Quantitative Proton Magnetic Resonance Spectroscopy To Detect Liver Fat Content, Methods Of Establishing And Liver Fat Content And Insulin Resistance In The Relationship Between

PartⅠEstablishment of the Method of Quantitively Measuring hepatic fat Content by ~1H magnetic Resonance Spectroscopy In VivoObjectiveTo establish the quantitive method of measuring liver fat content by ~1H magnetic resonance spectroscopy in high-fat diet induced fatty liver model of Wistar rats,and to evaluate the stability and accuracy in patients with NAFLD.Methods40 inbred line male Wistar rats were randomly divided into high-fat diet group and control group.The model group was fed high-fat diet(52%of energy from fat) for 16 weeks,and control group standard diet(12%of energy from fat).Five rats randomly chosen from control group and model group respectively at the fourth, eighth,twelfth,sixteenth weekend,were measured intrahepatic lipid peak and water peak by ~1H MRS in vivo.Peak areas for all resonances were obtained and lipid resonances were quantified with reference to water resonance.The ratio of the lipid peak area relative to water peak area was computed.Then all rats were sacrificed. Liver tissues were stained with SudanⅢfor neutral fat,and the percentage of liver fat content was measured by the software for analysis of medical images.14 patients with fatty liver disease were collected for this study.All the patients measured intrahepatic lipid peak and water peak by ~1H MRS(as above) in vivo and performed liver biopsy. The liver tissues were stained by hematoxylin eosin,and the percentage of liver fat content was measured by the same method as that in rats.ResultThe liver fat content of rats in high-fat diet group gradually increased during the follow-up period.Intrahepatic lipid peak and water peak were detected in all the rats of model group.The spectral line by ~1H MRS was stable and sensitive.Even the percentage of hepatic fat content was less than 1%,the change of lipid peak was observed.The lipid peak area and the ratio of the lipid peak area relative to water peak area in liver rised with increased liver fat content.There was a close positive correlation between the ratio of the lipid peak area relative to water peak area by ~1H MRS in vivo and liver fat content.by histologic measurement.[r=0.90;P＜0001;slope 0.145;y-intercept -0.005]in the rats.The method of quantitive measurement of hepatic fat content by ~1H MRS was established at the animal level.Fourteen patients with NAFLD were selected to measure liver fat content by ~1H MRS and performed liver biopsy.Intrahepatic lipid peak and water peak were detected in all the patients.The spectral line by ~1H MRS was more stable than that in the rats.The lipid peak area and the ratio of lipid peak area relative to water peak area increased with the increment of liver fat content.When the liver fat content was more than 75%,the water peak was inhibited by the lipid peak.There was a close positive correlation between the ratio of the lipid peak area relative to water peak area by ~1H MRS in vivo and liver fat content.by histologic measurement.[r=0.91;P＜0001;slope 0.487;y-intercept 0.065].The data of rats and patients was analyzed together,there was a close positive correlation[r=0.91;P＜0001;slope 0.565;y-intercept -0.037]between the ratio of the lipid peak area relative to water peak area and liver fat content.by histologic measurement.ConclusionIt was the first study that established successfully the method of measurement of liver fat content by ~1H MRS in China.It was a safe,stable,reproducible,sensitive and non-invasive method for relatively accurate quantification of intrahepatic lipid content, and had a good correlation with histologic results,which provided a new method for clinical and basic study of NAFLD. PartⅡThe Relationship of Liver Fat Accumulation with Insulin Resistance by different methods of evaluating liver fat contentObjectTo observe the association of liver fat content and insulin resistance in high-fat diet induced fatty liver model of rats and the patients with nonalcoholic fatty liver disease(NAFLD) by different methods of evaluating liver fat content.Materials and MethodsThe fatty liver model of rats was the same as that in the firs part of the study. Weight,fasting and random blood glucose and insulin were determined respectively every week.Five rats randomly chosen from control group and model group respectively at the fourth,eighth,twelfth,sixteenth weekend,was performed IPGTT. Then rats were sacrificed.Liver tissues were stained with SudanⅢfor neutral fat, and the percentage of liver fat content was measured as described in the first part.Clinical data of 374 outpatients with NAFLD and without known diabetes were collected in from Nov.2005 to Jul.2007,in which 133 subjects were performed calibrated computerized tomography(CT) scan and 15 subjects were measured liver fat content by percutaneous liver biopsy.The subjects were excluded in those with the history of chronic virus hepatitis,autoimmune hepatitis,hepatitis induced by drugs and genetics,male subjects with an alcohol consumption≥40g/d and female≥20g/d. Each subject received the standard interview.Anthropometrics(height,weight,waist circumference,hip circumference and blood pressure ) were measured.Laboratory tests included liver enzymes,lipid profile,HBsAg and HCVAb,Fasting and 2 hour plasma glucose and insulin level were tested in OGTT test in all subjects.Fasting, 30min,60min,120min,180min glucose and insulin in OGTT test in the 15 patients who performed liver biopsy were tested.MS was defined according to ATPⅢ. Differences were considered statistically significant if P＜0.05.ResultsFasting and random glucose of rats in the high fat diet group were significantly higher than those in the control group during the follow-up period.The areas under the curves of serum blood glucose levels(AUCg) and insulin levels(AUCi) and HOMA-IR were higher in the rats of high fat diet group which fed for 12 weeks than those of control group.The degree of insulin resistance worsen with the increment of liver fat content.The ratio of AUCi/AUCg was lower in the rats of high fat diet, which suggested there was dysfunction of insulin secretion in the beta cell.According to median of liver fat content(27%) determined by liver biopsy,15 subjects who measured liver fat content by liver biopsy and ~1H MRS were divided into two groups.Levels of BMI,waist circumference,WHR,blood pressure,liver enzymes,TG,serum glucose,insulin,HOMA-IR and AUCg in the group whose liver fat content≥27%were higher than that in the group whose liver fat content＜27%, but HDL-c,AUCi and the ratio of AUCi/AUCg were lower,possibly because of small case size,the results were no statistically significant differences.Liver fat content in all subjects tested by calibrated CT scan were divided into 4 groups according the quartile of liver fat content.BMI,waist circumference,diastolic blood pressure,serum ALT,AST,GGT level elevated respectively with increaseing of liver fat content,while serum HDL-c level was decreased.After adjusted age and sex, liver fat content showed statistically significant positive correlations with AST(r=0.534),ALT(r=0.525),GGT(r=0.348),waist circumference(r=0.304), BMI(r=0.232),diastolic blood pressure(r=0.292),systolic blood pressure(r=0.219), 2h glucose after OGTT(r=0.228)(all P＜0.05),negative correlation with HDL-c (r=-0.212)(P＜0.05).When liver fat content increased by quartile order,the percentage of MS was increased accordingly(P＜0.05).After adjusted for age and sex, Logistic multivariable stepwise regression analysis demonstrated that liver fat contend instead of waist circumference was the only significant risk factor of MS among parameters representing fat distribution(include BMI waist circumference and WHR) (OR=1.659,95%CI 1.132-2.430).Stepwise multiple linear regression analysis demonstrated that only liver fat content was the significant risk factor of HOMA-IR, while BMI and waist circumference were not the risk factor of it.Taking liver enzymes as the markers of liver fat accumulation,374 subjects with NAFLD without known diabetes were analyzed in this study.Liver enzymes' levels were found abnormal in 214 subjects(57.2%).Weight,BMI,blood pressure,TC,TG, fasting insulin,HOMA-IR of with abnormal liver enzymes were higher than that of subjects with normal liver enzymes,while level of HDL-c was lower(P＜0.05).Waist circumference and TG were the significant risk factors of abnormal liver enzymes after adjusted for age and sex[OR(95%CI):waist circumference 1.11(1.05-1.17), TG 1.36(1.09-1.70)].147 subjects(39.3%) were diagnosed MS using the ATPⅢcriteria.More subjects were diagnosed MS in the group with abnormal liver enzymes compared with those with normal liver enzymes(47.7%vs 28.1%,P＜0.001).After adjusted for age and sex,Logistic multivariable stepwise regression analysis demonstrated that level of liver enzymes decreased the contribution to MS of diastolic blood pressure,TG,HDL-c and fasting glucose,GGT was independently associated with the presence of MS[OR 1.52,95%CI(1.063-2.18)].ConclusionsWe found that liver fat content was associated with insulin resistance,and might affect the function of beta cell secretion in the fatty liver model of Wistar rats. Liver fat content was associated with most components of MS.Compared with BMI and waist circumference,liver fat content was a more sensitive affective factor of MS and insulin resistance,which suggested that visceral fat accumulation,particularly liver fat accumulation,was a more significant risk factor of MS.Liver enzymes as clinical markers of reflecting liver fat accumulation was associated closely to MS.