Quantification,of,liver,fat,deposition,in,obese,and,diabetic,patients:,A,pilot,study,on,the,correlation,with,myocardium,and,periapical,fat,content☆

Xi Chen , Hui-Qun Wen , Qing-Ling Li , Li-Shn Shen , Xio-Wen Luo , Bin Zhou ,Ruo-Mi Guo ,**

a Department of Radiology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China

b Department of VIP Medical Center, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China

c Department of Cardiology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China

Keywords:Cardiac fat content Diabetes mellitus Obesity Non-alcoholic fatty liver disease (NAFLD)Type 2 diabetes mellitus (T2DM)Magnetic resonance imaging (MRI)

ABSTRACT Backgroud and aim: Non-alcoholic fatty liver disease (NAFLD) is a worldwide health problem, which associated with systemic health problems and causes a higher risk of all-cause mortality. The leading causes of death in NAFLD patients are cardiac complications followed by NAFLD-related liver complications. This study aimed to quantitatively measure the contents of liver and cardiac fat with varying degrees of NAFLD in an obese group and a type 2 diabetes mellitus (T2DM) group to explore differences and correlations.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. It is becoming more common as people"s living standards improve in China. The situation is gaining more attention.1,2NAFLD-related liver diseases, such as non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma, have a significant morbidity and mortality rate.3,4Studies correlate NAFLD with metabolic heart diseases, with NAFLD-related heart diseases having a higher mortality rate than NAFLD-related liver diseases.5,6Therefore, it is important to provide precise early treatment for NAFLD-related heart diseases, especially asymptomatic cases.7,8Moreover, studies have shown that NAFLD caused by multisystem metabolic syndromes, such as type 2 diabetes mellitus (T2DM),obesity, hypertension, high cholesterol, and other metabolic diseases, is closely related to the pathophysiological factors of heart diseases.9-11T2DM and obesity,in particular,are closely related to myocardial structural changes and systolic cardiac dysfunction.12

The adipose tissue surrounding the myocardium and the major coronary branches closely associated with blood vessels is epicardial adipose tissue (EAT).13,14Increased visceral fat tissue plays a significant role in metabolic syndrome NAFLD and EAT are related to cardiometabolic disease.15,16EAT includes periapical adipose tissue, which is repeatedly associated with cardiovascular risk in various aspects. According to previous research, EAT is correlated with the development of atrial myopathy,including both coronary artery disease and plaque components, and the size of EAT may influence the left ventricular mass.17,18

Liver biopsy is the gold standard for NAFLD diagnosis and staging.However,it is invasive,with potential sampling errors,and cannot reflect the liver"s overall fat content and distribution.19Studies have revealed that magnetic resonance imaging (MRI) can be used to quantitatively measure the liver fat content in patients with fatty liver,thanks to advancements in MRI technologies.20-22Moreover, previous studies showed that an MRI sequence can be used to examine pericardial fat quantitatively. However, no research has examined the liver,myocardium,or periapical fat and their correlations.23,24As a quantitative and accurate MRI technique, iterative decomposition of water and fat with echo asymmetry and least square estimation-iron quantification (IDEAL-IQ)allows for precise quantitative measurement of the entire liver.IDEAL-IQ is the most accurate noninvasive method for quantitative measurement of organ fat contents. It minimizes multifrequency signal interference effects of protons in fat quantification by fat fraction (FF) mapping.25,26This is the first study to use IDEAL-IQ technology to quantitatively measure liver, myocardium, and periapical fat content in obese patients and T2DM patients with varying degrees of liver fat deposition, as well as to investigate differences in fat content and their correlations.

2.1. Ethical approval

This was a double-blinded controlled study approved by the Institutional Research Ethics Committee of The Third Affiliated Hospital of Sun Yat-sen University ([2022] 02-005-01). Written informed consent was obtained from all study participants.

2.2. Study population

The study retrospectively analyzed the data of 170 patients treated at our hospital between January 2017 and April 2021. The following were considered the exclusion criteria: a history of hepatitis or other chronic liver diseases, a history of liver cancer,drug or alcohol abuse, and a history of any liver or heart surgery.Meanwhile, the inclusion criteria for the T2DM group were as follows (defined by the World Health Organization criteria): hemoglobin A1c (HbA1c) 7-10%, age ≥18 years, and body mass index(BMI)18-27 kg/m2.And the inclusion criteria for the obesity group were as follows: BMI ＞27 kg/m2, HbA1c ＜7%, and age ≥18 years(Fig.1).27All patients completed liver and heart MRI in one session.

2.3. MRI scanning protocol

The patients fasted for 6 h before the scan. Before the scan,the patients were taught how to breathe properly to avoid breathing artifacts.They underwent MRI with a 3.0 T MR(Discovery 750 and Signa Architect, GE Healthcare, Milwaukee, WI, USA). Each patient was scanned in the supine position, with the head in the first position and the forearms crossed and raised.A 32-channel dedicated phased-array body coil was used to cover the heart and liver. The scan included the liver, the atrioventricular septum, and the heart below the atrioventricular septum.The MRI sequences included T1-weighted image(T1WI),T2-weighted image(T2WI),and IDEAL-IQ sequences at 3.0 T MR. The following were the T1WI parameters:repetition time (TR)/echo time (TE) = 450 ms/12 ms,matrix = 512 × 512, field of view (FOV) = 42 cm × 42 cm, slice thickness/slice spacing = 3 mm/1 mm, and number of excitations(NEX) = 2.00. Meanwhile, the following were the T2WI parameters:TR/TE=4000 ms/80 ms and slice thickness/slice spacing=3/1 mm;all other parameters were the same as those used for T1WI.

For the axial IDEAL-IQ breath-hold sequence, the heart and liver were scanned with a single breath-hold using the following parameters: TR/TE = 4 ms/1.8 ms, bandwidth = 125 kHz, flip angle = 3°, slice thickness = 3 mm, echo train length (ETL) = 6,FOV = 42 cm × 42 cm, matrix = 256 × 256, and NEX = 1.00. At the end of the IDEAL-IQ scan, an FF map was generated automatically.

2.4. MR image processing

Two experienced radiologists with more than 10 years of experience reviewed all participants’ clinical and imaging data in a double-blinded manner.The IDEAL-IQ images were transmitted to a workstation for postprocessing with Functool v6.3.1 software. The fat content(the percentage of fat in the organ mass)in the regions of interest (ROIs) was quantitatively measured using the FF map based on the IDEAL-IQ sequence. Each radiologist drew a round ROI of 80 mm2(deviation ＜10 mm2) in the right anterior liver lobe in the three adjacent slices of the second hepatic hilum, avoiding the blood vessels and intrahepatic bile ducts,and the liver capsule.The mean value was used as the liver fat content. On the FF map (Fig. 2a), a round ROI of 5 mm2(deviation ＜3 mm2) was drawn for the ventricular septum, left ventricular wall, and periapical fat using the T2WI sequence as a reference. On the liver FF map, the fat percentage in the liver mass was considered the fat content, which was classified as follows: ＜5%, normal liver; 5-14%, mild fatty liver; 14-28%,moderate fatty liver; and ＞28%, severe fatty liver.28,29Increased liver fat deposition in NAFLD patients may develop to systemic metabolic diseases and liver failure,29we directly measured NAFLD subgroup by the FF map for difference analysis with cardiac tissue fat deposition. Propensity score matching was used to choose patients on the 1:1 ratio for obese group and T2DM group, followed by subgroups. The 85 obese patients and 85 T2DM patients were separately divided into four subgroups based on liver fat content: the normal liver group (n = 25), mild fatty liver group (n = 20), moderate fatty liver group (n = 20),and severe fatty liver group (n = 20). The 95% consistency limits were -0.15-0.18 for obese patients and -0.15-0.17 for T2DM patients, demonstrating good interrater consistency between the two radiologists (Fig. 2b and c).

2.5. Statistical analysis

Fig.1. Flow diagram for the study cohort. Abbreviations: MR, magnetic resonance; MRI, magnetic resonance imaging.

The statistical methods and their application have been described in detail;GraphPad Prism 8 software(GraphPad Software Inc.,San Diego,CA,USA)was used for data analysis.FF values were quantitatively measured in ROIs on the FF map,and average values represented these values. The chi-square analysis was calculated for categorical variables and unpaired t-test for continuous variables. Multiple unpaired t-tests were used to compare the ventricular septum and left ventricular wall FF values, followed by multiple comparison analyses of variance (ANOVA) for multiple pairwise comparisons for multiple assessments between myocardial and periapical FF values in T2DM and obese patients. ANOVA examined the biomarkers for cardiovascular risk factors, creatine kinase (CK), creatine kinase isoenzyme (CK-MB), and lactate dehydrogenase (LDH), in two groups. The correlations between liver fat content and myocardial and periapical fat contents and whether liver fat content can predict myocardial and periapical fat contents were investigated using simple linear regression. P ＜0.05 was considered statistically significant. Bland-Altman analysis was conducted to assess the interrater consistency of the two radiologists when determining the FF values.

Fig.2. ROIs for cardiac tissue fat quantification and consistency analysis results.(a)ROIs selection for myocardial fat quantitation on the axial T2WI and FF map:the ventricular septum(red circle),the left ventricular wall(yellow circle),and periapical tissue(blue circle);(b-c)Bland-Altman analysis for quantifying the liver fat content in the normal liver group for the obese group (b) and diabetic group (c). Abbreviations: FF, fat fraction; ROI, region of interest; T2WI, T2-weighted image.

3.1. Participant characteristics

All images were clear and usable. Our study included a total of 170 patients, 85 of whom were obese and 85 of whom had T2DM.Each patient signed an informed consent form before participating in the study. The obesity group included 50 men and 35 women,with an average age of 45.48 years, whereas the T2DM group included 45 men and 40 women, 43.54 years. Table 1 shows the baseline characteristics of the patients in the obese group and T2DM group.

Table 1 Baseline characteristics of trial patients in the T2DM group and obese group.

3.2. Quantitative analyzed relationship between periapical fat and liver fat

The MRI FF map and pseudo-color map revealed that periapical fat increased with the liver fat content in both obese and T2DM patients (Figs. 3 and 4). As the liver fat content increased, the ventricular septum and left ventricle wall fat contents also increased in both obese and T2DM patients.However,there was no statistical significance for the fat contents of other myocardial components (P ＞0.05) (Fig. 5a and b). Linear regression analysis revealed that fatty liver severity positively correlated with myocardial fat deposition and periapical fat deposition in obese patients (ventricular septum, β = 0.05, P ＜0.01; left ventricular wall,β=0.06,P ＜0.01;periapical,β=0.17,P ＜0.01)(Fig.5c),and the same finding was also observed in T2DM patients (ventricular septum,β=0.10,P ＜0.01;left ventricular wall,β=0.09,P ＜0.01;periapical,β=0.18,P ＜0.01)(Fig.5d).Furthermore,the periapical fat content was significantly different only between the normal liver group and the mild fatty liver group in both obese patients and T2DM patients (obese group, P ＜0.01; T2DM group,P = 0.01) and was uncorrelated with fatty liver severity(P ＞0.05)(Fig.6a and d).

3.3. Quantitative analyzed relationship between myocardial fat and liver fat

Fig. 3. T2WI, FF map, and pseudo-color map of the periapical fat content in obese patients. Abbreviations: FF, fat fraction; T2WI, T2-weighted image.

Fig.4. T2WI, FF map, and pseudo-color map of the periapical fat content in T2DM patients. Abbreviations: FF, fat fraction; T2DM, type 2 diabetes mellitus; T2WI, T2-weighted image.

In the normal liver group,the myocardial fat content was higher in obese patients (ventricular septum, 2.04% ± 0.56%; left ventricular wall, 1.77% ± 0.44%) than in T2DM patients (ventricular septum,1.89% ± 0.62%; left ventricular wall,1.75% ± 0.54%). When the liver fat content reached the fatty liver standard,the ventricular septum and left ventricular wall fat contents were higher in T2DM patients than in obese patients.Multiple comparison results in the T2DM group revealed that the myocardial fat content (both the ventricular septum and left ventricular wall) increased with the liver fat content and was statistically significant (Fig. 6e and f,Table 2), but not in the obese group (Fig. 6b and c, Table 2). The myocardial fat content in obese patients was similar between the normal liver group and the non-severe fatty liver groups but considerably higher in the severe fatty liver group (ventricular septum, 3.65% ± 0.80%; left ventricular wall, 3.46% ± 0.84%;P ＜0.01)(Fig.6b and c).For T2DM patients,the fat contents of the ventricular septum and the left ventricular wall positively correlated with fatty liver severity, with the lowest fat contents in the normal liver group and highest fat contents in the severe fatty liver group(P ＜0.05)(Fig.6e and f).Of note,there was also a significant correlation between cardiac biomarkers and cardiac fat deposition.The assessment of CK, CK-MB, and LDH was at baseline in the normal liver group, but it reached the upper critical value in the severe fatty liver group.CK and LDH positively correlated with fatty liver severity in the T2DM group(P ＜0.01) (Table 2).

This study quantitatively measured and analyzed the liver fat content and myocardial and periapical fat contents of obese patients and T2DM patients with varying degrees of fatty liver and assessed liver fat contents to predict myocardial fat contents in the obese group and T2DM group.

The myocardium is a vital tissue that regulates cardiac systolic and diastolic functions. Patients with metabolic heart disease,which can lead to myocardial fibrosis, may experience changes in myocardial function.30,31Previous studies pointed out that under the premise of abdominal obesity, the myocardial triglyceride content was not correlated with diabetic obese subjects and nondiabetic obese subjects but was higher in diabetic subjects.32,33Based on prior studies,we divided T2DM and obese individuals into two distinct groups and evaluated whether myocardial fat deposition was positively correlated with NAFLD severity in T2DM patients. For obese patients, the myocardial fat content was significantly increased only in patients with severe fatty liver.Epidemiological studies show myocardial dysfunction in obese patients is closely related to metabolic disorders and less ectopic fat deposition in other organs and sites.34We reached a similar conclusion that there is no significant difference in myocardial fat content between patients with mild to moderate fatty liver and patients with a normal liver. Moreover, myocardial fat deposition was more severe in T2DM patients with fatty liver than in obese patients with fatty liver. The myocardial fat content was higher in T2DM patients than in obese patients with the same fatty liver severity. As myocardial enzymes for cardiac function, the CK, CKMB, and LDH also demonstrated significant positive correlations with myocardial fat deposition, consistent with previous studies’conclusion that NAFLD patients had a higher risk of left ventricular diastolic dysfunction.35Furthermore, the correction of the blood biochemical parameters with myocardial fat deposition in the T2DM group is statistically significant with the liver fat deposition.The result implies that increased liver fat content changes the myocardial fat content in diabetic patients and will affect cardiac function.

Both EAT and abdominal fat are brown fat tissues in the embryonic stage. Secreted adipokines are essential risk factors for major adverse cardiovascular events and are related to coronary heart disease.36,37We used IDEAL-IQ technology to quantitatively analyze liver fat contents and periapical fat contents and their correlations. We found that the periapical fat content increases with the liver fat content through a linear correlation in both obese and T2DM patients, regardless of fatty liver severity. Our findings showed that liver fat contents were statistically significant in the normal and mild liver groups, increasing periapical fat content,which is consistent with a previous study showing that EAT is a predictor of liver steatosis.38Under normal conditions, EAT regulates fatty acid accumulation and release, preventing lipotoxicity from meeting the energy demands of arteries and the myocardium.Enlarged fat cells secrete various inflammatory cytokines, such as C-reactive protein and interleukin-6,in response to increased liver fat deposition, resulting in fatty acid deregulation, excessive EAT deposition, and cardiovascular disease.39,40Based on previous studies,33,41we further showed that, although the periapical fat content increased with fatty liver severity in obese and T2DM patients, there was no statistical significance between fatty liver severity and periapical fat content. The scan covered only the atrioventricular septum and the heart below the atrioventricular septum. Therefore, the periapical fat content was used instead of EAT.

Fig. 5. The violin plots and relationship of different fat deposition. The ventricular septum fat content and left ventricular wall fat content in the obese group (a) and T2DM group(b).The linear relationship between the ventricular septum fat content,left ventricular wall,periapical fat content,and liver fat content in the obese(c)and T2DM groups(d).Solid lines represent the lines of the best fit with corresponding 95% confidence interval bands. Abbreviation: T2DM, type 2 diabetes mellitus.

Fig.6. The fat contents of periapical,ventricular septum and left ventricular wall in patients with different degrees of NAFLD.(a-c)For the obese group;(d-f)for the T2DM group. Abbreviations: NAFLD, non-alcoholic fatty liver disease; T2DM, type 2 diabetes mellitus.

In contrast to previous qualitative and semiquantitative measurement methods, IDEAL-IQ quantitatively measures the fatcontent of different tissues and organs via complete water-fat separation.42,43This study used IDEAL-IQ to precisely measure liver fat contents and myocardial and periapical fat contents while minimizing EAT-related measurement errors, resulting in the discovery of the connection between liver fat content and cardiac fat content using the new, effective, and noninvasive method.

Table 2 Biomarkers of cardiovascular risk factor in the obese and T2DM group for each fatty liver group.

The drawback of this study is that it did not analyze the correlations between liver fat content and the fat contents of the atrial septum, atrial wall, or myocardial surface; however, such analyses will be included in future studies.

This study uses the precise and noninvasive IDEAL-IQ technology to measure the liver,myocardium,and periapical fat content in T2DM and obese patients.It is useful for correlating liver fat content and periapical fat content, and different myocardial fat contents.The fat content trends were consistent in periapical fat in patients with varying degrees of fatty liver. The myocardial fat content increased with liver fat content, which is higher in T2DM patients than in obese patients, and the myocardial function in T2DM patients is affected by myocardial fat deposition.

Authors’ contributions

X.Chen and H.-Q.Wen contributed equally to this work.X.Chen and R.-M. Guo designed the study. R.-M. Guo and B. Zhou are the guarantors of integrity of the entire study.L.-S.Shen and X.-W.Luo contributed to literature research.Q.-L.Li,H.-Q.Wen contributed to clinical studies. R.-M. Guo and X. Chen were responsible for experimental studies and statistical analysis. X. Chen, Q.-L. Li, B.Zhou, and R.-M. Guo contributed to manuscript preparation. All authors have read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (No. 81801757) and Guangdong Basic and Applied Basic Research Foundation (No. 2019A1515012051,2022A1515010369) to R.-M. Guo.