Professional Documents
Culture Documents
Individuals
A Research Proposal
Bernido, Julevi P.
December 8, 2009
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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
the Filipino population). With these figures, Diabetes Mellitus ranks 9th among the 10 leading
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
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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
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
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
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
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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
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
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
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
confounder, since CRP is strongly associated with known causal risk factors for diabetes, such as
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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
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.
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,
1.3 Hypotheses
Based on the foregoing research questions identified, the researchers formulated the
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1. There is no significant relationship between the levels of CRP of the Type
Based on the foregoing research questions identified, the researchers formulated the
Smoking Status and Alcohol Drinking Status among Type 2 Diabetes Mellitus Diagnosed
Individuals
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Smoking and
Alcohol intake
of the type 2 DM
patients
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.
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Studies conducted by Pradhan et al., (2001) and Freeman (2002) further agreed that
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
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
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
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al., (1996) mentioned in their study that passive smoking worsen endothelial function in lipid-fed
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
& 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
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.
procedures, considerable interest has focused on CRP, a marker of inflammation that has been
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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
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
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).
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
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serum marker for both type 2 DM and coronary heart disease (CHD) and the effects of smoking
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
Exclusion Criteria
It was defined by Lindsey (2005) that CRP is one of the first acute phase proteins to rise in
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.
non-specific results.
2. Use of plasma rather than serum can lead to false positive results.
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3. CRP is found to be present after the first trimester of pregnancy and persists until
delivery.
immunosuppressive drugs, including steroids, unless the disease activity is affected and it
recommended that results of the test should be correlated with clinical findings to arrive at
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.
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
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Heavy Drinkers. As used in the study, refer to individuals who drink over 84 ounces per
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
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
caused by either lack of insulin secretion or decreased sensitivity of the tissues to insulin
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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
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
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
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
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decrease in insulin secretion in the pancreas. Moreover, the age of onset of type 2 DM is usually
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.
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)
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
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
The common laboratory tests done by the endocrinologist from SUMC is the fasting
plasma glucose.
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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.)
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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
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
Note: In the absence of unequivocal hyperglycemia and acute metabolic decompensation, these criteria
• Obesity (120% of desirable body weight or body mass index [BMI] of 27 kg/M2.
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• Hypertension (>140/90)
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
When ketoacid concentration rises, the kidneys excrete them into the urine (ketonuria)
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
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occurs because of loss of body protein and also by diminished utilization of carbohydrates for
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
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
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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
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
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
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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
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
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
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conditions. Such an association is consistent with the possibility that the development of diabetes
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
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
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
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
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
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
imaging), blood tests (lipid determinants, CRP), or cardiac function (brain natriuretic peptide),
cardiomyopathy). These tests are becoming more widely used (Braunwald, 2008).
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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
postulated that as an acute phase protein, elevation of plasma CRP may signal the underlying
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,
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dylipedemia, and hyperglycemia, play a proinflammatory role in the initiation of endothelial
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
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
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
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
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
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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
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 ,
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
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
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
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
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
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
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
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,
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
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
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
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
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
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
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
itself (Ross, 1999). Second, certain compounds of smoke such as free radicals (Tappia, 1995)
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).
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).
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).
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
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).
2
1.1 Study Site
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
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
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
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
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.
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
2
1.6 Data Collection and Presentation
All data in this study is based on incidental statistical results. For the purpose of this
Three statistical tables will be incorporated to show analysis of the three variants namely;
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.
Measures of relative variants which express the standard deviation or the result will be
X2 =
Σ (O-E)2
E
The homogeneity test will determine whether the distribution of a particular characteristic
Dummy Tables
Table 3.1.1. Cross Tabulation of Level of CRP and CHD Among Type 2 Diabetes mellitus Individuals
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
Table 3.1.3. Cross Tabulation of Level of CRP and Alcohol Status Among Type 2 Diabetes mellitus Individuals
Table 3.2.1. Cross Tabulation of Level of CRP and CHD Among Type 2 Diabetes mellitus Individuals
Table 3.2.2. Cross Tabulation of Level of CRP and Smoking Status Among Type 2 Diabetes mellitus Individuals
Table 3.2.3. Cross Tabulation of Level of CRP and Alcohol Status Among Type 2 Diabetes mellitus Individuals
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,
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,
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
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
Accessories
Glass slide with six reaction circles, Sample dispensing pipettes, Mixing sticks, Rubber
teat.
Stop watch, Test tubes, A high intensity direct light source, Isotonic saline.
1
TEST PROCEDURE
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
REMARKS
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.
immunosuppressive drugs, including steroids, unless the disease activity is affected and it
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
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.
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
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
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
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