Aplicación del principio de no confiscatoriedad en el procedimiento de

From the results it was clear that phytosterols had accumulated in highly significant concentrations in the plasma of infants receiving TPN, and in particular in those patients with severe liver dysfunction (26.3% of total sterols). These findings were in agreement with the data of Clayton et al. (1993) who reported that long-term infusion of Intralipid in neonates was associated with the accumulation of phytosterols. Importantly, it was found that large concentrations of these phytosterols had also been incorporated into the RBC membranes of these patients (21.2% of total sterols). This observation was consistent with experiments performed in rats which demonstrated that radiolabelled sitosterol rapidly moves from the chylomicron-like Intralipid particles into plasma lipoprotein fractions and then into red cell membranes (Kakis et a l, 1988).

Phytosterols are a major contaminant of lipid emulsions and are known to produce important effects on membrane properties. Since phytosterols have been shown to displace cholesterol from membranes (Bhadra and Subbiah, 1991), it might have been expected that there would be an reduction in the membrane cholesterol content. However, there was no evidence to indicate that this had occurred and indeed the cholesterol content of the membranes of the patients with severe liver dysfunction and higher phytosterol content were not significantly different from those in patients with mild liver dysfunction and normal controls. The results suggested that the phytosterol accumulation resulted in an increase in total sterol concentrations in the membranes.

A significant inverse correlation was found to exist between membrane fluidity and plasma total sterol/PC ratio. Although the corresponding relationship of membrane fluidity and membrane total sterol/PC ratio just failed to achieve statistical significance, this observation indicated that phytosterols appear to have a similar effect on

membranes as cholesterol i.e. they make membranes more rigid. This observation agrees with other findings in the literature. It has previously been shown that the alkylated side chains of stigmasterol and campesterol are particularly effective at ordering the two unsaturated fatty acyl chains of plant phospholipids and are able to reduce the fluidity of liposomal membranes containing plant lecithin (Schuler et a l,

1990). In terms of the effect of plant sterols on a mammalian-type membrane systems

one report has suggested that incorporation of a small proportion of sitosterol and campesterol into rat liver microsomal membranes leads to a reduction in fluidity (Leikin and Brenner, 1989). The situation with regard to the three patient groups in this study, however, was more complicated. For example, in the case of patients receiving TPN with severe liver dysfunction there was a significant increase of phytosterols in their RBC membranes. There was also evidence of an elevation in membrane PC

concentration. As a result two of these patients had very low total sterol/PC ratio values. These same two patients also had the greatest membrane fluidity. This suggested that the effect of the phospholipids was greater than that of the increased total sterol content.

The absolute concentrations of cholesterol and PC in the plasma of the TPN patients were rather surprising. Administration of lipid emulsion has previously been reported to result in the progressive accumulation of phospholipids and cholesterol in the plasma of neonates receiving TPN (Griffin et al., 1979). This rise in cholesterol typically begins with the onset of the lipid administration and increases with the duration of the

parenteral nutrition and with the amount of infused emulsion (Allardyce, 1982). Such an effect could conceivably alter the lipid profile of erythrocytes. However, in this study no significant differences in the plasma cholesterol or PC concentrations were found

between the three groups of subjects although the C/PC ratio of the patients with severe liver dysfunction was reduced.

This study was also unable to demonstrate any statistically significant increases in the cholesterol and PC concentrations of erythrocyte membranes in patients receiving TPN despite previous reports that long-term infusion of Intralipid in both rabbits (Goto et al., 1983) and rats (Innis, 1989) is associated with significant elevations in erythrocyte membrane cholesterol and PC content. Furthermore, in rabbits these changes were accompanied by an elevation in the membrane fluidity of their RBC (Goto et al., 1983). It is also well documented that patients with liver disease especially those in advanced stages have a disturbed lipid profile which can also affect the fluidity of erythrocyte membranes. Significant increases in both erythrocyte membrane PC and cholesterol content have previously been demonstrated to occur in patients with biliary obstruction

(Okano et al., 1978) and biliary atresia (Okano et al., 1979). Interestingly, it has been suggested that the excess cholesterol and PC incorporated into the RBC membranes may be derived following the net transfer of these lipids from the pathological lipoprotein, Lp-X (Verkleij e t a l , 1976).

There are a number of possible explanations for the failure of this study to demonstrate a statistically significant increase in PC and cholesterol concentrations in both the erythrocyte membranes and plasma of the neonates receiving TPN. Firstly, the patients in this study were administered 20% Intralipid as part of their TPN regime which has been shown to cause a much smaller increase in plasma lipid levels than other lipid emulsion preparations (Haumont et al., 1989). Secondly, the patients who were studied with severe TPN-associated liver disease had very severe liver dysfunction. It is widely recognised that a consequence of liver failure is a large reduction in plasma cholesterol concentrations (Bemuau and Benhamou, 1991). Thirdly, it is possible that some of the results failed to show statistical significance because of the small sample sizes of the study groups. Finally, the use of adult controls was not ideal. This is an unlikely

explanation, however, since previous studies have shown that no significant differences exist between the fluidity of erythrocytes ghosts from neonates and adults (Kehry et al.,

1977; Crespo et al., 1988).

In summary, the results showed that in patients receiving TPN who had severe liver dysfunction, significant concentrations of phytosterol accumulated in their plasma and RBC membranes. Although the increase in the membrane PC content of the patients with severe liver disease was not significantly different compared to controls; the results indicated that ÿ(e this rise in membrane PC more than negated the effect of phytosterol incorporation and led to an increase rather than a decrease in membrane fluidity.

Chapter 3

Developm ent o f an in vitro System to Exam ine the