DOI: 10.1111/eci.12610

ORIGINAL ARTICLE Association of serum amyloid A and oxidative stress with paraoxonase 1 in sarcoidosis patients Jasmina Ivani
sevi c*, Jelena Kotur-Stevuljevi c*, Aleksandra Stefanovic*, Slavica Spasic*, Violeta Vu
cini c † † Mihailovi c , Jelica Videnovi c Ivanov and Zorana Jelic-Ivanovic* * †

Department of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia, Clinic for Pulmonary Diseases and Tuberculosis, Clinical Centre of Serbia, Belgrade, Serbia

ABSTRACT Background It has been reported that high-density lipoprotein (HDL) particles have anti-inflammatory and antioxidant roles thanks to different enzymes such as paraoxonase 1 (PON1). Under inflammatory and oxidative stress conditions, HDL particles may lose their protective properties. Sarcoidosis is an inflammatory disease characterized by excessive oxidative stress. Serum amyloid A (SAA) is produced in liver and in granulomas, and its concentration increases in inflammatory conditions contributing to increased catabolism of HDL particles. The aim of our study was to determine PON1 activity, SAA concentration and their associations in patients with sarcoidosis. Materials and methods Inflammatory [high-sensitive C-reactive protein (hsCRP), angiotensin-converting enzyme (ACE), SAA], lipid [total cholesterol (TC), HDL-cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), triglycerides (TG)] oxidative stress status parameters [total oxidant status (TOS), malondialdehyde (MDA), pro-oxidant–antioxidant balance (PAB), sulfhydryl (SH) groups] and PON1 activities were determined in serum of 72 patients with sarcoidosis and 62 healthy subjects. Results HsCRP (P < 0
05), TC, LDL-c, TG, SAA, TOS, MDA and PAB (P < 0
001) were significantly higher, whereas HDL-c, SH groups and PON1 activity (P < 0
001) were significantly lower in patients with sarcoidosis when compared with controls. PON1 showed significant association with SAA, MDA and PAB. It was shown that 71% of decrease in PON1 activity may be explained by increase in TOS, PAB and SAA concentration. Conclusions We found decreased PON1 activity and increased SAA concentration in patients with sarcoidosis. Inflammatory condition presented by high SAA was implicated in impaired HDL functionality evident through dysregulated PON1 activity. Excessive oxidative stress was also involved in dysregulation of PON1 activity. Keywords HDL, oxidants, paraoxonase 1, sarcoidosis, serum amyloid A. Eur J Clin Invest 2016; 46 (5): 418–424

Introduction Besides the role in reverse cholesterol transport [1,2], HDL particles have been reported to have anti-inflammatory and antioxidant properties because of the presence of enzymes on their surface such as paraoxonase 1 (PON1), platelet activating factor acetylhydrolase (PAF-AH) and glutathione peroxidase (GPx), as well as apolipoproteins such as apolipoprotein A-I (apo A-I) [1,3]. Human PON1 is synthesized in the liver and secreted into the blood, where it is associated exclusively with HDLs [4]. One of the physiological roles of PON1 is metabolism of lipid mediators created in the state of elevated oxidative stress thus protecting low-density lipoprotein (LDL) particles from oxidation [2,5]. However, under inflammatory conditions, HDLs lose

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their ability to participate in reverse cholesterol transport and to protect LDLs from oxidation. These proinflammatory changes in HDLs are often caused by down-regulation of hepatic PON1 mRNA [6]. Decreased PON1 activity has been associated with increased risk for cardiovascular disease (CVD) development [2,6]. Sarcoidosis is an inflammatory disease characterized by hyperimmune response [7] contributing noncaseating epithelioid granuloma formation with activated CD4+ T cells and macrophages ultimately leading to fibrosis [8]. Local production of cytokines such as tumour necrosis factor (TNF), interleukin 1 (IL-1), IL-12, IL-18 and IL-10 has been found in disease [9]. Proinflammatory cytokines stimulate hepatic production of acute-phase proteins such as serum amyloid A (SAA) [7]. SAA is also produced by activated cells in granulomas and

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PARAOXONASE 1 ASSOCIATIONS IN SARCOIDOSIS

represents innate regulator of granulomatous inflammation in sarcoidosis [9]. Patients with sarcoidosis may be in either active disease or in resolution of disease depending on granuloma activity [8,10]. Several studies reported decreased HDL-cholesterol (HDL-c) concentration [11,12], elevated oxidative stress [11,13] and SAA levels [12,14] in pathogenesis of sarcoidosis. There are two described mechanisms for decrease HDL-c in inflammation: either proinflammatory cytokines may simultaneously up-regulate the expression of SAA and down-regulate the expression of apo A-I [15] or SAA may physically displace apoA-I from HDLs [12] contributing in both ways to increased catabolism of HDL particles [12,15]. In the only study published so far about PON1 in sarcoidosis, Uzun et al. [16] described down-regulation of enzyme activity but this article does not provide any connection between inflammation, oxidative stress and enzyme activity. The relationship between proinflammatory conditions represented by SAA concentration and antioxidative state reflected by PON1 activity had been studied for certain diseases [17]. To our knowledge, there are no data concerning the relationship between SAA and PON1 or relationship between PON1, SAA and disease activity in patients with sarcoidosis. Therefore, the aim of our study was to determine PON1 activity, SAA concentration, their possible interrelationships and relationships with lipid and oxidative stress status parameters in patients with sarcoidosis. PON1 and SAA relationships with markers of sarcoid activity or time to disease resolution have been also explored.

Materials and methods Subjects In this study, we included 72 patients with sarcoidosis (21 males and 51 females, mean age 51
6  10
80 years) from the Clinic for Pulmonary Diseases and Tuberculosis, Clinical Centre of Serbia, Belgrade whose diagnosis was established according to published criteria [8]. We also recruited 62 healthy subjects (27 males and 35 females, mean age 47
9  10
10 years). Patients and healthy subjects were matched according to age and gender. The blood samples were collected at patients regular check-up. Sarcoidosis activity in our group of patients was evaluated using the data of radiographic stage, pulmonary function test results, angiotensinconverting enzyme (ACE) activity, biopsy tests and clinical features, which are all recommended in establishing disease activity [8,10]. Patients were divided according to chest radiography results at 5 stages (0, I, II, III and IV). In our group of patients, there was no stage IV. Results are given in Table 1. According to the previous definition, there were 57 patients with active disease and 15 patients who went to disease

resolution. Mean time to disease resolution was 14  2 months. Exclusion criteria for patients were the presence of cardiovascular disease, pulmonary (any other pulmonary disease except sarcoidosis), neurological, renal, hepatic, endocrine or malignant disease. Including criteria for controls were the absence of any pulmonary, gastrointestinal, hepatic, renal, cardiovascular, malignant, endocrine disease. The study was of cross-sectional type. It was planned according to the ethical guidelines stated in Helsinki declaration. Written informed consent was obtained from all subjects prior to study entry. Research was approved by the local institutional committee (Ethics Board).

Methods After 12-h fasting period, venous blood samples were collected into serum tubes and centrifuged (2500 9 g, 15 min) to obtain

Table 1 Basic characteristics, lung function and lipid status parameters in sarcoidosis and control group

Parameter

Control group (N = 62)

Patients group (N = 72)

Age, year

47
9  10
1

51
6  10
8

0
135‡

Gender (m/f)

27/35

21/51

0
121§

FEV1 (%)†

93–117

43–127 (83  7)

93–112

50–153 (93  4)

DLCO (%)

90–110

41–107 (80  3)

Chest radiographic stage 0/I/II/III/IV (N)

/

22/21/26/3/0

Glucose (mmol/L)

5
26  0
58

5
41  2
33

0
636‡

Urea (mmol/L)

6
30  1
82

5
64  1
65

0
004‡

Creatinine (lmol/L)

88
7  13
2

79
7  14
9

< 0
001‡

Uric acid (lmol/L)

352  93

288  80

< 0
001‡

Total protein (g/L)

69
1  5
0

72
1  7
0

0
003‡

Albumin (g/L)

35
1  1
5

43
8  3
3

< 0
001‡

TC (mmol/L)

5
17  0
89

6
21  1
53

< 0
001‡

HDL-c (mmol/L)

1
63  0
44

1
31  0
43

< 0
001‡

LDL-c (mmol/L)

3
20  0
78

3
97  1
38

< 0
001‡

TG (mmol/L)*

1
23 (1
06–1
39)

1
89 (1
76–2
04)

< 0
001‡

†

FVC (%)

†

P

FEV1, forced expiratory volume in 1s; FVC, forced vital capacity; DLCO, diffuse capacity of the lung for carbon monoxide; TC, total cholesterol; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; TG, triglycerides; N, number of subjects. *Data shown as geometric mean and 95% confidence interval. † Data shown as range (mean  standard deviation). ‡ Student’s t-test. § Chi-square test.

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serum. Samples were aliquoted and stored at 80 °C. Aliquots of serum were thawed immediately before analyses. Forced expiratory volume in 1s (FEV1), forced vital capacity (FVC) and diffuse capacity of the lung for carbon monoxide (DLCO) as lung function parameters were determined using an appropriate method. FEV1 and FVC were measured with a pneumotachograph, DLCO by the single-breath method (Masterlab, Jaeger, W€ urzburg, Germany). The obtained values were presented as a percentage of those predicted based on age and gender. Acros Organics (Geel, Belgium) and Sigma-Aldrich (St. Louis, MO, USA) were suppliers for all the chemicals used. Serum glucose, total protein, albumin, urea, creatinine and uric acid were assayed by routine laboratory methods. The concentrations of lipid status parameters [total cholesterol (TC), LDL-c, HDL-c and triglycerides (TG)] were measured by standard laboratory procedures (ILAB 300+ analyser; Instrumentation Laboratory, Milan, Italy). Latex-enhanced immunoturbidimetry method (Quantex hsCRP kit; BIOKIT, Barcelona, Spain) was used to measure the concentration of high-sensitive C-reactive protein (hsCRP) on ILAB 600 analyser. ACE was determined by colorimetric method with p-hydroxybenzoyl-glycyl-L-hystidyl-L-leucine as a substrate. PON1 activity was determined towards two substrates (paraoxon and diazoxon). Activity towards paraoxon (POase activity) was assessed at 405 nm using ILAB 300+ analyser (Instrumentation Laboratory) while activity towards diazoxon (DZOase activity) was measured at 270 nm using UV/VIS spectrophotometer (Shimadzu, Japan) according to previously published procedure [18]. Paraoxon was purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and diazoxon was purchased from Chem Service (West Chester, PA, USA). The concentration of SAA was determined by commercially available two-site enzyme-linked immunosorbent assay (ELISA) kit (Immunology Consultants Laboratory, Portland, OR, USA). Malondialdehyde (MDA) concentration was determined in the assay previously published by Girotti et al. [19]. Ellman method [20] using 5
50 -dithiobis (2-nitrobenzoic acid) (DTNB) was used to measure the concentrations of sulfhydryl (SH) groups on ILAB 300+ analyser. Total oxidative status (TOS) was determined by Erel’s method [21]. The assay calibration was done using hydrogen peroxide (H2O2) and was performed on ILAB 300+ analyser. The results are expressed in terms of micromolar H2O2 equivalent per litre (lmol H2O2 equivalent/L). Pro-oxidant–antioxidant balance (PAB) was measured according to a previously published method [22]. Different proportions of 250 lmol/L H2O2 and 10 mmol/L uric acid were mixed to prepare standard solutions. Arbitrary HK units,

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which correspond to the percentage of H2O2 in the standard solution, were used to express PAB values.

Statistical analysis To describe the obtained data, we used means  standard deviation for normally distributed data, geometric means and 95% confidence intervals for log-normally distributed data and absolute frequencies for categorical variables. The normality of distribution of studied parameters was assessed using Kolmogorov–Smirnov test. Comparisons of continuous variables having normal distribution were performed using Student’s ttest. Differences between categorical variables were tested with chi-square test for contingency tables. Pearson’s correlation analysis was employed to determine possible correlations between PON1 and SAA and their correlations with oxidative stress, lipid status parameters, markers of sarcoid activity or time to disease resolution. The data obtained for disease activity were used to estimate correlations with PON1 and SAA in patients with active disease, whereas the data regarding disease resolution were used to calculate correlations with PON1 and SAA in patients with sarcoid resolution. For those variables, that followed normal distribution after logarithmic transformation, log-transformed data were used to calculate Pearson’s correlation coefficient. Multiple linear regression analysis (enter selection) was used to estimate the independent contribution of SAA concentration, oxidative stress status and lipid status parameters to PON1 activity. Models in multiple regression analysis included parameters whose P values for Pearson’s correlation coefficient were ≤ 0
1. Multicolinearity between variables was also tested for these models and none of the variables significantly influenced another variable in the models. All statistical analyses were performed using MS Excel, PASW Statistics Version 18
0 (SPSS software) and MEDCALC version 11
4 software. The 0
05 probability level was considered significant in all statistical tests.

Results Basic characteristics, lung function and lipid status parameters are shown in Table 1. FEV1 and DLCO were reduced in patients. FVC did not differ from controls (Table 1). Significantly lower urea, creatinine and uric acid concentrations were found in patients compared with controls. The levels of total protein and albumin were significantly higher in patients. TC, LDL-c and TG were significantly higher in patients than in controls while HDL-c was significantly lower. Inflammatory, oxidative stress status parameters and POase and DZOase activities of PON1 are presented in Table 2. Patients had significantly higher TOS, PAB, hsCRP and SAA when compared with controls and significantly lower POase

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Table 2 Inflammatory, oxidative stress status parameters and POase and DZOase activities of PON1

Parameter

Control group (N = 62)

TOS (lmol/L)* SH groups (mmol/L)

4
31 (3
81–4
88) 0
615  0
087

Patients group (N = 72) 9
78 (8
52–11
22) 0
461  0
146

P† < 0
001 < 0
001

MDA (lmol/L)*

0
32 (0
27–0
38)

1
19 (1
15–1
23)

< 0
001

PAB (HK units)

69
3  28
5

88
3  38
3

< 0
001

POase (U/L)* DZOase (U/L)

475 (404–559) 15089  4457

hsCRP (mg/L)* ACE (U/L)* SAA (lg/mL)*

1
11 (0
87–1
41) / 2
60 (2
03–3
33)

241 (197–295)

< 0
001

13732  4851

0
226

1
63 (1
37–1
93)

0
020

49
7 (43
5–53
3)

/

9
32 (7
22–12
03)

< 0
001

TOS, total oxidant status; SH groups, sulfhydryl groups; MDA, malondialdehyde; PAB, prooxidant antioxidant balance; POase, paraoxonase activity of PON1 (paraoxonase 1); DZOase, diazoxonase activity of PON1; hsCRP, high-sensitive C-reactive protein; ACE, angiotensin-converting enzyme; SAA, serum amyloid A; N, number of subjects. *Data shown as geometric mean and 95% confidence interval; † Student’s t-test.

and SH groups than controls. DZOase was lower in patients but the difference did not reach statistical significance. To determine the relationship between PON1 activities and lipids, oxidative stress status and inflammatory parameters, we have performed Pearson’s correlation analysis. In patients, POase activity was in significant positive association with TC, LDL-c and HDL-c. Also, we revealed that DZOase activity showed significant negative correlations with TOS, SAA, PAB and MDA. In the control group, POase activity correlated significantly with MDA (q = 0
293, P = 0
025), whereas we did not find any significant correlation between DZOase activity and other parameters (Table 3). Pearson’s correlation analysis was also performed to examine POase and DZOase activity of PON1 and SAA with markers of sarcoid activity or time to disease resolution. No significant correlations were obtained between these parameters (data not shown). As we noticed the decrease in PON1 in patients compared with controls, we performed multiple regression analysis to find possible independent influence of inflammatory, lipid and oxidative stress parameters to change in PON1 activity. Decrease in POase activity in patients with sarcoidosis was independently influenced by change in LDL-c concentration (b = 0
694, P = 0
040, adj. R2 = 0
330) when TC, HDL-c and LDL-c were included in the model.

To find which of examined parameters associated independently with decrease in DZOase activity, we created three different models. Model 1 included TC, HDL-c, SAA, TOS, PAB and MDA, model 2 consisted of lipids, SAA and TOS, whereas model 3 had SAA and oxidative stress parameters but without lipids. Decrease in DZOase activity was found to be associated with increase in SAA concentration for all three models (Table 4). According to our results, 71% of decrease in DZOase activity may be explained by increase in TOS, PAB and SAA concentration (model 1). Then, 48% of variation in DZOase could be due to the variations in TOS and SAA (model 2), whereas 46% of variation may originate from the variations in PAB and SAA (model 3). Multiple regression analysis showed that, in control group, only POase activity independently associated with MDA (b = 0
320, P = 0
008, adj. R2 = 0
220) when DZOase and MDA were inserted in model.

Discussion In our current study, we determined PON1 activity and SAA concentration in sarcoidosis. Both POase and DZOase activities of PON1 were lower, and SAA was higher in patients with sarcoidosis compared with controls (Table 2). In patients, significant correlation was noted among POase activity and lipid status parameters (Table 3). DZOase activity was significantly associated with oxidative stress status and SAA concentration (Table 3) with these parameters being independently associated with DZOase in multiple regression analysis (Table 4). Sarcoidosis is an inflammatory disease characterized by higher values of hsCRP, ACE [7,11,23] and SAA compared to controls [12,14]; these were findings also obtained in our study (Table 1). As it is partly mentioned previously, increased concentrations of SAA may be a consequence of increased production of proinflammatory cytokines that stimulate hepatic production of SAA [7]. Activated cells in granulomas also produce SAA that further regulates local granulomatous inflammation [9]. Increase in SAA synthesis in inflammatory processes may be accompanied by decreased apo A-I synthesis. These two processes may be regulated by inflammatory cytokines that simultaneously increase the expression of SAA and reduce expression of apo AI [15]. Also, physical displacement of apo AI by SAA may occur leading to reciprocal relationship between these markers [10,12]. Subsequently, no matter of mechanism, increased catabolism of HDL particles may be noticed [10,12,15]. Additionally, lipoprotein lipase activity may be dysregulated by inflammatory cytokines thereby promoting increase of LDL-c and TG concentrations [12]. These mechanisms, at least in part, may contribute to dyslipidemia found in our patients group and characterized by significantly lower

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Table 3 Pearson’s correlations of lipid, inflammatory, oxidative stress status parameters with POase and DZOase activity in patients group POase Parameter*

r

DZOase P

r

P

Model Model 1 (adj. R2 = 0
711)

TC

0
426

0
005

0
288

0
061

HDL-c

0
352

0
045

0
300

0
107

LDL-c

0
432

0
019

0
270

0
150

0
253

0
110

0
068

0
670

ACE

0
110

0
592

0
007

0
970

hsCRP

0
035

0
882

0
027

0
910

SAA

0
009

0
955

0
338

0
023

TOS

0
140

0
355

0
255

0
080

0
025

0
865

0
189

0
194

TG

SH groups MDA PAB

0
064

0
667

0
272

0
040

0
039

0
788

0
425

0
002

TC, total cholesterol; HDL-c, high-density lipoprotein cholesterol; LDL-c, lowdensity lipoprotein cholesterol; TG, triglycerides; ACE, angiotensin-converting enzyme; hsCRP, high-sensitive C-reactive protein; SAA, serum amyloid A; TOS, total oxidant status; SH groups, sulfhydryl groups; MDA, malondialdehyde; PAB, prooxidant antioxidant balance. *For those variables that followed normal distribution after logarithmic transformation, log-transformed data were used to calculate Pearson’s correlation coefficient.

HDL-c concentration and higher TC, LDL-c and TG when compared with controls (Table 1). Increased oxidative stress with diminished antioxidative protection is expected to be found in sarcoidosis as a result of ongoing inflammatory process [11,13]. Recruited inflammatory cells at disease sites are capable of producing reactive oxygen and nitrogen species contributing to imbalance between oxidants and antioxidants [24,25]. We also found higher levels of TOS, MDA and PAB and lower levels of SH groups in patients than in control group. The results of previous studies have shown that inflammation, altered lipoprotein profile [1,6] and increased oxidative stress [6,26] may be associated with decreased PON1 activity. It was also stated that apo AI could be modified by reactive oxygen species [6] indicating complex interrelationship among inflammation, oxidative stress and dyslipidemia. We found significantly lower POase activity in patients than in controls (Table 1). The only study presenting PON1 activity in sarcoidosis published so far reported similar result [16]. Then, we obtained somewhat lower DZOase activity in patients comparing to control group (Table 1). Coronary artery disease (CAD) also had decreased DZOase activity when

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Table 4 Association of DZOase activity with SAA, lipid and oxidative stress status parameters in patients with sarcoidosis according to multiple regression analysis Independent variables (b, significance level)*,† TOS (b = 0
504, P = 0
013) PAB (b = 0
485, P = 0
016) SAA (b = 0
525, P = 0
006) Model 2 (adj. R2 = 0
476)

SAA (b = 0
558, P = 0
007) TOS (b = 0
625, P = 0
003)

Model 3 (adj. R2 = 0
456)

SAA (b = 0
442, P = 0
003) PAB (b = 0
585, P = 0
001)

TOS, total oxidant status; PAB, prooxidant antioxidant balance; SAA, serum amyloid A. Model 1 included TC, HDL-c, TOS, MDA, PAB and SAA. Model 2 included TC, HDL-c, SAA and TOS. Model 3 included SAA, PAB, TOS and MDA. *Model included parameters whose P values for Pearson’s correlation coefficient was ≤ 0
1. † Variables from model that did not have significant association with appropriate parameter were not shown.

compared to controls [6]. To our knowledge, there is still no evidence about PON1 activity towards diazoxon in sarcoidosis. Systemic inflammation and oxidative stress are conditions found in both CAD [6] and sarcoidosis [7,11,13], so similar changes in PON1 activity may also show. POase activity was associated significantly with TC, LDL-c and HDL-c (Table 3). As PON1 is localized on HDL particles [2], it may be expected to find positive correlations between POase and HDL-c. Then, we may suppose that positive relationship between POase and LDL-c could arise from the possibility that as much LDL-c concentration is present, and higher POase activity is necessary to prevent oxidative modification of LDL particles. In our group of patients with sarcoidosis, DZOase was significantly associated with MDA, PAB and SAA (Table 3). To find whether inflammation and oxidative stress may be independently associated with decrease in DZOase activity, we performed multiple regression analysis (Table 4), which has shown that even 71% of variation in DZOase activity may be explained by variations in TOS, PAB and SAA concentration. SAA, either combined with oxidative stress parameters or both lipids and oxidative stress parameters, was independently associated with lower DZOase. Other parameters, such as TOS and PAB also showed independent association with DZOase but this association was less pronounced when compared with that obtained for SAA.

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We could speculate that decrease of PON1 activity in patients could be strongly influenced by elevated SAA concentration. Our finding of inverse correlation between DZOase and SAA was similar to the result observed in patients with metabolic syndrome [17] and rheumatoid arthritis [27] where higher SAA levels may contribute at least in part to lower PON1 activity. SAA and PON1 are both synthesized in liver, but during inflammatory reactions, production of SAA increases, whereas that of PON1 decreases [15]. However, in the study of Kappelle and coworkers [17] regarding metabolic syndrome, it remains unclear whether reciprocal regulation of SAA and PON1, as elicited by proinflammatory cytokines, is also operative in lowgrade systemic inflammation. Nonetheless, these authors [17] indicate that SAA may be implicated in impaired HDL functionality during low-grade systemic inflammation. As there were reports about PON1 stabilization by apo A-I in HDL particles [28] and about decrease of apo A-I synthesis [15] and its displacement from HDLs in inflammation [14,29], we could speculate that loss of apo A-I could contribute to decrease in PON1 activity too. Some studies suggested PON1 having Nterminal hydrophobic sequence that may facilitate its association with HDL-associated phospholipids [28,30]. Apo AI may either directly bind PON1 [30], or these two proteins may be interconnected by these phospholipids [28]. Besides apo A-I, hydrophobic environment of HDL may be crucial for PON1 activity [3,30]. As suggested by Tanimoto and coworkers [27], composition of HDL particles may be modified during inflammation leading to disturbance in the active site of PON1 and subsequent decreased activity. In addition, decreased PON1 activity may be also attributable to increased oxidative stress as PON1 has an antioxidative role and there are studies that concern different conditions, reporting exhausting of PON1 antioxidative capacity as a consequence of excessive oxidative stress [6,26,27]. Then, the absence of correlations between PON1 and inflammatory or oxidative stress markers in healthy subjects may indicate that inflammation, a key process occurring in sarcoidosis, may have a significant further impact on lipoprotein metabolism in these patients. Our study showed that PON1 activity and SAA did not have significant associations with disease activity markers or time to disease resolution. In the study of Koutsokera et al. [13], it was similarly demonstrated that levels of oxidative stress did not correlate significantly with lung function parameters, chest radiographic stage or dyspnoea. The possible explanation for this lack of significance, as stated in the previous study [13] could be attributed to the fact that oxidative stress could reflect the ongoing (during sample collection) pathophysiological status, whereas clinical parameters were consequences of previously occurring processes. Additionally, Bargagli et al. [14] showed that SAA correlated neither with lung function

parameters (with an exception of FEV1) nor radiographic stage of disease. Our study has the limitations in the way that it lacks replication cohort, so future investigations should firmly establish possible relationship between PON1 activity and inflammation, oxidative stress and lipid status as well as markers of disease activity. The replication has been proposed as a research practice that may help increase the proportion of true research findings [31]. In conclusion, we found decreased PON1 activity and increased SAA concentration in patients with sarcoidosis. Inflammation presented by high SAA concentration was implicated in impaired HDL functionality evident through dysregulated PON1 activity. Excessive oxidative stress was also involved in dysregulation of PON1 activity. Finally, decreased PON1 activity, observed in our group of patients, may be linked to potentially increased risk for CVD development in these patients. Acknowledgements This work was financially supported by the Ministry of Education, Science and Technological Development, Serbia (Project number 175035). Conflict of interest The authors declare that there is no conflict of interest. Contributions JI contributed to acquisition of data, conception and design, analysis and interpretation of data, drafting the manuscript; JKS contributed to acquisition of data, conception and design, drafting the manuscript; AS contributed to acquisition of data, conception and design, analysis and interpretation of data, drafting the manuscript; SS conception and design, critical revision of the manuscript; VVM contributed to acquisition of data, conception and design, critical revision of the manuscript; JVI contributed to acquisition of data; ZJI contributed to conception and design, critical revision of the manuscript. Address Department of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, 11221 Belgrade, Serbia (J. Ivani
sevic, J. K. Stevuljevic, A. Stefanovic, S. Spasic, Z. J. Ivanovic); Clinic for Pulmonary Diseases and Tuberculosis, Clinical Centre of Serbia, 11000 Belgrade, Serbia (V. V. Mihailovic, J. V. Ivanov). Correspondence to: Jasmina Ivani
sevic, Department of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia. Tel.: +381 11 39 51 265; fax: +381 11 39 72 840; e-mail: [email protected]

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