Improved Folch Method for Liver-Fat Quantification

Abstract

Fatty liver represents a significant metabolic pathology of excess intrahepatic fat in domestic animals and humans. Quantification of hepatic-fat content is therefore essential for diagnosis and investigation of liver and metabolic disease. However, the reproducibility of hepatic steatosis analysis is often low due to subjective and technical factors. We hypothesized that improvement in tissue-lipids extraction efficiency would contribute to the accuracy and precision of liver-fat determination. To test it, we investigated the effect of standardized tissue sonication on liver-fat quantification by the Folch method in sheep. Liver samples from grownup lambs of lean (n = 16) and fatty (n = 15) livers, and from pregnant ewes (n = 6) who died from pregnancy toxemia (PT), were used for hepatic-fat content determination with or without tissue sonication. In the grown lambs, an average hepatic-fat content of 6.6% was determined in sonicated compared to 5.1% in non-sonicated specimens (P = 0.0002). Similarly, in ewes with PT, an average of 12.5% was determined with sonication compared to 10.8% without it (P = 0.0006), and the reproducibility was higher with sonication (CV of 3.1 vs. 6.1%, respectively). Thus, tissue sonication improved the efficiency of liver-lipids extraction and was significant to the accuracy and precision of hepatic-fat determination. Enzymatic quantification of triglycerides was moderately correlated with the results obtained gravimetrically (r = 0.632, P < 0.005). The reported data provide reliable reference values for pregnancy toxemic sheep. The significant improvement in liver-fat quantification observed with the reported revised protocol is likely applicable to most mammals and humans.

Inulin Improves Diet-Induced Hepatic Steatosis and Increases Intestinal Akkermansia Genus Level

“…Liver fat was quantitated by the improved Folch method recently reported with minor modifications [50]. Briefly, 150 mg of liver tissue section was mechanically homogenized in chloroform:methanol (2:1) solution.…”

Hypocholesterolemic Effect of Milks Fermented with Specific Strains of Lactococcus lactis in Hypercholesterolemic Sprague–Dawley Rats

“…Total lipid content was determined in liver tissue and freeze-dried feces according to the modified Folch method. 26 200 mg of sample (liver or feces) was homogenized with 10 mL of chloroform:methanol (2:1) for two min using an automatic homogenizer (Bio-Gen Pro2000 Homogeneizer, ProScientific, Oxford, CT). The homogenized samples were sonicated for five min and incubated overnight in an orbital water bath shaker (450 rmp, 25 °C).…”

Hepatocyte-Secreted Autotaxin Exacerbates Nonalcoholic Fatty Liver Disease Through Autocrine Inhibition of the PPARα/FGF21 Axis

“…To measure liver triglyceride content, liver tissues (∼50 mg) were homogenized in chloroform/methanol (2:1 v/v) using a tissue homogenizer. Lipid extracts were prepared by the Folch method, as previously described, 77 and then were dried under nitrogen flow and further dissolved in ethanol. The FAO rate was measured using a commercial kit (E-141; Biomedical Research Service, University at Buffalo, Buffalo, NY) following the manufacturer’s instructions.…”