I= DEVELOPMENT AND CLINICAL APPLICATION OF ELETROPHORETIC ANALYSES IN DIAGNOSIS OF THE NATURE OF DIABETES MELLITUS
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Title of Thesis
DEVELOPMENT AND CLINICAL APPLICATION OF ELETROPHORETIC ANALYSES IN DIAGNOSIS OF THE NATURE OF DIABETES MELLITUS

Author(s)
Nabila Roohi
Institute/University/Department Details
Department of Zoology University of the Punjab, Lahore
Session
2004
Subject
Zoology
Number of Pages
255
Keywords (Extracted from title, table of contents and abstract of thesis)
DIABETES MELLITUS, diabetics, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SOS-PAGE, Glucose

Abstract
The present investigation was based on the assessment of serum protein profiles of type 1 and type 2 diabetics, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These were then compared with two groups of controls; the negative controls included the non diabetic subjects with negative clinical and family history of diabetes, whereas, the positive controls included the non diabetic subjects with positive family history of type 1 diabetes mellitus (positive controls type 10M) or the non diabetic subjects with positive family history of type 2' diabetes mellitus (positive controls type 2 DM). Moreover, the subjects included in the positive controls category showed either the normal glucose tolerance (NGT) or impaired glucose tolerance (IGT). The study was further elaborated to the assessment of serum protein profiles of five different families extended over three generations including the diabetics, normal glucose tolerance and impaired glucose tolerance subjects. In these families, both type 1 and type 2 OM was prevalent.

Healthy volunteers were contacted at their residences, for blood sampling, whereas, the affected subjects were sampled at the diabetic clinic of Social Security Hospital, Lahore where they had been visiting for the treatment of diabetes. The management of the said hospital also kindly cooperated to approach the families whose any member/s had already been visiting the hospital for the diagnosis and treatment of diabetes. Five of these families agreed to cooperate for blood sampling of all of their members, whether diabetic or non diabetic, extending over three generations.

All of the subjects sampled for the study were, first of all, assessed for their random and fasting glycemia to categorize them as healthy with NGT, IGT subjects and the diabetics according to the criteria presented by American Diabetes Association in 1997.

Sera samples were then electrophoresed. by SDS-PAGE, to resolve the protein fractions of high and low molecular weights thus to locate the fraction/s expressing any relationship of their appearance or disappearance and of a significant alteration. particularly, to the type of DM.

Following electrophoresis, the resolved protein fractions were quantified by gel documentation system which provided the data of molecular weight and percent raw volume for each of the fractions. The- data was analyzed statistically and employed in finding the enhancement/reduction and appearance/disappearance of particular protein fraction/s, for comparison among the healthy and the diabetics.

The prominent high molecular weight fractions expressed in the sera were 190, 160, 120, 78, 66, 54 and 43 kDa and notable low molecular weight fractions resolved were 28, 23, 20, 17, 14.5 and 14.2 kDa. Out of these, the fractions showing noticeable variations were electro eluted and run on isoelectric focusing gels, in second dimension, to determine their isoelectric points. The data of molecular weights and isoelectric points was then employed in the identification of protein fractions.

A noticeable enhancement in 120 kDa (C-reactive protein) fraction's expression was observed in most of the subjects of positive controls type 1 (both NGT and IGT) category and in type 1 diabetics. Type 2 diabetics and their positive controls (both NGT and IGT) also exhibited a trend of elevation of C-reactive protein (CRP). The elevation of CRP in most of the non diabetic first degree relatives of diabetics may indicate a sign towards the progression of diabetes in these subjects.

Serum albumin, recognized as 66 kDa protein fraction, was found to be the dominant fraction of the profile. The fraction exhibited a noticeable decline in both type 1 and type 2 diabetics, however, it did not vary considerably in the first degree relatives, termed as positive controls, of both type 1 and type 2 diabetics.

The 23 kDa protein fraction, detected as apolipoprotein A-1 (apo A-1) demonstrated a pronounced reduction in fraction's expression in both type I and type 2 diabetics as well as their non diabetic first degree relatives (both NGT and IGT subjects) indicating the chances for the future development of diabetes in these subjects.

The protein fraction of 20 kDa, identified to be the retinol binding protein (RBP), was found to be totally vanished in most of the type I diabetics and their first degree relatives considered as positive controls type 1 where fraction failed to manifest in all of the IGT and most of the NGT subjects. Hence, RBP may be regarded as the marker protein of type 1 diabetes and its disappearance in first degree relatives of type 1 diabetics strongly suggests that this protein could be employed in early detection of type 1 diabetes. Type 2 diabetics and their positive controls, on the other hand, did not indicate any significant alteration in fraction's expression further stressing upon this protein to be a marker for the identification as well as early prediction of type 1 diabetes.

The fraction of 14.5 kDa, detected as transthyrein, indicated either a very low expression or was totally unexpressed in most of the type I diabetics and their positive controls. Type 2 diabetics and their positive control IGT subjects also exhibited a similar trend with the exception of NGT positive controls type 2 where the fraction demonstrated its normal appearance. The fraction of 14.2 kDa could not be identified, however, exhibited its normal appearance in type 1 diabetics and their positive controls. In case of positive controls of type 2 DM, the NGT subjects did not show any significant variation, whereas, IGT subjects demonstrated very low levels of the fraction suggesting gradual progression of these subjects towards the development of type 2 DM. A pronounced decline in fraction's intensity and its absence in longer duration type 2 diabetics specified further the association of fraction's behavior with type 2 diabetes mellitus.

The present study investigates the correlation of symptomatic and also of asymptomatic diabetes mellitus and its various types to the resolved protein patterns by gel electrophoresis. in serum samples from the patients. The marker proteins detected with prove helpful in early diagnosis of the disease, in non affected relatives of the diabetics, and can allow timely remedial measures and better management in the affected subjects.

Download Full Thesis
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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
154.33 KB
2 1 Introduction 1
136.26 KB
3 2 Literature review 13
184.66 KB
4 3 Materials and methods 29
200.31 KB
  3.1 Total Plasma Glucose Estimation 37
  3.2 Some Dodecyl Slufate-polyacrylamide Gel Electrophoresis 38
  3.3 Two Dimensional Polyacryamide Gel Electrophoresis 42
  3.4 Estimation of serum total proteins and Fractions 47
5 4 Results 49
1664.68 KB
  4.1 Categorization of various forms of Glucose intolerances 49
  4.2 Finger printing proteins patterns in samples taken randomly from the general population 52
  4.3 Group comparisons 103
  4.4 Isoelectric Focusing 118
  4.5 Finger printing proteins patterns in families and their generations with diabetes prevalence 122
  4.6 Serum total proteins and fractions 146
6 5 Discussion 155
3111.34 KB
  5.1 References 194