You are on page 1of 58

Association of C-Reactive Protein (CRP) levels

with Cardiovascular Diseases, Smoking

Status and Alcohol Drinking Status among

Type 2 Diabetes Mellitus Diagnosed

Individuals

A Research Proposal

Bernido, Julevi P.

Batasinin, Candy Kaye G.

Dagoc, Praise Selah G.

Evangelista, Athena Heidi DC

Go, Irene Carla F.

December 8, 2009
2
Chapter I. THE PROBLEM: RATIONALE AND BACKGROUND

Worldwide, an estimated 177 million individuals suffer from diabetes mellitus in the year

2000. Based on the current trends, this number is believed to rise up to >360 million by the year

2030. The Center for Disease Control and Prevention (CDC) reported that 7% of the population

in the United States, accounting to 20.8 million persons, had diabetes in 2005 (~30% of this

number was undiagnosed). Approximately 1.5 million individuals (>20 years) were newly

diagnosed with diabetes in the same year (Powers, 2008).

In the Philippines, the number of individuals with DM is reported to be 13,922 (3.5% of

the Filipino population). With these figures, Diabetes Mellitus ranks 9th among the 10 leading

causes of mortality in the Philippines (Department of Health, 2002).

Diabetes mellitus as defined by Freeman (2005) is a syndrome of impaired carbohydrate,

fat, and protein metabolism caused by either lack of insulin secretion or decreased sensitivity of

the tissues to insulin. There are two general types of diabetes mellitus. Type 1 diabetes mellitus

(type 1 DM), also called insulin-dependent diabetes mellitus (IDDM), is caused by lack of

insulin secretion. Type 2 diabetes mellitus (type 2 DM), also called non-insulin-dependent

diabetes mellitus (NIDDM), is caused by decreased sensitivity of target tissues to the metabolic

effect of insulin. This reduced sensitivity to insulin is often referred to as insulin resistance

(Guyton, 2000).

The participants used in this study are diagnosed with Type 2 diabetes mellitus. It has

been reported that increased activities of liver enzymes are indicators of hepatocellular injury.

According to Wannamethee, et al., (2005) and Hanley, et al., (2005), increased activity of these

markers is associated with Type 2 diabetes mellitus.

1
On the other hand, disorders that affect the heart or blood vessels are called

cardiovascular disorders. These disorders are usually divided into heart disease and peripheral

blood vessel disorders. Heart disease affects the heart and the blood vessels that supply the heart

muscle. Peripheral blood vessel disorders affect the blood vessels of the arms, legs, and trunk

except those supplying the heart (Beers, et.al, 2003).

The most common cause of death worldwide is cardiovascular diseases (CVD). Today it

accounts for ~30% of deaths worldwide, including nearly 40% in high-income countries and

about 28% in low-income and middle-income countries (Gaziano and Gaziano, 2008).

According to World Health Organization (WHO) estimates, 16.7 million people around

the globe die of CVD each year. This is over twenty-nine percent (29%) of all deaths globally

(WHO, 2003). The current study focuses on a specific CVD in which is the Coronary Heart

Disease (CHD). Ingram (2005) defines CHD where there is a lack of nutrients and oxygen

reaching the heart muscle and resulting in myocardial ischemia.

The American Heart Association (AHA) and the Center for Disease Control (CDC) have

recommended that CRP be used as an adjunct for risk stratification in the prevention of CVD in

persons at intermediate risk for a CHD event. In these recommendations, cut-offs of 1.0 and 3.0

mg/L were recommended for categorization into low, intermediate, and high-risk groups

(Pearson, et al., 2003).

A number of prospective cohort studies also showed that higher levels of CRP are

associated with increased risk for CVD. Danesh and colleagues (2004) recently reported a meta-

analysis of 22 prospective studies published between 1996 and 2003 that examined CRP as a

predictor for CVD events. Participants in these studies had a mean age at entry of 57 years and

were followed for a mean of 12 years; 7068 participants developed coronary heart disease

(CHD). All the studies used high-sensitivity CRP assays, and almost all adjusted for smoking

2
and at least some other CHD risk factors. Studies published by Ridker, et al., (2005), Shlipak, et

al. (2005), and Pai et al. (2004), since this meta-analysis continues to show similar adjusted

relative risks for CRP. Thus, CRP is consistently, although weakly, associated with CHD after

adjustment for established risk factors.

As agreed by Pearson, et al., (2003) and Ridker, et al., (2002), CRP has been

recommended as the marker of choice to monitor cardiovascular risk, being a stronger predictor

of atherosclerosis. Libby (2002) and Ross (1999) also stated that inflammation is a prominent

feature of atherosclerosis, and it is postulated that as an acute-phase protein, elevation of plasma

CRP may signal the underlying atherosclerotic process.

Elevated CRP levels have been linked to an increased risk of later development of

diabetes according to Pradhan, et al., (2001) and Barzilay, et al, (2001). Furthermore, CRP levels

are higher in people with diabetes compared with those without diabetes (Ford, et al., 1999,

Grau, et al., 1996 and Wu, et al., 2002). Wu, et al., (2002) found that CRP is associated with

HbA1c levels; however, people with diabetes were excluded from the study. HbA1c was 5.4% in

people with low CRP and 5.5% in people with medium or high CRP (P<0.05). Another study

found an association between CRP and uncontrolled diabetes in 62 patients, but the study was

limited by a small sample size (Rodriguez-Moran, et al., 1999).

Previous studies, including a literature-based meta-analysis of ten studies involving 2,675

cases of diabetes, have shown a significant association between the risk of diabetes and higher

plasma levels of CRP. However, it remains uncertain whether CRP is causally involved in the

pathogenesis of diabetes. An alternative possibility to explain the association is that CRP is a

confounder, since CRP is strongly associated with known causal risk factors for diabetes, such as

obesity (Dehghan, et al., 2007).

2
A study conducted by Sattar, et al., (2004), showed that incorporating ALT and CRP

together with metabolic syndrome criteria, elevated ALT and CRP predicted incident diabetes.

Also mentioned by Pickup et al. (1997) that levels of CRP is increased in non-insulin-dependent

diabetes mellitus (NIDDM) compared with levels in control subjects.

Therefore, our study focuses mainly on how levels of CRP are affected by Type 2

diabetes mellitus with some associations with CHD, smoking status and alcohol drinking status.

This link will prove CRP to be a potential test in monitoring type 2 DM.

1.2 Statement of the Problem

The main problem of this study is to determine if CRP can be associated with CHD,

alcohol drinking status and smoking status among type 2 diabetes mellitus patients. Specifically,

this study aims to answer the following research questions:

1. Is there a significant relationship between the levels of CRP of the Type 2

diabetes mellitus patients and the presence of CHD?

2. Is there a significant relationship between the levels of CRP of Type 2

diabetes mellitus patients and their smoking status?

3. Is there a significant relationship between the levels of CRP of CRP of

Type 2 diabetes mellitus patients and their alcohol drinking status?

1.3 Hypotheses

Based on the foregoing research questions identified, the researchers formulated the

following null hypothesis:

3
1. There is no significant relationship between the levels of CRP of the Type

2 diabetes mellitus patients and the presence of CHD.

2. There is no significant relationship between the levels of CRP of Type 2

diabetes mellitus patients and their smoking status.

3. There is no significant relationship between the levels of CRP of Type 2

diabetes mellitus patients and their alcohol drinking status.

Based on the foregoing research questions identified, the researchers formulated the

following alternative hypothesis:

1. There is a significant relationship between the levels of CRP of the Type 2

diabetes mellitus patients and the presence of CHD.

2. There is a significant relationship between the levels of CRP of Type 2

diabetes mellitus patients and their smoking status.

3. There is a significant relationship between the levels of CRP of Type 2

diabetes mellitus patients and their alcohol drinking status.

1.4 Conceptual Framework

Association of C-reactive Protein (CRP) levels with Cardiovascular Diseases,

Smoking Status and Alcohol Drinking Status among Type 2 Diabetes Mellitus Diagnosed

Individuals

1
Smoking and
Alcohol intake
of the type 2 DM
patients

Type 2 Diabetes mellitus


Positive CRP test with Coronary
(atherosclerotic,
ischemic) heart disease

C-reactive protein (CRP), a marker of systemic inflammation, is emerging as an

independent risk factor for cardiovascular disease (Abdelmouttaleb et al., 1999; Ridker & Rifai,

2001). High CRP levels have been linked to an increased risk of thrombotic events including

myocardial infarction (Pradhan et al., 2001 & Barzilay, 2001). Furthermore, CRP levels are

higher in people with diabetes (Ford, 1999 & Goldberg, et al., 2000).

Recent research evidence supports a link between hyperglycemia and inflammation. CRP

is known to be higher in people with impaired glucose tolerance and frank diabetes (Ford, 1999

& Wu, 2002). Data was also reported by Fox et al., (2004) demonstrating that individuals with

cardiovascular risk factors are at increased risk of type 2 diabetes, which is only partially

mediated by insulin resistance or central adiposity. Thus, since cardiovascular risk factors predict

the appearance of type 2 diabetes, it is plausible that vascular damage precedes the clinical

manifestation of type 2 diabetes in which increased levels of CRP will highly be observed.

1
Studies conducted by Pradhan et al., (2001) and Freeman (2002) further agreed that

inflammation has been associated with the development of diabetes.

The possible intervening factors that may affect the levels of CRP in the blood are

alcohol intake and smoking behaviors of an individual. It is well known that there is a negative

correlation between alcohol intake and incidence of ischemic heart disease (Wannamethee, and

Shaper., 1998). The major mechanism underlying this proposed protective effect of alcohol on

ischemic heart disease is the action of ethanol on lipid metabolism: blood HDL cholesterol and

LDL cholesterol levels are increased and decreased, respectively, by alcohol drinking (Castelli et

al., 1992; Langer et al., 1992). In addition, alcohol drinking is also known to decrease blood

fibrinogen level (Krobot et al., 1992), resulting in retardation of atherosclerotic progression, and

to suppress thrombus formation. On the other hand, alcohol drinking induces hypertension

(Klatzky, 1996), and the incidence of cerebrovascular diseases, particularly cerebral hemorrhage

and subarachinoid hemorrhage, is known to be higher in moderate-to-heavy drinkers than in non-

drinkers (Hillbom, 1998). Thus, alcohol drinking has both preventive and accelerating effects on

atherosclerotic vascular diseases. Another study conducted by Howard, Arnsten and Gourevitch

(2004) that shows moderate alcohol consumption appears to be associated with a reduced risk for

diabetes, whereas some suggest that alcohol consumption may be associated with an increased

risk. Furthermore, among persons with diabetes, mild to moderate alcohol consumption is

associated with a decreased risk for cardiovascular events. Regarding levels of CRP and

alcoholism, this current study would then test the assumption that low levels of CRP might be

observed to those individuals who are in mild to moderate alcohol intake.

A study conducted by Djousse (2002) showed smoking and carotid atherosclerosis

consistent with published data and indicated detrimental effect of smoking on carotid

atherosclerosis as were also mentioned by Howard (1998) and de Waart (2000). Hutchinson et

2
al., (1996) mentioned in their study that passive smoking worsen endothelial function in lipid-fed

rabbits, leading to inappropriate vasoconstriction. Recently, Celermajer et al., (1996)

demonstrated this same adverse effect in healthy young adults: Passive smoking impaired

endothelium-dependent arterial dilation in the brachial artery. This effect may be a marker of

early vascular damage. Passive smoking accelerates atherosclerosis in rabbits and cockerels (Zhu

et al., 1993). Moreover, specific constituents of the secondhand smoke that accelerate

atherosclerosis are being identified; 1,3 butadiene, a vapor phase constituent of secondhand

smoke, accelerated atherosclerotic plaque development in cockerels at realistic exposures (Penn

& Snyder, 1996). Tracy et al., (1996) said that despite the powerful effect modification of

smoking status, there was no significant association of CRP values with smoking status itself,

although CRP was strongly related to lifetime smoking exposure. Moreover, lifetime exposure to

smoking, even in those who have stopped smoking, is strongly associated with CRP and affects

the association of CRP with other variables.

To provide further insight into the role of inflammation in cardiovascular disease and

type 2 diabetes mellitus, the researchers sought to elucidate the link between levels of CRP and

variables like smoking, alcohol intake in patients who have type 2 DM and CHD.

1.5 Significance of the Study

In an attempt to improve global cardiovascular disease and diabetes mellitus diagnostic

procedures, considerable interest has focused on CRP, a marker of inflammation that has been

shown in multiple prospective epidemiological studies to predict incident myocardial infarction,

peripheral arterial disease, sudden cardiac death and even hyperglycemia.

3
Cardiovascular disease (CVD) and type 2 diabetes mellitus pose great burdens to the

Filipino population as proven by their ranks, 1st and 9th respectively, in the ten leading causes of

mortality in the Philippines (WHO, 2008).

To address this issue, this current study will provide significant evidence against the

major causal effects of CRP on quantitative phenotypes related to diabetes and cardiovascular

risks. The study will soon provide the strongest argument yet against any material association of

plasma CRP levels with type 2 DM, heart problems and with their lifestyle (alcohol status and

smoking status). The purpose of this study is to investigate the relation between CRP and CHD

patients with type 2 DM, the effects of smoking and alcoholism in the levels of plasma CRP of

the type 2 DM patients. Therefore, the addition of CRP to standard cardiac problem and

metabolic disorders’ evaluation may thus provide a simple and inexpensive method to improve

monitoring and compliance with preventive approaches of diabetes mellitus complications.

Internationally, many studies projected positive results on the use of CRP as a serum

marker for CVD and Type 2 Diabetes mellitus. According to a study headed by Danesh,

prediction of coronary heart disease through the use of CRP needs to be further reviewed

(Danesh, 2004).

Furthermore, it is reported by Abdelmouttaleb et al., (1999), Ridker and Rifai., (2001)

and Ridker (1998) that C-reactive protein (CRP), a marker of systemic inflammation, is

emerging as an independent risk factor for cardiovascular disease (CVD). Moreover, Pradhan et

al., (2001) and Barzilay (2001) cited in their studies that elevated CRP levels have been linked to

an increased risk of later development of diabetes. Moreover, CRP levels are higher in people

with diabetes compared with those without diabetes (Ford et al., 1999; Grau et al., 1996);

Goldberg et al., 2000). All of these previous researches/claims mentioned by different authors

will be tested in this current study to provide more evidence of the efficiency of CRP to be a

2
serum marker for both type 2 DM and coronary heart disease (CHD) and the effects of smoking

and alcohol intake to the levels of CRP.

1.6 Scope and Limitations

Inclusion Criteria

The study will include all patients who are type 2 diabetes mellitus with FPG of > 7.0

mmol/L. The cardiovascular complication to be considered will be coronary heart disease (CHD)

or atherosclerosis or ischemia heart disease. Fasting plasma glucose level will be used to

determine the association of CRP, diabetes mellitus and CHD. The slide test for CHD will be

used which is a qualitative serologic test. Patients who smoke 10-19 sticks per day as well those

who drink over 84 ounces per week will be included as subjects.

Exclusion Criteria

It was defined by Lindsey (2005) that CRP is one of the first acute phase proteins to rise in

response to inflammatory disease. It is significantly elevated in acute rheumatic fever, bacterial

infections, rheumatoid arthritis, carcinomatosis, gout and viral infections. Considering this

conditions that would elevate CRP levels, patients that have these disorders will not be included

as study participants. In addition, those with type 1 diabetes mellitus will also be excluded. More

conditions like mentioned below will affect the levels of CRP thus, they will also be excluded.

The following information are from the Tulip Diagnostics (P) LTD.

1. Markedly lipemic, hemolysed and contaminated serum samples could produce

non-specific results.

2. Use of plasma rather than serum can lead to false positive results.

2
3. CRP is found to be present after the first trimester of pregnancy and persists until

delivery.

4. CRP levels increase in women who are on oral contraceptives.

5. CRP response is not affected by the commonly used anti-inflammatory or

immunosuppressive drugs, including steroids, unless the disease activity is affected and it

covers an exceptionally broad incremental range up to 3000 times.

6. Since CRP production is a non-specific response to tissue injury, it is

recommended that results of the test should be correlated with clinical findings to arrive at

the final diagnosis.

7. In cases where an increase in CRP levels is suspected, but the screening test

shows a negative result, semiquantitation should be done to rule out prozone effect.

Lindsey (2005) stated that CRP is significantly elevated in acute rheumatic fever,

bacterial infections, myocardial infarcts, rheumatoid arthritis, carcinomatosis, gout and viral

infections.

1.6 Definition of Terms

Coronary Heart Disease (CHD). Ingram (2005) defines CHD which is caused by a lack

of nutrients and oxygen reaching the heart muscle and resulting in myocardial ischemia.

Current Smokers. The median established for current smokers is 10-19 sticks per day

(Bazzano et al., 2003).

1
Heavy Drinkers. As used in the study, refer to individuals who drink over 84 ounces per

week (Maraldi et al., 2006).

CRP. Lindsey (2005) defines C-reactive protein (CRP) as one of the acute phase proteins

that is synthesized in the liver and appears in the blood of patients with diverse inflammatory

disease. Bernstein (2005) also mentioned that CRP increases dramatically under conditions of

sepsis, inflammation and infection.

Type 2 Diabetic Patients. Patients who are type 2 diabetes mellitus with FPG of > 7.0

mmol/L.

As used in the study, the positive CRP test would mean that there is agglutination when

serum is mixed with the RHELAX reagent.

Chapter II. REVIEW OF RELATED LITERATURE

2.1 Diabetes mellitus

Diabetes mellitus is a syndrome of impaired carbohydrate, fat, and protein metabolism

caused by either lack of insulin secretion or decreased sensitivity of the tissues to insulin

(Guyton and Hall, 2000).

2
According to the World Health Organization (2008), more than 180 million people

worldwide are diabetic, 90% of whom have type 2 DM. This number is expected to double by

2030. In the Philippines, an estimated 253,000 people have been diagnosed with diabetes. In

2004, it accounted for 16,552 deaths.

2.1.1 Classification

There are two broad types of diabetes established by the National Diabetes Data Group in

1979: Type 1, insulin-dependent diabetes mellitus (IDDM) and Type 2, non-insulin dependent

diabetes mellitus (NIDDM). However, the International Expert Committee on the Diagnosis and

Classification of Diabetes Mellitus proposed changes including the elimination of the older terms

of IDDM and NIDDM, retaining the categories of Type 1 and 2, with the adoption of Arabic

numerals instead of Roman numerals (Freeman, 2005).

Type 2 diabetes mellitus is characterized by hyperglycemia due to the individual’s

resistance to insulin, with or without an accompanying insulin secretory defect. This secretory

defect is only relative and not absolute. Most diabetic patients are constituted by those who have

type 2 diabetes mellitus. This type of diabetes usually goes undiagnosed for several years and is

strongly associated with genetics. Type 2 diabetes mellitus involves adult onset of the disease,

milder symptom than type 1 with a seldom occurrence of ketoacidosis. Patients with type 2 DM

are likely to go into a hyperosmolar nonketotic state and are at a higher risk of developing

macrovascular and microvascular complications (Freeman, 2005).

Funk, J.L. (2006) found out that type 2 DM differs from type 1 in several distinct ways. It

is 10 times more common; has a stronger genetic component; occurs most commonly in adults;

increases in prevalence with age (e.g. 20%) prevalence in individuals older than 65; and is

associated with increased resistance to the effects of insulin at its sites of action as well as a

2
decrease in insulin secretion in the pancreas. Moreover, the age of onset of type 2 DM is usually

>40 years of age. An epidemic of type 2 DM is occurring worldwide, particularly in non-

European populations. In addition, the great majority (80%) of type 2 DM are obese. Obesity,

particularly central obesity, is associated with increased insulin resistance. Furthermore, it was

Hak, et al., (1998) that claims obesity as the insulin resistance syndrome.

2.1.2 Risk Factors

2.1.2.1 Classification of Diabetes Mellitus in an Individual Patient

There are many causes of diabetes. Individuals with type 1 DM can have the following

characteristics: 1) effect of disease of people age 30 years old; 2) lean body habitus; 3)

requirement of insulin for therapy; 4) probability to develop ketoacidosis; 5) increased risk of

other autoimmune disorders such as autoimmune thyroid disease, adrenal insufficiency,

pernicious anemia, celiac disease and vitiligo. While individuals experiencing type 2 DM have

the following characteristics: 1) developing diabetes after the age of 30; 2) are usually obese

(80% are obese but elderly can be lean); 3) may or may not require insulin therapy; 4) may have

linked conditions such as insulin resistance by perfusion, cardiovascular disease, dyslipidemia of

PCOS. In type 2 DM, insulin resistance is connected with abnormal obesity and hyperglycemia.

There are many cases wherein individuals having type 2 DM are older. However, the age of

diagnosis is declining because of the sudden increase among overweight children and

adolescents (Powers, 2008).

2.1.3 Laboratory Diagnosis

The common laboratory tests done by the endocrinologist from SUMC is the fasting

plasma glucose.

2
Figure 2. 1. Spectrum of glucose homeostasis and diabetes mellitus (DM). The spectrum from normal

glucose tolerance to diabetes in type 1 DM, type 2 DM, other specific types of diabetes, and gestational

DM is shown from left to right. In most types of DM, the individual traverses from normal glucose

tolerance to impaired glucose tolerance to overt diabetes. Arrows indicate that changes in glucose

tolerance may be bi-directional in some types of diabetes. For example, individuals with type 2 DM may

return to the impaired glucose tolerance category with weight loss; in gestational DM diabetes may revert

to impaired glucose tolerance or even normal glucose tolerance after delivery. The fasting plasma glucose

(FPG) and 2-h plasma glucose (PG), after a glucose challenge for the different categories of glucose

tolerance, are shown at the lower part of the figure. These values do not apply to the diagnosis of

gestational DM. Some types of DM may or may not require insulin for survival, hence the dotted line.

(Conventional units are used in the figure.) (Adapted from the American Diabetes Association, 2007.)

1
Table 2.1. Criteria for the Diagnosis of Diabetes Mellitus

Symptoms of diabetes plus random blood glucose concentration 11.1 mmol/L (200

mg/dL)aor

Fasting plasma glucose 7.0 mmol/L (126 mg/dL)bor

Two-hour plasma glucose 11.1 mmol/L (200 mg/dL) during an oral glucose

tolerance testc

a
Random is defined as without regard to time since the last meal.
b
Fasting is defined as no caloric intake for at least 8 h.
c
The test should be performed using a glucose load containing the equivalent of 75 g anhydrous glucose

dissolved in water; not recommended for routine clinical use.

Note: In the absence of unequivocal hyperglycemia and acute metabolic decompensation, these criteria

should be confirmed by repeat testing on a different day.

Source: Adapted from American Diabetes Association, 2007.

Additional Criteria for the Diagnosis of Diabetes Mellitus (Freeman, 2005).

• Obesity (120% of desirable body weight or body mass index [BMI] of 27 kg/M2.

• Family history of diabetes in a first degree relative.

• Membership in a high-risk of minority population (eg, African American, Hispanic

American, Native American, or Asian American).

• History of GDM or delivering a baby >9 lb (>4.1 kg).

2
• Hypertension (>140/90)

• Low high-density lipoprotein (HDL) cholesterol concentrations (eg, <35mg/dL)

• Elevated triglyceride concentrations (eg, >250 kg/dL)

• A history of impaired fasting glucose /impaired glucose tolerance.

Signs and Symptoms of Diabetes mellitus

Glucose is the key regulator of insulin secretion by the pancreatic beta cell. (Berkow,

1997). The deficiency in insulin secretion would decrease the utilization of tissue glucose and

would result to starvation (Crawford, 1997).

When ketoacid concentration rises, the kidneys excrete them into the urine (ketonuria)

and thus leading to polyuria. (Guyton & Hall, 1996).

Glucose also spills into the blood once tissue utilization decreases, resulting to high blood

sugar levels or hyperglycemia (Crawford, 1997). When blood sugar level rises above 160 to 180

mg/dl, it exceeds the capability of the renal threshold to reabsorb the glucose. With this

condition, the kidneys begin to spill over glucose into the urine (glycosuria or glucosuria)

(Pagana, & Pagana,, 2002). The glycosuria induces an osmotic diuresis and thus polyuria,

resulting to loss of water and electrolytes (Crawford, 1997). Due to excessive urination, the body

must compensate the loss of fluids and thus triggering the thirst glands leading to polydipsia.

Despite eating vast amounts of food, the person still remains in the state of starvation and loses

weight due to insulin deficiency and so there is no efficient tissue glucose utilization. This

condition has a tendency towards polyphagia (excessive hunger). Asthenia or lack of energy also

1
occurs because of loss of body protein and also by diminished utilization of carbohydrates for

energy (Guyton & Hall, 2000).

2.1.4 Association of C-reactive protein with Diabetes mellitus

According to Lindsey (2005), C-reactive protein (CRP) is synthesized in the liver and

appears in the blood of patients with diverse inflammatory diseases. Considering this claim,

Capuzzi and Freeman (2007) also stated that CRP is considered to be a major inflammatory

cytokine that functions as a non-specific defense mechanism in response to tissue injury or

infection. Synthesized primarily in the liver, CRP activity is stimulated by other cytokines,

especially interleukin, (IL)-6, IL-1ß, and tumor necrosis factor-α (TNF-α). CRP binds to variety

of molecules, particularly liposomes and lipoproteins, such as LDL and VLDL cholesterol.

According to Remaley, McNamara and Warnick (2005), high level of cholesterol and/or

triglycerides (carried most by LDL and VLDL) in most people, however, as a result of increased

consumption of foods rich in fat and cholesterol, smoking and lack of exercise, or to other

disorders or disease states that affect lipid metabolism such as diabetes, hypertension,

hypothyroidism, obesity, other hormonal imbalances, liver and kidney diseases. This proves that

CRP might have its significant role in type 2 diabetes diagnoses accompanied with obesity as it

binds to LDL and VLDL. However, Ford (1999) claimed that even though the results of his

study about CRP and diabetes confirm cross-sectional findings from previous studies that show

elevated CRP concentrations among individuals who are obese or have diabetes, the implications

of those findings remain unclear. Moreover, Capuzzi & Freeman (2007) also assert that the

conditions under which CRP indicates or mediates inflammatory processes remain undefined.

Much evidence indicates that inflammation may drive the development of insulin resistance, type

2
2 diabetes. The interaction among various cytokines and cell types in these pathogenic processes

remains unclear.

But still, a growing body of data reinforces the concept that inflammation also plays an

important role in the pathogenesis of type 2 diabetes mellitus and links diabetes with

concomitant conditions with inflammatory components (Festa, A., & Haffner, S.M., 2005).

Much evidence exists that inflammatory mechanisms play a major role in the cascade of events

that results in rupture of atherosclerotic plaque. Upregulation of receptors for advanced glycation

end products has been associated with enhanced inflammatory reactions. Increased expression of

these receptors has been found to be associated with impaired glycemic control and may be a

contributory factor on the complex array of mechanisms that leads to accelerated atherosclerosis

in patients with diabetes (Cippollone, et al., 2003).

Barzilay, et al., (2001) found that the Cardiovascular Health Study whose participants

have circulating levels of inflammatory markers were determined both at baseline and after 3-4

years of follow-up those who developed diabetes and higher measured levels of CRP than those

who remained euglycemic. In addition, those with elevated levels of CRP were found more

likely to develop diabetes over the course of the study. In a national survey study, respondents

with haemoglobin A1c (A1C) levels >9% had a significantly higher rate elevated levels of CRP

than those with A1C levels <7%. This suggests an association between diminished glycemic

control and systemic inflammation in people with established diabetes.

Another study in the USA carried out as part of the Women’s Health Study among

initially nondiabetic participants who developed diabetes over the course of the study, median

baseline levels of IL-6 and CRP were significantly higher among the case than among control

subjects (P<0.001), and increasing levels both markers were associated with a higher risk of

developing diabetes. In this study, increased CRP levels predicted the onset of diabetes even

2
after adjustment for obesity, coronary risk factors and fasting insulin levels (Pradhan, et al.,

2001). Similar results were gathered by Dehghan, et al., (2007) in their population-based cohort

study and the meta-analysis showed that serum CRP is associated with risk of diabetes

independent of obesity.

The prospective study conducted by Pradhan, et al., (2001) further explained that their

prospective data support a possible role for inflammation in diabetogenesis and are in accord

with previous hypotheses originated by Pickup, et al. (1997) that type 2 DM may be a

manifestation of an ongoing cytokine-mediated acute phase response initiated by the body’s

innate immune system. With particular relevance to their results, CRP is thought to exhibit

several characteristics that imply a fundamental role in natural host defense. Specifically, CRP is

a member of the pentraxin family of oligometric proteins involve in innate immunity.

Another prospective nested, case-control study of inflammatory markers as predictors of

type 2 diabetes was done among 32, 826 women in 11 US states. This study conducted by Hu, et

al., (2004) showed that the data gathered support the role of inflammation in the pathogenesis of

type 2 DM. Elevated CRP levels are a strong independent predictor of type 2 diabetes. As also

confirmed by Yudkin, et al., (1999) and Perticone, et al., (2008) they found out that there is a

significant relationship between CRP and both endothelial dysfunction and type 2 DM,

supporting the idea that endothelial function contributes to the development of type 2 diabetes

mellitus.

Several studies suggest that inflammation plays a role in the pathogenesis of some

glucose disorders in adults. Concerning this, a study was done by Barzilay, et al., (2001) which

showed that elevated baseline CRP levels were associated with the development of diabetes over

a 3 to 4 year period. This association was present after adjustment for known baseline predictors

of FG (normal fasting glucose) status change and was confounded by other inflammatory

3
conditions. Such an association is consistent with the possibility that the development of diabetes

may in part be inflammatory.

According to King, et al., (2003) and Soinio, et al., (2006), CRP was significantly higher

in patients with type 2 diabetes. It was suggested that hyperglycaemia contributes to the

simultaneous inflammation, endothelial dysfunction, and insulin resistance. The simultaneous

inflammation was brought by the oxidative stress on the endothelium. A number of studies

conducted by Schmidt et al., (1999), Festa, et al., (2000), Pradhan, et al., (2001), Barzilay, et al.,

(2001) and Wu, et al., (2002) further confirm that inflammatory markers such as CRP have been

related to the development of insulin resistance and type 2 DM.

2.2 Cardiovascular diseases

According to Gaziano & Gaziano (2008), cardiovascular diseases (CVD) include diseases

of the heart as well as vascular lesions of the central nervous system. CVD comprises diseases

such as congenital heart disease, rheumatic heart disease, bacterial infective endocarditis,

syphilitic heart disease, coronary (atherosclerotic, ischemic) heart disease, myocardial infarction,

hypertensive heart disease, congestive heart failure, and pericardial disease. Since CVD

comprises a lot of diseases, the study only focuses on coronary (atherosclerotic, ischemic) heart

disease.

The most common cause of death worldwide is due to cardiovascular diseases (CVD).

Today it accounts for ~30% of deaths worldwide, including nearly 40% in high-income countries

and about 28% in low-income and middle-income countries (Gaziano & Gaziano, 2008).

Random glucose concentration is described as without regard to time since the last meal.

Fasting is defined as no caloric intake for at least 8 hours. The two-hour plasma glucose test

should be done using a glucose load having the equivalent of 75g anhydrous glucose dissolved in

4
water. This test is not recommended for routine clinical use. In the absence of unequivocal

hyperglycemia and acute metabolic decomposition, these criteria should be confirmed by repeat

testing on a different day (American Diabetes Association, 2007).

2.2.1 Criteria for the Diagnosis of Cardiovascular Diseases

According to the New York Heart Association, the factors of a full cardiac diagnosis

must consist of some systematic considerations which include the underlying etiology, the

anatomic abnormalities, the physiologic disturbances and functional disability. The underlying

etiology of the disease determines the origin of the disease. The study will then only focus of

hypertensive etiology of CVD. Anatomic abnormalities include which chambers are involved

and whether they are hypertrophied or dilated or both, the affected valves and whether they are

regurgitant and/or stenotic, pericardial involvement and occurrence of myocardial infarction.

Physiologic disturbances look at the presence of arrhythmia and proof of congestive heart failure

or myocardial ischemia. Functional disability describes how exhausting the physical activity

required to bring forth symptoms.

Complete diagnosis has to be linked to the patient’s history and physical examination.

Clinical examination is a basis of diagnosis of a wide variety of disorders. There are five types of

laboratory tests and these include ECG, non-invasive imaging examinations (chest

roentgenogram, echocardiogram, radionuclide, computer tomographic and magnetic resonance

imaging), blood tests (lipid determinants, CRP), or cardiac function (brain natriuretic peptide),

occasionally specialized invasive examinations (cardiac catheterization and coronary

arteriography and genetic tests to identify monogenic cardiac diseases (hypertrophic

cardiomyopathy). These tests are becoming more widely used (Braunwald, 2008).

2
2.2.2 Association of C-reactive protein with Cardiovascular Diseases

Various studies have already been conducted on the correlation of CRP to incidence and

risk to cardiovascular diseases. Many of these studies reported that CRP is a strong marker for

CVD mainly because of its characteristics. In fact, CRP has been recommended as the marker of

choice to monitor CVD risk, being a stronger predictor of atherosclerosis than even plasma LDL

concentration. This is explained by CRP’s ability to bind to a large number of autologous and

extrinsic ligands, including native and modifies plasma lipoprotein, phospholipids, and apoptotic

cells, which are present in the atherosclerotic lesions. When bound to ligands, CRP activates the

classic pathway of complement, a majorplayer in the immune and inflammatory response, and

reacts with Fcγ receptors on phagocytic cells. Both CRP and complement are known to

colocalise in human atherosclerotic lesions, which suggests that CRP, by activating the

complement, may be an active participant in atherosclerosis development (Paul, et al., 2004).

It has been found that inflammation is a prominent feature of atherosclerosis, and it is

postulated that as an acute phase protein, elevation of plasma CRP may signal the underlying

atherosclerotic process. Moreover, researchers observed that an inflammatory reaction is a

common response of the brain parenchyma to various forms of insult. Cytokines play an

important role during the cerebral ischemia and are involved in carotid atherosclerosis thus,

making CRP a practical candidate for inclusion among CVD inflammatory markers (Cesari, et

al., 2003).

Moreover, according to Ross (1999) & Ridker et al. (2002), whether CRP can mediate as

well as predict the progression of atherosclerosis is under active investigation and is likely in

some clinical conditions. A wealth of data suggests that chronic inflammation is a major risk

factor that drives the progression of atherosclerosis and atherothrombosis. Edward, Yeh and

Palusinski (2003) said that diverse cardiovascular risk factors, such as smoking, hypertension,

1
dylipedemia, and hyperglycemia, play a proinflammatory role in the initiation of endothelial

dysfunction and atherosclerotic plaque formation.

Modest elevations of CRP can be found even in apparently healthy people (Yeh, 2003).

Ballantyne et al., (2004) also mentioned that a progressive rise in CRP can reflect augmented

stages of vascular inflammation, but the specific clinical conditions under which this occurs are

incompletely understood. Reports that CRP levels are elevated during acute cardiovascular and

cerebrovascular events suggest that CRP has value in predicting the subsequent occurrence of

such events (Ridker et al., 1998 & Crea et al., 2002).

Several clinical studies have shown that markers of inflammation associate with coronary

risk. Plasma levels of CRP for instance prospectively predict risk of myocardial infarction. Also,

CRP levels show a relationship with outcome of patients with acute coronary syndromes.

Elevated levels of CRP may be a sign of ongoing inflammation rather than a direct etiologic role

for CRP in coronary artery disease. Although some studies have shown that the causality of some

biomarkers that predict cardiovascular risk, such as C-reactive protein (CRP) remains uncertain

(Libby, 2008).

A recent study conducted by Kerner, et al in 2005 showed that higher CRP levels were

observed in subjects with a high ALT level compared with subjects with normal ALT levels,

indicating the role of systemic inflammatory state consequences in the liver and the development

of coronary atherosclerosis. The results of the study of Adibi (2007) were also similar.

In a 10-year prospective study conducted by Mary Cushman (2005), she and her

colleagues found that coronary heart disease (CHD) risk increased with increasing CRP in men

and women ≥65 years of age. When recent clinical guidelines were applied, intermediate CRP

concentrations (1 to 3 mg/L) were weakly related to future CHD, and elevated CRP (>3.0 mg/L)

was associated with a 1.45-fold increased risk of CHD, with adjustment for other vascular risk

2
factors. They also reported that correaction of elevated CRP could eliminate up to 11% of

incident CHD in this age group.

Most of the studies conducted were done in vitro. Thus, Antoni Paul and his colleagues

(2004), conducted a study that examines CRP’s proatherogenic action in vivo. In this study, they

used laboratory mice as their sample. They found that, in male apoE-1- mice, transgenic

expression of human CRP accelerates aortic atherosclerosis development. We observed a very

substantial and significant effect, ie, a 34% to 48% increase in atherosclerotic lesion area in male

mice that express human CRP compared with those that do not. There is also a similar but not as

significant trend observed in female mice. CRP expression causes accelerated atherosclerosis

progression in apoE-1- mice. Therefore, they concluded that CRP is not merely a risk marker but

also an active participant of atherogenesis in vivo (Paul, et al., 2004).

In addition, it has been found by Hage and Szalai (2007) that the rise in blood CRP after

tissue insult or injury is rapid and robust, with levels increasing by as much as 1,000-fold above

baseline within 24 hours. This data supports the notion that blood CRP is an ideal clinical marker

of a patient’s general health status. The recent introduction of “high sensitivity CRP” has

resulted in accumulation of vast amounts of data linking blood CRP to various kinds of

cardiovascular diseases.

According to Paul Ridker (2003), the addition of CRP to standard cholesterol evaluation

may provide a simple and inexpensive method to improve global risk prediction and compliance

with preventive approaches. He added that in most clinical settings, a single CRP assessment is

likely to be adequate as long as levels less than 10 mg/L are observed. Because major infections,

trauma, or acute hospitalizations can elevate CRP levels (usually 100-fold or more), levels

greater than 10 mg/L should initially be ignored and the test repeated at a future date when the

patient is clinically stable.

2
Moreover, he mentioned that there is no need to obtain fasting blood samples for CRP

assessment since CRP levels are stable over long periods of time, are not affected by food intake,

and demonstrate almost no circadian variation. Despite being an acute phase reactant, the

variability in CRP levels in given individuals is quite similar to that associated with cholesterol

screening, as long as the CRP levels are within the clinical range (Ridker, 2003).

2.2.3 Association of C-reactive protein with both Diabetes Mellitus and Cardiovascular

Diseases

Hyperglycemia and abnormal lipid metabolism are major factors that cause diabetic

complications. These complications can be classified into microvascular (neuropathy,

nephropathy and retinopathy) and macrovascular (cardiovascular diseases, cerebrovascular

diseases and gangrene). Macrovascular complications are characterized by the thickening of the

blood vessels or the formation of plaques (artherosclerosis), which results to the poor oxygen and

blood supply that would later on develop to chronic diseases such as cardiovascular disease ,

cerebrovascular diseases and gangrene (Powers, 2008).

In a study conducted by Soinio in 2006, she proposed that the increased CRP levels and

inflammation has been found related to the insulin resistance syndrome, which may partly

explain the high incidence of cardiovascular disease in patients with type 2 diabetes. There are

mechanisms that would explain how elevated hs-CRP would enhance artherosclerosis, which

would later on progress to CVD. Elevated hs-CRP would lead to the adhesion of monocytes to

the arterial wall that would result to endothelial dysfunction. It was also suggested that hs-CRP

would enhance the entry of LDL into macrophages (Soinio, et al., 2006). Monocyte attachment

to the endothelium, migration into the intima, and maturation to form lipid-laden macrophages

1
thus represent key steps in the formation of the fatty streak, the precursor of fully formed

atherosclerotic plaques (Libby, 2008).

Paul Ridker (2003) examined CRP’s advantages over other newly discovered risk factors

of CVD and Diabetes. He based his report on studies conducted by other researchers and some

he conducted himself. According to his report, CRP is not the only inflammatory biomarker that

has been shown to predict myocardial infarction and stroke. More sophisticated measures of

cytokine activity, cellular adhesion, and immunologic function (such as interleukin-6,

intercellular adhesion molecule-1, macrophage inhibitory cytokine-1, and soluble CD40 ligand)

have all been shown to be elevated among those at increased vascular risk. However, these

approaches are not practical for clinical use because the assays required for their assessment are

either inappropriate for routine clinical use or the protein of interest has too short a half-life for

clinical evaluation. Measures for fibrinogen, a biomarker involved in both inflammation and

thrombosis, remain poorly standardized, and methodological issues limit use of this parameter

despite consistent population based data. Other broad measures of systemic inflammation, such

as the white blood cell count of the erythrocyte sedimentation rate, have proven unreliable in

clinical settings.

By contrast, high sensitivity assays for CRP have been standardized across many commercial

platforms. Moreover, CRP is highly stable, allowing measures to be made accurately in both

fresh and frozen plasma without requirements for special collecting procedures. This is due in

part to the stable pentraxin structure of CRP and its long plasma half-life of 18 to 20 hours. In

selected patients, such as those with markedly premature and unexplained atherosclerosis,

evaluation of other markers, such as lipoprotein(a) and homocysteine, may have clinical utility.

However, the relative magnitude of these biomarkers has been small in direct comparison to

CRP in the available population-based studies. Recent data also indicate that CRP is a stronger

2
predictor of risk than nuclear magnetic resonance-based evaluation of LDL particle size and

concentration (Ridker, 2003).

2.2.4 Association of Alcohol with C-reactive protein Levels, Cardiovascular Diseases and

Diabetes Mellitus

A number of studies have shown that light to moderate alcohol consumption is associated

with a reduced mortality rate. This finding has been attributed to the reduced risk of

cardiovascular disease (CVD), primarily because of the protective effect of moderate alcohol

consumption on coronary heart disease (CHD) (Gaziano, et al., 2000). It has been suggested that

favorable changes in blood lipids and in the haemostatic profile might mediate the

atheroprotective effect of moderate alcohol (Rimm, et al., 1999); nevertheless, the underlying

mechanisms remain unclear. However, there is strong evidence that the increased levels of

inflammatory marker, including C-reactive protein (CRP), predict the onset of cardiovascular

events and mortality, identifying subjects at increased CVD risk (Cesari, et al., 2003). In line

with this, recent studies have shown that light to moderate alcohol intake is associated with lower

levels of C-reactive protein, along with other acute-phase markers. These findings suggest that

the protective effect of moderate alcohol consumption on health-related outcomes may be

mediated through an anti-inflammatory effect. Moreover, some experts hypothesized that the

effect of moderate alcohol consumption may be modified by the level of inflammatory markers

(Maraldi, et al., 2006).

Volpato et al found that weekly alcohol intake had a J-shaped association with CRP

levels and its major regulator, interleukin-6. As moderate alcohol intake has also been associated

2
with decreased risk of a number of adverse health outcomes, all-cause mortality, cardiovascular

diseases, insulin resistance and diabetes, and dementia, all of which inflammation has been

proposed as a key pathogenic component, Volpato and his colleagues proposed that the

relationship between alcohol intake and the level of inflammatory markers would offer an

additional biologically credible explanation. They concluded that light alcohol intake may have

an anti-inflammatory effect that may reflect a direct effect of ethanol on interleukin-6

metabolism and eventually CRP (Volpato, et al., 2004). Furthermore, a study by Albert et al

observed that a J- or U- shaped relationship between alcohol intake and cardiovascular mortality

supports the hypothesis that, in part, the effect of alcohol on cardiovascular mortality may have

links in inflammation (Albert, et al., 2003).

However, in a study conducted by Alho et al, heavy alcohol consumption was found to

result to increased serum C-reactive protein levels, in contrast with the effects of light to

moderate drinking, thus reflecting a pro-inflammatory effect. He and his colleagues found that

among the 494 participants in their study, increase of CRP levels in the top and bottom halves of

the alcohol misusers compared the control groups’ statistics were observed (Alho, et al., 2003).

This reversal is believed to be caused by the damage in several tissues, including the liver,

among heavy drinkers (Imhof, et al., 2004).

On the contrary, Maraldi and her colleagues (2006) suggested that the protective effect of

light to moderate alcohol consumption may not be mediated by its beneficial effects on lipids

and inflammatory profile, which includes C-reactive protein, although they were able to prove

that light to moderate alcohol consumption is associated with a 26% reduced risk of all-cause

mortality and almost 30% reduced risk of cardiac events. The aim of their study was to

investigate the relationship between alcohol consumption, all-cause mortality, and cardiac events

(CHD and Heart Failure) in a sample of well- functioning, CVD-free older persons, and to

2
evaluate whether this relationship is mediated or modified by serum inflammatory marker (IL-6

and CRP) levels (Maraldi, et al., 2006). Maraldi’s conclusion were found to be consistent with

that of Mukamal et al, who found no significant effect of potential mediators, including CRP, on

the relationship between alcohol intake and CHD (Mukamal, et al., 2006).

On the other hand, moderate alcohol consumption has also been found to be protective

against type 2 diabetes (Howard, et al., 2004). In studies done on the male population by

Tsumara et al, Ajani et al, and Hu et al, light to moderate drinking were suggested to be inversely

associated with the development of type 2 diabetes mellitus (Tsumara, et al., 1999, Ajani, et al.,

2000, and Hu, et al., 2001). Moreover, Wannameethee et al (2003) observed that all women who

drank 5 g or more a day had a lower risk of diabetes mellitus than nondrinkers, irrespective of

the pattern of drinking, although moderate intake of alcohol (5.0 – 29.9 grams/day), when taken

regularly (4 – 7 days/week), was associated with a lower risk than when the same amount was

consumed over 1 to 3 days/week. Worsening glucose tolerance was accompanied by more

abstaining, less frequent intake of alcohol, and smaller amounts consumed (Ogge, et al., 2006).

This finding is mostly explained by the observation that low to moderate amounts of

alcohol intake may increase insulin sensitivity and slow glucose uptake from a meal (Lazarus, et

al., 1997). However, recent studies suggest that anti-inflammatory effects of moderate alcohol

consumption may also be involved in this risk reduction (Sierksma, et al., 2002). Moreover, a

recent cross-sectional study conducted by Festa et al showed that elevated CRP levels correlate

significantly with features of the metabolic syndrome (insulin resistance syndrome), including

adiposity, hyperinsulinemia, insulin resistance, hypertriglyceridemia, and low HDL (high density

lipoprotein) cholesterol (Festa, et al., 2000). Another explanation involves adinopectin, a plasma

protein expressed in adipocytes, which might link type 2 diabetes, insulin resistance, and

inflammation. This protein has insulin-sensitizing and anti-inflammatory properties (Spranger, et

3
al., 2003). In relation to this, recent reports have shown that moderate alcohol intake was

associated with high adiponectin concentrations in diabetic men and in nondiabetic subjects

(Shai, et al., 2004, and Thamer, et al., 2004).

2.2.5 Association of Smoking with C – reactive protein Levels, Cardiovascular Disease

and Diabetes Mellitus

A number of studies suggested that smoking is associated with excess morbidity and

mortality of diabetes patients. In a study conducted by Foy and his associates, current smokers

displayed a significantly higher incidence of diabetes at 5 years than never smokers (odds ratio

[OR] 2.06, P= 0.006). With the exception of age being associated with higher incidence of

diabetes (1.03, P = 0.01), there were no other significant effects for any of the covariates (Foy, et

al., 2005). Experts suggest that this may be due to circulatory and cardiovascular disease. It was

found that smoking can also cause transient elevations in blood glucose concentrations and may

also influence insulin sensitivity (Sargeant, 2000). In addition, the combination of smoking and

diabetes may appear to intensify the development of macrovascular complications (Yudkin,

1999).

In a study conducted by Rimm and his colleagues (1995), it was proven that there are

biological mechanisms that smoking may contribute to the development of diabetes through

alternations in fat distribution, which is associated with insulin resistance and through a direct

toxic effect on pancreatic tissue.

Smoking is also a documented risk for the development and progression of

atherosclerosis in diabetic patients (Haire-Joshu, 1999). Furthermore, it was also proven that

smoking causes a chronic, increased inflammatory response. It also appeared that CRP

4
elevations is caused by underlying subclinical atherothrombotic disease and may reflect the

ongoing process of thrombin generation (Tracy, 1997).

In a few studies compiled by Frohlich and his colleagues, the mechanisms were explained

on how smoking may be associated with systemic markers of inflammation. First, acute phase

proteins may be surrogates for smoking-induced chronic inflammatory processes such as

periodontitis (Fredriksson, 1999), airway inflammation (Maestrelli, 2001) and atherosclerosis

itself (Ross, 1999). Second, certain compounds of smoke such as free radicals (Tappia, 1995)

and phenol-rich glycoproteins (Francus, 1993) directly exert an inflammatory stimulus on

macrophages which may trigger the production of inflammatory cytokines such as TNF, IL-1,

and IL-6 (Tappia, 1995). And third, it is conceivable that there is an indirect effect by nicotine-

induced catecholamine release which modulates the systemic and local cytokine balance

(Elenkov, 2002).

It is becoming apparent that inflammation is associated with atherosclerosis. This

association with CVD is complex in nature (Tracy, 1997). A number of studies have shown that

smoking has been a major modifiable risk of cardiovascular disease, including coronary heart

disease, stroke, peripheral vascular disease, and congestive heart failure (Bazzano, 2003). It was

suggested that smoking is associated with increased inflammatory markers (Bahkru, 2005).

The serum level of CRP is used clinically as an indicator of inflammation. Elevations in

CRP have been shown to be positively associated with acute MI and sudden cardiac-related death

in patients with unstable angina, linking inflammation and acute coronary events (Tracy, 1997).

C-reactive protein in particular is also increasingly being implicated in the pathogenesis of

atherosclerosis. It was previously believed to be synthesized by the liver; evidence suggests that

C-reactive protein is also produced at the site of atherosclerosis by smooth muscle cells. Thus, C-

5
reactive protein is increasingly described not only as a marker of the inflammatory response, but

also as a mediator in the pathogenesis of atherosclerotic cardiovascular disease (Bahkru, 2005).

There are different mechanisms by which smoking may initiate and accelerate cardiovascular

disease. In a study conducted by Frohlich and his colleagues, it was proven that smoking causes

coagulation and lipid abnormalities, and circulatory effects due to enhanced catecholamine

release have been reported. Furthermore, in a study conducted by Waters et al., smoking

increases the production of thromboxane that increases platelet aggregation, increases the plasma

viscosity, and it enhances the amount of fibrinogen. Smoking would also lead to carbon

monoxide-induced hypoxic injury to endothelial cells. (Thus, it would result to decrease oxygen

delivery that would lead to cardiovascular diseases (myocardial infarction) (Waters, 1996).

Chapter III. METHODOLOGY

2
1.1 Study Site

The study subjects are patients recommended by an endocrine specialist of Silliman

University Medical Center (SUMC). They are currently diagnosed as type 2 DM patients and

CHD patients from whose data we hope to be able to determine CRP activity and other factors

that elevate CRP. Through statistical data from these patients this study hopes to reach a

conclusive analysis on the viability of CRP associated with DM II and CHD.

1.2 Research Design

This study is using the retrospective control-case study to determine the predominance of

the association of people who have type 2 diabetes mellitus, with CHD, smoking and alcohol

status. As defined by Elsevier (2009), this type of research is a non-experimental research design

using an epidemiologic approach in which previous cases of the condition are used in lieu of new

information gathered from a randomized population. A group of patients with a particular disease

is compared with a control group of persons who do not have that medical problem. An

incidental sampling of patients in the SUMC will provide the field data for statistical analysis.

This population of incidental respondents will be subjected to laboratory exams and close

monitoring of their status to confirm CRP activity.

In obtaining survey results, two surveys will be conducted—the initial survey and follow-

up survey. Both surveys will measure associations with consistent variable values, both within

identical time frames and conditions. The initial survey hopes to present early observations hopes

to present early observations and the follow-up will present a confirmation of these observations.

This particular study aims to prove the viability of our hypotheses using the Chi-square

test of homogeneity as the standard statistical measure and its analysis. The comprehensive result

3
of this analysis published in this research paper hopes to allow for a narrowing down of possible

directions for follow up research to take.

1.3 Determination of Sample Size

The exact data concerning the number of in-patients and out-patients with Type 2

diabetes mellitus in Silliman University Medical Center (SUMC) is not available. Due to this

limitation, the determination of sample size is based on the number of tests that can be performed

in one bottle of RHELAX CRP latex reagent. This reagent, which will be used in this study in

determining CRP levels, has a maximum of 100 tests per bottle. Therefore, sample size that will

be used is between 50-100 individuals diagnosed with Type 2 diabetes mellitus.

1.4 Sampling Design and Technique

Analysis from statistical data is achieved through summary of the three most commonly

used values of central tendency which are the mean, median and the mode. The measure of

dispersion employed in this study should consistently relate to the three standard variants of

CVD, smoking and alcohol consumption throughout the whole observation process.

1.5 Research Instrument

A person-to-person interview will be conducted between the researchers and the study

participants. This type of research instrument is often regarded as the industry standard because

of the high response rate. The interviewer being at the scene, can make additional observations

regarding subtle aspects of the interviewee’s behavior; this may be used to help validate the

interview (Kuzma & Bohnenblust, 2001). The interview questions will include the study

participant’s smoking and alcohol drinking status.

2
1.6 Data Collection and Presentation

All data in this study is based on incidental statistical results. For the purpose of this

proposal, all statistical data included here are of assumed values.

Three statistical tables will be incorporated to show analysis of the three variants namely;

CVD, smoking and alcohol consumption.

Corresponding statistical tables will show the incidence of association between type 2

DM and CVD, type 2 DM with smoking and type 2 DM with alcohol consumption.

1.7 Data Processing Method

Measures of relative variants which express the standard deviation or the result will be

based on the chi-square homogeneity test.

The formula of this test is shown below.

X2 =
Σ (O-E)2

E
The homogeneity test will determine whether the distribution of a particular characteristic

is similar for the various groups involved.

Dummy Tables

INITIAL SURVEY RESULTS (1st week of January to 3rd week of January)

Table 3.1.1. Cross Tabulation of Level of CRP and CHD Among Type 2 Diabetes mellitus Individuals

Test Results CHD Total

1
With CHD w/o CHD
Positive for CRP
Negative for CRP
Total

Table 3.1.2. Cross Tabulation of Level of CRP and Smoking Status Among Type 2 Diabetes mellitus Individuals

Test Results Smoking Status Total


Smokers Nonsmokers
Positive for CRP
Negative for CRP
Total

Table 3.1.3. Cross Tabulation of Level of CRP and Alcohol Status Among Type 2 Diabetes mellitus Individuals

Test Results Alcohol Status Total


Heavy Drinker Nondrinker
Positive for CRP
Negative for CRP
Total

FOLLOW-UP SURVEY RESULTS (1st week of February to 3rd week of February)

Table 3.2.1. Cross Tabulation of Level of CRP and CHD Among Type 2 Diabetes mellitus Individuals

Test Results CHD Total


With CHD w/o CHD
Positive for CRP
Negative for CRP
Total

Table 3.2.2. Cross Tabulation of Level of CRP and Smoking Status Among Type 2 Diabetes mellitus Individuals

Test Results Smoking Status Total


Smokers Nonsmokers
Positive for CRP
Negative for CRP
Total

Table 3.2.3. Cross Tabulation of Level of CRP and Alcohol Status Among Type 2 Diabetes mellitus Individuals

Test Results Alcohol Status Total

1
Heavy Drinker Nondrinker
Positive for CRP
Negative for CRP
Total

3.8 Methods

The procedure that will be used in the study will be from the Tulip Diagnostics (P) LTD.

C-reactive protein (CRP) is a serum protein which is synthesized in the liver. Its rate of

synthesis and secretion increases within hours of an acute injury or the onset of inflammation

and may reach as high as 20 times the normal levels. Elevated serum concentration of CRP is an

unequivocal evidence of an active tissue damage process and CRP measurement thus provides a

simple screening test for organic disorders. Apart from indicating inflammatory disorders,

standard microbiological investigations are difficult.

Its use in post operative surveillance is of great importance. CRP levels invariably rise

after major surgery but fall to normal within 7-10 days. Absence of this fall is indicative of

possible septic or inflammatory post operative complications. Serum CRP measurement also

provides useful information patients with myocardial infarction there being an excellent

correlation between peak levels of CRP and Creatine phosphokinase (CPK) (Tulip Diagnostics,

2008). Lindsey (2005) defines C-reactive protein (CRP) as one of the acute phase proteins that

is synthesized in the liver and appears in the blood of patients with diverse inflammatory disease.

Bernstein (2005) also mentioned that CRP increases dramatically under conditions of sepsis,

inflammation and infection.

Principle of CRP Test

1
RHELAX CRP slide test for detection of CRP is based on the principle of agglutination.

The test specimen (serum) is mixed with RHELAX CRP latex reagent and allowed to react. If

CRP concentration is greater than 0.6 mg/dl a visible agglutination is observed. If CRP

concentration is less than 0.6 mg/dl, then no agglutination is observed.

Specimen Collection and Preparation

There will be no special preparation of the patient is required prior to specimen collection

by approved techniques. Only serum will be used for testing. The sample will be stored at 2-8°C

if there will be any delay in testing. Samples then can be stored for up to a week. Hemolysed

serum will not be used.

Material Provided With the Kit Reagent

RHELAX CRP latex reagent, Positive control, Negative control.

Accessories

Glass slide with six reaction circles, Sample dispensing pipettes, Mixing sticks, Rubber

teat.

Additional Material Required

Stop watch, Test tubes, A high intensity direct light source, Isotonic saline.

1
TEST PROCEDURE

1 drop of specimen on slide

Added with 1 drop of RHELAX CRP latex


reagent on the first drop

Mix test specimen and the latex


reagent uniformly

king the slide gently, back and forth, observing for agglutination
macroscopically at two minutes

3
INTERPRETATION OF TEST RESULTS

Qualitative Method

Agglutination is a positive test result and indicates the presence of detectable levels of

CRP in the test specimen. No agglutination is a negative test result and indicates the absence of

detectable levels of CRP in the test specimen.

REMARKS

1. Markedly lipemic, hemolysed and contaminated serum samples could produce

non-specific results.

2. Use of plasma rather than serum can lead to false positive results.

3. CRP is found to be present after the first trimester of pregnancy and persists until

delivery.

4. CRP levels increase in women who are on oral contraceptives.

5. CRP response is not affected by the commonly used anti-inflammatory or

immunosuppressive drugs, including steroids, unless the disease activity is affected and it

covers an exceptionally broad incremental range up to 3000 times.

6. Do not read results beyond indicated testing time limits.

3
7. Since CRP production is a non-specific response to tissue injury, it is

recommended that results of the test should be correlated with clinical findings to arrive at

the final diagnosis.

8. In cases where an increase in CRP levels is suspected, but the screening test

shows a negative result, semiquantitation should be done to rule out prozone effect.

3.9 Statistical Treatment

The x2 for dichotomous variables determined significant univariate associations (Eason,

et al., 2005). According to Kuzma and Bohnenblust (2001) chi square is performed to determine

whether there is some association between the two variables. This current study will be using this

type of statistical tool in each association between CRP levels and CHD; CRP levels and

smoking status; and CRP levels and alcohol drinking status of the Type 2 diabetes mellitus study

participants. The dependent variable will be the levels of CRP and the independent variable will

be the underlying disease and behavior of the study participants. The following independent

variables that will be included are the study participant’s smoking and alcohol drinking status as

well as the presence of CHD.

3.10Data Analysis and Interpretation

The assumed computed values in this study determine the association between type 2 DM

and the three variants of this study, namely: Type 2 DM with CHD; type 2 DM and smoking;

and type 2 DM and alcohol consumption. The assumed computed chi-square values per variant

of 50 falls above the critical region at 0.01 level of significance. So, we conclude that the

variants affect the levels of CRP of the type 2 DM patients.

1
Based on the study of Ridker et al., (2002) on 15, 745 of study participants in the

Women’s Health Study in the United States, the observed frequencies has a 77% possibility of

LDL cholesterol and CRP levels which is associated with CHD existence.

Alho et al., (2004) conducted a survey of participants that are with type 2 DM and who

were heavy alcohol drinkers are 494 respondents. The inflammatory markers CRP increased in

the bottom and top halves of alcohol misusers compared to control groups’ statistics.

Foy and Associates (2005) concluded that current smokers displayed a significantly

higher type 2 DM at five (5) years than never smokers. With the exception of age being

associated with higher incidence of DM, there were no other significant effects of the co-

variants.

2
4
References

Powers, A.C. (2008). Diabetes Mellitus. In Fauci, A.S., Kasper, D.L., Longo, D.L., Braunwald,

E., Jameson, J.L., & Loscalzo, J. (Eds.), Harrison’s principles of internal medicine, (17th

ed.). (p. 2275). USA: The McGraw-Hill Companies, Inc.

Department of Health (2002). [www.doh.gov.ph]. (Accessed July 2009).

Freeman, V.S. (2005). Diabetes Mellitus. In Bishop, M.L, Fody, E.P., & Schoeff, L.E. (Eds.),

Clinical chemistry: Principles, procedures and correlations, (5th ed.). (pp. 268 – 273). PA:

Lippincott Williams and Wilkins.

Guyton, A., & Hall, J.E. (2000). Diabetes Mellitus. Textbook of Medical Physiology, (10th ed.).

(p. 894). PA: W.B. Saunders Company.

Wannamethee, S.G., et al. (2005). Hepatic enzymes, the metabolic syndrome, and the risk of

type 2 diabetes in older men. Diabetes Care, 28, 2913 – 2918.

Hanley, A.J., et al. (2005). Liver markers and development of the metabolic syndrome: the

insulin resistance atherosclerosis study. Diabetes, 54, 3140 – 3147.

Beers, M. H., et al. (2003). The Merck manual: Medical information, 2nd home edition.USA:

Simon-Schuster Aviacom Company.

Gaziano, J.M, & Gaziano, T.A. (2008). Epidemiology of cardiovascular disease. In Fauci, A.S.,

Kasper, D.L., Longo, D.L., Braunwald, E., Jameson, J.L., & Loscalzo, J. (Eds.), Harrison’s

principles of internal medicine, (17th ed.). (ch. 218). USA: The McGraw-Hill Companies,

Inc.

World Health Organization (2003). [http://www.who.int] (Accessed: July 2009).

Pearson, T.A., et al. (2003). Markers of inflammation and cardiovascular disease: application to

clinical and public health practice: A statement for healthcare professionals from the Centers

5
for Disease Control and Prevention and the American Heart Association. Circulation, 107,

499 – 511.

Ingram, (2005). In Bishop, M.L, Fody, E.P., & Schoeff, L.E. (Eds.), Clinical chemistry:

Principles, procedures and correlations, (5th ed.). (p. 501). PA: Lippincott Williams and

Wilkins.

Danesh, J., et, al. (2004). C - reactive protein and other circulating markers of inflammation in

the prediction of coronary heart disease. New England Journal of Medicine, 350, 1387 - 97.

Ridker, P.M., et al. (2005). Non-HDL cholesterol, apolipoproteins A-I and B100, standard lipid

measures, lipid ratios, and CRP as risk factors for cardiovascular disease in women. JAMA,

294, 326 – 333.

Shlipak, M.G., et al. (2005). Cardiovascular mortality risk in chronic kidney disease: comparison

of traditional and novel risk factors. JAMA, 293, 1734 – 1745.

Pai, J.K., et al. (2004). Inflammatory markers and the risk of coronary heart disease in men and

women. New England Journal of Medicine, 351, 2599 – 2610.

Ridker, P.M., et al. (2002). Comparison of C-reactive protein and low-density lipoprotein

cholesterol levels in the prediction of first cardiovascular events. New England Journal of

Medicine, 347, 1557 – 1565.

Libby, P., et al. (2002). Inflammation and atherosclerosis. Circulation. 105, 1135 – 1143.

Ross, R. (1999). Atherosclerosis – an inflammatory disease. N Engl J Med, 340, 115 – 126.

Pradhan, A.D., et al. (2001). C - reactive protein, Interleukin 6, and risk of developing type 2

diabetes mellitus. JAMA, 286(3), 327 – 334.

Barzilay, J.I., et al. (2001). The relation of markers of inflammation to the development of

glucose disorders in the elderly: The cardiovascular health study. Diabetes, 50, 2384 – 2389.

6
Ford, E.S. (1999). Body mass index, diabetes, and C-reactive protein among U.S. Adults.

Diabetes Care, 22, 1971 – 1977.

Grau, A.J., et al. (1996). The association of leukocyte count, fibrinogen and C-reactive protein

with vascular risk factors and ischemic vascular diseases. Thromb Res, 82, 245 – 255.

Wu, T., et al. (2002). Associations of serum C-reactive protein with fasting insulin, glucose, and

glycosylated hemoglobin. American Journal of Epidemiology, 155, 65 – 71.

Rodriguez- Moran, M., et al. (1999). Increased levels of C-reactive protein in noncontrolled type

II diabetic subjects. J Diabetes Complications, 13, 211 – 215.

Dehghan, A., et al. (2007). Genetic variation, C - reactive protein levels, and incidence of

diabetes. Diabetes, 56, 872 – 878.

Sattar, N., et al. (2004). Elevated Alanine Aminotransferase predicts new-onset of type 2

diabetes independently of classical risk factors, metabolic syndrome, and C - reactive

protein in the west of Scotland coronary prevention study. Diabetes, 53, 2855 - 2860.

Pickup J.C., et al. (1997). NIDDM as a disease of the innate immune system: Association of

acute-phase reactants and interleukin-6 with metabolic syndrome X. Diabetologia, 40 (11),

1286 – 1292.

Abdelmouttaleb, I., et al. (1999). C-reactive protein and coronary artery disease: additional

evidence of the implication of an inflammatory process in acute coronary syndromes.

American Heart Journal, 137, 346 – 351.

Ridker, P.M., & Rifai, N. (2001). Novel risk factors for systemic atherosclerosis: a comparison

of C-reactive protein, fibrinogen, homocysteine, lipoprotein(a), and standard cholesterol

screening as predictors of peripheral arterial disease. JAMA, 285, 2481 – 2485.

Fox, C.S., et al. (2004). Trends in cardiovascular complications of diabetes. JAMA, 292(20),

2495-9.

7
Freeman, D.J., et al. (2002). West of Scotland Coronary Prevention Study: C-reactive protein is

an independent predictor of risk for the development of diabetes in the West of Scotland

Coronary Prevention Study. Diabetes, 51, 1596 – 1600.

Wannamethee, S. G. and Shaper, A. G. (1998) Alcohol, coronary heart disease and stroke: an

examination of the J-shaped curve. Neuroepidemiology, 17, 288–295.

Castelli W.P., et al. (1992). Lipids and risk of coronary heart disease: the Framingham Study.

Ann Epidemiol, 2, 23–8.

Langer, R. D., Criqui, M. H. and Reed, D. M. (1992) Lipoproteins and blood pressure as

biological pathways for effect of moderate alcohol consumption on coronary heart disease.

Circulation, 85, 910–915.

Krobot, K., et al. (1992). Determinants of plasma fibrinogen: Relation to body weight, waist-to-

hip ratio, smoking, alcohol, age, and sex. Results from the second MONICA Augsburg

survey 1989 – 1990. Arteriosclerosis and Thrombosis, 12, 780 – 788.

Klatzky, A.L. (1996). Alcohol and hypertension. Clinical Chimica Acta, 246, 91 – 105.

Hillbom, M. (1998). Alcohol consumption and stroke: benefits and risks. Alcoholism: Clinical

and Experimental Research . 22 (7 Suppl), 352S–358S.

Howard, A.A., Arnsten, J.H., and Gourevitch, M.N. (2004). Effect of alcohol consumption on

diabetes mellitus: a systematic review. Ann Intern Med, 140, 211 – 219.

Djousse, L., et al. (2002). Influence of Apolipoprotein E, smoking, and alcohol intake on carotid

atherosclerosis: National Heart, Lung, and Blood Institute Family Heart Study. Stroke, 33,

1357 – 1361.

Howard G., et al. (1998). Cigarette smoking and progression of atherosclerosis: the

Atherosclerosis Risk in Communities (ARIC) Study. JAMA, 279, 119-124.

8
de Waart, F.G., et al. (2000). Smoking characteristics, antioxidant vitamins, and carotid artery

wall thickness among life-long smokers. Journal of Clinical Epidemiology, 53, 707 – 714.

Hutchinson, S., et al. (1996). L-arginine restores normal endothelium: medicated relaxation in

hypercholesterolemic rabbits exposed to environmental tobacco smoke. J Am Coll Cardiol.,

27, 39.

Celermajer, D., et al. (1996). Passive smoking and impaired endothelium-dependent areterial

dilation in healthy young adults. New England Journal of Medicine, 334, 150 – 154.

Zhu, B-Q, et al., (1993). Passive smoking increases experimental atherosclerosis in cholesterol-

fed rabbits. J Am Coll Cardiol., 21, 225 – 232.

Penn, A., & Snyder, C. (1996). 1,3 Butadiene, a vaporphase component of environmental

tobacco smoke, accelerates atherosclerotic plaque development. Circulation, 93, 552 – 557.

Tracy, et al. (1996). C-reactive protein and incidence of cardiovascular disease in older women:

the Rural Health Promotion Project and the Cardiovascular Health Study. Circulation, 93,

622.

World Health Organization (2008). [http://www.who.int] (Accessed: July 2009).

Ridker, P.M., et al. (1998). C-reactive protein adds to the predictive value of total and HDL

cholesterol in determining risk of first myocardial infarction. Circulation, 97, 2007 – 2011.

Goldberg, R.B., et al. (2000). Cardiovascular disease in diabetic patients. Med Clin North Am,

84, 81 – 93.

Lindsey, B.J. (2005). In Bishop, M.L, Fody, E.P., & Schoeff, L.E. (Eds.), Clinical chemistry:

Principles, procedures and correlations, (5th ed.). (p. 198). PA: Lippincott Williams and

Wilkins.

9
Maraldi, C.M., et al. (2006). Impact of inflammation on the relationship among alcohol

consumption, mortality, and cardiac events: The health, aging, and body composition study.

Arch Intern Med. 166, 1490 - 1495.

Tulip Diagnostics (2008). Slide Test CRP.

Bernstein, L.H. (2005). In Bishop, M.L, Fody, E.P., & Schoeff, L.E. (Eds.), Clinical chemistry:

Principles, procedures and correlations, (5th ed.). (p. 636). PA: Lippincott Williams and

Wilkins.

Funk, J. L. (2006) Diabetes Mellitus. In McPhee, S.J., & Ganong, W.F. (Eds.), Pathophysiology

of Disease: An Introduction to Clinical Medicine, (5th ed.). (pp. 521 – 527).USA: The

McGraw-Hill Companies, Inc.

Hak, E.A., et al. (1998). Associations of C - reactive protein with measures of obesity, insulin

resistance, and subclinical atherosclerosis in healthy, middle-aged women. Arterioscler

Thromb Vasc Biol, 19, 1986 – 1991.

Berkow, R. (1997). The Merck Manual of Medical Information. (pp.717-723). Division of Merck

& Co., Inc. Whitehouse Station, New Jersey.

Crawford, J. (1997). Basic Pathology, (6th ed.). (pp. 563-574). W.B. Saunders Company.

Pagana, K., & Pagana, T. (2002). Moby’s Manual of Diagnostic and Laboratory Test, (2nd ed.).

(pp.110, 186, 195-196, 246-247, 372, 424, 866-867, 897, 900). United States of America:

Mosby Inc.

Capuzzi, D.M., & Freeman, J.S. (2007). C - reactive protein and Cardiovascular Risk in the

Metabolic Syndrome and Type 2 Diabetes: Controversy and Challenge. Clinical Diabetes,

25, 16 – 22.

10
Remaley, A.T., McNamara, J.R., & Warnick, G.R. (2005). Diabetes Mellitus. In Bishop, M.L,

Fody, E.P., & Schoeff, L.E. (Eds.), Clinical chemistry: Principles, procedures and

correlations, (5th ed.). (p. 294). PA: Lippincott Williams and Wilkins.

Festa, A., & Haffner, S.M. (2005). Inflammation and cardiovascular disease in patients with

diabetes: Lessons from the diabetes control and complications trial. Circulation, 111, 2414 –

2415.

Cippollone, F., et al., (2003). The receptor RAGE as a progression factor amplifying

arachidonate-dependent inflammatory and proteolytic response in human atherosclerotic

plaques: role of glycemic control. Circulation, 108, 1070 – 1077.

Hu, F.B., et al. (2004). Inflammatory markers and risk of developing type 2 diabetes in women.

Diabetes, 53, 693 – 700.

Yudkin, J.S., et al. (1999). C - reactive protein in healthy subjects: Associations with obesity,

insulin resistance, and endothelial dysfunction: A potential role for cytokines originating

from adipose tissue? Arterioscler Thromb Vasc Biol., 19, 972 – 978.

Perticone, F., et al. (2008). Endothelial dysfunction and C - reactive protein are risk factors for

diabetes in essential hypertension. Diabetes, 57, 167 – 171.

King, D.E., et al. (2003). C - reactive protein and glycemic control in adults with diabetes.

Diabetes Care, 26(5), 1535 – 1539.

Soinio, M., et al. (2006). High-sensitivity C – reactive protein and coronary heart disease

mortality in patients with type 2 diabetes: A 7-year follow-up study. Diabetes Care, 29, 329

– 333.

Schmidt, M.I., et al. (1999). Markers of inflammation and prediction of diabetes mellitus in

adults (Atherosclerosis risk in communities study): A cohort study. Lancet, 353, 1649 –

1652.

11
Festa, A., et al. (2000). Chronic subclinical inflammation as part of the insulin resistance

syndrome: The insulin resistance atherosclerosis study. Circulation, 102, 42 – 47.

American Diabetes Association. (2007). National Diabetes Fact Sheet, 2007.

[http://www.cdc.gov/diabetes/pubs/pdf/ndfs_2007.pdf]. (Accessed July 2009).

Braunwald, E. (2008). In Fauci, A.S., Kasper, D.L., Longo, D.L., Braunwald, E., Jameson, J.L.,

& Loscalzo, J. (Eds.), Harrison’s principles of internal medicine, (17th ed.). (ch. 217). USA:

The McGraw-Hill Companies, Inc.

Paul, A., et al. (2004). C-reactive protein accelerates the progression of atherosclerosis in

Apolipoprotein E-deficient mice. Circulation, 109, 647 – 655.

Cesari, M., et al. (2003). Inflammatory markers and onset of cardiovascular events: Results from

the Health ABC Study. Circulation, 108, 2317 - 2322.

Kerner, A., et al. (2005). Association between elevated liver enzymes and C-reactive protein:

possible hepatic contribution to systemic inflammation in the metabolic syndrome.

Arterioscler Thromb Vasc Biol, 25, 193 – 197.

Ballantyne, C.M., et al. (2004). Lipoprotein-Associated Phospholipase A2, High-Sensitivity C-

Reactive Protein, and risk for incident coronary heart disease in middle-aged men and

women in the Atherosclerosis Risk in Communities (ARIC) Study. Circulation, 109, 837 –

842.

Adibi, P., et al. (2007). Prediction of Coronary Atherosclerotic Disease With Liver Transaminase

Level. Liver International, 27(7), 895 – 900.

Cushman M., et al. (2005). C-reactive protein and the 10-year incidence of coronary heart

disease in older men and women: The Cardiovascular Health Study. Circulation, 112, 25-31

Hage, F.G., & Szalai, A.J. (2007). C-reactive protein gene polymorphisms, C-reactive protein

blood levels, and cardiovascular disease risk. J Am Coll Cardiol, 50, 1115 – 1122.

12
Edward, T.H., Yeh, M.D. & Palusinski, R.P. (2003). C-reactive protein: the pawn has been

promoted to queen. Current Atherosclerosis Reports, 5, 101–105.

Yeh, E.T.H, & Palusinski, R.P. (2003). C-reactive protein: the pawn has been promoted to

queen. Curr Atheroscler Rep, 5, 101 – 105.

Crea, F., et al. (2002). Inflammatory predictors of mortality in the Scandinavian Simvastatin

Survival Study. Clin Cardiol, 25, 461 – 466.

Libby, P., et al. (2008). The Pathogenesis, Prevention and Treatment of Atherosclerosis. In

Fauci, A.S., Kasper, D.L., Longo, D.L., Braunwald, E., Jameson, J.L., & Loscalzo, J. (Eds.),

Harrison’s principles of internal medicine, (17th ed.). (ch. 235). USA: The McGraw-Hill

Companies, Inc.

Ridker, P.M., et al. (2003). C-reactive protein, the metabolic syndrome, and risk of incident

cardiovascular events: An 8-year follow-up of 14 719 initially healthy American women.

Circulation, 107, 391 – 397.

Gaziano, J.M., et al. (2000). Light to moderate alcohol consumption and mortality in the

Physician's Health Study enrollment cohort. J Am Coll Cardiol., 35, 96 – 105.

Rimm, E.B., et al. (1999). Moderate alcohol intake and lower risk of coronary heart disease:

Meta-analysis of effects on lipids and haemostatic factors. Brit Med J., 319, 1523 – 1528.

Volpato, S., et al. (2004). Relationship of alcohol intake with inflammatory markers and

plasminogen activator inhibitor-1 in well-functioning older adults: The health, aging, and

body composition study. Circulation, 109, 607 - 612.

Albert, M.A., Glynn, R.J., & Ridker, P.M. (2003) Alcohol consumption and plasma

concentration of C-reactive protein. Circulation, 107, 443 - 447.

Alho, H., et al. (2004). Alcohol Misuse increases serum antibodies to oxidized LDL and C-

reactive protein. Alcohol & Alcoholism, 39 (4), 312 - 315.

13
Imhof, A., et al. (2004) Overall alcohol intake, beer, wine, and systemic markers of inflammation

in Western Europe: Results from three MONICA samples (Augsburg, Glasglow, Lille).

European Heart Journal, 25, 2092 - 2100.

Mukamal, K.J., et al. (2006). Alcohol consumption and risk of coronary heart disease in older

adults: the Cardiovascular Health Study. J Am Geriatr Soc., 54, 30 - 37.

Tsumara, K., et al. (1999). Daily alcohol consumption and the risk of type 2 diabetes in Japanese

men. Diabetes Care, 22, 1432 - 1437.

Ajani, U.A., et al. (2000). Alcohol consumption and risk of of type 2 diabetes mellitus among US

male physicians. Arch Intern Med. 50, 2390 - 2395.

Hu, F.B., et al. (2001). Diet, lifestyle, and the risk of type 2 diabetes mellitus in women. New

England Journal of Medicine, 345, 790 - 797.

Wannamethee, S.G., et al. (2003). Alcohol drinking patterns and risk of type 2 diabetes mellitus

among younger women. Arch Intern Med., 163, 1329 - 1336.

Ogge, L.E., et al. (2006). Alcohol consumption in relation to metabolic regulation, inflammation,

and adiponectin in 64-year-old caucasian women: a Population-based study with a focus on

impaired glucose regulation. Diabetes Care, 29, 908 - 913.

Lazarus, R., et al. (1997). Alcohol intake and insulin levels: the Normative aging study.

American Journal of Epidemiology, 145, 909 - 916.

Sierksma, A., et al. (2002). Moderate alcohol consumption reduces plasma C-reactive protein

and fibrinogen levels; a randomized, diet-controlled intervention study. Eur J Clin Nutr., 56,

1130 - 1136.

Spranger, J., et al. (2003). Adiponectin and protection against type 2 diabetes mellitus. Lancet,

361, 226 - 228.

14
Shai, I., et al. (2004). Moderate alcohol intake and markers of inflammation and endothelial

dysfunction among diabetic men. Diabetologia, 47, 1760 - 1767.

Thamer, C., et al. (2004). Relationship between moderate alcohol consumption and adiponectin

and insulin sensitivity in a large heterogenous population. Diabetes Care, 27, 1240.

Foy, C.G., et al. (2005). Smoking and incidence of diabetes among US adults: findings from the

Insulin Resistance Atherosclerosis Study. Diabetes Care, 28, 2501 – 2507.

Sargeant, L. A., et al. (2001). Cigarette smoking and glycaemia: the EPIC-Norfolk Study.

International Journal of Epidemiology, 30, 547-554.

Rimm E, et al. (1995). Prospective study of cigarette smoking, alcohol use, and the risk of

diabetes in men. B M J, 310, 555–559.

Haire-Joshu, D., Glascow, R.F., & Tibbs, T.L. (1999) Smoking and Diabetes. Diabetes Care, 22,

1887 - 1898.

Tracy, R.P., et al. (1997). Lifetime Smoking Exposure Affects the Association of C-Reactive

Protein with Cardiovascular Disease Risk Factors and Subclinical Disease in Healthy

Elderly Subjects. Arteriosclerosis, Thrombosis, and Vascular Biology, 17, 2167-2176.

Fröhlicha, M., et al. (2003). Independent association of various smoking characteristics with

markers of systemic inflammation in men. European Heart Journal, 24(14), 1365-1372.

Fredriksson M.I., et al. (1999).Effect of periodontitis and smoking on blood leukocytes and

acutephase proteins. J Periodontol, 70, 1355–60.

Maestrelli P, et al. (2001). Remodeling in response to infection and injury. Airway inflammation

and hypersecretion of mucus in smoking subjects with chronic obstructive pulmonary

disease. Am J Respir Crit Care Med, 164, S76–80.

Tappia P.S., et al. (1995). Cigarette smoking influences cytokine production and antioxidant

defenses. Clin Sci, 88,485–9.

15
Francus T., et al. IL-1, IL-6, and PDGF mRMA expression in alveolar cells following

stimulation with a tobacco-derived antigen. Cell Immunol, 145, 156–74.

Elenkov I.J., & Chrousos G.P. (2002). Stress hormones, proinflammatory and antiinflammatory

cytokines, and autoimmunity. Ann NY Acad Sci, 966,290–303.

Bazzano, L.A., et al (2003). Relationship between Cigarette Smoking and Novel Risk Factors for

Cardiovascular Disease in the United States. Ann Intern Med. 138, 891-897.

Bakhru, A., & Erlinger, T.P. (2005). Smoking Cessation and Cardiovascular Disease Risk

Factors: Results from the Third National Health and Nutrition Examination Survey. PLoS

Medicine, 2, 0528-0536

Waters, D., et al. (1996). Effects of Cigarette Smoking on the Angiographic Evolution of

Coronary Atherosclerosis Intervention Trial (CCAIT) Substudy. Circulation, 94, 614 – 621.

Eason, S.L., et al. (2005). Diabetes mellitus, smoking, ad the risk for asymptomatic peripheral

arterial disease: whom should we screen? J Am Board Fam Pract, 18, 355 – 361.

Kuzma, J.W., & Bohnenblust, S.E. (2001). The Health Survey and the Research Report. Basic

statistics for health science, (4th ed.). (p. 288). US: McGraw-Hill Education (Asia).

…The Chi-Square Test. (pp. 187 - 203).

16