Distinguishing nonalcoholic steatohepatitis from fatty liver: serum-free fatty acids, insulin resistance, and serum lipoproteins

Abstract: Objectives: The prognosis of nonalcoholic fatty liver disease is determined by liver biopsy; steatohepatitis can be progressive whereas fatty liver is benign. Insulin resistance and increased hepatic-free fatty acids are central to the
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  , Clinical Studies Distinguishing nonalcoholic steatohepatitisfrom fatty liver: serum-free fatty acids,insulin resistance, and serum lipoproteins Ian D. Bookman 1 , J. Pham 1 ,M. Guindi 2 and E. J. Heathcote 1 1 Departments of Medicine,  2 Pathology,University Health Network, University ofToronto, Toronto, Canada Bookman ID, Pham J, Guindi M, Heathcote EJ. Distinguishing nonalcoholicsteatohepatitis from fatty liver serum-free fatty acids, insulin resistance, andserum lipoproteins.Liver International 2006: 26: 566–571. r Blackwell Munksgaard 2006 Abstract:  Objectives:  The prognosis of nonalcoholic fatty liver disease isdetermined by liver biopsy; steatohepatitis can be progressive whereas fattyliver is benign. Insulin resistance and increased hepatic-free fatty acids arecentral to the pathophysiology of this disorder. Our objective was to assesswhether serum-free fatty acids, lipoproteins, and insulin resistance areincreased in steatohepatitis compared with fatty liver and healthy controls,and thus may be potential noninvasive markers for liver disease severity. Methods:  Fifteen subjects with biopsy proven nonalcoholic steatohepatitis,15 with histological fatty liver, and 15 healthy controls were enrolled. Fastingserum glucose and insulin levels, serum-free fatty acids, HDL, LDL, andcholesterol were collected from each subject. Insulin resistance was calculatedusing the homeostasis assessment model.  Results:  Insulin resistance, LDL,and cholesterol-to-HDL ratio values were significantly higher insteatohepatitis, whereas HDL was significantly lower compared with bothfatty liver and controls. Free fatty acids were similar in all groups. Conclusions:  Along with insulin resistance, serum LDL, and cholesterol-to-HDL ratio values increase with worsening severity of liver histology, andserum HDL values decline. Free fatty acids, however, do not vary betweengroups. Keywords: fatty liver – free fatty acids – HDL –insulin resistance – LDL – NASHIan Bookman, MD, BSc, FRCPC,3200 Dufferin Street,Suite 505,Toronto, ON,Canada M6A 2B3e-mail: ian.bookman@utoronto.caReceived 6 July 2005,accepted 2 February 2006  Nonalcoholic fatty liver disease (NAFLD) is nowthe most common cause of abnormal liver bio-chemistry in North America (1). The spectrum of liver involvement in NAFLD includes simplesteatosis or fatty liver, nonalcoholic steatohepati-tis (NASH) with hepatic fat deposition associatedwith inflammation and hepatocellular damage,and cirrhosis, which may be complicated by liverfailure and primary hepatocellular carcinoma.The prognosis of NAFLD is quite variableacross the spectrum. Steatosis is a benign condi-tion characterized by macrovesicular fatty infil-tration of the liver without evidence of inflammation or fibrosis. Epidemiological evi-dence suggests that most individuals with simplesteatosis have a benign course (2), whereas pro-gression to cirrhosis in NASH occurs in up to26% of subjects over a 7-year follow up (3). A‘two hit hypothesis’ suggests that individuals withNAFLD have factors that predispose them tosimple steatosis; however, a second insult isrequired for progression to NASH (4). The pre-cise etiology of NASH is not completely under-stood. The two metabolic abnormalities moststrongly associated with NAFLD are insulinresistance and an increased supply of fatty acidsto the liver (5, 6). As adipose tissue becomesresistant to insulin, serum lipoprotein levels shift,and flux of free fatty acids to the liver increases (7,8). The cumulative effects of insulin resistanceand increased circulating free fatty acids act inconcert to channel fatty acids into storage ratherthan secretory and degradative pathways (9, 10).As these storage pathways become saturated, theintrahepatic free fatty acids are more available tointracellular microsomes where they undergo li-pid peroxidation. By-products of lipid peroxida-tion include reactive oxygen species and cytokinesthat can impair mitochondrial function and pro-mote hepatic inflammation (11). Liver International 2006: 26: 566–571  r 2006 Blackwell Munksgaard DOI: 10.1111/j.1478-3231.2006.01256.x 566  Currently, the only way to distinguish betweensimple steatosis and NASH is on liver biopsy.Despite this criteria, most studies use only ima-ging modalities to determine the presence and/orextent of fatty liver disease (12–14). In addition,studies of biopsy-proven NAFLD rarely stratifypatients into simple steatosis vs NASH, andalmost never include a healthy control group(15, 16). Investigations of noninvasive markersof NAFLD histology have focussed on distin-guishing different grades of NASH fibrosis (17– 20). Often, a positive marker signifies advanceddisease. To date, no study has attempted toidentify a clinical tool that would differentiatepatients with benign simple fatty liver from thosewith NASH.The objective of this study was to characterizethe prevalence of elevated circulating free fattyacids and serum lipoproteins along with insulinresistance in patients with biopsy-proven NASHvs those with simple steatosis and healthy con-trols. We hypothesized that the severity of freefatty acid levels, insulin resistance, and lipopro-teins carry a higher risk of NASH and could thuspotentially be used as noninvasive measures todistinguish NASH from simple fatty liver. Methods Population Participants with fatty liver or NASH were pa-tients who had been referred to the liver clinic atthe Toronto Western Hospital for investigationof abnormal liver biochemical tests. A detailedhistory was taken to determine whether excessivealcohol intake of more than 20g/day was present.If so, these patients were excluded. The diagnosisof steatosis or NASH was based on liver pathol-ogy from biopsy samples obtained between Jan-uary 2002 and December 2003. Subjects withconcomitant liver disease, such as viral hepatitis,autoimmune liver disease, Wilson disease,  a 1 -antitrypsin deficiency, or hemochromatosis werealso excluded. Patients with NASH and fatty liverwere matched for age, gender, and body massindex (BMI).Control subjects were nondiabetic volunteerswho were not on any medications and had nothad bowel surgery. The controls were matched tothe fatty liver and NASH subjects with respect toage, but not gender or BMI.This study was approved by the Review EthicsBoard of the University Health Network inaccordance with the Helsinki Declaration of 1975. All subjects recruited gave their writteninformed consent to the study. Liver histology Hepatic fat may account for up to 5% of theweight of a normal liver (21). On the basis of thisstatistic and the lack of an established definitionof simple fatty liver, we arbitrarily defined un-complicated fatty liver as a minimum of 10%macrovescular intrahepatic fat accumulation inthe absence of the other histological features.NASH was defined histologically as the pre-sence of fat accumulation in addition to balloon-ing degeneration, Mallory’s hyaline or sinusoidalfibrosis (22). The Brunt et al. (23) standardizedhistological grading system was used to assessseverity of inflammation and extent of fibrosis inliver biopsies.In general, as steatosis vs steatohepatitis wasthe primary distinction in the biopsies (fibrosisgrade secondary), a cut off of a minimum of 1.0cmwas selected as representing adequate length.All liver biopsies were reviewed by one experi-enced hepatopathologist (M. G.). Insulin resistance and other noninvasive measurements All measurements were collected within a max-imum 2 months duration from liver biopsy.Height and weight were measured for each recruitand their BMI was calculated.The index of insulin resistance was calculatedon the basis of three fasting insulin and glucoseblood samples collected at 5-min intervals fromeach subject, according to the homeostasis modelassessment method (24). Insulin resistance, asdetermined by this method, correlates closelywith more complex techniques, such as the eu-glycemic clamp method (25). Control HOMA-IRvalues were obtained from our 15 nonobese,nondiabetic, healthy volunteers (25–64 years)without a family history of diabetes. On the basisof these results, subjects with NASH or fatty liverwere categorized as insulin resistant if theHOMA-IR was greater than 1.95 m Ummol/l(corresponding to the 90th centile).Fasting-free fatty acid levels and serum lipo-protein levels were collected along with the firstinsulin measurement. Statistics Statistical analysis of each variable across allthree groups (NASH, fatty liver, and controls)was conducted using one-way analysis of variancewith  a 5 0.05. Smaller comparisons between twogroups were performed by the Games-Howell orBonferroni method depending on homogeneity of  567 Distinguishing nonalcoholic steatohepatitis resistance, and serum lipoproteins  variance, as determined by Levene’s test forequality of variance. Statistical significance wasconsidered at  P o 0.05. All statistical analyseswere conducted with SPSS software.Our sample size of 15 subjects with NASH and15 subjects with simple fatty liver was derivedfrom publications of similar projects. Sanyal et al.(10) recorded statistically significant differencesin insulin resistance using a sample size of sixsubjects in each group (i.e. simple fatty liver andNASH). Thus, our sample size is larger than thatneeded previously to achieve statistical signifi-cance with regard to insulin resistance. Results A total of 45 subjects were investigated. Ourstudy included 15 subjects with simple fatty liver,15 subjects with NASH, and 15 healthy controls(Table 1). The size of the biopsy length wassimilar in each group (fatty liver: range 0.8– 3cm, mean 1.8cm; NASH: range 1.0–3.1cm,mean 1.9cm;  P 5 0.57). The average age was 39years old in the NASH group, 47 years old in thefatty liver subjects, and 43 years old in thecontrols ( P 5 0.28). The majority of patientswith NASH and simple steatosis was male (80%and 67%, respectively), whereas only 47% of controls were male ( P 5 0.17). The BMI wassimilarly increased for NASH and simple steato-sis, but significantly lower in the controls( P o 0.001).Insulin resistance was statistically significantacross all three groups (ANOVA,  P 5 0.0001),and was significantly higher in subjects withNASH than fatty liver (Games-Howell, P 5 0.047) (Fig. 1). Fasting serum insulin andglucose levels were also significant by ANOVA( P 5 0.0001 and 0.002, respectively), but couldnot distinguish between NASH and fatty liver(Bonferonni,  P 5 0.064 and 0.292, respectively)(Table 2).For the serum lipoproteins, fasting HDL,LDL, and cholesterol-to-HDL ratio values wereall significant by ANOVA ( P 5 0.048, 0.041, and0.0005, respectively) (Fig. 2).Fasting serum-free fatty acid levels were similarin all three groups (ANOVA,  P 5 0.395) (Fig. 3).All patients regardless of liver histology werewithin the normal reference range for fastingserum-free fatty acid levels. Discussion NAFLD is a very common form of liver diseaseand may affect up to 20% of the population (26).The natural history of simple fatty liver is likely Table1. Demographics of NASH, fatty liver and control subjects NASH( n  5 15)Fatty liver( n  5 15)Controls( n  5 15)Age (years) 39.3    12.3 46.9    9.4 42.6    16.1Male (%) 80 67 47BMI 29.9    3.9 31.6    6.5 23.2    2.9Mean    standard deviation. NASH, nonalcoholic steatohepatitis; BMI,body mass index. 76543210    I  n  s  u   l   i  n   R  e  s   i  s   t  a  n  c  e   (  u   U   *  m  m  o   l   ) P  =0.0467 P  =0.0001 P  =0.0001NASHFatty LiverControl Fig.1 . Mean insulin resistance.ANOVA  P 5 0.0001. Table2. Noninvasive biochemical parameters Laboratory test (reference range)NASH( n  5 15)Fatty liver( n  5 15)Controls( n  5 15) ANOVA,  P  Fasting glucose (3.8–6.0mmol/l) 6.12    0.96 5.79    0.63 5.13    0.38 0.002 n Fasting insulin (50–250pmol/l) 147    59 109    50 47    15  o 0.0001 n Insulin resistance ( o 1.95 m Ummol) 5.78    2.8 3.98    1.7 1.49    0.5  o 0.0001 n Free Fatty Acids (280–700Umol/l) 448    158 527    226 536    171 0.395Cholesterol (2.0–5.19mmol/l) 4.94    0.8 5.31    1.0 4.64    0.9 0.160Triglycerides ( o 1.7mmol/l) 1.86    1.1 1.70    1.7 1.27    0.8 0.445HDL ( 4 1.1mmol/l) 1.1    0.2 1.30    0.3 1.43    0.5 0.048 n LDL (1.5–2.99mmol/l) 3.04    0.6 2.8    0.6 2.63    0.7 0.041 n Cholesterol to HDL ratio ( o 4.4–4.9) 4.64    1.0 4.19    0.7 3.40    0.7 0.0005 n Values expressed as mean    standard deviation.  n Considered statistically significant at  P  o 0.05. NASH, nonalcoholic steatohepatitis. 568 Bookman et al.  benign (2); however, NASH may ultimately resultin cirrhosis and liver failure. Thus, it would beideal to distinguish simple fatty liver from NASHwithout relying on an invasive test such as liverbiopsy.The pathogenesis of NASH is not understoodand several mechanisms may be involved.Although studies have demonstrated an associa-tion between insulin resistance and NASH, therelationship between free fatty acids, lipoproteinsand insulin resistance, and severity of histo-pathology remains unclear. Most studies havecompared NASH subjects with controls, andhave focussed on distinguishing between differentgrades of fibrosis in NASH. In our study, wecompared patients with NASH to simple fattyliver as well as controls. The unique aim of ourstudy was to clarify the distinction betweenNASH and simple steatosis, and to explore thepotential use of measurements of insulin resis-tance, free fatty acid levels, and serum lipopro-teins to predict the severity of disease. Our goal isto find noninvasive markers of NAFLD severity,thus sparing subjects from repeated liver biopsies.All three groups were similar in age, but BMIwas significantly lower in the control group. Thisdifference was the result of an intentional selec-tion bias: liver biopsies were not performed onour control subjects, but the fact that they wereleaner decreased the likelihood of fatty liverdisease. The control group was also screened fordiabetes, because recent studies report that in-sulin resistance and hyperinsulinemia are asso-ciated with fatty liver, independent of BMI or fatdistribution (27).The level of insulin resistance we observed inpatients with NASH was significantly higher thanthose with simple steatosis. Insulin resistance washigher in both NASH and fatty liver patients thanhealthy controls. These finding are consistentwith other published studies (15, 16). A receiveroperating characteristic curve for insulin resis-tance in patients with NASH vs fatty liver has anarea under the curve of 0.695 (Fig. 4). A lowercut-off value of 2.65 m Ummol/l gives a sensitivityof 87% and a specificity of 36%, whereas a highercut-off value of 5.1 m Ummol/l provides a sensi-tivity of 67% and a specificity of 79% (Fig. 4).Fasting serum lipoproteins HDL and LDLwere both statistically significant. The choles- 54.543.532.521.510.55 NASH FL C    H   D   L ,   L   D   L ,   C   h  o   l   /   H   D   L   R  a   t   i  o   (  m  m  o   l   /   L   )  HDLLDLChol/HDL Fig.2 . Serum lipoprotein levels: HDL, LDL, and total choles-terol:HDL ratio.ANOVA  P 5 0.048 (HDL),  P 5 0.041(LDL),and  P 5 0.0005 (TC:HDL). 7006005004003002001000    F  r  e  e   F  a   t   t  y   A  c   i   d   L  e  v  e   l  s   (   U  m  o   l   /   L   ) NASHFatty LiverControl Fig.3 . Free fatty acids.ANOVA  P 5 0.395. Markers on  Y  -axis denote upper and lowerlimits of normal reference range. 0.2 0.4 0.6 0.8 1.0    S  e  n  s   i   t   i  v   i   t  y 1-SpecificityAUC 0.695(5.1)(2.65) Fig.4 . Receiver operating characteristic curve for insulin resis-tance in NASH vs fatty liver.AUC, area under the curve. Alower cut-off value of 2.65 m Ummol/l gives a sensitivity of 87%and a specificity of 36%. A higher cut-off value of 5.1 m Ummol/l provides a sensitivity of 67% and a specificity of 79%. 569 Distinguishing nonalcoholic steatohepatitis resistance, and serum lipoproteins  terol-to-HDL ratio, an independent predictor of coronary artery disease risk (28), was also foundto be statistically significant by ANOVA. Thesefindings highlight the correlation between meta-bolic changes and hepatic histopathology.We also compared serum-free fatty acid levels,and found comparable values across all threegroups. One possible reason for this findingmay be that the predominant increased flux of fatty acids in NAFLD occurs between visceral fatand the liver via the portal circulation. Measure-ments of free fatty acids in the systemic circula-tion may not accurately reflect levels in the portalcirculation that are being presented to the liver.Alternatively, the mechanism for maintaininghepatic steatosis in NAFLD may be more relatedto increased hepatic fatty acid synthesis anddecreased triglyceride export from the liver. Ourresults differ from Kelley et al. (29, 30) who founda significant correlation between plasma-freefatty acids, insulin sensitivity, and hepatic stea-tosis. One explanation for this discrepancy maybe that all of the patients in Kelley’s studies hadtype 2 diabetes, and thus perhaps greater degreesof insulin resistance than our subjects. Further-more, in Kelley’s papers, hepatic steatosis wasdiagnosed only with imaging modalities, andinvestigations were not done to exclude othercauses of chronic liver disease. It therefore re-mains unclear as to the accuracy of their diag-nosis of pure fatty liver.There are several potential pitfalls to ouranalysis. Despite a lean BMI and negative screen-ing history of risk factors for fatty liver disease,our control patients did not undergo an imagingstudy for confirmation. As well, although ourcontrols were matched for age, they were notmatched for gender or BMI. Furthermore, alarger sample size would increase the power of our study, especially in the setting of negativefindings. However, despite these limitations, ourstudy is unique in comparing healthy controls tohistologically proven simple fatty liver andNASH.In summary, we were able to show a significantcorrelation between liver histopathology and in-sulin resistance, as well as HDL, LDL, andcholesterol-to-HDL ratio. Serum-free fatty acidlevels were normal in subjects with NASH, simplefatty liver and healthy controls, and this findingmay be an example of the inherent flaw inmeasuring parameters of systemic vs portal cir-culation to reflect states of liver pathophysiology.Thus, a combination of various noninvasivemarkers, including insulin resistance, HDL,LDL, and cholesterol-to-HDL ratio, may serveuseful as a clinical tool to diagnose and follow-uppatients with simple steatosis vs NASH, sparingunnecessary biopsies, and highlighting potentialfoci of directed therapy. Acknowledgement This project was supported by a PSI grant. Reference 1.  Sheth S G ,  Gordon F D ,  Chopra S  Nonalcoholicsteatohepatitis. Ann Intern Med 1997; 126: 137.S.2.  James O F ,  Day C P  Non-alcoholic steatohepatitis(NASH): a disease of emerging identity and importance.J Hepatol 1998; 29(3): 495–501.3.  Powell EE ,  CooksleyWG , Hanson R , et al. The naturalhistory of nonalcoholic steatohepatitis: a follow up study of forty-two subjects for up to 21 years. Hepatology 1990; 11:74.4.  Day CP , James OFW.  Steatohepatitis: a tale of two ‘hits’?Gatroenterology 1998; 114: 842–5.5.  Luyckx F H ,  Lefebvre P J ,  Scheen A J.  Non-alcoholicsteatohepatitis: association with obesity and insulin resis-tance, and influence of weight loss. Diabetes Metab 2000;26: 98–106.6.  Cortez-Pinto H ,  Camilo M E ,  Baptista A , et al. Non-alcoholic fatty liver: another feature of the metabolicsyndrome? Clin Nutr 1999; 18: 353–8.7.  Campbell PJ ,  Carlson M G ,  Nurjhan N.  Fat metabo-lism in human obesity. Am J Physiol 1994; 266: E600–5.8.  Gibbons G F ,  Islam K ,  Pease R J.  Mobilisation of triacyglycerol stores. Biochem Biophys Acta 2000; 1483:37–57.9.  Wiggins D ,  Gibbons GF.  The lipolysis/esterification cycleof hepatic triacylglycerol. Its role in the secretion of very-low-density lipoprotein and its response to hormones andsulphonylureas. Biochem J 1992; 284: 457–62.10.  Gibbons G F ,  Wiggins D.  Intracellular triacylglycerollipase: its role in the assembly of hepatic very-low densitylipoprotein (VLDL). Adv Enzyme Regul 1995; 35: 179–98.11.  Sanyal A J ,  Campbell-Sargent C ,  Mirshahi F , et al.Nonalcoholic steatohepatitis: association of insulin resis-tance and mitochondrial abnormalities. Gastroenterology2001; 120(5): 1183–92.12.  Marchesini G ,  Brizi M ,  Morselli-Labate A M , et al.Association of nonalcoholic fatty liver disease with insulinresistance. Am J Med 1999; 107: 450–5.13.  GotoT ,  OnumaT ,  Takebe K , et al. The influence of fattyliver on insulin clearance and insulin resistance in non-diabetic Japanese subjects. Int J Obes Relat Metab Disord1995; 19(12): 841–5.14.  Lee JH , Rhee PL , Lee JK , et al. Role of hyperinsulinemiaand glucose intolerance in the pathogenesis of nonalcoholicfatty liver in subjects with normal body weight. Gastro-enterology 1986; 24(8): 403–15.15.  Chitturi S ,  Abeygunasekera S ,  Farrell G C , et al.NASH and insulin resistance: insulin hypersecretion andspecific association with the insulin resistance syndrome.Hepatology 2002; 35(2): 373–9.16.  Marchesini G ,  Bugianesi E ,  Forlani G , et al. Nonalco-holic fatty liver, steatohepatitis, and the metabolic syn-drome. Hepatology 2003; 37(4): 917–23.17.  Bugianesi E ,  Manzini P D ,  Antico S , et al. Relativecontribution of iron burden, HFE mutations, and insulinresistance to fibrosis in nonalcoholic fatty liver. Hepatology2004; 39(1): 179–87. 570 Bookman et al.
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