Journal of the American Society of Hypertension
-(-)
(2014) 1–5
Research Article
Adiponectin is better predictor of subclinical atherosclerosis than liver function tests in patients with nonalcoholic fatty liver disease Elena Omelchenko, MDa, Dov Gavish, MDa,c, and Marina Shargorodsky, MDb,c,* a
b
Department of Medicine, Wolfson Medical Center, Holon, Israel; Department of Endocrinology, Wolfson Medical Center, Holon, Israel; and c Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Manuscript received January 16, 2014 and accepted March 6, 2014
Abstract Adiponectin has recently been considered as a possible link between liver dysfunction and atherosclerosis in patients with nonalcoholic fatty liver disease (NAFLD). The present study was designed to evaluate the relation between circulating adiponectin and arterial stiffness parameters, such as pulse wave velocity (PWV) and aortic augmentation index (AI), in patients with hepatic steatosis. The study group consisted of 52 subjects with NAFLD. PWV and AI were performed using SphygmoCor (version 7.1, AtCor Medical, Sydney, Australia). Metabolic parameters, homeostasis model assessment-insulin resistance, and adiponectin levels were determined. Adiponectin was significantly, positively associated with AI (r ¼ 0.467; P < .0001) and with PWV (r ¼ 0.348; P ¼ .011). No association between arterial stiffness parameters and liver function tests was observed. In a multiple linear regression analysis, adiponectin remained a significant predictor of PWV even after controlling for age, gender, and MAP. Serum adiponectin levels were significantly associated with indices of subclinical atherosclerosis, such as PWV and AI in patients with NAFLD. This association was independent of age, gender, and blood pressure level and suggests an active role of adiponectin in the pathophysiology of vascular disease in this particular population group. J Am Soc Hypertens 2014;-(-):1–5. Ó 2014 American Society of Hypertension. All rights reserved. Keywords: Adiponectin; nonalcoholic fatty liver disease; pulse wave velocity; aortic augmentation index.
Introduction Insulin resistance has been identified as a potential pathogenic mechanism for the initiation and progression of atherosclerosis in patients with nonalcoholic fatty liver disease (NAFLD).1–4 Recently, circulating adiponectin, an endogenous insulin-sensitizing hormone, which is highly specific to adipose tissue, has been considered as a possible link between liver dysfunction and atherosclerotic vascular disease in patients with NAFLD.5 Although, it has been shown that adiponectin level is inversely associated with hepatic steatosis and predicts its Conflict of interest: There is no conflict of interest. This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. *Corresponding author: Marina Shargorodsky, MD, Wolfson Medical Center, POB 5, Holon, 58100, Israel. Tel.: 972 3 5028372; fax: 972 3 5028375. E-mail: [email protected]
grade and severity,6,7 beneficial effects of adiponectin could paradoxically disappear in people with advanced liver disease. Recently, it was shown that as hepatic fat declines with advanced fibrosis, adiponectin levels progressively rise, independent of its usual metabolic associations such as insulin resistance, leptin, and body mass index.8 While the relationship between adiponectin levels and different steatosis grades has been investigated, data has revealed the vascular impact of circulating adiponectin in patients with NAFLD is limited. The vascular adverse changes in NAFLD patients may be assessed noninvasively by arterial pulse-wave contour analysis. These techniques can be regarded as a valid marker of early, preclinical atherosclerosis, as well as a predictor of cardiovascular morbidity and mortality.9–11 Previously, it has been shown than various liver enzymes, such as ALT, AST, ALP, and GGT were significantly associated with pulse wave velocity (PWV) in subjects with NAFLD; however, significant correlation between adiponectin and PWV was not found.12 Since adiponectin has recently been considered as a
1933-1711/$ - see front matter Ó 2014 American Society of Hypertension. All rights reserved. http://dx.doi.org/10.1016/j.jash.2014.03.002
2
E. Omelchenko et al. / Journal of the American Society of Hypertension
possible link between liver dysfunction and atherosclerosis in patients with NAFLD, who have exceptionally high risk of cardiovascular disease, it is critical to understand the impact of adoponectin on vascular function in this population. The present study was designed to investigate a possible association between serum adiponectin levels and atherosclerotic vascular changes, as determined by PWV and aortic augmentation index (AI) in patients with nonalcoholic fatty liver disease.
Methods Subjects In this single-center study, 52 patients (25 males and 27 females) diagnosed with NAFLD were recruited from the outpatient clinic at the Wolfson Medical Center to participate in the study. The diagnosis of NAFLD was based on the results of abdominal ultrasonography and exclusion of viral, autoimmune, or drug-induced liver disease, as well as any alcohol intake of more than 20 g/day. Fatty liver disease was diagnosed on the basis on four sonographic criteria: a diffuse hyperechoic echotexture (bright liver), increased echotexture compared with the kidneys, vascular blurring, and deep attenuation.13 Screening procedures included physical examination, complete blood chemistry, complete blood count, urinalysis, and electrocardiography. Patients with a history of unstable angina, myocardial infarction, cerebrovascular accident, or major surgery within the 6 months preceding the study were excluded. All concomitant medications were kept to a consistent routine for up to 3 months to prevent possible effects on the study variables. The patients were instructed to consult the study physician if any change in medical treatment was suggested by another physician. The study was approved by the Institutional Review Board, and the patients signed a full informed consent before participation.
Biochemical Parameters Blood sampling for full chemistry and metabolic parameters, including fasting glucose, fasting insulin, lipid profile, C-reactive protein, liver function tests, and plasma adiponectin was performed. Glucose was measured using the Aeroset chemistry system (Abbott Diagnostics); highdensity lipoprotein cholesterol (HDL) and triglycerides were assayed using an Aeroset automated analyzer (Abbott Diagnostics, Berkshire, UK); low-density lipoprotein cholesterol (LDL) was calculated using Friedewald’s formula; and insulin was measured using an immunometric assay specific for human insulin (Invitron, Monmouth, UK). Adiponectin was determined by a commercial sandwich enzyme immunoassay technique (R&D Systems,
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(2014) 1–5
Minneapolis, MN, USA [catalog number DRP300]) with 2.8% intra-assay and 6.5% inter-assay variability. Homeostasis model assessment-insulin resistance (HOMA-IR) was calculated by the following formula: fasting plasma insulin (mU/mL) fasting plasma glucose (mg/dL)/405.
Blood Pressure and PWV Measurement Blood pressure was measured using an automated digital oscillometric device (Omron model HEM 705-CP, Omron Corporation, Tokyo, Japan), and a mean of three readings was taken. The radial pressure waveform was recorded and subsequently transformed by using a validated generalized transfer function incorporated in the SphygmoCor (version 7.1, AtCor Medical, Sydney, Australia) to give an estimate of the corresponding central ascending aortic pulse wave. With the integral software, the central augmented pressure was calculated as the difference between the early and late systolic peaks of the estimated central pressure waveform. Central AI was calculated as the augmented pressure expressed as a percentage of the pulse pressure. PWV was measured by simultaneous recording of the right carotid and the right radial artery pulse waveforms by two pressure transducers using the SphygmoCor Vx PWV System. This technique, which has been validated for its reproducibility and used extensively, is able to estimate the PWV between the two artery sites.14
Statistical Analysis Analysis of data was carried out using SPSS 9.0 statistical analysis software (SPSS Inc, Chicago, IL, USA; 1999). For continuous variables, such as hemodynamic and biochemistry measures, descriptive statistics were calculated and reported as mean standard deviation. Distributions of continuous variables were assessed for normality using the Kolmogorov-Smirnov test (cut-off at P ¼ .01). Associations between continuous variables with approximately normal distributions, including anthropometric, hemodynamic, and arterial stiffness parameters, were described using Pearson’s correlation coefficients. Associations between continuous variables with distributions significantly deviating from normal were described using Spearman’s rho coefficients. PWV and, separately, AI, were modeled using multiple linear regression analysis. All tests are two-sided and considered significant at P < .05.
Results The clinical characteristics of the study groups are presented in Table 1. The study population was comprised of 27 (52%) females, mean age 53.5 13 years with diagnosed NAFLD.
E. Omelchenko et al. / Journal of the American Society of Hypertension Table 1 Demographic and clinical characteristics of study patients
PWV 25/27 53.5 31.1 5 (10%) 25 (48%) 29 (56%) 12 (23%) 138.5 76.8 98.6 65.6 110.1 5.9 181.3 110.4 47.6 146.9 0.5 26.4 29.5 64.1 37.3 104.6 0.9 31.2 8019.3 21.9 6.6 6.1 25.9
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Table 2 Correlations
Variables Male/female Age (y) Body mass index (kg/m2) Current smokers, n (%) Hypertension, n (%) Dyslipidemia, n (%) Diabetes mellitus, n (%) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Mean pressure (mm Hg) Heart rate (beats/min) Fasting glucose (mg/dL) HbA1C Total cholesterol (mg/dL) LDL cholesterol (mg/dL) HDL-cholesterol (mg/dL) Triglycerides (mg/dL) C-reactive protein (mg/dL) AST (U/l) ALT (U/l) ALP (U/l) GGT (U/l) LDL (U/l) Creatinine (mg/dL) Urea (mg/dL) Adiponectin (ng/mL) Fasting insulin (IU) HOMA-IR PWV (m/sec) AI (%)
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13.1 5.2
19.6 9.3 12.4 10.0 23.9 0.8 32.4 37.9 15.1 80.9 0.5 9.0 16.1 17.8 38.5 24.2 0.2 9.6 4922.2 20.6 8.9 0.9 10.4
AI, aortic augmentation index; HDL, high-density lipoprotein; HOMA-R, homeostasis model assessment-insulin resistance; LDL, low-density lipoprotein; PWV, pulse wave velocity.
As can be seen in Table 2, PWV was significantly, positively associated with adiponectin (r ¼ 0.348; P ¼ .011). AI was significantly, positively associated with adiponectin (r ¼ 0.467; P < .0001) as well. Significant positive association between circulating adiponectin and aortic systolic pressure (ASP), as well as aortic pulse pressure (APP), was observed (r ¼ 0.295; P ¼ .034 and r ¼ 0.309; P ¼ .026, respectively). PWV, as well as AI, was not associated with any of the liver function tests, such as ALT, AST, ALP, and g-GT. Multiple linear regression analysis (Table 3) was arrived at using a backward, stepwise approach with probability of F ¼ 0.05 for entry and 0.15 for removal from the model. Backward elimination was performed to identify variables independently associated with PVW and AI. Multiple linear regression analysis of PWV and, separately, AI, included age, gender, and mean arterial pressure (MAP) as covariates. In this model, adiponectin remained a significant predictor of PWV even after controlling for age, gender, and
Systolic BP r-value P-value Diastolic BP r-value P-value Mean BP r-value P-value Pulse pressure r-value P-value Heart rate r-value P-value Fasting glucose r-value P-value HbA1C r-value P-value Creatinine r-value P-value HDL cholesterol r-value P-value LDL cholesterol r-value P-value Triglycerides r-value P-value C-reactive protein r-value P-value Fibrinogen r-value P-value ALP r-value P-value GGT r-value P-value ALT r-value P-value AST r-value P-value Adiponectin r-value P-value
AI
0.016 0.912
0.389 0.004
0.037 0.796
0.291 0.036
0.051 0.722
0.477 0.000
0.039 0.784
0.294 0.035
0.039 0.784
0.377 0.006
0.092 0.518
0.039 0.784
0.048 0.735
0.271 0.052
0.157 0.265
0.120 0.395
0.364 0.009
0.315 0.026
0.112 0.444
0.021 0.888
0.300 0.030
0.158 0.263
0.028 0.844
0.023 0.869
0.323 0.021
0.226 0.111
0.165 0.242
0.166 0.240
0.157 0.278
0.012 0.936
0.104 0.464
0.224 0.110
0.017 0.903
0.045 0.750
0.348 0.011
0.467 0.000 (continued on next page)
E. Omelchenko et al. / Journal of the American Society of Hypertension
4 Table 2 (continued )
Fasting Insulin r-value P-value HOMA-IR r-value P-value
PWV
AI
0.038 0.790
0.240 0.870
0.052 0.714
0.166 0.240
AI, aortic augmentation index; BP, blood pressure; HDL, highdensity lipoprotein; HOMA-R, homeostasis model assessmentinsulin resistance; LDL, low-density lipoprotein; PWV, pulse wave velocity.
MAP. The overall model was significant (P < .002) and explained 25.2% of the variability in PWV. In multiple linear regression analysis of AI, adiponectin remained a significant predictor of AI after controlling for age, gender, and MAP. The overall model was significant (P < .0001) and explained 64.5% of the variability in AI.
Discussion The present study demonstrates that circulating adiponectin levels were significantly associated with indices of subclinical atherosclerosis, such as PWV and AI in patients with NAFLD. This association was independent of age, gender, blood pressure, and liver enzyme level and suggests an active role of adiponectin in the pathophysiology of vascular disease in this population. Findings of the present study concur with recently published study showed that gamma-glutamyl transferase (g-GT) activity, although moderately correlated to insulin resistance, was not different on the basis of the hepatosteatosis severity in obese patients. Moreover, high g-GT levels were clearly associated with hypertension, but not with increased carotid intima media thickness, which is considered a surrogate for systemic atherosclerotic disease burden as well as predictor of cardiovascular mortality.15 While adiponectin has recently been considered as a possible link between liver dysfunction and atherosclerosis in patients with NAFLD, who have exceptionally high risk of cardiovascular disease, the impact of plasma adiponectin Table 3 Multiple linear regression analysis Variables
Corrected model Intercept Age Gender Mean arterial pressure Adiponectin
PWV
AI
F
P-value
F
P-value
4.442 7.440 .596 .735 .258 8.965
.002 .009 .444 .396 .614 .004
19.556 .479 21.383 4.014 1.537 4.397
.000 .492 .000 .051 .221 .042
AI, aortic augmentation index; PWV, pulse wave velocity.
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levels on the earliest stage of atherosclerosis in this population is limited. To the best of our knowledge, the present study is the first to estimate a relation of serum adiponectin levels to early vascular changes as determined by PWV and AI in patients with diagnosed NAFLD. Although the association between hypoadiponectinemia and increasing hepatic steatosis grade has been shown, this relationship could paradoxically disappear in people with advanced liver disease. Recently, it was shown that as hepatic fat declines with advanced fibrosis, circulating adiponectin levels progressively rise.8,15 The potential explanations for these findings are: adiponectin resistance, adiponectin receptors down-regulation, and an imbalance between adiponectin production and hepatic extraction. Additional mechanisms have been hypothesized to explain the relative elevation of adiponectin levels in advanced nonalcoholic steatohepatitis patients, such as elevation in circulating bile acids and hepatocyte-adipocyte crosstalk mediated by bile acids and their receptors as well as alteration of physiological regulation of adiponectin.16–19 Previously published study found that serum adiponectin is positively associated with NAFLD and various liver enzymes, such as ALT, AST, ALP, and GGT, especially in females; however, significant correlation between adiponectin and PWV was not found.12 In the present study, PWV, as well as AI, was not associated with any of the liver function tests. These findings concur with those of previous studies that compared histological findings in NAFLD patients with normal and persistently abnormal liver enzymes and found a similar grading of steatosis and staging of fibrosis. Moreover, some patterns of hepatocellullar damage, such as ballooning degeneration and glycogenated-nuclei, were more common among patients with silent NAFLD.20 The findings of the present study support previous reports that have identified the ultrasonographically detected NAFLD as a component of the diagnostic criteria for metabolic syndrome21 and suggest that adiponectin has an active role in the pathophysiology of atherosclerotic vascular disease in NAFLD subjects. Our study has several limitations, being that it has focused on patients with NAFLD: therefore, the correlation of our findings to other patient populations remains uncertain. Additionally, because serum levels of total adiponectin were assayed in the present study, the impact of different multimers of adiponectin (low, middle, and high molecular weight form) on vascular pathophysiology remains unclear. It also lacks a parallel control group, and as such, it is not possible to neutralize the effect of additional factors on relations found in the present study. However, factors known to influence arterial stiffness parameters, including blood pressure and concomitant medications, were kept stable for up to 3 months to prevent possible effects on the study variables. In conclusion, serum adiponectin levels were significantly associated with indices of subclinical atherosclerosis, such
E. Omelchenko et al. / Journal of the American Society of Hypertension
as PWV and AI, in patients with NAFLD. This association was independent of age, gender, blood pressure, and liver enzyme levels such as ALT, AST, ALP, and g-GT. The precise mechanisms for these vascular effects, as well as overall clinical impact of increased adiponectin levels on cardiovascular outcomes in patients with advanced NAFLD, deserve further investigation.
References 1. Bugianesi E, Gastaldelli A, Vanni E, Gambino R, Cassader M, Baldi S, et al. Insulin resistance in nondiabetic patients with non-alcoholic fatty liver disease: sites and mechanisms. Diabetologia 2005;48:634–42. 2. Targher G, Bertolini L, Padovani R, Zenari L, Zoppini G, Falezza G. Relation of nonalcoholic hepatic steatosis to early carotid atherosclerosis in healthy men: role of visceral fat accumulation. Diabetes Care 2004;7:1498–500. 3. Brea A, Mosquera D, Martin E, Arizti A, Cordero JL, Ros E. Nonalcoholic fatty liver disease is associated with carotid atherosclerosis: a case-control study. Arterioscler Thromb Vasc Biol 2005;25:1045–500. 4. Targher G, Bertolini L, Padovani R, Rodella S, Zoppini G, Zenari L, et al. Relations between carotid artery wall thickness and liver histology in subjects with nonalcoholic fatty liver disease. Diabetes Care 2006;29:1325–30. 5. Ozcelik F, Yuksel C, Arslan E, Genc S, Omer B, Serdar MA. Relationship between visceral adipose tissue and, adiponectin, inflammatory markers and thyroid hormones in obese males with hepatosteatosis and insulin resistance. Arch Med Res 2013;44:273–80. 6. Turer AT, Browning JD, Ayers CR, Das SR, Khera A, Vega GL, et al. Adiponectin as an independent predictor of the presence and degree of hepatic steatosis in the Dallas Heart Study. J Clin Endocrinol Metab 2012;97:E982–6. 7. Finelli C, Tarantino G. What is the role of adiponectin in obesity related non-alcoholic fatty liver disease? World J Gastroenterol 2013;19:802–12. 8. van der Poorten D, Samer CF, RamezaniMoghadam M, Coulter S, Kacevska M, Schrijnders D, et al. Hepatic fat loss in advanced nonalcoholic steatohepatitis: are alterations in serum adiponectin the cause? Hepatology 2013;57:2180–8. 9. Zieman SJ, Melenovsky V, Kass DA. Mechanisms, pathophysiology, and therapy of arterial stiffness. Arterioscler Thromb Vasc Biol 2005;25:932–43.
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10. Schiffrin EL. Vascular stiffening and arterial compliance. Am J Hypertens 2004;17:39–48. 11. Gomez-Marcos MA, Recio-Rodrıguez JI, PatinoAlonso MC, Agudo-Conde C, Gomez-Sanchez L, Rodriguez-Sanchez E, et al. Yearly evolution of organ damage markers in diabetes or metabolic syndrome: data from the LOD-DIABETES study. Cardiovasc Diabetol 2011;10:9. 12. Kim SG, Kim HY, Seo JA, Lee KW, Oh HJ, Kim NH, et al. Relationship between serum adiponectin concentration, pulse wave velocity, and nonalcoholic fatty liver disease. Europ J Endocrinol 2005;152:225–31. 13. Joy D, Thava VR, Scott BB. Diagnosis of fatty liver disease: is biopsy necessary? Eur J Gastroenterol Hepatol 2003;15:539–43. 14. O’Rourke M, Staessen JA. Clinical application of arterial stiffness definitions and reference values. Am J Hypertens 2002;15:426–44. 15. Tarantino G, Finelli C, Colao A, Capone D, Tarantino M, Grimaldi E, et al. Are hepatic steatosis and carotid intima media thickness associated in obese patients with normal or slightly elevated gamma-glutamyl-transferase? J Transl Med 2012;10:50. 16. Claudel T, Trauner M. Adiponectin, bile acids, and burnt-out nonalcoholic steatohepatitis: new light on an old paradox. Hepatology 2013;57:2106–9. 17. Hui JM, Hodge A, Farrell GC, Kench JG, Kriketos A, George J. Beyond insulin resistance in NASH: TNFalpha or adiponectin? Hepatology 2004;40:46–54. 18. Hui CK, Zhang HY, Lee NP, Chan W, Yueng YH, Leung KW, et al. Serum adiponectin is increased in advancing liver fibrosis and declines with reduction in fibrosis in chronic hepatitis B. J Hepatol 2007;47: 191–202. 19. Tacke F, Wustefeld T, Horn R, Luedde T, Srinivas Rao A, Manns MP, et al. High adiponectin in chronic liver disease and cholestasis suggests biliary route of adiponectin excretion in vivo. J Hepatol 2005;42: 666–73. 20. Sorrentino P, Tarantino G, Conca P, Perrella A, Terracciano ML, Vecchione R, et al. Silent nonalcoholic fatty liver disease-a clinical-histological study. J Hepatol 2004;41:751–7. 21. Tarantino G, Finelli C. What about non-alcoholic fatty liver disease as a new criterion to define metabolic syndrome? World J Gastroenterol 2013;19:3375–84.
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Research Article
Adiponectin is better predictor of subclinical atherosclerosis than liver function tests in patients with nonalcoholic fatty liver disease Elena Omelchenko, MDa, Dov Gavish, MDa,c, and Marina Shargorodsky, MDb,c,* a
b
Department of Medicine, Wolfson Medical Center, Holon, Israel; Department of Endocrinology, Wolfson Medical Center, Holon, Israel; and c Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Manuscript received January 16, 2014 and accepted March 6, 2014
Abstract Adiponectin has recently been considered as a possible link between liver dysfunction and atherosclerosis in patients with nonalcoholic fatty liver disease (NAFLD). The present study was designed to evaluate the relation between circulating adiponectin and arterial stiffness parameters, such as pulse wave velocity (PWV) and aortic augmentation index (AI), in patients with hepatic steatosis. The study group consisted of 52 subjects with NAFLD. PWV and AI were performed using SphygmoCor (version 7.1, AtCor Medical, Sydney, Australia). Metabolic parameters, homeostasis model assessment-insulin resistance, and adiponectin levels were determined. Adiponectin was significantly, positively associated with AI (r ¼ 0.467; P < .0001) and with PWV (r ¼ 0.348; P ¼ .011). No association between arterial stiffness parameters and liver function tests was observed. In a multiple linear regression analysis, adiponectin remained a significant predictor of PWV even after controlling for age, gender, and MAP. Serum adiponectin levels were significantly associated with indices of subclinical atherosclerosis, such as PWV and AI in patients with NAFLD. This association was independent of age, gender, and blood pressure level and suggests an active role of adiponectin in the pathophysiology of vascular disease in this particular population group. J Am Soc Hypertens 2014;-(-):1–5. Ó 2014 American Society of Hypertension. All rights reserved. Keywords: Adiponectin; nonalcoholic fatty liver disease; pulse wave velocity; aortic augmentation index.
Introduction Insulin resistance has been identified as a potential pathogenic mechanism for the initiation and progression of atherosclerosis in patients with nonalcoholic fatty liver disease (NAFLD).1–4 Recently, circulating adiponectin, an endogenous insulin-sensitizing hormone, which is highly specific to adipose tissue, has been considered as a possible link between liver dysfunction and atherosclerotic vascular disease in patients with NAFLD.5 Although, it has been shown that adiponectin level is inversely associated with hepatic steatosis and predicts its Conflict of interest: There is no conflict of interest. This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector. *Corresponding author: Marina Shargorodsky, MD, Wolfson Medical Center, POB 5, Holon, 58100, Israel. Tel.: 972 3 5028372; fax: 972 3 5028375. E-mail: [email protected]
grade and severity,6,7 beneficial effects of adiponectin could paradoxically disappear in people with advanced liver disease. Recently, it was shown that as hepatic fat declines with advanced fibrosis, adiponectin levels progressively rise, independent of its usual metabolic associations such as insulin resistance, leptin, and body mass index.8 While the relationship between adiponectin levels and different steatosis grades has been investigated, data has revealed the vascular impact of circulating adiponectin in patients with NAFLD is limited. The vascular adverse changes in NAFLD patients may be assessed noninvasively by arterial pulse-wave contour analysis. These techniques can be regarded as a valid marker of early, preclinical atherosclerosis, as well as a predictor of cardiovascular morbidity and mortality.9–11 Previously, it has been shown than various liver enzymes, such as ALT, AST, ALP, and GGT were significantly associated with pulse wave velocity (PWV) in subjects with NAFLD; however, significant correlation between adiponectin and PWV was not found.12 Since adiponectin has recently been considered as a
1933-1711/$ - see front matter Ó 2014 American Society of Hypertension. All rights reserved. http://dx.doi.org/10.1016/j.jash.2014.03.002
2
E. Omelchenko et al. / Journal of the American Society of Hypertension
possible link between liver dysfunction and atherosclerosis in patients with NAFLD, who have exceptionally high risk of cardiovascular disease, it is critical to understand the impact of adoponectin on vascular function in this population. The present study was designed to investigate a possible association between serum adiponectin levels and atherosclerotic vascular changes, as determined by PWV and aortic augmentation index (AI) in patients with nonalcoholic fatty liver disease.
Methods Subjects In this single-center study, 52 patients (25 males and 27 females) diagnosed with NAFLD were recruited from the outpatient clinic at the Wolfson Medical Center to participate in the study. The diagnosis of NAFLD was based on the results of abdominal ultrasonography and exclusion of viral, autoimmune, or drug-induced liver disease, as well as any alcohol intake of more than 20 g/day. Fatty liver disease was diagnosed on the basis on four sonographic criteria: a diffuse hyperechoic echotexture (bright liver), increased echotexture compared with the kidneys, vascular blurring, and deep attenuation.13 Screening procedures included physical examination, complete blood chemistry, complete blood count, urinalysis, and electrocardiography. Patients with a history of unstable angina, myocardial infarction, cerebrovascular accident, or major surgery within the 6 months preceding the study were excluded. All concomitant medications were kept to a consistent routine for up to 3 months to prevent possible effects on the study variables. The patients were instructed to consult the study physician if any change in medical treatment was suggested by another physician. The study was approved by the Institutional Review Board, and the patients signed a full informed consent before participation.
Biochemical Parameters Blood sampling for full chemistry and metabolic parameters, including fasting glucose, fasting insulin, lipid profile, C-reactive protein, liver function tests, and plasma adiponectin was performed. Glucose was measured using the Aeroset chemistry system (Abbott Diagnostics); highdensity lipoprotein cholesterol (HDL) and triglycerides were assayed using an Aeroset automated analyzer (Abbott Diagnostics, Berkshire, UK); low-density lipoprotein cholesterol (LDL) was calculated using Friedewald’s formula; and insulin was measured using an immunometric assay specific for human insulin (Invitron, Monmouth, UK). Adiponectin was determined by a commercial sandwich enzyme immunoassay technique (R&D Systems,
-(-)
(2014) 1–5
Minneapolis, MN, USA [catalog number DRP300]) with 2.8% intra-assay and 6.5% inter-assay variability. Homeostasis model assessment-insulin resistance (HOMA-IR) was calculated by the following formula: fasting plasma insulin (mU/mL) fasting plasma glucose (mg/dL)/405.
Blood Pressure and PWV Measurement Blood pressure was measured using an automated digital oscillometric device (Omron model HEM 705-CP, Omron Corporation, Tokyo, Japan), and a mean of three readings was taken. The radial pressure waveform was recorded and subsequently transformed by using a validated generalized transfer function incorporated in the SphygmoCor (version 7.1, AtCor Medical, Sydney, Australia) to give an estimate of the corresponding central ascending aortic pulse wave. With the integral software, the central augmented pressure was calculated as the difference between the early and late systolic peaks of the estimated central pressure waveform. Central AI was calculated as the augmented pressure expressed as a percentage of the pulse pressure. PWV was measured by simultaneous recording of the right carotid and the right radial artery pulse waveforms by two pressure transducers using the SphygmoCor Vx PWV System. This technique, which has been validated for its reproducibility and used extensively, is able to estimate the PWV between the two artery sites.14
Statistical Analysis Analysis of data was carried out using SPSS 9.0 statistical analysis software (SPSS Inc, Chicago, IL, USA; 1999). For continuous variables, such as hemodynamic and biochemistry measures, descriptive statistics were calculated and reported as mean standard deviation. Distributions of continuous variables were assessed for normality using the Kolmogorov-Smirnov test (cut-off at P ¼ .01). Associations between continuous variables with approximately normal distributions, including anthropometric, hemodynamic, and arterial stiffness parameters, were described using Pearson’s correlation coefficients. Associations between continuous variables with distributions significantly deviating from normal were described using Spearman’s rho coefficients. PWV and, separately, AI, were modeled using multiple linear regression analysis. All tests are two-sided and considered significant at P < .05.
Results The clinical characteristics of the study groups are presented in Table 1. The study population was comprised of 27 (52%) females, mean age 53.5 13 years with diagnosed NAFLD.
E. Omelchenko et al. / Journal of the American Society of Hypertension Table 1 Demographic and clinical characteristics of study patients
PWV 25/27 53.5 31.1 5 (10%) 25 (48%) 29 (56%) 12 (23%) 138.5 76.8 98.6 65.6 110.1 5.9 181.3 110.4 47.6 146.9 0.5 26.4 29.5 64.1 37.3 104.6 0.9 31.2 8019.3 21.9 6.6 6.1 25.9
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Table 2 Correlations
Variables Male/female Age (y) Body mass index (kg/m2) Current smokers, n (%) Hypertension, n (%) Dyslipidemia, n (%) Diabetes mellitus, n (%) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Mean pressure (mm Hg) Heart rate (beats/min) Fasting glucose (mg/dL) HbA1C Total cholesterol (mg/dL) LDL cholesterol (mg/dL) HDL-cholesterol (mg/dL) Triglycerides (mg/dL) C-reactive protein (mg/dL) AST (U/l) ALT (U/l) ALP (U/l) GGT (U/l) LDL (U/l) Creatinine (mg/dL) Urea (mg/dL) Adiponectin (ng/mL) Fasting insulin (IU) HOMA-IR PWV (m/sec) AI (%)
-(-)
13.1 5.2
19.6 9.3 12.4 10.0 23.9 0.8 32.4 37.9 15.1 80.9 0.5 9.0 16.1 17.8 38.5 24.2 0.2 9.6 4922.2 20.6 8.9 0.9 10.4
AI, aortic augmentation index; HDL, high-density lipoprotein; HOMA-R, homeostasis model assessment-insulin resistance; LDL, low-density lipoprotein; PWV, pulse wave velocity.
As can be seen in Table 2, PWV was significantly, positively associated with adiponectin (r ¼ 0.348; P ¼ .011). AI was significantly, positively associated with adiponectin (r ¼ 0.467; P < .0001) as well. Significant positive association between circulating adiponectin and aortic systolic pressure (ASP), as well as aortic pulse pressure (APP), was observed (r ¼ 0.295; P ¼ .034 and r ¼ 0.309; P ¼ .026, respectively). PWV, as well as AI, was not associated with any of the liver function tests, such as ALT, AST, ALP, and g-GT. Multiple linear regression analysis (Table 3) was arrived at using a backward, stepwise approach with probability of F ¼ 0.05 for entry and 0.15 for removal from the model. Backward elimination was performed to identify variables independently associated with PVW and AI. Multiple linear regression analysis of PWV and, separately, AI, included age, gender, and mean arterial pressure (MAP) as covariates. In this model, adiponectin remained a significant predictor of PWV even after controlling for age, gender, and
Systolic BP r-value P-value Diastolic BP r-value P-value Mean BP r-value P-value Pulse pressure r-value P-value Heart rate r-value P-value Fasting glucose r-value P-value HbA1C r-value P-value Creatinine r-value P-value HDL cholesterol r-value P-value LDL cholesterol r-value P-value Triglycerides r-value P-value C-reactive protein r-value P-value Fibrinogen r-value P-value ALP r-value P-value GGT r-value P-value ALT r-value P-value AST r-value P-value Adiponectin r-value P-value
AI
0.016 0.912
0.389 0.004
0.037 0.796
0.291 0.036
0.051 0.722
0.477 0.000
0.039 0.784
0.294 0.035
0.039 0.784
0.377 0.006
0.092 0.518
0.039 0.784
0.048 0.735
0.271 0.052
0.157 0.265
0.120 0.395
0.364 0.009
0.315 0.026
0.112 0.444
0.021 0.888
0.300 0.030
0.158 0.263
0.028 0.844
0.023 0.869
0.323 0.021
0.226 0.111
0.165 0.242
0.166 0.240
0.157 0.278
0.012 0.936
0.104 0.464
0.224 0.110
0.017 0.903
0.045 0.750
0.348 0.011
0.467 0.000 (continued on next page)
E. Omelchenko et al. / Journal of the American Society of Hypertension
4 Table 2 (continued )
Fasting Insulin r-value P-value HOMA-IR r-value P-value
PWV
AI
0.038 0.790
0.240 0.870
0.052 0.714
0.166 0.240
AI, aortic augmentation index; BP, blood pressure; HDL, highdensity lipoprotein; HOMA-R, homeostasis model assessmentinsulin resistance; LDL, low-density lipoprotein; PWV, pulse wave velocity.
MAP. The overall model was significant (P < .002) and explained 25.2% of the variability in PWV. In multiple linear regression analysis of AI, adiponectin remained a significant predictor of AI after controlling for age, gender, and MAP. The overall model was significant (P < .0001) and explained 64.5% of the variability in AI.
Discussion The present study demonstrates that circulating adiponectin levels were significantly associated with indices of subclinical atherosclerosis, such as PWV and AI in patients with NAFLD. This association was independent of age, gender, blood pressure, and liver enzyme level and suggests an active role of adiponectin in the pathophysiology of vascular disease in this population. Findings of the present study concur with recently published study showed that gamma-glutamyl transferase (g-GT) activity, although moderately correlated to insulin resistance, was not different on the basis of the hepatosteatosis severity in obese patients. Moreover, high g-GT levels were clearly associated with hypertension, but not with increased carotid intima media thickness, which is considered a surrogate for systemic atherosclerotic disease burden as well as predictor of cardiovascular mortality.15 While adiponectin has recently been considered as a possible link between liver dysfunction and atherosclerosis in patients with NAFLD, who have exceptionally high risk of cardiovascular disease, the impact of plasma adiponectin Table 3 Multiple linear regression analysis Variables
Corrected model Intercept Age Gender Mean arterial pressure Adiponectin
PWV
AI
F
P-value
F
P-value
4.442 7.440 .596 .735 .258 8.965
.002 .009 .444 .396 .614 .004
19.556 .479 21.383 4.014 1.537 4.397
.000 .492 .000 .051 .221 .042
AI, aortic augmentation index; PWV, pulse wave velocity.
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levels on the earliest stage of atherosclerosis in this population is limited. To the best of our knowledge, the present study is the first to estimate a relation of serum adiponectin levels to early vascular changes as determined by PWV and AI in patients with diagnosed NAFLD. Although the association between hypoadiponectinemia and increasing hepatic steatosis grade has been shown, this relationship could paradoxically disappear in people with advanced liver disease. Recently, it was shown that as hepatic fat declines with advanced fibrosis, circulating adiponectin levels progressively rise.8,15 The potential explanations for these findings are: adiponectin resistance, adiponectin receptors down-regulation, and an imbalance between adiponectin production and hepatic extraction. Additional mechanisms have been hypothesized to explain the relative elevation of adiponectin levels in advanced nonalcoholic steatohepatitis patients, such as elevation in circulating bile acids and hepatocyte-adipocyte crosstalk mediated by bile acids and their receptors as well as alteration of physiological regulation of adiponectin.16–19 Previously published study found that serum adiponectin is positively associated with NAFLD and various liver enzymes, such as ALT, AST, ALP, and GGT, especially in females; however, significant correlation between adiponectin and PWV was not found.12 In the present study, PWV, as well as AI, was not associated with any of the liver function tests. These findings concur with those of previous studies that compared histological findings in NAFLD patients with normal and persistently abnormal liver enzymes and found a similar grading of steatosis and staging of fibrosis. Moreover, some patterns of hepatocellullar damage, such as ballooning degeneration and glycogenated-nuclei, were more common among patients with silent NAFLD.20 The findings of the present study support previous reports that have identified the ultrasonographically detected NAFLD as a component of the diagnostic criteria for metabolic syndrome21 and suggest that adiponectin has an active role in the pathophysiology of atherosclerotic vascular disease in NAFLD subjects. Our study has several limitations, being that it has focused on patients with NAFLD: therefore, the correlation of our findings to other patient populations remains uncertain. Additionally, because serum levels of total adiponectin were assayed in the present study, the impact of different multimers of adiponectin (low, middle, and high molecular weight form) on vascular pathophysiology remains unclear. It also lacks a parallel control group, and as such, it is not possible to neutralize the effect of additional factors on relations found in the present study. However, factors known to influence arterial stiffness parameters, including blood pressure and concomitant medications, were kept stable for up to 3 months to prevent possible effects on the study variables. In conclusion, serum adiponectin levels were significantly associated with indices of subclinical atherosclerosis, such
E. Omelchenko et al. / Journal of the American Society of Hypertension
as PWV and AI, in patients with NAFLD. This association was independent of age, gender, blood pressure, and liver enzyme levels such as ALT, AST, ALP, and g-GT. The precise mechanisms for these vascular effects, as well as overall clinical impact of increased adiponectin levels on cardiovascular outcomes in patients with advanced NAFLD, deserve further investigation.
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