Glycogen as a Proxy Marker for Non-Alcoholic Fatty Liver Disease: Histochemical and Spectroscopic Insights

Background and Objective: Glycogen storage in the liver is a natural physiological function; however, its accumulation may also signify pathological states, such as Non-Alcoholic Fatty Liver Disease (NAFLD). This circumstance underscores the importance of employing the Periodic acid-Schiff (PAS) staining technique for its visualization. The objective of this research is to utilize Fourier Transform Infrared (FTIR) spectroscopy in conjunction with PAS staining to provide an objective and diagnostic quantification of glycogen levels to discriminate between NAFLD and normal liver. Materials and Methods: A total of 20 archival human samples from fatty liver cases and 10 normal livers were retrieved and sectioned at 4μm. The resulting sections underwent staining with Hematoxylin and Eosin, as well as PAS, both with and without diastase pre-treatment. A FTIR spectra were recorded from the sections of liver, organized into diastase-treated and untreated categories. The spectral intensities corresponding to glycogen at 1030 cm^-1 were statistically analyzed using t-tests, with a predefined significance threshold of p<0.05. The Receiver Operating Characteristic (ROC) Curve method was applied to assess the specificity and sensitivity of the FTIR techniques. Results: Notable distinctions in glycogen concentration were observed between digested and undigested normal liver sections (0.79±0.01 vs 1.15±0.03, p = 0.002) and between pre-digested and undigested NAFLD liver sections (0.55±0.02 vs 1.35±0.06, p = 0.003), and between normal liver and NAFLD sections (1.15±0.03 vs 1.35±0.06, p = 0.01) validated by PAS photomicrographs. A FTIR analysis revealed a sensitivity of 100% and a specificity less than 80% in differentiating between pre-digested and undigested normal and pathological liver sections. Conclusion: A FTIR spectroscopy holds promise for the standardization of glycogen detection methodologies, such as PAS, and as an adjunct technique to discriminating NAFLDs from normal liver by offering a more objective and reproducible approach to measurement.