Phospholipids in Dialysate and the Peritoneal Surface Layer

Jian-hui Zhong,1 Qun-ying Guo,1 Ren-gao Ye,1 Bengt Lindholm,2 Tao Wang1,2

Dialysate concentration of phospholipids has been used to monitor peritoneal membrane status. However, we recently found that the peritoneum has a surface layer in which phospholipids may be the main constituent. Therefore, in this study, we compared the phospholipids composition of peritoneal dialysate and of the peritoneal surface in rats.

Eight male Sprague–Dawley rats were used in the study. Five rats received an intraperitoneal injection of 25 mL 4.25% glucose dialysis solution. After four hours, the rats were killed, and the dialysate was drained completely. Then 20 mL of Folch solution was infused into the peritoneal cavity for 30 seconds and drained completely. The other three rats received the Folch solution without dialysis. The effluent and Folch solution were then processed for phospholipids analysis using high performance thin-layer chromatography (HPTLC).

The total phospholipids content was ten times higher in the surface layer than in the dialysate effluent. In the effluent, four clearly different components were seen: lysophosphatidylcholine (LPC), sphingomyelin (SM, 29%), phosphatidylcholine (PC, 66%), and phosphatidylinositol (PI, 4.5%). However, in the surface layer, as well as LPC, SM (20.6%), PHC (47%), and PI (6.3%), two additional components were seen, phosphatidylserine (PS, 17.1%) and phosphatidylethanolamine (PE, 8.9%). The quantity of phospholipids in the peritoneal surface of non dialyzed rats was similar to the total quantity of phospholipids (in effluent and in the peritoneal surface) of dialyzed rats.

Our results suggest that: (1) a surface layer is present on the peritoneum; this layer could well be extracted by Folch solution; and, with appropriate incubation time, one can separate the surface layer without damaging the mesothelial cells; (2) the composition of phospholipids in the effluent is different from that in the peritoneal surface layer, which contains membrane phospholipids (PS and PE); (3) shielding from the peritoneal surface may be the main reason for the presence of phospholipids in the dialysate.

Key words

Peritoneum, phospholipids

Introduction

Surface-active phospholipids similar to those demonstrated by Hills et al in pleural fluid and pericardial fluid were identified in peritoneal fluid by Grahame et al (1). The main constituent of the phospholipids in peritoneal dialysate effluent is phosphatidylcholine, long recognized for the surface activity it displays at the alveolar type II cells as lamellar bodies (2). Lamellar bodies have also been identified within peritoneal mesothelial cells (3).

The role of surface-active phospholipids in peritoneal dialysis is not clear. However, several studies have demonstrated that phospholipids, especially phosphatidylcholine, could decrease the peritoneal fluid absorption rate and increase peritoneal fluid removal (4–7). The observed effect was attributed to phospholipids forming a water-repellent layer around the mesothelial cells (8,9). Grahame et al speculated that any situation that altered the amount of phospholipids in the peritoneal membrane might be reflected in changes in the physiology of the membrane, particularly with respect to water repellency (1). Di Paolo et al first observed a relationship in patients between loss of ultrafiltration and low levels of phosphatidylcholine in the effluents (5). They also demonstrated that adding 50 mg/L phosphatidylcholine to the dialysate could significantly increase peritoneal fluid removal in patients with peritoneal ultrafiltration failure. Furthermore, Krack et al demonstrated that, even in patients with normal ultrafiltration, adding phosphatidylcholine to the dialysate could significantly increase peritoneal fluid removal (6). Although these positive results have not been not confirmed by other studies (10), phospholipids are believed to reduce peritoneal fluid absorption (4).

In a previous study, we demonstrated that a surface layer is present on the peritoneal mesothelial cell surface and that this layer consists mainly of phospholipids (11). However, it is not clear whether the composition of phospholipids in the dialysate effluent differs from that in the peritoneal surface layer. Therefore, the purposes of this study were (a) to evaluate a method that could extract phospholipids from the membrane surface layer without damaging the peritoneal mesothelial cell membrane, and (b) to evaluate and compare the constituents of phospholipids in the peritoneal dialysate effluent and in the peritoneal surface layer.

Materials and methods
Reagents

Standard phospholipids—including phosphatidylcholine (PC), lysophosphatidylcholine (LPC), sphingomyelin (SM), phosphatidylinositol (PHI), phosphatidylserine (PS), and phosphatidylethanolamine (PE)—were purchased from Sigma (St. Louis, MO, U.S.A.). Chloroform, methanol, and acetone of analytical grade were purchased from Shantou Guanghua Chemical Company (Shantou, China). Normal-phase silica gel high-performance thin-layer chromatography (HPTLC) plates (20 × 20 cm) were purchased from Merck (Darmstadt, Germany). Phosphin E was purchased from Kasei Co. (Tokyo, Japan).

Membrane lipid extraction

Normal samples of peritoneal tissue were taken from two male Sprague–Dawley rats. The tissues were immediately immersed in Folch solution (chloroform:methanol, 2:1) for 0 seconds (control), 30 seconds (FS1), 1 minute (FS2), and 2 minutes (FS3). All tissues were then washed with phosphate-buffered saline (PBS, pH 7.4) and frozen at –80°C. The frozen transverse sections were stained with hematoxylin and eosin (HE) or with a hydrophobic probe, phosphin E, and were observed under light microscopy or epifluorescence microscopy.

Dialysate and membrane collection and lipid extraction

Eight male Sprague–Dawley rats were used. Five rats received an intraperitoneal injection of 25 mL 4.25% glucose dialysis solution. After 4 hours, the rats were killed and the dialysate was drained completely. Then 20 mL of Folch solution was infused into the peritoneal cavity for 30 seconds and drained completely. The other three rats received the Folch solution without dialysis. The effluents were extracted by Folch solution using a ratio of dialysate to Folch solution of 1:5.

Phospholipids determination

Phospholipids were estimated as the phosphorus content of lipid extractable by Folch solution following a method modified from Rouser et al in which chloroform-soluble phosphorus is converted to phosphate by digestion with perchlorate. The phosphate thus produced was estimated calorimetrically with ammonium molybdate indicator, recording light at a wavelength of 830 nm on a spectrophotometer (12).

HPTLC

The HPTLC plates were pre-cleaned by developing them with 2-propanol and then air drying them. The plates were activated before use by heating for 40 minutes in an oven at 110°C. The plates were then stored over silica gel with auxiliary P2O5 in a desiccator. Preparative amounts of extract in the range of 5 – 10 mg (in 5 mL) were spotted on an HPTLC plate. The samples were applied 2 cm from the bottom of the plate. After the spots were fully dried, one-dimensional TLC was run in chloroform:methanol:acetic acid:water (53:31:2:1). The plates were then fully dried and thoroughly sprayed with phosphomolybdic acid (10%) in 20% ethanol (1). The density of spots was quantitatively determined at 500 nm with a TLC densimeter (CS-9000: Shimadzu, Kyoto, Japan).

Results
Membrane lipids extraction and membrane damage

Under epifluorescence microscope and phosphin E staining, a golden-green continuous layer was present on the surface of normal peritoneal mesothelial cells (Figure 1). In the FS1 group, this layer was almost completely lost (Figure 2), but the mesothelial cells remained intact (HE staining, Figure 3). In the FS2 group, the surface layer was completely lost, and part of the mesothelial cell membrane was damaged (Figure 4). In the FS3 group, the surface layer and the mesothelial cells were both lost (Figure 5).

Phospholipids in dialysate effluent and in peritoneal membrane

The total phospholipid content was ten times higher in the surface layer than in the dialysate effluent: 1.121 ± 0.219 mg from the peritoneal surface versus 0.122 ± 0.023 mg from dialysate effluent, p < 0.01. In the effluent, four clearly different components were seen: lysophosphatidylcholine (LPC), sphingomyelin (SM, 29%), phosphatidylcholine (PC, 66%), and phosphatidylinositol (PI 4.5%). However, in the surface layer, besides LPC, SM (20.6%), PC (47%), and PI (6.3%), two additional components were seen: phosphatidylserine (PS, 17.1%) and phosphatidylethanolamine (PE, 8.9%). The quantity of phospholipids in the peritoneal surface of non dialyzed rats was 1.265 ± 0.211 mg, similar to the total quantity of phospholipids (in effluent and in the peritoneal surface) of dialyzed rats.

Discussion

Surface-active substance (surfactant) was first identified in peritoneal dialysis effluent in 1985 by Grahame and co-workers. This observation has since raised great interest in the peritoneal dialysis community, owing to the special physical properties of surfactants. The present study shows that a large reservoir of phospholipids exists in the peritoneal surface and that the constituents of phospholipids in the effluent are different from those in the peritoneal surface layer.

Surfactant consists of a mixture of phospholipids (67%), proteins (8%), neutral lipids (21%), carbohydrates (2%), and minor constituents (2%) (13). In general, besides lowering the surface tension of alveolae, surfactants have been shown to play important roles in local host defense, in tissue permeability and in maintaining the fluid balance in alveolae (14,15). However, despite extensive studies in surfactant physiology, the function of the individual constituents of surfactant, and especially the role of various phospholipids in surfactant is, to a large extent, unknown (16).

As mentioned, evidence suggests that besides lubrication of the peritoneal cavity, surfactant, and especially its content of phosphatidylcholine, may play an important role in peritoneal transport. The present study compared for the first time the composition of phospholipids in the peritoneal dialysis effluent and in the peritoneal surface layer. The reason for the presence of two additional constituents of phospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), in the peritoneal surface as compared to the dialysate effluent is not clear from the present study. A previous study applied an approach similar to the one in the present study to extract phospholipids from the pleura by dipping the tissue into Folch solution for 2 minutes. That study found no significant change in tissue histology (17). Although we found in the in vitro study that incubation of the peritoneal tissue with Folch solution for 2 minutes destroyed mesothelial cells, shortening the incubation time to 30 seconds removed the surface layer but left the mesothelial cell layer intact. It is therefore unlikely that the PS and PE come from mesothelial cell membrane itself.

The differences between the dialysate and the peritoneal surface layer in terms of phospholipids is worth speculating on. In eukaryotic cells, the phospholipids in the lipids bilayer of the membrane have been found to possibly be asymmetrically distributed. While the choline-containing phospholipids—such as sphingomyelin and phosphatidylcholine—are predominantly located in the outer monolayer of the membrane, most aminophospholipids—phosphatidylserine and phosphatidylethanolamine—are confined to the membrane’s inner leaflet (18). This asymmetrical distribution of phospholipids in eukaryotic cells is important for normal cell function; disturbance of the asymmetry may be an important step for pathological processes such as apoptosis (19). Whether the phospholipids in the peritoneal surface layer are also asymmetrically distributed—and therefore whether the outer layer consists of mainly phosphatidylcholine and sphingomyelin that can easily be washed out by the dialysis procedure—needs further study to confirm.

Dialysate concentration of phospholipids has been used to monitor peritoneal membrane status (1,20,21). However, the present study suggests that the composition of phospholipids in the dialysate and the surface layer may be different and that the difference should be taken into account when interpreting the results of dialysate phospholipids measurement. Furthermore, in support of our recent finding that the surface layer was unexpectedly thick and consisted mainly of phospholipids (11), our results show that the quantity of phospholipids in the peritoneal surface layer is at least ten times higher than that in the dialysate effluent. This observation suggests that slight changes in phospholipid concentration in the dialysate may not necessarily have a significant impact on peritoneal transport. Also, the equilibrium of phospholipids between the peritoneal dialysate and the peritoneal surface layer should also be taken into account when interpreting the relationship between dialysate phospholipid concentration and peritoneal transport rate (21).

Existing evidence suggests that the phospholipids content in the peritoneal dialysate effluent, as well as in the peritoneal surface, is derived from peritoneal mesothelial cells (3,9). The similar total quantity of phospholipids in the non dialyzed peritoneal membrane and in the dialyzed membrane plus effluent further supports this idea. The exact mechanism by which phospholipids bind to the peritoneal surface is not clear so far. However, it has been proposed that surfactant binds to a solid surface by a process called adsorption or chemisorption for stronger ionic binding, more characteristic of cationic surfactants, especially those incorporating the strongly positively charged terminal quaternary ammonium (QA) ion. All mammalian biosurfaces, such as the peritoneal surface, are now well known to be negatively charged—with membrane-bound carbohydrates introducing carboxyl and sulfonyl groups—and, therefore, most conducive to adsorption of cationic surfactants. When binding to hydrophilic surfaces via their polar groups, the nonpolar groups of surfactant molecules are oriented outwards, rendering the surface less compatible with water and, therefore, more hydrophobic. In excess, surfactants often build up more layers parallel to the adsorbed monolayer as an oligolamellar coating, those with QA ions often forming structures alternating lipid bilayers with water layers invoking chelate binding, as seen in vivo in the lung (13).

We did not quantify the amounts of lysophosphatidylcholine in the dialysate and in the peritoneal surface layer because, in the present study, we found that the visualization of the lysophosphatidylcholine standard was not ideal with ammonium molybdate staining. Note that the phospholipids found in the dialysate in the present study differed from those found in the study by Grahame, in that PS and PE were present in the previous study, which used peritoneal dialysis patients (1). Whether this difference is related to species (rat versus human) or to the difference in time on dialysis (acute study versus chronic dialysis study) is currently under investigation in our laboratory.

Conclusion

Our results suggest that: (a) a surface layer is present on the peritoneal mesothelium; this layer could well be extracted by Folch solution; and, with appropriate incubation time (30 seconds), the surface layer can be separated without damaging the mesothelial cells; (b) the composition of phospholipids in the effluent is different from that in the peritoneal surface layer (the surface layer also contains membrane phospholipids, phosphatidylserine and phosphatidylethanolamine); (c) shielding from the peritoneal surface may be the major reason for the presence of phospholipids in the dialysate.

Acknowledgment

This study was supported by a grant from Sun Yat-sen University of Medical Sciences (No. 98051) and by a grants from the China Medical Board and from Baxter Healthcare Corporation, McGaw Park, IL, U.S.A.

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Corresponding author:

T. Wang, md, Department of Nephrology, 1st Affiliated Hospital, Sun Yat-sen University of Medical Sciences, 58 Zhongshan Road 2, Guangzhou 510080 P.R. China.

From: 1Department of Nephrology, 1st Affiliated Hospital, Sun Yat-sen University of Medical Sciences, Guangzhou, P.R. China and 2Divisions of Baxter Novum and Renal Medicine, Karolinska Institute, Huddinge University Hospital, Sweden.