**Bio-Chemical Aspects, Pathophysiology of Microalbuminuria and Glycated Hemoglobin in Type 2 Diabetes Mellitus**

Manjunatha B. K. Goud, Sarsina O. Devi, Bhavna Nayal and Saidunnisa Begum

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50398

## **1. Introduction**

18 Pathophysiology and Complications of Diabetes Mellitus

Biophys Chem, 59(3):315–328.

necrotic treatments, Apoptosis10(1):201–208.

[46] Rello S, Stockert JC, Moreno V, Gámez A, Pacheco M,Juarranz A, Cañete M, Villanueva A, (2005) Morphological criteria to distinguish cell death induced by apoptotic and

[47] Pollack GH, (1996)Phase transitions and the molecular mechanism of contraction,

Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia and derangement in protein and fat metabolism [1]. The worldwide prevalence of diabetes was approximately 2.8% in 2000 and is estimated to grow to 4.4% by 2030. Approximately 40% of patients with type 1 diabetes and 5 - 15% of patients with type 2 diabetes eventually develop end stage renal disease (ESRD), although the incidence is substantially higher in certain ethnic groups [2, 3]. The main risk factors for the development of diabetes are ethnic variations, changes in the food habits, obesity and altered lifestyles. However in type 2 diabetic patient additional factors, related or unrelated to diabetes plays an important role in causation of diabetic nephropathy such as hypertension, dyslipidemia, obesity and it has been named as metabolic syndrome [4]. There are mainly three types of diabetes which include Type 1 diabetes, Type 2 diabetes including a related condition called pre-diabetes and gestational diabetes. The occurrence of diabetic nephropathy varies with type of diabetes and highest risk indiviusuals are type 1 diabetics, but also type 2 diabetics have significant risk. The studies have shown that incidence of renal failure in type 1diabetes may be decreasing due to better preventive measures. However the incidence of renal complications in type 2 diabetes showed uprising [5-8] because type 2 diabetes accounts for at least 90% of all patients with diabetes. Thereby number of type 2 patients with nephropathy and ESRD exceeds those with type 1 diabetes overall.

Diabetic nephropathy is one of the most serious complication of diabetes and the most common cause of end stage renal disease. Advanced diabetic nephropathy is also the leading cause of glomerulosclerosis and end-stage renal disease worldwide. 20% to 40% of

© 2012 Goud et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Goud et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

patients with diabetes ultimately develop nephropathy, although the reason why not all patients with diabetes develop this complication is unknown [9]. The combination of hypertension and diabetes is an especially dangerous clinical situation; both are risk factors as singly or in combination for micro vascular and macro vascular complications of diabetes and for diabetes-related mortality. It's unfortunate that most of diabetics at the time of diagnosis will have hypertension and studies have shown that 50% of patients with diabetes and hypertension results in a sevenfold increase in mortality [10]. Concomitant nephropathy in patients with diabetes and hypertension results in a 37-fold increase in mortality.

Bio-Chemical Aspects, Pathophysiology of

Microalbuminuria and Glycated Hemoglobin in Type 2 Diabetes Mellitus 21

Early detection of nephropathy through screening of diabetic patients allows early intervention and better control of progression of nephropathy and cardiovascular events

Type 2 diabetes is the commonest form of diabetes constituting 90% of the diabetic population in any country and prevalence of diabetes is estimated to increase from 4% in 1995 to 5.4% by the year 2025 (23). The countries with the largest number of diabetic subjects are India, China and U.S. and in the former two countries diabetes occurs mostly in the age range of 45-64yrs, in contrast with an age of >65 in the developed countries. Epidemiological studies conducted in India showed that not only was the prevalence high in urban India but it was also increasing [24-26]. This is mainly attributed to life style changes and genetic

The period between1989-95 showed a 40% rise in the prevalence and subsequently a further increase of 16.4% was seen in the next 5 years. A national survey of diabetes conducted in six major cities in India in the year 2000 showed that the prevalence of diabetes in urban adults was 12.1%. The prevalence of impaired glucose tolerance (IGT) was also high (14.0%). A younger age at onset of diabetes had been noted in Asian Indians

In the national study, onset of diabetes occurred before the age of 50 years in 54.1% of cases, implying that these subjects developed diabetes in the most productive years of their life and had a greater chance of developing the chronic complications of diabetes. The recent studies found that the occurrence of diabetic nephropathy with respect to age is been

Table 1 shows the prevalence of the vascular complications observed in a study by the

Microvascular Macrovascular

Prevalence of retinopathy is high among the Indian type 2 diabetic subjects. Another study done in 1996 in South India showed a prevalence of 34.1% of retinopathy [29]. The prevalence of nephropathy in India was less (8.9% in Vellore) [30]. 5.5% in Chennai [28] when compared with the prevalence of 22.3% in Asian Indians in the UK in the study by

Cardiovascular disease 11.4 Peripheral vascular disease 4.0 Cerebrovascular accidents 0.9 Hypertension 38.0

and mortality.

**1.1. Socio-economic burden of diabetes in India** 

decreasing and most of people affected in early ages.

Retinopathy 23.7 Background 20.0 Proliferative 3.7 Nephropathy 5.5 Polyneuropathy 27.5

**Table 1.** Prevalence () of vascular complications in type 2 diabetes

predisposition in Indian population.

in several studies [26, 27].

Diabetes Research Centre [28].

Samanta et al in 1991[31].

The main treatment dialysis and renal transplantation are costly [11, 12] and most of the poor patients cannot afford the same. Patients with type 2 diabetes undergoing maintenance dialysis require significantly higher financial resources than those suffering from nondiabetic end-stage renal diseases. Furthermore, this group of patients has a very poor prognosis on maintenance dialysis owing to extremely high mortality due to various cardiovascular events [13].

Is the diabetic nephropathy preventable, the answer is yes as diabetic nephropathy progresses from subclinical disease, through the earliest clinically detectable stage characterized by microalbuminuria i.e., urinary albumin 30 to 300mg/day to overt nephropathy with macroalbuminuria [14-16].The combination of strict glycemic control and various biochemical parameters in the form of microalbuminuria, glycated hemoglobin have decreased the occurrence of nephropathy.

Various sensitive tests are available to identify patients with renal involvement early in the clinical course and clinicians should have the knowledge about diabetic nephropathy in the form of its onset, prevention, progression, and treatment in their patients.

Detection of microalbuminuria identifies not only individuals who are at risk of developing renal diseases [(17, 18] but also cardiovascular events and death [19] in these patients. Up to 30% of people with newly diagnosed type 2 diabetes will already have abnormally high urine albumin levels I.e. macroalbuminuria which indicates that many may have overt diabetic nephropathy at the time of diagnosis.

Renal disease is strongly linked to heart disease and the presence of microalbuminuria is a predictor of worse outcomes for both in renal and cardiac patients. Microalbuminuria does not directly cause cardiovascular events; it serves as a marker for identifying those who may be at increased risk. Microalbuminuria is caused by glomerular capillary injury and so may be a marker for diffuse endothelial dysfunction. According to Steno hypothesis, albuminuria might reflect a general vascular dysfunction and leakage of albumin and other plasma macromolecules such as low density lipoproteins into the vessel wall that may lead to inflammatory responses and in turn start the atherosclerotic process [20, 21].

Recently, it has been suggested that microalbuminuria may be a risk factor for the development of cardiovascular disease in non-diabetics and may therefore have a role in screening programs [22].

Early detection of nephropathy through screening of diabetic patients allows early intervention and better control of progression of nephropathy and cardiovascular events and mortality.

## **1.1. Socio-economic burden of diabetes in India**

20 Pathophysiology and Complications of Diabetes Mellitus

cardiovascular events [13].

decreased the occurrence of nephropathy.

diabetic nephropathy at the time of diagnosis.

screening programs [22].

patients with diabetes ultimately develop nephropathy, although the reason why not all patients with diabetes develop this complication is unknown [9]. The combination of hypertension and diabetes is an especially dangerous clinical situation; both are risk factors as singly or in combination for micro vascular and macro vascular complications of diabetes and for diabetes-related mortality. It's unfortunate that most of diabetics at the time of diagnosis will have hypertension and studies have shown that 50% of patients with diabetes and hypertension results in a sevenfold increase in mortality [10]. Concomitant nephropathy

The main treatment dialysis and renal transplantation are costly [11, 12] and most of the poor patients cannot afford the same. Patients with type 2 diabetes undergoing maintenance dialysis require significantly higher financial resources than those suffering from nondiabetic end-stage renal diseases. Furthermore, this group of patients has a very poor prognosis on maintenance dialysis owing to extremely high mortality due to various

Is the diabetic nephropathy preventable, the answer is yes as diabetic nephropathy progresses from subclinical disease, through the earliest clinically detectable stage characterized by microalbuminuria i.e., urinary albumin 30 to 300mg/day to overt nephropathy with macroalbuminuria [14-16].The combination of strict glycemic control and various biochemical parameters in the form of microalbuminuria, glycated hemoglobin have

Various sensitive tests are available to identify patients with renal involvement early in the clinical course and clinicians should have the knowledge about diabetic nephropathy in the

Detection of microalbuminuria identifies not only individuals who are at risk of developing renal diseases [(17, 18] but also cardiovascular events and death [19] in these patients. Up to 30% of people with newly diagnosed type 2 diabetes will already have abnormally high urine albumin levels I.e. macroalbuminuria which indicates that many may have overt

Renal disease is strongly linked to heart disease and the presence of microalbuminuria is a predictor of worse outcomes for both in renal and cardiac patients. Microalbuminuria does not directly cause cardiovascular events; it serves as a marker for identifying those who may be at increased risk. Microalbuminuria is caused by glomerular capillary injury and so may be a marker for diffuse endothelial dysfunction. According to Steno hypothesis, albuminuria might reflect a general vascular dysfunction and leakage of albumin and other plasma macromolecules such as low density lipoproteins into the vessel wall that may lead to

Recently, it has been suggested that microalbuminuria may be a risk factor for the development of cardiovascular disease in non-diabetics and may therefore have a role in

form of its onset, prevention, progression, and treatment in their patients.

inflammatory responses and in turn start the atherosclerotic process [20, 21].

in patients with diabetes and hypertension results in a 37-fold increase in mortality.

Type 2 diabetes is the commonest form of diabetes constituting 90% of the diabetic population in any country and prevalence of diabetes is estimated to increase from 4% in 1995 to 5.4% by the year 2025 (23). The countries with the largest number of diabetic subjects are India, China and U.S. and in the former two countries diabetes occurs mostly in the age range of 45-64yrs, in contrast with an age of >65 in the developed countries. Epidemiological studies conducted in India showed that not only was the prevalence high in urban India but it was also increasing [24-26]. This is mainly attributed to life style changes and genetic predisposition in Indian population.

The period between1989-95 showed a 40% rise in the prevalence and subsequently a further increase of 16.4% was seen in the next 5 years. A national survey of diabetes conducted in six major cities in India in the year 2000 showed that the prevalence of diabetes in urban adults was 12.1%. The prevalence of impaired glucose tolerance (IGT) was also high (14.0%). A younger age at onset of diabetes had been noted in Asian Indians in several studies [26, 27].

In the national study, onset of diabetes occurred before the age of 50 years in 54.1% of cases, implying that these subjects developed diabetes in the most productive years of their life and had a greater chance of developing the chronic complications of diabetes. The recent studies found that the occurrence of diabetic nephropathy with respect to age is been decreasing and most of people affected in early ages.

Table 1 shows the prevalence of the vascular complications observed in a study by the Diabetes Research Centre [28].


**Table 1.** Prevalence () of vascular complications in type 2 diabetes

Prevalence of retinopathy is high among the Indian type 2 diabetic subjects. Another study done in 1996 in South India showed a prevalence of 34.1% of retinopathy [29]. The prevalence of nephropathy in India was less (8.9% in Vellore) [30]. 5.5% in Chennai [28] when compared with the prevalence of 22.3% in Asian Indians in the UK in the study by Samanta et al in 1991[31].

## **1.2. The main health problems related to diabetes are**

Diabetes can have a significant impact on quality of life by increasing risk for a variety of complications mainly long standing. These include:

Bio-Chemical Aspects, Pathophysiology of

Microalbuminuria and Glycated Hemoglobin in Type 2 Diabetes Mellitus 23

poor glycemic or blood pressure control or may have additional independent genetic basis

Diabetic siblings of patients with diabetes and renal disease are five times more likely to develop nephropathy than diabetic siblings of diabetic patients without renal disease. Even this has been proved by histo-pathological studies in twins with type 1 diabetics [40, 41]. Genetic factors may play an important role in diabetic nephropathy and/or may be clustered with genes influencing other cardiovascular diseases. There is ongoing research in identifying genetic loci for diabetic nephropathy susceptibility through genomic screening and candidate gene approaches [42-44]. A recent genome scan for diabetic nephropathy in African Americans identified susceptibility loci on chromosomes 3q, 7p and 18q [45] and in

Diabetic nephropathy and hypertension are multifactorial disorders resulting from both environmental and genetic factors, which make it complex and difficult to identify at the genetic level what confers susceptibility to diabetic kidney disease. Gene polymorphism play's an important role for example in renin–angiotensin system, nitric oxide (NO), aldose reductase, glucose transporter 1 (GLUT-1), and lipoproteins which are potentially involved in the genetic predisposition to hypertension, vascular reactivity, and

A recent study has shown that the strong association between a polymorphism in the 5′-end of the aldose reductase gene and the development of diabetic nephropathy in type 1 diabetic

ESRD is known to be more prevalent in certain ethnic groups—Native Americans, Mexican Americans, and African Americans—than in Caucasian Americans. Certainly, there is reason for special vigilance for early signs of nephropathy in these high-risk populations,

The factors which contribute for the development of diabetic nephropathy are shown in

Aldose reductase (AR)/ Polyol pathway

Transforming growth factor β (TGF- β)

 Vascular endothelial growth factor (VEGF) Platelet-derived growth factor (PDGF)

Growth hormone (GH) and insulin –like growth

whose members presumably have a genetic predisposition to nephropathy.

Metabolic factors Advanced glycation end products (AGEs)

 Endothelin Nitric oxide

Factors (IGFs)

**Table 3.** Factors involved in development of diabetic nephropathy

Hemodynamic factors Angiotensin 2 / renin – angiotensin system (RAS)

Intracellular factors Diacyglycerol (DAG) – protein kinase C (PKC) pathway

Pima Indians it has been identified on chromosome 7 [46].

[38, 39].

insulin resistance [47].

patients [47].

Table 3.

Growth factors and

cytokines


**Table 2.** Main chronic complications in diabetes

## **2. Diabetic nephropathy**

The abnormal glycemic status of diabetes is closely related to the development of microvascular complications. However, in humans the evidence of a straightforward causal relationship between hyperglycemia and renal disease is less compelling than in animal models. The development of diabetic nephropathy is characterized by a progressive increase in the excretion of albumin, continued increase in blood pressure and decline in glomerular function which later leads to end stage renal failure. The patients with diabetes are more prone for this condition due to associated factors like hyperlipidemia and hypertension. The mortality and morbidity is high and it's mainly due to a cardiovascular event [32, 33].

There are various factors which lead to diabetic nephropathy like biochemical, hormonal, immunological and rheological.


## **2.1. Genetic and ethnic role**

Although we know that all patients with diabetes will not develop ESRD this is due to the good glycemic and blood pressure control. In addition to the risks of poor glycemic control and hypertension, a subset of patients may be at greater risk for nephropathy based on inherited factors. Familial clustering of patients with nephropathy may result from similarly poor glycemic or blood pressure control or may have additional independent genetic basis [38, 39].

22 Pathophysiology and Complications of Diabetes Mellitus

**1.2. The main health problems related to diabetes are** 

Blindness(Mainly cataract and retinopathy)

complications mainly long standing. These include:

Cardiac Disease and Stroke

Nervous System Disease Pregnancy complications

**Table 2.** Main chronic complications in diabetes

**Chronic complications**

Renal Disease Hypertension

Amputations

**2. Diabetic nephropathy** 

immunological and rheological.

membrane thickening [36].

**2.1. Genetic and ethnic role** 

[34].

diabetes [35].

Diabetes can have a significant impact on quality of life by increasing risk for a variety of

The abnormal glycemic status of diabetes is closely related to the development of microvascular complications. However, in humans the evidence of a straightforward causal relationship between hyperglycemia and renal disease is less compelling than in animal models. The development of diabetic nephropathy is characterized by a progressive increase in the excretion of albumin, continued increase in blood pressure and decline in glomerular function which later leads to end stage renal failure. The patients with diabetes are more prone for this condition due to associated factors like hyperlipidemia and hypertension. The

mortality and morbidity is high and it's mainly due to a cardiovascular event [32, 33].

There are various factors which lead to diabetic nephropathy like biochemical, hormonal,

Biochemical factors include long standing hyperglycemia and glycosylation process

Studies have shown that growth hormone promotes basement membrane thickening in

Both exogenous and endogenous insulin autoantibodies, IAA contributed in basement

The red blood cell deformity due to glycosylation and fibrin deposition results in

Although we know that all patients with diabetes will not develop ESRD this is due to the good glycemic and blood pressure control. In addition to the risks of poor glycemic control and hypertension, a subset of patients may be at greater risk for nephropathy based on inherited factors. Familial clustering of patients with nephropathy may result from similarly

altered permeability and hypercoagulability in diabetic patients [37].

Diabetic siblings of patients with diabetes and renal disease are five times more likely to develop nephropathy than diabetic siblings of diabetic patients without renal disease. Even this has been proved by histo-pathological studies in twins with type 1 diabetics [40, 41]. Genetic factors may play an important role in diabetic nephropathy and/or may be clustered with genes influencing other cardiovascular diseases. There is ongoing research in identifying genetic loci for diabetic nephropathy susceptibility through genomic screening and candidate gene approaches [42-44]. A recent genome scan for diabetic nephropathy in African Americans identified susceptibility loci on chromosomes 3q, 7p and 18q [45] and in Pima Indians it has been identified on chromosome 7 [46].

Diabetic nephropathy and hypertension are multifactorial disorders resulting from both environmental and genetic factors, which make it complex and difficult to identify at the genetic level what confers susceptibility to diabetic kidney disease. Gene polymorphism play's an important role for example in renin–angiotensin system, nitric oxide (NO), aldose reductase, glucose transporter 1 (GLUT-1), and lipoproteins which are potentially involved in the genetic predisposition to hypertension, vascular reactivity, and insulin resistance [47].

A recent study has shown that the strong association between a polymorphism in the 5′-end of the aldose reductase gene and the development of diabetic nephropathy in type 1 diabetic patients [47].

ESRD is known to be more prevalent in certain ethnic groups—Native Americans, Mexican Americans, and African Americans—than in Caucasian Americans. Certainly, there is reason for special vigilance for early signs of nephropathy in these high-risk populations, whose members presumably have a genetic predisposition to nephropathy.


The factors which contribute for the development of diabetic nephropathy are shown in Table 3.

**Table 3.** Factors involved in development of diabetic nephropathy

## **2.2. Natural history of diabetic nephropathy (Table 3) and renal changes in diabetic nephropathy**

Diabetic nephropathy is a spectrum of progressive renal lesions secondary to diabetes mellitus ranging from renal hyper-filtration to end stage renal disease. The earliest clinical evidence of nephropathy is the presence of microalbuminuria. It occurs in 30% of type 1 diabetics 5 to 15 years after diagnosis but may be present at diagnosis in type 2 diabetics as the time of onset of type 2 diabetes is often unknown. The microalbuminuria progresses to overt proteinuria over the next 7 to 10 years. Once overt proteinuria develops, renal function progressively declines and end stage renal disease is reached after about 10 years.

Bio-Chemical Aspects, Pathophysiology of

Microalbuminuria and Glycated Hemoglobin in Type 2 Diabetes Mellitus 25

albumin in the urine is a cardinal sign of renal damage, especially glomerular disease, and is

Various studies have shown different factors play a role in microalbuminuria. The two important factors plays a role in urinary albumin excretion are trans glomerular passage of albumin and tubular reabsorption. The glomerular and tubular proteinuria can be distinguished by simultaneously measuring the urinary β2-microglobulin and albumin [49, 50]. Rodicio et.al., in their article has put forward the causes of microalbuminuria in

Reflects the existence of intrinsic glomerular damage leading to changes in the

 May be the result of a tubular dysfunction in normal reabsorption of filtered albumin. It may be the renal manifestation of a generalized, genetically conditioned vascular endothelial dysfunction which may therefore link urinary albumin excretion and

There is a general belief that increased urine albumin excretion in diabetic nephropathy is mostly glomerular in origin. For albumin to appear in the urine it must cross the glomerular filtration barrier, which consists of fenestrated glomerular endothelial cells, the glomerular

It has been seen that increased intraglomerular pressure, loss of negatively charged glycosaminoglycan's in the basement membrane and, later, increased basement membrane pore size, all contribute to the albuminuria. The earliest morphological change of diabetic nephropathy is expansion of the mesangial area [52] and is caused by an increase in extracellular matrix deposition and mesangial cell hypertrophy. After a short period of proliferation, mesangial cells exposed to hyperglycemia become arrested in the G1-phase of the cell cycle and is mediated by p27 Kip1, an inhibitor of cyclin-dependent kinases [53, 54].

Hyperglycemia activates the mitogen-activated protein kinases (MAPKs) which lead to a

In addition, ANG II further enhances p27 Kip1 induction and blockade of ANG II attenuates high glucose mediated mesangial cell hypertrophy [54]. Thickening of the GBM is progressive over years; both increased extracellular matrix synthesis and impaired removal

There is a decrease in the expression of heparin sulphate and the extent of sulphation followed by increase in collagen type IV deposition. The type of collagen expressed in GBM mainly contains α 3, α 4, and α 5 chains and mesangial matrix has α 1 and α 2 of type IV

collagen and increased expression is seen in diabetic populations [56, 57].

not detectable by screening techniques using urinary dipsticks.

hypertension which is invariably associated with diabetes as follows: -

glomerular barrier filtration.

elevated risk of cardiovascular diseases [51].

basement membrane, and glomerular epithelial cell or podocyte.

post-transcriptional increase in p27 Kip1 expression [55].

contribute to GBM thickening.

Can be a consequence of an augmented intraglomerular capillary pressure.

**3.1. Structural abnormalities seen during increased excretion of albumin** 


Adapted from SIGN Guidelines (48)

**Table 4.** Evolution of diabetic renal disease

Renal changes are characterized by specific renal morphological and functional alterations which include:


## **3. Pathophysiology of microalbuminuria**

Normal human urine contains only very small quantities of albumin, less than 30 mg of albumin being excreted by healthy adults in 24 hours. The appearance of large amounts of albumin in the urine is a cardinal sign of renal damage, especially glomerular disease, and is not detectable by screening techniques using urinary dipsticks.

Various studies have shown different factors play a role in microalbuminuria. The two important factors plays a role in urinary albumin excretion are trans glomerular passage of albumin and tubular reabsorption. The glomerular and tubular proteinuria can be distinguished by simultaneously measuring the urinary β2-microglobulin and albumin [49, 50].

Rodicio et.al., in their article has put forward the causes of microalbuminuria in hypertension which is invariably associated with diabetes as follows: -

Can be a consequence of an augmented intraglomerular capillary pressure.

24 Pathophysiology and Complications of Diabetes Mellitus

**diabetic nephropathy** 

**2.2. Natural history of diabetic nephropathy (Table 3) and renal changes in** 

progressively declines and end stage renal disease is reached after about 10 years.

Stage 2 "Silent phase" (structural changes on biopsy but no clinical

Stage 4 Overt "dipstick positive" proteinuria (macroalbuminuria) : AER >

Requiring dialysis or transplant to maintain life

and renal hypertrophy, increased urinary albumin excretion (UAER).

Stage 1 Glomerular hypertension and hyper filtration

manifestations) Normoalbuminuria

200µg/min Hypertension

Stage 5 End stage renal failure

**Table 4.** Evolution of diabetic renal disease

**3. Pathophysiology of microalbuminuria** 

Adapted from SIGN Guidelines (48)

which include:

laminin.

Stage 3 Microalbuminuria: AER 20 – 200µg/min Normal serum creatinine Increased blood pressure

progression of nephropathy

Raised GFR, normal serum creatinine

Diabetic nephropathy is a spectrum of progressive renal lesions secondary to diabetes mellitus ranging from renal hyper-filtration to end stage renal disease. The earliest clinical evidence of nephropathy is the presence of microalbuminuria. It occurs in 30% of type 1 diabetics 5 to 15 years after diagnosis but may be present at diagnosis in type 2 diabetics as the time of onset of type 2 diabetes is often unknown. The microalbuminuria progresses to overt proteinuria over the next 7 to 10 years. Once overt proteinuria develops, renal function

Normoalbuminuria: urinary albumin excretion rate (AER) <20 µg/min

Serum creatinine may be normal Increase in serum creatinine with

Renal changes are characterized by specific renal morphological and functional alterations

Features of early diabetic changes in the form of glomerular hyper filtration, glomerular

 Increased basement membrane thickness (BMT) and mesangial expansion with the accumulation of extracellular matrix (ECM) proteins such as collagen, fibronectin and

 Advanced diabetic nephropathy is characterized by proteinuria, a decline in renal function, decreasing creatinine clearance, glomerulosclerosis and interstitial fibrosis.

Normal human urine contains only very small quantities of albumin, less than 30 mg of albumin being excreted by healthy adults in 24 hours. The appearance of large amounts of


#### **3.1. Structural abnormalities seen during increased excretion of albumin**

There is a general belief that increased urine albumin excretion in diabetic nephropathy is mostly glomerular in origin. For albumin to appear in the urine it must cross the glomerular filtration barrier, which consists of fenestrated glomerular endothelial cells, the glomerular basement membrane, and glomerular epithelial cell or podocyte.

It has been seen that increased intraglomerular pressure, loss of negatively charged glycosaminoglycan's in the basement membrane and, later, increased basement membrane pore size, all contribute to the albuminuria. The earliest morphological change of diabetic nephropathy is expansion of the mesangial area [52] and is caused by an increase in extracellular matrix deposition and mesangial cell hypertrophy. After a short period of proliferation, mesangial cells exposed to hyperglycemia become arrested in the G1-phase of the cell cycle and is mediated by p27 Kip1, an inhibitor of cyclin-dependent kinases [53, 54].

Hyperglycemia activates the mitogen-activated protein kinases (MAPKs) which lead to a post-transcriptional increase in p27 Kip1 expression [55].

In addition, ANG II further enhances p27 Kip1 induction and blockade of ANG II attenuates high glucose mediated mesangial cell hypertrophy [54]. Thickening of the GBM is progressive over years; both increased extracellular matrix synthesis and impaired removal contribute to GBM thickening.

There is a decrease in the expression of heparin sulphate and the extent of sulphation followed by increase in collagen type IV deposition. The type of collagen expressed in GBM mainly contains α 3, α 4, and α 5 chains and mesangial matrix has α 1 and α 2 of type IV collagen and increased expression is seen in diabetic populations [56, 57].

The recent evidence shows that an alteration in structure and function of podocytes occurs early in diabetic nephropathy. The podocytes which are adhering to GBM through integrin's are altered due to hyperglycemia.

Bio-Chemical Aspects, Pathophysiology of

Microalbuminuria and Glycated Hemoglobin in Type 2 Diabetes Mellitus 27

Increased systemic capillary permeability has also been linked with microalbuminuria in healthy populations and recent study shows that endothelial dysfunction leads to impaired

Therefore, microalbuminuria may be a marker of generalized vascular disease, as the formation of atherosclerotic thrombi is related to endothelial dysfunction in arteries. Thus in addition to being an early marker of incipient diabetic nephropathy, urinary albumin excretion may represent common pathways for the development of both large and small vessel disease making microalbuminuria as a possible marker for cardiovascular

Abnormalities of many cellular processes have been described in the kidney cells of experimental and/or human diabetes. Most work so far has been focused on the glomerular endothelial and mesangial cells. Direct effects of hyperglycemia per se (glucose toxicity), glycation, formation of advanced glycation products, increased flux through the polyol and hexosamine pathways have all been implicated in the pathogenesis of diabetic

Recently it has been suggested that the central abnormality linking all of these pathways is oxidative stress, a defect in the mitochondrial electron transport chain resulting in overproduction of reactive oxygen molecules which stimulate each of the above pathways [69].

Increased activity of a large number of growth factors has been demonstrated in diabetes

Transforming growth factor ß-1 and connective tissue growth factor: May be involved

Growth hormone and insulin like growth factor-1 (IGF-1) appear to be associated with

 Vascular endothelial growth factor (Synthesized by the podocyte): Plays a major role in maintaining the fenestrae in glomerular endothelial cells, has pressor effects leading to

 Glucose itself also stimulates some signaling molecules, leading to the increased intra glomerular pressure. Several isoforms of protein kinase C, diacyl glycerol, mitogenic

The glomerular hemodynamic changes in the form of hyper filtration and hyper perfusion results in decreased resistance in both afferent and efferent arterioles of the glomerulus. Many diverse factors including prostanoids, nitrogen oxide (NO), atrial natriuretic factor, growth hormone, glucagon, insulin, angiotensin II (ANG II), and others have been

kinases, and transcription factors are all activated in diabetic nephropathy.

in the fibrotic changes seen in mesangium and interstitium.

the glomerular hyper filtration and hypertrophy.

constriction of the efferent glomerular arterioles.

**3.5. Hemodynamic abnormalities** 

**3.3. Changes in endothelial function** 

**3.4. Cellular and molecular mechanisms** 

diseases.

nephropathy.

[70].

insulin action as well as to capillary leakage of albumin [67, 68].

In addition, renal biopsies from Pima Indians showed a broadening in podocyte foot processes and a concomitant reduction in the number of podocytes per glomerulus [58] in type 2 diabetic patients.


The structural abnormalities seen are:

**Table 5.** Main structural abnormalities in diabetic nephropathy

### **3.2. Glomerular and tubular mechanisms**

The alterations in glomerular function and tubular reabsorption play an important role in microalbuminuria. The glomeruli receive 25% of cardiac output per day. Of the 70kg of albumin that passes through the kidneys every 24hr, less than 0.01% reaches the glomerular ultra filtrate and hence enters the renal tubules [59, 60, and 61]. Almost all filtered albumin is reabsorbed by proximal tubule via a high affinity, low capacity endocytic mechanism with only 10-30mg/24hour appearing in the urine [62].

The passage of albumin through glomeruli depends on two main factors, charge and size. The negative charge on the glomerular membrane repels the anionic proteins thereby preventing the passage of albumin molecules through glomeruli normally. The loss of glomerular charge selectivity has been found in both diabetics and non-diabetic population with microalbuminuria [63, 64].

Established microscopic abnormalities include thickening of the glomerular basement membrane, accumulation of mesangial matrix, and increase in the numbers of mesangial cells with disease progression there is a close relationship between mesangial expansion and declining glomerular filtration [65].

Mesangial expansion also correlates inversely with capillary filtration surface area, which itself correlates with glomerular filtration rate. Changes in the tubulointerstitium, including thickening of tubular basement membrane, tubular atrophy, interstitial fibrosis and arteriosclerosis, have been well described. Interstitial enlargement correlates with glomerular filtration, albuminuria, and mesangial expansion. It has been suggested that the accumulation of protein in the cytoplasm of proximal tubular cells causes an inflammatory reaction which leads to tubulointerstitial lesions [65]. Similarly, rise in blood pressure plays an important role by altering the fraction of plasma filtered by the glomerulus.

## **3.3. Changes in endothelial function**

26 Pathophysiology and Complications of Diabetes Mellitus

are altered due to hyperglycemia.

The structural abnormalities seen are:

Glomerulosclerosis (diffuse, nodular) Basement membrane thickening (glomerular

**3.2. Glomerular and tubular mechanisms** 

only 10-30mg/24hour appearing in the urine [62].

with microalbuminuria [63, 64].

declining glomerular filtration [65].

Mesangial expansion Fibrin cap lesion

Arteriosclerosis Tubular atrophy Capsular drop lesion Interstitial fibrosis Interstitial inflammation Podocyte abnormalities

**Table 5.** Main structural abnormalities in diabetic nephropathy

and tubular) Endothelial foam cells

type 2 diabetic patients.

The recent evidence shows that an alteration in structure and function of podocytes occurs early in diabetic nephropathy. The podocytes which are adhering to GBM through integrin's

In addition, renal biopsies from Pima Indians showed a broadening in podocyte foot processes and a concomitant reduction in the number of podocytes per glomerulus [58] in

The alterations in glomerular function and tubular reabsorption play an important role in microalbuminuria. The glomeruli receive 25% of cardiac output per day. Of the 70kg of albumin that passes through the kidneys every 24hr, less than 0.01% reaches the glomerular ultra filtrate and hence enters the renal tubules [59, 60, and 61]. Almost all filtered albumin is reabsorbed by proximal tubule via a high affinity, low capacity endocytic mechanism with

The passage of albumin through glomeruli depends on two main factors, charge and size. The negative charge on the glomerular membrane repels the anionic proteins thereby preventing the passage of albumin molecules through glomeruli normally. The loss of glomerular charge selectivity has been found in both diabetics and non-diabetic population

Established microscopic abnormalities include thickening of the glomerular basement membrane, accumulation of mesangial matrix, and increase in the numbers of mesangial cells with disease progression there is a close relationship between mesangial expansion and

Mesangial expansion also correlates inversely with capillary filtration surface area, which itself correlates with glomerular filtration rate. Changes in the tubulointerstitium, including thickening of tubular basement membrane, tubular atrophy, interstitial fibrosis and arteriosclerosis, have been well described. Interstitial enlargement correlates with glomerular filtration, albuminuria, and mesangial expansion. It has been suggested that the accumulation of protein in the cytoplasm of proximal tubular cells causes an inflammatory reaction which leads to tubulointerstitial lesions [65]. Similarly, rise in blood pressure plays

an important role by altering the fraction of plasma filtered by the glomerulus.

Increased systemic capillary permeability has also been linked with microalbuminuria in healthy populations and recent study shows that endothelial dysfunction leads to impaired insulin action as well as to capillary leakage of albumin [67, 68].

Therefore, microalbuminuria may be a marker of generalized vascular disease, as the formation of atherosclerotic thrombi is related to endothelial dysfunction in arteries. Thus in addition to being an early marker of incipient diabetic nephropathy, urinary albumin excretion may represent common pathways for the development of both large and small vessel disease making microalbuminuria as a possible marker for cardiovascular diseases.

## **3.4. Cellular and molecular mechanisms**

Abnormalities of many cellular processes have been described in the kidney cells of experimental and/or human diabetes. Most work so far has been focused on the glomerular endothelial and mesangial cells. Direct effects of hyperglycemia per se (glucose toxicity), glycation, formation of advanced glycation products, increased flux through the polyol and hexosamine pathways have all been implicated in the pathogenesis of diabetic nephropathy.

Recently it has been suggested that the central abnormality linking all of these pathways is oxidative stress, a defect in the mitochondrial electron transport chain resulting in overproduction of reactive oxygen molecules which stimulate each of the above pathways [69].

Increased activity of a large number of growth factors has been demonstrated in diabetes [70].


#### **3.5. Hemodynamic abnormalities**

The glomerular hemodynamic changes in the form of hyper filtration and hyper perfusion results in decreased resistance in both afferent and efferent arterioles of the glomerulus. Many diverse factors including prostanoids, nitrogen oxide (NO), atrial natriuretic factor, growth hormone, glucagon, insulin, angiotensin II (ANG II), and others have been

implicated as agents causing hyperperfusion and hyper filtration [71].Hyperglycemia itself stimulates the synthesis of angiotensin II, which leads to various hemodynamic changes in the form of trophic, inflammatory and profibrogenic effects.

Bio-Chemical Aspects, Pathophysiology of

Microalbuminuria and Glycated Hemoglobin in Type 2 Diabetes Mellitus 29

Long term prospective studies are required in all major ethnic groups to establish more precisely the glucose and HbA1c levels predictive of micro vascular and macro vascular complications. A working group should be established to examine all aspects of HbA1c and

The diagnosis of diabetes in an asymptomatic person should not be made on the basis of a single abnormal plasma glucose or HbA1c value. At least one additional HbA1c or plasma glucose test result with a value in the diabetic range is required, fasting, a random (casual)

The main long term vascular complications are coronary artery disease, stroke, renal failure etc. The measurement of glycosylated hemoglobin (GHb) is one of the well-established means of monitoring glycemic control in patients with diabetes mellitus [80]. In 1968 Bookchin and Gallop subsequently reported that the largest of these minor fractions, designated HbA1c, had a hexose moiety linked to the N-terminus of the β-globin chain [81].The functions of many proteins depend upon post translational modification, hemoglobin is one such protein [82]. Hemoglobin (Hb) is composed of four globin chains and adult hemoglobin (HbA) is the most abundant form in most adults and consists of two α and two β chains. Fetal hemoglobin (HbF), which is predominantly present at birth, consists of two α and two γ chains. HbF is a minor form in normal adults. HbA2 is minor Hb after birth and consists of two α and two δ chains. The most common Hb variants worldwide in descending order of prevalence are HbS, HbE, HbC and HbD. All of these hemoglobin's have single amino acid substitutions in the β chain. Normal adult hemoglobin consists primarily of hemoglobin's A (90-95%), A2 (2-3%), F (0.5%), Ala (1.6%), Alb (0.8%), and A1c (3-6%). Glycosylated hemoglobin's (GHb) are the minor hemoglobin molecules separable by chromatographic techniques into three major components: Ala, Alb, and A1c.

Important perspective studies on chronic complications of Diabetes mellitus allowed us to establish with absolute certainty the role of glycosylated hemoglobin (HbA1c) as a marker of evaluation of long term glycemic control in diabetic patients and the strict relationship between the risk for chronic complications and HbA1c levels. Diabetes Control and Complication Trial (DCCT), a great extent study, has demonstrated that the 10% stable reduction in HbA1c determines a 35% risk reduction for retinopathy, a 25- 44% risk reduction

Glycosylation is a non-enzymatic reaction between free aldehyde group of glucose and free amino groups of proteins. A labile aldiminic adduct (Schiff base) forms at first, then,

In the hemoglobin, the preferential glycosylation site is the amino-terminal valine of the β chain of the globin (about 60% of glycosylated globin). Other sites are: lysin 66 and 17 of the β chain, valine 1 of the α chain. The term HbA1c refers to the hemoglobin fraction of the

through a molecular rearrangement, a stable ketoaminic product slowly accumulates.

glucose measurement methodology.

sample, or the oral glucose tolerance test (OGTT) report.

Hemoglobin Al refers to a combination of these three components [83].

for nephropathy and a 30% risk reduction for neuropathy [84].

glucose bound stably (ketoamine) to beta terminal of valines.

**4.1. Glycosylation process** 

The vascular endothelial growth factors (VEGFs), and cytokines, such as transforming growth factor β (TGF-β), may mediate hyper filtration by dilatation of the afferent vessels by inhibiting calcium transients [72]. Furthermore, TGF- β increases NO production in early diabetes, probably by up-regulation of endothelial NO synthase (eNOS) mRNA expression and by enhancing arginine resynthesis [72]. Thus, TGF- β could clearly play a role in diabetic vascular dysfunction [74].

The studies have shown that shear stress and mechanical strain causes hemodynamic alterations by inducing the autocrine and/or paracrine release of cytokines and growth factors.

The factors contributed are:


## **4. Role of glycated hemoglobin in diabetes**

Glycated hemoglobin (HbA1c), a marker of average glycaemia, is a predictor of micro vascular complications in diabetic individuals. However, it is not yet clear whether the HbA1c is an indicator of the risk of the macro vascular complications associated with diabetes mellitus.

HbA1c is the product of non-enzymatic reaction between glucose and free amino groups of hemoglobin. This reaction, called glycosylation, involves lots of other proteins, too and it is the principal mechanism through which glucotoxicity occurs. Other mechanism involved s is: oxidative stress, activation of the polyols pathway, activation of protein kinase-C, endothelial damage, hemodynamic and coagulative changes [75].

HbA1c reflects average plasma glucose over the previous 8 to 12 weeks as the life span of RBC's is 80-120days [76]. It can be performed at any time of the day and does not require any special preparation such as fasting. These properties have made it the preferred test for assessing glycemic control in diabetics. More recently, there has been substantial interest in using it as a diagnostic test for diabetes and as a screening test for persons at high risk of diabetes [77]. The use of HbA1c can avoid the problem of day-to-day variability of glucose values, and importantly it avoids the need for the person to fast and to have preceding dietary preparations. These advantages have implications for early identification and treatment which have been strongly advocated in recent years.

However, HbA1c may be affected by a variety of genetic, hematologic and illness-related factors [78]. The most common important factors worldwide affecting HbA1c levels are hemoglobinopathies (depending on the assay method employed), certain anemia's, and disorders associated with accelerated red cell turnover such as malaria [79].

Long term prospective studies are required in all major ethnic groups to establish more precisely the glucose and HbA1c levels predictive of micro vascular and macro vascular complications. A working group should be established to examine all aspects of HbA1c and glucose measurement methodology.

The diagnosis of diabetes in an asymptomatic person should not be made on the basis of a single abnormal plasma glucose or HbA1c value. At least one additional HbA1c or plasma glucose test result with a value in the diabetic range is required, fasting, a random (casual) sample, or the oral glucose tolerance test (OGTT) report.

The main long term vascular complications are coronary artery disease, stroke, renal failure etc. The measurement of glycosylated hemoglobin (GHb) is one of the well-established means of monitoring glycemic control in patients with diabetes mellitus [80]. In 1968 Bookchin and Gallop subsequently reported that the largest of these minor fractions, designated HbA1c, had a hexose moiety linked to the N-terminus of the β-globin chain [81].The functions of many proteins depend upon post translational modification, hemoglobin is one such protein [82]. Hemoglobin (Hb) is composed of four globin chains and adult hemoglobin (HbA) is the most abundant form in most adults and consists of two α and two β chains. Fetal hemoglobin (HbF), which is predominantly present at birth, consists of two α and two γ chains. HbF is a minor form in normal adults. HbA2 is minor Hb after birth and consists of two α and two δ chains. The most common Hb variants worldwide in descending order of prevalence are HbS, HbE, HbC and HbD. All of these hemoglobin's have single amino acid substitutions in the β chain. Normal adult hemoglobin consists primarily of hemoglobin's A (90-95%), A2 (2-3%), F (0.5%), Ala (1.6%), Alb (0.8%), and A1c (3-6%). Glycosylated hemoglobin's (GHb) are the minor hemoglobin molecules separable by chromatographic techniques into three major components: Ala, Alb, and A1c. Hemoglobin Al refers to a combination of these three components [83].

Important perspective studies on chronic complications of Diabetes mellitus allowed us to establish with absolute certainty the role of glycosylated hemoglobin (HbA1c) as a marker of evaluation of long term glycemic control in diabetic patients and the strict relationship between the risk for chronic complications and HbA1c levels. Diabetes Control and Complication Trial (DCCT), a great extent study, has demonstrated that the 10% stable reduction in HbA1c determines a 35% risk reduction for retinopathy, a 25- 44% risk reduction for nephropathy and a 30% risk reduction for neuropathy [84].

### **4.1. Glycosylation process**

28 Pathophysiology and Complications of Diabetes Mellitus

diabetic vascular dysfunction [74].

The factors contributed are:

diabetes mellitus.

Renin-angiotensin system

factors.

the form of trophic, inflammatory and profibrogenic effects.

**4. Role of glycated hemoglobin in diabetes** 

endothelial damage, hemodynamic and coagulative changes [75].

treatment which have been strongly advocated in recent years.

disorders associated with accelerated red cell turnover such as malaria [79].

implicated as agents causing hyperperfusion and hyper filtration [71].Hyperglycemia itself stimulates the synthesis of angiotensin II, which leads to various hemodynamic changes in

The vascular endothelial growth factors (VEGFs), and cytokines, such as transforming growth factor β (TGF-β), may mediate hyper filtration by dilatation of the afferent vessels by inhibiting calcium transients [72]. Furthermore, TGF- β increases NO production in early diabetes, probably by up-regulation of endothelial NO synthase (eNOS) mRNA expression and by enhancing arginine resynthesis [72]. Thus, TGF- β could clearly play a role in

The studies have shown that shear stress and mechanical strain causes hemodynamic alterations by inducing the autocrine and/or paracrine release of cytokines and growth

Glycated hemoglobin (HbA1c), a marker of average glycaemia, is a predictor of micro vascular complications in diabetic individuals. However, it is not yet clear whether the HbA1c is an indicator of the risk of the macro vascular complications associated with

HbA1c is the product of non-enzymatic reaction between glucose and free amino groups of hemoglobin. This reaction, called glycosylation, involves lots of other proteins, too and it is the principal mechanism through which glucotoxicity occurs. Other mechanism involved s is: oxidative stress, activation of the polyols pathway, activation of protein kinase-C,

HbA1c reflects average plasma glucose over the previous 8 to 12 weeks as the life span of RBC's is 80-120days [76]. It can be performed at any time of the day and does not require any special preparation such as fasting. These properties have made it the preferred test for assessing glycemic control in diabetics. More recently, there has been substantial interest in using it as a diagnostic test for diabetes and as a screening test for persons at high risk of diabetes [77]. The use of HbA1c can avoid the problem of day-to-day variability of glucose values, and importantly it avoids the need for the person to fast and to have preceding dietary preparations. These advantages have implications for early identification and

However, HbA1c may be affected by a variety of genetic, hematologic and illness-related factors [78]. The most common important factors worldwide affecting HbA1c levels are hemoglobinopathies (depending on the assay method employed), certain anemia's, and

Vasoactive hormones such as nitric oxide, prostacyclin, Endothelin -1,Urotensin

Glycosylation is a non-enzymatic reaction between free aldehyde group of glucose and free amino groups of proteins. A labile aldiminic adduct (Schiff base) forms at first, then, through a molecular rearrangement, a stable ketoaminic product slowly accumulates.

In the hemoglobin, the preferential glycosylation site is the amino-terminal valine of the β chain of the globin (about 60% of glycosylated globin). Other sites are: lysin 66 and 17 of the β chain, valine 1 of the α chain. The term HbA1c refers to the hemoglobin fraction of the glucose bound stably (ketoamine) to beta terminal of valines.

## **4.2. Other proteins which undergo glycosylation**

Albumin, α2 macroglobulin, antithrombin III, fibrinogen, ferritin, HDL, LDL, transferrin; all of them are short half-life proteins. The glycosylation process of short half-life proteins stops at the formation of the stable ketoamine adduct.

Bio-Chemical Aspects, Pathophysiology of

Microalbuminuria and Glycated Hemoglobin in Type 2 Diabetes Mellitus 31

Cardiovascular diseases- Independent

of diabetes

Immunochemical strip test is specific for albumin. Albumin in the sample is bound by soluble conjugate of antibodies and the β-galactosidase enzyme marker. Conjugate-albumin complexes are separated and the β galactosidase enzyme reacts with a substrate to produce

a red dye. The intensity of the color produced is

complex reaction. This causes a reduction in the intensity of light as it passes through the solution. Turbidimetry is the measurement of this loss in

intensity because of scattering, absorption or reflection of the incident light in the angle/direction of the incident

4'-dih ydroxy-5, 5'-di nitrophenyl)-3,4,5,6-

proportional to the albumin concentration in the urine.

method. It uses the high affinity dye bis (3,3'-diiodo- 4,

tetrabromosulfonephthalein. At a constant pH, the strip turns blue in the presence of albumin, and color is directly related to albumin concentration in the urine

 Early morning mid-stream urine sample is usually rather concentrated and using this sample has good correlation between the excretion rate and concentration of albumin.

Many conditions can give a false positive value. Some of these common conditions are

Acute hyperglycemia Urinary tract infection

catabolism and altered renal circulation Febrile condition and Stress

Clinitec Microalbumin The test strip is based on dye binding by albumin

sample.

light.

Immunoturbidimetry In this process turbidity is produced by an immune

Contamination with seminal or menstrual fluid- Which has more amount of albumin

shown in table 6:

Hypertension- Independently causes

Heavy exercise- Due to increased protein

**Table 6.** Various factors affecting microalbumin estimation

Principle

microalbuminuria

**Semi quantitative methods**

strip

**Quantitative** 

Micral microalbumin urine test

## **4.3. Advanced Glycosylation End products (AGE)**

The long half-life proteins such as actin, collagen, fibronectin, myelin, nucleoproteins, spectrin, and tubulin can also be glycosylated. These long half-life proteins (myelin and collagen) undergo a complex and irreversible rearrangement process, with the formation of Advanced Glycosylation End products (AGE). AGE form a family with many compounds, only partially identified; they accumulate in the structural proteins modifying the function of them. They bind to specific macrophage receptors inducing a release of hydrolytic enzymes, cytokines and growth factors able to promote the synthesis of fundamental substance and, acting at intracellular level, to determine a damage of the nucleic acids [85, 86].

Three mechanisms have been postulated that explain how hyperglycemia causes tissue damage: nonenzymatic glycosylation that generates advanced glycosylation end products, activation of PKC, and acceleration of the aldose reductase pathway. Oxidative stress seems to be a common to all three pathways.

## **5. Note on laboratory aspects**

## **5.1. Microalbuminuria estimation**

## *5.1.1. Sample handling*

The collection of sample is very important when you are measuring MA. As many factors will alter the value and errors may occur due to improper aseptic precautions, improper storage and handling. After the collection it is preferable to measure on the same day and if urine albumin is not estimated immediately then urine can be stored at 4°C. Alternatively, 2ml of 50 g /L sodium azide can be added per 500ml of urine. Specimens are stable for at least 2 weeks at 4°C and 5 months at -70°C. Freezing samples may decrease albumin but mixing immediately before assay eliminates this effect [87].

Albumin excretion varies with physiological factors like exercise posture, diuresis. Thus samples should not be collected after exercise, in the presence of urinary tract infection, during acute illness, immediately after surgery or after an acute fluid overload.

The following are considered acceptable [88]:


 Early morning mid-stream urine sample is usually rather concentrated and using this sample has good correlation between the excretion rate and concentration of albumin.

Many conditions can give a false positive value. Some of these common conditions are shown in table 6:


Contamination with seminal or menstrual fluid- Which has more amount of albumin


30 Pathophysiology and Complications of Diabetes Mellitus

at the formation of the stable ketoamine adduct.

to be a common to all three pathways.

**5. Note on laboratory aspects** 

**5.1. Microalbuminuria estimation** 

*5.1.1. Sample handling* 

**4.2. Other proteins which undergo glycosylation** 

**4.3. Advanced Glycosylation End products (AGE)** 

intracellular level, to determine a damage of the nucleic acids [85, 86].

mixing immediately before assay eliminates this effect [87].

occur due to improper sample collection and transport.

The following are considered acceptable [88]:

Overnight (8 - 12 hour) urine sample collection

Albumin, α2 macroglobulin, antithrombin III, fibrinogen, ferritin, HDL, LDL, transferrin; all of them are short half-life proteins. The glycosylation process of short half-life proteins stops

The long half-life proteins such as actin, collagen, fibronectin, myelin, nucleoproteins, spectrin, and tubulin can also be glycosylated. These long half-life proteins (myelin and collagen) undergo a complex and irreversible rearrangement process, with the formation of Advanced Glycosylation End products (AGE). AGE form a family with many compounds, only partially identified; they accumulate in the structural proteins modifying the function of them. They bind to specific macrophage receptors inducing a release of hydrolytic enzymes, cytokines and growth factors able to promote the synthesis of fundamental substance and, acting at

Three mechanisms have been postulated that explain how hyperglycemia causes tissue damage: nonenzymatic glycosylation that generates advanced glycosylation end products, activation of PKC, and acceleration of the aldose reductase pathway. Oxidative stress seems

The collection of sample is very important when you are measuring MA. As many factors will alter the value and errors may occur due to improper aseptic precautions, improper storage and handling. After the collection it is preferable to measure on the same day and if urine albumin is not estimated immediately then urine can be stored at 4°C. Alternatively, 2ml of 50 g /L sodium azide can be added per 500ml of urine. Specimens are stable for at least 2 weeks at 4°C and 5 months at -70°C. Freezing samples may decrease albumin but

Albumin excretion varies with physiological factors like exercise posture, diuresis. Thus samples should not be collected after exercise, in the presence of urinary tract infection,

24 hour collection is preferred by some centers but this is cumbersome and errors may

during acute illness, immediately after surgery or after an acute fluid overload.

Short term urine collection i.e. 1-2 hour collection (in laboratory or clinic)



Bio-Chemical Aspects, Pathophysiology of

Type 2 diabetes sample

Microalbuminuria and Glycated Hemoglobin in Type 2 Diabetes Mellitus 33

Urine Urine and blood


In the last 20 years improved techniques in laboratory and new electrophoretical, chromatographic and immunological methods available, gave us a greater reliability on results. However the use of different methods, the lack of a common calibration concerning the same method and the variability of instrumentation do not make reproducible results yet in different laboratories. For this reason studies and procedures of standardization are going on [89]. Methods of GHb assays have primarily evolved around three basic methodologies:

assayed

Marker Type 1 diabetes sample

Glomerular transferrin Urine Urine Fibronectin Plasma Urine Serum laminin P1 - Serum Urine laminin P1 Urine Urine

Heparan sulfate proteoglycan Urine -

Retinol-binding protein Urine and serum Urine Tamm-Horsfall protein Urine - Alpha1-microglobulin Urine Urine

Cholinesterase - Urine Gamma-glutamyl transpeptidase - Urine Alanine aminopeptidase - Urine

Tubular proteins beta-2

Tubular antigens,brush-border

**Table 8.** Newer markers of diabetic nephropathy [89]:

1. Based on difference in ionic charge. 2. Based on structural characteristics. 3. Based on chemical reactivity.

Cation exchange chromatography

 Electrophoresis/Electroendosmosis Electrospray mass spectrometry

High performance liquid chromatography

Affinity chromatography

The main methods are,

 Isoelectric focusing Radioimmunoassay Spectrophotometric assay

**6. Methods of estimation of glycated hemoglobin** 

microglobulin

antigen

assayed

Type 4 collagen - Serum and urine

N-acetyl-beta-D-glucoseaminidase Urine and serum Urine and serum

Note: Advantages of both RIA and CLIA are highly sensitivity, specificity and reproducible. Disadvantages are unavailability, cost factor; proper infrastructure needed, radioactive hazards, Government permission for use of radioactive materials is the limiting factors.

**Table 7.** Methods of estimation [87, 88]

Bio-Chemical Aspects, Pathophysiology of Microalbuminuria and Glycated Hemoglobin in Type 2 Diabetes Mellitus 33


**Table 8.** Newer markers of diabetic nephropathy [89]:

## **6. Methods of estimation of glycated hemoglobin**

In the last 20 years improved techniques in laboratory and new electrophoretical, chromatographic and immunological methods available, gave us a greater reliability on results. However the use of different methods, the lack of a common calibration concerning the same method and the variability of instrumentation do not make reproducible results yet in different laboratories. For this reason studies and procedures of standardization are going on [89]. Methods of GHb assays have primarily evolved around three basic methodologies:


The main methods are,

32 Pathophysiology and Complications of Diabetes Mellitus

Chemiluminescent immunoassay

**Table 7.** Methods of estimation [87, 88]

(CLIA)

Most colorimeters and spectrophotometers can measure turbidity with good precision and accuracy. This is the most widely used test as it can be done on most semi auto chemistry analyzers. It can even be done on

turbidimetry it measures scattered light at 90° to the incident light. The instrument is called a nephelometer.

between radio labelled and unlabelled molecules of antigen to high affinity, specific antibody. The amount of unlabelled antigen present in the specimen is measured by its competitive effect on the labelled antigen for limited antibody sites. It involves the use of radio isotopes like tritium (3H), 131I or 125I as labels. It has high sensitivity and specificity. The sample values are determined by comparison with a calibration curve. The advantages are sensitivity and precision, whereas the disadvantage is short shelf life and radioactivity of the

Chemiluminescence is a chemical reaction that emits

immunoassay technology, the light produced by the reaction indicates the amount of analyte in a sample.

Luminescent Immunoassay (LIA): Here the labelled and unlabelled antigen competes for the limited binding sites on the labelled antibody. An inverse relationship exists between concentration of labelled antibody bound

Immuno Chemiluminometric assay (ICMA): This is a sandwich assay in which unlabelled antigen is

sandwiched between antibody bound to paramagnetic particles and antibody labelled Acridinium ester (AE). A direct relationship exists between the concentration of antigen in the patient sample and the amount of light

energy in the form of light. When used with

to the antigen and the unlabelled antigen.

emitted during oxidation of the AE.

This again is of two types:

automated chemistry analyzers.

It is more sensitive than turbidimetry.

Nephelometry This assay is also based on scatter detection but unlike

Radio immunoassay (RIA) This assay procedure involves competitive binding

reagents.

Note: Advantages of both RIA and CLIA are highly sensitivity, specificity and reproducible. Disadvantages are unavailability, cost factor; proper infrastructure needed, radioactive hazards,

Government permission for use of radioactive materials is the limiting factors.


## **6.1. Specimen collection**

Handling of specimens before the assay is important as short period of hyperglycemia before blood is taken, leads to an acute increase in the formation of aldimine which may increase the concentration of glycosylated hemoglobin by 10-20%--for example, from 9% to 11% of total hemoglobin-thus reducing the reliability of the test as a measure of long term diabetic control.

Bio-Chemical Aspects, Pathophysiology of

Microalbuminuria and Glycated Hemoglobin in Type 2 Diabetes Mellitus 35

Administration of erythropoietin, Iron,

 Reticulocytosis, Chronic liver disease. Certain hemoglobinopathies, increased intra-

hemoglobinopathies, splenomegaly,

rheumatoid arthritis and drugs such as antiretrovirals, ribavirin, dapsone therapy.

interfere.

Affected by

binding sites.

 Variable interference from hemoglobinopathies, HbF and carbamylated Hb but the current ion exchange assays correct for HbF and carbamylated Hb does not

 Measures not only glycation of N-terminal valine on beta chain, but also beta chains glycated at other sites and glycated alpha chains.

> hemoglobinopathies with altered amino acids on

Decreased erythrocyte life span:

False decreases may result when HbS (Ex: Sickle cell disease) or other positively

Hemolytic anemia and chronic blood loss result in decreased red cell life span and

Vitamin B12,

erythrocyte pH.

Hypertriglyceridemia.

**6.2. Factors and clinical conditions affecting glycated hemoglobin levels are** 

**Increased glycated hemoglobin Decreased glycated hemoglobin** 

**6.3. Advantages and disadvantages of various HbA1c assay methods** 

Can inspect chromograms for Hb

**Method Advantages Disadvantages**

Measurements with great

Minimal interference from

Not affected by HbE, HbD or

 Relatively easy to implement under many different formats.

carbamylated Hb.

carbamylated Hb

hemoglobinopathies, HbF and

variants.

precision.

charged variants are present

Iron and Vitamin B12 deficiency

 Chronic renal failure decreased intraerythrocyte pH Increased erythrocyte life span:

**shown below [78]** 

Alcoholism

**Table 9.**

**Ion Exchange Chromatography** 

**Boronate Affinity** 

**Immunoassays** 

**Table 10.**

Splenectomy Hyperbilirubinemias Large doses of aspirin Chronic opiate use

therefore lower glycosylated hemoglobin levels.

Blood samples should therefore be treated to remove the aldimine residues before the assay [91]. In measurement of HbA1c the prevalence of the most common hemoglobin variants (HbS, HbC, and HbD) depends on the genetic background of the population being analysed. There are many Hb variants that result in false low HbA1c level in diabetes. More than 700 Hb variants are known and about half of these variants are clinically silent, their presence may falsely interfere with the measurement of HbA1c by HPLC. Hence, the identification of Hb variants is important to avoid inaccurate HbA1c results [92].

Recent reports have shown that the concentration of total glycosylated hemoglobin measured by commonly used methods may change significantly over a period of hours. This reflects the short term fluctuations in glucose concentration. It is now realized that these rapid changes will depend on the synthesis or dissociation of the labile fraction of HbA1c, which is not separable from the stable form of HbA1c, by most routine methods. In most cases, the labile fraction constitutes approximately 10% of the total glycosylated hemoglobin. This may increase to 25% when plasma glucose concentrations are high, as in poor glycemic control.

These day to day variations in glycosylated hemoglobin concentration secondary to changes in serum glucose are negligible in stable diabetics, but are very wide in unstable diabetics and are almost entirely dependent on the prevailing plasma glucose concentration. Thus, during poor glycemic control there will be large swings in plasma glucose levels, a single HbA1c measurement may be misleading as an index of long term control. It would therefore make sense to measure the stable fraction of glycosylated hemoglobin. However, this is not routinely available because most laboratories measure total HbA1 or HbA1c, which includes both labile and stable components. Therefore, in unstable diabetics, HbAl measurements should be interpreted in relation to the simultaneous glucose concentration. To minimize the contribution of the labile fraction, glycosylated hemoglobin should be measured when the plasma glucose concentration is within or near the normal range.

Physicians should be aware of the expected variation in HbA1c during conditions such as [93],


## **6.2. Factors and clinical conditions affecting glycated hemoglobin levels are shown below [78]**


**Table 9.**

34 Pathophysiology and Complications of Diabetes Mellitus

Handling of specimens before the assay is important as short period of hyperglycemia before blood is taken, leads to an acute increase in the formation of aldimine which may increase the concentration of glycosylated hemoglobin by 10-20%--for example, from 9% to 11% of total hemoglobin-thus reducing the reliability of the test as a measure of long term

Blood samples should therefore be treated to remove the aldimine residues before the assay [91]. In measurement of HbA1c the prevalence of the most common hemoglobin variants (HbS, HbC, and HbD) depends on the genetic background of the population being analysed. There are many Hb variants that result in false low HbA1c level in diabetes. More than 700 Hb variants are known and about half of these variants are clinically silent, their presence may falsely interfere with the measurement of HbA1c by HPLC. Hence, the identification of

Recent reports have shown that the concentration of total glycosylated hemoglobin measured by commonly used methods may change significantly over a period of hours. This reflects the short term fluctuations in glucose concentration. It is now realized that these rapid changes will depend on the synthesis or dissociation of the labile fraction of HbA1c, which is not separable from the stable form of HbA1c, by most routine methods. In most cases, the labile fraction constitutes approximately 10% of the total glycosylated hemoglobin. This may increase to 25% when plasma glucose concentrations are high, as in poor glycemic control.

These day to day variations in glycosylated hemoglobin concentration secondary to changes in serum glucose are negligible in stable diabetics, but are very wide in unstable diabetics and are almost entirely dependent on the prevailing plasma glucose concentration. Thus, during poor glycemic control there will be large swings in plasma glucose levels, a single HbA1c measurement may be misleading as an index of long term control. It would therefore make sense to measure the stable fraction of glycosylated hemoglobin. However, this is not routinely available because most laboratories measure total HbA1 or HbA1c, which includes both labile and stable components. Therefore, in unstable diabetics, HbAl measurements should be interpreted in relation to the simultaneous glucose concentration. To minimize the contribution of the labile fraction, glycosylated hemoglobin should be measured when the

Physicians should be aware of the expected variation in HbA1c during conditions such as [93], False increases in HbA1c levels may occur in the presence of HbF (Ex: Hereditary

 HbA1c levels may also be increased in patients with renal insufficiency, caused by hemoglobin carbamylation resulting from condensation of urea with the same site to

Increased levels can be seen in people with a longer red blood cell lifespan, such as with

Increased HbA1c occurs with advanced malignancy and iron deficiency anemia.

Hb variants is important to avoid inaccurate HbA1c results [92].

plasma glucose concentration is within or near the normal range.

which glucose attaches.

Vitamin B12 or folate deficiency.

persistence of fetal Hb) and other negatively charged hemoglobin's

Splenectomy can result in elevated levels of glycosylated hemoglobin

**6.1. Specimen collection** 

diabetic control.

#### **6.3. Advantages and disadvantages of various HbA1c assay methods**


**Table 10.**

## **7. Diagnosis of diabetes, its complications and management**

Diabetes screening is recommended for:


Bio-Chemical Aspects, Pathophysiology of

Microalbuminuria and Glycated Hemoglobin in Type 2 Diabetes Mellitus 37

Podiatry if any evidence of neuropathy or breakdown of skin integrity, and nutrition, if

**Type 1:** Annual screening for type 1 diabetes should begin at puberty and for those patients

**Note**: Microalbuminuria is urinary albumin excretion between 30-300 mg per day without an alternative explanation (e.g. urinary tract infection, heart failure, exercise in past 48 hours and blood glucose > 200 mg/dL). If no protein is found in a urine analysis, then a 24 hour urine collection for microalbumin or a spot urine albumin-creatinine ratio may be used

 For patients with type 1 diabetes who are 13 years of age or older and who have had the disease for 5 years, a baseline screening examination is recommended, and yearly

For patients with type 2 diabetes, a baseline screening examination is recommended at

Diabetic retinopathy is the leading cause of legal blindness among Americans, aged 20-74. It is highly correlated with patient age and duration of diabetes. Visual loss secondary to diabetic retinopathy is largely preventable if screening is universal and appropriate

 All patients who demonstrate microalbuminuria should be prescribed ACE inhibitors to slow the progression of nephropathy whether they are hypertensive or normotensive. Patients with type 1 who are hypertensive and do not demonstrate microalbuminuria should be prescribed ACE inhibitors. Such patients usually develop microalbuminuria in concert with hypertension and are best served by controlling blood pressure initially

Regular physical activity: Helps in movement of blood glucose into tissues.

**Type 2:** Initial testing at diagnosis and thereafter annual screening needed.

(abnormal if > 30 mg albumin/ g creatinine) for screening.

the time diagnosis and yearly thereafter.

dietary non-compliance is suspected.

who have had the disease for 5 years.

**7.2. Microalbumin testing** 

**7.3. Retinopathy screening** 

treatment follows screening.

 Pneumovaz every five years. Influenza vaccine annually.

Baseline Screening:

thereafter.

**7.4. Vaccines** 

**7.5. ACE inhibitors** 

with ACE inhibitors.

Recommendations:

The diagnosis of diabetes is mainly done by using Oral Glucose Tolerance Test (OGT) and the values are shown in Table:


#### **Table 11.**

### **7.1. Frequency of visits and laboratory testing**

The recommended frequency of follow-up is 3-6 months for patients with type 1 diabetes and for type 2 diabetes patients depending on the glycemic status.


#### **Table 12.**

The frequency of laboratory assessment is subject to flexibility, based on clinical judgment, patients' current control of diabetes, and past laboratory values.

Podiatry if any evidence of neuropathy or breakdown of skin integrity, and nutrition, if dietary non-compliance is suspected.

## **7.2. Microalbumin testing**

36 Pathophysiology and Complications of Diabetes Mellitus

Diabetes screening is recommended for:

Overweight adults (BMI greater than 30)

Overweight children

Adults over age 45

the values are shown in Table:

DIABETES IN ADULTS

glucose tolerance test.

Glycosylated hemoglobin

approach)

**Table 12.**

 Electrolytes, BUN and creatinine Physical examination including foot

present:

diabetes.

**Table 11.**

NEW CRITERIA FOR DIAGNOSING

One or more of the following must be

1. Fasting plasma glucose level of > 126 mg/dL on at least two separate occasions.

2. Random plasma glucose level of > 200 mg/dL with signs and symptoms of

3. Fasting plasma glucose level < 126 mg/dL but 2 hour glucose concentration of > 200 mg/dL during a 75-gram oral

**7.1. Frequency of visits and laboratory testing** 

**Every 3 - 6 months Yearly**

examination by filament testing (Carville

and for type 2 diabetes patients depending on the glycemic status.

patients' current control of diabetes, and past laboratory values.

**7. Diagnosis of diabetes, its complications and management** 

 Family history of diabetes along associated risk factors such smoking, hypertension etc. The diagnosis of diabetes is mainly done by using Oral Glucose Tolerance Test (OGT) and

ADULTS

After 75g oral glucose

load

The recommended frequency of follow-up is 3-6 months for patients with type 1 diabetes

The frequency of laboratory assessment is subject to flexibility, based on clinical judgment,

TSH

Fasting

NORMAL PLASMA GLUCOSE VALUES FOR

< 115 mg/dL (6.4 mM)

< 200 mg/dL (11.1 mM) < 200 mg/dL (11.1 mM) < 200 mg/dL (11.1 mM) < 140 mg/dL (7.8 mM)

Time Zero

30 min

60 min

90 min

120 min

 U/A or urine for microalbumin Complete chemistry panel (lipids, LFT, electrolytes, BUN & creatinine)

 Ophthalmology examination Podiatry and nutrition

**Type 1:** Annual screening for type 1 diabetes should begin at puberty and for those patients who have had the disease for 5 years.

**Type 2:** Initial testing at diagnosis and thereafter annual screening needed.

**Note**: Microalbuminuria is urinary albumin excretion between 30-300 mg per day without an alternative explanation (e.g. urinary tract infection, heart failure, exercise in past 48 hours and blood glucose > 200 mg/dL). If no protein is found in a urine analysis, then a 24 hour urine collection for microalbumin or a spot urine albumin-creatinine ratio may be used (abnormal if > 30 mg albumin/ g creatinine) for screening.

## **7.3. Retinopathy screening**

Baseline Screening:


Diabetic retinopathy is the leading cause of legal blindness among Americans, aged 20-74. It is highly correlated with patient age and duration of diabetes. Visual loss secondary to diabetic retinopathy is largely preventable if screening is universal and appropriate treatment follows screening.

#### **7.4. Vaccines**


Regular physical activity: Helps in movement of blood glucose into tissues.

### **7.5. ACE inhibitors**

Recommendations:

	- ACE inhibitors should not be used in pregnant women due to the risk of fetal morbidity and mortality.

Bio-Chemical Aspects, Pathophysiology of

Microalbuminuria and Glycated Hemoglobin in Type 2 Diabetes Mellitus 39

Since, early detection of microalbuminuria can help in early diagnosis of diabetic

Further studies have to be conducted to find better markers for chronic complications. Regular health education programs has to be conducted at an regular intervals, so that

The authors are thankful to Dr. Raghuveer CV, Director and Dean, Srinivas Institute of Medical and Health Sciences, Mangalore and Dr. Ullas Kamath, Dean, Melaka Manipal

[1] Aaron & Vinik. Diabetes and macrovascular disease. Journal of diabetes and its

[2] Gaede P, Vedel P, Larsen N, Jensen GV, Parving HH, Pedersen O. Multifactorial intervention and cardiovascular disease in patients with type 2 diabetes. N Engl J Med

[3] DeFronzo R. Diabetic nephropathy: etiologic and therapeutic considerations. Diabetes

[4] Remuzzi G, Schieppati A, Ruggenenti P. Nephropathy in patients with type 2 diabetes.

[5] Ismail N, Becker B, Strzelczyk P, Ritz E. Renal disease and hypertension in non-insulin-

[6] Bojestig M, Arnqvist H, Hermansson G, Karlberg B, Ludvigsson J. Declining incidence of nephropathy in insulin-dependent diabetes mellitus. N Engl J Med 1994; 330:15-18.

nephropathy, adequate care and precautions has to be taken while estimating it.

 and Saidunnisa Begum *Department of Biochemistry, Ras Al Khaimah Medical and Health Sciences,* 

*Department of Nursing, Vidya Nursing College, Udupi, Karnataka, India* 

*Department of Pathology, KMC, Manipal University, Manipal, Karnataka, India* 

Medical College, Manipal, Karnataka for their support and guidance.

dependent diabetes mellitus. Kidney Int 1999; 55:1-28.

**9. Further research** 

patients lead a better life.

Manjunatha B. K. Goud\*

**Acknowledgement** 

**10. References** 

 \*

2003; 348:383–93.

Rev 1995; 3:510-64.

Corresponding Author

complications 2001;16:235-245.

N Engl J Med 2002;346: 1145–51.

*University, Ras Al Khaimah, U.A.E* 

**Author details** 

Sarsina O. Devi

Bhavna Nayal

NOTE:

ACE inhibitors should be titrated as high as the patient tolerates without orthostatic symptoms, hyperkalemia and /or increasing renal insufficiency.

## **7.6. Oral hypoglycemic drugs and/or insulin therapy needed**

Some of the drugs used are,

Sulphonylureas


#### **Table 13.**


7.7. Life style changes such as cessation of smoking is necessary.


7.10. Management of diabetic complications.

## **8. Conclusion**

The most common chronic complication in diabetic patients is ESRD and several factors contribute to the development of renal damage such as genetic factors, hypertension and hyperglycemia.

The suspected cases of diabetic nephropathy will also invariably have diabetic retinopathy and more predisposed for cardiovascular events and mortality. That's why it's very important to prevent the condition then providing treatment. This is possible, as the progression of diabetic nephropathy is slow and can be detected at an early stage. Microalbuminuria is an early indicator of diabetic nephropathy and urine examination for micro albumin is routinely done to detect and monitor the progression of nephropathy. As many factors can interfere with the estimation of micro albumin, it is very important that high standards are maintained while estimating the MA levels. Other than microalbuminuria various newer markers have come, which still have to be studied for their probable role in its prevention of diabetic nephropathy.

Since, early detection of microalbuminuria can help in early diagnosis of diabetic nephropathy, adequate care and precautions has to be taken while estimating it.

## **9. Further research**

38 Pathophysiology and Complications of Diabetes Mellitus

symptoms, hyperkalemia and /or increasing renal insufficiency.

**7.6. Oral hypoglycemic drugs and/or insulin therapy needed** 

**First generation Second generation**

7.7. Life style changes such as cessation of smoking is necessary.

Tolbutamide Glipizide Tolazamide Glyburide Acetohexamide Glibenclamide Chlorpropamide Glimepiride

**Meglitinides:**Repaglinide, Nateglinide etc

7.8. Health education programs should be started. 7.9. Self-monitoring of plasma glucose using glucometer.

probable role in its prevention of diabetic nephropathy.

7.10. Management of diabetic complications.

 Thiazolidinediones: Pioglitazone etc  **Alpha-glucosidase:** Acarbose, Miglitol etc

 **Biguanides:** Metformin etc

and mortality.

Some of the drugs used are,

Sulphonylureas

NOTE:

**Table 13.**

**8. Conclusion** 

hyperglycemia.

ACE inhibitors should not be used in pregnant women due to the risk of fetal morbidity

ACE inhibitors should be titrated as high as the patient tolerates without orthostatic

The most common chronic complication in diabetic patients is ESRD and several factors contribute to the development of renal damage such as genetic factors, hypertension and

The suspected cases of diabetic nephropathy will also invariably have diabetic retinopathy and more predisposed for cardiovascular events and mortality. That's why it's very important to prevent the condition then providing treatment. This is possible, as the progression of diabetic nephropathy is slow and can be detected at an early stage. Microalbuminuria is an early indicator of diabetic nephropathy and urine examination for micro albumin is routinely done to detect and monitor the progression of nephropathy. As many factors can interfere with the estimation of micro albumin, it is very important that high standards are maintained while estimating the MA levels. Other than microalbuminuria various newer markers have come, which still have to be studied for their Further studies have to be conducted to find better markers for chronic complications. Regular health education programs has to be conducted at an regular intervals, so that patients lead a better life.

## **Author details**

Manjunatha B. K. Goud\* and Saidunnisa Begum *Department of Biochemistry, Ras Al Khaimah Medical and Health Sciences, University, Ras Al Khaimah, U.A.E* 

Sarsina O. Devi *Department of Nursing, Vidya Nursing College, Udupi, Karnataka, India* 

Bhavna Nayal *Department of Pathology, KMC, Manipal University, Manipal, Karnataka, India* 

## **Acknowledgement**

The authors are thankful to Dr. Raghuveer CV, Director and Dean, Srinivas Institute of Medical and Health Sciences, Mangalore and Dr. Ullas Kamath, Dean, Melaka Manipal Medical College, Manipal, Karnataka for their support and guidance.

## **10. References**


<sup>\*</sup> Corresponding Author

	- [7] Ritz E, Rychlik I, Miltenberger-Miltenyi G. Optimizing antihypertensive therapy in patients with diabetic nephropathy. J Hypertens 1998; Suppl. 16:S17-22.

Bio-Chemical Aspects, Pathophysiology of

Microalbuminuria and Glycated Hemoglobin in Type 2 Diabetes Mellitus 41

[25] Ramachandran A, Snehalatha C, Latha E, Vijay V, Viswanathan M. Rising prevalence of

[26] Ramachandran A, Snehalatha C, Kapur A, Vijay V, Mohan V, Das AK, Rao PV, Yajnik CS, Prasanna KS, Nair JD. For the Diabetes Epidemiology Study Group in India (DESI). High prevalence of diabetes and impaired glucose tolerance in India: National Urban

[27] Ramaiya KL, Kodali VR, Alberti KGMM. Epidemiology of diabetes in Asians of the

[28] Ramachandran A, Snehalatha C, Satyavani K, Latha E, Sasikala R, Vijay V. Prevalence of vascular complications and their risk factors in type 2 diabetes. J Assoc Phy India

[29] Rema M, Ponnaiya M, Mohan V. Prevalence of retinopathy in non insulin dependent diabetes mellitus at a diabetes centre in Southern India. Diab Res Clin Prac 1996; 34: 29-

[30] John L, Sundar Rao PSS, Kanagasabapathy AS. Prevalence of diabetic nephropathy in

[31] Samanta A, Burden AC, Jagger C. A comparison of the clinical features and vascular complications of diabetes between migrant Asians and Caucasians in Leicester, U.K.

[32] Cooper ME. Pathogenesis, prevention, and treatment of diabetic nephropathy. Lancet

[33] Alberti KGMM. Problems related to definitions and epidemiology of type 2 DM.

[34] Olgemoller B, Schleicher E. Alterations of glomerular proteins in the pathogenesis of

[38] Krolewski A, Fogarty D, Warram J. Hypertension and nephropathy in diabetes mellitus: what is inherited and what is acquired? Diabetes Res Clin Pract 1998; 39 (Suppl):S1-14. [39] Strojek K, Grzeszczak W, Ritz E. Risk factors for development of diabetic nephropathy:

[40] Seaquist E, Goetz F, Rich S, Barbosa J. Familial clustering of diabetic kidney disease: evidence for genetic susceptibility to diabetic nephropathy. N Engl J Med 1989;

[41] Fioretto P, Steffes M, Barbosa J, Rich S, Miller M, Mauer M. Is diabetic nephropathy inherited? Studies of glomerular structure in type 1 diabetic sibling pairs. Diabetes 1999;

[42] Rudofsky Jr G, Isermann B, Schilling T, Schiekofer S, Andrassy M, Schneider JG et al . A 63 bp deletion in the promoter of RAGE correlates with a decreased risk for

[35] Flybjerg A. Growth factors and diabetic complications. Diabet Med 1990;7:387-393. [36] Raskin P, Posenstock J. The genesis of diabetic susceptibility. In: Alberti KGMM, Zimmer P, Defronzo RA, Keen H. Eds International textbook of diabetes mellitus.2nd

[37] Lowe GDO. Clinicla blood rheology. Boca Raton, FL:CRC Press Inc, 1988.

a review. Nephrol Dial Transplant 1997; 12 (Suppl 2):24-26.

NIDDM in urban population in India. Diabetologia 1997; 40: 232-237.

Indian Sub continent. Diabetes Metabolism Rev 1990; 6: 125-146.

non-insulin dependent diabetics. Indian J Med Res 1991; 94: 24-29.

Diabetes Survey. Diabetologia 2001; 44: 1094-1101.

1999; 47: 1152-1156.

1998;352:213–219.

320:1161-65.

48:865-69.

Diab Res Clin Prac 1991;14: 205-214.

diabetic nephropathy. Clin Invest 1993;71:13-19.

eds. Chichester:Wiley;1997.p.1225-1244.

Diabetologia 1993; 36:948-984.

36.


[25] Ramachandran A, Snehalatha C, Latha E, Vijay V, Viswanathan M. Rising prevalence of NIDDM in urban population in India. Diabetologia 1997; 40: 232-237.

40 Pathophysiology and Complications of Diabetes Mellitus

Drugs 1998; 56:189-202.

55.

1994; 3:558-563.

1992;41: 836-839.

1999;40:40-45.

proteins in diabetes. Diabetes 1996;45:488-95.

ratio. J Am Soc Nephrol 1996;7:930-937.

diabetes mellitus. Kidney Int 2000;58:1228-1237.

observational study.BMJ 1994;314:783-788.

Examination Survey. Am J Kid Dis 2003; 41: 580-587

body adiposity. Diabetes Care 1992; 15:1348- 1355.

Canada: Canadian Institute for Health Information;2001.

–stage renal disease (ESRD). Clin Invest Med 1995;8:455-464.

[7] Ritz E, Rychlik I, Miltenberger-Miltenyi G. Optimizing antihypertensive therapy in

[8] Ritz E, Keller C, Bergis K, Strojek K. Pathogenesis and course of renal disease in IDDM/NIDDM: differences and similarities. Am J Hypertens 1997; 10:202-207S. [9] Makino H, et al. Phenotypic modulation of the mesangium reflected by contractile

[10] MacLeod M, McLay J. Drug treatment of hypertension complicating diabetes mellitus.

[11] Canadian Organ Replacement Registry (CORR).2001 Annal report.Ottawa, ON,

[12] Goeree R, Manalich J, Grootendorst P, et al. Cost analysis of dialysis treatments for end

[13] Parving HH. Diabetic nephropathy: prevention and treatment. Kidney Int 2001;60:2041–

[14] Mathiesen ER, Ronn B, Storm B, et al. The natural course of microalbuminuria in insulin-dependent diabetes :a 10- year prospective study. Diabet Med 1995;12:482-487. [15] Warram JH, Gearin G, Laffel L et al. Effect of duration of type 1 diabetes on thr prevalence of stages of diabetic nephropathy defined by urinary albumin/creatinine

[16] Lemley KV, Abdulla I, Myers BD et al. Evolution of incipient nephropathy in type 2

[17] Marre M, Bouhanick B, Berrut G. Microalbuminuria. Curr Opin Nephro Hypertens

[18] Gall MA, Hougaard P et al. Risk factors for development of incipient and overt diabetic nephropathy in patients with non-insulin dependent diabetes mellitus: prospective,

[19] Messent JWC, Elliott TG, Hill RD et al. Prognostic significance of microalbuminuria in insulin –dependent diabetes mellitus:a twenty-threeyear follow up study. Kidney Int

[20] Wrone EM, Carnethon MR, Panaliappan LP, et al. Association of dietary protein intake and microalbuminuria in healthy adults: Third National Health and Nutrition

[21] Deckert T, Feldt-Rasmussen B, Borch-Johnsen K, et al. Albuminuria reflects widespread

[22] Gerstein HC, Mann JEF et al. The validity of random urine specimen albumin measurement as a screening test for diabetic nephropathy. Yonesi Med J 1999;

[23] King H, Aubert RE, Herman WH (1998) Global burden of diabetes 1995-2025;

[24] Ramachandran A, Snehalatha C, Daisy Dharmaraj, Viswanathan M. Prevalence of glucose intolerance in Asian Indians. urban rural difference and significance of upper

Prevalence, numerical estimates and projection. Diabetes Care. 21: 1414-31.

vascular damage. The Steno hypothesis. Diabetologia 1989; 32:219-226.

patients with diabetic nephropathy. J Hypertens 1998; Suppl. 16:S17-22.


nephropathy in patients with type 2 diabetes. Exp Clin Endocrinol Diabetes2004;.112:135–41.

Bio-Chemical Aspects, Pathophysiology of

Microalbuminuria and Glycated Hemoglobin in Type 2 Diabetes Mellitus 43

[61] Deen WM, Myers BD, Brenner BM The glomerular barrier to macromolecules: theoretical and experimental considerations. In: Brenner BM, Stein JA,eds. Nephrotic

[62] Park CH, Maack T. Albumin absorption and catabolism by isolated perfused

[63] Gosling P, Beevers DG. Urinary albumin excretion in the general population. Clin Sci

[64] West JN, Gosling P, Dimmit SB, Littler WA. Non-diabetic microalbuminuria in clinical practice and its relationship to posture, exercise and blood pressure. Clin Sci

[65] Osterby R, Parving HH, Hommel E, et al. Glomerular structure and function in diabetic

[66] Gilbert RE, Cooper ME. The tubulo-interstitium in progressive diabetic kidney disease:

[67] Felt- Raemussen B. Increased transcapillary escape rate of albumin in type 1 diabetic

[68] Yudkin JS. Hyperinsulinaemia, insulin resistance, microalbuminuria and risk of

[69] Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature

[70] C Gnudi L, Gruden G, Viberti GC. Pathogenesis of diabetic neprhopathy. IN PICKUP JC & WILLIAMS G (Eds.) Textbook of diabetes. 3rd ed. Oxford, Blackwell Science

[71] Hostetter TH. Hyperfiltration and glomerulosclerosis. Semin Nephrol 2003;23:194–9. [72] Chen S, Wolf G, Ziyadeh FN. The renin-angiotensin system in diabetic nephropathy.

[73] Sharma K, Deelman L, Madesh M, Kurz B, Ciccone E, Siva S et al. Involvement of transforming growth factor-beta in regulation of calcium transients in diabetic vascular

[74] Tsuchida K, Cronin B, Sharma K. Novel aspects of transforming growth factor-beta in

[75] Lorenza Calisti, Simona Tognetti. Measure of glycosylated hemoglobin. Acta Biomed

[76] Nathan DM, Turgeon H, Regan S. Relationship between glycated hemoglobin ;levels

[77] International expert committee report on the role of the glycated hemoglobin assay in

[78] Gallagher EJ, Bloomgarden ZT, Le Roith D. Review of glycated hemoglobin in the

[79] Roberts WL, De BK, Brown D Et al. Effects of hemoglobin C and S traits on eight glycol

smooth muscle cells. Am J Physiol Renal Physiol 2003;285:F1258–70.

and mean glucose levels overtime. Diabetologia 2007; 50:2239-2244.

the diagnosis of diabetes. Diabetes care 2009;32:1327-1334.

mangagement of diabetes. Journal of Diabetes 2009;1:9-17.

hemoglobin methods. Clin Chem 2002; 48:383-385.

more than an aftermath of glomerular injury? Kidney Int 1999; 56:1627-1637.

Syndrome. New York: Churchill Livingstone 1982;.8-9.

nephropathy. Diabetes 1990; 39:1057-1063.

coronary heart disease. Ann Med 1996;28:433-438.

diabetic kidney disease. Nephron 2002;92:7–21.

1989;76:39-42.

1991;81:373-377.

2001; 414: 813-820.

Contrib Nephrol 2001;135:212–21.

2005;76; Suppl. 3: 59-62.

Ltd.2003.

convoluted tubules of the rabbit. J Clin Invest 1984;73:767-777.

patients with microalbuminuria. Diabetologia 1989; 32:219-226.


[61] Deen WM, Myers BD, Brenner BM The glomerular barrier to macromolecules: theoretical and experimental considerations. In: Brenner BM, Stein JA,eds. Nephrotic Syndrome. New York: Churchill Livingstone 1982;.8-9.

42 Pathophysiology and Complications of Diabetes Mellitus

diabetes. Diabetes 2004; 53:1570–6.

Diabetes2004;.112:135–41.

26.

1998; 47:821–30.

Int 1998; 68: 551-54.

2003;.200:537–46.

99:342–8.

35:755-765.

1999; 22(suppl 2):B53–B58.

nephropathy in patients with type 2 diabetes. Exp Clin Endocrinol

[43] Hansen TK, Tarnow L, Thiel S, Steffensen R, Stehouwer CD, Schalkwijk CG et al. Association between mannose-binding lectin and vascular complications in type 1

[44] Susztak K, Sharma K, Schiffer M, McCue P, Ciccone E, Böttinger EP. Genomic strategies

[45] Bowden DW, Colicigno CJ, Langefeld CD, Sale MM, Williams A, Anderson PJ et al. genome scan for diabetic nephropathy in African Americans. Kidney Int 2004; 66:1517–

[46] Imperatore G, Hanson RL, Pettitt DJ. Sib-pair linkage analysis for susceptibility genes for microvascular complications among Pima Indians with type 2 diabetes. Diabetes

[47] Marre M. Genetics and the prediction of complications in type 1 diabetes. Diabetes Care

[48] Scottish Intercollegiate Guidelines Network. SIGN 11: Management of diabetic renal disease – a National Clinical Guideline recommended for use in Scotland. March 1997.

[51] Rodicio LJ, Campo C, Ruilope ML. Microalbuminuria in essential hypertension. Kidney

[52] Mauer SM, Steffes MW, Ellis EN, Sutherland DER, Brown DM, Goetz FC. Structuralfunctional relationships in diabetic nephropathy. J Clin Invest 1984; 74:1143–55. [53] Wolf G, Schroeder R, Ziyadeh FN, Thaiss F, Zahner G, Stahl RAK. High glucose stimulates expression of p27Kip1 in cultured mouse mesangial cells: relationship to

[54] Wolf G, Wenzel U, Ziyadeh FN, Stahl RAK. Angiotensin converting-enzyme inhibitor treatment reduces glomerular p16INK4 and p27Kip1 expression in diabetic BBdp rats. [55] Wolf G, Reinking R, Zahner G, Stahl RAK, Shankland SJ. Erk 1,2 phosphorylates p27Kip1: functional evidence for a role in high glucose-induced hypertrophy of

[56] Tsilibary EC. Microvascular basement membranes in diabetes mellitus. J Pathol

[57] Zeisberg M, Ericksen MB, Hamano Y, Neilson EG, Ziyadeh F, Kalluri R. Differential expression of type IV collagen isoforms in rat glomerular endothelial and mesangial

[58] Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke H et al. Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest 1997;

[60] Waller KV, Ward KM, Maken JD et al. Current concepts in proteinuria. Clin Chem 1989;

[59] De Wardener HE . The Kidney. Edinburgh: Churchill Livingstone 1985;:49-50.

[49] Laterre EC, Heremans JF. Proteins in normal and pathological urine. Basle 1970;45.

for diabetic nephropathy. J Am Soc Nephrol 2003; 14:S271–8.

[50] Peterson PA., Ervin PE, Bergard JF. Clin Invest 1969; 48: 1189.

hypertrophy. Am J Physiol 1997;.273(Renal Physiol 42):348–56.

mesangial cells. Diabetologia 2003; 46:1090–9.

cells. Biochem Biophys Res Commun 2002; 295:401–7.


	- [80] Diabetic Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long term complications in insulin dependent diabetes mellitus. N. Engl J Med 1993; 329:977-986.

**Chapter 3** 

© 2012 Liuzzo et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Liuzzo et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Diabetes mellitus (DM) is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels (Expert

Diabetes is one of the most common chronic diseases in the world. It is thought that more than 360 million persons will be affected by this disease in 2030 (Wild et al., 2004). Prevalence of diabetes is higher in western countries because of the increasing of population age, physical inactivity and obesity, however it is rapidly spreading also in developing countries due to the socio-economic growth with progressive urbanization and changes in

Cardiovascular disease (CVD) in diabetic patients is characterized by microvascular damage, associated with the development of diabetic retinopathy, nephropathy, and neuropathy, and macrovascular complications linked to the accelerated course of atherosclerosis shown in these patients. Coronary heart disease (CHD) remains the principal cause of morbidity and mortality, in association with an increased risk of developing

cerebrovascular disease, peripheral vascular disease and heart failure.

Committee on the Diagnosis and Classification of Diabetes Mellitus, 1997, 2003)

**Type 2 Diabetes, Immunity** 

**and Cardiovascular Risk:** 

**A Complex Relationship** 

Daniela Pedicino, Ada Francesca Giglio,

Francesco Trotta and Giovanna Liuzzo

Additional information is available at the end of the chapter

Vincenzo Alessandro Galiffa,

http://dx.doi.org/10.5772/50611

**1. Introduction** 

**2. Epidemiology** 

lifestyle.


**Chapter 3** 
