1 Service de Néphrologie et Hémodialyse, 2 Laboratoire de Biochimie, Hôpital Saint André and 3 Laboratoire de Biochimie, Hôpital Pellegrin, Bordeaux, France
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Abstract |
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Methods. We measured plasma PCT levels in 62 patients on maintenance haemodialysis (30 M/32 F, age 61.8±17.1 years, on dialysis for 75±93 months, 12 h/week, with a Kt/V of 1.53±0.31, high-flux membrane being used in 25 patients and low-flux in 37 patients, without reuse). PCT levels were compared with other markers of inflammation and nutritional status, including C-reactive protein (CRP), fibrinogen, interleukin-6 (IL-6), leukocytes, urea, creatinine, albumin, prealbumin, normalized protein catabolic rate (nPCR), haemoglobin (Hb), and epoetin (Epo) doses. Patients were divided into different groups according to their infectious and vascular status.
Results. PCT plasma levels before dialysis were 0.69±0.81 ng/ml. Fifty-seven per cent of PCT values were higher than the upper normal limit of 0.5 ng/ml. CRP and PCT concentrations were high in patients with a current infection, while IL-6 values were elevated in all patients regardless of infection status. Plasma CRP concentrations before dialysis were 21.2±31.4 mg/l, and 70% of these values were higher than the upper normal limit. CRP, PCT, IL-6, and fibrinogen were positively correlated with each other and were all negatively correlated with albumin. Prealbumin was negatively correlated with CRP and IL-6. In the 43 patients treated with Epo, haemoglobin was negatively correlated with IL-6 and Epo doses, while Epo doses were positively correlated with IL-6 but not with CRP or PCT. The 23 patients with both elevated PCT and CRP plasma levels had the lowest Hb, albumin, and prealbumin concentrations, and the highest fibrinogen concentrations and Epo doses.
Conclusion. PCT in haemodialysis patients is positively correlated with currently used markers of inflammation such as CRP and fibrinogen, and negatively correlated with markers of nutritional status such as albumin. The concomitant elevations in PCT and CRP could be more sensitive in the evaluation of inflammation than each marker separately.
Keywords: acute-phase proteins; albumin; C-reactive protein; haemodialysis; inflammation; interleukin 6; procalcitonin
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Introduction |
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The ideal biological marker of inflammation should: (i) be obtained by minimally invasive techniques, (ii) reflect inflammatory status and acute-phase response, (iii) distinguish infectious diseases from non-infectious inflammatory diseases, and (iv) correlate with nutritional markers to provide a predictive parameter of mortality.
Procalcitonin (PCT), a polypeptide of 116 amino acids (MW 13 kDa), is the precursor of calcitonin. The main sites of PCT production remain unknown but there is evidence that they may be in leukocytes or neuroendocrine cells in the bronchial epithelium and the liver [9]. High serum levels of calcitonin prohormone were first described in 1993 by Assicot et al. [10] in patients with sepsis and infection. Since this first report, numerous studies have confirmed PCT as a strong marker (or perhaps a mediator) of inflammation in the fields of infectious diseases, paediatrics and critical care [1113].
PCT has not yet been studied extensively in haemodialysis patients. The objective of this study was to evaluate PCT in haemodialysis patients and its correlation to other traditional inflammatory and nutritional markers such as C-reactive protein, interleukin 6, fibrinogen, albumin, and prealbumin.
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Patients and methods |
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Dialysis procedure
Haemodialysis treatment was performed using bicarbonate-buffered dialysate, glucose at 1 g/l and enoxaparin as an anticoagulant. Haemodialyser membranes were classified as low permeability, n=37 (polysulphone F6 and F7, Fresenius; Caprin-polymer Ambiowet 750, Asahi Medical) or high permeability, n=25 (polyamide Polyflux 14, Gambro; cellulose triacetate Tricea 150 or 190, Baxter), and were not reused. The dialysis dose as Kt/V was calculated according to DOQI guidelines [14]. The mean urea reduction ratio was 72±6%, the mean Kt/V was 1.53±0.31, and the 10th percentile was 1.22. The median Kt/V was 1.33.
The water treatment system consisted of a simple reverse osmosis, disinfected weekly. All dialysis generators were disinfected after each dialysis procedure, two or three times a day. The chemical and biological analyses of dialysis water, performed once per month, were made according to the recommendations of the European Pharmacopoeia (endotoxin <0.25 EU/ml, colony-forming units <100/ml).
Forty-three patients (69%) received Epo, three injections per week, administered subcutaneously at the end of the dialysis procedure. Patients were classified into two subgroups: those treated with Epo (Epo+) and those not treated (Epo-). Other medications included steroid treatment with prednisolone 5 mg/day (four patients, amyloid arthropathy) or 10 mg/day (two patients, lupus erythematosus). No patient was being prescribed other immunosuppressive drugs.
Classification of the patients
On the day of the study, patients were examined and classified into different groups according to their vascular or infectious status.
Infectious status
Patients were divided in two groups according to the presence of a current bacterial infection (Inf+, 18%) or no infection (Inf-). Criteria to define current infection were: temperature before dialysis >38 °C and suspected source of infection (including vascular access) and leukocyte count >12 000/mm3 and antibiotic therapy or positive bacteriological diagnosis (blood, vascular access withdrawal, or sputum or urinary culture). Patients with chronic viral infection were not included in this group (chronic hepatitis B (n=3) or C (n=5); no patient received treatment with interferon and only one patient was HIV positive and received antiretroviral therapy).
Cardiovascular history
Past history of cardiovascular events was evaluated from medical records, identifying two subgroups showing presence (CV+, 63%) or absence (CV) of cardiovascular events. Cardiovascular events noted from medical records were: myocardial infarction (physical examination, enzymatic and ECG changes), coronary artery disease (coronary angiography, previous coronary artery bypass grafting, coronary artery angioplasty), ischaemic stroke (computed tomography), and peripheral vascular disease (claudication, ultrasonography or arteriography). Smoking habits, hypertension, interdialytic weight gain, and diabetes were also evaluated as traditional vascular risk factors, identifying two subgroups (presence or absence).
Other proinflammatory conditions
Conditions that could promote the synthesis of acute-phase reactants were evaluated with appropriate diagnostic techniques, and included cancer (6%), amyloid arthropathy (24%), and vasculitis or connective tissue disease (3%). These were used to identify two subgroups (presence or absence).
Left ventricular mass (LVM)
LVM was evaluated with transthoracic echocardiography, using Devereux's formula [15], and patients were classified as having left ventricular hypertrophy (LVM+) if left ventricular mass was >120 g/m2 (69%).
Analytical procedures
Blood samples were taken from the arterial site of the vascular access before starting dialysis and prior to heparin administration, in the midweek session after a 48-h interval.
PCT
Blood samples were immediately centrifuged. We used the Lumitest PCT kit (Brahms Diagnostica, Berlin, Germany), an immunoluminometric assay for specific measurement of PCT in serum. Two specific monoclonal antibodies bind the PCT at two different sites (calcitonin and katacalcin segments). One of these antibodies is luminescence labelled. After 90 min incubation, luminescence was measured and patients were identified as P+ if detection showed >0.5 ng/ml, and as P- if <0.5 ng/ml. The inter-assay and intra-assay coefficients of variation were respectively 2.5 and 5.3% for PCT values varying from 1.3 to 66.4 ng/ml.
C-reactive protein
C-reactive protein was measured by a nephelometric assay (BN II, Behring) and patients were divided into two groups based on the concentration of CRP being >5 (C+) or <5 mg/l (C-).
Interleukin 6
IL-6 concentrations were measured by enzyme-linked immunosorbent assay (Medgenix IL-6 Easia kit, Biosource, Belgium) using blood samples that had been centrifuged immediately after withdrawal. The upper limit of normal was 10 pg/ml, and two subgroups were identified: IL-6+ (>10) and IL-6- (<10 pg/ml).
Other inflammatory and nutritional parameters
Fibrinogen was measured by a chronometric assay (STA Diagnostica Stago, Fibrinomat, Biomerieux, France), albumin was determined by a bromocresol green dye binding colorimetric assay (RXL, Dade, Behring), and prealbumin by a nephelometric assay (BNII, Behring). Other standard biochemical and haematological laboratory tests were measured routinely. Protein catabolic rate was calculated according to DOQI guidelines [14]. Body weight and body mass index (BMI) were calculated from dry weight on the day of the study.
Statistical analysis
Results and figures are given as means±SD. Comparisons between groups were performed using analysis of variance and Fischer's exact test as appropriate (Statview 5TM, Abacus Concept, Berkeley, CA) for variables with normal distribution, and MannWitney and KruskallWallis tests were used for variables with non-normal distribution, i.e. CRP, IL-6, and PCT. Relationships between parameters were studied by a Pearson's correlation matrix, and significance was calculated using the Fischer's z-test. For non-normally distributed variables, logarithmic transformations were used. All analyses were performed with two-tailed tests, with P=0.05 as the level of significance.
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Results |
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Other inflammatory proteins
CRP serum concentration was 21.2±31.4 mg/l, over 5 mg/l in 70% of the population, and over 10 mg/l in 50%. IL-6 values were 32.1±27.2 pg/ml, and 94% of these concentrations were higher than the upper limit of 10 pg/ml. Patients with a current infectious process had higher CRP levels (42±41 vs 16.7±27 mg/l, P=0.015), but IL-6 concentrations were not significantly different from those in patients without infection.
For other clinical parameters we found no relationships between mean CRP and IL-6 levels and history of cardiovascular diseases, left ventricular hypertrophy, Epo treament, types of nephropathy, types of vascular access (central catheter, fistula or implant) or vascular risk factors such as smoking habits, hypertension, interdialytic weight gain, and diabetes. Nevertheless, patients with peripheral vascular disease were more often classified into the CRP-positive group (2 test, P=0.001), and IL-6 was significantly higher in patients with peripheral vascular disease (P=0.01). No differences in these parameters were found when the various dialysis membranes were compared, or when patients were grouped according to high or low permeability.
Nutritional markers
The main biological results are shown in Tables 1 and 3
. BMI was 22.5±5.1 kg/m2; the 10th percentile was 18.3, and the 90th percentile was 28.9. Serum albumin was 34.0±4.3 g/l and prealbumin was 0.34±0.1 g/l. nPCR was 1.08±0.27 g/kg/day and the 10th percentile was 0.72 g/kg/day. Therefore very few patients could be considered as severely malnourished.
Correlations between biological variables
Table 6 shows the Pearson's intercorrelation matrix between inflammatory, nutritional and haematological parameters. Logarithmic transformations of CRP, PCT, and IL-6 were used.
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Other inflammatory proteins including CRP, IL-6, and fibrinogen were positively intercorrelated and were all negatively correlated with albumin levels. Prealbumin was negatively correlated with CRP and IL-6 only.
Haemoglobin levels were negatively correlated with PCT and IL-6. In the group of 43 patients treated with Epo, haemoglobin was negatively correlated with IL-6 and Epo doses, while Epo doses were positively correlated with IL-6 but not with CRP or PCT (r=0.43, P=0.005).
Sensitivity of the different markers
In order to examine the relationship between the different inflammatory markers and the nutritional or the haematological parameters, we grouped patients into four categories according to their PCT and CRP levels (normal or elevated):
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Discussion |
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Biological markers of inflammation in patients with chronic renal failure
As in previously published studies, we found a high frequency of sub-clinical inflammation in HD patients, with CRP higher than 5 mg/l in 70% of the patients and higher than 10 mg/l in 50% of the patients. Thirty-five per cent of the 1054 patients studied by Owen and Lowrie [8] presented CRP concentrations higher than the upper limit of normal and CRP levels were higher than 8 mg/l in 46% of the patients studied by Zimmerman et al. [6].
IL-6 concentrations were higher than the upper limit of normal in 94% of our patients. Similar results have been found in other studies: IL-6 was higher than normal in 89 of 90 patients in the study of Bologa et al. [7], and in 19 of 32 dialysed patients tested by Herbelin et al. [17]. Moreover, in this latter study, IL-6 was elevated in 10 of 16 not yet-dialysed patients vs only 1 of 23 of control subjects.
The distribution of nutritional markers was the same as in other studies: 53% of patients had albumin levels under 35 g/l and 28% had prealbumin levels under 0.3 g/l. These results accord with recent data obtained in French haemodialysis patients [18]. Markers of inflammation and of nutrition both have been reported to be predictors of mortality in patients with end-stage renal failure. Both CRP and IL-6 serum concentrations were good predictors of death. Bologa et al. [7] have shown that a rise of 1 pg/ml in IL-6 concentration increased the relative risk of death by 4.4%. Similar results were observed with nutritional markers such as albumin or prealbumin [1921] or more sensitive markers like total body nitrogen or reactance [22,23]. Nevertheless, all these studies demonstrated large variations for each marker.
PCT as a new marker of inflammation and infection
Previous studies have shown that PCT is a sensitive marker of systemic inflammation [24]. Experimental kinetic studies have demonstrated that the secretion of PCT occurs within less than 4 h after initiation of sepsis. PCT, which is probably synthesized in the neuroendocrine system, seems to be stimulated by TNF or IL-6 secretion since these cytokines peak before the appearance of PCT in plasma. Very high PCT levels have been described in acute infections, and show a positive correlation with the severity of the sepsis [25].
In chronic inflammatory diseases such as connective tissue diseases, elevated levels have also been described, although at lower concentrations. In these situations there is a discrepancy between CRP and PCT levels, particularly when an infection occurs. In CRF, very few studies have been published. No relationship has been described between serum creatinine and PCT [26]. In addition, little is known about PCT in patients treated with renal replacement therapy. In 43 haemodialysed patients, infection and not the haemodialysis procedure itself seemed to be responsible for raised PCT levels [27]. In another study, Ulrich et al. [28] found moderately elevated PCT levels in haemodialysis patients who showed no signs of systemic bacterial infection, and slightly higher levels with low-flux dialysers compared with high-flux membranes. In their cohort of 63 patients, and contrary to our results, these authors found no relationship between PCT and other inflammatory parameters such as CRP or IL-6 [28]. Concerning PCT elimination by dialysis, the clearance seems to become constant at 2.3 to 3.4 ml/min during a 5 h to 24 h haemodiafiltration session [29].
In this study we found a close relationship between PCT and CRP concentrations, PCT being increased in 57% of our 62 patients. As with CRP, PCT levels were higher in infected patients. Infections could be local or systemic but there was no sign of severe sepsis or septic shock. The well-known correlation between PCT levels and severity of sepsis probably explains why values were slightly higher in the infected group compared with the non-infected group [25]. As well as for CRP, PCT levels remained also higher than the upper limit of the normal in most of the non-infected patients.
Association between PCT and CRP
In the present study, positive associations between various inflammatory markers such as CRP, PCT, IL-6 and fibrinogen, and negative associations with nutritional markers were found (except between PCT and prealbumin). One of the main interests of this study was the complementary use of PCT and CRP. The use of the two markers increased the sensitivity to detect inflammation and strengthened the relationship with nutritional and other inflammatory markers. The subgroup of 23 patients with concomitant elevations of CRP and PCT concentrations had a worse nutritional status, characterized by decreased albumin and prealbumin, and also by decreased haemoglobin levels. Numerous studies have reported a strong association between inflammation, nutritional status, and mortality in dialysis patients [6,8,30]. As shown in Figure 1, albumin and prealbumin levels were higher in patients with isolated PCT increases (P+C-) than in patients with elevated CRP, with or without concurrently elevated PCT (P+C+ and P-C+). Prealbumin levels were also highest in patients with isolated PCT increases. Although these observations must be considered with caution because of the small size of our population, they could suggest that PCT may be a better marker of inflammation than malnutrition, or that PCT could be considered as an acute-phase protein associated with the early phase of inflammatory processes before the occurrence of impaired nutritional status.
Given the influence of inflammation in the pathophysiology of atherosclerosis, [31], the combined evaluation of PCT and CRP could be of major interest as a predictive index of vascular risk. However, we did not find any association between various markers of inflammation and past history of vascular disease or left ventricular hypertrophy; the best way to analyse such relationships would be a prospective study. Contrary to findings reported by other authors [24] we did not find any influence of neoplasm or connective tissue diseases on plasma PCT levels, but our patient groups were too small to draw firm conclusions.
Epo
Inflammation is one of the major factors for Epo resistance. Recently, Gunnel et al. [32] showed in 92 haemodialysed patients that the best predictors of the Epo/haematocrit ratio were albumin, CRP, and ferritin. Barany et al. [33] observed similar results but only when CRP values were higher than 20 mg/l. In our study there was a negative correlation between Epo and sensitive markers of acute-phase inflammation (IL-6 and PCT but not CRP) amongst the 43 Epo-treated patients. The association between PCT and CRP seems to be more reliable since the Epo doses were significantly higher in the P+C+ group than in any other group.
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Conclusion |
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Notes |
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References |
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