We also found that cytochrome a content, another good marker of mitochondrial content (Larsen et al., 2012), did not differ in isolated mitochondria between experimental groups, suggesting that the content of electron transport system complexes does not change acutely between topor and IBE.
Even though citrate synthase activity is widely used as a marker of mitochondrial content [ 2, 18– 20], it may become a subject of oxidative damage [ 21] and therefore it may not reliably reflect the mitochondrial density.
In the diaphragm muscle, chymoptrysin-like and cathespin B+L activities peaked at day 4. Citrate synthase activity, a marker of mitochondrial content, was minimal at day 4 both in gastrocnemius and diaphragm muscles.
Tissue homogenates were incubated with O-dianisidine and H2O2 and light absorbance at 460 nm was measured as a marker of myeloperoxidase content.
Cytochrome C level, as a marker of mitochondrial content, was measured comparably using a rabbit polyclonal cytochrome c antibody (BD PharMingen, Woerden, NL).
To address the effect of experimental preparation on mitochondrial respiration, mitochondrial O2 consumption was measured during a sequential substrate addition protocol (schematically illustrated in Figure 3A) and normalized for a marker of mitochondrial content (complex IV activity).
The protein-to-DNA ratio, a marker of protein content, was significantly reduced in the EDL following 24 h LPS administration (23%; P<0.05), but was no different from controls in the myocardium.
Since intestinal samples contained both enterocytes and myocytes, we also measured H-FABP as a marker of muscle content with a direct non-competitive sandwich-type ELISA using monoclonal antibodies obtained from Hycult biotechnology (HK 403; Uden, the Netherlands) as described previously [76].
Skin autofluorescence (AF) is a validated marker of tissue content of AGEs.
MPO activity is a marker of neutrophil content and is upregulated in inflammatory tissue.
