Original Article
Ks Shivaprasad, DM1; Manoj Kumar, MD1; Deep Dutta, DM, DNB2; Bineet Sinha, MD3; Samim Ali Mondal, MSc4; Indira Maisnam, DM5; Satinath Mukhopadhyay, DM1; Subhankar Chowdhury, DM, MRCP1 ABSTRACT Objective: Cardiovascular events are the most common cause of mortality in Cushing syndrome (CS). This study aimed to evaluate the impact of novel factors on atherosclerosis in endogenous CS. Methods: A total of 22 female patients with CS and 33 normal female controls underwent evaluation of fibrinogen, high-sensitivity C-reactive protein (hsCRP), inflammatory cytokines (interleukin [IL]-6, IL-1β, soluble tumor necrosis factor receptor [sTNFR]-1, and sTNFR2), glutathione peroxidase (GPx; measure of oxidative stress), carotid intima media thickness (CIMT; a measure of atherosclerosis), and percent change in flow-mediated vasodilation (%FMV) of the brachial artery, a measure of endothelial dysfunction. Stepwise multiple linear regressions were done after adjusting for variables in models 1 through 3 to evaluate their role in predicting CIMT and %FMV. Model 1 consisted of age and body mass index
Submitted for publication August 23, 2014 Accepted for publication October 20, 2014 From the 1Department of Endocrinology & Metabolism, Institute of PostGraduate Medical Education & Research (IPGMER) and Seth Sukhlal Karnani Memorial (SSKM) Hospital, Calcutta, India, 2Department of Endocrinology & Metabolism, Post-Graduate Institute of Medical Education & Research (PGIMER) & Dr. Ram Manohar Lohia (RML) Hospital, New Delhi, India, 3Department of Cardiology, IPGMER and SSKM Hospital, Calcutta, India, 4Department of Biochemistry, Dr. B.C. Roy Post-Graduate Institute of Basic Medical Education and Research, IPGMER & SSKM Hospital, Calcutta, India, and 5Department of Medicine, Dr. Rath Govinda (RG) Kar Medical College & Hospital, Calcutta, India. Address correspondence to Dr. Deep Dutta, Assistant Professor, Department of Endocrinology & Metabolism, Post-Graduate Institute of Medical Education & Research (PGIMER) & Dr. Ram Manohar Lohia (RML) Hospital, Baba Kharak Singh Marg, Connaught Place, New Delhi-110001, India. E-mail: [email protected]. Published as a Rapid Electronic Article in Press at http://www.endocrine practice.org on November 4, 2014. DOI: 10.4158/EP14399.OR To purchase reprints of this article, please visit: www.aace.com/reprints. Copyright © 2015 AACE.
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(BMI). Model 2 consisted of model 1 plus blood pressure (BP), fasting blood glucose (FBG), and 2-hour postglucose blood glucose (2hPGBG). Model 3 consisted of model 2 plus triglycerides and low- and high-density lipoprotein. Results: Females with CS had significantly higher BMI, BP, FBG, 2hPGBG, total cholesterol, triglycerides, fibrinogen, IL-6, IL-1β, sTNFR1, and GPx. CIMT and %FMV were significantly higher and lower, respectively, in CS patients. Regression analyses revealed sTNFR1 to be a consistent predictor of CIMT after adjusting for model 1 (β = 0.656; P = .004), model 2 (β = 0.571; P = .047), and model 3 (β = 0.683; P =.026). GPx was a predictor of CIMT after adjusting for model 1 (β = 0.565; P = .033) and model 3 (β = 0.756; P = .038). Conclusion: This study highlights increased CIMT and endothelial dysfunction in CS, associated with an altered inflammatory milieu. sTNFR1 and GPx may predict CIMT in females with CS. (Endocr Pract. 2015;21:286-295) Abbreviations: 2hPGBG = 2-hour postglucose blood glucose; ACTH = adrenocorticotropic hormone; BMI = body mass index; BP = blood pressure; CIMT = carotid intima medial thickness; CS = Cushing syndrome; CV = coefficient of variation; FBG = fasting blood glucose; FMV = flow-mediated vasodilation; GPx = glutathione peroxidase; hsCRP = high-sensitivity C-reactive protein; IL = interleukin; ONDST = overnight dexamethasone suppression test; sELISA = sandwich enzyme-linked immunosorbent assay; sTNFR = soluble tumor necrosis factor receptor; UFC = urine free cortisol INTRODUCTION Cardiovascular events (myocardial infarction, cardiac failure, and stroke) are the most important causes of morbidity in Cushing syndrome (CS), responsible for a 4-fold increased mortality as compared to general population (1). Carotid intimal media thickness (CIMT) has been
287
demonstrated to be a good indicator for early detection of atherosclerosis and is a strong risk factor for future cardiovascular events (2). Increased CIMT and atherosclerotic plaques have been documented in CS (3,4). Increased prevalence of atherosclerosis in CS is believed to be the consequence of persistent systemic hypercortisolemia, which leads to increased visceral adiposity, dyslipidemia, dysglycemia, hypertension, and thrombotic diathesis, all of which are well-documented (classical) risk factors of atherosclerosis (5). Impaired flow-mediated vasodilation (FMV) is a reliable measure of endothelial dysfunction (6). In the past few years, several novel risk factors in the development of atherosclerosis have been discovered, including fibrinogen, high-sensitivity C-reactive protein (hsCRP), oxidative stress, and a plethora of inflammatory cytokines (7,8). Soluble tumor necrosis factor receptor (sTNFR)1 and sTNFR2 modulate the activity of circulatory tumor necrosis factor (TNF)-α and are increasingly being recognized as playing a role in the pathogenesis of several disorders, including rheumatoid arthritis, lupus, Behcet’s disease, Graves orbitopathy, inflammatory bowel disease, inflammatory myositis, osteomyelitis, and endometriosis (9-12). sTNFR1, along with cytokines such as interleukin (IL)-6 and IL-8 have been documented to be increased in CS (13,14). Glutathione peroxidase (GPx), a ubiquitous anti-oxidant enzyme, uses reduced glutathione (GSH) to catalyze the reduction of various organic hydroperoxides, is a major component of the human anti-oxidant defense system (15). A robust anti-oxidant system is essential for maintenance of integrity of cells against reactive oxygen species, and oxidative stress. Functional variants in the gene encoding GPx-1 have been reported to be associated with increased CIMT and peripheral vascular diseases in type 2 diabetes (16). GPx activity is usually raised as a compensatory mechanism in the presence of excess oxidative stress (15). GPx alterations in CS have not been previously evaluated. Hence, the aim of this study was to evaluate the impact of novel atherosclerotic factors viz. fibrinogen, hsCRP, IL-6, IL-1β, sTNFR1, sTNFR2, and GPx on CIMT and FMV in endogenous CS. METHODS Consecutive patients with high pretest clinical probability of CS, admitted in the wards of the Department of Endocrinology and Metabolism were considered. The study protocol was explained, and only those who gave informed written consent were included. The study duration was from January 2012 until July 2014. The institutional ethics committee approved the study protocol before the study was initiated. All considered patients underwent detailed clinical evaluation. Height (to ±0.1 cm) was measured using a Charder HM200PW wall-mounted stadiometer (calibrated using a 36-inch calibration rod; Perspective
Enterprise, Portage, MI), and body weight (to ±100 g) was measured using an electronic calibrated scale (Tanita, Japan, Model HA521, Lot number-860525). The patients underwent evaluation of 8 am cortisol and 11 pm (midnight) sleeping cortisol 72 hours after admission. Midnight sleeping cortisol sample was obtained from a precannulated vein. A sample of urine was collected for assessment of 24-hour urine free cortisol (UFC). Thereafter, an overnight dexamethasone suppression test (ONDST) was done by administration of 1 mg of dexamethasone at 11 pm and assessment of cortisol from the next day’s 8 am blood sample. A low-dose dexamethasone suppression test (LDDST) was done by administration of 0.5 mg of dexamethasone every 6 hours for 48 hours starting at 8 am, and a blood sample was collected for cortisol assay 48 hours later at 8 am. Serum and urine cortisol were estimated using a solid phase enzyme-labeled chemiluminescent immunoassay (Immulite 1000, Siemens, Gwynedd, UK; analytical sensitivity, 0.2 µg/dL; range, 1 to 50 µg/dL; coefficient of variation [CV], 4.7 to 6.3%). Endogenous hypercortisolism was defined as midnight cortisol >2 µg/dL or ONDST cortisol >2 µg/dL or LDDST cortisol >2 µg/dL or 24-hour UFC greater than the upper limit of normal of the laboratory (Table 1) (17). Patients with typical clinical features suggestive of CS along with ≥2 tests positive for endogenous hypercortisolism were included in the study (17). Patients with systemic infections, chronic inflammatory conditions, on drugs (glucocorticoids, estrogen/progestogen hormone preparations, statins, fibrates, or niacin), chronic liver disease, kidney disease, coronary artery disease, or any severe comorbid state were excluded. Plasma adrenocorticotropic hormone (ACTH) was measured in all included patients with CS to differentiate ACTH-dependent CS (ACTH ≥10 pg/mL) from ACTH-independent CS (ACTH
Ks Shivaprasad, DM1; Manoj Kumar, MD1; Deep Dutta, DM, DNB2; Bineet Sinha, MD3; Samim Ali Mondal, MSc4; Indira Maisnam, DM5; Satinath Mukhopadhyay, DM1; Subhankar Chowdhury, DM, MRCP1 ABSTRACT Objective: Cardiovascular events are the most common cause of mortality in Cushing syndrome (CS). This study aimed to evaluate the impact of novel factors on atherosclerosis in endogenous CS. Methods: A total of 22 female patients with CS and 33 normal female controls underwent evaluation of fibrinogen, high-sensitivity C-reactive protein (hsCRP), inflammatory cytokines (interleukin [IL]-6, IL-1β, soluble tumor necrosis factor receptor [sTNFR]-1, and sTNFR2), glutathione peroxidase (GPx; measure of oxidative stress), carotid intima media thickness (CIMT; a measure of atherosclerosis), and percent change in flow-mediated vasodilation (%FMV) of the brachial artery, a measure of endothelial dysfunction. Stepwise multiple linear regressions were done after adjusting for variables in models 1 through 3 to evaluate their role in predicting CIMT and %FMV. Model 1 consisted of age and body mass index
Submitted for publication August 23, 2014 Accepted for publication October 20, 2014 From the 1Department of Endocrinology & Metabolism, Institute of PostGraduate Medical Education & Research (IPGMER) and Seth Sukhlal Karnani Memorial (SSKM) Hospital, Calcutta, India, 2Department of Endocrinology & Metabolism, Post-Graduate Institute of Medical Education & Research (PGIMER) & Dr. Ram Manohar Lohia (RML) Hospital, New Delhi, India, 3Department of Cardiology, IPGMER and SSKM Hospital, Calcutta, India, 4Department of Biochemistry, Dr. B.C. Roy Post-Graduate Institute of Basic Medical Education and Research, IPGMER & SSKM Hospital, Calcutta, India, and 5Department of Medicine, Dr. Rath Govinda (RG) Kar Medical College & Hospital, Calcutta, India. Address correspondence to Dr. Deep Dutta, Assistant Professor, Department of Endocrinology & Metabolism, Post-Graduate Institute of Medical Education & Research (PGIMER) & Dr. Ram Manohar Lohia (RML) Hospital, Baba Kharak Singh Marg, Connaught Place, New Delhi-110001, India. E-mail: [email protected]. Published as a Rapid Electronic Article in Press at http://www.endocrine practice.org on November 4, 2014. DOI: 10.4158/EP14399.OR To purchase reprints of this article, please visit: www.aace.com/reprints. Copyright © 2015 AACE.
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(BMI). Model 2 consisted of model 1 plus blood pressure (BP), fasting blood glucose (FBG), and 2-hour postglucose blood glucose (2hPGBG). Model 3 consisted of model 2 plus triglycerides and low- and high-density lipoprotein. Results: Females with CS had significantly higher BMI, BP, FBG, 2hPGBG, total cholesterol, triglycerides, fibrinogen, IL-6, IL-1β, sTNFR1, and GPx. CIMT and %FMV were significantly higher and lower, respectively, in CS patients. Regression analyses revealed sTNFR1 to be a consistent predictor of CIMT after adjusting for model 1 (β = 0.656; P = .004), model 2 (β = 0.571; P = .047), and model 3 (β = 0.683; P =.026). GPx was a predictor of CIMT after adjusting for model 1 (β = 0.565; P = .033) and model 3 (β = 0.756; P = .038). Conclusion: This study highlights increased CIMT and endothelial dysfunction in CS, associated with an altered inflammatory milieu. sTNFR1 and GPx may predict CIMT in females with CS. (Endocr Pract. 2015;21:286-295) Abbreviations: 2hPGBG = 2-hour postglucose blood glucose; ACTH = adrenocorticotropic hormone; BMI = body mass index; BP = blood pressure; CIMT = carotid intima medial thickness; CS = Cushing syndrome; CV = coefficient of variation; FBG = fasting blood glucose; FMV = flow-mediated vasodilation; GPx = glutathione peroxidase; hsCRP = high-sensitivity C-reactive protein; IL = interleukin; ONDST = overnight dexamethasone suppression test; sELISA = sandwich enzyme-linked immunosorbent assay; sTNFR = soluble tumor necrosis factor receptor; UFC = urine free cortisol INTRODUCTION Cardiovascular events (myocardial infarction, cardiac failure, and stroke) are the most important causes of morbidity in Cushing syndrome (CS), responsible for a 4-fold increased mortality as compared to general population (1). Carotid intimal media thickness (CIMT) has been
287
demonstrated to be a good indicator for early detection of atherosclerosis and is a strong risk factor for future cardiovascular events (2). Increased CIMT and atherosclerotic plaques have been documented in CS (3,4). Increased prevalence of atherosclerosis in CS is believed to be the consequence of persistent systemic hypercortisolemia, which leads to increased visceral adiposity, dyslipidemia, dysglycemia, hypertension, and thrombotic diathesis, all of which are well-documented (classical) risk factors of atherosclerosis (5). Impaired flow-mediated vasodilation (FMV) is a reliable measure of endothelial dysfunction (6). In the past few years, several novel risk factors in the development of atherosclerosis have been discovered, including fibrinogen, high-sensitivity C-reactive protein (hsCRP), oxidative stress, and a plethora of inflammatory cytokines (7,8). Soluble tumor necrosis factor receptor (sTNFR)1 and sTNFR2 modulate the activity of circulatory tumor necrosis factor (TNF)-α and are increasingly being recognized as playing a role in the pathogenesis of several disorders, including rheumatoid arthritis, lupus, Behcet’s disease, Graves orbitopathy, inflammatory bowel disease, inflammatory myositis, osteomyelitis, and endometriosis (9-12). sTNFR1, along with cytokines such as interleukin (IL)-6 and IL-8 have been documented to be increased in CS (13,14). Glutathione peroxidase (GPx), a ubiquitous anti-oxidant enzyme, uses reduced glutathione (GSH) to catalyze the reduction of various organic hydroperoxides, is a major component of the human anti-oxidant defense system (15). A robust anti-oxidant system is essential for maintenance of integrity of cells against reactive oxygen species, and oxidative stress. Functional variants in the gene encoding GPx-1 have been reported to be associated with increased CIMT and peripheral vascular diseases in type 2 diabetes (16). GPx activity is usually raised as a compensatory mechanism in the presence of excess oxidative stress (15). GPx alterations in CS have not been previously evaluated. Hence, the aim of this study was to evaluate the impact of novel atherosclerotic factors viz. fibrinogen, hsCRP, IL-6, IL-1β, sTNFR1, sTNFR2, and GPx on CIMT and FMV in endogenous CS. METHODS Consecutive patients with high pretest clinical probability of CS, admitted in the wards of the Department of Endocrinology and Metabolism were considered. The study protocol was explained, and only those who gave informed written consent were included. The study duration was from January 2012 until July 2014. The institutional ethics committee approved the study protocol before the study was initiated. All considered patients underwent detailed clinical evaluation. Height (to ±0.1 cm) was measured using a Charder HM200PW wall-mounted stadiometer (calibrated using a 36-inch calibration rod; Perspective
Enterprise, Portage, MI), and body weight (to ±100 g) was measured using an electronic calibrated scale (Tanita, Japan, Model HA521, Lot number-860525). The patients underwent evaluation of 8 am cortisol and 11 pm (midnight) sleeping cortisol 72 hours after admission. Midnight sleeping cortisol sample was obtained from a precannulated vein. A sample of urine was collected for assessment of 24-hour urine free cortisol (UFC). Thereafter, an overnight dexamethasone suppression test (ONDST) was done by administration of 1 mg of dexamethasone at 11 pm and assessment of cortisol from the next day’s 8 am blood sample. A low-dose dexamethasone suppression test (LDDST) was done by administration of 0.5 mg of dexamethasone every 6 hours for 48 hours starting at 8 am, and a blood sample was collected for cortisol assay 48 hours later at 8 am. Serum and urine cortisol were estimated using a solid phase enzyme-labeled chemiluminescent immunoassay (Immulite 1000, Siemens, Gwynedd, UK; analytical sensitivity, 0.2 µg/dL; range, 1 to 50 µg/dL; coefficient of variation [CV], 4.7 to 6.3%). Endogenous hypercortisolism was defined as midnight cortisol >2 µg/dL or ONDST cortisol >2 µg/dL or LDDST cortisol >2 µg/dL or 24-hour UFC greater than the upper limit of normal of the laboratory (Table 1) (17). Patients with typical clinical features suggestive of CS along with ≥2 tests positive for endogenous hypercortisolism were included in the study (17). Patients with systemic infections, chronic inflammatory conditions, on drugs (glucocorticoids, estrogen/progestogen hormone preparations, statins, fibrates, or niacin), chronic liver disease, kidney disease, coronary artery disease, or any severe comorbid state were excluded. Plasma adrenocorticotropic hormone (ACTH) was measured in all included patients with CS to differentiate ACTH-dependent CS (ACTH ≥10 pg/mL) from ACTH-independent CS (ACTH
