# Chemical Compositional Changes in Over-Oxidized Fish Oils
## Discussion
### Specific Groups of Secondary Oxidation Products and Biological Activity
#### 4.4.3 Oxysteros  
We also addressed if oxysterols might be formed in hoki liver oil during the prolonged over-oxidation induced by exposure to oxygen and light. Three oxysterols, 5\(\alpha\),6\(\alpha\)-epoxycholesterol, 5\(\beta\),6\(\beta\)-epoxycholesterol, and 7-hydroxycholesterol, proved to be very sensitive markers of the oxidation process, showing increases in level already at day 5. That these correspond to two epoxides and a primary alcohol suggests that these oxysterols reflect primary oxidation, which is analogous to the formation of fatty acid hydroperoxides. Interestingly, the two 5,6-epoxy derivatives, which are highly sensitive to oxidative conditions, were also the most abundant oxysterols detected in the oil after the oxidation process.  
We did not determine the content of cholesterol of the oils used in the present study. However, the level of cholesterol in refined hoki liver oil has been reported to be 1.41 g/kg [92]. Refined anchovy oil that has been refined, bleached, deodorized, and further processed by molecular distillation contains cholesterol in the range of 3-6 g/kg [93]. Thus, the levels of cholesterol in these two refined oils are likely to be present in a similar concentration range, and our initial hypothesis that cholesterol level would be substantially higher in hoki liver oil than in anchovy oil does not hold. Thus, the availability of cholesterol as an oxidizable substrate is not the explanation for the higher formation of oxysterols in hoki liver oil than in the anchovy oil, although understanding the relative contributions of esterified and free cholesterol to oxysterol formation merits further research. The main difference remains the large difference in susceptibility to oxidation, which is mainly defined by the difference in antioxidant protection. Antioxidants such as tocopherols are able to reduce the levels of oxysterols in a variety of foods [94]. The finding that specific oxysterols can be formed during the over-oxidation of fish oil has not been reported previously. Nevertheless, the levels are low (nmol per kg oil range). Interestingly, both unoxidized hoki liver oil and anchovy oil already contained low levels of various oxysterols, some of which are unique to each oil type.  
The formation of oxysterols has been associated with the onset and progression of a range of disorders [95; 96]. The pathophysiological activities of oxysterols are inter-related with the multiple biological functions of cholesterol, including being a precursor molecule for the synthesis of steroid hormones, neuroactive steroids, and bile acids. Many oxysterols are cytotoxic toward endothelial and smooth muscle cells at low micromolar concentrations, including 5,6-epoxycholesterol and 7-hydroxycholesterol, which increased markedly in the over-oxidized hoki liver oil [97; 98; 99]. The addition of oxidized cholesterol to salmon oil has been shown to place a marked stress on antioxidant defenses in rat liver [100]. Oxysteros may also mediate compromised insulin sensitivity, resulting from the consumption of cholesterol-rich diets, as well as toxic effects on the central nervous system [101; 102]. Several oxysterols, such as 25-hydroxycholesterol, 7-ketocholesterol, and 22-hydroxycholesterol, can impair the differentiation and fusion of term trophoblast cells and alter their secretion of chorionic gonadotropin, via activating nuclear liver X receptors (LXR) and inhibition of Sterol Response Element Binding Protein-2 (SREBP-2), which may have important implications for placental development and function [103; 104]. Although specific research related to perinatal morbidity has not been carried out,there are multiple possibilities regarding how specific oxysteros might compromise the health of rat pups and dams following the exposure to excessive amounts of dietary oxysterols.