4.3. Objetivo Específico N° 03:
4.3.3. Critica al extorno del ingreso como recaudación
Single flasks of confluent Hep G2 cells were incubated with 10ml supplemented Intralipid medium in 95% air-5% CO2 at 37°C for between 30 minutes and four hours. Control solutions of either 10ml growth medium + 20% Intralipid containing 10% (v/v) soybean oil alone or growth medium alone were incubated with cells for 4 hours. After being rinsed thoroughly five times with 10ml isotonic lOmM PBS pH7.4, the Hep G2 cells were harvested and sonicated as described earlier in section 3.9.2. Cell protein was determined by the BCA method (section 2.6.3) and phytosterol concentrations were measured in a 200pl aliquot of cell lysate as previously described (section 2.6.5).
3.11.3 Results
A 30 minute incubation with Intralipid + 5mM phytosterols resulted in only 0.43pmol/g cell protein of sitosterol and no campesterol being taken up. Even after 4 hours only small amounts of phytosterols (sitosterol, 0.51 and campesterol, 0.48|iimol/g cell protein) could be detected compared to cellular cholesterol concentration of
86.5|uimol/g cell protein. No phytosterols were taken up by cells incubated with IL + soy oil or medium alone. Thus, the results demonstrated that 20% Intralipid was unable to load significant quantities of phytosterols into Hep G2 cells.
3.12 Discussion
These experiments have demonstrated the difficulty in developing an in vitro system to examine the role of phytosterols in TPN-associated complications. From the results it was clear that HPC was able to load significant concentrations o f phytosterols into RBC, however, HPC was found to mediate cholesterol effiux and haemolysis o f the cells, an observation supported by the findings of others (Irie et al., 1982; Ohtani et al.,
1989). HPC was shown to mediate a dose-dependent and rapid effiux of [^"^C]cholesterol from both erythrocytes and Hep G2 cells.
Physiological acceptors involved in reverse cholesterol transport such as HDL have been shown to promote a relatively constant release of cholesterol from cultured cells throughout an 8 hour incubation (Kilsdonk et al., 1995). It is believed that this occurs by an aqueous diffusion mechanism whereby the cholesterol molecules move from the membrane into the aqueous medium before being sequestered by the acceptor molecule (Lang et al., 1983). Yancey et al. (1996) have suggested that HPC can promote a significantly more efficient cholesterol effiux because cholesterol molecules are able to diffuse directly from the cell membrane into the hydrophobic core of a cyclodextrin molecule without prior desorption in the aqueous phase.
The RBC experiments also showed tj^ t HPC-induced haemolysis as demonstrated in Figure 3-5. Although the relationship between cholesterol efflux and RBC haemolysis was not linear the results indicated that the haemolysis may have been produced from the removal of membrane cholesterol from RBC by the cyclodextrin. It is conceivable that a threshold concentration of cholesterol has to be extracted from the erythrocyte membrane by the HPC (Figure 3-14). This would certainly account for the dramatic increase in haemolysis observed with the larger concentrations of HPC. One finding that was clear was that the amount of cellular cholesterol effiux and haemolysis was reduced when the HPC had been pre-saturated with phytosterols. This result suggested that the phytosterols competed with the cholesterol for encapsulation within the cyclodextrin. Thus, the removal of membrane cholesterol would only have arisen from free HPC in the solution.
X 3 E LU 'Ô cr — O u u T|- 'Ô3 U <u S o ü Dh 7 5 i 60- 45- 30- 15- 0 10 20 30 40 Percentage RBC Haemolysis o
In recent years attempts have been made to synthesise cyclodextrin derivatives which possess lower membrane disrupting abilities (Shiotani et al., 1994; 1995). When
negatively charged polar groups such as sulphur or sulphur-butyl groups are introduced the haemolytic activity of P-cyclodextrins can be significantly lowered. These
polysulphated cyclodextrins can even protect erythrocytes from chlorpromazine- induced haemolysis; unfortunately attachment of these groups to the cyclodextrin also severely affects the ability of the cyclodextrin to solubilise lipids. Cyclodextrins have been used to deliver small concentrations of lipophilic compounds without adverse effects on cells. Awad et al. (1996) recently described the supplementation ofHT-29 cells with 16|liM sitosterol using 0.8% (w/v) HPC. However, the use of HPC at this concentration is much too small to solubilise the large quantities of phytosterols required for this study. There was, therefore, a need to find another form of delivery system which had the capacity to load the cells with significant levels of plant sterols.
Attempts were made to dissolve phytosterols in organic solvents prior to addition to PCS. The results o f the study demonstrated that an inverse relationship existed between the polarity index of solvents and their phytosterol solubility. In particular diethyl ether and chloroform proved to be highly effective at dissolving millimolar concentrations of phytosterols. However, hexane which possessed the lowest polarity index value of 0, was found to dissolve low concentrations of the plant sterols. A possible explanation for this finding was that hexane was more non-polar than the phytosterols and, therefore, interacted less strongly with these compounds than some of the other solvents (Flynn et al., 1979). This observation is fully consistent with the finding o f low cholesterol solubility in mineral oil, an apolar solvent (Wright and Presberg, 1964) and adds support to the suggestion that moderate solvent polarity favours the solubility of sterols.
On addition to PCS it was found that more phytosterols were taken up from chloroform than from diethyl ether. The difference in phytosterol uptake from the two solvents may be related to their volatilities. Diethyl ether (b.p. 34.6“C) is much more volatile than chloroform (b.p. 61°C) and thus when added to the PCS the diethyl ether would have evaporated from the solution more readily than the chloroform. Accordingly, the
phytosterols may have precipitated out of solution more rapidly. It is, therefore, likely that despite their similar concentrations, the plant sterols in the diethyl ether were unable to interact with the lipoproteins for as long as the plant sterols dissolved in chloroform. Nonetheless, only relatively small concentrations of phytosterols were transferred from chloroform and these were unevenly distributed within the PCS which clearly suggested that the phytosterols had not been properly incorporated into the PCS.
It was also found that the lipid emulsion (20% Intralipid) even when supplemented with levels of phytosterol as high as 5mM was unable to load significant quantities o f the plant sterols into Hep G2 cells. There are several reasons which might explain this finding. Firstly, the cells may not have been exposed for sufficiently long to the
phytosterols for them to be loaded from the lipid emulsion. Secondly, the lipid emulsion may have been toxic to the cells thereby affecting their function and growth and hence uptake of a range of nutrients from the medium. Thirdly, the amount of lipoprotein in the medium may have been insufficient for phytosterol uptake (assuming phytosterol uptake into the cells utilises the same pathway as uptake of cholesterol by hepatocytes). Finally, it is possible that the mechanism by which cholesterol enters the hepatocytes is simply not available to phytosterols. This is the situation in enterocytes. In man, phytosterols are only taken up to a very small degree by enterocytes and they in fact inhibit the uptake of cholesterol (Glover and Morton, 1958). This is the basis of the use of phytosterols to lower cholesterol absorption from the diet (Lees et a l, 1977). If phytosterols inhibit cholesterol uptake into liver cells, this might reduce the amount of cholesterol available for bile acid synthesis and reduce the amount available for
incorporation into bile. These properties could contribute to cholestasis and the effect of phytosterols on cholesterol uptake has been studied in more detail in chapter 4.
Another possible way in which phytosterols could potentially contribute to cholestasis would be via inhibition of 7a-hydroxylase, the rate limiting enzyme of bile acid
synthesis (Shefer et al., 1988). It has been shown that Hep G2 cells are able to
synthesise bile acids. Of course if phytosterols are unable to enter cells it is unlikely that any inhibition would be observed. This study indicated that Hep G2 cells produced low
concentrations of the C27 bile acid THCA. This suggested that the cells possessed a defect in peroxisomal side chain oxidation of THCA a finding consistent with the data of Everson and Polokoff (1986). Significant quantities of chenodeoxycholic and cholic acids were also synthesised and secreted by the Hep G2 cells. The results showed that the ratio of chenodeoxycholic acid to cholic acid ranged between 4 .1 and 6; 1 which was considerably smaller than the value of 40:1 quoted by Everson and Polokoff (1986). Interestingly, these values were closer to the normal physiological ratio of 0.5-2 and moreover were similar to those reported by Javitt et al. (1989) who failed to
demonstrate any peroxisomal defects in Hep G2 cells.