chapter ii review of literature 2.1 non communicable...

Chapter II Review of Literature

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2.1 Non Communicable Diseases (NCD) and CVD

NCDs are a plethora of conditions which are non-infectious and non-transmissible.

63% of 57 million deaths in 2008 were due to NCD1. CVD which is a component of NCD

accounts for a majority of deaths and disabilities (48%) 1. CVDs include the diseases of

heart, Vessels of the heart, brain and the peripheral vessels 53

. The different types of CVDs

are,

1. CVDs due to atherosclerosis:

CHD or Ischemic heart disease or coronary artery disease (e.g. heart attack)

Cerebrovascular disease (e.g. stroke)

Diseases of the aorta and arteries (e.g. hypertension and peripheral vascular

disease).

2. Other CVDs

Congenital heart disease

Rheumatic heart disease

Cardiomyopathies

Cardiac arrhythmias.

2.1.1 CVD - Global Scenario

CVD accounted for more than 17 million deaths in 2008. 80% of these deaths

occurred in low and middle income countries. More than 3 million of these deaths occurred

before the age of 60 years. The percentage of premature deaths from CVD ranges from 4%

in high-income countries, to 42% in low-income countries. CHD accounted for 7.3 million

deaths and strokes, 6.2 million of the 17 million deaths 53

. The Disability-Adjusted Life


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Year (DALY) is a measure of overall disease burden, expressed as the number of years lost

due to ill-health, disability or premature death. CVD is reported to be responsible for 1,51,

377 million DALYs 53

.

2.1.2 CVD - Indian Scenario

CVD is increasingly becoming a big cause of concern among the health care

fraternity in India as the incidence is skyrocketing every year. By 2015, India is predicted

to have the largest burden of CVD in the world 54

. With CHD being the larger player in

CVD, the government is marshaling the resources to combat this emerging menace. It is

estimated that by 2020, 2.6 million Indians would die due to CHD, and this would

constitute more than half of all CVD deaths. Nearly half of these deaths are likely to occur

in young and middle aged individuals (30-69 years), cutting down the productive life years

of the people 55

.

2.2 Coronary Heart Diseases

CHD is narrowing or a blockage of the arteries that supply the heart resulting in

acute coronary syndrome. CHD is a manifestation of atherosclerosis. Atherosclerosis,

which was once considered a benign condition associated with lipid storage that reduces

the arterial lumen is now believed as a chronic inflammatory condition that starts at a very

young age 56

. The greatest danger with the atheromatous plaque, is its thrombogenic

potential and not just the magnitude of stenosis 57

.

2.2.1 Pathogenesis of Atherosclerosis: Role of Inflammation

Endothelium, the inner most lining of the vessel, in a healthy state will prevent

adhesion of leucocytes and promote fibrinolysis. Hypercholesterolemia, hypertension,


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smoking and insulin resistance can disrupt the endothelium and result in endothelial

dysfunction, which predisposes the arteries to the formation of an atheroma 58

. Oxidized

and glycated LDL-C can gain entry into the arterial wall damaging the endothelial lining

and vascular smooth muscles 56

. Leucocyte adhesion molecules induced by the mediators

associated with this trapped LDL-C, enhance monocytes in blood to adhere to the

endothelial surface. Macrophages (matured monocytes) engulf the oxidized LDL-C and

proliferate inside the intima. This enhances the inflammatory process by releasing

cytokines and enzymes that can damage the extracellular matrix of the artery.

In addition to macrophages, T lymphocytes are also found in the atheroma. T

lymphocytes, one of the major components of cell mediated immunity, gain entry into the

vessel wall by binding to Vascular Cell Adhesion Molecule-1 (VCAM-1), a mediator

involved in the initiation of atherogenesis. T lymphocytes have a vital role in atherogenesis

by releasing pro-inflammatory cytokines. Thus, it is evident that inflammation plays a

critical role in all the stages of atherosclerosis.

2.3 Inflammatory Markers of CHD

Inflammation associated with CHD, is subclinical in nature and can be diagnosed

using various inflammatory markers. The inflammatory markers can be grouped into three

categories namely, cytokines and chemokines, soluble adhesion molecules and acute phase

reactants 16

.

Cytokines are signaling molecules involved in inter-cellular communication and

chemokines are cytokines which has the ability to induce chemotaxis. Interleukin-1β (IL-


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1β), IL-6, IL-8, IL-10, Tumor Necrosis Factor-α (TNF-α) and Monocyte Chemoattractant

Protein (MCP-1) are the substances that have been evaluated for an association with CHD.

E-selectin, P-selectin, Soluble Intracellular Adhesion Molecule-1 (SICAM-1) and

Soluble Vascular Adhesion Molecule-1 (SVCAM-1) are the substances investigated in this

category for an association with CHD.

Acute phase reactants are proteins synthesized by the liver, in response to the

release of IL-6. CRP, SAA and fibrinogen, are the substances investigated in this category

for a possible association with CHD 16

.

2.4 CRP and hs CRP

It is a novel inflammatory marker that is very sensitive for systemic inflammation,

infection, tissue damage and neoplasms 13

. CRP is named after its ability to precipitate the

“C” polysaccharide derived from pneumococcal cell wall 59

. High sensitivity CRP refers

to the assay that has the ability to measure CRP, below the measurement range of most

conventional assays.

2.4.1 Structure and Function

CRP is a nonglycosylated protein that belongs to a family named pentraxins. It

contains five noncovalently bound protomers arranged around a central pore. The

molecular weight of CRP is 118000 Da14

.

The function of CRP is related to its role in innate immunity, where it binds to

phosphocholine expressed on the surface of dead or dying cells, in order to activate the


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complement system. By binding phosphocholine, CRP enhances phagocytosis by

macrophages and enhances the clearance of necrotic and apoptotic cells 60

. Concentration

of CRP can increase by 10,000 fold rapidly and peaks around 48 hours after a stimulus.

With the half- life period hovering around 19 hours, the concentration of CRP is largely

influenced by rate of synthesis.

2.4.2 Clinical use of hsCRP:

Recently the national academy of clinical biochemistry USA, published its practice

guidelines on the emerging biomarkers for primary prevention of cardiovascular diseases.

An expert panel of this organization performed a review on the emerging biomarkers for its

clinical use, predictive value and analytical methods. They identified 10 markers which are

commonly investigated for a potential association with CHD. The markers were hsCRP,

fibrinogen, leucocytes, lipoprotein (Lp) subclasses and particle concentration, lipoprotein

(a), apolipoprotein A-I (apoA-I) and apo B, homocysteine, B-type natriuretic peptide

(BNP), N-terminal pro BNP (NT-pro BNP), and markers of renal function. The results of

the review, showed that hsCRP was the most powerful marker, eligible for clinical use in

predicting cardiovascular risk 11

. This recommendation reinforces the statement for health

professionals by the centre for disease control and American heart association published in

2003 12

.

2.4.2.1 Stability of hsCRP:

Stability of hsCRP over a period of time, was investigated by Glynn and

colleagues, on 8901 participants who were allocated to the placebo group in the JUPITER


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trial. CRP measurements were repeated at 13 weeks, 1, 2, 3 and 4 years. Investigators

found that the concentration remained high throughout the trial duration 61

.

Shemesh and colleagues investigated the reproducibility of hsCRP, with an

intention to determine if this marker can be used as a marker of future CVD in community

based risk screening. They performed the test on an Australian cohort of 70 people

dwelling in the community. Participants were followed up after 829 days. At follow up, it

was observed that the concentration remained stable, and the correlation was better than for

other traditional CVD risk factors 62

. Both trials confirm that the concentration of hsCRP is

consistent and stable for many years.

2.4.3 Measurement of hs CRP

Several methods have been used to measure hsCRP. Radioimmunoassay,

immunonephelometry, immunoturbidimetry, immunoluminometry and Enzyme Linked

Immuno Sorbent Assay (ELISA) are the methods 16

. The national academy of clinical

biochemistry recommends clinicians and researchers to express hsCRP results in mg/L 11

.

The Centre for disease control and prevention along with American heart association has

recommended the following guidelines for the measurement of hsCRP,

a. HsCRP can be measured both in fasting and non- fasting state

b. When the test values are more than 10 mg/L the results should be discarded and

test has to be done after 2 weeks.

c. For a reliable estimate, an average of two assays performed 2 weeks apart

should be considered

d. When hsCRP levels are > 10 mg/L on two consecutive tests, search for other

infections and inflammation has to be performed 12

.


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2.4.3.1 Immunoturbidimetry:

The method used in this trial to measure hsCRP is immunoturbidimetry. This is based

on the principle of light scattering. After adding an assay reagent, antibody and antigen

cluster, a monochromatic light is passed through the solution. Some light gets absorbed,

some scattered and some pass through the solution. Turbidimetry measures the decrease in

the intensity of the incident beam. This decrease is proportional to the concentration of the

substance.

2.4.4 Factors affecting concentration of CRP

There are a variety of factors that can influence the concentration of hsCRP. Elevated

blood pressure, obesity, smoking, diabetes, metabolic syndrome, dyslipidemia, hormone

use are the individual characteristics that can increase levels of hsCRP, along with chronic

infection or inflammation. Moderate alcohol consumption, improved fitness, weight loss

and medications like statins, fibrates, niacin, aspirin, and NSAIDs can decrease the level of

hsCRP 12

. The list of factors mentioned here is not exhaustive and there may be other

factors that can potentially influence hsCRP concentration. Extraneous factors which could

cause variation in hsCRP concentration are,

2.4.4.1 Seasonal Variation

A couple of cross sectional studies indicate that there could be seasonal variation in

the concentration of hsCRP 63,64

with an increase during fall and winter as compared to

summer. A longitudinal trial by Chiriboga et.al on 641, white, married and overweight

participants (mean age- 45 years) measured HsCRP at baseline and repeated the

measurement every three months till one year. Mean hsCRP of 1.72 mg/L varied by about


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9% across seasons. The highest values were witnessed in the month of November and

December and the lowest was found in June 65

. Another trial performed on healthy

Japanese workers also had similar results, but hsCRP was expressed in mg/dl as against

mg/L 66

.

2.4.4.2 Diurnal Influence

IL-6 is a cytokine responsible for the synthesis of CRP and has been proven to have

diurnal variation. They were found to be high before bed-time and low in the morning 67,68

.

Thus it was of interest to Meier-Ewer and colleagues to determine if CRP also would

exhibit similar variation. 13 healthy participants (10 males and 3 females) in the age group

of 21-35 years were observed for 24 hours, with eight hours of sleep at night. Blood

samples were withdrawn on an hourly basis and were analyzed. Results of the analysis

revealed that there was no time of the day variation 69

. Clinical implications of this result

would be that, hsCRP can be measured at any time of the day without any concern for

circadian variation.

2.4.4.3 Age and Gender Influence

Age and gender difference in hsCRP concentration has been observed by many

cross sectional studies 70-72

. CRP has been found to increase with age and has been found

to be more in women. The third National Health and Nutrition Evaluation Survey

(NHANES III) data of more than 22,000 individuals show that there is a linear relationship

between age and CRP 70

. As the relationship was found to be strong, the authors generated

an equation to estimate the upper limit of CRP based on age. The equations provided were:

age/50 for males, and age/50 + 0.6 for females. The equations clearly suggest high levels

in women and an increase with age 70

. However, the national academy of clinical


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biochemistry, centre for disease control and prevention and American heart association in

their guidelines do not mention about age adjusted or gender adjusted normal values. This

could possibly be because of a difference that is clinically not significant.

2.4.4.4 Racial Differences in concentration

Data from the Dallas Heart Study, which has 6,101 multi-ethnic participants

showed that black subjects had higher values of hsCRP as compared to whites. This has

been supported by the data from NHANES III, which revealed that black participants had

higher values as compared to Mexican-Americans and non-Hispanic whites 73

.

A systematic review of population based studies by Nazmi and Victora 74

, included

15 studies to assess the ethnic difference in CRP. Fourteen out of 15 trials found an ethnic

variation and the results clearly show that Hispanics and South Asians had the highest CRP

among all ethnicities.

2.4.4.5 HsCRP in Indians:

There is enough evidence gathered from large observational studies suggesting that

the concentration of hsCRP is high in Indians 51,75-78

. Indian Asians living in the United

Kingdom has been found to have 17% higher CRP values as compared to Europeans 75

, the

same has been found on Indians living in the United States as compared to Caucasians 51

and in Singapore as compared to Chinese and Malays 76

.

A recent population based trial, recruited 334 participants with metabolic syndrome

and 342 healthy urban citizens of Chennai to investigate the relationship between

inflammatory markers in people with and without metabolic syndrome. The concentration


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of hsCRP in healthy individuals was found to be high at 2.19 mg/L and was even higher

for people with metabolic syndrome 78

.

2.5 CRP – A Predictor of Risk

Elevated hsCRP has been associated with many non-communicable diseases like

CHD, ischemic stroke, diabetes, hypertension, metabolic syndrome and peripheral artery

disease. The most extensively studied area is the association with risk for CHD. There are

about 25 large observational studies published since the late 90’s which confirm the

independent predictive ability of hsCRP in estimating the risk of CHD.

2.5.1 CRP – Independent Predictive Ability

A landmark study that made hsCRP a powerful predictor of cardiovascular events

was performed by Ridker and colleagues 24

. CRP and LDL were measured at baseline for

29,939 American women. The participants were followed for eight years for

cardiovascular events. The investigators determined relative risk for CVD according to

quintiles of CRP and LDL. Relative risks reported according to increasing quintiles

compared against the lowest quintile of CRP were 1.4, 1.6, 2.0 and 2.3. Similar relative

risks were estimated using LDL too; the values were 0.9, 1.1, 1.3 and 1.5. The differences

between the relative risks measured were statistically significant, suggesting that CRP is a

more powerful marker of risk than LDL.

A meta-analysis of all the large observational studies was done to pool the data and

determine the predictive ability of hsCRP. Investigators found that the people in the top of

the quartile have an odds ratio of about 1.5 for cardiovascular events compared to people at

the lowest quartile 26

.


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2.5.2 CRP in Global Cardiovascular Risk Tools:

Framingham CHD risk score, Systematic Coronary Risk Evaluation (SCORE) and

Reynolds risk score are the tools to evaluate an individual’s cardiovascular risk. These

tools estimate the 10 year absolute risk for cardio vascular events based on the traditional

risk factors (age, gender, diabetes, dyslipidemia, smoking and hypertension) and stratify

the patients to “low risk”, “intermediate risk” and “high risk”. Guidelines for low and high

risk are very clear but not for intermediate risk (people who have a risk of 10-20%).

Moreover, it has been noted that a significant number of people could not be classified

appropriately based on the established risk factors alone 79

. 40% of deaths due to CHD

happen in people with normal lipid profile 10

and a few develop cardiovascular events with

one or less than one risk factor. Thus the search for newer markers of risk continues

unabated. Given the proven ability of CRP as an independent risk predictor, researchers

were keen to investigate if addition of CRP to the global risk assessment scores would

enhance the tool’s sensitivity to identify people at risk.

Two large cohort studies consisting of 3435 and 3006 participants, report that the

addition of hsCRP as a variable in the Framingham risk score, enhances the predictive

ability and help categorize people at different risk categories better 32,80

. Similar results

were observed in the predictive ability of Reynolds risk score too. 10,724 American, non-

diabetic men were followed up for about 11 years and 1294 cardiovascular events occurred

during the study period. Two prediction models, one with traditional risk factors and the

other model that has hsCRP and parental history along with traditional risk factors were

analyzed. The model which had hsCRP and parental history improved the predictive ability

of the tool 31

.


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2.6 CRP – A Potential Risk Factor

Numerous risk factors for CVD have been identified by observational studies and

the number continues to grow. One risk factor that has emerged from being a marker of

CHD to potentially mediate atherosclerosis is hsCRP. Accumulating empirical evidence

indicates that CRP could mediate atherosclerosis at various steps, along with other

traditional risk factors 33

. The following are the potential mechanisms explaining the

atherogenic capability of CRP,

In Vitro analysis proved that elevated levels of CRP, inhibits nitric oxide

synthesis and its bioactivity in human endothelial cells 35

. Inhibition of

nitric oxide can set a cascade of events leading to atherosclerosis.

CRP has been shown to promote monocyte chemotaxis and tissue factor

expression 81,82

. Tissue factor is a potent procoagulant, which can lead to

disseminated intravascular coagulation and ultimately to thrombosis during

inflammatory states.

CRP induces the production of Monocyte Chemoattractant Protein-1 (MCP-

1) in endothelial cells 83

. MCP-1 is a protein responsible for the migration

of monocytes to the site of lesion 5.

Devaraj and colleagues observed that elevated CRP, induces PAI-

1expression in human aortic endothelial cells. PAI-1is a protease inhibitor

that regulates fibrinolysis by inhibiting tissue plasminogen activator.

Increased PAI-1 indicates lowered fibrinolysis and this will lead to

atherogenesis 36,84

.


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A few Mendelian studies question the causative role of CRP in atherogenesis

demonstrating that genetic variation in the CRP gene is associated with lifelong increased

CRP levels, but not with increased risk of atherothrombosis85,86

. However, association

between CRP gene variants and risk of CHD could not be ascertained by the authors of this

study 33

. Till this is established, CRP could be considered a causative factor for

atherosclerosis and a target for therapy.

2.7 Association of CRP with Other Risk Factors

Risk factors of CHD like adiposity, physical inactivity and aerobic capacity have

been studied for association with hsCRP. Association between these variables are briefly

explained in separate sections under this heading

2.7.1 Adiposity and hsCRP:

Adiposity is measured clinically by measuring BMI, waist circumference, waist hip

ratio and fat percentage. BMI was reported to have a positive correlation with hsCRP in

many trials 46,47,87-90

with the correlation coefficient ranging from 0.24 to 0.55. Waist

circumference and waist hip ratio (measures of central obesity) had a similar relationship,

with the coefficient ranging from 0.27 to 0.47 and 0.00 to 0.33 respectively. Association

between fat percentage and hsCRP was also investigated in two trials. The strength of the

relationship was reported to be 0.60 and 0.6147,91

.

An editorial review by John Hopkins centre for prevention of heart disease, that

included 14 cross sectional trials reported that the strength of the association between

hsCRP and adiposity ranges from 0.40 – 0.6187

. In summary, the measure that had

consistently good association with hsCRP was fat percentage, followed by BMI and waist


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circumference. The measure that had a poor correlation was waist hip ratio. Measures that

had good correlation can be considered as surrogate markers of systemic inflammation.

Three out of six trials mentioned above were done in India, and results from a cumulative

sample of 850 Indians is no different than the one reported.

2.7.2 Physical activity and hsCRP:

Cross sectional studies that evaluated the relationship between these two variables

consistently report that increased physical activity is associated with lower levels of CRP.

Plaisance and Grandjean reviewed 12 observational trials and found that CRP was 6 – 35%

lower in individuals with high levels of physical activity 42

. Albert and colleagues

categorized men and women based on the frequency of physical activity, as those involved

in activity less than once a week, 2 -3 times a week and more than 4 days a week. They

found decreasing levels of CRP with increasing frequency 92

. This relationship is further

strengthened by another large multi-ethnic study on 1335 post-menopausal women, which

concluded a strong inverse relationship between these two variables 93

. However, Rawson

and colleagues did not find any association (r = 0.02) between these two variables 46

. This

is the only trial to have discordant results; one of the probable reasons for not eliciting the

expected response could be that it had only 109 participants as against large observational

studies.

2.7.3 Aerobic Capacity and hsCRP:

Aerobic capacity or cardiorespiratory fitness, is measured using exercise testing

and is expressed as Vo2 max or Vo2 peak (Maximal or peak oxygen uptake) or as

Metabolic Equivalents (METs). Higher Vo2 max / Vo2 peak or METs indicate greater


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capacity. It has been demonstrated, that aerobic capacity has an inverse relationship with

CHD and All-cause mortality through numerous epidemiological trials. Information

pertaining to association

between hsCRP and aerobic capacity is not as exhaustive as the former. However, all the

studies have consistently reported that CRP values were lower in individuals with higher

aerobic capacity 48,49,91,94-96

. This association persisted even after correcting for age, BMI

and other risk factors of CHD.

2.8 Exercise in CHD Prevention

Prevention of CHD is being delivered at three levels. Primordial (prevention of risk

factors), primary (prevention of the first event by treating risk factors) and secondary

(prevention of recurrence) are the levels of prevention. A multifactorial approach that

includes but is not limited to, diet, exercise, medications and psycho-social support has

been recommended at all levels of prevention. Improved physical activity has been shown

to independently reduce the risk of cardiovascular events by 35% 39

.

Exercise is a planned and structured physical activity that involves repetitive bodily

movement that is done to improve or maintain one or more components of physical fitness

97. Each MET improvement that happens with exercise training has the potential to reduce

the risk of cardiovascular events by 15% 40

. Exercise exerts its cardio-protective influence

through a variety of mechanisms 43,98

, one of the mechanisms hypothesized was an anti-

inflammatory effect, which in turn is anti-atherosclerotic.


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2.8.1 Exercise and CRP – Acute Response

Exercise is a physiologic stimulus that has a potential to disrupt homeostasis. The

human body responds in a variety of ways to maintain or restore homeostasis. This section

discusses the acute changes in the concentration of CRP with exercise.

Search strategy used for retrieving information regarding this section is:

acute[All Fields] AND response[All Fields] AND ("exercise"[MeSH

Terms] OR "exercise"[All Fields]) AND CRP[All Fields].

Changes in the concentration of CRP with a single session of exercise was of

interest to researchers to ascertain if the reduction found in the longitudinal studies

happened because of training or the result of the last exercise bout before measurement.

CRP has been documented to increase slightly with acute exercise 99

. This slight increase

has been independent of the changes in IL-6. Cytokine IL-6 has been shown to increase to

as high as 25 folds with strenuous exercise but CRP does not seem to increase

proportionately with it 100-102

. Plaisance et.al reported that the concentration of CRP

remained unaltered after a single session of exercise 103

.

A recent trial by Mendham and colleagues which investigated the effect of mode

and intensity of exercise reported that vigorous intensity exercise altered the CRP levels

marginally but not the mode of exercise. Another important finding by Tomaszewski et.al

was that ICAM-1 and E-selectin (substances that enhance monocyte adhesion to

endothelium) did not increase with an increase in CRP. This finding has a clinical value as

it implies safety during and after exercise. Acute exercise has been shown to increase the


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levels of anti-inflammatory cytokine IL-10 and cytokine inhibitors like IL-1receptor

antagonist which balances the increase in inflammatory cytokines 99

.

2.8.2 CRP Response to Exercise Training:

Longitudinal studies to investigate the effects of aerobic exercise training on

hsCRP have been conducted ever since CRP was thought to mediate atherosclerosis. We

categorized the trials to randomized and non-randomized controlled trials. The

characteristics of non-randomized trials are interpreted in the following table.

2.8.2.1 Summary of Non-randomized Controlled Trials

Table 2.1 Summary of non-randomized trials

Trials Participants Hs CRP

measurement Exercise training Results

Smith

et.al104

1999

52 participants

with a mean age

of 49 years that

included men

and women

Radial

immunodiffusion

6 months of supervised

exercise program

↓ of CRP by

35%

Okita

et.al105

2004

227 healthy

women with a

mean age of 52

years

Immuno-

nephelometry

Bicycle or treadmill

exercise at 60-80% peak

heart rate for 30-60

minutes, twice a week for

2 months

↓ of CRP

Lakka

et.al106

2004

652, sedentary

and healthy

white men and

women with a

mean age of 35

years

Chemiluminiscent

immunometry

20 week program with an

intensity between 55-75%

maximum oxygen uptake

for 30-50 minutes

↓ of CRP by

1.3 mg/L in

people with

higher

baseline

Kondo

et.al 107

2006

96 healthy

Japanese

women between

the age group of

18-23 years

Commercial assay

7 months exercise

program at an intensity of

60-70% HR reserve for

30-60 min/day for 4-5

days/week

↓ of CRP


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It is very clear from table 2.1 that exercise training appears to lower hsCRP;

however caution must be exercised in interpreting these test results as they were drawn for

non-randomized trials. As with any non-randomized trial the results could have been

influenced by weak internal validity.

2.8.2.2. Summary of randomized controlled trials

The results of the randomized trials are conflicting regarding the reduction of CRP.

A large longitudinal trial which administered 16 weeks of aerobic training on healthy

young women reduced CRP levels significantly 108

. The result of this trial is in line with

the results of Campbell et.al, who found that moderate intensity exercise training

orchestrated a similar reduction in CRP 109

. Barring these two trials most of the other

randomized trials did not observe a notable difference in exercisers as compared to

controls 110-115

. Two recent trials found that exercise training reduces CRP, only if weight

loss is achieved 116,117

. Interestingly another non randomized trial which found a reduction

in CRP with exercise induced weight loss observed that the quartile of patients who had

lost lesser body weight had greater reduction in CRP compared to the quartile which had a

greater reduction in weight loss 105

.

Adding to the uncertainties that prevail over the efficacy of exercise training, two

systematic reviews also paint different pictures. Kasapis et.al 45

in a review of three

prospective trials noted that there is a reduction in CRP but the review by Kelley and

Kelley on five similar trials did not observe any reduction on meta-analysis of the results.

Given the contradictory results from the reviews and the growing number of randomized


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trials we performed a systematic review of all the randomized controlled trials published

till date.

2.8.2.2.1Types of Studies included:

We included all randomized controlled trials which compared the effect of any

mode of aerobic exercise training (walking, jogging, running, cycling rowing or cross

training) with no exercise, other forms of exercise, diet or education. Moreover we

included studies which had training for more than 4 weeks.

2.8.2.2.2 Types of Studies Excluded:

We excluded all trials if they had included patients with systemic illness or an

athletic population. Moreover we excluded trials if they had chosen a method of non-high

sensitivity CRP measurement or has expressed CRP in any other units other than mg/L.

2.8.2.2.3 Search strategy for identification of studies:

The following search strategy was used to run searches in PUBMED, SCIENCE

DIRECT, CINAHL and OVID SP.

"Exercise"[All Fields] AND "C Reactive Protein"[All Fields] AND

("humans"[MeSH Terms] AND (Clinical Trial[ptyp] OR Meta-Analysis[ptyp]

OR Randomized Controlled Trial[ptyp] OR Review[ptyp]) AND English[lang]

AND "adult"[MeSH Terms])

Methodological quality of included trials was assessed by the Cochrane’s “Risk of

bias tool”. Six potential sources of bias which can influence the validity of results

identified by Cochrane collaboration are random sequence generation, allocation


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concealment, blinding of participants and personnel, blinding of outcome assessment,

incomplete outcome data and selective reporting. These sources can introduce selection,

performance, detection, attrition and reporting bias respectively. These sources were

assessed individually and reported as “Low risk”, “High risk” and “Unclear risk”.

Justification for this judgement is also mentioned along with. Data was extracted from

each included trial. When sufficient information was unavailable in the studies, authors

were contacted for further clarification. We used Review Manager 5.1 endorsed by the

Cochrane Collaboration for this systematic review.

2.8.2.2.4 Summary of the results of systematic review

13 randomized controlled trials were included for this review. Characteristics of

every study is mentioned in brief from table 2.2 to table 2.14 and its risk of bias table with

justification is displayed from table 2.2.1 to 2.14.1. Risk of bias graph and summary of all

the trials are included in the final paragraph of this section. One of the limitations of this

review is, the identification of studies, data extraction and risk of bias assessment were

performed by a single researcher.


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Table 2.2 Rauramaa et.al 2004 118

Methods 6 year randomized, active controlled, outcome assessor blind, two arm,

repeated measures trial. Participants 140 middle aged men (mean age – 57 years) were randomly allocated to

exercise and control group Intervention Experimental group

Frequency: First 3 months - 3 days/week and progressed to 5 days/week

Intensity: 40-60% VO2 max

Duration: First 3 months - 30-45 minutes and later 45-60 minutes

Mode: Walking, jogging, cross country skiing, swimming, cycling

Control group: Maintained the usual level of physical activity Outcomes Primary outcome measure: Carotid artery intima media thickness

Secondary outcome measure: CRP (commercial immunoassay), Ventilatory

threshold Notes Period of trial: 1994-1995 to 2001-2002

Setting: University of Kuopio and Kuopio university hospital

Table 2.2.1 Risk of Bias Table for Rauramaa et.al

Bias Authors' judgement Support for judgement

Random sequence

generation Low risk

Not mentioned, but allocation

concealment was explained adequately

Allocation concealment Low risk "Sealed opaque envelope"

Blinding of participants and

personnel Low risk

Not mentioned. Not likely to introduce

bias

Blinding of outcome

assessment Low risk

Outcome assessor was blinded

Incomplete outcome data High risk 20 men lost to follow up. Intention to

treat principle was employed, thus less

likely to result in bias

Selective reporting Low risk Trial protocol not available

Other bias Low risk Appears to be free of other bias


P a g e |28

Table 2.3 Hammett et.al 2004 111

Methods Randomized, active controlled, two arm trial

Participants 61 healthy elderly participants of both gender (mean age of 66 years) were

randomly allocated to exercise and control groups Intervention Experimental group

Frequency: 4 days/ week for 6 months

Intensity: Started gradually and progressed to 80% VO2 max

Duration of session: up to 45 minutes

Mode of training: Not mentioned

Control group: Maintained the usual level of physical activity

Outcomes Primary outcome measure: CRP (High sensitivity assay-Dade Behring Inc)

Secondary outcome measures:VO2 max, Body fat, Trunk fat, Glucose and

LDL-Cholesterol Notes Period of trial: not mentioned

Setting: Green lane cardiovascular services (Auckland city hospital)

Table 2.3.1 Risk of Bias Table for Hammett et.al:

Bias Judgement Support for Judgement

Random sequence generation High risk

Less likely it was performed as another

trial published by the same author had

the same information missing

Allocation concealment High risk

Less likely it was performed as another

trial published by the same author had

the same information missing


personnel Low risk

Not mentioned, however this is not

likely to introduce any bias to the

outcome

Blinding of outcome

assessment Low risk

Not mentioned, however because there

was no change in CRP post

intervention. This could not have

influenced the results

Incomplete outcome data Low risk

Two participants lost to follow up, but

less likely to introduce bias

Selective reporting Low risk All outcomes were reported



P a g e |29

Table 2.4 Hammett et.al 2006 112

Methods Randomized, active controlled, parallel group, two arm trial

Participants 166 women (mean age of 38 years) were randomly allocated to exercise and

health education group

Intervention Exercise training group

Frequency: 3 days per week for 12 weeks

Intensity: 60 - 70% estimated maximal heart rate

Duration of session: 45 minutes

Mode of training: Walking/cycling/rowing

Health education group: Three 45 minutes session each week consisting of

group support and general lifestyle advice

Outcomes Primary Outcome Measures: C-reactive protein, soluble intercellular

adhesion molecule 1, white blood cell count, fibrinogen and soluble CD40

ligand

Secondary Outcome Measures: Maximal oxygen uptake, body weight and

physical activity

Notes Period of trial: not mentioned

Setting: Green lane cardiovascular services (Auckland city hospital)

Table 2.4.1 Risk of Bias Table for Hammett et.al

Bias Judgement Support for judgement

Random sequence

generation High risk

Not mentioned. It appears that it was not

performed as the number of participants in

the two arms were not equal and there is

heterogeneity at baseline

Allocation concealment High risk Not mentioned.

Blinding of personnel Low risk Not mentioned and not needed either

Blinding of outcome

assessment Unclear risk

Not mentioned, however because there was

no change in CRP post intervention.

Incomplete outcome data High risk Significant loss to follow up was present in

both arms

Selective reporting Unclear risk Trial protocol unavailable



P a g e |30

Table 2.5 Huffman et.al 2006 114

Methods Randomized, active controlled, parallel group, multiple arm trial

Participants 193 Sedentary, overweight to mildly obese, dyslipidemic men and women

between 40 - 70 years were allocated to control or one of the three exercise

groups

Intervention Groups and Intensity: Three aerobic exercise groups are low amount-

moderate intensity (40%-55% peak Vo2), low amount-high intensity (65%-80%

peak Vo2) and high amount-high intensity (65%-80% peak Vo2)

Duration: 6 months

Mode: Not mentioned

Control Group: Continued to remain inactive

Outcomes Primary outcome measure: CRP (Enzyme Linked Immunoassay)

Secondary outcome measure: Maximal oxygen consumption, visceral

adiposity and subcutaneous fat, fasting insulin and fasting glucose Notes Period of trial: January 1999 - April 2003

Setting: Duke and East Carolina University

Table 2.5.1 Risk of Bias Table for Huffman et.al


Random sequence generation High risk Randomly assigned to the groups but

there is heterogeneity in the number of

participants in the groups

Allocation concealment High risk Not mentioned. Probably not performed

for the reasons cited above.

Blinding of personnel Low risk Not mentioned and not needed either.

Blinding of outcome

assessment Low risk

Blood samples sent for analysis were

anonymous with respect to treatment

group

Incomplete outcome data High risk Out of 334 participants randomized, 193

was taken for analysis

Selective reporting High risk CRP was not published as an outcome

measure of the STRRIDE study in any of

the previously published literature



P a g e |31

Table 2.6 Stewart et.al 2007 119

Methods 12 Weeks Randomized, active controlled, parallel group, four arm trial

Participants 29 younger (18-35 years) and 31 older (65-85) individuals (men and women)

were randomized to one of the four groups : Young physically active (control),

young physically inactive, old physically active (control) and old physically

inactive

Intervention Exercise Groups: Aerobic training at 70-80% heart rate reserve in the form of

treadmill walking/jogging and resistance training at 70-80% 1 RM, 8

repetitions x 2 set of leg extension, leg flexion, leg press, hip adduction, hip

abduction, chest press, seated row, and lat pull down

Control groups: Continued the same level of physical activity

Outcomes Primary Outcome measure: IL-6, TNF- alpha and CRP

Secondary outcome measures: Weight, body composition and 1 RM

Notes Period of trial: Not mentioned

Setting: Division of exercise physiology. Louisiana State University

Table 2.6.1 Risk of Bias Table for Stewart et.al


Random sequence generation High risk Not mentioned. Probably not done as a

study by the same author in 2010 also

had no mention about sequence

generation.

Allocation concealment Unclear risk Not mentioned. Probably not done,

however this is lesslikely to introduce

bias as the groups were comparable at

baseline.


personnel Unclear risk

Not mentioned

Blinding of outcome


Not mentioned

Incomplete outcome data Unclear risk Loss to follow up not mentioned.

Selective reporting Low risk Appears comprehensive



P a g e |32

Table 2.7 Campbell et.al 2008 110

Methods 12 months Randomized, active controlled, outcome assessor blind, two arm

trial

Participants 202 sedentary men and women (mean age- 55 years) were allocated to aerobic

exercise and control group

Intervention Aerobic exercise group

Frequency: six days in a week

Intensity: 60% - 85% Maximal Heart Rate

Duration of session: 60 minutes per day


Control group: asked not to change their dietary and exercise habits

Outcomes Primary Outcome Measure: CRP (High sensitivity assay on nephelometry)

Secondary outcome measures: Weight, body composition (DEXA) and Vo2

max


Setting: Fred Hutchinson cancer research center, University of Washington

Table 2.7.1 Risk of Bias Table for Campbell et.al


Random sequence generation Unclear risk Not explained clearly

Allocation concealment Unclear risk Not explained clearly

Blinding of participants and personnel Low risk Not performed, not needed

either

Blinding of outcome assessment Low risk Outcome assessor was blind

Incomplete outcome data Low risk Appears to be without loss to

follow up

Selective reporting Unclear risk Study protocol not available


except that there is no

progression in exercise intensity

or duration


P a g e |33

Table 2.8 Campbell et.al 2009 109

Methods One year, Randomized, active controlled, parallel group, two arm trial

Participants 162 post-menopausal women (mean 60 years) were randomly allocated to

aerobic exercise or control group


Frequency: 5 days in a week for one year


Duration of session: at least 45 minutes per day

Mode of training: Supervised and unsupervised walking

Control group: 45 minutes stretching session once weekly

Outcomes Primary Outcome Measures: CRP and Serum Amyloid A (Latex enhanced

nephelometer)

Secondary Outcome Measure: Body weight, BMI and Waist circumference


Setting: Fred Hutchinson cancer research center, University of Washington

Table 2.8.1 Risk of Bias Table for Campbell et.al


Random sequence generation High risk Not explained. Probably not

performed as a similar trial by

the same authors too had the

same inadequacy

Allocation concealment Unclear risk Not explained. Probably not

performed as a similar trial by

the same authors too had the

same inadequacy

Blinding of participants and personnel Unclear risk Not explained

Blinding of outcome assessment Unclear risk Not explained

Incomplete outcome data Unclear risk Very minimal loss to follow up

Selective reporting Unclear risk Study protocol unavailable



P a g e |34

Table 2.9 Camhi et.al 2010 120

Methods One year randomized, active controlled, parallel group, multiple arm trial

Participants 278 participants (149 men with mean age of 49 years and 125 postmenopausal

women) were randomly assigned to control, diet, physical activity and diet

plus physical activity groups

Intervention Control group: Maintained their usual lifestyle

Diet group: Diet based on National Cholesterol Education Program

PA group: Frequency: Three days in a week


Duration of session: 20 minutes later progressed to 45 - 60 minutes

Mode of training: Brisk walking, jogging and running

Diet plus PA group: Received diet and PA as individual treatments

Outcomes Primary outcome measure: CRP (Immunoturbidimetry)

Secondary outcome measures: Body weight and fat percentage

Notes Period of trial: 1992 - 1993

Setting: Stanford Medical School's Prevention Research Centre

Table 2.9.1 Risk of Bias Table for Camhi et.al


Random sequence generation Low risk "Computer generated randomization"

Allocation concealment Low risk Modified Efron procedure was used


personnel Low risk

Not mentioned

Blinding of outcome

assessment Low risk

Not mentioned, however it appears to be

free of detection bias

Incomplete outcome data High risk There is loss to follow up of 99

participants from the pool of 377

Selective reporting Unclear risk This literature appears to be free of

reporting bias but CRP was not

mentioned in the parent article published

in 1998



P a g e |35

Table 2.10 Church et.al 2010 116


Participants 44 men and 118 women who were sedentary were randomly allocated to

aerobic exercise and control group


Frequency: 3 - 5 sessions per week for 4 months

Intensity: 60 - 80% vo2 max (16 K.cal per kg body weight per week)

Duration: 150 - 210 minutes per week

Mode of training: Walking, Cycling

Control group: Maintained the current level of physical activity

Outcomes Primary outcome measure: CRP (Chemiluminiscent immunoassay)

Secondary outcome measures: Vo2 max, BMI, Visceral adiposity and

subcutaneous fat

Notes Period of trial: March 2005 - August 2006

Setting: Pennington Biomedical Research Center, Louisiana State University.

Table 2.10.1 Risk of Bias Table for Church et.al


Random sequence generation Low risk "Computer generated"

Allocation concealment Low risk "Sequentially Numbered Opaque,

Sealed Envelopes"


personnel Low risk

Statistician performed the whole

process of randomization

Blinding of outcome assessment Unclear risk Not mentioned

Incomplete outcome data Low risk 25 participants were lost to follow up,

however intention to treat principle was

used to deal with this bias

Selective reporting High risk Secondary outcome measures

mentioned in study protocol

(interleukin-6 (IL-6), Tumor Necrosis

Factor-alpha (TNF-alpha), and heart

rate variability were not reported.



P a g e |36

Table 2.11 Donges et.al 2010 113

Methods Semi-randomized, active controlled, Multiple arm trial

Participants 102 middle aged me and women were semi-randomly allocated to aerobic

exercise(n=41), resistance exercise (n=35) and control group (n=26)

Intervention Aerobic exercise group: Frequency: not mentioned

Intensity: 70% MHR for the first four weeks and progressed to 75% MHR

Mode and Duration of session: Cycling for 30 minutes for first two weeks

and progressed by 5 minutes every two weeks till 50 minutes

Resistance exercise group: 70% 1RM for four weeks and progressed to 75%

1 RM X 2 sets and 10 repetitions with the rest duration of 120 seconds for the

following exercises: Chest press, shoulder press, lat pull down, seated row, leg

press, leg curl and lunges bilaterally

Control group: Remained sedentary

Outcomes Primary outcome measure: CRP (Turbidimetric immunoassay), IL-6 and

Body composition

Secondary outcome measures: Glucose, Total cholesterol, Triglycerides,

HDL-C, LDL-Cholesterol, TC/HC ratio, BMI, Waist girth, Hip girth, Insulin

and HbA1C

Notes Period of trial: March 2005 - August 2006

Setting: Charles sturt University. Australia.

Table 2.11.1 Risk of Bias Table for Donges et.al


Random sequence


Mentioned as "Semi - Randomized"

Allocation

concealment High risk

Not performed. Reinforced by the variation in the

number of participants among groups

Blinding of

participants and

personnel

Low risk Not performed, but less likely to influence

outcome.

Blinding of outcome

assessment High risk

Mentioned as " all measures were conducted by

the same research team"

Incomplete outcome

data High risk

Loss to follow up not mentioned

Selective reporting Unclear risk Study protocol not available



P a g e |37

Table 2.12 Stewart et.al 2010 115

Methods Six months Randomized, active controlled, parallel group, multiple arm trial

Participants 421 Sedentary, overweight/obese post-menopausal women (mean 57 years)

were randomly allocated to exercise and control group

Intervention Experimental Groups: There are 3 exercise groups with varying intensities

(4, 8, 12 Kcal.kg.week)

Duration: Based on caloric goal

Frequency: Based on caloric goal

Mode: Cycle ergometer and treadmill

Control Group: No change in the level of physical activity

Outcomes Primary Outcome Measure: CRP High sensitivity assay on nephelometer

Secondary outcome measures: Blood glucose, lipids, heart rate variability,

blood pressure, quality of life and body composition

Notes Period of trial: January 2001 to December 2006

Setting: The Cooper Institute Dallas. Texas

Table 2.12.1 Risk of Bias Table for Stewart et.al


Random sequence


Not mentioned. Probably not done

Allocation concealment High risk Not mentioned, probably not done as there

is significant heterogeneity at baseline

Blinding of participants

and personnel Low risk

Not mentioned

Blinding of outcome


Not mentioned

Incomplete outcome data High risk Out of 464 participants recruited, data was

taken only from 421 participants

Selective reporting Low risk Outcome measures were not completely

reported, however the authors have given

the reference for other published data.

Protocol available (NCT00011193)



P a g e |38

Table 2.13 Arikawa et.al 2011108


Participants 391 healthy young women with a mean age of 25 years were allocated to

exercise training and control groups through stratified randomization

Intervention Exercise training group: Frequency: Five days in a week for 16 weeks

Intensity: 65 - 70% MHR for the first 4 weeks, then progressed to 70 -

75 % for the next 4 weeks, 75 - 80% from weeks 8 - 12 and 80 - 85%

from 12 - 16 weeks.

Duration of session: 30 minutes per day


Control group: asked not to change their physical activity or engage in any

new exercise program

Outcomes Primary outcome measure: CRP, Serum Amyloid A, and Leptin (Multiplex

bead array assays)

Secondary outcome measures: Body weight, BMI, Body composition and

METs


Setting: Department of food science and nutrition. University of Minnesota

Table 2.13.1 Risk of Bias Table for Arikawa et.al


Random sequence

generation Low risk

Not mentioned clearly, however the execution

of randomization appears to be good.

Allocation concealment Low risk Stratified randomization performed to achieve

60:40 (treatment - control) in each stratum.


and personnel Unclear risk

Not mentioned and not needed either.

Blinding of outcome

assessment Low risk

Not mentioned, however the analysis was

performed in the biochemistry lab and none of

the authors is a biochemist.

Incomplete outcome

data High risk

There is significant loss to follow up (46 in the

exercise training group and 26 in the control

group)

Selective reporting Unclear risk Study protocol is not available



P a g e |39

Table 2.14 Fisher 2011 et.al 117

Methods Randomized, active controlled, parallel group, three arm trial

Participants 126 Healthy, overweight, premenopausal women were allocated to one of the

three intervention groups: diet only, diet and aerobic training and diet and

resistance training

Intervention Diet only group: 800 kcal diet to meet all nutrient requirement

Diet and aerobic training group: Diet same as the diet only group

Frequency, duration and mode: Walking/running for 3 days per week

for ~ 50 minutes

Intensity: Started gradually at 65% MHR and progressed to 80% MHR

Diet and resistance training group: 10 exercises of the major muscle groups,

three sessions per week at an intensity of 60% 1RM gradually increased to 80%

1 RM. One set of 10 repetitions for the first 4 weeks and progressed to 2 sets

Outcomes Outcome Measures: Body composition and CRP (High sensitivity ELISA kit)


Setting: Bell training facility, University of Alabama in Birmingham

Table 2.14.1 Risk of Bias Table for Fisher et.al


Random sequence

generation Unclear risk

Not mentioned

Allocation concealment Unclear risk Not mentioned


and personnel Unclear risk

Not required

Blinding of outcome

assessment Low risk

It appears that the biochemist was blinded

to the groups

Incomplete outcome data High risk Out of 213 healthy volunteers, results from

126 participants alone were analyzed

Selective reporting Unclear risk Study protocol is not available



P a g e |40

Fig 2.1 Risk of bias graph: Judgement about each risk of bias item presented as

percentages across all included studies

It is obvious from Fig.1 that the existing trials are plagued by one or the other risk

of bias. More than 50% of the included trials have high risk of bias for sequence generation

and incomplete outcome data. About 30% of the trials have high risk of bias for allocation

concealment. Most of the trials have low risk of detection bias as the biochemist was

blinded. Fig. 2 indicates judgement passed on for each trial. From these two figures one

can understand that there is heterogeneity in the methodological rigor of the trials.


P a g e |41

Fig 2 .2 Risk of bias

summary: Judgement

about each risk of bias

item for each included

study


P a g e |42

2.9 Summary of the Review of Literature

CHD is gaining a dubious distinction of becoming the leading cause of death

worldwide and more so in India. Inflammation has been proven to play a critical role in all

the stages of atherosclerosis. HsCRP, a marker of inflammation is now recognized as the

most powerful marker for predicting cardiovascular risk. A growing strength of evidence

through large observational studies suggest that the concentration hsCRP is high in

Indians.

Accumulating empirical evidence indicates that CRP could mediate atherosclerosis

at various steps and thus is considered a therapeutic goal in cardiovascular prevention and

rehabilitation. One of the mechanisms of athero-protective effects of exercise is its anti-

inflammatory function and thus was hypothesized to cool down the flames of

inflammation. The results of longitudinal trials which investigated the effects of exercise

training on hsCRP are conflicting as witnessed by the systematic review we performed.

Adding to the uncertainty the methodological weakness of the existing trials warrant

rigorously controlled clinical trials. Moreover, the parameters of exercise training like

intensity, used in the existing trials are very heterogeneous. So it is unknown, if there is a

dose response relationship between exercise intensity and hsCRP. This information is

crucial to know if there is a threshold dose and an optimal dose of exercise for reducing

hsCRP.


P a g e |43

It would be interesting to observe how exercise training influences sub-clinical

inflammation on young Indian population (who have higher levels of hsCRP and have an

increased propensity to develop CHD early). It has been observed that hsCRP has a linear

relationship with adiposity and physical inactivity and is inversely related to aerobic

capacity. This information is also scant in Indian population.

chapter ii review of literature 2.1 non communicable...

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