Retinol Binding Protein-4 in Type 2 Diabetic Patients with and without Liver Cirrhosis

Tesis de Máster , 2017 , 128 Páginas , Calificación: C

Medicina - Medicina general, fundamentos

The aim of the work is to assess the level of Retinol Binding Protein 4 in type 2 diabetic patients with and without liver cirrhosis.

Diabetes mellitus is a common disease despite the fact that only half or two thirds are diagnosed, the figure is around 189 million people in 2003, and this may reach 324 million by 2025. Diabetes mellitus result from a lack or diminished effectiveness of endogenous insulin. Hyperglycemia is just one aspect of a far reaching metabolic derangement, which may cause serious microvascular and macrovascular complications. Etiological wise diabetes mellitus is classified into type 1, type 2 and other causes, with type 2 accounting for 90% of cases globally.

The prevalence of type 2 diabetes continues to rise at alarming rate, significant defect in glucose homeostasis and fuel metabolism are detectable long before overt diabetes occurs. One of the earliest derangements in metabolism is insulin resistance. It is defined as impaired glucose disposal in a hyperinsulinemic –euglycemic study. Fasting hyperinsulinemia, a compensatory mechanism to maintain euglycemia in setting of insulin resistance, is also present before overt diabetes occurs. Integrated fuel homeostasis relies among interactions among numerous tissues in widely varying states, including the fed state, fasting, and exercise.
This harmony in various tissues is maintained by communication between them, and is signaled by glucose and its various metabolites.

When this communication between tissues (mainly pancreas, adipose tissues, and liver) is lost insulin resistance and diabetes mellitus occurs. Retinol binding protein 4 (RBP4) a 21-KDa protein synthesized in the liver and adipose tissues, formerly recognized for its role as a specific transport for vitamin A, now it is known that it plays a crucial role in insulin resistance and diabetes mellitus. Adipose RBP4 expression and serum RBP4 level are elevated in mouse model of insulin resistance, elevated circulating RBP4 increase blood glucose by inhibiting insulin signaling in skeletal muscle and upregulating hepatic gluconeogenesis.

2

Acknowledgement

First and foremost, I am thankful to Allah, for without His grace, this work

would have never been accomplished as we feel his great care, support and guidance

in every step in our life.

I am genuinely and deeply indebted to Dr. Nehad Shokry Shoeib Professor of

Internal Medicine and Endocrinology, Faculty of Medicine, Ain shams University for her

constant guidance, gentle encouragement, foresight and faith in me. Thank you for your

help and unfailing support pushing me forward all the time, to overcome every obstacle in

the way.

I would like to express my deepest appreciations to Dr. Salwa Seddik Hosny

Assistant professor of Internal Medicine and Endocrinology, Faculty of Medicine, Ain

Shams University for her keen supervision, and valuable advice that guided me all the

way through the various phases of this work, and for teaching me through her

tremendous experience.

My special thanks and cardinal appreciations to Dr. Maram Mohamed Maher

Mahdy Lecturer of Internal Medicine and Endocrinology, Faculty of medicine, Ain

Shams University for her continuous interest, precious suggestions and inexhaustible

patience.Really,I owe to her much more than can be expressed. God blessing all of her

faithful efforts.

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3

Content

ListofAbbreviations

3

Introduction

6

DiabetesMellitus

8

Classificationofdiabetesmellitus:

10

Diagnosisofdiabetesmellitus:

17

Criteriafordiabetesmellitusdiagnosis:

18

Prediabetes

23

Type2diabetesmellitus:

25

Riskfactorsoftype2diabetesmellitus:

26

Pathogenesisoftype2diabetes:

28

Type2DiabetesMellitusandInsulinResistance:

28

KineticofInsulinSecretion

29

Mechanismsofglucoseinducedinsulinsecretion:

31

Alterationsofinsulinsecretion:

32

InsulinActionattheCellularLevel:

33

RoleofinsulinsignalingsystemsinInsulinResistance:

34

Themetabolicsyndrome

35

Type2diabetesmellitusandtheliver

38

TheRoleoftheLiverinGlucoseHomeostasis:

38

Spectrumofliverdiseaseindiabetics:

39

Nonalcoholicfattyliverdiseases(NAFLD)

39

PathogenesisofNAFLD

41

Diabetesmellitusandhepatocellulacarcinoma:

42

Livercirrhosis

44

Definition:

44

Classificationoflivercirrhosis:

45

Retinolbindingprotein4

53

Adiposetissueinhealthanddisease

53

RetinoidReceptors:

56

RetinolBindingProtein4andInsulinResistance:

57

Defectsintheabilityoffatcellstotransportglucosearelinkedtoinsulinresistanceinmuscle

andlivers:

58

SubjectsandMethods

59

Specimencollectionandpreparation:

61

Statisticalanalysis

63

Results

64

Discussion

98

Summary

106

Recommendations

109

References

110

L

ISTOF

A

BBREVIATIONS

ADAG

AlC-derived average glucose

ALB

Albumin

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4

ALT

Alanine amino transferase

AST

Aspartate amino transferase

ATP Adenosine triphosphate

BMI Body mass index

CGM Continuous glucose monitor

CHOL

Cholesterol

CVD

Cardiovascular disease

DCCT Diabetes control and complication trial

DM

Diabetes mellitus

DPP

Diabetes prevention program

DSME Diabetes self management education

FPG

Fasting plasma glucose

GDM Gestational diabetes mellitus

HAPO

Hyperglycemia and adverse pregnancy outcomes

HbA1c Haemoglobin A1c

HCV Hepatitis C virus

HDL High density lipoprotein

HNF

Hepatic nuclear factor

IDDM

Insulin - dependent diabetes mellitus

IFC

International federation of clinical chemistry

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5

IFG

Impaired fasting glucose

IGT

Impaired glucose tolerance

IRSI

Insulin Receptor substrate I

LADA

Latent autoimmune diabetes in adults

LDL

Low density lipoprotein

MODY

Maturity onset diabetes in the young

NAFLD

Non alcohol fatty liver disease

NASH

Non Alcohol steato hepatitis

NGSP

National glycol hemoglobin standarzation

program

NHANES

National health and nutrition examination

survey

OGTT

Oral glucose tolerance test

PPG

Postprandial plasma glucose

PT

Prothrombin time

RBP4

Retinol binding protein 4

SMBG

Self monitoring of blood glucose

TG

Triglycerides

UKPDS

United kingdom prospective diabetes study

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6

I

NTRODUCTIONI

Diabetes mellitus is a common disease despite the fact that only half or two

thirds are diagnosed, the figure is around 189 million people in 2003, and this

may reach

324

million by 2025 (Helen and John., 2009).

Diabetes mellitus result from a lack or diminished effectiveness of endogenous

insulin. Hyperglycemia is just one aspect of a far reaching metabolic

derangement, which may cause

serious

microvascular and macrovascular

complications (Murry., 2007). Etiological wise diabetes mellitus is classified

into type 1, type 2 and other causes, with type 2 accounting for 90% of cases

globally (Henry and Shalomo., 2008).

The prevalence of type 2 diabetes continues to rise at alarming rate, significant

defect in glucose homeostasis and

fuel

metabolism are detectable long before

overt diabetes occurs. One of the earliest derangements in metabolism is insulin

resistance.It is defined as impaired glucose disposal in a hyperinsulinemic ­

euglycemic study (Mokdad., 2003).

Fasting hyperinsulinemia, a compensatory mechanism to maintain euglycemia

in setting of insulin resistance, is also present before overt diabetes occurs

(Storlien et al., 2004).

Integrated fuel homeostasis relies among interactions among numerous tissues

in widely varying states, including the fed state, fasting, and exercise. This

harmony in various tissues is maintained

by

communication between them, and

is signaled by glucose and its various metabolites. When this communication

between tissues (mainly pancreas, adipose tissues, and liver) is lost insulin

resistance and diabetes mellitus occurs (Von Eynatten and Humpert., 2007).

Retinol binding protein 4 (RBP4) a 21-KDa protein synthesized in the liver and

adipose tissues, formerly recognized for its role as a specific transport for

vitamin A, now it is known that it plays a crucial role in insulin resistance and

---

7

diabetes mellitus. Adipose RBP4 expression and serum RBP4 level are elevated

in mouse model of insulin resistance, elevated circulating RBP4 increase blood

glucose by inhibiting insulin signaling in skeletal muscle and upregulating

hepatic gluconeogenesis (Koch et al., 2010).

Recent finding also showed that RBP4 is an independent risk factor in type 2

diabetes, impaired glucose

tolerance

, and in healthy individuals with strong

family history of diabetes. Hyperinsulinemia and insulin resistance are found in

nearly all patient with liver cirrhosis, and insulin resistance is a risk factor for

both survival and disease progression in patient with liver cirrhosis (Bugianesi et

al., 2007).

Approximately 70% of persons with type 2 diabetes

mellitus have a fatty liver

and the disease follows a more aggressive course with necroinflammtions and

fibrosis. This

potentially

severe complication is frequently over looked, and

patients may benefit from it's early diagnosis and management (Kenneth., 2009).

Aim of

the

work

To assess the level of Retinol Binding Protein 4 in type 2 diabetic

patients

with

and without liver cirrhosis.

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8

D

IABETES

M

ELLITUS

Diabetes mellitus 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. Several pathogenic processes are involved in the

development of diabetes. These range from autoimmune destruction of the

-

cells of the pancreas with consequent insulin deficiency to abnormalities that

result in resistance to insulin action. The basis of the abnormalities in

carbohydrate, fat and protein metabolism in diabetes is deficient action of

insulin on target tissues (American Diabetes Association., 2009).

Diabetes mellitus (DM) comprises a group of common metabolic disorders

sharing the phenotype of hyperglycemia. Several distinct types of DM exist

caused by interaction of genetics, environmental factors, and life style choices.

Factors contributing to hyperglycemia may include reduced insulin secretion,

decreased glucose utilization, and increase glucose production (Larry., 2006).

The resulting hyperglycemia is associated with disorder of carbohydrate, fat, and

protein metabolism and can lead to long term organ dysfunction (Michael.,

2009).

Diabetes is a chronic illness that requires continuing medical care and ongoing

patient self management education and support to prevent acute complications

and to reduce the risk of long-term complications (American Diabetes

Association., 2010).

Epidemiology of Diabetes Mellitus:

Diabetes mellitus is one of the most common endocrine disorders, the

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9

prevalence of this chronic metabolic disease is increasing (Townsend., 2000).

In 1985, 30 million people around the world were diagnosed with diabetes; in

2000, that figure rose to over 150 million (Vincent., 2008).

In 2006, according to the world health organization, at least 171 million people

suffered from diabetes, and it is estimated that by the year 2030, this number

will double (Jones et al., 2006).

The International Diabetes Federation states "every ten seconds two people are

diagnosed with diabetes somewhere in this world" (Vincent., 2008).

Type 2 DM is the predominant form of diabetes worldwide accounting for 90-

95% of cases globally. The prevalence of type 2 DM is expected to rise more

rapidly in the future because of increasing obesity and reduced activity levels

(Kasper and Fuaci., 2005)

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10

ighest number of estimated cases of diabetes

List of countries with h

Table (1):

for 2030.

Country

People with diabetes (Million)

India

79.4

China

42.3

United states

30.3

Indonesia

21.3

Pakistan

13.9

Brazil

11.3

Bangladesh

11.1

Japan

8.9

Philippines

7.8

Egypt

6.7

(Roglic., 2004)

C

LASSIFICATIONOFDIABETESMELLITUS

:

There is increasing interest in the classification of sub types of diabetes,

which is assisting in the personalization of treatment for affected individuals.

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11

This classification has now replaced the earlier, clinical classification into

`insulin- dependent diabetes mellitus' (IDDM) and `non- insulin dependent

diabetes mellitus' (NIDDM), which was based on the need for insulin treatment

at diagnosis. IDDM is broadly to type 1 diabetes and NIDDM to type 2 diabetes

(Bious and Donnelly., 2010).

Calssification of diabetes mellitus:

Table (2):

I. Type 1 diabetes (Beta-cell destruction, usually leading to absolute insulin

deficiency)

A. Immune-mediated

B. Idiopathic

II. Type 2 diabetes (may range from predominantly insulin resistance with

relative insulin deficiency to a predominantly insulin secretory defect with

insulin resistance).

III. Other specific types of diabetes

A. Genetic defects of Beta-cell function characterized by mutations in:

1. Hepatocyte nuclear transcription factor (HNF) 4

(MODY 1)

2. Glucokinase (MODY 2)

3. HNF-1

(MODY 3)

4. Insulin promoter factor-1 (IPF-1) (MODY 4)

5. HNF-1 (MODY 5)

6. NeuroD1 (MODY 6)

7. Mitochondrial DNA

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12

8. Subunits of ATP-sensitive potassium channel

9. Proinsulin or insulin conversion

B. Genetic defects in insulin action

1. Type A insulin resistance

2. Leprechaunism

3. Rabson-Mendenhall syndrome

4. Lipodystrophy syndromes

C. Diseases of the exocrine pancreas--pancreatitis, pancreatectomy, neoplasia,

cysticfibrosis, hemochromatosis, fibrocalculous pancreatopathy, mutations in

carboxyl ester lipase

D. Endocrinopathies--acromegaly, Cushing's syndrome, glucagonoma,

pheochromocytoma, hyperthyroidism, somatostatinoma, aldosteronoma.

E. Drug- or chemical-induced--Vacor, pentamidine, nicotinic acid,

glucocorticoids, thyroid hormone, diazoxide, B-adrenergic agonists, thiazides,

phenytoin, -interferon, protease inhibitors, clozapine.

F. Infections--congenital rubella, cytomegalovirus, coxsackie.

G. Uncommon forms of immune-mediated diabetes--"stiff-person" syndrome,

anti-insulin receptor antibodies

H. Other genetic syndromes sometimes associated with diabetes--Down's

syndrome, Klinefelter's syndrome, Turner's syndrome, Wolfram's syndrome,

Friedreich's ataxia, Huntington's chorea, Laurence-Moon-Biedl syndrome,

myotonic dystrophy, porphyria, Prader-Willi syndrome.

IV. Gestational diabetes mellitus (GDM).

(Bious and Donnelly., 2010)

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13

Type I DM: result from destruction of the beta cells of the pancreas due to

autoimmunity and associated with genetic susceptibility, and poorly understood

environmental

factors that initiate the disease process. It is believed that this

process starts along time before the disease actually begins. This may allow

future prevention of the disease. Autoimmune antibodies like islet cell

antibodies antiglutamate decarboxylase and anti inhibitor of apoptosis protein's

are present (Tim and Sudhesh., 2010).

Type I DM is divided into:

Autoimmune diabetes:

-

A

It was previous called type 1 diabetes, (IDDM) or juvenile onset diabetes. It

commonly occurs in childhood and adolescence, but it can occur at any age,

even in the 8

th

and 9

th

decades of life. It accounts for only 5-10% of those with

diabetes. It results from a cellular-mediated autoimmune destruction of the

-

cells of the pancreas. Markers of the immune destruction of the

-cell include

islet cell auto-antibodies (ICAs), auto-antibodies to insulin, auto-antibodies to

glutamic acid decarboxylase (GAD), and auto-antibodies to the tyrosine

phosphatase IA-2 and IA-2

. The rate of -cell destruction is quite variable,

being rapid in some individuals (mainly infants and children) and slow in others

(mainly adults) (David and Dolores., 2007).

Idiopathic diabetes:

-

B

This form of diabetes is strongly inherited, lacks immunological evidence

for

-cell autoimmunity and is not HLA associated. Only a minority of patients

with type-1 diabetes falls into this category. Individuals with this form of

diabetes suffer from episodic ketoacidosis and exhibit varying degree of insulin

deficiency between episodes, so an absolute requirement for insulin replacement

therapy in the affected patients may be needed (American Diabetes Association.,

2009).

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14

1- Type 2 DM: Is a complex heterogeneous condition and recent genetic

studies revealed many subtypes, up to 50% of children presenting with DM

are now presenting with type 2 DM in some areas due to increasing

prevalence of obesity. Latent autoimmune diabetes in adults (LADA)

comprise about 5% of patients with type 2 DM and have auto antibodies,

but their presentation is some what like type 2 DM. (Tim and Sudhesh.,

2010).

2- Specific types of diabetes:

3- Monogenic diabetes (previously referred to as maturity onset diabetes in the

young, (MODY). This is a collection of autosomal dominant conditions

resulting from single gene mutations usually presenting before 25 years of

age.

4- MODY I: Due to mutation in hepatocytes nuclear transcription factor(

HNF)4. These patients display a progressive decline in B cell function and

develop chronic complications of diabetes comparable to type 2 diabetes,

and fair better with insulin.

MODY 2: Due to reduction of glucokinase activity leading to reduced insulin

secretion. Homozygotes developed insulin deficiency at birth,

while heterozygotes have a bengin course that respond well to diet

or oral therapy.

MODY 3: This is the most common type Due to mutation in hepatocytes nuclear

transcription factor( HNF)1. The course is similar to type 2

diabetes but no insulin resistance.

MODY 4: Due to mutation in hepatocytes nuclear transcription factors(

HNF)1. In this type homozygotes develop agenesis of the

pancneas, while heterozygotes develop a mild late onset disease

(median age 35 years).

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15

MODY 5: A moderately severe form with congenital kidney defects and

nephropathy.

MODY 6: A milder form similar to mody 4.

(David and Dolores., 2007)

Gestational Diabetes Mellitus (GDM):

For many years, GDM was defined as any degree of glucose intolerance

with onset or first recognition during pregnancy, whether or not the condition

persisted after pregnancy, and not excluding the possibility that unrecognized

glucose Intolerance may have antedated or begun concomitantly with the

pregnancy. This definition facilitated a uniform strategy for detection and

classification of GDM, but its limitations were recognized for many years. As

the ongoing epidemic of obesity and diabetes has led to more type 2 diabetes in

women of childbearing age, the number of pregnant women with undiagnosed

type 2 diabetes has increased (Lawrence et al., 2008).

GDM carries risks for the mother and neonate. The Hyperglycemia and

Adverse Pregnancy Outcomes (HAPO) study, a large-scale (~25,000 pregnant

women) multinational epidemiologic study, demonstrated that risk of adverse

maternal, fetal, and neonatal outcomes continuously increased as a function of

maternal glycemia at 24-28 weeks, even within ranges previously considered

normal for pregnancy. These results have led to careful reconsideration of the

diagnostic criteria for GDM. (International Association of Diabetes., 2010).

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Screening for and diagnosis of GDM.

Table (3):

Perform a 75-g OGTT, with plasma glucose measurement

fasting and at 1 and 2 h, at 24-28 weeks of gestation in women

not previously diagnosed with overt diabetes.

The OGTT should be performed in the morning after an

overnight fast of at least 8 h.

The diagnosis of GDM is made when any of the following

plasma glucose values are exceeded:

Fasting >92 mg/dl (5.1 mmol/1)

1 h > 180 mg/dl (10.0 mmol/1)

2 h >153 mg/dl (8.5 mmol/1)

(Diabetes care., 2011)

These new criteria will significantly increase the prevalence of GDM,

primarily because only one abnormal value, not two, is sufficient to make the

diagnosis (Diabetes care., 2011).

It is important to note that 80-90% of women in both of the mild GDM

studies (whose glucose values overlapped with the thresholds recommended

herein) could be managed with lifestyle therapy alone (Landon et al., 2009).

Women with a history of GDM have a greatly increased subsequent risk for

diabetes and should be followed up with subsequent screening for the

development of diabetes or prediabetes, as outlined in testing for diabetes (Kim

and Choi., 2002)

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Clinical features of type 1 and type 2 diabetes.

Table (4):

Type 1 diabetes

Type 2 diabetes

Sudden onset with severe symptoms of

thirst and ketoacidosis (vomiting,

hyperventilation, dehydration.

Usually insidious onset of

tiredness, thirst, polyuria,

nocturia.

Spontaneous ketosis.

No ketoacidosis.

Recent, marked weight loss. Usually lean. Usually overweight or obese;

often no recent weight loss.

Life-threatening; needs urgent insulin

replacement.

Frequent infections, e.g. urine,

skin, chest.

Absent C ­ peptide.

C - peptide detectable

Markers of autoimmunity present (e.g.

islet cell antibodies).

Often other features of

`metabolic syndrome', e.g.

hypertension.

Symptoms may be minimal

and/or ignored by patient.

(Bious and Donnelly., 2010)

D

IAGNOSISOFDIABETESMELLITUS

:

The diagnosis of diabetes rests on the measurement of plasma glucose level.

Because plasma glucose concentration range as a continuum, the criteria are

based on the threshold for complications of diabetes.

The primary end point used to evaluate the relationship between glucose level

and complications was retinopathy. There is also association between fasting

plasma glucose (FPG) and 2 hours plasma glucose and risk of macrovascular

and cardiovascular disease. Undiagnosed type 2 DM is common, with estimated

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18

lag of 5 to 7 years between the onset of diabetes and diagnosis. It is estimated

that up to 30% of patients are undiagnosed, coupled with greater like hood of

having dyslipidemia, hypertension and obesity, it is important for the clinician to

screen for diabetes in a cost effective manner in subjects who demonstrate major

risk factors for diabetes (Henry and Shalomo., 2008).

C

RITERIAFORDIABETESMELLITUSDIAGNOSIS

:

A) Normal glucose tolerance

Fasting glucose 100 mg/dl.

Random glucose 140 mg/dl.

2 hours post 75g glucose load 140 mg/dl.

B) Impaired fasting glucose

Fasting glucose 100 mg/dl- 126 mg/dl .

C) Impaired glucose tolerance

2 hours post 75 gram load glucose 140mg- 200mg/dl

D) Diabetes (Non pregnant adult)

Fasting glucose 126 mg/ dl

2 hours post 75 glucose load 200 mg/dl.

Diagnosis requires confirmatory test on separate day.

(American Diabetes Association., 2010)

Recent research suggest that non-invasive spectroscopic measurements of

advanced glycation end products in the skin may be useful, accurate, and less

invasive. Advanced glycation end products are biomarkers for diabetes and are

closely associated with and predictive of diabetes complications specially

retinopathy and nephropathy and are more sensitive at diagnosing diabetes than

fasting glucose or HbA (Trisha and Glenn., 2008).

The use of HbA1c for the diagnosis of diabetes was not recommended due

to lack of uniformity of the assay world wide. There was world wide effort for

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19

international standerdization suggesting the use of international federation of

clinical chemistry reference system. In June 2009 the international committee of

expert announced their consensus that the HbA1c assay is an accurate way to

diagnose diabetes mellitus in adult and children but not in pregnant women. The

international committee advices that anyone with reading of 6.5% or greater

should be considered to have diabetes, waiting for major diabetes groups to

agree (Leonid., 2009).

Epidemiologic datasets show a similar relationship between A1C and risk

of retinopathy as has been shown for the corresponding FPG and 2-h plasma glu-

cose thresholds. The A1C has several advantages to the FPG and OGTT,

including greater convenience, since fasting is not required; evidence to suggest

greater pre-analytical stability; and less day-to-day perturbations during periods

of stress and illness. These advantages must be balanced by greater cost, the

limited availability of A1C testing in certain regions of the developing world,

and the incomplete correlation between A1C and average glucose in certain

individuals. In addition, A1C levels can vary with patients' ethnicity as well as

with certain anemias and hemoglobinopathies. For patients with an abnormal

hemoglobin but normal red cell turnover, such as sickle cell trait, an A1C assay

without interference from abnormal hemoglobins should be used. For conditions

with abnormal red cell turnover, such as pregnancy, recent blood loss or

transfusion, or some anemias, the diagnosis of diabetes must employ glucose

criteria exclusively (Ziemer et al., 2010).

The established glucose criteria for the diagnosis of diabetes (FPG and 2-h

PG) remain valid as well. Just as there is less than 100% concordance between

the FPG and 2-h PG tests, there is not perfect concordance between A1C and

either glucose-based test. Analyses of National Health and Nutrition Examina-

tion Survey (NHANES) data indicate that, assuming universal screening of the

undiagnosed, the A1C cut point of 6.5% identifies one-third fewer cases of

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20

126 rng/dl (7.0

undiagnosed diabetes than a fasting glucose cut point of

mmol/1) (Cowie et al., 2010).

e diagnosis of diabetes.

Criteria for th

Table (5):

6.5%. The test should be performed in a laboratory

A1C

using a method that is NGSP certified and standardized to the

DCCT assay.*

or

126 mg/dl (7.0 mmol/1). Fasting is defined as no caloric

FPG

intake for at least 8h.*

or

200 mg/dl (11.1 mmol/1) during an

sma glucose

h pla

-

2

OGTT. The test should be performed as described by the

World Health Organization, using a glucose load containing

the equivalent of 75 g anhydrous glucose dissolved in water.

or

In a patient with classic symptoms of hyperglycemia or

200 mg/dl

hyperglycemic crisis, a random plasma glucose

(11.1 mmol/1).

* In the absence of unequivocal hyperglycemia, result should be confirmed by repeat testing.

(Diabetes care., 2011)

As with most diagnostic tests, a test result diagnostic of diabetes should be

repeated to rule out laboratory error, unless the diagnosis is clear on clinical

grounds, such as a patient with a hyperglycemic crisis or classic symptoms of

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21

preferable that

200 mg/dl. It is

mia and a random plasma glucose

hyperglyce

the same test be repeated for confirmation,

since there will be a greater likelihood of concurrence in this case. For

example, if the A1C is 7.0% and a repeat result is 6.8%, the diagnosis of

diabetes is confirmed. However, if two different tests (such as A1C and FPG)

are both above the diagnostic thresholds, the diagnosis of diabetes is also

confirmed (Diabetes care., 2011).

On the other hand, if two different tests are available in an individual and

the results are discordant, the test whose result is above the diagnostic cut point

should be repeated, and the diagnosis is made on the basis of the confirmed test.

6.5%)

terion of the A1C (two results

That is, if a patient meets the diabetes cri

but not the FPG (126 mg/dl or 7.0 mmol/1), or vice versa, that person should

be considered to have diabetes) (Sacks et al., 2002).

Because A1C is thought to reflect average glycemia over several months

and has strong predictive value for diabetes complications (Staratton et al.,

2000), A1C testing should be performed routinely in all patients with diabetes,

at initial assessment and then as part of continuing care. Measurement

approximately every 3 months determines whether a patient's glycemic targets

have been reached and maintained. Some patients with stable glycemia well

within target may do well with testing only twice per year, while unstable or

highly intensively managed patients (e.g., pregnant type 1 women)

may be tested more frequently than every 3 months.

The availability of the A1C result at the time that the patient is seen (point-

of-care testing) has been reported to result in increased intensification of therapy

and improvement in glycemic control (Miller et al., 2003).

The A

1

C test is subject to certain limitations. Conditions that affect

erythrocyte turnover (hemolysis, blood loss) and hemoglobin variants must be

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22

considered, particularly when the A1C result does not correlate with the patient's

clinical situation. In addition, A1C does not provide a measure of glycemic

variability or hypoglycemia. For patients prone to glycemic variability

(especially type 1 patients, or type 2 patients with severe insulin deficiency),

glycemic control is best judged by the combination of results of self monitoring

of blood glucose (SMBG) testing and the A1C. The A1C may also serve as a

check on the accuracy of the patient's meter (or the patient's reported SMBG

results) and the adequacy of the SMBG testing schedule (Sacks et al., 2002).

Table 5 contains the correlation between A1C levels and mean plasma glu-

cose levels based on data from the international AlC-Derived Average Glucose

(ADAG) trial utilizing frequent (SMBG) and continuous glucose monitor

(CGM) in 507 adults (83 % Caucasian) with type 1, type 2, and no diabetes

(Nathan et al., 2008).

Correlation of A1C with average glucose.

Table (6):

Mean plasma glucose

AIC (%)

Mg/dI

MmoI/I

6

126

7.0

7

154

8.6

8

183

10.2

9

212

11.8

10

240

13.4

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23

11

269

14.9

12

298

16.5

(Nathan., 2008)

For patients in whom AlC/estimated average glucose (eAG) and measured

blood glucose appear discrepant, clinicians should consider the possibilities of

hemoglobinopathy or altered red cell turnover, and the options of more frequent

and/or different timing of SMBG or use of continuos glucose monitor CGM.

Other measures of chronic glycemia such as fructosamine are available, but their

linkage to average glucose and their prognostic significance are not as clear as is

the case for A1C (Diabetes care., 2011).

P

REDIABETES

In 1997, The Expert Committee on Diagnosis and Classification of Diabe-

tes Mellitus recognized an intermediate group of individuals whose glucose

levels, although not meeting criteria for diabetes, are nevertheless too high to be

considered normal. These persons were defined as having impaired fasting glu-

cose (1FG) (FPG levels 100-125 mg/dl [5.6-6.9 mmol/1]) or impaired glucose

tolerance (IGT) (2-h PG values in the OGTT of 140-199 mg/dl [7.8-11.0

mmol/1]) (Diabetes care., 1997).

Individuals with IFG and/or IGT have been referred to as having

prediabetes, indicating the relatively high risk for the future development of

diabetes, IFG and IGT should not be viewed as clinical entities in their own

right but rather risk factors for diabetes as well as cardiovascular disease (CVD).

IFG and IGT are associated with obesity (especially abdominal or visceral

obesity), dyslipidemia with high triglycerides and/or low HDL cholesterol, and

hypertension

(Genuth et al., 2003).

---

24

In a systematic review of 44, 203 individuals from 16 cohort studies with a

follow-up interval averaging 5,6 years (range 2.8-12 years), those with an A1C

between 5.5 and 6.0% had a substantially increased risk of diabetes with 5-year

incidences ranging from 9-25%. An A1C range of 6.0-6.5% had a 5-year risk of

developing diabetes between 25-50% and relative risk 20 times higher compared

with an A1C of 5.0%

(Zhang et al., 2010).

Diagnosis of prediabetes:

Table (7):

FPG 100-125 mg/dl (5.6-6.9 mmol/1): IFG

or

2-h plasma glucose in the 75-g OGTT 140-199 mg/dl (7.8-11.0 mmol/1):

IGT

or

A1C 5.7-6.4%

*For all three tests, risk is continuous, extending below the lower limit of the range and becoming

disproportionately greater at higher ends of the range.

(Diabetes care., 2011)

As is the case for individuals found to have IFG and IGT, individuals with

an A1C of 5.7-6.4% should be informed of their increased risk for diabetes as

well as CVD and counseled about effective strategies to lower their risks. As

with glucose measurements, the continuum of risk is curvilinear--as A1C rises,

the risk of diabetes rises disproportionately (American Diabetes Associatio.,

2009).

Criteria for testing for diabetes in asymptomatic adult individuals

1. Testing should be considered in all adults who are overweight (BMI

25kg/m2) and

have additional risk factors:

---

25

Physical inactivity.

First-degree relative with diabetes.

Women who delivered a baby weighing 4kg.

Hypertension (140/90mmHg or on therapy for hypertension).

HDL cholesterol level 35mg/dl (0.9mmol/l) and/or a triglyceride level

250mg/dl (2.82mmol/l)

Women with polycystic ovary syndrome.

HbA1c 5.7%, IGT, or IFG on previous testing.

Other clinical condition associated with insulin resistance (e.g., severe

obesity, acanthosis nigricans).

History of cardiovascular disease.

2. In absence of the above criteria, testing diabetes should begin at age 45 years.

3. If results are normal, testing should be repeated at least at 3-year intervals.

(American Diabetes Association., 2010)

T

YPE

2

DIABETESMELLITUS

:

It is the commonest type of diabetes, various clinical risk factors are

associated with the disease such as obesity, increasing age, family history of

diabetes and ethnic and geographical variations in its frequency give clues to the

etiology and pathophysiology of type 2 diabetes (Abo Romia., 2004).

It was previously called (NIDDM) or adult-onset diabetes this form of

diabetes accounts for 90-95% of those with diabetes. It is characterized by

disorder of insulin action and insulin secretions either of which may be

predominant feature (American Diabetes Association., 2009).

Type 2 diabetes is currently thought to occur in genetically predisposed

individuals who are exposed to series of environmental influences that

precipitate the onset of the clinical disease. Type 2 diabetes has a strong genetic

---

26

component, although the major genes that predispose to this disorder have not

yet identified

(Kasper and Fuaci., 2001).

Type 2 frequently goes undiagnosed for many years because the

hyperglycemia develops gradually and at earlier stages is often not severe

enough for the patient to notice any of the classic symptoms of diabetes.

Ketoacidosis seldom occurs spontaneously in this form of diabetes when seen it

usually arises in association with the stress of another illness such as infection

(Diabetes Care., 2003).

Most patients with this form of diabetes are obese, and obesity itself causes

some degree of insulin resistance. Patients who are not obese by traditional

weight criteria may have an increased percentage of body fat distributed

predominantly in the abdominal region (Buse and Polonsky., 2003).

The principle siqnaling pathway involves sequential acivation of the insulin

receptor, insulin receptor substrates (IRS), phosphatidiinositol -3- kinase (PI3K),

AKT and prtien kinase C isoform (Youssef et al., 2011).

R

ISKFACTORSOFTYPE

2

DIABETESMELLITUS

:

The greater the number of risk factors in an individual the greater the

chance of developing diabetes (Kriska et al., 2003).

Age 45 years.

Overweight (BMI 25kg/m

2

).

Family history of diabetes (i.e., parents or siblings with diabetes).

Habitual physical inactivity.

Race/ethnicity (e.g., African-Americans, Hispanic-Americans, Asian-

Americans, and pacific Islanders).

Previously identified Impaired Fasting Glucose or Impaired Glucose

Tolerance

---

27

History of Gestational diabetes or delivery of an overweight baby weighting

4kg.

Hypertension (140/90mmHg in adults).

HDL cholesterol 35mg/dl or triglyceride level 250mg/dl.

Polycystic ovary syndrome.

History of vascular disease

(Kriska et al., 2003)

Tim and Sudhesh.,

d 10 lean men fare on the Journey through life (Joslin, 1941) (

How 10 obese men an

Fig. (1):

2010).

---

28

P

ATHOGENESISOFTYPE

2

DIABETES

:

The key hormonal defects responsible for hyperglycemia in type 2 diabetes

are decreased insulin secretion and elevated glucagon secretion but also the

secretion and action of the gut incretin hormones, glucagon-like peptide-1 and

glucose-dependent insulinotropic polypeptide are altered in type 2 diabetes

(Farech et al., 2009).

Persons with type 2 diabetes consistently demonstrate three cardinal

abnormalities:

I. Resistance to the action of insulin in peripheral tissues particularly muscle, fat

and also the liver.

II. Defective insulin secretion, particularly in response to glucose stimulus.

III. Increased glucose production by the liver.

(Powers., 2001)

T

YPE

2

D

IABETES

M

ELLITUSAND

I

NSULIN

R

ESISTANCE

:

Insulin is a protien consisting of 51 amino acids contained within an Alpha

chain (21 amino acid) and Beta chain (30 amino acid) joined by disulfide

bridges. Insulin is secreted by Beta cells of islets of pancreas which comprises

about 1- 1.5% of pancreatic volume. A precursor molecule, preproinsulin is

cleaved to proinsulin and then to insulin by microsomal enzymes, 50% of

secreted insulin is degraded in a single pass through the liver (David and

Dolores., 2007).

---

29

(Leclercq et al., 2007).

Insulin signaling

Fig. (2):

Insulin is an anabolic hormone secreted by pancreatic -cells that is

required for the maintenance of glucose homeostasis.

It

inhibits hepatic glucose

production and increases peripheral glucose uptake and glycogen synthesis

(Leclercq et al., 2007).

K

INETICOF

I

NSULIN

S

ECRETION

Glucose and other secretagogues affect insulin release by acting on multiple ionic

and metabolic mechanisms. Beta-cells are sensitive to the rate of change as well

as the actual concentration of a secretagouge. Characteristics of insulin

release in

response to a rapid-onset stimulation by glucose are as follows:

First phase:

Initial response is a transient, rapid rise in release (firstphase), which

terminates in 5-10 minutes. The -cells release their contents in an all-or-none

---

30

fashion when their thresholds are exceeded by the ambient glucose (Bokvist et

al., 1995).

Impaired first-phase release predicts impending development of type 1

diabetes and is an early feature of impaired ß-cell function in type 2 diabetes.

This lack of first-phase response, or acute insulin response (AIR), in persons

with type 2 diabetes has been observed by many investigators (DeFronzo and

Ferrannini., 1982).

Second phase:

This is followed by a progressively increasing (second phase) in which glucose

continually amplifies its own signal.

It is produced by a potentiating factor (P), which in response to constant

glucose, gradually increases. In contrast to first-phase release, P is not depleted

during insulin secretion, but is degraded gradually with a half-life of 20-60

minutes. These characteristics of P metabolism create the memory (priming) in

the -cell (Grodsky., 1996).

Third phase:

If the islets are exposed to elevated glucose levels for periods 15

minutes, they become primed or potentiated so that, after a brief rest period,

their response to the same stimulus is greater than seen initially. This memory

component can persist for 1 hour and is referred to as time-dependent

potentiation (Byrne et al., 1995).

If the islets are further stimulated by glucose beyond 1-3 hours, insulin

release spontaneously declines to a low level, which can be maintained for up

to 48 hours (thirdphase) (Bolaffl et al., 1990).

---

31

Chronic exposure of ß -cells to glucose (hyperglycemic environment) for

1-3 hours results in spontaneous decreased insulin secretion. This phenomenon

has been referred to as glucose toxicity (Weir and Leahy., 1994).

The mechanisms responsible for these changes have still not yet been

defined, but several biochemical alterations have emerged. Perhaps the most

striking abnormality is the profound reduction in the Beta-cells glucose

transporter GLUT-2 found in cases of hyperglycemia (Ogawa et al., 1995).

Glucokinase plays a critical role in regulating the rate of insulin secretion

and changes in the intrinsic activity of this enzyme are found in hyperglycemic

states (Chen et al., 1994).

M

ECHANISMSOFGLUCOSE

INDUCEDINSULINSECRETION

:

(1) Ion Flux:

Glucose inhibits ATP-sensitive K

+

channels (presumably through its

stimulation of ATP production), thereby causing membrane depolarization;

depolarization activates the L-type voltage-dependent Ca

++

channels resulting

in Ca

++

entry (influx) and an increase in cytosolic Ca

++

. It was found that the

predominant effect of ion flux is on first-phase release (Ammala et al., 1993).

(2) cAMP:

Glucose stimulates adenyl cyclase and cAMP levels that mediate protein

kinase A activity and phsphorylation of intracellular proteins. Although an

increase in cAMP alone elicits only a small nonphasic insulin release, it

enhances both phases in the presence of glucose. An effect on first-phase

release is consistent with observations that cAMP facilitates flux and Ca

++

uptake into the -cell (Ma et al., 1993).

---

32

(3) Phosphoinositol Metabolism:

Activation of phospholipase C results in phosphoinositol hydrolysis,

causing production of both diacylglycerol and inositol phosphates.

Diacylglycerol phosphate, in turn, activates protein kinase C and

phosphorylation of protein substrates different from those

phosphorylated by cAMP. The current evidence indicates that this

pathway is an important positive contributor to the regulation of second-phase

insulin secretion (Kelley et al., 1994).

A

LTERATIONSOFINSULINSECRETION

:

(1) Obesity:

Hypersecretion of insulin is an early feature in nondiabetic obese subjects.

This hypersecretion is necessary to compensate for metabolic insulin resistance

secondary to obesity and overeating. Primary hypersecretion of insulin as the

cause of insulin resistance and obesity has been proposed, but never proven.

(Vaag et al., 1995).

The expression of both the GLUT-2 gene and the glucokinase gene is

increased in obese persons, which may explain why hyperinsulinemia may

develop despite normal or near-normal plasma glucose values (Chen et al., 1994).

It was found that insulin deficiency developed years before hyperglycemia

in genetically predisposed subjects. This may indicate that, for genetic reasons,

the -cells in obese diabetics may be unable to compensate for insulin resistance

in the long term (Vaag et al., 1995).

In the general population, peripheral insulin resistance, IGT and

hyperinsulinemia are metabolic changes observed

with aging. Physical inactivity

and obesity appear to be important factors in causing the reduction in insulin

sensitivity in the elderly population. On completion of exercise training

---

33

programs, elderly subjects demonstrated marked improvements in insulin

sensitivity from baseline, which was associated with a corresponding reduction

in Beta-cell secretory responses to glucose (Muller et al., 1996).

I

NSULIN

A

CTIONATTHE

C

ELLULAR

L

EVEL

:

Circulating insulin rapidly reaches the target tissues, where it interacts

with its receptor. The insulin receptor (IR) is a transmembrane tyrosine kinase

that is expressed as a tetramer in an 2 2 configuration (Ullrich et al., 1985).

Insulin binding to specific regions of the

-subunit leads to a rapid

configurational change in the receptor that eventuates in autophosphorylation

of specific tyrosine residues of the intracellular region of the -subunits

resulting in activation of the tyrosine kinase activity of the receptor (Lee et al.,

1997).

In the inactive state, the catalytic site of the tyrosine kinase is occluded

by the activation-loop preventing access of ATP. Autophosphorylation of

tyrosine residues in the activation-loop causes a conformational change that

allows ATP to reach the catalytic site (Hubbard., 1997).

The activated insulin receptor kinase phosphorylates substrate proteins on

tyrosine residues and these phosphorylated tyrosine residues serve as docking

sites for downstream effectors (White., 1998).

Insulin-receptor signaling involves two major pathways: the mitogen-

activated protein (MAP) kinase and the phosphatidylinositol 3-kinase (PI3-K).

When the MAP kinase pathway is activated, it mediates the growth-promoting

effects of insulin by phosphorylating transcription factors leading to the

induction of genes (Marshall., 1995).

The metabolic response to insulin is primarily mediated via the PI3-K

pathway. When the PI3-K pathway is activated, it results in production of

---

34

phospatidylinositol 3,4,5 phosphate which activates the PI3-K dependent kinase

which has been implicated in regulating the translocation of GLUT 4, an insulin-

sensitive glucose transporter expressed by muscle and fat cells, to the cell

surface (Czech and Corvera., 1999).

Stimulation of glycogen synthesis is another key metabolic effect of

insulin. Glycogen synthase kinase-3 (GSK-3) mediates the activation of

glycogen synthase in response to insulin. Insulin induces the phsphorylation and

inactivation of GSK-3, rendering it incapable of inhibiting glycogen synthase

activity, thereby enhancing protein synthesis. Insulin can also activate protein

synthesis at the translational level (Cross et al., 1995).

R

OLEOFINSULINSIGNALINGSYSTEMSIN

I

NSULIN

R

ESISTANCE

:

Insulin resistance is a common pathologic state in which target cells fail to

respond to ordinary levels of circulating insulin. It is frequently associated with

a number of diseases, including obesity and type 2 diabetes (Virkamaki et al.,

1999).

At the molecular level, impaired insulin signaling results from mutations

or post-translation modifications of the insulin receptor itself or any of its

downstream effector molecules (Taylor and Arioglu., 1998).

In some cases, insulin resistance could be accounted for by a defect in

insulin binding to its receptor; however, insulin resistance is most often

attributed to a post binding defect (Roach et al., 1994).

A marked reduction in the receptor kinase activity was observed in patients

with extreme insulin resistance, but with normal insulin binding. However, these

events are rare and do not play an important role in the pathophysioloy of typical

type 2 diabetes or obesity (Krook and O'Rahilly., 1996).

---

35

Glycogen synthesis is markedly reduced in the muscle of type 2 diabetic

patients. Recent studies, using nuclear

magnetic resonance, demonstrated that

one of the defects resulting in these changes was a reduction in glucose

transport, as evidenced by reduced intracellular levels of glucose and glucose-6

phosphate (Krook et al., 2000).

Insulin resistance is present in the majority of patients with type 2 diabetes,

their first degree relatives, individuals with impaired glucose tolerance and

obesity. For years debates raged about whether insulin resistance or insulin

secretion was more important in the pathogenesis of diabetes (Golin et al.,

1996).

An important concept was the nature of the relationship between insulin

resistance and insulin secretion in maintaining normal glucose tolerance, thus

mismatches of Beta-cell function relative to insulin requirements were predicted

to result in hyperglycemia and the development of diabetes (Hermanns et al.,

2005).

Insulin resistance has been associated with multiple metabolic

abnormalities, several of which are traditional cardiovascular risk factors (high

LDL cholesterol, hypertension, obesity and diabetes), while others are known as

non traditional risk factors like increase reactive proteins, interleukin 6, increase

fibrinogen, plasminogen activator inhibitor, increase homocysteine, uric acid

and presence of microalbuminurea. Because of the link between insulin

resistance and these cardiovascular risk factors, it has been attractive that

insulin resistance is the underlying pathophysiological cause of increase

cardiovascular disease in diabetes and obesity (

Maheux et al., 1997).

T

HEMETABOLICSYNDROME

---

36

A constellation of metabolic derangements in patient with insulin resistance

and diabetes millitus are associated with increase risk of cardiovascular disease,

and has been called the metabolic syndrome (Henry and Shalomo., 2008).

Individuals with this syndrome has significantly increase risk for

cardiovascular disorders, for example men with this syndrome are 3-4 times

more likely to die of cardiovascular disease (Pamela et al., 2005).

The metabolic syndrome strongly predict diabetes mellitus type 2. Indeed,

metabolic syndrome accounts for up to half of new case of diabetes in the

Framingham offspring study among those who did not have diabetes as base line

and were followed for 8 years (

Ford et al., 2008).

In 2008 expert recommended that metabolic syndrome could be used to

identify an early likehood of developing cardiovascular disease. The presence of

any of metabolic syndrome parameter should raise the awareness to determine

10 years absolute risk for cardiovascular disease and treatment initiated based on

this (

Rosenzweig et al., 2008)

.

Diabetes mellitus increase the death from myocardial infraction, stroke, and

non ischemic cardiovascular disease. Its estimated that 80% of diabetes mellitus

die of cardiovascular disease, and has synergistic effect with other factor such as

smoking, hypertension, dyslipidemia and insulin resistance as measured by

elevated fasting insulin level (Henry and Shalomo., 2008).

In 2005, the International Diabetes Foundation proposed a new definition

for metabolic syndrome for clinical use (set out in Table 9) that avoids the

technical difficulties associated with insulin measurement.

---

37

iabetes Federation definition of metabolic

International D

Table (8):

syndrome

.

Parameter

Qualification

Central obesity (waist

circumference

In all cases:

Central obesity (Waist

circumference)

In men:

94cm in Europids, Sub-Saharan

Africans and Arabs.

90cm in South Asians and Chinese

85cm in Japanese

In women:

80cm in all populations, except

90cm in Japanese

Any 2 of the following 4:

Raised triglyceride level

1.7mmol/l

OR

Specific treatment for this lipid

abnormality

Reduced HDL-C levels

0.9 mmol/l in males or _1.1 mmol/l

in female

OR

Specific treatment for this lipid

abnormality

Raised blood pressure

Systolic 130mmHg

or diastoiic 80 mmHg

OR

treatment of previously diagnosed

hypertension

---

38

Raised fasting plasma glucose

5.6 mmol/l

(OGTT recommended to confirm

diabetes)

OR

previously diagnosed type 2 diabetes

(Steven and Richard., 2008)

Insulin resistance is accompanied by many other alterations that are not

included in the diagnostic criteria for the metabolic syndrome. Increases in apo

B and C-III, uric acid, prothrombotic factors (fibrinogen, plasminogen activator

inhibitor 1), serum viscosity, asymmetric dimethylarginine, homocysteine, white

blood cell count, pro-inflammatory cytokines, the presence of microalbuminuria,

non-alcoholic fatty liver disease and/or non-alcoholic steatohepatitis, obstructive

sleep apnoea, and polycystic ovarian disease are all associated with insulin

resistance (Eckel and Grundysun., 2005).

T

YPE

2

DIABETESMELLITUSANDTHELIVER

T

HE

R

OLEOFTHE

L

IVERIN

G

LUCOSE

H

OMEOSTASIS

:

The liver plays important roles in the homeostasis of glucose metabolism since it

acts as a major target organ for insulin and a site for gluconeogenesis and

glycogen storage. Diabetes mellitus, commonly develops in patients with liver

cirrhosis as the result of hepatocyte dysfunction, is known as hepatogenous

diabetes mellitus (Kim and Choi., 2006).

An appreciation of the role of the liver in the regulation of carbohydrate

homeostasis is essential to understanding the many physical and biochemical

alterations that occur in the liver in the presence of diabetes and to

understanding how liver disease may affect glucose metabolism. The liver uses

glucose as a fuel and also has the ability to store it as glycogen and synthesize it

from non-carbohydrate precursors (gluconeo-genesis) (Levinthal and Tavill.,

1999).

---

39

Glucose absorbed from the intestinal tract is transported via the portal vein to

the liver. Although the absolute fate of this glucose is still controversial, some

authors suggest that most of the absorbed glucose is retained by the liver so that

the rise in peripheral glucose concentration reflects only a minor component of

postprandial absorbed glucose. Therefore, it is possible that the liver plays a

more significant role than does peripheral tissue in the regulation of systemic

blood glucose levels following a meal (Bjorntorp and sjostrom., 1978).

While Katz et al. (1983) suggested that

most

absorbed glucose is not taken up by

the liver but is rather metabolized via glycolysis in the peripheral tissues.

Insulin is metabolized by insulinase in the liver, kidney, and placenta. About 50%

of insulin secreted by the pancreas is removed by first-pass extraction in the liver.

Insulin promotes glycogen synthesis (glyco-genesis) in the liver and inhibits its

breakdown (glyco-genolysis).It promotes protein, cholesterol, and triglyceride

synthesis and stimulates formation of very-low-density lipoprotein cholesterol. It

also inhibits hepatic gluconeogenesis, stimulates glycolysis, and inhibits

ketogenesis. The liver is the primary target organ for glucagon action, where it

promotes glyco-genolysis, gluconeogenesis, and ketogenesis (Karem and

Forsham., 1994).

In type 2 diabetes, excessive hepatic glucose output contributes to the fasting

hyperglycemia. Increased gluconeogenesis is the predominant mechanism

responsible for this increased glucose

output

, while glycogenolysis has not been

shown to be increased in patients with type 2 diabetes. Hyper-glucagonemia has

been shown to augment increased rates of hepatic glucose output, probably

through enhanced gluconeogenesis (Consoli et al., 1989).

Liver disease is an important cause of death in type 2 diabetes.

In the population-

based Verona Diabetes Study, cirrhosis

was the fourth leading cause of death

and accounted for 4.4%

of diabetes-

related

deaths. The standardized mortality

ratio, i.e., the relative rate of an event compared with the

background rate, for

cirrhosis was 2.52 compared with 1.34 for

cardiovascular disease (CVD). In

another prospective cohort

study, cirrhosis accounted for 12.5% of deaths in

patients

with diabetes (Tolman et al., 2007).

S

PECTRUMOFLIVERDISEASEINDIABETICS

:

N

ONALCOHOLICFATTYLIVERDISEASES

(NAFLD)

Nonalcoholic fatty liver disease is a chronic liver condition characterized by

insulin resistance and

hepatic

fat accumulation, in the absence of other

---

40

identifiable causes of fat accumulation, such as alcohol abuse, viral hepatitis,

autoimmune hepatitis, alpha-1 antitrypsin deficiency, medications like

corticosteroids and estrogens, and other conditions (Ali and Cusi., 2009).

The perception of nonalcoholic fatty liver disease as an uncommon and benign

condition is rapidly

changing

. Approximately 70% of persons with type 2 diabetes

mellitus have a fatty liver and the disease follows a more aggressive course with

necroinflammation and fibrosis (i.e. nonalcoholic steatohepatitis) in diabetes.

New evidence suggests that it is not steatosis per se but the development of

lipotoxicity induced mitochondrial dysfunction and activation of inflammatory

pathways that leads to progressive liver damage. Nonalcoholic steatohepatitis is

a leading cause of end-stage liver disease and contributes to cardiovascular

disease in patients with type 2 diabetes mellitus (Kenneth., 2009).

Diabetes, dyslipidemia, hypertension, and cardiovascular disease (CVD) occur

more frequently in individuals with NAFLD (Targher et al., 2008).

This is of concern as the prevalence of NAFLD is rapidly increasing in children

and adolescents, particularly in Hispanics. As with adults, metabolic syndrome

(MetS) is common in children with NAFLD (Schwimmer et al., 2008).

Obesity and type 2 DM share a `metabolic soil' that promotes hepatocyte

lipotoxicity: adipose tissue insulin resistance, subclinical inflammation,

hyperinsulinemia, and abnormal glucose metabolism (Cusi., 2008). Bland

steatosis has a relatively benign course. Follow up of patients with hepatic

steatosis alone has revealed that 3% will eventually develop Cirhosis overtime.

NASH in contrast has a progressive course, where a sizable subset of patients

will develop fibrosis and cirrhosis (Matteoni et al., 1999).

Patients with NASH who develop cirrhosis have 30-40% liver related mortality

over 10 years follow up period similar to patients with chronic hepatitis C

related cirrhosis and there is an even higher mortality rate in obese patients.

Patients who present with cryptogenic cirrhosis, which is a possible presentation

of end stage NAFLD, can also develop hepatocellular carcinoma (Ratziu et al.,

2002).

The vast majority of patients with cryptogenic cirrhosis have features of

metabolic syndrome in particular obesity and type 2 diabetes, leading to the

belief that these patients in effect have end stage NAFLD.

Type 2 diabetes is an independent risk factor for the development of NAFLD.

The prevalence of type 2 diabetes is 30- 50% in patients with NAFLD, and 62%

of type 2 diabetes met ultrasound criteria for NAFLD (Jimba et al., 2005).

---

41

Furthermore, type 2 diabetes is an independent risk factor for increase liver

related mortality in a case- cohort study of patients with NAFLD (Younossi et

al., 2004).

More advanced liver disease was seen in those patients with morbid obesity with

type 2 diabetes as compared to those with normal glucose level (Papadia et al.,

2004). The presence of obesity and type 2 diabetes in patients with metabolic

syndrome has an additive effect on the prevalence and severity of chronic liver

disease (Marchesini et al., 2004).

P

ATHOGENESISOF

NAFLD

patitis C infection and in

Potential role of RBP4 in mediating the progression of chronic he

Fig. (3):

the metabolic pathway to NASH. RBP4 was suggested as a novel circulating marker for HCV-induced

steatosis and could in addition play a

role

in the metabolic pathway from visceral obesity to insulin

resistance and fatty liver disease (Matthias et al., 2008).

The most commonly accepted hypotheses of the pathogenesis of NASH is two-

hit hypothesis (

Day and James, 1998)

. The first hit is insulin resistance, which

leads to accumulation of fat in the liver

(Li et al., 2002).

There is a second hit possible in the form of endotoxin or another insult to the

hepatic steatosis triggering oxidative stress, which generates reactive oxygen

species resulting in lipid peroxidation and cytokines relies ultimately leading to

inflammation and hepatic fibrosis (

Yang

et al., 1997).

---

42

Tumor necrosis factor is increased in obese patients. This cytokines along with

interleukin-6 is believed to result in insulin resistance by producing the up

regulation of suppressor of cytokines signaling proteins (Katsuki et al., 1998).

Central obesity along with increase lipolysis of visceral tissue result in increase

delivery of free fatty acids to the liver leading to hepatic steatosis, the first hit in

the development of NAFLD. Insulin resistance decreases the uptake of glucose

to muscle and increase lipolysis of visceral fat, resulting in increased delivery of

free fatty acids to the liver (

Nielsen

et al., 2004).

There is also increase hepatic lipogenesis and decrease export of lipid from the

liver despite increased beta oxidation of fat, with net accumulation of fat in the

liver (

Diraison

et al., 2003).

Free fatty acids, besides increasing free fatty acid oxidation and lipid

peroxidation, can directly increase tumor necrosis factor this leads to and

further promoting insulin resistance leading to a higher prevalence of type 2

diabetes in NAFLD associated cirrhosis (Feldstein et al., 2004).

The most accurate means of diagnosing NAFLD is a liver biopsy specially in

patients with the metabolic syndrome (

Nugent

and Younossi., 2007) abdominal

imaging studies like abdominal ultrasound, computed tomography and magnetic

resonance imaging cannot differentiate between benign hepatic steatosis and

NASH (

Saadeh

et al., 2002).

Because nonalcoholic steatohepatitis may develop even in the presence of

normal liver transaminases, a liver biopsy is still necessary for a definitive

diagnosis. However, new imaging methods and plasma biomarkers are emerging

as alternative diagnostic tools (Kenneth., 2009).

Raised serum leptin and resistin and decreased adiponectin levels, may

differentiate between bland hepatic steatosis and NASH and may render the

biopsy obsolete (Gawrieh et al., 2004).

D

IABETESMELLITUSANDHEPATOCELLULACARCINOMA

:

Most diabetic patients are non-insulin dependent and are characterized by

hyperinsulinemia. High insulin level or its precursors may interact with liver

cells to stimulate mitogenesis or carcinogenesis (Moore et al., 1998).

Alternatively, the metabolic effects of diabetes may increase the risk of

hepatocellular carcinoma through non Alcoholic steato hepatitis and

Cryptogenic cirrhosis (Marceau et al., 1999).

---

43

Patients

with diabetes have a high prevalence of liver disease and patients

with

liver disease have a high prevalence of diabetes (Trombetta et al., 2005).

The negative impact of diabetes on the retina, renal, nervous and cardiovascular

system is well recognized, yet little is known about it's effect on the liver

(Liane., 2010).

Existing guidelines do not advocate screening for liver related complications

among persons with diabetes, making the liver a potentially neglected target

organ. Although diabetic hepatopathy is potentially less common, it may be

appropriate for addition to the list of target- organ conditions related to diabetes.

When the liver fail, there is no equivalent form of management, such as

haemodialysis or photocoagulation. Annual screening for liver disease might be

accomplished by means of a simple biochemical analysis such as alanine amino

transferase (Goessling et al., 2008).

Individual with type 2 diabetes have an increased prevalence of cirrhosis, and a

proportion of patients with acute and chronic liver disease develop diabetes

mellitus (Petrides., 1994).

Recent evidence has even suggested that elevated level of aminotransferase may

be a marker of future risk of diabetes (Orasanu and Plutzky., 2009)

Virtually the entire spectrum of liver disease is seen in patients with type 2

diabetes. This includes abnormal liver enzymes, nonalcoholic

fatty liver disease

(NAFLD), cirrhosis, hepatocellular carcinoma,

and acute liver failure. In

addition, there is an unexplained

association of diabetes with hepatitis C

(Trombetta et al., 2005).

Also, Patients with various forms of liver disease can be predisposed to impaired

glucose tolerance because of corticosteroid and hydrochlorothiazide therapy or

haemo-chromatosis (Niedereau et al., 1985).

The management of diabetes in patients with liver disease is

theoretically

complicated by liver-related alterations in drug

metabolism, potential

interactions between the drugs, and a

low, albeit real, incidence of hepatotoxicity

(Tolman et al., 2007).

---

44

L

IVERCIRRHOSIS

D

EFINITION

:

Cirrhosis is defined as the histological development of regenerative nodules

surrounded by fibrous bands in response to chronic liver injury which lead to

portal hypertension and end-stage liver disease (Schuppan and Afdhal., 2008).

It is an irreversible alteration of the liver architecture and consist of diffuse

fibrosis of the hepatic parenchyma resulting in nodule formation (Friedmann

and Keeffe., 1998).

Fibrosis is not synonymous with cirrhosis, fibrosis may be in acinar zone 3 in

heart failure or in zone 1 in bile duct obstruction and congenital hepatic fibrosis

or interlobular in granulomatous liver disease but without a true cirrhosis.

Nodule formation without fibrosis as in partial nodular transformation is not

cirrhosis (Sherlok and Dooly., 2002a).

Cirrhosis has followed hepatic cellular necrosis. Although the causes are

multiple the end result is the same. In all cirrhotic patients the triad of necrosis,

regeneration and scarring is present (Conn and Atterbury., 1987).

World Health Organization definition of cirrhosis is a diffuse process

characterized by fibrosis and the conversion of normal liver architecture into

structurally abnormal nodules that lack normal lobular organization. This

definition distinguishes cirrhosis from other types of liver disease that have

either nodule formation or fibrosis, but not both. These hepatic disorders

may be characterized by portal hypertension in the absence of cirrhosis.

Nodular regenerative hyperplasia, for example, is characterized by diffuse

nodularity without fibrosis, whereas chronic schistosomiasis is characterized

by Symmers' pipestem fibrosis with no nodularity (Lowarence and Emmet.,

2012).

The consequence of cirrhosis includes vascular disturbances such as intrahepatic

shunting of blood, obstruction to portal venous blood flow (resulting in portal

venous hypertension) and impaired parenchymal function including protein

synthesis, hormone metabolism and excretion of bile (Millward and Wright.,

1979).

Structural changes in the liver may cause impairment of hepatic function

manifested as (Jaundice, portal hypertension and varices, ascites, hepatorenal

syndrome, spontaneous bacterial peritonitis, hepatic encephalopathy and

progressive hepatic failure) (Lowarence and Emmet., 2012).

---

45

C

LASSIFICATIONOFLIVERCIRRHOSIS

:

A- Morphological classification:

1.

Micronodular cirrhosis: Characterized by thick, regular septa, regenerating

small nodules less than 3mm in diameter, uniform and by involvement of every

lobule (Sherlock and Dooley., 2002a).

Micronodular liver may represent impaired capacity for growth as in: alcoholism,

hemochromatosis, biliary obstruction, hepatic venous outflow obstruction and

Indian childhood cirrhosis (Friedmann and Keeffe., 1998).

Macronodular cirrhosis: Is characterized by septa and nodules of variable

sizes more than 3mm in diameter. Regeneration is reflected by large cells with

large nuclei and by cell plates of varying thickness (Sherlock and Dooley.,

2002a), such as in chronic hepatitis C, chronic hepatitis B, alpha-1-antitrypsin

deficiency and primary biliary cirrhosis (Friedmann and Keeffe., 1998).

Mixed cirrhosis [Micronodular and Macronodular cirrhosis]: It is a

compromise term used when both macro and micronodules are present with

equal frequency (Anthony et al., 1978).

Regeneration in micronodular cirrhosis often converts to macronodular

(Sherlock and Dooley, 2002a).

B- Histological classification:

It confirms the morphologic diagnosis and subdivides it into histological and

sometimes etiological categories. According to histological classification, liver

cirrhosis is classified in to the following types:

Portal cirrhosis.

Post-hepatic cirrhosis.

Post-necrotic cirrhosis.

Biliary cirrhosis.

Cardiac cirrhosis.

(Conns and Atterbury., 1987)

C- Functional classification:

In any case of cirrhosis the following should be considered:

Degree of development of cirrhosis:

---

46

Activity: indicating tendency towards progression which depends mostly on the

activity of the attendant chronic hepatitis as reflected by

hypergammaglobulinemia and by inflammation and cell necrosis.

Degree of hepatocyte alteration: (steatosis, hepatic cholestasis) as a basis of

presenting hepatic symptoms indicated by liver function testes.

Presence of complications: as portal hypertension and its sequelae.

Liver biopsy is required for the evaluation of the first three considerations

(Winkel et al., 1976).

D- Aetiological classification:

.

cirrhosis according to etiology

of

sification

Clas

Table (9) :

(Anand., 1999)

1.

delta; C viral hepatitis with extensive necrosis

types B;

:

Viral hepatitis

can progress directly to post necrotic cirrhosis and chronic active hepatitis may

progress to cirrhosis but it is not clear how often does this sequence of events

occur (Schalm., 1997).

Hepatitis B, C and D represent the major viral agents currently shown to cause

chronic hepatitis. Long term complications are cirrhosis with attendant problems

resulting from hepatic synthetic failure, portal hypertension and hepatocellular

carcinoma (Sherlok and Dooly., 2002a).

The World Health Organization has declared hepatitis C a global health

problem, with approximately 3% of the world's population (roughly 170-200

million people) infected with HCV. In the United States , approximately 3

million people are chronically infected, many of whom are still undiagnosed. In

---

47

Egypt the situation is quite worse. Egypt has a population of 81 million and

contains the highest prevalence of hepatitis C in the world. The national

prevalence rate of HCV antibody positivity has been estimated to be between

10-13% (Mohamed., 2004).

Chronic hepatitis C infection may lead to cirrhosis and hepatocellular carcinoma

and is a leading cause of liver transplantation in the United States (Alter et al.,

1992).

In addition to these known risk factors, there is now emerging evidence to

suggest that hepatitis C virus (HCV) infection may also contribute to the

development of diabetes for example, glucose intolerance is observed more

often in patients with hepatitis C virus infection compared with controls with

liver disease (Allison et al., 1994).

There is also increase frequency of genotype 2a among diabetic patients with

hepatitis C infection in contrast to genotype 1 which is the most common form

of infection in the general population (Andrew., 1999).

Experimental data are compatible with direct interference of HCV with the

insulin signaling cascade. This was first suggested by a study in which liver

specimens obtained from 42 non-obese, non-diabetic, HCV-infected individuals

and 10 non-HCV-infected subjects matched for age and BMI were exposed in

vivo to insulin, and examined for the contents and phosphorylation/activation

status of some insulin signaling molecules (Aytug et al., 2003).

Insulin-stimulated IRS-1 tyrosine phosphorylation was decreased by two-fold in

HCV-infected patients compared to non-HCV-infected ones, and this was

paralleled by significant reductions in IRS-1/p85 phosphatidylinositol 3-kinase

association, IRS-1-associated phosphatidylinositol 3-kinase enzymatic activity

and insulin-stimulated Akt phosphorylation (Aytug et al., 2003).

-

2

Alcoholic liver cirrhosis is serious sequale of the chronic abuse

Alcohol:

of ethanol. The development of liver damage is related to the amount and

duration of alcohol abuse (Boyer., 1985).

Recent study done on sixteen European countries showed that alcohol

consumption has negative impact on health in terms of liver cirrhosis mortality,

also no difference was found between different types of alcohol. Indeed beer has

the most serious consequences on health than other types (Jan and Valdmer.,

2010).

---

48

3-Biliary cirrhosis:

Due to long standing intermittent or partial biliary obstruction. It may be

primary or secondary:

a) Primary biliary cirrhosis: (PBC).

It has been called chronic non suppurative destructive cholangitis (Burroughs

and Westaby, 2002).

It is the most common autoimmune liver disease affecting up to 1 in 1000

women over 40 years of age (Kaplan and Gershwin, 2005).

It is characterized by destruction of intrahepatic bile ducts and high titers of

antimitochondrial antibodies in patients sera (Akiyoshi and Murri, 1998).

The granulomatous destruction of interlobular bile ducts that characterized PBC

is almost always associated with antimitochondrial antibodies specific for the E2

subunit of the pyruvate dehydrogenase complex (Gershwin and Mackay, 2008).

b) Secondary biliary cirrhosis:

It is a sequal of long standing obstruction of the biliary tract for more than one

year before cirrhosis develops (Tartakovsky and Hagg, 1995).

4) Wilson's disease:

Wilson disease (WD) is an autosomal recessive disorder of copper transport that

results in accumulation of copper primarily in the liver, brain and cornea. WD is

the most common inherited liver disease with the prevalence of 1: 37,000 in the

pediatric population in Korea. Mutations in the ATP7B gene cause failure of

copper excretion in to the bile and a defective incorporation of copper into

ceruloplasmin (Seo, 2006).

It is a rare inherited disease predominantly of young people that is characterized

by cirrhosis of the liver, bilateral softening degeneration of basal ganglia and

greenish brownish pigmented rings in the periphery of the cornea (Kayser

---

49

Fleischer rings) (Sherlock and Dooley, 2002a).

It should be suspected in any individuals between ages 3-40 years with:

Unexplained serum aminotransferase elevation, fulminant hepatic failure,

chronic hepatitis or cirrhosis.

- Neurological features of unexplained a etiology.

- Psychiatric disorders with sings of hepatic or neurological disease or patients

who are refractory to therapy.

- Kayser-Fleischer rings on routine eye examination.

- Unexplained acquired coombs-negative hemolytic anemia.

- A sibling or a parent carrying the diagnosis of Wilson disease.

(Wisam et al., 1998)

Investigations of WD:

- Low serum ceruloplasmin 20mg/dl.

- High 24hrs urine copper 100 microgram.

- High hepatic copper content 250 microgram/g of dry liver.

- Kayser-Fleischer rings were found in 96% on routin eye examination.

- A combination of any two of the above finding is strong support for

diagnosis WD (Seo, 2006).

5- Alpha-1-antitrypsin deficiency:

Alpha 1-antitrypsin deficiency is an inherited metabolic disorder that

predisposes the affected individual to chronic pulmonary disease, in addition to

chronic liver disease, cirrhosis and HCC. Just over one third of genetically

susceptible adult patients with the most severe phenotype, develop clinically

significant liver injury (Fairbanks and Tavill, 2008).

It is the most common metabolic deficiency. It should be considered in the

differential diagnosis in any adult who present with chronic hepatitis or cirrhosis of

---

50

unknown a etiology. The incidence of liver disease in adolescent with alpha-1-

antitrypsin deficiency is 2% by age 20-40 years, 5% by age of 40-50 years and 15%,

thereafter with slight male predominance (Hugo, 1998).

6) Haemochromatosis:

Hereditary haemochromatosis is a common disorder with a prevalence of up to

1/250-300 in persons of northern European descent. It was first described in

1886 as Bronze diabetes; it is an autosomal recessive metabolic disorder in

which there is an increased iron absorption over many years.

The disease is often recognized and diagnosed only in the setting of advanced

liver disease. With increased awareness of the disease, the most common

presenting symptoms are:

- Incidental diagnosis because of elevated serum iron markers.

- Chronic liver disease.

- Arthritis.

It is to be noted that liver biopsy with determination of hepatic iron

concentration is the best method for establishing a definitive diagnosis

(Freidmann and Keeffe, 1998).

7- Budd- chiari syndrome (BCS):

BCS is characterized by hepatic venous outflow obstruction at any level from

the small hepatic veins to the atriocaval junction (Horton et al., 2008).

Hepatic vein occlusion or BCS is an disorder characterized by abdominal pain,

hepatomegaly and ascites.

The majority of patients with BCS have an underlying thrombotic diathesis. In

less than 30% of cases the disorder is idiopathic. Disorders associated with BCS

include the following:

---

51

a) Hematological:

Polycythemia rubra vera.

Myeloproliferative disorders.

Paroxysmal nocturnal haemoglobulinuria.

Antiphospholipid syndrome.

b) Inherited thrombotic diathesis:

Protein C deficiency.

Protein S deficiency.

Antithrombin III deficiency.

Factor V Leiden mutation.

(Horton et al., 2008)

8- Immunologic:

a- Autoimmune hepatitis.

b- Graft-versus-host disease.

(Horton et al., 2008)

Autoimmune hepatitis:

It is unresolving inflammation of the liver of unknown aetiology that is

characterized by at least periportal hepatitis on histologic examination,

autoantibodies in serum and hypergammaglobuli-naemia. Because there are no

pathognomonic features, the diagnosis requires the exclusion of chronic viral

hepatitis, Wilson disease, alpha-1-anti-trypsin deficiency, haemochromatosis,

drug induced hepatitis, alcoholic and non alcoholic hepatitis and other

immunologic condition such as autoimmune cholangitis, primary biliary

cirrhosis and primary sclerosing cholangitis (Cazaja et al., 2000).

Now is recognized as a multisystem disorder that can occur in males and females of

all ages. This condition can coexist with other liver diseases (e.g., chronic viral

hepatitis) and chemicals (e.g minocycline) (Vento and Cainelli, 2004).

---

52

Pathophysiology:

Liver cells injury in autoimmune hepatitis patients results from cell mediated

immunologic attack. This attack is directed against genetically predisposed

hepatocytes. Aberrant display of human leucocyte antigen (HLA) class II on the

surface of hepatocytes facilitates the presentation of normal cell membrane

constituents to antigen processing cells. These activated cells stimulate clonal

expansion of autoantigen sensitized cytotoxic T-lymphocytes. Cytotoxic T-

lymphocytes infilterate liver tissue, release cytokines and help to destroy liver

cells (Seki et al., 1990).

Autoantibodies:

Three types of autoimmune hepatitis have been recognized:

Type I with antibodies:

I. Antinuclear.

II. Anti-smooth muscle (Anti-actin).

Type II with antibodies:

Anti-liver/kidney microsomal (Anti-LKM1). Most frequently in girls and young

women.

Type III with soluble liver antigen:

Same as type I (Burroughs and Westaby, 2002).

Diagnosis of liver cirrhosis:

Diagnosis of liver cirrhosis is based on, history, clinical examination looking

for:

1- Stigmata of chronic liver disease like, spider naevi, gynaecomastia and

testicular atrophy.

2- Features of protal hypertension like ascites and spleno megaly.

---

53

3- Features of hepatic encepahlopathy like flapping tremer.

Laboratory investigations:

1- Full blood count.

2- Liver function tests.

Special tests looking for the under lying causes.

Imaging studies like abdominal ultrasound, computed tomography and

magnetic resonance imaging.

The gold standard for diagnosis is liver biopsy.

(Lowrence S and Emmet., 2012)

R

ETINOLBINDINGPROTEIN

4

A

DIPOSETISSUEINHEALTHANDDISEASE

Adipose tissue is found in the skin as subcutaneous fat, and within the body

cavity surrounding the heart, in the mesentery and in the retroperitonium. This

portal fat drains directly into the portal circulation and has been linked to

morbidities, such as cardiovascular disease and type 2 DM. Adipose tissue has a

considerable effect on glucose homeostatis by secreting adipokines, some like

leptin, adiponectin has anti diabetic action while others like tumor necrosis

factor, interleukin 6 and retinol binding protein 4 has diabetogenic actions

(Masur et al., 2008).

The adipose tissue is an important endocrine organ that secretes a variety of

proteins termed adipocytokines which are biologically active polypeptides that

are produced either exclusively or substantially by the adipocytes and act by the

endocrine, paracrine, or autocrine mechanism.The adipokines appear to be

involved in a wide range of physiological processes; these include haemostasis,

---

54

lipid metabolism, blood pressure regulation, insulin sensitivity and angiogenesis

(Petersen et al., 2007).

Inflammatory cytokines are key players in the pathogenesis and severity of

atherosclerosis and its complications (Fonseca et al., 2009).

Retinol-binding protein (RBP)-4 was reported in 2005 by Yang

et al as a new

fat-derived adipokine that specifically binds to retinol, that has been proposed as

an adipokine involved in the regulation of systemic glucose metabolism and

pathogenesis of insulin resistance, and that it may provide a link between

obesity and insulin resistance. It has been demonstrated that RPB4 is secreted by

human adipose tissue and that it is expressed almost exclusively in mature

adipocytes. The expression of RBP4 has been demonstrated in subcutaneous

and, in visceral adipose tissue in humans

(Weiping et al., 2007).

More than 50 adipokines has been Identified leptin, adiponectin, resistin and

retinol binding protein 4 among others. Obesity has been associated with a

chronic inflammatory response, characterized by elevated circulating levels of

tumor necrosis factor, interleukin 6 and c reactive protein (Engelis et al., 2003).

RBP4 is predominantly secreted by visceral adipose tissue and the liver. It is the

only specific transport protein for retinol and by interacting with nuclear retinal

x receptor (RXR), it takes part in control of metabolic and proliferative cell

functions (Youssef., 2011).

A large number of studies in rodents and also in humans confirm an association

of increased level of RBp4 with obesity, insulin resistance, type 2 diabetes,

dyslipidemia, hypertension and metabolic syndrome (Rabe et al., 2008).

Retinol binding protein4 (RBP4) a 21-KDa protein synthesized in the liver and

adipose tissues, formerly recognized for its role as a specific transport for

vitamin A, now it is known that it plays a crucial role in insulin resistance and

---

55

diabetes mellitus. Adipose RBP4 expression and serum RBP4 level are elevated

in mouse model of insulin resistance, elevated circulating RBP4 increase blood

glucose by inhibiting insulin signaling in skeletal muscle and up regulating

hepatic gluconeogenesis (Koch et al., 2010).

Fig. (4): Retinol binding protein 4 (Rask et al., 1987).

Vitamin A circulates in human plasma as retinol (Vitamin A alcohol) bound to a

specific transport protein. This protein differs from the known low and high density

plasma lipoproteins. There appears to be one binding site for retinol per molecule

of RBP. Solutions of RBP are fluorescent (characteristic of retinol). There are no

fatty acids or fatty aryl chains in purified RBP. The usual concentration of RBP

in plasma is of the order of 4-15 mg/100 ml (Kanai et al., 1968).

---

56

R

ETINOID

R

ECEPTORS

:

Retinoid X receptor (RXR) is a member of the nuclear hormone receptor super

family of ligand-controlled transcription factors. These receptors modulate gene

expression by binding coperatively as dimers to sequences called hormone

responsive elements. RXR forms homodimers and is involved in 9-cis retinoic

acid (a naturally occurring retinoid isomer) (9c RA)-mediated gene activation. It

also forms heterodimers with other receptors, including all-trans retinoic acid

receptor (RAR) and peroxisome proliferators activated receptor (PPAR)

(Mangelsdorf and Evans., 1995).

There are three RXR isotypes, RXR , , and , among them, RXR is

abundantly expressed in the liver and plays important role in regulating cell

proliferation and differentiation. In addition, PPAR ­ RXR a heterodimer is

deeply involved in lipid metabolism (Youssef et al., 2011).

More over RBP4 gene expression in humans was associated with inflammatory

markers and RBP4 levels were associated with inflammatory response in obese

patients (Blagobal et al., 2007).

Increased serum RBP4 levels impair post receptor insulin signaling at the level

of phosphoinositide-3-Kinase in muscle and enhance the expression of

phosphoenolpyruvate carboxykinase in liver (Yang et al., 2005) and increases

basal glucose production, and reduce insulin action to suppress glucose

production in hepatocytes (Shin et al., 2002).

Therefore, increased serum RBP4 levels in humans might contribute to impaired

insulin stimulated glucose uptake in muscle and elevated hepatic glucose

production, both of which are characteristic of type 2 diabetes (Kahn et al.,

1998). Regions near the RBP4 locus on human chromosome 10q have been

linked to hyperinsulinemia or early onset of type 2 diabetes, a finding consisting

with a pathogenic role for RBP4 in insulin resistance and type 2 diabetes (Meigs

et al., 2002).

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57

Berry and Noy., 2009 showed that binding of retinol-bound RBP(holo-RBP) to

it's receptors induces phosphorylation and regulates certain genes that control

adipocyte lipid homeostasis and insulin responses.

Fig. (5): RBP4 and insulin interactions at cellular level (Newcomer et al., 1984).

R

ETINOL

B

INDING

P

ROTEIN

4

AND

I

NSULIN

R

ESISTANCE

:

Mouse and human studies have highlighted a pathogenic link among 1R,

diabetes, and high serum and adipose levels of RBP4, identifying RBP4 as a

novel adipocytokine. There is clinical evidence that circulating RBP4 levels are

related to the severity of 1R and to the various features of the metabolic

syndrome (Von Eynatten and Humpert., 2007). Specifically, raised serum

levels of RBP4 have been linked to NAFLD (non alcoholic fatty liver disease),

assessed by ultrasonography in a cohort of subjects with and without diabetes

(Janke et al., 2006).

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58

D

EFECTSINTHEABILITYOFFATCELLSTOTRANSPORTGLUCOSE

ARELINKEDTOINSULINRESISTANCEINMUSCLEANDLIVERS

:

Liver, skeletal muscle and adipose tissue are the three major targets for the

metabolic actions of insulin. Insulin regulates glucose output and by increasing

the rate of glucose uptake by skeletal muscle and adipose tissue. Stimulation of

glucose uptake into muscle cells and adipocytes by insulin depends largely on

translocation of the glucose transporter GLUT4 from an intracellular

compartment to the cell surface (Shepherd and Kahn., 1999).

The expression of GLUT4 is reduced in adipocytes, but not in skeletal muscle,

in obesity and type 2 diabetes (Shepherd and Kahn., 1999). Given that skeletal

muscle is the major sink for glucose disposal, the hyperglycemia associated with

obesity type 2 diabetes can not be explained by the decreased uptake of

glucose into adipose tissue that is attributable to the decreased expression of

GLUT4 in adipocytes. However, over expression of GLUT4 specifically in

adipose tissue enhances systemic glucose tolerance and insulin sensitivity in

mice (Shepherd et al., 1993).

Furthermore, adiposity-selective ablation of GLUT4 results in a degree of

systemic insulin resistance similar to that induced by muscle selective GLUT4

ablation. In addition, insulin resistance is apparent in both the liver and skeletal

muscle of mice lacking GLUT4 in adipose tissue (Specifically).These findings

suggested the possibility that expression of GLUT4 in adipocytes regulates

whole ­ body insulin sensitivity through an unknown mechanism (Abel et al.,

2001).

Barbara kahn and her colleagues predicted the existence of a factor secreted

from adipose tissue (an adipokine) that induces insulin resistance in the liver and

skeletal muscle (Abel et al., 2001) In nature, (Yang et al., 2005) have now

identified RBP4 as such a factor. These researchers found that expression of the

gene encoding RBP4- a protein whose only function was thought to be the

delivery of retinol to tissues was increased in adipose tissue of mice with

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59

adipocyte specific ablation of GLUT4. Over expression of RBP4 or injection of

recombinant RBP4 in normal mice induced insulin resistance, whereas mice

heteroszygous or homozygous for knockout of the gene encoding RBP4 showed

increased insulin sensitivity compared with wild-type mice. Furthermore, the

synthetic retinoid fenretinide, which reduced the serum level of RBP4 through

urinary excretion, ameliorated insulin resistance in mice fed a high-fat diet.

S

UBJECTSAND

M

ETHODS

This cross- sectional study was conducted on 120 subjects recruited from

endocrinology outpatient clinic and hepatology clinics and wards, Ain Shams

University Hospital.

The subjects were divided into 30 diabetic patients, 30 patients with liver

cirrhosis, 30 patients with diabetes and liver cirrhosis, and 30 control subjects

matched for age and sex.

Study Design:

Subjects were divided into 4 groups:

Group 1: 30 diabetic patients.20 of them were females and 10 were males.

Group 2: 30 cirrhotic patients.18 of them were males and 12 were females.

Group 3: 30 diabetic and cirrhotic.16 of them were males and 14 were

females.

Group 4: 30 control subjects. 16 of them were males and 14 were females.

Patients with early liver cirrhosis were selected according to Childs

classification. (table 10)

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60

Table (10): Modified child's classification (Pugh).

Clinical variable

1 Point

2 Point

3 Point

Encephalopathy

Absent

Stage 1-2

Stage 3-4

Ascitis

Absent

Slight

Moderate

Bilirubin(mg/dl)

2

2-3

3

Bilirubin in PBC or PSC (mg/dl)

4

4-10

10

Albumin (gm/dl)

3.5

2.8-3.5

2.8

Prothrombin time

(seconds above normal) or INR

4 Sec

or INR 1.7

4-6 Sec

or INR 1.7-2.3

6 Sec

or INR 2.3

(Mullen and Dasarthy., 1999)

Exclusion criteria:

1- Pregnant ladies.

2- Renal dysfunction.

3- Critically ill patients.

4- Late stage liver cirrhosis.

All cases enrolled into the study were submitted to:

1- Full medical history.

2- Thorough clinical examination.

3- Anthropometric examination:

- Weight, measurement in kg.

- Height in cm

- Body mass index (BMI); = Wt. (kg) /Ht.(m)

2

.

- Waist circumference in cm.

4- Blood Samples were taken to measure the following:

a) Fasting plasma glucose.mg/dl

b) HbA

1c

%.

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61

c) Lipid profile including total cholesterol, LDL HDL, and TG.

Mg/dl.

d) Liver function tests including ALT, AST,in u/l serum albumin in

g/dl and prothrombin time in seconds.

e) Retinol binding protien 4(RBP4) in mg/dl by Elisa.

5- Radiological examination; Abdominal ultrasound was done for all

patients and control subjects to assess liver morphology,size,

echopattern, and to detect the presence of ascites.

S

PECIMENCOLLECTIONANDPREPARATION

:

A fasting morning serum sample should be obtained, 8 hours for glucose

and 12 hours for lipid profile. The blood should be in a plain red top

venipuncture tube without additions or gel barrier. Allow the blood to clot.

Centrifuge the specimen to separate the serum from the cells.

Samples refrigerated in deep freeze -20 °C.

Samples for prothrombin time was collected in a blue top venipuncture

tube, samples for HbA

1c

% was collected in pink top venipuncture tube.

HbA

1

C

%;

estimation of HbA

1c

% was done by glycohemoglobin reagent

set from pointe scientific Inc. normal range: 4.2 - 6.2 %. (Bates,. 1978).

Lipid profile : after 12 to 14 hours overnight fasting:

o

Cholesterol ; estimation of cholesterol an triglyceride by cholesterol

enzyme colorimetric assay.

Normal range: cholesterol : 150 - 240 mg/dl

Triglyceride: 150 - 200 mg/dl (Brain and Eric., 2009).

o

High density lipoprotein (HDL); estimation of HDL by

precipitation with dextran sulphate normal range: 40 mg/dl low

60 mg/dl high (Brain and Eric., 2009).

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62

o

Low density lipoprotein (LDL); was calculated according to

Edward formula :

LDL= total cholesterol ­ TG /5 ­ HDL.

Normal range: LDL= 100-160 mg/d (Brain and Eric., 2009).

Fasting plasma Glucose: after 8 hours overnight fasting.

It was done by the timed endpoint method using hexokinase enzyme, the

change in absorbance is measured at 340 nm. It was performed on Syn chron

CX9 autoanalyzer.

Normal range = 70-100 mg/dl (Gaede et al., 2008).

o

Assay of serum RBP4:

Measurement of serum RBP4 was done by enzyme ­linked

immunosorbent assay (ELISA). The AssayMax Human RBP4 ELISA Kit,

Immundiagnostic AG, Stubenwald-Allee 8a, D 64625 Bensheim desgined

for detection of human RBP4 in serum. This assay employs a quantitive

sandwitch enzyme immunoassay technique that measures RBP4. Blood

samples were incubated in duplicate wells of micro titer plates.Which had

been recoated with antibodies specific for the analyte RBP4.During this

incubation,the analyte present. In the standards samples was bound by

immobilized antibodies in the respective assay plates. After repeated

washing and aspiration to remove all unbound substances, an enzyme ­

linked polyclonal antibody specific for the analyte was added to the wells

of the wells of the assay plates.Unbound enzyme conjugate streptavidin­

peroxidase conjugate was removed by repeated washing and substrate

solution was added to the wells of the assay plates,with color developing in

proportion to the amount of the analyte bound in the initial step. Color

devolpment was stopped with the addition of an acid solution,and the

intensity of color was read using a programmable spectrophotometer. The

concentration of RBP4 in samples were determined by interpolation from

individual standard curves.

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63

Normal range 4-15mg/dl (Graham et al., 2006).

S

TATISTICALANALYSIS

Data were statistically described in terms of mean

standard deviation (SD),

range, or frequencies (number of cases) and percentages when appropriate.

Comparison of numerical variables between the study groups was done using

one way analysis of variance (ANOVA) test with posthoc multiple 2-group

comparisons. For comparing categorical data, Chi square (

2) test was

performed. Exact test was used instead when the expected frequency is less than

5. All statistical calculations were done using computer programs SPSS

(Statistical Package for the Social Science; SPSS Inc.,Chicago, IL,USA)

version15 for Microsoft Windows.

Significant level (P) was express as follows:

P value 0.05 =Non Significant (N.S)

P value0.05 = Significant (Sig)

a) P value 0.001 = Highly Significant (H.S)

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64

R

ESULTS

The present study was conducted on 120 subjects divided into four groups:

group (1) included 30 diabetic patients, group (2) included 30 patients with liver

cirrhosis, group (3) included 30 patients with diabetes and liver cirrhosis and

group (4) included 30 control subjects. They were selected from patients

attending the Endocrinology Clinic and Hepatology clinics and wards, Ain

Shams University Hospital.

Group (I): (DM) included (30) thirty diabetic patients they were 10 (33.3%)

males, and 20(66.7%) females. Their age ranged from 30- 60 with a

mean of (51.97+7.52) years. The mean body mass index was (28.5 +

6.17) kg/m

2

. The mean waist circumference was (103.7± 17.76) cm.

The

mean HbA

1

c% was (10.35 + 2.83). The mean fasting plasma glucose was

(142.43 +23.38) mg/dl. The mean cholesterol was (209.97 + 28.10)

mg/dl. The mean LDL was (151.13 + 22.32) mg/dl. The mean HDL was

(34.83 + 4.13) mg/dl. The mean triglyceride was (115.53 + 21.27) mg/dl.

The mean ALT was (18.1 + 5.37) u/l. The mean AST was (18.73 + 5.55)

u/l. The mean serum albumin was (4.17 + 0.52) g/dl. The mean

prothrombin time was (10.30 +2.35) The mean Retinol binding protein

was (20.1± 8.31) mg/dl.

Group (2): Included 30 cirrhotic patients they were 18 (60%) males and 12

(40%) females. Their age ranged from 30- 60 with a mean of

(46.93+9.90) years. The mean body mass index was (25.6+ 4.15) kg/m.

The mean waist circumference was (83.43+ 7.40) cm. The mean

HbA

1C

% was (5.92 + 0.68). The mean fasting plasma glucose was (97.87

+ 8.68) mg/dl. The mean cholesterol was (237.53 + 23.87) mg/dl. The

mean LDL was (169.89 + 25.52) mg/dl. The mean triglyceride was

(122.3± 15.25) mg/dl. The mean HDL was (37.97 + 6.17) mg/dl. The

mean ALT was (33.00 + 18.35) u/l. The mean AST was (33.00 + 18.35)

u/l. The mean Albumin was (2.39 + 0.64) g/dl. The mean prothrombin

---

65

time was (18.33 + 3.99) seconds. The mean RBP4 was (9.31 + 3.99)

mg/dl.

Group (3): Included 30 patients with diabetes and liver cirrhosis they were 16

(53.3%) males and 14 (46.7%) females. Their age ranged from 30- 60

with a mean of (51.47 + 6.80) years. The mean body mass index was

(29.1+4.1) kg/m

2

. The mean waist circumference was (88.33+6.00) cm.

the mean HbA

1C

% was (7.40+2.60).

The mean fasting plasma glucose was (123.77 +24.45) mg/dl the mean

cholesterol was (228.67 +31.87)

mg/dl. The mean LDL was (159.57 +28.05) mg/dl. The mean HDL was

(36.83+5.37) mg/dl. The mean Triglycerides was (119.83 + 21.29) mg/dl.

The mean ALT was (28.33+8.63) u/l. The mean AST was (38.33 + 37.58)

u/l. The mean albumin was (2.33+0.49) g/dl. The mean prothrombin time

was (17.13±3.66) seconds. The mean RBP4 was (8.59 ±7.17) mg/dl.

Group (4): Include 30 control subjects they were 16 (53.3%) males and 14

(46.7%) females. Their age ranged from 30- 60 with a mean of (47.83 ±

8.74) years. The mean body mass index was (22.5±2.53) kg/m2. The

mean waist circumference was (79.73 ±8.98) cm. The mean HbA1c%

was (5.8 ± 0.46). The mean fasting plasma glucose was (95.93 ± 7.52)

mg/dl. The mean cholesterol level was (198.97 ±17.58) mg/dl. The mean

LDL was (138.83 ± 28.35) mg/dl. The mean HDL was (38.17 ± 5.03)

mg/dl. The mean triglycerides was (98.33 ±11.29) mg/dl. The mean ALT

was (19.33±4.90) u/l. The mean AST was (19.33±0.46) u/l. The mean

serum Albumin was (4.19 ± 0.46) g/dl. The mean prothrombin time was

(10.0±1.7) seconds. The mean RBP4 was (13.5± 3.97) mg/dl.

As regard the clinical and laboratory data of different groups, the data were

arranged in tables according to the groups and results.

---

66

1- BMI (body mass index) kg/m

2

)

On comparing the 4 groups as regards BMI the mean BMI of group 1 (28.5

±6.17), group 2 (25.6 ±4.15), group 3 (29.1± 4.1), group 4 (22.5±2.53).

There is a highly statistical significant difference between group 4 (Control)

(22.5±2.53) and group I (diabetic)(28.5 ±6.17) P= 000, group 4 (control) (22.5±

2.53) and group 2 (liver cirrhosis) (25.6 ±4.15) P=0.047, group 4 (control) and

group 3 (diabetes and liver cirrhosis) a significant difference was also found p=

000,between group 2 (cirrhosis) (25.6± 4.15) and group 3 (diabetes and liver

cirrhosis) (29.1± 4.1) p=0.02.

It was found that there is no significant difference between group I (diabetes)

(28.5± 6.17) and group 2(liver cirrhosis) (25.6± 4.15) p= 0.08, and group 3

(diabetes and liver cirrhosis) (29± 4.1) p= 1.

2- Waist circumference (cm):

It was found that there is a high significant statistical difference between group 4

(controls) (79.9 ± 8.9) and groupI (diabetic) (103.07± 17.76) p= 000, group

3(DM and liver cirrhosis) (88.33±6.00) p = 0.000. A significant difference was

also found between group 1 (DM) (103.07± 17.76) , group 2 (liver cirrhosis)

(83.43± 7.40) p= 0.000, and group 3 (DM and liver cirrhosis) (88.33±6.00)

p=0.001.

It was found that there is no significant difference between group 4 (controls)

(79.73± 8.98), and group 2 (liver cirrhosis) (83.43+ 7.40) p=0.413.

3- FPG (fasting plasma glucose) (mg/dl):

It was found that there is a high significant statistical difference between group 4

(control) (95.93± 7.52), group I (diabetes) (142.43± 23.38) p= 000, and group 3

(diabetes and liver cirrhosis) (123.77± 24.45).

---

67

It was found that there is no significant difference between group 4 (control)

(95.93± 7.52) and group 2 (liver cirrhosis) (97.87± 8.68).

4- HbA

1C

%

It was found that there is a high significant statistical difference between group 4

(controls) (5.80± 0.46), group I (diabetes) (10.35± 2.83) p= 000, and group 3

(diabetes and liver cirrhosis) (7.40.0.62) p = 0.000.

It was also found that there is a high significant statistical difference between

group 1 (DM) (10.35± 2.83), and group 2 (liver cirrhosis) (5.92± 0.68) p= 000.

It was found that there is no significant difference between group 4 (control)

(5.80± 0.46) and group 2 (liver cirrhosis) (5.92± 0.68).

It was found that there is no significant difference between group 1 (diabetes)

(10.35±2.83) and group 3 (DM and liver cirrhosis) (7.40±0.62), p = 0.523.

2 (liver cirrhosis) (169.89± 25.52) p= 0.00 and group 3 (diabetes and liver

cirrhosis) (159.57± 28.05) p= 0.016.

It was found that there is no statistically significant difference between group I

(diabetes) (151.13± 22.32), group 2 (cirrhosis) (169.89± 25.52) p 0.42, group 3

(diabetes and liver cirrhosis) (159.57± 28.05) p=1.00 and group 4 control

(138.83 ± 28.35) p= 0.42.

There was also no significant difference between group 2 (liver cirrhosis)

(169.89± 25.52) and group 3 (diabetes and liver cirrhosis) (159.57± 28.05) p=

0.941.

9- Alanine aminotransferase (ALT u/l):

It was found that there is a high significant statistical difference between group 4

(controls) (19.33±4.90) , group 2 liver cirrhosis) (33.00± 18.35) p = 0.00,and

group 3 (diabetes and liver cirrhosis) (28.33±8.63) p= 0.00

---

68

It was found also that there is a high siginificant statistical between group I

(diabetes) (18.10 ± 5.37), group 2 (liver cirrhosis) (33.00±18.35) p= 0.001, and

group 3 (diabetes and liver cirrhosis) (28.33±8.63) p= 0.00.

It was found that there is no significant difference between group I diabetes

(18.10 ± 5.37) and group 4 (control) (19.33±4.90) p=0.924.There was also no

significant

5- Cholesterol mg/dl:

It was found that there is a high significant statistical difference between group 1

(diabetes) (209.97± 28.1) group 2 (liver cirrhosis) (237.53± 23.87) p= 000 and

group 3 (diabetes and liver cirrhosis) (228.67± 31.87) p= 0.031.

There was also statistically significant difference between group 4 (control)

(198.97± 17.58), group 2 (liver cirrhosis) (237.53± 23.87) p= 000 and group 3

(diabetes and liver cirrhosis) (228.67± 31.87) p= 000.

It was found that no statistically significant difference between group 2

(cirrhosis) (237.53± 23.87) and group 3 (diabetes and liver cirrhosis) (228.67±

31.87) p=1. There was no difference between group 4 (control) (198.97± 17.58)

and group 1 (diabetes) (209.97± 28.1) p= 0.576.

6-High density lipoprotein mg/dl;

It was found that there is no statistically significant group 1 (diabetes)

(34.83±4.13), group 2 (liver cirrhosis) (37.97±6.17), group 3 (diabetes and liver

cirrhosis) (36.83±5.37), and group 4 (control) (38.17±5.03).p=0.058

7- Triglycerides mg/dl:

It was found that there is a high siginificant statistical difference between group

4 (control) (98.33± 11.29), group 2 (liver cirrhosis) (122.30± 15.25) p=0.00 and

group 3 (diabetes and liver cirrhosis) (119.83± 21.29) p= 0.00

---

69

It was found that there is no significant difference between group 1 (diabetes)

(115.53± 21.27), group 2 (cirrhosis) (122.3± 15.25) p 0.877 and group 3

(diabetes and liver cirrhosis) (119.83±21.29) p= 1.00 No statistically significant

difference was found between group 2 (cirrhosis) (122.33± 15.25) and group 3

(diabetes and liver cirrhosis) (119.83± 21.29) p= 1.00

8- Low density lipoproteins:

It was found that there is a high significant statistical difference between group 4

(control) (138.83± 28.35), group2 (liver cirrhosis) (169.89± 25.52) p= 0.00 and

group 3 (diabetes and liver cirrhosis) (159.57± 28.05) p= 0.016.

It was found that there is no statistically significant difference between group I

(diabetes) (151.13± 22.32), group 2 (cirrhosis) (169.89± 25.52) p 0.42, group 3

(diabetes and liver cirrhosis) (159.57± 28.05) p=1.00 and group 4 control

(138.83 ± 28.35) p= 0.42.

There was also no significant difference between group 2 (liver cirrhosis)

(169.89± 25.52) and group 3 (diabetes and liver cirrhosis) (159.57± 28.05) p=

0.941.

9- Alanine aminotransferase (ALT u/l):

It was found that there is a high significant statistical difference between group 4

(controls) (19.33±4.90) , group 2 liver cirrhosis) (33.00± 18.35) p = 0.00,and

group 3 (diabetes and liver cirrhosis) (28.33±8.63) p= 0.00

It was found also that there is a high siginificant statistical between group I

(diabetes) (18.10 ± 5.37), group 2 (liver cirrhosis) (33.00±18.35) p= 0.001, and

group 3 (diabetes and liver cirrhosis) (28.33±8.63) p= 0.00.

It was found that there is no significant difference between group I diabetes

(18.10 ± 5.37) and group 4 (control) (19.33±4.90) p=0.924.There was also no

---

70

significant difference between group 2 (liver cirrhosis) (33.00±18.35) and group

3 (diabetes and liver cirrhosis) (28.33±8.63) p=0.751.

10- Aspartate Aminotransferase (AST u/l).

It was found that there is statistically significant difference between group 3

(diabetes and liver cirrhosis) (38.33± 37.58), group I (diabetes) (18.73± 5.55),

group 2 (liver cirrhosis) (33.00± 18.35) and group 4 (control) (19.33± 4.90)

p=0.04.

It was found that there is no significant difference between group 1 (diabetes)

(18.73± 5.55), group 2 (liver cirrhosis) (33.00± 18.35), and group 4 (control)

(19.33± 4.90) p= 0.63.

11- Serum Albumin g/dl.

It was found that there is a high significant statistical difference between group I

(diabetes) (4.17± 0.52), group 2 (liver cirrhosis) (2.39± 0.64) and group 3

(diabetes and liver cirrhosis) (2.33 ±0.49) p= 0. 00

There was also a high significant statistical between group 4 (control) (4.19±

0.46), group 2 (liver cirrhosis) (2.39± 0.64) and group 3 (diabetes and liver

cirrhosis) (2.33± 0.49). P= 0.00

There was no significant difference between group 4 (control) (4.19± 0.46)

group 1(diabetes) (4.17 ± 0.52) p=0.1

There was also no significant difference between group 2 (liver cirrhosis) (2.39±

0.64) and group 3 (diabetes and liver cirrhosis) (2.33± 0.49) p= 0.1

12- Prothrombin time/ seconds:

It was found that there is a high significant statistical difference between group I

(diabetes) (10.30± 2.35), group 2 (liver cirrhosis) (18.33± 3.99) and group 3

(diabetes and liver cirrhosis) (17.13± 3.66) p=0.00

---

71

It was found that there is a high significant statistical between group 4 (control)

(10.00+ 1.70), group 2 (liver cirrhosis) (18.33± 3.99) and group 3 (diabetes and

liver cirrhosis) (17.13± 3.66) p= 0.00

It was found that there is no significant difference between group I (diabetes)

(10.30± 2.35) and group 4 (control) (10.10± 1.70) p= 0.81

13- Retinol Binding Protein4 mg/dl:

It was found that there is a significant statistical difference between group 4

(control) (13.5± 3.97), group I diabetes (20.10± 8.31) p= 0.002 and group 3

(diabetes and liver cirrhosis) (8.59± 7.17) p= 0.045.

It was found that there is a high significant statistical difference between group I

(diabetes) (20.10± 8.31), group 2 (liver cirrhosis) (9.31±7.63) and group 3

(diabetes and liver cirrhosis) (8.59± 7.17) p= 0.00

A statistically significant difference was found between group 4 (control) (13.5±

3.97) and group 2 (liver cirrhosis) 9.31± 3.99) P =0.02.

No difference was found comparing group 2 (cirrhosis) (9.31± 3.99) and group 3

(diabetes and liver cirrhosis (8.59± 7.17) p=1.

RBp4 was highest in group1 (20.10 ± 8.31) followed by group p 4 (13.5 ± 3.97)

and group 2 (9.31 ± 3.99) and lastly group 3 (8.59 ± 7.17).

---

72

Table (11): Post hoc test to detect LSD between group 1 (DM) and group 2

(liver cirrhosis) as regarding all clinical and laboratory

parameters in the studied groups.

Group 1

(DM)

Group 2

(liver cirrhosis)

P value Sig

Age (years)

51.97 ±7.52

46.93 ±9.90

0.05

S

BMI kg/m

2

28.5±6.17

25.6±4.15

0.05

N.S

Waist

circumference (cm)

103.07±17.76 83.43±7.40

0.001 H.S

HbA

1

C %

10.35

2.83 5.92

0.68

0.001 H.S

FPG (mg/dl)

142.43

23.38

97.87

8.68

0.001 H.S

LDL (mg/dl)

151.13

22.32

169.89

25.52

0.05

N.S

HDL (mg/dl)

34.83

4.13

37.97

6.17

0.05

N.S

TG (mg/dl)

115.53

21.27

122.3

15.25

0.05

N.S

CHOL (mg/dl)

209.97

28.1

237.53

23.87

0.05

N.S

ALT (u/l)

18.10

5.37

33.00

18.35

0.001 H.S

AST (u/l)

18.73

5.55

33.00

18.35

0.05

N.S

ALB (g/dl)

4.17

0.52

2.39

0.64

0.001 H.S

PT (sc)

10.3

2.35

18.33

3.99

0.001 H.S

RBP4 (mg/dl)

20.1

8.31

9.31

3.99

0.001 H.S

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

---

73

On comparing group 1(DM) and group 2(liver cirrhosis), a high statistical

significant difference was found (p0.001) regarding FPG, waist circumference,

HbA

1C

%, cholesterol, ALT, serum albumin, PT and RBP4. NO statistical

significant difference as regards BMI, LDL, HDL, TG and AST. (table 11)

Table (12): Post hoc test to detect LSD between group 1 (DM) and group 3

(DM liver cirrhosis) as regarding all clinical and laboratory

parameters in the studied groups.

Group 1 (DM)

Group 3

(DM liver

cirrhosis)

P value Sig

Age (years)

51.97 ±7.52

51.47 ± 6.80

0.05

S

BMI (kg/m

2

)

28.5±6.17

29.1±4.1

0.05

N.S

Waist

circumference (cm)

103.07±17.76

88.33±6.00

0.001

H.S

HbA

1C

%

10.35

2.83

7.40

0.62

0.05

N.S

FPG (mg/dl)

142.43

23.38

123.77

24.45

0.001 H.S

LDL (mg/dl)

151.13

22.32

159.57

28.05

0.05

N.S

HDL (mg/dl)

34.83

4.13

36.83

5.37

0.05

N.S

TG (mg/dl)

115.53

21.27

119.83

21.29

0.05

N.S

CHOL (mg/dl)

209.97

28.1

228.67

31.87

0.05

N.S

ALT (mg/dl)

18.10

5.37

28.33

8.63

0.001 H.S

AST (mg/dl)

18.73

5.55

38.33

37.58

0.05

N.S

Alb (g/dl)

4.17

0.52

2.33

0.49

0.001 H.S

---

74

PT( sc)

10.3

2.35

17.13

3.66

0.001 H.S

RBP4 (mg/dl)

20.1

8.31

8.59

7.17

0.001 H.S

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

On comparing group 1(DM) and 3 (DM and liver cirrhosis), a high statistical

significant difference was found (P0.001) regarding FPG, ALT, serum

albumin, PT and RBP4 level but no statistical significant difference was found

as regards BMI waist circumference, HbA

1C

%, LDL, HDL, TG, cholesterol

level, AST.(table 12)

Table (13): Post hoc test to detect LSD between group 1 (DM) and group 4

(Controls) as regarding all clinical and laboratory parameters

in the studied groups.

Group 1 (DM)

Group

4(Controls)

P value Sig

Age (years)

51.97 ±7.52

47.83 ± 8.74

0.05

S

BMI (kg/m

2

)

28.5±6.17

22.5±2.53

0.001 H.S

Waist circumference

(cm)

103.07±17.76

79.73±8.98

0.001 H.S

HbA

1

C %

10.35

2.83

5.80

0.46

0.05

Sig

FPG (mg/dl)

142.43

23.38 95.93

7.52

0.001 H.S

LDL (mg/dl)

151.13

22.32 138.83

28.35 0.05

N.S

---

75

P value Non 0.05 =Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

On comparing group 1(DM) and group 4 (controls) a high statistical significant

difference was found (P0.001) regarding BMI, waist circumference, HbA

1C

%,

FPG, TG, and RBP4 level but no statistical significant differences was found as

regards LDL, HDL, cholesterol level, AST, ALT, serum albumin, and PT.(table

13)

HDL (mg/dl)

34.83

4.13

38.17

5.03

0.05

N.S

TG (mg/dl)

115.53

21.27 98.33

11.29

0.001 H.S

CHOL (mg/dl)

209.97

28.1

198.97

17.58 0.05

N.S

ALT (mg/dl)

18.10

5.37

19.33

4.90

0.05

N.S

AST (mg/dl)

18.73

5.55

19.33

4.9

0.05

N.S

ALB (g/dl)

4.17

0.52

4.19

0.46

0.05

N.S

PT (sc)

10.3

2.35

10.00

1.7

0.05

N.S

RBP4 (mg/dl)

20.1

8.31

13.5

3.97

0.001 H.S

---

76

Table (14): Post hoc test to detect LSD between group 2 (liver cirrhosis) and

group 3 (DM liver cirrhosis) as regarding all clinical and

laboratory parameters in the studied groups.

Group 2

(liver

cirrhosis)

Group 3

(DM liver cirrhosis)

P value Sig

Age(years)

46.93 ±9.90

51.47 ± 6.80

0.05

S

BMI (kg/m

2

)

25.6±4.15

29.1±4.1

0.05

N.S

Waist

circumference

(cm)

83.43±7.40

88.33±6.00

0.05

N.S

HbA

1C

%

5.92

0.68

7.40

0.62

0.001

H.S

FPG (mg/dl)

97.87

8.68

123.77

24.45

0.001

H.S

LDL (mg/dl)

169.89

25.52 159.57

28.05

0.05

N.S

HDL (mg/dl)

37.97

6.17

36.83

5.37

0.05

N.S

TG (mg/dl)

122.3

15.25

119.83

21.29

0.05

NS

CHOL (mg/dl)

237.53

23.87 228.67

31.87

0.05

N.S

ALT (u/l)

18.10

5.37

28.33

8.63

0.05

N.S

AST (u/l)

33.00

18.35

38.33

37.58

0.05

N.S

ALB (g/dl)

2.39

0.64

2.33

0.49

0.05

N.S

PT (sc)

18.33

3.99

17.13

3.66

0.05

N.S

RBP4 (mg/dl)

9.31

3.99

8.59

7.17

0.05

N.S

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

On comparing group 2 (liver cirrhosis) and 3 (DM and liver cirrhosis), apart

from FPG, and HbA

1c

%, no statistical significant difference was found regarding

all variables. (table 14)

---

77

Table (15): Post hoc test to detect LSD between group 3 (DM liver

cirrhosis) and group 4 (controls) as regarding all clinical and

laboratory parameters in the studied groups.

Group3 (DM

liver cirrhosis)

Group

4(Controls)

P value Sig

Age (years)

51.47 ± 6.80

47.83 ± 8.74

0.05

S

BMI (kg/m

2

)

29.1±4.1

22.5±2.53

0.001

H.S

Waist

circumference

(cm)

88.33±6.00

79.73±8.98

0.001

H.S

HbA

1

C %

7.40

0.62

5.8

0.46

0.001

H.S

FPG (mg/dl)

123.77

24.45

95.93

7.52

0.001

H.S

LDL (mg/dl)

159.57

28.05

138.83

28.35

0.001

H.S

HDL (mg/dl)

36.83

5.37

38.17

5.03

0.05

N.S

TG (mg/dl)

119.83

21.29

98.33

11.29

0.001

H.S

CHOL (mg/dl) 228.67

31.87

198.97

17.58

0.001

H.S

ALT (u/l)

28.33

8.63

19.33

4.90

0.001

H.S

AST (u/l)

38.33

37.58

19.33

4.90

0.001

H.S

ALB (g/dl)

2.33

0.49

4.19

0.46

0.001

H.S

PT (sc)

17.13

3.66

10.0

1.7

0.001

H.S

RBP4 (mg/dl)

8.59

7.17

13.5

3.97

0.001

H.S

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

On comparing group 3 (DM and liver cirrhosis) and 4 (controls) a high

statistical significant difference was found (P0.001) regarding BMI, waist

circumference, FPG, LDL, TG, cholesterol level, ALT, AST, PT, serum albumin

and RBP4 level.(table 15)

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78

Table (16): Post hoc test to detect LSD between group 2 (liver cirrhosis)

and group 4 (controls) as regarding all clinical and laboratory

parameters in the studied groups.

Group 2

(liver cirrhosis)

Group 4

(Controls)

P value

Sig

Age (years)

46.93 ±9.90

47.83 ± 8.74

0.05

S

BMI (kg/m

2

)

25.6±4.15

22.5±2.53

0.001

H.S

Waist circumference

(cm)

83.43±7.40

79.73±8.98

0.05

N.S

HbA

1C

%

5.92

0.68

5.8

0.46

0.05

N.S

FPG (mg/dl)

97.87

8.68

95.93

7.52

0.05

N.S

LDL (mg/dl)

169.89

25.52

138.83

28.35

0.05

N.S

HDL (mg/dl)

37.97

6.17

38.17

5.03

0.05

N.S

TG (mg/dl)

122.3

15.25

98.33

11.29 0.05

NS

CHOL (mg/dl)

237.53

23.87

198.97

17.58

0.05

N.S

ALT (u/l)

33.00

18.35

19.33

4.90

0.05

S

AST (u/l)

33.00

18.35

19.33

4.90

0.05

N.S

ALB (g/dl)

2.39

0.64

4.19

0.46

0.001

H.S

PT (sc)

18.33

3.99

10.0

1.70

0.001

H.S

RBP4 (mg/dl)

9.31

3.99

13.5

3.97

0.05

S

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

---

79

P value 0.001 = Highly Significant (H.S).

On comparing group 2 (liver cirrhosis) and 4 (controls) a high statistical

significant difference was found, (P001) regarding BMI, PT and serum

albumin. A statistical significant was found as regard ALT and RBP4. No

statistical significant difference was found as regards waist circumference,

HbA

1

c%, FPG, LDL, HDL, TG, cholesterol level, and AST.(table 16)

On comparing all groups with each other there was a high statistical significant

difference (p0.001) regarding BMI, waist circumference, HbA

1

C%

,

FPG,

cholesterol level, TG, ALT, AST, serum albumin PT and RBP4 level. No

statistical significant difference was found (P0.05) as regards HDL.

On comparing RBP4 between all groups a high stasistically significant

difference was found between group 4 (controls), group I (DM), and group 3

(DM and liver cirrhosis) P 0.001. A statistical significant difference was found

between group 4 (control) and group 2 (liver cirrhosis) p 0.05.

Table (17): Correlation coefficient between RBP4 and all clinical and

laboratory parameters in the studied groups.

Pearson correlation

R

Sig

Waist circumferance

(cm)

0.227

0.021 Sig

HBA

1

C%

0.189

0.031 Sig

FPG (mg/dl)

0.268

0.004 Sig

LDL (mg/dl)

- 0.119

0.196 N.S

HDL (mg/dl)

-0.370

0.689 N.S

---

80

TG (mg/dl)

-0.145

0.115 N.S

Chol (mg/dl)

-0.187

0.04

Sig

ALT (u/l)

-0.174

0.058 N.S

AST (u/l)

-0.172

0.063 N.S

Alb (g/dl)

0.410

000

H.S

**.Correlation is significant at the 0.01 level (2-tailed).

*. Correlation is significant at the 0.05 level (2-tailed).

The correlation study showed that there is a highly significant direct correlation

between RBP4, serum albumin, HbA

1

C %, waist circumference, FPG and

cholesterol but was indirect with , LDL, HDL, TG,AST, and ALT. (table 17)

Table (18): Descriptive data of clinical parameter among the 4 groups.

Group 1

(DM)

Group 2

(liver

cirrhosis)

Group 3

(DM Liver

cirrhosis)

Group 4

(Controls)

P

value Sig

Age (years)

51.97± 7.52

46.93 ± 9.90

51.47 ± 6.80

47.83 ± 8.74

0.044 Sig

Body mass

index (kg/m

2

)

28.5± 6.17

25.6 ± 4.15

29.1 ± 4.1

22.5 ± 2.53

0.00

H.S

Waist

circumference

cm

103.07 ± 17.76 83.43 ± 7.40

88.33 ± 6.00

79.73 ±8.98

0.00

H.S

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

Table (19): Comparison between pateints and controls as regards sex.

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81

Cases

Controls

P value

Number

90

30

0.67

(N.S)

no

%

no

%

Males

44

48.88%

16

53.3%

Females

46

51.12%

14

46.7%

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

---

82

Table (20): Descriptive data of laboratoryfindings among the 4 groups.

Group 1

(DM)

Group 2

liver

cirrhosis

Group 3 DM

and liver

cirrhosis

Group 4

controls

Anova

Variables

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

F

P

Value

sig

HBA

1

C%

10.35 ± 2.83

5.92 ±0.68

7.40±0.62

5.80 ±0.46

2.189

0.093

N.S

FPG

(mg/dl)

142.43±23.38

97.87±8.68

123.77±24.45

95.93±7.52

46.569

0.000

H.S

LDL mg/dl

151.13±22.30

169.89±25.52

159.57±28.05

138.83±28.35

7.273

0.000

H.S

HDL mg/dl

34.83±4.13

37.97±6.17

36.83±5.37

38.17±5.03

2.560

0.058

N.S

TG mg/dl

115.53±21.27

122.3±15.25

119.83±21.29

98.33±11.29

10.930

0.000

H.S

CHOLmg/dl 209.97±28.1

237.53±23.87

228.67±31.87

198.97±17.58

14.276

0.000

H.S

ALT (u/l)

18.10±5.37

33.00±18.35

28.33±8.63

19.33±4.90

15.826

0.000

H.S

AST u/l

18.73±5.55

33.0±18.35

38.33±37.58

19.33±4.9

6.453

0.000

H.S

ALB g/dl

4.17±0.52

2.39±0.64

2.33±0.49

4.19±0.46

116.134

0.000

H.S

PT sc

10.3±2.35

18.33±3.99

17.13±3.66

10.0±1.7

60.785

0.000

H.S

BRP4 mg/dl

20.1±8.31

9.31±3.99

8.59±7.17

13.5±3.97

17.182

0.000

H.S

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

Table (21): Shows sex distribution in the 4 groups.

Group

1 DM

Group 2

Cirrhosis

Group 3

DM+Cirrhosis

Group

4

Control

Total

Sex F

Count

20

12

14

14

60

%within

Group

66.7%

40.0%

46.7%

46.7%

50.0%

M Count

10

18

16

16

60

%within

Group

33.3%

60.0%

53.3%

53.3%

50.0%

Total

Count

30

30

30

30

120

---

83

%within

Group

100.0% 100.0%

100.0%

100.0% 100.0%

Fig. (7): Sex distribution among the studied groups.

Table (22): Shows comparison between the 4 groups as regards waist

circumference (cm).

Mean ±SD

P

value

Waist

Circumference

(cm)

Group 1 (DM)

103.07 ± 17.76

0.000

(H.S)

Group 2 (Liver cirrhosis)

83.43±7.40

Group 3 (DMliver cirrhosis) 88.33 ±6.00

Group 4 ( controls)

79.73 ±8.98

P value 0.05 = Non Significant (N.S)

66.7

33.3

46.7

53.3

46.7

60

40

53.3

0

10

20

30

40

50

60

70

P

ercentage

DM

Control

Cirrhosis

DM+Cirrhosis

Female

Male

---

84

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

Fig. (8): Comparison between the 4 groups as regards waist circumference (cm).

Table (23): Shows comparison between the 4 groups as regards body mass

index kg/m

2

.

Mean ±SD

P

value

Body mass

index

kg/m

2

Group 1 (DM)

28.5 ± 6.17

0.000

(H.S)

Group 2 (Liver cirrhosis)

25.6 ± 4.15

Group 3 (DMliver cirrhosis)

29.1 ± 4.1

Group 4 ( controls)

22.5 ± 2.53

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

0

20

40

60

80

100

120

Datenreihen1

---

85

Fig. (9): Comparison between the 4 groups as regards body mass index kg/m2.

Table (24): Shows comparison between the 4 groups as regards HbA

1

C %

Mean ±SD

P value

HbA

1

C

%

Group 1 (DM)

10.35 ± 2.83

0.05

Sig

Group 2 (Liver cirrhosis)

5.92 ± 0.68

Group 3 (DMliver cirrhosis)

7.40 ± 0.62

Group 4 ( controls)

5.8 ± 0.46

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

---

86

Fig. (10): Comparison between the 4 groups as regards HbA

1

C %.

Table (25): Shows comparison between the 4 groups as regards fasting

plasma glucose mg/dl.

Mean ±SD

P

value

Fasting

plasma

glucose

(mg/dl)

Group 1 (DM)

142.43±23.38

0.000

(H.S)

Group 2 (Liver cirrhosis)

97.87 ± 8.68

Group 3 (DM liver cirrhosis) 123.77 ± 24.45

Group 4 (controls)

95.93 ± 7.52

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

.

0

2

4

6

8

10

12

Group 1

(DM)

Group 2

(Liver

cirrhosis)

Group 3

(DMliver

cirrhosis)

Group 4 (

controls)

Datenreihen1

---

87

Fig. (11): Comparison between the 4 groups as regards fasting plasma glucose mg/dl.

Table (26): Shows comparison between the 4 groups as regards low density

lipoprotein mg/dl.

Mean ±SD

P

value

Low density

lipoprotein

LDL

mg/dl

Group 1 (DM)

151.13 ± 22.32

0.000

(H.S)

Group 2 (Liver cirrhosis)

169.89 ±25.52

Group 3 (DM liver cirrhosis) 159.57±28.05

Group 4 (controls)

138.83±28.35

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

---

88

Fig. (12): Comparison between the 4 groups as regards low density lipoprotein mg/dl.

Table (27): Shows comparison between the 4 groups as regards high density

lipoprotein mg/dl.

Mean ±SD

P value

High

density

lipoprotein

mg/dl

Group 1 (DM)

34.83±4.13

0.058

N.S

Group 2 (Liver cirrhosis)

37.97±6.17

Group 3 (DM liver cirrhosis) 36.83±5.37

Group 4 (controls)

38.17±5.03

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

---

89

Fig.(13): Comparison between the 4 groups as regards high density lipoprotein mg/dl.

Table (28): Shows comparison between the 4 groups as regards total

cholesterol mg/dl.

Mean ±SD

P

value

Total

cholesterol

mg/dl

Group 1 (DM)

209.97±28.10

0.000

(H.S)

Group 2 (Liver cirrhosis)

237.53±23.87

Group 3 (DM liver cirrhosis) 228.67±31.87

Group 4 (controls)

198.97±17.58

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

---

90

Fig. (14): Comparison between the 4 groups as regards total cholesterol mg/dl.

Table (29): Shows comparison between the 4 groups as regards triglyceride

mg/dl .

Mean ±SD

P value

Triglyceride

mg/dl

Group 1 (DM)

115.53±21.27

0.000

(H.S)

Group 2 (Liver cirrhosis)

122.3±15.25

Group 3 (DM liver cirrhosis) 119.83±21.29

Group 4 (controls)

98.33±11.29

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

---

91

Fig. (15): Shows comparison between the 4 groups as regards triglyceride mg/dl.

Table (30): Shows comparison between the 4 groups as regards alanine

aminotransferase (ALT) U/L.

Mean ±SD

P value

Alanine

aminotransferase

(u/l)

Group 1 (DM)

18.10±5.37

0.000

(H.S)

Group 2 (Liver cirrhosis)

33.00±18.35

Group 3 (DM liver

cirrhosis)

28.33±8.63

Group 4 (controls)

19.33±4.90

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

---

92

Fig. (16): Comparison between the 4 groups as regards alanine aminotransferase (ALT) U/L.

Table (31): Shows comparison between the 4 groups as regards aspartate

aminotransferase (AST) U/L.

Mean ±SD

P

value

Aspartate

aminotransferase

(AST).u/l

Group 1 (DM)

18.73±5.55

0.000

(H.S)

Group 2 (Liver cirrhosis)

33.00±18.35

Group 3 (DM liver cirrhosis) 38.33±37.58

Group 4 (controls)

19.33±4.9

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

0

5

10

15

20

25

30

35

Group 1

(DM)

Group 2

(Liver

cirrhosis)

Group 3

(DM

liver

cirrhosis)

Group 4

(controls)

Datenreihen1

---

93

Fig. (17): Comparison between the 4 groups as regards.aspartate aminotransferase (AST) U/L.

Table (32): Shows comparison between the 4 groups as regards serum

albumin g/dl.

Mean ±SD

P value

Serum

albumin

g/dl

Group 1 (DM)

4.17±0.52

0.000

(H.S)

Group 2 (Liver cirrhosis)

2.39±0.64

Group 3 (DM liver cirrhosis) 2.33±0.49

Group 4 (controls)

4.19±0.46

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

---

94

Fig. (18): Comparison between the 4 groups as regards serum albumin g/dl.

Table (33): Shows comparison between the 4 groups as regards

prothrombin time / seconds.

Mean ±SD

P value

Prothrombin

time

(seconds)

Group 1 (DM)

10.30±2.35

0.000

(H.S)

Group 2 (Liver cirrhosis)

18.33±3.99

Group 3 (DM liver cirrhosis)

17.13±3.66

Group 4 (controls)

10.0±1.7

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

---

95

Fig. (19): Comparison between the 4 groups as regards prothrombin time / seconds.

Table (34): Shows comparison between the 4 groups as regards serum

retinol binding protein mg/dl.

Mean ±SD

P value

Serum retinol

binding

protein 4

Group 1 (DM)

20.1±8.31

0.000

(H.S)

Group 2 (Liver cirrhosis)

9.31±3.99

Group 3 (DM liver

cirrhosis)

8.59±7.17

Group 4 (controls)

13.5±3.97

P value 0.05 = Non Significant (N.S)

P value 0.05 = Significant (Sig)

P value 0.001 = Highly Significant (H.S)

---

96

Fig. (20): Comparison between the 4 groups as regards serum retinol binding protein mg/dl.

Fig. (21): Correlation between RBP4 and total cholesterol.

---

97

Fig. (22):

Correlation between RBP4 and serum albumin.

Correlation between RBP4 and waist circumference

---

98

Fig. 24 correlation between RBP4 and HbA1c

D

ISCUSSION

Type 2 DM is the predominant form of diabetes worldwide accounting for 90-

95% of cases globally. The prevalence of type 2 DM is expected to rise more

rapidly in the future because of increasing obesity and reduced activity levels

(Kasper and Fauci., 2005).

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. The basis of the abnormalities in carbohydrate, fat and

protein metabolism in diabetes is deficient action of insulin on target tissues

(American Diabetes Association., 2009).

Liver disease is an important cause of death in type 2 diabetes.In the population-

based Verona Diabetes Study, cirrhosis was the fourth leading cause of death

and accounted for 4.4% of diabetes-related deaths (Marceau et al., 1999).

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99

RBP4 is predominantly secreted by visceral adipose tissue and the liver. A large

number of studies in rodents and also in humans confirm an association of

increased level of RBP4 with obesity, insulin resistance, type 2 diabetes,

dyslipidemia, hypertension and metabolic syndrome (Rabe et al., 2008).

Hyperinsulinemia and insulin resistance are found in nearly all patient with liver

cirrhosis, and insulin resistance is a risk factor for both survival and disease

progression in patient with liver cirrhosis. Recent finding also showed that

RBP4 is an independent risk factor in type 2 diabetes, impaired glucose

tolerance, and healthy individuals with strong family history of diabetes

(Bugianesi et al., 2005).

Exercise training reduced RBP4 levels in patients whose insulin resistance

improved with exercise. In a mouse model, mice lacking adipocyte glucose

transporter 4 (GLUT4) had increased levels of RBP4 and RBP4 was shown to

cause insulin resistance in mouse muscle and liver. An inverse relationship

between GLUT4 in adipocytes and serum RBP4 was demonstrated in the human

study, as well. Whether RBP4 in humans causes, or is correlated with, insulin

resistance has not been determined. (Graham et al,. 2006).

The aim of this study was to estimate RBP4 level in patients with type 2

diabetes mellitus, with and without liver cirrhosis.

The study was conducted on 120 adult persons age range from 30-60 years

they were divided into 4 groups,

Group 1: 30 diabetic patients.

Group 2: 30 cirrhotic patients.

Group 3: 30 diabetic and cirrhotic.

Group 4: 30 control subjects.

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100

Both patients and controls were subjected to full medical history, clinical

examination, anthropometric measurements, and laboratory tests, (FPG,

HBA1c%, Lipid profile, AST, ALT, serum Albumin, prothrombin time, RBP4

and abdominal ultrasound).

With regard to sex; males predominate in group 2 (liver cirrhosis), 18 (60%) of

them were males and 12 (40%) were females, group 3 (DM and liver cirrhosis)

16 (53.3%) of them were males and 14 (46.7%) were females, and group 4

(controls), 16 (53.3%) were males and 14 (46.7%) were females. This is

consistent with those of (Camma et a. 2006), who conducted a study on 311

patients with chronic liver disease and found that males are more than females.

Also in a population based, matched retrospective study done by Liane to study

the relationship between diabetes and chronic liver disease, males were more

than females and is inconsistent with the present study regarding group 1(DM)

we found that 10 (33.3%) were males, and 20(66.7%) were females.(Liane.,

2010), because the sample was small in our study.

In our study we found that in group 1 (DM), body mass index (28.5±6.17)

kg/m

2

, and waist circumference (103.07±17.76) cm, were high, because DM is

associated with central obesity. These findings are in accordance with (Young et

al., 2009) who studied diabetic patients and found the same results concerning

body mass index and waist circumference .

In group 2 (liver cirrhosis) and group 3 (DM and liver cirrhosis), our study

showed a highly significant difference regarding ALT (33.00±18.35) u/l and

(28.33±8.63) respectively compared to controls (21.90±3.22) u/l, this results

were matched with (EL-Tabey et al., 2011) who found a highly statistical

difference (p0.001) between cases and controls regarding liver enzymes.

The cirrhotic patients in the present study group 2 (liver cirrhosis) and 3(DM

and liver cirrhosis) had a significant lower albumin (2.39±0.64) g/dl and

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101

(2.33±0.49) g/dl respectively, and prolonged prothrombin time (18.33±3.99)

seconds and (17.13±3.66) seconds respectively compared to controls

(10.00±1.7) seconds , these results are in agreement with (Erlanger and

Benhamou., 1999).who found low serum albumin and prolonged prothrombin

time in patients with liver cirrhosis .

In the present study there was a highly significant difference in RBP4 level

mean value was (20.10±8.31) mg/dl, waist circumference (103.07 ±17.76) cm,

and body mass index (28.5±6.17) kg/m

2

between group 1 (DM) and group 4

(controls) mean value was (13.53.97) mg/dl, (79.73±8.98) cm, (22.5±2.53)

kg/m

2

respectively, due to insulin resistance, similarly this finding is consist

with Graham who conducted a study on 60 patients 20 obese, 20 diabetic and

20 lean. He measured serum RBP4, insulin resistance, and components of the

metabolic syndrome in these groups. Measurements were repeated after exercise

training in one group. GLUT4 protein was measured in isolated adipocytes, and

he found that serum RBP4 levels correlated with the magnitude of insulin

resistance in subjects with obesity, impaired glucose tolerance, or type 2

diabetes and in nonobese, nondiabetic subjects with a strong family history of

type 2 diabetes. Elevated serum RBP4 was associated with components of the

metabolic syndrome, including increased body-mass index, waist-to-hip ratio,

serum triglyceride levels, and systolic blood pressure and decreased high-density

lipoprotein cholesterol levels. Exercise training was associated with a reduction

in serum RBP4 levels only in subjects in whom insulin resistance improved.

Adipocyte GLUT4 protein and serum RBP4 levels were inversely correlated.

Graham concluded that RBP4 is an adipocyte-secreted molecule that is elevated

in the serum before the development of frank diabetes and appears to identify

insulin resistance and associated cardiovascular risk factors in subjects with

varied clinical presentations. These findings provide a rationale for antidiabetic

therapies aimed at lowering serum RBP4 levels. (Graham et al,. 2006).

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102

Our study also going with (Yang et al,.2005) who conducted a cohort study in

obesity and type 2 diabetes and showed that serum RBP4 levels are elevated in

insulin-resistant mice and humans with obesity and type 2 diabetes, RBP4 levels

are normalized by rosiglitazone, an insulin-sensitizing drug, and transgenic

overexpression of human RBP4 or injection of recombinant RBP4 in normal

mice causes insulin resistance. Conversely, genetic deletion of RBP4 enhances

insulin sensitivity. Fenretinide, a synthetic retinoid that increases urinary

excretion of RBP4, normalizes serum RBP4 levels and improves insulin

resistance and glucose intolerance in mice with obesity induced by a high- fat

diet. Increasing serum RBP4 induces hepatic expression of the gluconeogenic

enzyme phosphoenolpyruvate carboxykinase and impairs insulin signalling in

muscle. They concluded that, RBP4 is an adipocyte-derived 'signal' that may

contribute to the pathogenesis of type 2 diabetes, and lowering RBP4 could be a

new strategy for treating type 2 diabetes.

Sussane, studied women with polycystic ovary syndrome and impaired glucose

metabolism and showed similar result to our study (Sussane, 2007) since

polycystic ovary syndrome is associated with insulin resistance.

In another study on patients with normal glucose tolerance, impaired glucose

tolerance and type 2 diabetes mellitus, Plasma Plasma RBP4 concentrations

were found to be high in impaired fasting glucose median (18.9), microgram/ ml

and diabetic patients median (20.9) microgram/ ml compared to normal glucose

tolerance (18.1) microgram/ ml and is in accordance with our study (Young et

al., 2009).

Contrary to the result of the present study, (Jurgen., 2006) showed in a cross

sectional study done on 30 obese menopausal Caucasian women during weight

loss protocol and found that no relationship between RBP4 level and gene

expression in adipose tissue but Jurgen study was on subcutaneous fat and not

visceral fat. Because visceral fat drains directly in the portal circulation and has

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103

a high number of macrophages which liberates adipokines like RBP4 and

cytokines like tumor necrosis factors (TNF ) and interleukin 6 which are

important in the pathogenesis of insulin resistance.

In the present study there was significant difference between patients with group

2 (liver cirrhosis) and group 3(DM and liver cirrhosis) mean value (9.31± 3.99)

mg/dl and (8.5±1.3) mg/dl respectively, and controls, mean value (13.5±0.7)

mg/dl p 0.05 as regards RBP4, This result came also in agreement with Koch

et al who stated that on his prospective single centered study on 123 critically ill

patients followed for 3 years, RBP4 is reduced in liver cirrhotic patients and is a

marker of bad prognosis.( Koch et al,.2010)

This results is also consistent with Yagmour who found in their study RBP4

levels were significantly lower in 111 patients with liver cirrhosis as compared

to 99 age and sex matched healthy controls. In addition, in animal model study a

high gene expression of RBP4 in hepatic tissue was reduced after induction of

experimental cirrhosis (Yagmour et al., 2007).

Our study is consistant with (Frank,. 2008) who conducted a study on patients

with chronic liver disease and concluded that serum retinol binding protien 4 is

reduced in liver cirrhosis.

It was reported that in 19 HCV infected patients in advanced stages, RBP4 is

decreased compared to 19 healthy controls because of reduced hepatic

production as the liver the major source of RBP4.(Bahr et al., 2009).

Huang found that RBP4 is inversely correlated with disease severity, they

found in their HCV infected patients a significant decreasing linear trend of

RBP4 dependent on histological grading (Huang et al., 2008).

Similar to the current study, (Iwaza et al., 2009) found in their HCV

infected patients that plasma levels of RBP4 were lower in patients than

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104

controls p 0.001. They found higher RBP4 levels were linked to normal

liver function.

Similar to the present study, is Kwon et al., study done on 573 chronic liver

disease patients including hepatitis C patients, RBP4 was assayed and

hyaluronic acid (marker for liver fibrosis); serum RBP4 levels were significantly

reduced and hyaluronic acid increased in patients in comparison to controls.

They concluded that RBP4 is a serologic marker for disease severity in patients

with chronic liver diseases (Kwon et al., 2009).

Contrary to the present study, (Petta et al., 2008) showed that serum RBP4 is

high in patients with liver cirrhosis, but Petta study was on hepatitis C virus and

patients with steatohepatitis, and he explained that HCV is directly incorporated

into the hepatocytes and increase RBP4 level.

A high significant correlation was found between RBP4, fasting plasma glucose

R = 0.004 and serum albumin R= 0.00, while a significant correlation was found

between RBP4, waist circumference R=0.013, HbA

1

c% R= 0.039, and total

cholesterol R= 0.04.

In agreement with our study Iwaza et al conducted a study on 311 patients with

chronic liver disease and reported that RBP4 is reduced and positively correlated

with FPG and total cholesterol. Iwaza also concluded that RBP4 levels return to

normal after interferon therapy. (Iwaza et al,. 2009)

These results are also consistent with (Sussane., 2007) who conducted a study

on obese diabetic females with polycystic ovary sundrome and found that there

is a significant direct correlation between RBP4 and FPG.

Our statistical analysis showed that there was a highly significant direct

correlation between RBP4 and serum albumin, R = (0.00), and also there was a

significant direct correlation between RBP4, HbA

1

C %, R = (0.031), and total

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105

cholesterol R=(0.04), and is in accordance with an epidemiologic cohort study,

conducted on 273 subjects with FPG 110 mg/dl and 234 patients with type 2

diabetes, by Naoyuki and found that RBP4 significantly correlates with

HbA

1

C%, total cholesterol, and serum albumin (Naoyuki., 2006).

On the other hand our study is not goining with (John., 2008) who conducted a

study on 285 subjects with and without diabetes mellitus attending a hospital

lipid disorders clinic he found that there is no correlation between RBP4 and

FPG. These results can be explained by different patients, different medications

(lipid lowering drugs), or samples collection.

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106

S

UMMARY

Diabetes mellitus is a common disease despite the fact that only half or two

thirds are diagnosed, the figure is around 189 million people in 2003, and this

may reach 324 million by 2025.

Egypt is expected to be among ten countries with the highest number of

estimated cases of diabetes for 2030.

The prevalence of type 2 DM continues to rise at alarming rate. Significant

defect in glucose homeostasis and fuel metabolism are detectable long before

overt diabetes occurs. One of the earliest derangements in metabolism is insulin

resistance.

The liver plays important roles in the homeostasis of glucose metabolism since it

acts as a major target organ for insulin and a site for gluconeogenesis and

glycogen storage. Diabetes mellitus, commonly develops in patients with liver

cirrhosis as the result of hepatocyte dysfunction, is known as hepatogenous

diabetes mellitus.

Retinol binding protein4 (RBP4) a 21-KDa protein synthesized in the liver and

adipose tissues, formerly recognized for its role as a specific transport for

vitamin A, now it is known that it plays a crucial role in insulin resistance and

diabetes mellitus.

Recently attention has been paid to the role of retinol ­binding protein 4 (RBP4)

in the pathogenesis of insulin resistance.

The aim of this study is to estimate RBP4 level in type 2 diabetic patients

with and without liver cirrhosis. Fasting plasma glucose was high in

group 1 (DM) (142.43±23.38) mg/dl and group 3 (DM and liver cirrhosis)

(123.77±24.45) mg/dl and normal in group 2 (liver cirrhosis)

(97.87±8.68) compared with group 4 (controls) (95.93±7.52).

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107

As regards HbA

1

C% it was high in group 1 (DM) (10.35±2.83) and group

3 (DM and liver cirrhosis) (7.40±0.62) and normal in group 2 (liver

cirrhosis) (5.92±0.68) compared with group 4 (controls) (5.80±0.46).

Concerning BMI, it was high in group 3 (DM and liver cirrhosis)

(29.1±4.10) kg/m

2

followed by group 1 (DM) (28.5±6.17) kg/m

2

and

group 2 (liver cirrhosis) (25.60±4.15) kg/m

2

compared with group 4

(controls) (22.50±2.35) kg/m

2

.

Waist cicumference found to be high in group 1 (DM) (103.07±17.76) cm

followed by group 3 (DM and liver cirrhosis) (88.33±6.00) cm and group

2 (liver cirrhosis) (83.43±7.40) cm compared with group 4 (controls)

(79.73±8.98) cm.

The study was conducted on 120 of adult persons age range from 30-60

years, they where divided into 4 groups:

Group 1: 30 diabetic patients.

Group 2: 30 cirrhotic patients.

Group 3: 30 diabetic and cirrhotic.

Group 4: 30 control subjects.

The data were statistically analysed and it is found that:

RBP4 was highest among group 1 (DM) (20.10±8.31) mg/dl compared to

group 4 (controls) (13.50±3.97) mg/dl and was low in group 2 (liver

cirhosis) (9.31±3.99) mg/dl and group 3 (DM and liver cirrhosis)

(8.59±7.17) mg/dl.

Toatl cholsterol was found (209.97±28.10) mg/dl in group 1 (DM),

(237.53±23.87) mg/dl in group 2 (liver cirrhosis), (228.67±31.87) mg/dl

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108

in group 3 (DM and liver cirrhosis) and (198.97±17.58) mg/dl in control

group.

Low density lipoprotien LDL, was found to be (151.13±22.32) mg/dl in

group1(DM), (169.89±25.52) mg/dl in group 2 (liver cirrhosis), and

(159.57±28.05) mg/dl in group 3 (DM and liver cirrhosis), compared with

group 4 (controls) (138.83±28.35) mg/dl.

High density lipoprotein HDL was found to be low in group 1 (DM)

(34.83±4.13) mg/dl, group 2 (liver cirrhosis) (37.97±6.17) mg/dl, and

group 3 (DM and liver cirrhosis) (36.83±5.37) mg/dl compared with

group 4 (controls) (38.17±5.03) mg/dl.

Triglyceride was found to be (115.53±21.27) mg/dl in group 1 (DM),

(122.3±15.25) mg/dl in group 2 (liver cirrhosis), and (119.83±21.29)

mg/dl in group 3 (DM and liver cirrhosis) compared with group 4

(controls) (98.33±11.29) mg/dl.

ALT and AST, were found be (18.10±5.37) u/l and (18.73±5.55) u/l

respectively in group 1 (DM), (33.00 ±18.35) u/l and (33.00±18.35) u/l

respectively in group 2 (liver cirrhosis) u/l, and (28.33±8.63) u/l and

(38.33±37.58) u/l respectively in group 3 (DM and liver cirrhosis)

compared with group 4 (controls) (21.90±3.22) u/l and (19.33±4.90) u/l

respectively.

While serum albumin was low in group 2 (liver cirrhosis) (2.39±0.64) g/dl

and group 3 (DM and liver cirrhosis) (2.33±0.49) g/dl and normal in

group 1 (DM) (4.17±0.52) g/dl compared with group 4 (controls)

(4.19±0.46) g/dl.

The prothrombin time was found to be high in group 2 (liver cirrhosis)

(18.33±3.99) seconds, and group 3 (DM and liver cirrhosis) (17.13±3.66)

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109

seconds, and normal in group 1(DM), (10.30 ±2.35) seconds, compared to

group 4 (controls) (10.00±1.70) seconds.

In this study on comparing the four groups, a high significant difference

was found as regards BMI, waist circumference, FPG, HBA

1

c%, total

cholesterol, LDL, TG, ALT, AST, serum albumin and no significant difference

was found regarding HDL.

A high significant correlation was found between RBP4, fasting plasma

glucose R = 0.004 and serum albumin R= 0.00, while a significant correlation

was found between RBP4, waist circumference R=0.013, HbA

1

c% R= 0.039,

and total cholesterol R= 0.04.

Conclusions:

From the previous results our study suggests that:

1. Serum RBP4 concentrations are significantly increased in type 2 diabetic

patients.

2. The liver is the major source of circulating RBP4 in humans, and

therefore the hepatic biosynthetic capacity may greatly influence serum

RBP4.

R

ECOMMENDATIONS

1. RBP4 can be used as a new marker of insulin resistance.

2. Serum concentrations of RBP4 may be used for follow up of patients with

type 2 diabetes mellitus aiming to reduce its level.

3. Future studies are to be done to assess the relation between RBP4 as a

marker of insulin resistance and it's association with different liver

diseases such as NASH and HCV positive patients.

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110

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The chapters are:\ ListofAbbreviations\ Introduction\ DiabetesMellitus\ Classificationofdiabetesmellitus:\ Diagnosisofdiabetesmellitus:\ Criteriafordiabetesmellitusdiagnosis:\ Prediabetes\ Type2diabetesmellitus:\ Riskfactorsoftype2diabetesmellitus:\ Pathogenesisoftype2diabetes:\ Type2DiabetesMellitusandInsulinResistance:\ KineticofInsulinSecretion\ Mechanismsofglucoseinducedinsulinsecretion:\ Alterationsofinsulinsecretion:\ InsulinActionattheCellularLevel:\ RoleofinsulinsignalingsystemsinInsulinResistance:\ Themetabolicsyndrome\ Type2diabetesmellitusandtheliver \ TheRoleoftheLiverinGlucoseHomeostasis:\ Spectrumofliverdiseaseindiabetics:\ Nonalcoholicfattyliverdiseases(NAFLD)\ PathogenesisofNAFLD\ Diabetesmellitusandhepatocellulacarcinoma:\ Livercirrhosis\ Definition:\ Classificationoflivercirrhosis:\ Retinolbindingprotein4\ Adiposetissueinhealthanddisease \ RetinoidReceptors:\ RetinolBindingProtein4andInsulinResistance:\ Defectsintheabilityoffatcellstotransportglucosearelinkedtoinsulinresistanceinmuscle\ andlivers:\ SubjectsandMethods \ Specimencollectionandpreparation:\ Statisticalanalysis\ Results\ Discussion\ Summary\ Recommendations\ References

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The author expresses gratitude to Allah, Dr. Nehad Shokry Shoeib, Dr. Salwa Seddik Hosny, and Dr. Maram Mohamed Maher Mahdy for their support, guidance, and contributions to the work.

What Abbreviations are in this language preview?

The list of abbreviations are:\ ADAG\ ALB\ ALT\ AST\ ATP\ BMI\ CGM\ CHOL\ CVD\ DCCT\ DM\ DPP\ DSME\ FPG\ GDM\ HAPO\ HbA1c\ HCV\ HDL\ HNF\ IDDM\ IFC\ IFG\ IGT\ IRSI\ LADA\ LDL\ MODY\ NAFLD\ NASH\ NGSP\ NHANES\ OGTT\ PPG\ PT\ RBP4\ SMBG\ TG\ UKPDS

• Calificación: C
• Autor: Hyder Mirghani (Autor)
• Año de publicación: 2017
• Páginas: 128
• No. de catálogo: V385499
• ISBN (Ebook): 9783668617902
• ISBN (Libro): 9783668617919
• Idioma: Inglés
• Etiqueta: retinol, binding, protein-4, type, diabetic, patients, liver, cirrhosis