DETETCTION LEVELS OF CRPhs IN CHILDREN WITH JUVENILE RHEUMATOID ARTHRITIS
I. V. Romankevych
P.L.Shupik National Medical Academy of Postgraduate Education, Kiev, Ukraine
CRPhs is well recognized marker of vascular damage. He causes in vascular wall numbers of proatherogenic effects. Endothelial dysfunction is a first stage of atherosclerosis. detection for signs of endothelial activation and injury is important because cardiovascular pathology is the leading cause of death in patients with rheumatoid arthritis (RA). In children with juvenile rheumatoid arthritis (JRA) possible presence of endothelial dysfunction in connection with the presence of systemic inflammatory process. Early prevention will reduce the risk of atherosclerosis and prolong life in patients with RA. Determining the level CRPhs performed in 22 children with JRA and 19 control healthy children. CRPhs in healthy children was 0.0084±0.0072 mg/L vs 0.07725±0.05 mg/L in children with JRA (р=0.01). In children with I degree of disease activity CRPhs was around average 0,08±0,035 mg/L, II and III 0,089±0,076 mg/L and 0,144±0,00063 mg/L respectively. In patients with absence of activity of the process level CRPhs responsible 0.0095±0.007 mg/L. CRPhs level is 0.140±0.055 mg/l in children with polyarthritis vs 0.067±0.045 mg/l (р=0.01) in petients with oligoarthritis and 0.12±0.005 mg/l in systemic form vs 0.086±0.04 mg/l in non-systemic disease (р=0.01). Disease duration, age, gender don`tinfluence on CRPhs level.Endothelial dysfunction (endothelium dependent dilation of the brachial artery) was associated with CRPhs (r=-0.92) in children with JRA.
Key words: CRPhs, juvenile rheumatoid arthritis, endotheium, inflammation.
C-reactive protein (CRP) — protein of acute phase of inflammation that plays important role in inflammatory reactions of the body and the immune system. CRP in human body released in two forms: native and monomer. With monomeric CRP associated all properties of acute-phase protein . Among the important biological effects of CRP are the following: activation of the classical complement cascade system, enhance phagocytosis of antigens through connections with Fcreceptors, increased concentration of components of active inflammatory response and activation of immune cells, increased phagocytosis [3,7]. The level of CRP may rise during tissue necrosis, viral and bacterial infections, sepsis, tumor metastasis and rheumatologic diseases .
Intense exercise, sleep disorders, chronic fatigue syndrome, depression, later stages of pregnancy, alcohol consumption, oral contraceptives and hormone replacement drugs may also lead to rising of protein level .
CRP is a very sensitive marker of inflammation. Its concentration can be increased more than 100 times after the onset of inflammation and decreased by 50% during the day while improving condition of the patient .
Since the use of CRP as a marker of inflammation in human body limits of using this laboratory value expanded and acquired new meanings.
Now in general medical practice, acute-phase indicators, including CRP and high sensitivity CRP (CRP high sensitivity, CRPhs) are used not only in the diagnosis of inflammatory processes but also to determine the risk and progression of cardiovascular disease [18,24,34]. Subsequently, the level of CRP was considered as independent sign of cardiovascular risk lesions [14,32]. CRP is used as a biomarker of atherosclerosis . Important is the fact that the protein may determine the risk of cardiac events in asymptomatic individuals with average cholesterol levels in the blood .
The great number of studies have found increased CRP in patients with hypertension, insulin resistance and type 2 diabetes, myocardial infarction, heart failure, ischemic stroke [16,21,28]. Also there is evidence of independent association CRP and atherosclerosis. It is believed that it has a key role in the development of local inflammation in the vascular wall and involved in formation, scarring and destabilization of atherosclerotic plaque [11,23].
Recently actively revised regulations on the risk of vascular damage depending on level of CRPhs. Thus, the level >1 mg/l was associated with atherosclerosis, and people with CRPhs <1 mg/l had the risk of cardiovascular disease. However, studies have revealed that CRPhs provides future risks even if content in blood is <1 mg/l, which increased proportionally with increasing CRPhs .
The basic place of protein synthesis is the liver . Interleukin-6 (IL-6) is the strongest inducer of CRP synthesis in human body, also but in lesser extent, this makes IL-1 . There is little scientific evidence that show that the endothelial cells can produce CRP after stimulation by IL-6 and IL-1 . In atheroma concentration of mRNA CRP more than 10 times higher than the concentration in blood . Consequently, not all amount of acute phase protein «comes» in the vessels from the liver.
The endothelium plays a key role in the interaction between inflammation and development of cardiovascular disease .
Endothelial dysfunction is the first stage of atherosclerotic vascular lesions and initiates its development [2,6,35]. The first step in the development of endothelial dysfunction is activation and damage of endothelial cells . Endothelial dysfunction is manifested not only as disorders of bioavailability of NO (reduction of its synthesis, increased destruction, reducing number of receptors on the surface of endothelial activation which leads to increased production of NO), but also increased synthesis of vasoconstrictor substances, coagulating and pro-atherogenic factors which leads to generalized endothelial dysfunction with development of a violation of its integrity and constrictive vascular changes .
Among the effects of CRP on endothelial cells can be distinguished their activation, dysfunction and damage. V. Pasceri, J.T. Willerson et al. showed 10-fold increased synthesis of adhesion molecules ICAM-1 by endothelial cell after 24 hours of incubation and statistical increasing levels of VCAM-1 and E-selectin after 6-hour incubation in CRP solution with a concentration 10 mg / ml (2000) . By the force of effect CRP on endothelial cells did not differ from the effects of interleukin-1â.
CRP activates endothelial cells via interaction with surface proteoglycans and surface protein FcãRs; further through interaction with lipid cholesterol crosslinks, it enhances the synthesis of cell cytokines and active forms of oxygen . Protein also involved in synthesis by activated endothelial cells monocytic chemo-attractant factor 1 (causing migration, adhesion of monocytes to the intima and penetrating the vessel wall), Vilebranta factor, interleukin-8 .
When various concentrations of CRP in blood may be different effects on vascular endothelium: >5 mg/l have a direct pro-inflammatory effect on endothelium, at concentrations >15 mg/l suppresses differentiation of endothelial progenitor cells and their «survival» .
In addition to local proinflammatory effects CRP reduces NO bioavailability and reduces activity of NO-synthase (NOS) and endothelium-dependent dilatation of blood vessels. Protein inhibits excretion of basal and stimulated NO . Travis W. Heina, Uma Singh et al. during the research of influence of CRP on the functional state of vessels showed a significant reduction of NO, tetra-hydro-pterine, the ratio of eNOS dimer / monomer forms and cGMP and NOS activity.
As damaging action mechanisms authors consider accumulation of superoxide, membrane translocation of p47phox and as a result development activity disorders of NADP oxidase (2009) . However, in study, B.R. Clapp, G.M. Hirschfield et al. found that purified human CRP increases NO synthesis and expression of
GMP-cyclohydrolase-1 in vitro (2005) . CRP also contributes to the enhanced absorption of lipids in the vascular wall macrophages with formation of foam cells, increases the activation of angiotensin II receptor type on smooth muscle cells in vascular wall with increasing of their migration and proliferation, reducing the concentration of prostacyclin and tissue plasminogen activator [11,31].
The regular correlation between CRP and endothelial function detected not only in adults but also in pediatric populations. M.J. Jarvisalo, A. Harmoinen et al. found a significant decrease in endothelial dependent dilation of brachial artery and increasing the thickness of intima-media complex (IMC) of common carotid arteries in healthy children with increasing CRP in blood (2002) . Endothelium-dependent vasodilation of brachial artery, IMC common carotid along with vascular stiffness and calcification of the coronary arteries are recognized markers of vascular damage .
Violation of endothelial function extensively studied in patients with rheumatoid arthritis. It found increasing CRP in adult patients with rheumatoid arthritis . The level of CRP is a sensitive predictor of premature death from cardiovascular disease in patients with arthritis, regardless of disease severity . Discovered a direct connection between levels of CRP and endothelial dilatation of brachial artery in children with juvenile rheumatoid arthritis (JRA) . Also, establishe relation between CRP and thickness of IMC common carotid arteries in patients with RA .
M. Urban, E. Pietrewicz et al. found raising of CRP in children with juvenile idiopathic arthritis compared with healthy children, respectively 1.97 mg/l vs. 0.08 mg/l (2009). Additionally, was established a direct correlation between the value of CRP and IMC common carotid arteries in these patients. In another study, was discovered connection between endothelial brachial artery dilation and CRP levels in children with juvenile
rheumatoid arthritis .
There are sufficient data on the onset of vascular lesions in childhood and young age, which manifests after 30 years of life . In children with JRA additionally present a strong and independent factor for atherosclerosis — chronic inflammation. Therefore, this pathology requires careful studying at early stages for development timely preventive measures. In children with juvenile rheumatoid arthritis data on vascular lesions and communication with their level CRPhs are very limited. Most of studies used only CRP as a marker of inflammation. Practically no research works on CRPhs as a factor of assess state of vascular channel.
Purpose of research: investigate the level of serum marker SRPhs as an independent factor of cardiovascular disease in children with JRA and compare the results with the performance of traditional qualitative determination of protein levels and the value endothelial dependent dilation of the brachial artery.
Materials and methods
We examined 43 children — 24 patients with JRA and 19 healthy. The control group consisted of 10 girls
and 9 boys, the average age 13±1,91 years. Among patients with JRA was 13 (54.2%) girls and 11 (45.8%) boys, the average age of the group was 11,7±2,25 years.
Preferably articular form of JRA was observed in 22 patients (91.7%) with oligoarthritis in 6 (26%) and polyarthritis in 16 (73%), joint and visceral in 2 (8.3%) children. The first level of activity was in 6 (25%), second in 6 (25%), third in 4 (16.6%) children, the lack of activity of the process was observed in 8 (35.4%) children (Figure 1). The average disease duration was 3,26±2,7 years. There was involvement of the knee, ankle, elbow and wrist joints, small joints of the hand. Sick children receive basic treatment drugs: methotrexateand prednisolone, folic acid, NSAID (Voltaren, Movalis). In the absence of inflammatory activity four children (16.6%) did not receive basic treatment drugs.
Determining the level CRPhs was carried using a set of high-sensitivity ELISA for the quantitative determination of human serum CRP production of DIAMEB (USA). Qualitative determination of CRP was conducted by agglutination reaction using latex diagnostics. Additionally, endothelium-dependent dilatation of brachial artery was studied by ultrasonic scanner En Visor 5000 (Philips) according to the recommendations of Noninvasive Assessment of Subclinical Atherosclerosis in Children and Adolescents, Recommendations for Standard Assessment for Clinical Reserch: A Scientific Statement Froom the American Heart Association. Statistical analysis was performed using Microsoft Excel Statistics.
Results and discussion
CRPhs level in healthy children was 0.0084±0.0072 mg/l against 0.07725±0.05 mg/l in children with JRA (p=0.01). The highest protein levels were found in children with III level of JRA activity — an average of 0.144±0.00063 mg/l. In II level of activity averaged protein levels 0.089±0.076 mg/l, I level of activity - 0.08±0.035 mg/l. For inactive process level SRPhs was 0.0095±0.007 mg/l (Fig. 2). We have not found significant differences between the observed CRP in healthy children and patients without activity of inflammatory process (p=0.9), but in patients with I level of activity protein level was statistically higher compared with children without activity or healthy (p=0.0004).
The results showed influence on protein levels not only disease activity, but number of affected joints. So, in children with polyarthritis protein level in averaged was 0.140±0.055 mg/l, and in children with oligoarthritis and monoartrit — 0.067±0.045 mg/l (p=0.01). In children with systemic disease course CRPhs concentration was 0.12±0.005 mg/l, in patients with preferably articular form was at a lower level and reached 0.086±0.04 mg/l (p=0.01).
We didn't establish correlation between CRPhs levels and duration of disease, age and gender. Was also found strong negative connection (r = —0.92) between CRPhs level and magnitude of endothelial dependent vasodilatation of brachial artery in children with JRA, which was on average 8.4%, while in healthy children — 13.1% (p=0.02).
According to recent works of P.H. Dessein, G.R. Norton et al. (2007), who studied the patterns of CRP and CRPhs in patients with RA, in nearly 40% of patients with common test systems can't detect increasing level of protein, which may be below sensitivity level.
The study found increasing CRPhs over threshold determination only in 52% of patients and 8% of patients with remission its level was >8 mg/l .
One of the objectives of our study was to investigate the ratio of CRPhs and qualitative determination of CRP, which is most widely used in medical practice in Ukraine. Qualitative determination of CRP reveals its increase more than 10 mg/ml and with modern immunoturbidimetric techniques — 6 mg/ml in blood serum [4,5].
We divided patients with JRA into four groups on the quantitative level of CRP, expressed in «+», regardless of the form and disease activity. In the first group protein was not detected, in second its level corresponded to «+», in third — «+ +», in fourth — «+ + +».
First group consisted of 14 (53.8%) average value of CRPhs was 0.0388±0,04 mg/l with and minimum value from 0.0015 mg/l to 0.112 mg/l. The second group consisted of 4 (15.4%) children with average CRPhs level 0,0625±0,0361 mg/l (p=0.04). The third group consisted of 2 (7.7%) children with average CRPhs 0,1±0,007 mg/l (p=0.019), the fourth group consisted of 6 (23%) children with average CRPhs 0,1439±0,001 mg/l (p=0.003) (Fig. 3).
As seen from the results in sick children with negative CRP determination of quality method revealed statistically increasing CRPhs compared with the control group, despite the absence an active inflammatory process by results of traditional laboratory research.
1. In children with JRA statistically significant increasing SRPhs compared with healthy children.
2. Increasing CRPhs observed in patients even in the absence and with minimal disease activity.
3. Found a strong negative correlation between CRPhs and development of endothelial dysfunction in children with JRA.
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