International Journal of Rheumatic Diseases 2014
ORIGINAL ARTICLE
Serum soluble toll-like receptor 2: a novel biomarker for systemic lupus erythematosus disease activity and lupus-related cardiovascular dysfunction Maha E. HOUSSEN,1 Rasha H. EL-MAHDY2 and Dina A. SHAHIN3 1
Biochemistry Department, Faculty of Pharmacy, Damanhour University, Damanhour, Egypt, 2Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, Mansoura, Dakahlia, Egypt, and 3Internal Medicine Department, Rheumatology and Immunology Unit, Faculty of Medicine, Mansoura University, Mansoura, Dakahlia, Egypt
Abstract Aim: To assess the serum levels of soluble toll-like receptor (sTLR2) as an endogenous negative regulator of TLR2 signaling in systemic lupus erythematosus (SLE) patients, to investigate the correlation between sTLR2 and SLE disease activity index (SELDAI), SLE-related cardiovascular risk factors and ventricular dysfunction and to evaluate the effect of different therapeutic regimens on serum sTLR2 levels. Methods: Ninety-six SLE patients, along with 30 healthy controls, were enrolled in the study. Echocardiography measurements were performed. Serum levels of (sTLR2) were measured using enzyme-linked immunosorbent assay (ELISA). Serum lipid profiles, uric acid and creatinine were also detected. Results: Mean serum levels of sTLR2 in SLE patients was 3.98 4.4 ng/mL, which was significantly decreased as compared with that of the control group (11.3 4.9 ng/mL; P < 0.0001). sTLR2 was negatively correlated with SELDAI, low-density lipoprotein (LDL) and left ventricular diastolic dysfunction. sTLR2 levels were increased in patients receiving hydroxychloroquine, statins and corticosteroids. Conclusion: Serum sTLR2 can attenuate disease activity and negatively impact left ventricular diastolic dysfunction and hypercholersterelemia in SLE patients. Statins, corticosteroids and chloroquine increase sTLR2 levels. Key words: lupus-related cardiovascular risk factors, systemic lupus erythematosus, TLR2 signaling (sTLR2).
INTRODUCTION Systemic lupus erythematosus (SLE) is an inflammatory disease that mainly affects women and is characterized by the production of antibodies that affect multiple organ systems, including the kidneys, skin, lung, heart, the hematopoeitic system and the brain.1,2 Systemic inflammation and impaired renal function in the
Correspondence: Dr Maha E. Houssen, Associate Professor of Biochemistry, Faculty of Pharmacy, Damanhour University, Egypt. Email: [email protected]
setting of SLE increase morbidity and mortality from cardiovascular diseases.3 Signaling through toll-like receptors (TLRs) mediates the immediate response to microbial challenge through the production of a variety of proinflammatory, cytotoxic and immunoregulatory molecules. However, the excessive TLR-mediated release of some proinflammatory molecules, either by overactivation of the receptor or by dysregulation of endogenous TLR-signaling inhibitory mechanisms, may lead to chronic inflammatory conditions, such as autoimmunity and myocardial dysfunction.4,5 Dynamic regulation of TLR signaling is necessary to prevent chronic inflammation and tissue destruction.5
© 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
M. E. Houssen et al.
A number of negative regulatory mechanisms controlling TLR responses have been reported, including cell membrane-bound TLR suppressors and the naturally soluble TLRs (sTLRs).6 sTLR2, which consists of most of the TLR2 extracellular domain, is released by normal monocytes and is present in plasma and breast milk.7 It is hypothesized that sTLR2 may serve as a negative first-line regulator of TLR2-mediated responses.8 On this basis the aims of the present study were to assess the serum levels of sTLR2 among SLE patients, investigate the correlation between serum sTLR SLE disease activity, lupus-related cardiovascular risk factors and ventricular dysfunction as assessed by echocardiography measurements, and to evaluate the effect of different therapeutic regimens on serum sTLR2 levels.
PATIENTS AND METHODS Study design In a cross-sectional case control analysis, SLE patients who were consecutively seen in or referred to the Rheumatology and Immunology Unit (inpatient and outpatient departments), Mansoura University Hospital, Egypt, were included in the study. The diagnosis of SLE was based on the 1997 America College of Rheumatology (ACR) criteria for SLE classification.9 The systemic lupus disease activity was assessed using the Systemic Lupus Erythematosus disease activity index (SLEDAI).10 Thirty apparently healthy volunteers, non-smokers, non-pregnant, proportionally matched for age and sex to the patient group, were enrolled in the study as controls. The clinical assessment and the echocardiographic examination of the study population were performed at the time of blood sampling, by a rheumatologist and a cardiologist, respectively. The study was approved by the ethics committee of Mansoura Faculty of Medicine, every study participant gave informed consent.
Exclusion criteria Subjects with a history of smoking, diabetes, concomitant heart failure, previous history of myocardial infarction, hepatic or endocrine diseases, were excluded from the study. Pregnant females were not eligible for the present study.
Echocardiographic examination Conventional 2-dimensional Doppler and M mode echocardiography were performed with a MEDISON (Medison Co. Ltd. Seoul, South Korea), model-Sonoace X6,
2
power 100–120/200–240 V-0.8/5A, 50/60 Hz equipped with 2.5–5 MHz transducer. The echocardiographic examination was performed by a single investigator who had achieved level 3 training in echocardiography. The examiner was blinded to the clinical and laboratory characteristics of the participants. All patients were examined in the left lateral decubitus position and data were obtained during post-expiratory apnea. Echocardiographic data were calculated as mean values of three measurements of the cardiac cycle. Standard left parasternal long and short axis and apical two-, four- and five-chamber views were performed in all patients.11 The following morphological parameters were analyzed: left ventricular performance parameters;12 end diastolic diameter (EDD); end systolic diameter (ESD); ejection fraction (EF); left ventricular posterior wall thickness (PWT); interventricular septal thickness (IVST); left atrial diameter (LAD) (end-systolic diameter); left ventricular mass (LVM) utomatic; and LVM index (LVMI). The diastolic function was assessed by analyzing the mitral flow velocity curves: the peak flow velocity in early (E) and late (A) diastole, and their ratio (E/A), the deceleration time of E wave and the isovolumetric relaxation time (IVRT).13
Sample collection Five milliliters of venous blood was withdrawn after 12–14 h overnight fasting from each subject in the study. Each blood sample was divided into three aliquots. The first citrated blood portion was used for determination of Westergren erythrocyte sedimentation rate (ESR). The second aliquot of 1 mL ethylenediaminetetraacetic acid whole blood portion was used for complete blood count (CBC) measurement using an automatic counter. The third aliquot was centrifuged at 2860 g for 10 min to obtain serum. The resulting sera were divided into two aliquots. The first serum aliquot was of 1.5 mL and used for immediate estimation of cholesterol, triglycerides, high-density lipoprotein (HDL), creatinine, uric acid, antinuclear antibodies (ANA), double-stranded DNA (dsDNA) and complement levels. The remaining serum was stored at –70°C till time of assay of sTLR2.
Biochemical analyses Serum triglyceride levels were determined according to the method of Wahlefeld14 using kits provided by Biocon Diagnostic (Hamburg, Germany; detection limit of 0.7 mg/dL). Serum total cholesterol levels were determined according to the method of Allain et al.15 using kits provided by Spinreact (S.A.U. Santa Coloma, Spain with detection limit of 0.6 mg/dL). Serum HDL choles-
International Journal of Rheumatic Diseases 2014
SLE and soluble TLR 2
terol levels were determined according to the method of Assmann et al.16 using Biocon Diagnostic with detection limit of 1.57 mg/dL. Serum low-density lipoprotein (LDL) cholesterol levels were calculated using the formula of Friedewald et al.17 Serum sTLR2 levels were detected by enzyme-linked immunosorbent assay (ELISA) technique18 using bluegene (Shanghai, China with detection limit 0.1 ng/mL). Serum uric acid levels were determined according to the method of Bahram and Trinder19 using kits provided by Spinreact with detection limit 0.03 mg/dL. Serum creatinine levels were determined using kits provided by bioMerieux (Craponne, France with detection limit 0.283 mg/dL.20
Statistical analysis Data entry and statistical analyses were done using SPSS software package version 17 (SPSS Inc., Chicagp, IL, USA). Data were expressed as means SD and frequencies. Comparisons between groups were conducted using independent t-test, one-way analysis of variance and Fisher’s exact test and v2 test when applicable. Pearson’s correlation analysis was used for estimating correlation between variables. Probability levels < 0.05 were considered significant.
RESULTS Clinical and laboratory characteristics of SLE patients with different therapeutic regimens Patients with lupus nephritis totalled 39 (40.6%) from whole SLE group; patients with pericarditis/effusion and those with pleurisy/pleural effusion totalled 22 patients (22.9%) and seven patients (7.3%), respectively (Table 1). Pulmonary hypertension and Raynaud’s phenomenon were found in 26 (27.1%) and 21 (21.9%), respectively. Arthritis and arthralgia manifested in 23 patients (24%). Alopecia was diagnosed in eight patients (8.2%). Patients with psychosis, deep vein thrombosis, cerebral stroke and left ventricular thrombus totalled two (2.1%) for each. Mitral regurgitation and aortic regurgitation were detected in 57 (59.4%) and 12 patients (12.5%), respectively. Meanwhile, tricuspid regurgitation and pulmonary regurgitation were found in 30 (31.3%) and 36 (37.5%) of SLE patients, respectively (Table 1). Laboratory characteristics Elevated titers of ANA and anti-dsDNA were detected in the serum of 82 (85.4%) and 72 (75%) SLE patients, respectively. Hypocomplementenemia was shown in 26 (27.1%) SLE patients (Table 1).
International Journal of Rheumatic Diseases 2014
Table 1 Clinical, immunological and therapeutic characteristics of the studied systemic lupus erythematosus (SLE) patients Comorbidities Nephritis Pericarditis/pericardial effusion Pleurisy/pleural effusion Antiphospholipid syndrome Pulmonary hypertension Raynaud’s Discoid Oral ulcers Arthritis/arthralgia Alopecia Psychosis Deep vein thrombosis Cerebral stroke Left ventricular thrombus Mitral regurge Aortic regurge Tricuspid regurge Pulmonary regurge Laboratory parameters Antinuclear antibody Anti-double-stranded DNA Hypocomplementenemia Therapeutic regimens (current therapy) Steroid therapy 60 mg Hydroxychloroquine Azathioprine Cyclophosphamide pulse
SLE patients n = 96 (100%) 39 (40.6%) 22 (22.9%) 7 (7.3%) 22 (22.9%) 26 (27.1%) 21 (21.9%) 2 (2.1%) 8 (8.2%) 23 (24%) 8 (8.2%) 2 (2.1%) 2 (2.1%) 2 (2.1%) 2 (2.1%) 57 (59.4%) 12 (12.5%) 30 (31.3%) 36 (37.5%) 82 (85.4%) 72 (75%) 26 (27.1%)
26 (27.1%) 40 (41.7%) 30 (31.3%) 32 (33.3%) 21 (21.9%) 20 (20.8%)
Therapeutic regimens Patients enrolled in the study were assigned to different therapeutic regimens. Those receiving steroid therapy with doses < 40 mg totalled 26 (27.1%) SLE patients. Patients receiving steroids with a dose 40– 60 mg, and > 60 mg totalled 40 (41.7%) and 30 (31.3%), respectively. Patients receiving hydroxychloroquine azathioperine and cyclophosphamide totaled 32 (33.3%), 21 (21.9%) and 20 (20.8%) respectively (Table 1).
Clinical characteristics and echo findings in SLE patients and controls SLE patients showed significant increase in left atrium size, interventricular septum, PWT and left ventricular end diastolic diameter (LVEDD) compared to controls
3
M. E. Houssen et al.
Parameters
Patients (n = 96) Mean SD
Left atrium size (cm) SLEDAI Interventricular septum (cm) Posterior wall thickness (cm) Left ventricular mass (g) Left ventricular mass index (g/m2) LVEDD (cm) LVESD (cm) Ejection fraction % E (m/s) A (m/s) E/A ratio DT (ms) IVRT (ms)
3.4 17.5 1.2 1.1 194.4 73.5 4.7 3 61.6 0.9 0.8 1.2 296.6 86.8
0.5 6.3 0.2 0.2 49.6 17.6 0.5 0.6 9.1 0.3 0.3 0.5 139.3 17.3
Control (n = 30) Mean SD
P-value
2.9 0.3 – 0.9 0.1 0.9 0.1 192.7 18 69.9 5.1 3.9 0.3 3 0.3 65.3 6 0.8 0.1 0.5 0.1 1.6 0.2 191.4 17.3 75.3 8.8
ORIGINAL ARTICLE
Serum soluble toll-like receptor 2: a novel biomarker for systemic lupus erythematosus disease activity and lupus-related cardiovascular dysfunction Maha E. HOUSSEN,1 Rasha H. EL-MAHDY2 and Dina A. SHAHIN3 1
Biochemistry Department, Faculty of Pharmacy, Damanhour University, Damanhour, Egypt, 2Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, Mansoura, Dakahlia, Egypt, and 3Internal Medicine Department, Rheumatology and Immunology Unit, Faculty of Medicine, Mansoura University, Mansoura, Dakahlia, Egypt
Abstract Aim: To assess the serum levels of soluble toll-like receptor (sTLR2) as an endogenous negative regulator of TLR2 signaling in systemic lupus erythematosus (SLE) patients, to investigate the correlation between sTLR2 and SLE disease activity index (SELDAI), SLE-related cardiovascular risk factors and ventricular dysfunction and to evaluate the effect of different therapeutic regimens on serum sTLR2 levels. Methods: Ninety-six SLE patients, along with 30 healthy controls, were enrolled in the study. Echocardiography measurements were performed. Serum levels of (sTLR2) were measured using enzyme-linked immunosorbent assay (ELISA). Serum lipid profiles, uric acid and creatinine were also detected. Results: Mean serum levels of sTLR2 in SLE patients was 3.98 4.4 ng/mL, which was significantly decreased as compared with that of the control group (11.3 4.9 ng/mL; P < 0.0001). sTLR2 was negatively correlated with SELDAI, low-density lipoprotein (LDL) and left ventricular diastolic dysfunction. sTLR2 levels were increased in patients receiving hydroxychloroquine, statins and corticosteroids. Conclusion: Serum sTLR2 can attenuate disease activity and negatively impact left ventricular diastolic dysfunction and hypercholersterelemia in SLE patients. Statins, corticosteroids and chloroquine increase sTLR2 levels. Key words: lupus-related cardiovascular risk factors, systemic lupus erythematosus, TLR2 signaling (sTLR2).
INTRODUCTION Systemic lupus erythematosus (SLE) is an inflammatory disease that mainly affects women and is characterized by the production of antibodies that affect multiple organ systems, including the kidneys, skin, lung, heart, the hematopoeitic system and the brain.1,2 Systemic inflammation and impaired renal function in the
Correspondence: Dr Maha E. Houssen, Associate Professor of Biochemistry, Faculty of Pharmacy, Damanhour University, Egypt. Email: [email protected]
setting of SLE increase morbidity and mortality from cardiovascular diseases.3 Signaling through toll-like receptors (TLRs) mediates the immediate response to microbial challenge through the production of a variety of proinflammatory, cytotoxic and immunoregulatory molecules. However, the excessive TLR-mediated release of some proinflammatory molecules, either by overactivation of the receptor or by dysregulation of endogenous TLR-signaling inhibitory mechanisms, may lead to chronic inflammatory conditions, such as autoimmunity and myocardial dysfunction.4,5 Dynamic regulation of TLR signaling is necessary to prevent chronic inflammation and tissue destruction.5
© 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
M. E. Houssen et al.
A number of negative regulatory mechanisms controlling TLR responses have been reported, including cell membrane-bound TLR suppressors and the naturally soluble TLRs (sTLRs).6 sTLR2, which consists of most of the TLR2 extracellular domain, is released by normal monocytes and is present in plasma and breast milk.7 It is hypothesized that sTLR2 may serve as a negative first-line regulator of TLR2-mediated responses.8 On this basis the aims of the present study were to assess the serum levels of sTLR2 among SLE patients, investigate the correlation between serum sTLR SLE disease activity, lupus-related cardiovascular risk factors and ventricular dysfunction as assessed by echocardiography measurements, and to evaluate the effect of different therapeutic regimens on serum sTLR2 levels.
PATIENTS AND METHODS Study design In a cross-sectional case control analysis, SLE patients who were consecutively seen in or referred to the Rheumatology and Immunology Unit (inpatient and outpatient departments), Mansoura University Hospital, Egypt, were included in the study. The diagnosis of SLE was based on the 1997 America College of Rheumatology (ACR) criteria for SLE classification.9 The systemic lupus disease activity was assessed using the Systemic Lupus Erythematosus disease activity index (SLEDAI).10 Thirty apparently healthy volunteers, non-smokers, non-pregnant, proportionally matched for age and sex to the patient group, were enrolled in the study as controls. The clinical assessment and the echocardiographic examination of the study population were performed at the time of blood sampling, by a rheumatologist and a cardiologist, respectively. The study was approved by the ethics committee of Mansoura Faculty of Medicine, every study participant gave informed consent.
Exclusion criteria Subjects with a history of smoking, diabetes, concomitant heart failure, previous history of myocardial infarction, hepatic or endocrine diseases, were excluded from the study. Pregnant females were not eligible for the present study.
Echocardiographic examination Conventional 2-dimensional Doppler and M mode echocardiography were performed with a MEDISON (Medison Co. Ltd. Seoul, South Korea), model-Sonoace X6,
2
power 100–120/200–240 V-0.8/5A, 50/60 Hz equipped with 2.5–5 MHz transducer. The echocardiographic examination was performed by a single investigator who had achieved level 3 training in echocardiography. The examiner was blinded to the clinical and laboratory characteristics of the participants. All patients were examined in the left lateral decubitus position and data were obtained during post-expiratory apnea. Echocardiographic data were calculated as mean values of three measurements of the cardiac cycle. Standard left parasternal long and short axis and apical two-, four- and five-chamber views were performed in all patients.11 The following morphological parameters were analyzed: left ventricular performance parameters;12 end diastolic diameter (EDD); end systolic diameter (ESD); ejection fraction (EF); left ventricular posterior wall thickness (PWT); interventricular septal thickness (IVST); left atrial diameter (LAD) (end-systolic diameter); left ventricular mass (LVM) utomatic; and LVM index (LVMI). The diastolic function was assessed by analyzing the mitral flow velocity curves: the peak flow velocity in early (E) and late (A) diastole, and their ratio (E/A), the deceleration time of E wave and the isovolumetric relaxation time (IVRT).13
Sample collection Five milliliters of venous blood was withdrawn after 12–14 h overnight fasting from each subject in the study. Each blood sample was divided into three aliquots. The first citrated blood portion was used for determination of Westergren erythrocyte sedimentation rate (ESR). The second aliquot of 1 mL ethylenediaminetetraacetic acid whole blood portion was used for complete blood count (CBC) measurement using an automatic counter. The third aliquot was centrifuged at 2860 g for 10 min to obtain serum. The resulting sera were divided into two aliquots. The first serum aliquot was of 1.5 mL and used for immediate estimation of cholesterol, triglycerides, high-density lipoprotein (HDL), creatinine, uric acid, antinuclear antibodies (ANA), double-stranded DNA (dsDNA) and complement levels. The remaining serum was stored at –70°C till time of assay of sTLR2.
Biochemical analyses Serum triglyceride levels were determined according to the method of Wahlefeld14 using kits provided by Biocon Diagnostic (Hamburg, Germany; detection limit of 0.7 mg/dL). Serum total cholesterol levels were determined according to the method of Allain et al.15 using kits provided by Spinreact (S.A.U. Santa Coloma, Spain with detection limit of 0.6 mg/dL). Serum HDL choles-
International Journal of Rheumatic Diseases 2014
SLE and soluble TLR 2
terol levels were determined according to the method of Assmann et al.16 using Biocon Diagnostic with detection limit of 1.57 mg/dL. Serum low-density lipoprotein (LDL) cholesterol levels were calculated using the formula of Friedewald et al.17 Serum sTLR2 levels were detected by enzyme-linked immunosorbent assay (ELISA) technique18 using bluegene (Shanghai, China with detection limit 0.1 ng/mL). Serum uric acid levels were determined according to the method of Bahram and Trinder19 using kits provided by Spinreact with detection limit 0.03 mg/dL. Serum creatinine levels were determined using kits provided by bioMerieux (Craponne, France with detection limit 0.283 mg/dL.20
Statistical analysis Data entry and statistical analyses were done using SPSS software package version 17 (SPSS Inc., Chicagp, IL, USA). Data were expressed as means SD and frequencies. Comparisons between groups were conducted using independent t-test, one-way analysis of variance and Fisher’s exact test and v2 test when applicable. Pearson’s correlation analysis was used for estimating correlation between variables. Probability levels < 0.05 were considered significant.
RESULTS Clinical and laboratory characteristics of SLE patients with different therapeutic regimens Patients with lupus nephritis totalled 39 (40.6%) from whole SLE group; patients with pericarditis/effusion and those with pleurisy/pleural effusion totalled 22 patients (22.9%) and seven patients (7.3%), respectively (Table 1). Pulmonary hypertension and Raynaud’s phenomenon were found in 26 (27.1%) and 21 (21.9%), respectively. Arthritis and arthralgia manifested in 23 patients (24%). Alopecia was diagnosed in eight patients (8.2%). Patients with psychosis, deep vein thrombosis, cerebral stroke and left ventricular thrombus totalled two (2.1%) for each. Mitral regurgitation and aortic regurgitation were detected in 57 (59.4%) and 12 patients (12.5%), respectively. Meanwhile, tricuspid regurgitation and pulmonary regurgitation were found in 30 (31.3%) and 36 (37.5%) of SLE patients, respectively (Table 1). Laboratory characteristics Elevated titers of ANA and anti-dsDNA were detected in the serum of 82 (85.4%) and 72 (75%) SLE patients, respectively. Hypocomplementenemia was shown in 26 (27.1%) SLE patients (Table 1).
International Journal of Rheumatic Diseases 2014
Table 1 Clinical, immunological and therapeutic characteristics of the studied systemic lupus erythematosus (SLE) patients Comorbidities Nephritis Pericarditis/pericardial effusion Pleurisy/pleural effusion Antiphospholipid syndrome Pulmonary hypertension Raynaud’s Discoid Oral ulcers Arthritis/arthralgia Alopecia Psychosis Deep vein thrombosis Cerebral stroke Left ventricular thrombus Mitral regurge Aortic regurge Tricuspid regurge Pulmonary regurge Laboratory parameters Antinuclear antibody Anti-double-stranded DNA Hypocomplementenemia Therapeutic regimens (current therapy) Steroid therapy 60 mg Hydroxychloroquine Azathioprine Cyclophosphamide pulse
SLE patients n = 96 (100%) 39 (40.6%) 22 (22.9%) 7 (7.3%) 22 (22.9%) 26 (27.1%) 21 (21.9%) 2 (2.1%) 8 (8.2%) 23 (24%) 8 (8.2%) 2 (2.1%) 2 (2.1%) 2 (2.1%) 2 (2.1%) 57 (59.4%) 12 (12.5%) 30 (31.3%) 36 (37.5%) 82 (85.4%) 72 (75%) 26 (27.1%)
26 (27.1%) 40 (41.7%) 30 (31.3%) 32 (33.3%) 21 (21.9%) 20 (20.8%)
Therapeutic regimens Patients enrolled in the study were assigned to different therapeutic regimens. Those receiving steroid therapy with doses < 40 mg totalled 26 (27.1%) SLE patients. Patients receiving steroids with a dose 40– 60 mg, and > 60 mg totalled 40 (41.7%) and 30 (31.3%), respectively. Patients receiving hydroxychloroquine azathioperine and cyclophosphamide totaled 32 (33.3%), 21 (21.9%) and 20 (20.8%) respectively (Table 1).
Clinical characteristics and echo findings in SLE patients and controls SLE patients showed significant increase in left atrium size, interventricular septum, PWT and left ventricular end diastolic diameter (LVEDD) compared to controls
3
M. E. Houssen et al.
Parameters
Patients (n = 96) Mean SD
Left atrium size (cm) SLEDAI Interventricular septum (cm) Posterior wall thickness (cm) Left ventricular mass (g) Left ventricular mass index (g/m2) LVEDD (cm) LVESD (cm) Ejection fraction % E (m/s) A (m/s) E/A ratio DT (ms) IVRT (ms)
3.4 17.5 1.2 1.1 194.4 73.5 4.7 3 61.6 0.9 0.8 1.2 296.6 86.8
0.5 6.3 0.2 0.2 49.6 17.6 0.5 0.6 9.1 0.3 0.3 0.5 139.3 17.3
Control (n = 30) Mean SD
P-value
2.9 0.3 – 0.9 0.1 0.9 0.1 192.7 18 69.9 5.1 3.9 0.3 3 0.3 65.3 6 0.8 0.1 0.5 0.1 1.6 0.2 191.4 17.3 75.3 8.8
