Formation of Oxysterols

3 (3-Hydroxycholest-5-en-7-peroxyl radical Cholesterol 7-peroxyl radical CHOO  

---

Extensive studies in vitro from many groups have confirmed that exposure of LDL to a variety of pro-oxidant systems, both cell-free and cell-mediated, results in the formation of lipid hydroperoxides and peroxidation products, fragmentation of apoprotein Bioo, hydrolysis of phospholipids, oxidation of cholesterol and cholesterylesters, formation of oxysterols, preceded by consumption of a-tocopherol and accompanied by consumption of 8-carotene, the minor carotenoids and 7-tocopherol. 

Patel, R.P., Diazfalusy, U., Dzeletovic, S., Wilson, M.T., Darley-Usmar, V.M. 1996. Formation of oxysterols during oxidation of low-density lipoprotein by peroxynitrite, myoglobin and copper. J. Lipid Res. 37, 2361-2371. 

As summarized in the previous paragraph, LDL cholesterol is oxidized to various oxysterols due with high cytotoxicity and promoting vascular injury [63]. Oleuropein and a mixture of phenols extracted firom virgin oil inhibited dose-dependently the formation of oxysterols and prevented apoprotein modification in UV irradiated LDL. IC50 are shown in table 4 ... 

Additionally, cholesterol oxidation can occur in in vivo by enzymes or by lipid peroxidation. Enzymatic formation of oxysterols can be divided into two classes (a) direct enzymatic action on cholesterol or another related sterol and (b) enzymatic activity leading to the formation of radicals, which in turn attack cholesterol. All reactions of the first type seem at present to be cytochrome P-450 dependent [16]. 

In our above proposed hypothesis for the in vivo formation of oxysterols, we attribute their occuiTence to the various phenomena that make it possible, (i) for various almost water-insoluble (at 25°C cholesterol solubility is approximately 0.2 mg/dl or 4.7 xM) cholesterol species to occur in serum, an aqueous medium [15], and (ii) to allow these species as lipoprotein entities to circulate in blood throughout our bodies. 

As mentioned earlier, oxidation of LDL is initiated by free radical attack at the diallylic positions of unsaturated fatty acids. For example, copper- or endothelial cell-initiated LDL oxidation resulted in a large formation of monohydroxy derivatives of linoleic and arachi-donic acids at the early stage of the reaction [175], During the reaction, the amount of these products is diminished, and monohydroxy derivatives of oleic acid appeared. Thus, monohydroxy derivatives of unsaturated acids are the major products of the oxidation of human LDL. Breuer et al. [176] measured cholesterol oxidation products (oxysterols) formed during copper- or soybean lipoxygenase-initiated LDL oxidation. They identified chlolcst-5-cnc-3(3, 4a-diol, cholest-5-ene-3(3, 4(3-diol, and cholestane-3 3, 5a, 6a-triol, which are present in human atherosclerotic plaques. 

The health impairing and toxic elfects of oxidation of lipids are due to loss of vitamins, polyenoic fatty acids, and other nutritionally essential components formation of radicals, hydroperoxides, aldehydes, epoxides, dimers, and polymers and participation of the secondary products in initiation of oxidation of proteins and in the Maillard reaction. Dilferent oxysterols have been shown in vitro and in vivo to have atherogenic, mutagenic, carcinogenic, angiotoxic, and cytotoxic properties, as well as the ability to inhibit cholesterol synthesis (Tai et ah, 1999 Wpsowicz, 2002). 

Formation of mevalonate. The conversion of acetyl CoA to acetoacetyl CoA and then to 3-hydroxy-3-methylglutaryl CoA (3-HMG CoA) corresponds to the biosynthetic pathway for ketone bodies (details on p. 312). In this case, however, the synthesis occurs not in the mitochondria as in ketone body synthesis, but in the smooth endoplasmic reticulum. In the next step, the 3-HMG group is cleaved from the CoA and at the same time reduced to mevalonate with the help of NADPH+H 3-HMG CoA reductase is the key enzyme in cholesterol biosynthesis. It is regulated by repression of transcription (effectors oxysterols such as cholesterol) and by interconversion... 

Dzeletovic, S., Babiker, A., Lund, E., Diczfalusy, U. 1995. Time course of oxysterol formation during in vitro oxidation of low density lipoprotein. Chem. Phys. Lipids 78, 119-128. 

Table 4. IC50 of phenolic components of olive oil for the inhibition of oxysterol formation in hnman LDL ...

The final component of the project involves correlating the analytical data with clinical information obtained by the contributing physicians. This could provide new insights regarding the role of oxysterols in the pathogenesis of atherosclerosis. Because specimens will be acquired from a variety of sources, a standardized format or questionnaire would be used to normalize the quality of the clinical information obtained from the different sources. 

After absorption, oxysterols react with blood plasma lipoproteins to form complexes that can initiate formation of atherosclerotic deposits in the vascular wall. However, the important oxidation reaction from the physiological point of view is the transformation of provitamins D (ergosterol and 7-dehydrocholesterol) into vitamins D2 and Dj, respectively. 

Brown, A.J., Dean, R.T., and Jessup, W., 1996, Free and esteiified oxysterol formation during copper-oxidation of low density hpoprotein and uptake by macrophages, /. Lipid Res. 37 320-335. 

There is experimental evidence that suggests that some oxysterols, but not pure cholesterol, are the prime cause of atherosclerotic lesion formation (162). Upon cholesterol feeding, a strong relationship was seen between plasma oxysterols and aortic wall oxysterols. One may speculate that the deposition of pure lipids, such as cholesterol and its esters, may be merely a secondary process in response to oxysterol-induced endothelial cell injury. Cell injury/dysfunction and the subsequent disruption of endothelial barrier function by oxysterols (163, 164) could initiate the early events in atherosclerosis. Such injury could allow increased uptake... 

Excess cholesterol can also be metabolized to CE. ACAT is the ER enzyme that catalyzes the esterification of cellular sterols with fatty acids. In vivo, ACAT plays an important physiological role in intestinal absorption of dietary cholesterol, in intestinal and hepatic lipoprotein assembly, in transformation of macrophages into CE laden foam cells, and in control of the cellular free cholesterol pool that serves as substrate for bile acid and steroid hormone formation. ACAT is an allosteric enzyme, thought to be regulated by an ER cholesterol pool that is in equilibrium with the pool that regulates cholesterol biosynthesis. ACAT is activated more effectively by oxysterols than by cholesterol itself, likely due to differences in their solubility. As the fatty acyl donor, ACAT prefers endogenously synthesized, monounsaturated fatty acyl-CoA. 

---