Arcilla Chacko ( Hidralgirita )

This project was concerned with examining the hypothesis that a component(s) of the lipid emulsion used in TPN may play a role in the development of TPN-associated cholestasis. The aim was to establish in vitro cell systems to examine the individual and cumulative effects of these components on membranes and on cellular function and to discover whether or not there were any effects which could contribute to hepatobiliary dysfunction and specifically to impaired bile flow (cholestasis).

The fluidity of biological membranes is mainly determined by their lipid composition. Lipids from lipoproteins are known to exchange with their counterparts in red blood cell membranes. Thus, changes in plasma lipid composition are associated with corresponding changes in the composition of the erythrocyte membrane. One could predict that infusing patients with lipid emulsions with a lipid profile very different to that of normal plasma could affect the fluidity of the membrane and thereby influence the structure and function of the cell. Examination of the lipid composition of

erythrocytes from neonates receiving TPN suggested that the elevated levels of phospholipids and phytosterols in their red cell membranes exerted a significant effect on membrane fluidity. The membrane fluidity could be related to sterol/PC ratios and the results indicated that patients on long term TPN who had increased membrane PC showed an elevation in their erythrocyte membrane fluidity. This suggested that

accumulation of PC was a more potent influence than accumulation of (phyto)sterols in the membrane.

The phospholipid profile of intravenous lipid emulsions was qualitatively characterised using ESI-MS/MS. The method was capable of demonstrating clear differences between the two lipid emulsions, 20% Intralipid which is emulsified with egg

phospholipids and 20% Lipofundin S which is emulsified with soybean phospholipids. The phospholipid composition of 2 0% Intralipid was found to be closer to that of normal human tissues. Phospholipids are a major constituent of bile. The phospholipids which are synthesised by the liver and secreted into bile are PC and typically contain palmitic acid (16:0) at 5«-l and oleic or linoleic acids (18:1 or 18:2) at sn-2. The exogenous phospholipids from lipid emulsions, if very different to normal tissues, could alter this synthesis and/or lead to the accumulation in the hepatocytes of phospholipids

which are not efficiently secreted into bile by the MDR3 phospholipid translocase. A

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genetic model has recently been described which highlights such a mechanisnj^the pathogenesis of cholestatic liver disease (Smit et al., 1993). Using a knockout model it has been shown that the homozygous disruption of the murine gene which encodes the phospholipid translocase, leads to the complete absence of biliary phospholipids and development of liver disease similar to neonatal cholestasis.

A number of approaches were undertaken to develop an in vitro system to examine more closely the effects of phytosterols on cellular function. In children with TPN- associated cholestasis, phytosterols comprise 20-25% of plasma and erythrocyte total sterol concentration with cholesterol making up the remaining 75-80%. It proved difficult to incorporate such amounts into cells in vitro without having adverse effects on the cells. For example, although 2-hydroxyropyl-P-cyclodextrin was able to load phytosterols at a maximum concentration equivalent to 25% of the erythrocyte total sterol it was found that the vehicle itself removed cholesterol from the red cell membrane.

Attempts to load cells with phytosterols by using phytosterol enriched Intralipid produced some interesting findings. The results showed that the lipid emulsion had a profound effect on the uptake of cholesterol by hepatocytes. Rather than loading cells with phytosterols, Intralipid inhibited the net cholesterol uptake by Hep G2 cells. A very striking effect of the Intralipid-induced inhibition of cholesterol uptake was that the inhibition was greatest if Intralipid was incubated with PCS for 24 hours. This suggested that PCS and Intralipid might interact in such a way that the cholesterol becomes progressively less available for uptake with time. The inhibition also differed with different Intralipid preparations and the data suggested that inhibition of

cholesterol uptake increased with increasing PC and/or the phytosterol concentrations of the fat emulsions. Addition of phytosterols alone to a cell system, however, failed to produce any significant alteration in the uptake of cholesterol by Hep G2 cells. This suggested that phospholipids may be the more important mediator of the inhibition.

Whatever the mechanism, it can be concluded that if the administration of Intralipid

mediated such a profound inhibition of cholesterol uptake by the liver it could have clear pathological implications in the development of TPN-associated cholestasis. It is possible that the phospholipids and phytosterols of lipid emulsions exist in a particle which is responsible for mediating the inhibitory effects on cholesterol uptake. For example, the cholesterol may be incorporated into an Lp-X-like particle which is poorly taken up by the hepatocytes. If the delivery of cholesterol to the hepatocyte is impeded it could seriously affect the mechanisms involved in bile flow. Such effects could then conceivably lead to the stagnation of bile i.e. cholestasis. Furthermore, the bile

components might then leak into the blood stream resulting in the progressive formation of more Lp-X and exacerbate the associated complications of the patient. Further work will be required to clarify the role played by specific components from lipid emulsions in the inhibition of cholesterol uptake. This will involve determining the presence of Lp-X in the cell medium using density gradient ultracentrifugation, affinity column

chromatography and agar gel electrophoresis. Studies will also have to be carried out to investigate the effects of supplementation of the cell media with phospholipid alone.

In conclusion, it has been shown that Intralipid may alter normal cellular function of hepatocytes and erythrocytes in vitro. The reduction in cholesterol uptake observed in the Hep G2 cell line may well be important in the pathogenesis of cholestasis. Whether the phospholipid and/or phytosterol component of the lipid emulsion are responsible for these effects or whether the formation of Lp-X is an important factor remains to be established.