 2.2%, implying that 850,000 individuals in India had overt nephropathy.1 Various biochemical markers such as microalbuminuria, glycated hemoglobin and renal function tests help in screening, diagnosis, prognosis and treatment of diabetic nephropathy. However, search is on for laboratory tests which detect nephropathy at early stages and correlate with clinical stages of the disease. 1

Trace elements play vital role in human health and disease. Copper and iron might play a key role in the etiopathogenisis of diabetic complications due to their involvement in oxidative stress, antioxidant mechanisms, and inflammation. There is paucicity of studies on change in blood levels of trace elements and their binding proteins, and their correlation with the glycemic status and clinical progression of diabetic nephropathy. 2

A study will therefore be conducted on clinically diagnosed diabetic nephropathy   patients to assess the serum levels of copper, ceruloplasmin, iron, total Iron binding capacity and total antioxidant capacity, in comparison to diabetic individuals without complications and normal healthy controls.

6.2 Review of Literature:

        Etiopathogenisis of diabetic nephropathy involves multiple molecular phenomena such as glycation of proteins and formation of advanced glycation end products, activation of polyol pathway and generation of reactive oxygen species and oxidative stress. The pathology of diabetic nephropathy manifests histologically as glomerulosclerosis and is characterized by glomerular basement membrane thickening and mesangial expansion with 

increased extracellular matrix deposition. A direct correlation was observed between the degree of mesangial expansion, magnitude of proteinuria, severity of hypertension and degree of renal impairment. 2,3

The earliest clinical evidence of nephropathy is the appearance of low but abnormal levels (≥ 30 mg/day or 20 μg/min) of albumin in the urine, referred to as microalbuminuria. Patients with microalbuminuria are referred to as having incipient nephropathy. Without specific interventions, about 80% of subjects with diabetes who develop sustained microalbuminuria have their urinary albumin excretion increase at a rate of about 10–20% per year to the stage of overt nephropathy or clinical albuminuria (≥300 mg/24 h or ≥200 μg/min) over a period of 10–15 years, and gradually the glomerular filtration rate (GFR) falls over a period of several years. 3

Copper plays an important role as a cofactor of key enzymes of metabolism such as cytochrome oxidase, ascorbate oxidase, cytosolic superoxide dismutase and ceruloplasmin. Hence it is involved in many physiological processes like energy metabolism, melanin synthesis and maturation of connective tissue proteins. Thus copper has a pro-oxidant role. Ceruloplasmin , the ferroxidase , binds 90% of total copper in plasma. It oxidizes ferrous iron to ferric, helping in incorporation of iron into transferrin for transport, thus ceruloplasmin is an antioxidant. 2

A study by Sarkar et al showed that there was significant increase in fasting plasma glucose and copper, and decrease in ceruloplasmin in type 2 Diabetes Mellitus cases compared to healthy controls. 4

        Iron is an essential component of hemoglobin, myoglobin, cytocromes, peroxidases, catalases and iron-sulphar proteins. Iron is known to be toxic when accumulated in tissues due to its role in generation of free radicals and induction of oxidative stress. Iron-binding protein transferring is shown to be decreased in kidney diseases. Increased levels of iron in the serum of humans with kidney disease suggest its possible role in the pathogenesis of diabetic nephropathy. Swaminathan et al observed that an increased amount of iron has been 

shown in the kidneys of both animals and humans with kidney disease, evidence of increased urinary iron in patients with diabetic nephropathy and prevention of progression either by an iron-deficient diet or by iron chelators. 5

Diabetes mellitus is a proinflammatory state with increased oxidative stress, which could trigger and tremendously increase the microvascular and macrovascular complications. Lipid peroxidation, is an important index for oxidative stress, lipid peroxide levels increased significantly in diabetic patients as compared to normal controls. In addition, a significant increase in lipid peroxide levels was found among the groups of the diabetic patients with macroalbuminuria and microalbuminuria when compared to normoalbuminuria. Oxidative stress was increased in diabetes and the overproduction of reactive oxygen species in diabetes was a direct consequence of hyperglycemia. Diabetes patients have more severe oxidative stress than normal persons. Oxidative stress is higher in diabetic nephropathy when compared to diabetic patients without complications. 6

A study by Kavitha et al showed significantly decreased total antioxidant capacity in diabetic patients and the imbalance in oxidant-antioxidant status correlated with severity of the diabetic nephropathy. 6

 6.3 Objectives of the study

1.      To estimate serum levels of copper, ceruloplasmin, iron, total iron binding capacity, total antioxidant capacity in patients with diabetic nephropathy, in comparison to diabetic individuals without complications and normal healthy controls.

2.      To assess the correlation among serum levels of copper, ceruloplasmin, iron, total iron binding capacity and total antioxidant  capacity in patients with diabetic nehropathy

Material and methods:

7.1 Source of data

This study will be done at Father Muller Medical College Hospital, Mangalore. Patients with diabetic nephropathy and diabetic individuals without complications and normal healthy controls will be the subjects of the study.

7.2 Method of collection of data ( including sampling procedure, if any)

Study type:  Case control study.

Sample and sampling technique

Inclusion criteria:

Group I: Thirty, clinically diagnosed diabetic nephropathy patients (age: 25-70years) admitted in Nephrology unit of Father Muller Medical College and hospital, Mangalore.

Group II: Thirty, diabetic individuals (age 25-70years) without any complications

Group III: Thirty, normal, healthy, age and sex-matched volunteers (age:25-70years)

Exclusion criteria: Chronic alcoholics, smokers, tobacco chewers, pregnancy, and other systemic illness (infections, malignancy, hepatobiliary  diseases, renal failure, cardiovascular diseases)

Collection of samples: Blood samples will be collected with aseptic precautions as per requirement and processed accordingly. Random urine samples will also be collected.

Assays to be done: Following biochemical parameters will be analyzed using standard spectrophotometric methods. 2

A. In plasma: 1. Fasting plasma glucose      2. Post prandial glucose, estimated by hexokinase method. 2

B. In whole blood: Glycated hemoglobin (HbA1c) estimated by Immunoturbidometry. 2

C. In serum:

1. Copper: spectrophotometric method by using Sodium diethyl dithio carbamate method. 7
2. Ceruloplasmin: by using p-Phenylenediamine Oxidase activity. 8
3. Iron: by using ferrozine reagent. 9
4. Total iron binding capacity (TIBC): unsaturated iron binding capacity (UIBC) is estimated and TIBC is calculated by adding iron and UIBC values. 2
5. Total antioxidant capacity:  spectrophotometric method by using method of Korosovic et al. 10

 D. Urine: 1. Microalbuminuria: Immunoturbidometry. 2

2. Urine protein/creatinine ratio:urine protein is estimated by using pyrogallol red

dye binding method; creatinine by Jaffe’s method. 2

 Plan for data analysis:  collected data will be analyzed by Student’s “t†test, Chi- square test, ANOVA (for significance of results), and Karl Pearson’s correlation analysis.

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, blood samples are collected with aseptic precautions after obtaining informed consent from patients.

 7.4 Has ethical clearance been obtained from your institution in case of 7.3

Yes.