NAFLD is the leading cause of chronic liver disease worldwide with its growing prevalence paralleling increasing cases of metabolic syndrome. While early stages of hepatic steatosis may be quite benign, it may progress to NASH and eventually cirrhosis [1–6]. Most cases will not develop high-grade NASH and cirrhosis; however, given its high prevalence, even small percentages translate to significant numbers. Pharmacologic treatment of NAFLD, specifically NASH-related fibrosis, is under extensive research; nonetheless, risk factor modification in early stages and preventing further progression remains the most appropriate measure. Therefore, developing non-invasive, accessible, simple methods of screening and diagnosis is of utmost importance. Liver biopsy remains the gold standard of diagnosis, unsuitable for screening due its invasive nature, potential complications and cost. Serum biomarkers have proven to significantly underestimate presence of NAFLD. The most commonly implemented radiologic methods are ultrasound and magnetic resonance spectroscopy (MRS); however, the cost-effectiveness of screening the general population by them is still under debate . Our study indicates that rest phase heart/liver uptake ratio of 99mTc-MIBI is significantly higher in patients with NAFLD; proposing it as a simple screening method in patients already undergoing MPI to rule out myocardial ischemia, who are at a higher risk for NAFLD as well, with no additional cost, radiation burden or complication.
As expected we observed a gradual rise in Heart/Liver ratios in either groups, which is explained by the physiologic clearance of 99mTc-MIBI from the liver . NAFLD patients demonstrated significantly higher Heart/Liver ratios, regardless of measuring time, including 1 hour post injection, which is the usual time point when rest phase acquisition is performed. Our findings are in accordance with findings of Masuda K et al, who evaluated 26 patients with biopsy-proven NAFLD by performing laboratory tests and a 99mTc-MIBI liver scintigraphy. They acquired a 2 minute planar image of thoraco-abdominal region in anterior view, 10 minutes after bolus injection of 600 MBq 99mTc-MIBI in rest phase. Mean count per pixel liver/heart ratios were calculated by defining ROIs in the right upper lobe of liver and the heart. They categorized the patients into non-NASH/simple hepatic steatosis (n=4), borderline NASH (n=11) and NASH (n=11) and reported that the relative hepatic uptake was significantly lower in NASH and borderline NASH compared to the non-NASH group, also compared to the healthy population (based on existing literature) and observed a significant correlation between the liver/heart ratio and degree of NASH . Aside the small number of patients and not having a control group with the same exclusion criteria, a prominent limitation of this study is that the effect of cardiac uptake, which may be significantly reduced in patients with CAD and other cardiomyopathies, is not accounted for, as the patients did not undergo a stress phase MPI scan [13, 14]. 99mTc-MIBI uptake portrays the mitochondrial function of cells  and NAFLD has been established as a disease of the mitochondria, with functional alterations of the mitochondria present even in the early stages of hepatic steatosis. These functional alterations lead to gradual impairment of fatty acid oxidation, resulting in oxidative stress and in turn leading to inflammation (NASH), hepatocyte apoptosis and fibrosis in advanced stages [9, 10], justifying the observed decreased hepatic uptake of 99mTc-MIBI in NAFLD patients compared to healthy individuals and possibly a correlation between relative uptake ratio and disease severity.
We developed a model to predict NAFLD based on patient’s demographic, laboratory and scintigraphy (at 0.5, 1 and 2 hrs.) data. The model's strength while excluding laboratory data, which may not be always available, decreases to 0.84 AUROC, which is still sufficient enough for a screening tool. Employing single time point scintigraphy measurement results at 1 hour post tracer injection, the time rest phase MPI acquisitions usually take place, yields a comparable accuracy (AUROC = 0.85), alleviating the need for multiple acquisitions, with heart/liver uptake ratios higher than the threshold of 1.0 warranting evaluation for potential NAFLD (Fig. 3). Naturally, implementing the model in larger populations with result in a more accurate threshold. Several studies have focused on evaluation and comparison of the diagnostic performance of non-invasive methods for screening NAFLD. Bril et al, evaluated the accuracy of liver ultrasound for the diagnosis of nonalcoholic fatty liver disease in overweight and obese patients. They found performance of US (parenchymal echo alone) rather modest, and significantly worse than MRS (AUROC: 0.82 [0.69–0.94] vs. 0.96 [0.90–1.00]; P = 0.04). Implementing echography parameters improved the AUROC (0.89 [0.83–0.96]) . Another study assessed the accuracy of hepatic ultrasound score, based on hepatic attenuation and the anteroposterior diameter of the right hepatic lobe, in predicting NAFLD. Its best performance (cutoff point ≥ 1 point) had AUROC of 0.85 . The results were less favorable in children, at optimum screening cut-points aiming for a specificity ≥ 80%, US had only mediocre performance with AUROC of 0.70 . Fatty liver index (FLI), NAFLD liver fat score (NAFLD-LFS), hepatic steatosis index (HSI), visceral adiposity index (VAI), triglyceride × glucose (TyG) index and Zhejiang University (ZJU) index are noninvasive and non-imaging indexes, based on a complex list of laboratory data, employed to predict NAFLD [18, 19]. The AUROCs of FLI, LFS, HSI, VAI and TyG for predicting hepatic steatosis has been determined 0.83, 0.80, 0.81, 0.92 and 0.9; respectively. However, they had relatively poor performance in distinguishing moderate and severe steatosis, with fibrosis and inflammation identified as significant confounding factors . Another study evaluated HSI, ZJU index and FLI for predicting NAFLD, with their AUROC determined 0.874, 0.886, and 0.884, respectively . Considering all these data, the accuracy of our scintigraphy-based model for predicting NAFLD (AUROC of 0.85) is acceptable and comparable to other commonly used US and laboratory-based indexes. Of course, we are not implying it as an independent screening method competing with liver ultrasound; but when applied in patients already undergoing MPI to rule out myocardial ischemia, it has the prominent advantage of it being a secondary finding of MPI, with no additional cost, radiation burden or side-effect.
Our study demonstrated no significant difference in age, gender distribution, AST, ALT, serum Albumin and PT levels among the groups; while BMI, TG and total cholesterol levels were expectedly higher in NAFLD patients. These findings are in line with existing literature as liver function tests are proven to be neither sensitive nor specific in screening for NAFLD and are normal in about 50% of NAFLD patients [20, 21]. On the other hand hyperlipidemia is reported to be significantly associated with NAFLD. Nakahara et al, reported hyper-LDL cholesterolemia and hypo-HDL cholesterolemia in 37.5 and 19.5% of biopsy-proven NAFLD patients  and another study has determined TG as the strongest predictor of NAFLD among other parameters .
This study has some limitations that should be considered. While liver ultrasound is currently recommended as the first line screening method for NAFLD; liver biopsy remains the gold standard of diagnosis, despite being rarely performed for this sole purpose. Regarding its invasive nature, possible complications and significant cost, acquiring pathological correlation for all included patients in this study was not feasible, nor ethical. The relatively small number of patients as well as limited accuracy of ultrasound did not allow us to further categorize NAFLD patients based grade of disease. To prevent the interfering effect of possibly reduced hepatic uptake and rest phase myocardial activity on relative hepatic uptake ratios, both due to micro-vascular disease, diabetic patients were excluded from the study. Whether similar results would be obtained in these excluded patients should be assessed in future studies. To ensure better signal to noise ratios, the relative hepatic uptake was calculated on an additional 2-minute planar acquisition, future studies are required to assess the feasibility of using the routine SPECT projections, omitting the need for additional acquisitions, for this measurement.
New Knowledge Gained
The main novelties of our manuscript are the following:
1. To our knowledge, this is the first study which assessed the feasibility of determining relative hepatic uptake of 99mTc-MIBI during rest phase MPI scan in screening patients for NAFLD, alongside evaluating the effect of different acquisition timings on the results.
2. To our knowledge, this is the first study which developed a model to predict NAFLD during MPI, based on patient’s demographic, laboratory and multiple time point scintigraphy data
3. In comparison with the single reported previous study addressing this matter, comparing relative hepatic uptake of 99mTc-MIBI in steatohepatitis versus simple hepatic steatosis, our study compromised a significantly larger population including a control group, enabling assessing the value of relative 99mTc-MIBI hepatic uptake as a screening tool in differentiating NAFLD (regardless of degree) from non-NAFLD patients. Also, our study was performed in MPI setting with all participants undergoing stress phase as well, including only patients with normal myocardial perfusion. Therefore, the interfering effect of possibly reduced myocardial uptake due to any simultaneous cardiomyopathy was accounted for and the results could be attributed to routine MPI studies.