Fig 1.
Effect of sample processing on GLDH quantification.
GLDH is located within the mitochondrial matrix and it has been hypothesized that failure to remove intact mitochondria during blood processing for serum/plasma can alter GLDH quantifications. Therefore, we processed blood samples collected from mice in multiple ways to determine if mitochondrial removal impacted measurements (A). All blood samples initially underwent a standard low speed spin to remove red blood cells and debris and to isolate plasma. An aliquot from each of these samples underwent an additional high-speed spin that was forceful enough to pellet any intact mitochondria that may have been present in the standard plasma to obtain post-mitochondrial supernatant (16,000xg plasma). GLDH was then quantified in the fresh samples with the expectation that if the assay reagents can penetrate the mitochondrial membrane, removal of intact mitocondria would result in a reduction in GLDH levels. However, if the reagents cannot penetrate the undamaged membrane, the presence of intact mitochondria in plasma will not be consequential until these mitochondria are lysed (B). We performed three freeze/thaw cycles on our samples which would presumably lyse any mitochondria that remained in the plasma samples. This lysis should liberate GLDH from damaged mitochondria and result in a rise in GLDH levels if the assay reagents cannot detect GLDH in intact mitochondria.
Fig 2.
Effect of APAP on liver histology and biomarkers.
Male C57BL/6J mice received a single intraperitoneal administration of vehicle (Veh; 0.9% saline) or APAP (300 mg/kg). Liver tissue and blood were collected 24h post-dosing. H&E staining revealed APAP-induced hepatocellular necrosis. Representative photomicrographs at 10X magnification are shown for vehicle- (A) and APAP-dosed mice (B). ALT, AST and GLDH were quantified in freeze/thawed plasma samples from Veh (white) and APAP-treated (black) mice. Significance was *p<0.05, **p<0.01, or ***p<0.001 (C). Pearson’s r was calculated to explore the correlation between ALT and GLDH quantities measured in plasma (D).
Fig 3.
Effect of FS on liver histology and biomarkers.
Male C57BL/6J mice received a single intraperitoneal administration of vehicle (Veh) or FS (400 mg/kg). Liver tissue and blood were collected post-dosing. H&E staining revealed FS-induced hepatocellular necrosis and inflammatory cell infiltration. Representative photomicrographs at 10X magnification are shown for vehicle- (A) and FS-treated mice (B). ALT, AST, and GLDH were quantified in freeze/thawed plasma samples from vehicle (white) and FS-treated (black) mice. Significance was **p<0.01 or ***p<0.001 (C). Pearson’s r was calculated to explore the correlation between ALT and GLDH quantities measured in plasma (D).
Fig 4.
Effect of high-speed spin and freeze/thaw on GLDH levels after APAP administration.
Plasma from APAP-dosed mice was isolated with only a standard low-speed spin (white) or with the addition of a high-speed spin (gray). GLDH was quantified in “fresh,” unfrozen samples (triangles) and again in samples that had undergone three freeze/thaw cycles (squares). Some fresh samples froze during transit and could not be measured, resulting in missing symbols. Each tick on the X-axis represents an individual APAP-dosed mouse and each mouse’s samples are separated by a vertical dotted line.
Fig 5.
Effect of high-speed spin and freeze/thaw on GLDH levels after FS administration.
Plasma from FS-dosed mice was isolated with only a standard low-speed spin (white) or with a subsequent high-speed spin (gray). GLDH was quantified in “fresh,” unfrozen samples (triangles) and again in samples that had undergone three freeze/thaw cycles (squares). Some fresh samples froze during transit and could not be measured, resulting in absent symbols. Each tick on the X-axis represents an individual FS-treated mouse and each mouse’s samples are separated by a vertical dotted line.
Fig 6.
Mitochondrial integrity after compound treatment.
The integrity of mitochondria in the liver were assessed by EM and photomicrographs were taken at 10,000X (A-D, bar = 600nm or 15,000X (E,F, bar = 400 nm) magnification. Representative images from APAP-vehicle (A), APAP (B), FS-vehicle (C) and FS (D) dosed mice are shown. Both APAP and FS-dosed animals exhibited megamitochondria and electron dense areas in the mitochondrial matrix (arrow head). FS dosed animals also exhibited swollen mitochondria with dispersion of mitochondrial matrix and blunted cristae (E) and mitochondria within vesicles (F). Abbreviations: M, mitochondria; RER, rough endoplasmic reticulum.