Standardized Ultrasound Hepatic/Renal Ratio and Hepatic Attenuation Rate to Quantify Liver Fat Content: An Improvement Method

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1 nture publishing group Open Stndrdized Ultrsound Heptic/Renl Rtio nd Heptic Attenution Rte to Quntify Liver Ft Content: An Improvement Method Ming-Feng Xi 1, Hong-Mei Yn 1, Wn-Yun He 2, Xio-Ming Li 1, Cho-Lun Li 2, Xiu-Zhong Yo 3, Ruo-Kun Li 3, Meng-Su Zeng 3 nd Xin Go 1 Accurte mesures of liver ft content re essentil for investigting the role of heptic stetosis in the pthophysiology of multiple metbolic disorders. No trditionl imging methods cn ccurtely quntify liver ft content. [ 1 H]-mgnetic resonnce spectroscopy (MRS) is restricted in lrge-scle studies becuse of the prcticl nd technologicl issues. Previous ttempts on computer-ided ultrsound quntifiction of liver ft content vried in method, nd the ultrsound quntittive prmeters mesured from different ultrsound mchines were hrdly comprble. We imed to estblish nd vlidte simple nd propgble method for quntittive ssessment of liver ft content bsed on the combintion of stndrdized ultrsound quntittive prmeters, using [ 1 H]-MRS s gold stndrd. Totlly 127 prticipnts were exmined with both ultrsonogrphy (US) nd [ 1 H]-MRS. Ultrsound heptic/renl echo-intensity rtio (H/R) nd ultrsound heptic echo-intensity ttenution rte (HA) were obtined from ordinry ultrsound imges using computer progrm. Both prmeters were stndrdized using tissue-mimicking phntom before nlysis. Stndrdized ultrsound H/R nd HA were positively correlted with the liver ft content by [ 1 H]-MRS (r = 0.884, P < nd r = 0.711, P < 0.001, respectively). Liner regression nlysis showed ultrsound H/R could modestly predict the mount of liver ft (djusted explined vrince 78.0%, P < 0.001). The ddition of ultrsound HA slightly improved the djusted explined vrince to 79.8%. Difference of estimted liver ft contents between different ultrsound mchines nd opertors ws resonbly well. Thus, computer-ided US is vlid method to estimte liver ft content nd cn be pplied extensively fter stndrdiztion of ultrsound quntittive prmeters. Obesity (2011) 20, doi: /oby Introduction Nonlcoholic ftty liver disese (NAFLD) is rpidly becoming the most common liver disese worldwide (1). The prevlence of NAFLD in the generl popultion is estimted to be 20 30% in western countries (2,3) nd 15% in Asin countries (4,5). Though the prevlence is prtly depended on the method used to dignose NAFLD, NAFLD hs been undoubtedly n importnt globl disese burden not only for its high nd fst incresing prevlence, but lso for its severe metbolic complictions. NAFLD ssocites with insulin resistnce, obesity, hypertension, type 2 dibetes mellitus (T2DM), hyperlipidemi (6,7) nd my precede T2DM development nd crdiovsculr disese (CVD (8)). Recently, crotid therosclerosis hs been detected in ptients with NAFLD even with no ltertions in liver enzyme tests (9). Thus, n ccurte ssessment of heptic ft infiltrtion would hve profound significnce in the clinicl setting. Liver biopsy is the gold stndrd for quntifiction of heptic stetosis. However, it is hrd to be ccepted by most ptients for its invsiveness nd significnt degree of smpling error (10). Moreover, most ptients with heptic stetosis re symptomtic (11), nd only 50% of ptients with NAFLD (12) will hve elevted lnine trnsminses. Therefore, n ccurte, cost-effective nd noninvsive imging method tht cn quntittively mesure liver ft content is idelly needed. [ 1 H]-mgnetic resonnce spectroscopy (MRS) directly mesures protons in cyl groups of liver tissue triglycerides (13), nd the vlues obtined using [ 1 H]-MRS correlte well with the histologicl liver ft content (14 16), which provides n ccurte, sensitive, nd noninvsive method to quntify liver ft content. Our previous study lso showed close positive correltion between the heptic triglyceride content by [ 1 H]-MRS nd pthologicl mesurements of liver ft content The first two uthors contributed eqully to the work. 1 Deprtment of Endocrinology nd Metbolism, Zhongshn Hospitl, Fudn University, Shnghi, Chin; 2 Deprtment of Ultrsonogrphy, Zhongshn Hospitl, Fudn University, Shnghi, Chin; 3 Deprtment of Rdiology, Zhongshn Hospitl, Fudn University, Shnghi, Chin. Correspondence: Xin Go (go.xin@zs-hospitl.sh.cn) Received 29 April 2011; ccepted 28 August 2011; published online 20 October doi: /oby VOLUME 20 NUMBER 2 februry

2 (r = 0.878, P < (17)). The upper limit of norml for heptic triglyceride content by [ 1 H]-MRS ws determined to be 5.56% in the Dlls Hert Study (18). However, [ 1 H]-MRS is expensive nd fr less vilble thn other imging exmintions, which limits its use in clinicl prctice nd lrge-scle epidemiologicl studies. Ultrsonogrphy (US) is n ppeling technique to detect ftty infiltrtion of the liver becuse of its simplicity, low-cost, noninvsive nture, nd widespred vilbility. However, its ppliction is limited by the interobserver nd introbserver vribility (19), poor sensitivity in detecting mild heptic stetosis (20), nd ultimtely it is unble to provide n ccurte mesurement of liver ft content. Recently severl ttempts hve been mde to estblish methods for quntittive ssessment of liver ft content by US. Edens et l. showed the fesibility of ultrsound liver ft content quntifiction by using combintion of computer-ssisted ultrsound mesures from routine ultrsound imges (21). However, these ultrsound prmeters were obtined with specilly developed softwre progrm nd somewht complex for prcticl clinicl ppliction. Menwhile, studies by Webb et l. (22) nd Mncini et l. (23) reported tht computer-ided mesurement of US heptic/renl echo-intensity rtio (H/R) were highly correlted with liver ft content determined by histology nd [ 1 H]-MRS, respectively. These two studies indicted US heptic/renl rtio s suitble quntittive prmeter to quntittively reflect liver ft content, but the vlue rnges of the US H/R rtios from the two studies differed gretly, which were totlly not comprble. Thus, stndrdiztion of the US H/R rtio is necessry before its widespred clinicl ppliction. In ddition, one recent pilot study lso ttempted to use phntom-clibrted, computermesured ultrsound heptic ttenution coefficients to ssess the severity of heptic stetosis in diry cttles (24). However, none of these studies hs clerly provided reltive ccurte, stble, nd reproducible US quntittive method for liver ft content yet. In this study, we improved the method for US H/R rtio nd US heptic echo-intensity ttenution rte (HA) mesurement by introducing tissue-mimicking phntom for stndrdiztion to mke them more comprble mong different ultrsound mchines. Furthermore, we lso ttempted to estblish nd vlidte predictive lgorithm for liver ft content with stndrdized US H/R nd HA, using [ 1 H]-MRS s stndrd. Methods nd Procedures Subjects US nd [ 1 H]-MRS exmintions were performed in 127 prticipnts (ge rnge, yers; BMI rnge, kg/m 2 ) who were dignosed with no or different degrees of heptic stetosis by routine US exmintions from the outptient deprtment of endocrinology nd physicl exmintion center of Zhongshn Hospitl, Shnghi, Chin. Of the 127 prticipnts, 35 were dignosed with no heptic stetosis, 64 with mild heptic stetosis, nd 28 with moderte or severe heptic stetosis. The prticipnts did not hve history, clinicl symptoms, or signs of other liver or renl disese; nor did they hve history of dibetes or excess lcoholic drinking ( 20 g/dy for men nd 10 g/dy for women (25)). They were not tking hypolipidemic drug, liver protectnt, or drugs tht could cuse stetosis. All prticipnts hd negtive heptitis B virus surfce ntigen nd heptitis C virus ntibody, norml renl function, nd bsence of proteinuri in spot urine collection. The protocol for the study ws pproved by the ethics committee of Zhongshn Hospitl, Shnghi. Written informed consent ws obtined from ll prticipnts. Anthropometric, ultrsound, nd [ 1 H]-MRS mesurements were performed t the sme dy, nd fsting venous blood smples were drwn for determintion of blood routine, liver nd renl function, nd heptitis virus indiction. Anthropometric mesurements The mesurement of height nd weight required the subjects wore light clothing without shoes. Body mss index ws clculted s weight (kg) divided by height (m) squred. Wist circumference ws mesured with soft tpe on stnding subjects midwy between the lowest rib nd the ilic crest. Hip circumference ws ssessed t the level of the greter trochnters. Blood pressure ws mesured on right rm with subjects in sitting position fter 5-min rest. Biochemicl nlysis Biochemicl tests for liver enzymes, serum lipid profile, nd fsting plsm glucose were performed using n utonlyzer (Hitchi 7300, utomtic nlyzer, Tokyo, Jpn). Serum heptitis B virus surfce ntigen nd heptitis C virus ntibody were tested by the Enhnced Electrochemiluminescence method. US All exmintions were performed with GE Vivid7 ultrsound mchine (GE Helthcre, Horten, Norwy) equipped with GE 4C curved rry trnsducer (GE H4904PC). Twenty-six of the subjects lso ccepted second US exmintion within 12 h on nother GE Logiq P5 ultrsound mchine (GE Helthcre, Milwukee, WI). US studies were performed by two experienced rdiologists (WH nd CL) who were unwre of the ptient s clinicl detils nd lbortory findings. All the instrument settings, including gin, depth, nd time-gin compenstion, were fixed for ech mesurement. For ssessment of US H/R rtio, ultrsound imges with both liver nd right kidney clerly visulized were obtined in the sgittl liver/right kidney view in the lterl position (Figure 1). US heptic echo-intensity ttenution rte ws ssessed in right intercostls view t nterior xill line in the supine position (Figure 1b). Ultrsound imge nlysis All imges were trnsferred to personl computer nd reviewed by one of the two rdiologists involved in scnning. Anlysis of digitized ultrsound imges ws performed by using NIHimge softwre (ImgeJ 1.41o, Ntionl Institutes of Helth, Bethesd, MD). US heptic/renl echo-intensity rtio In sgittl liver/right kidney view, region of interest (ROI) of cm (1,296 pixels) in the liver prenchym ws selected. The ROI hd to be s uniform s possible, excluding blood vessels, bile ducts, nd other focl hypo/hyperechogenicity. Another ROI of cm (144 pixels) ws identified in the right renl cortex with no lrge vessels, renl sinus or medull. To void the interference of depth-depended echo-intensity ttenution nd the borderline echo distorting effects, the boundry between liver nd right kidney re should be plced ner the center of the imge, nd the liver nd right kidney ROIs were selected t the sme depth of the ultrsound imges. The gry scle men vlue of the pixels within the two ROIs ws used s mesurement of echo intensity (Figure 1). Then we divided the verge heptic gry scle by the verge renl cortex gry scle to clculte the US heptic/renl rtio. US heptic echo-intensity ttenution rte In right intercostls view t nterior xill line, tngent line of the sector ultrsound imge ws drwn nd the ultrsound wve trnsmission line ws determined, strting from the point of tngency obesity VOLUME 20 NUMBER 2 februry

3 Where Δd is the distnce between the ner-field nd fr-field ROIs, nd other prmeters re defined in eqution (2). b nd perpendiculr to the tngent line. Two ROIs of cm (1,296 pixels) were selected in liver homogeneous regions long the ultrsound trnsmission line ner the liver nterior mrgin (depth 4 6 cm) nd the liver posterior mrgin, respectively. The liner distnce between the two ROIs ws lso mesured (Figure 1b). The echo intensity of ultrsound wve ws ttenuted exponentilly, shown s the following eqution (26,27): fd A = A (1) d 0 e Where A 0 nd A d re ultrsound echo intensity t the sound source nd the liver prenchym t specific depth, respectively; is the ttenution coefficient of the liver prenchym; f is the frequency of the ultrsound detector; d is the depth of ROI. The rtio of the verge echo intensity in the liver ner-field ROI to liver fr-field ROI ws then clculted bsed on eqution (1): A n 6.64 cm Figure 1 Ultrsound imges of liver in () sgittl liver/right kidney view nd (b) right intercostls view in 53-yer-old mn show grphic representtion of region of interest (ROI) rectngles. ROI-1, ROI-2, ROI-3, nd ROI-4 show the gry scle distribution of the pixels in the selected liver, right kidney cortex, liver ner-field, nd liver fr-field region, respectively. The liner distnce between the top left corners of ner-field nd fr-field liver ROIs ws lso mesured. A f df dn f = e ( ) (2) Where A n nd A f re verge ultrsound echo intensity in the ner-field ROI nd the fr-field ROI, respectively; nd f hve been defined in eqution (1); dn nd df re the depth of liver ner-field nd fr-field ROIs. Then the formul for ultrsound heptic echo-intensity ttenution rte ws deduced from eqution (2): = ( ln An ln Af)/( d f ) (3) Stndrdiztion of ultrsound quntittive prmeters To stndrdize the mesured vlues of US H/R rtio nd heptic echointensity ttenution rte mong different ultrsound mchines, 3D bdominl phntom (Model 057; Computerized Imging Reference Systems, Norfolk, VA), contining mimic bdominl orgns, ws used for stndrdiztion in this reserch. The ultrsound imges of the phntom were obtined under both of the ultrsound mchines (GE Vivid7 nd GE Logiq P5), nd the phntom s H/R rtio nd heptic ttenution rte were mesured with the sme protocol for ultrsound imge nlysis described bove. Then we divided the mesured ultrsound H/R rtios by the phntom s H/R rtio nd subtrcted the phntom s heptic ttenution rte from the ptients heptic ttenution rtes to obtin the stndrdized US H/R rtio nd stndrdized heptic ttenution rte (Supplementry Figure S1 online). Vlidtion of ultrsound quntifiction of liver ft content To ssess the greement in mesuring stndrdized US H/R nd heptic ttenution rte between different opertors nd different ultrsound mchines, 26 prticipnts ccepted US exmintions on two different ultrsound mchines (GE Vivid7 nd GE Logiq P5) by the sme rdiologist within 12 h, nd 23 prticipnts were exmined seprtely by two rdiologists within 12 h. Mesurement of heptic triglyceride content using [ 1 H]-MRS [ 1 H]-MRS mesurements were performed on 1.5-T mgnetic resonnce (MR) scnner (Siemens Avnto, Erlngen, Germny) equipped for proton spectroscopy cquisitions. Sgittl, coronl, nd xil slices covering the whole liver were preliminrily cquired for positioning of the spectroscopy cquisition voxel. A single voxel of 8 cm 3 (2 2 2 cm) ws plced within the right lobe voiding mjor vsculr structures nd subcutneous ft tissue. The proton spectrum ws cquired using the body coil fter shimming over the volume of interest by mens of point-resolved spectroscopy (PRESS) sequence with the following prmeters: repetition time = 1,500 ms, echo time = 135 ms. Signl intensities of wter pek t 4.8 ppm (Sw) nd the ft pek t 1.4 ppm(sf) were mesured, nd heptic ft percentge ws clculted using the formul 100 Sf/(Sf + Sw), s described by our group previously (28). Sttisticl nlysis All sttisticl nlyses were performed using SPSS softwre version 13.0 (SPSS, Chicgo, IL). The dt re presented s men ± s.d. except for skewed vribles, which re presented s medin (interqurtile rnge 25 75%). One-wy ANOVA ws used for comprisons mong groups. Multivrite liner stepwise regression nlysis ws used to estblish predictive lgorithm for liver ft content with the ultrsonic quntittive prmeters, using [1H]-MRS s stndrd. Receiver operting chrcteristic curve nlysis ws used to determine the pproprite cutoff vlue for ultrsoundestimted liver ft content to dignose NAFLD. The optiml cutoff vlues were obtined from the Youden index (mximum (sensitivity + specificity 1 (29))). Person s correltion nlysis ws used to explore the ssocition of ultrsound liver ft content with ll mesured metbolism-relted prmeters. Interobserver vrition, s well s the vrition between different ultrsound mchines, ws ssessed using intrclss correltion (ICC) coefficient nd Blnd Altmn sttistics (30). Vlues for P < 0.05 were considered sttisticlly significnt for ll nlyses. Results Study popultion The study popultion consisted of 71 men nd 56 women, with n ge rnge of yer, BMI of kg/m 2, nd wist-to-hip rtio of Liver ft content determined by [ 1 H]-MRS nlysis rnged from 3.0 to 70.9% (men 20.2%). 446 VOLUME 20 NUMBER 2 februry

4 By ppliction of the current criteri for dignosis of stetosis by [ 1 H]-MRS, 81.1% of the subjects (103/127) hd liver ft content exceeding 5.56%. The originl ultrsound heptic/ renl rtios nd heptic echo-intensity ttenution rtes were (men 1.39) nd /MHz/cm /MHz/ cm (men /MHz/cm), respectively. After stndrdiztion, the heptic/renl rtios were djusted to be , nd the djusted heptic echo-intensity ttenution rtes rnged from to /MHz/cm (Tble 1). Estimtion of liver ft content by [ 1 H]-MRS using US quntittive prmeters Both US heptic/renl rtio nd US heptic ttenution rte were highly positively ssocited with the liver ft content by [ 1 H]-MRS, with r = 0.884, (P < 0.001) nd r = 0.711, (P < 0.001), respectively (Figure 2,b). Multivrite liner regression nlysis ws performed to investigte min fctors ssocited with liver ft content from ll common nthropometric nd US quntittive prmeters. The gretest contribution to the prediction Tble 1 Chrcteristics of the study ptients [ 1 H]-MRS heptic ft content (%) Chrcteristic < Totl P vlue* Number of prticipnts Age (yer) 56(50 58) 52(46 57) 54(49 61) 53(44 59) 52(40 57) 53(44 59) Sex (mle/femle) 4/12 12/8 19/8 21/18 15/10 71/ Anthropometric mesures Weight (kg) 64.9 ± ± ± ± ± ± Height (cm) ± ± ± ± ± ± BMI (kg/m 2 ) 24.4 ± ± ± ± ± ± Wist circumference (cm) 82.1 ± ± ± ± ± ± 0.9 <0.001 Hip circumference (cm) 93.5 ± ± ± ± ± ± Wist-to-hip rtio 0.88 ± ± ± ± ± ± 0.01 <0.001 SBP (mm Hg) ± ± ± ± ± ± DBP (mm Hg) 71.9 ± ± ± ± ± ± Liver enzymes ALT (IU/l) 17 (12 20) 18 (14 25) 28 (19 45) 34 (21 60) 42 (33 85) 28 (18 50) <0.001 AST (IU/l) 19 (15 23) 19 (18 23) 23 (18 27) 24 (17 40) 32 (23 40) 23 (18 30) ALP (IU/l) 64.4 ± ± ± ± ± ± γ-gt (IU/l) 20 (12 37) 30 (23 50) 28 (22 69) 35 (22 57) 51 (28 61) 34 (22 57) Lipid profile Triglycerides (mmol/l) <0.001 ( ) ( ) ( ) ( ) ( ) ( ) Cholesterol (mmol/l) 4.95 ± ± ± ± ± ± HDL cholesterol (mmol/l) 1.43 ± ± ± ± ± ± LDL cholesterol (mmol/l) 3.00 ± ± ± ± ± ± Apo-A (g/l) 1.48 ± ± ± ± ± ± Apo-B (g/l) 0.91 ± ± ± ± ± ± Apo-E (mg/dl) 37 (30 42) 36 (30 42) 42 (35 52) 45 (38 57) 47 (38 53) 41 (36 50) Uric Acid (umol/l) ± ± ± ± ± ± Blood glucose Fsting glucose (mmol/l) 5.2 ( ) 5.6 ( ) 6.1 ( ) 6.2 ( ) 6.0 ( ) 5.9 ( ) h postlod glucose (mmol/l) 7.1 ± ± ± ± ± ± US mesures (stndrdized) Heptic/renl rtio 0.56 ± ± ± ± ± ± 0.01 <0.001 Heptic ttenution rte (cm 1 MHz 1 ) ± ± ± ± ± ± Dt re men ± s.e.m. or medin (interqurtile rnge) for continuous vribles. DBP, distolic blood pressure; HDL, high-density lipoprotein; [ 1 H]-MRS, proton mgnetic resonnce spectrum; LDL, low-density lipoprotein; SBP, systolic blood pressure. *P vlues for the overll comprisons mong groups with different heptic ft content. <0.001 obesity VOLUME 20 NUMBER 2 februry

5 of liver ft content cme from the US H/R rtio, nd it could be used independently to clculte liver ft content (djusted explined vrince to 78.0%, P < 0.001). (Model 1, Tble 2). The ddition of US heptic ttenution rte to the US heptic/renl rtio further improved the explined vrince in liver ft content from 78.0 to 79.8%. This ws reflected in the 4.2% reduction in root men squred error. (Model 2, Tble 2). The lgorithm derived from the second predictive model ws s follows: Liver ft content (%) = US heptic/renl rtio US heptic ttenution rte Quntittive vs. qulittive US Vlidity of the bove ultrsound quntittive lgorithm, in comprison with qulittive US method used in clinicl cre, b Heptic ft content by 1H-MRS (%) Heptic ft content by 1H-MRS (%) y = 0.736x r = 0.884, P < Stndrdized US Heptic/renl rtio 80 y = 6.226x r = 0.711, P < Stndrdized US heptic ttenution rte Figure 2 () Liner correltion between liver ft contents by [ 1 H]-MRS nd () US heptic/renl rtio (r = 0.884, P < 0.001) nd (b) US heptic ttenution rte (r = 0.711, P < 0.001). is shown in Tble 3. Ptients with liver ft content mesured by [ 1 H]-MRS of t lest 5.56% were dignosed s heptic stetosis. Receiver operting chrcteristic curve nlysis showed tht the optiml cutoff vlue for ultrsound-estimted liver ft content to dignose heptic stetosis ws 9.15%. Using cutoff vlue of 9.15%, the sensitivity nd specificity for quntittive US to dignose heptic stetosis were 95.1% nd 100%, respectively, better thn the qulittive US, whose sensitivity nd specificity were 82.5 nd 83.3%, respectively. In the 63 subjects with liver ft content by [ 1 H]-MRS less thn 15%, the quntittive US lso yielded very high sensitivity (82.6%) nd specificity (100%), but the sensitivity nd specificity of trditionl US were only 47.8 nd 83.3%, respectively (Tble 3). Correltion between ultrsound-estimted liver ft content nd ll other metbolism-relted prmeters The Person correltion coefficients between ultrsound-estimted liver ft content nd other common nthropometric nd biochemicl prmeters re given in Tble 4. After djustment for ge, sex, nd BMI, liver ft content estimted by ultrsound ws positively ssocited with wist circumference, hip circumference, serum ALT, AST, triglycerides, Apo-E, nd fsting glucose levels nd negtively ssocited with high-density lipoprotein cholesterol (HDL-C) levels. Relibility There is excellent interobserver greement for US heptic/ renl rtio nd US heptic ttenution rte (ICC = nd ICC = 0.942, respectively). Excellent greement between different ultrsound mchines ws lso obtined for US heptic/ renl rtio nd US heptic ttenution rte (ICC = nd ICC = 0.861, respectively). Blnd nd Altmn nlysis suggested tht if 95% of the difference were within the limits of greement then this denoted good greement between the two sets of mesurements (Figure 3). According to the Blnd Altmn method, the 95% limits of interobserver difference of US heptic/renl rtio nd US ttenution rte were ( % of totl US heptic/renl rtio rnge) nd /cm/MHz ( % of totl US heptic ttenution rte rnge), respectively. The 95% limits of difference between different US mchines were ( % of rnge) for US heptic/renl rtio, nd /cm/MHz ( % of rnge) for US ttenution rte, respectively. If we tolerte difference smller thn 5% for liver ft content estimtion, s shown by the interrupted lines in Figure 4, 21 (91.3%) nd 18 (69.2%) ptients Tble 2 Prediction models for heptic ft content by [ 1 H]-MRS using nthropometric nd ultrsound quntittive prmeters Heptic ft content (%) Model US heptic/ renl rtio B ± s.e. US heptic ttenution rte (MHz 1 cm 1 ) Constnt Model P vlue Model dj. R 2 RMSE ± ± < % ± ± ± < % 5.93 B, expected chnge in heptic ft content per unit increse in the covrite; RMSE, root men squred error; US, ultrsound. Model: cndidte predictors included for nlysis were ge, gender, BMI, weight, wist circumference, wist-to-hip rtio, US heptic/renl rtio, nd US heptic ttenution rte. Skewed vribles were log trnsformed to norml distribution before liner regression nlysis. 448 VOLUME 20 NUMBER 2 februry

6 Tble 3 Comprison of dignostic performnce between quntittive nd qulittive ultrsound exmintions Dignostic criteri Ftty liver dignosed by [ 1 H]-MRS Sensitivity (%) Specificity (%) PPV(%) NPV(%) All prticipnts Quntittive ultrsound (estimted LFC 9.15%) Qulittive ultrsound Prticipnts with [ 1 H]-MRS LFC <15% Quntittive ultrsound (estimted LFC 9.15%) Qulittive ultrsound LFC, liver ft content; NPV, negtive predictive vlue; PPV, positive predictive vlue. would hve the difference of clculted liver ft contents from different opertors or different US mchines within the rnge of ± 5% liver ft content (Figure 4). Discussion In this study, we found tht computer-ssisted US heptic/ renl rtio nd US heptic ttenution rte from ordinry US heptic nd right kidney imges were highly positively ssocited with liver ft content by [ 1 H]-MRS. Combintion of US heptic/renl rtio nd US heptic ttenution rte could be used to simply nd reltive ccurtely estimte liver ft content, nd our improved US quntittive method lso showed higher sensitivity thn trditionl US qulittive method in detecting mild heptic stetosis. As for the relibility of our improved US quntittive method for liver ft content, the reproducibility between different opertors nd different US mchines ws resonbly well fter stndrdiztion by tissue-mimicking phntom, s the difference of the US-estimted liver ft content between different opertors nd different ultrsound mchines mostly fell in the rnge of ± 5% liver ft content. Heptic stetosis cn be ssessed by some chrcteristics on ultrsound imges, including (i) hyperechogenity of liver tissue ( bright liver ) s often compred to hypoechogenity of the kidney cortex, (ii) fll of echo mplitude with depth (posterior bem ttenution), (iii) fine, tightly pcked echoes, (iv) loss of echoes from the wlls of the portl veins (20). However, these criteri re qulittive, so the trditionl US dignosis of heptic stetosis ws highly depended on the subjective interprettion of the exminer, which cn led to limittion of the method reproducibility, nd not lest to dignostic errors. In contrst, the computer-ided mesurement of US H/R rtio nd US heptic echo-intensity ttenution rte relized the objective quntifiction of US imge chrcteristics of heptic stetosis nd showed enormous dvntges over trditionl qulittive US. Using the ultrsound quntittive prmeters, we were ble to identify the miniml bright liver echo chnges on ultrsound imges, which were impossible to distinguish by nked eyes. Thus, the low sensitivity of trditionl US to detect mild stetosis (20) could be remrkbly resolved Tble 4 Correltion of heptic ft content by improved US with nthropometric, biochemicl, nd ultrsonic prmeters in 127 subjects Vribles Ultrsound liver ft content Ultrsound heptic ft content (ge, sex, nd BMI djusted) r P r P Age BMI Sex Weight Height Wist circumference < Hip circumference < Wist-to-hip rtio SBP DBP ALT < AST ALP γ-gt Triglycerides < <0.001 Cholesterol HDL cholesterol LDL cholesterol Apo-A Apo-B Apo-E Uric cid Fsting glucose h postlod glucose [ 1 H]-MRS heptic ft content < <0.001 HDL, high-density lipoprotein; LDL, low-density lipoprotein; US,ultrsonogrphy. Vribles were significntly skewed nd log trnsformed to norml distribution when tken into Person correltion nlysis. by the US quntittive method, nd in the current study we lso confirmed the high sensitivity nd specificity of US quntittive method in dignosing mild heptic stetosis. On the other hnd, the ultrsound quntittive method is objective nd less dependent on opertors subjective impression, so it could overcome the subjective error of US exmintion results to certin degree. The men inter- nd introbserver greement rtes for trditionl US dignosis of heptic stetosis were 72% nd 76% (19). In comprison, the interobserver difference of US-quntified liver ft content in our current study is bout ± 5% liver ft content, which corresponds to 95.6% greement rtes for the qulittive dignosis of heptic stetosis. More importntly, our US quntittive method formultes reltive ccurte quntifiction of the liver ft content, rdiclly obesity VOLUME 20 NUMBER 2 februry

7 Difference in US heptic/renl rtio from Vivid7 nd LP5 0.2 Upper 95% limit 0.1 Men difference Lower 95% limit Men US heptic/renl rtio from Vivid7 nd LP5 b Difference in US heptic ttenution rte from Vivid7 nd LP Upper 95% limit Men difference Lower 95% limit Men US heptic ttenution rte from Vivid7 nd LP5 c Difference in US heptic/renl rtio from different opertors d Difference in US heptic ttenution rte from different opertors Upper 95% limit Men difference Lower 95% limit Men US heptic/renl rtio from different opertors Upper 95% limit Men difference Lower 95% limit Men US heptic ttenution rte from different opertors Figure 3 Blnd Altmn nlysis for greement of US quntittive prmeters between mesurements with different ultrsound mchines (,b) nd by different opertors (c,d). () mesurement of US heptic/renl rtio from GE Vivid7 US mchine minus mesurement from GE Logiq P5 US mchine; (b) mesurement of US heptic ttenution rte from GE Vivid7 US mchine minus mesurement from GE Logiq P5 mchine; (c) mesurement of US heptic/renl rtio by opertor 1 minus mesurement by opertor 2; nd (d) mesurement of US heptic ttenution rte by opertor 1 minus mesurement by opertor 2. LP5, Logiq P5. b Difference in predicted HFC from Vivid7 nd LP5 (%) Difference in predicted HFC from different opertors (%) Men predicted HFC from Vivid7 nd LP5 (%) Men predicted HFC from different opertors (%) Figure 4 Blnd Altmn nlysis for greement of liver ft content estimted with US quntittive prmeters mesured from () different ultrsound mchines nd (b) different opertors. In, 18 of 26 (69.2%) points fll within the rnge of ± 5% liver ft content; in b, 21 of 23 (91.3%) points fll within the rnge of ± 5% liver ft content. chnges the qulittive nture of trditionl US, nd provides n idel tool for quntifiction of liver ft content in lrgescle clinicl studies nd determintion of tretment efficcy for heptic stetosis. Therefore, the ultrsound quntittive method for liver ft content bsed on the combintion of US quntittive prmeters might be cliniclly vluble s n esy nd reltive ccurte method to dignose heptic stetosis. On the bsis of the previous ttempts on ultrsound quntifiction of liver ft content (22 24), we further improved the US quntittive method by tking the difference mong different ultrsound mchines into considertion nd introduced stndrdiztion procedure for the US imge nlysis. It is noticeble tht the originl US quntittive prmeters mesured with different ultrsound mchines might vry tremendously. The originl heptic/renl rtio rnged from 0.88 to 2.17 (men 1.39) in our study, while the US heptic/renl rtios reported previously were (men 2.5 (23)) nd (men 1.65 (22)), respectively. The gret discrepncy might be cused by the different postprocess when ultrsound scnner utomticlly trnsltes echo mplitude vlues to the brightness on the US imges. Different US mchines hve different forms of dptive contrst or texture enhncement (31,32), so the initil US quntittive prmeters from different US mchines vry gretly, nd this bdly restricts its prcticlity nd ppliction forecst. To djust for the difference mong ultrsound devices, we introduced tissue-mimicking phntom for stndrdiztion in the current study, nd the stndrdiztion procedure remrkbly improved the comprbility of US quntittive prmeters mong different US mchines nd incresed the ppliction nd extension vlue of the ultrsound quntittive method for liver ft content. Our study lso showed tht the optiml US-estimted liver ft content cutoff of 9.15% yielded very high sensitivity (95.1%) nd specificity (100%), enbling us to ttin good positive 450 VOLUME 20 NUMBER 2 februry

8 predictive vlue (PPV) nd negtive predictive vlue (NPV) (100% nd 82.2%, respectively) for the dignosis of heptic stetosis. Noticebly, the cutoff vlue for US-estimted liver ft content to dignose heptic stetosis is higher thn tht of [ 1 H]-MRS. This result ws explicble if we noticed tht the liver ft content estimted by US ws ctully not strictly linerly relted to [ 1 H]-MRS liver ft content. When liver ft content by [ 1 H]-MRS ws between 5% nd 40%, the US-estimted liver ft content greed well with the liver ft content by [ 1 H]-MRS. However, when the liver ft content by [ 1 H]-MRS decresed pproximtely below 5%, the US-estimted liver ft content showed steep decline from 5% 10% to 0%, nd when the liver ft content incresed over 40%, the US-estimted liver ft content tended to rech pltform. Therefore, the US heptic ft quntifiction method might slightly overestimte rel liver ft content when the liver ft content is ner 5%, nd the US-estimted liver ft content cutoff vlue for heptic stetosis ws lso higher thn tht of [ 1 H]-MRS correspondingly (Supplementry Figure S2 online). Even though it ws still n pproximte estimtion of liver ft content, the US quntittive method is undoubtedly n dvnce in imging dignosis of heptic stetosis. Becuse the US quntittive method is esy for opertion, sensitive in detection of mild heptic stetosis, nd reltively ccurte in reflecting the severity of heptic stetosis, it is especilly suitble for lrge-scle quntittive study on heptic stetosis nd clinicl trils on the follow-up nd determintion of tretment efficcy for heptic stetosis. One limittion of our study is tht spectroscopy, rther thn histologic exmintion, ws used s the reference. Pthologic exmintion of biopsied specimens from the liver remins the criterion stndrd in current clinicl prctice to estblish the dignosis of stetosis. However, liver biopsy is n invsive procedure with morbidity rte of 3% nd mortlity rte of 0.03% (33). It hs been demonstrted tht histology correltes well with [ 1 H]-MRS heptic triglyceride content (15). Severl clinicl trils (34,35) on NAFLD hve used spectroscopic mgnetic resonnce s n outcome mesure. Therefore, spectroscopy my be more pproprite reference stndrd thn histology in ccurtely ssessing ft content. In summry, our current improved US quntittive method integrted the merits of previous studies on ultrsound liver ft quntifiction, estblished stndrdiztion procedure for mesurement of US quntittive prmeters, nd provided mthemticl lgorithm for liver ft content estimtion with the US quntittive prmeters. Further nlysis on the vlidity of the improved US quntittive method confirmed the ccurcy, reproducibility, nd populriztion vlue of this method, so this improved ultrsound quntittive method could be considered s simple, reltive ccurte, reproducible, nd low-cost nlytic tool in the clinicl evlution of heptic stetosis. SUPPLEMENTARY MATERIAL Supplementry mteril is linked to the online version of the pper t The Obesity Society Acknowledgments This work ws supported by grnts from Ntionl Key Technologies R&D Progrm (grnt no BAI52B03 nd grnt no. 2009BAI80B01 to X. G.), the Mjor Project of Subject Construction of Shnghi Bureu of Helth (grnt no. 08GWZX0203 to X. G.), the Science nd Technology Commission of Shnghi Municiplity (grnt no to X. G. nd grnt no to H.-M. Y.) nd the Mjor Progrm of Shnghi Municiplity for Bsic Reserch (grnt no. 08dj to X. G.). Disclosure The uthors declred no conflict of interest. REFERENCES 1. Clrk JM, Brncti FL, Diehl AM. Nonlcoholic ftty liver disese. 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