223
Clinica Chimica Acta, 63 (1975) 223-225 0 Elsevier Scientific Publishing Company,
PLASMA LYSOLECITHIN
W. SCHARDT
Amsterdam
- Printed
in The Netherlands
IN URAEMIA
and E.N. WARDLE
Wellcome Newcastle
Research Laboratories, Department upon Tyne, NE1 4LP (U.K.)
(Received
March 15, 1975)
of
Medicine,
Royal
Victoria
Infirmary.
Introduction Besterman and Gillett [ 1,2] have shown that exposure of blood platelets to lysolecithin inhibits irreversible platelet aggregation. A role of decreased plasma lysolecithin as a factor contributing to an increased risk of thromboembolic disease has therefore been postulated. In uraemic patients with poor renal function there is often a bleeding tendency due to poor platelet function [3--51, and yet paradoxically these patients are prone to thromboses including cerebral and coronary thrombosis. These events may in fact be more frequent in those who are also nephrotic, for such patients have even higher serum lipids than other patients with chronic renal failure [6,7]. We therefore decided that it would be worth investigating the effect of uraemia on plasma lysolecithin levels. Materials and methods The blood urea and plasma lysolecithin were estimated in 16 patients with uraemia and in 23 control patients. Since 6 of the patients with uraemia had acute renal failure, 10 repeat estimations were made after the patients had recovered to the stage of normal renal function. The blood samples were collected in EDTA tubes and were transported in ice. For lipid extraction 0.5 ml plasma was added to 2.0 ml methanol with shaking and the mixture was then allowed to stand for five minutes with occasional agitation. Then 4.0 ml chloroform and 1 ml of 0.7% saline were added and mixed, and allowed to stand for a further five minutes. The specimens were then centrifuged at 3500 r.p.m. for five minutes so that the contents were separated into two phases with a compact disc of protein at the interface. The upper layer was discarded and the lower was decanted and evaporated to dryness under a nitrogen jet. The lipid material was redissolved in 0.25 ml chloroform
224
(2 vols) to methanol (1 vol) and 50 1-11 aliyuots were applied at 1 c’m horizontal streaks at the bottom of 25 cm x 25 cm silica gel plates, which had been preheated at 120°C for half an hour to ‘activate’ them. The solvent system LW~ for chromatography was chloroform (50 parts)/methanol (30 pa&)/acetic acid (8 parts)/water (4 parts). The plates were run to within one inch of the top, and they were then dried and were stained in a tank (*ontainii~~ iodine vapour. The spots corresponding to Iysoletithin and lecithin were removed for phosphatcl determination and a similar sized lipid free area was taken for the blank estimation. In order to determine phosphorus the silica gel spots were placed in heavy glass tubes with 0.5 ml 10 N sulphuric acid and antibumping granules, and were heated at 230°C for 30 minutes. After cooling 2 drops of hydrogen peroxide were added and they were then reheated for yet another ten minutes until the contents were white, On retooling 1 drop of 5% urea was added and the tubes were then heated at 230°C for five minutes to decompose the peroxide. An extra four tubes in each batch were similarly treated for later use for the standards. On cooling 4.4 ml of water was added to each silica gel containing tube and to two of the blank tubes. To the remaining two tubes were added 0.5 ml standard phosphate solution and 3.9 ml water. Finally 0.2 ml ammonium molybdate and 0.2 ml ANS solution (~~-anlino-3-hydroxynaphthalene-l-sulphonic acid) were added to each tube and they were heated on a boiling water bath for seven minutes. After centrifugation the absorbance of the supernatants was measured at 830 nm. The reaction was shown to give a linear relationship between phosphate concentration and optical density. The absorbance measurements were corrected for blank values and were used to calculate the amount of ‘phosphate’ in lysolecithin and lecithin of the original plasma, as mg per dl of plasma. The total phosphate of each lipid extract was also measured and the results were finally expressed as lysole~ithin phosphate/total lipid phosphate (LL/TLP). Results and discussion The important finding was that in some patients with a high blood urea the plasma lysolecithin values were low, as shown in Fig. 1 which revealed a logarithmic relationship. On the other hand plasma lecithin values were completely independent of the blood urea. The normal range for lysolecithin phosphate was 0.0255-0.0925, when expressed as a fraction of the total lipid phosphate. The results of the three patient categories (normal, uraemic and recovery groups) were therefore expressed as an ‘index’ (the log of that value times ten) which was then plotted against the level of blood urea as in the figure. Although some patients with a high blood urea had low lysolecithin values, this did not apply to all uraemic patients. However it is significant that with adequate recovery of renaf function the lysolecithin/total lipid phosphorus indices of the acute renal failure patients returned to the normal range. The patients who were uraemic but who had normal lysolecithin values were mainly chronic renal failure patients who were receiving treatment by intermittent haemodialysis. The findings are the more significant when set against the fact that plasma lecithin values were independent of the blood urea.
225
I
I
100
0
Blood
we0
203
I 300
(mg/Ul)
Fig. 1. The ‘lysolccithin index’ (the ratio of log plasma lysolecithin phosphorus to total lipid phosphorus times ten) has been plotted against blood urea for uraemic patients (triangles), acute renal failure cases after recovery (open circles) and controls (black dots).
The likely explanation is that in advanced uraemia there is a defect of the enzyme lecithin-cholesterol acyltransferase (LCAT). This enzyme normally engages in a reaction whereby LDL or HDL cholesterol is esterified at the expense of lecithin, which in turn is converted to lysolecithin. In previous reports all the profound reductions of LCAT have been found in patients with liver disease. Studies of this enzyme in uraemia now seem worth while. Acknowledgements Our thanks are due to Mr G.A. Cheyne of the MRC Growth and Reproduction unit, Princess Mary Hospital for help with the technique and to Dr A. Cassels-Smith of Newcastle General Hospital for the facilities. References 1 2 3 4 5 6 7
E.M.M. Besterman and M.P.T. Gillett, Atherosclerosis. 14 (1971) 323 E.M.M. Besterman and M.P.T. Gill&t, Nature New Biology, 241 (1973) 223 HI. Horowitz, B.D. Cohen, P. Martinez and M.F. Papogoanou. Blood, 30 (1967) 331 HI. Horowitz. I.M. Stein, B.D. Cohen and .J.G. White, Am. J. Med., 49 (1970) 336 S.F. Rabiner and F. Molinas. Am. J. Med., 49 (1970) 346 E.N. Wardle. W. Schardt and P.R. Uldall. Post-grad. Med. J., 50 (1974) 737 E.N. Wardle, IS. Menon and J. Anderson. Q. J. Med.. 41 (1971) I5
Clinica Chimica Acta, 63 (1975) 223-225 0 Elsevier Scientific Publishing Company,
PLASMA LYSOLECITHIN
W. SCHARDT
Amsterdam
- Printed
in The Netherlands
IN URAEMIA
and E.N. WARDLE
Wellcome Newcastle
Research Laboratories, Department upon Tyne, NE1 4LP (U.K.)
(Received
March 15, 1975)
of
Medicine,
Royal
Victoria
Infirmary.
Introduction Besterman and Gillett [ 1,2] have shown that exposure of blood platelets to lysolecithin inhibits irreversible platelet aggregation. A role of decreased plasma lysolecithin as a factor contributing to an increased risk of thromboembolic disease has therefore been postulated. In uraemic patients with poor renal function there is often a bleeding tendency due to poor platelet function [3--51, and yet paradoxically these patients are prone to thromboses including cerebral and coronary thrombosis. These events may in fact be more frequent in those who are also nephrotic, for such patients have even higher serum lipids than other patients with chronic renal failure [6,7]. We therefore decided that it would be worth investigating the effect of uraemia on plasma lysolecithin levels. Materials and methods The blood urea and plasma lysolecithin were estimated in 16 patients with uraemia and in 23 control patients. Since 6 of the patients with uraemia had acute renal failure, 10 repeat estimations were made after the patients had recovered to the stage of normal renal function. The blood samples were collected in EDTA tubes and were transported in ice. For lipid extraction 0.5 ml plasma was added to 2.0 ml methanol with shaking and the mixture was then allowed to stand for five minutes with occasional agitation. Then 4.0 ml chloroform and 1 ml of 0.7% saline were added and mixed, and allowed to stand for a further five minutes. The specimens were then centrifuged at 3500 r.p.m. for five minutes so that the contents were separated into two phases with a compact disc of protein at the interface. The upper layer was discarded and the lower was decanted and evaporated to dryness under a nitrogen jet. The lipid material was redissolved in 0.25 ml chloroform
224
(2 vols) to methanol (1 vol) and 50 1-11 aliyuots were applied at 1 c’m horizontal streaks at the bottom of 25 cm x 25 cm silica gel plates, which had been preheated at 120°C for half an hour to ‘activate’ them. The solvent system LW~ for chromatography was chloroform (50 parts)/methanol (30 pa&)/acetic acid (8 parts)/water (4 parts). The plates were run to within one inch of the top, and they were then dried and were stained in a tank (*ontainii~~ iodine vapour. The spots corresponding to Iysoletithin and lecithin were removed for phosphatcl determination and a similar sized lipid free area was taken for the blank estimation. In order to determine phosphorus the silica gel spots were placed in heavy glass tubes with 0.5 ml 10 N sulphuric acid and antibumping granules, and were heated at 230°C for 30 minutes. After cooling 2 drops of hydrogen peroxide were added and they were then reheated for yet another ten minutes until the contents were white, On retooling 1 drop of 5% urea was added and the tubes were then heated at 230°C for five minutes to decompose the peroxide. An extra four tubes in each batch were similarly treated for later use for the standards. On cooling 4.4 ml of water was added to each silica gel containing tube and to two of the blank tubes. To the remaining two tubes were added 0.5 ml standard phosphate solution and 3.9 ml water. Finally 0.2 ml ammonium molybdate and 0.2 ml ANS solution (~~-anlino-3-hydroxynaphthalene-l-sulphonic acid) were added to each tube and they were heated on a boiling water bath for seven minutes. After centrifugation the absorbance of the supernatants was measured at 830 nm. The reaction was shown to give a linear relationship between phosphate concentration and optical density. The absorbance measurements were corrected for blank values and were used to calculate the amount of ‘phosphate’ in lysolecithin and lecithin of the original plasma, as mg per dl of plasma. The total phosphate of each lipid extract was also measured and the results were finally expressed as lysole~ithin phosphate/total lipid phosphate (LL/TLP). Results and discussion The important finding was that in some patients with a high blood urea the plasma lysolecithin values were low, as shown in Fig. 1 which revealed a logarithmic relationship. On the other hand plasma lecithin values were completely independent of the blood urea. The normal range for lysolecithin phosphate was 0.0255-0.0925, when expressed as a fraction of the total lipid phosphate. The results of the three patient categories (normal, uraemic and recovery groups) were therefore expressed as an ‘index’ (the log of that value times ten) which was then plotted against the level of blood urea as in the figure. Although some patients with a high blood urea had low lysolecithin values, this did not apply to all uraemic patients. However it is significant that with adequate recovery of renaf function the lysolecithin/total lipid phosphorus indices of the acute renal failure patients returned to the normal range. The patients who were uraemic but who had normal lysolecithin values were mainly chronic renal failure patients who were receiving treatment by intermittent haemodialysis. The findings are the more significant when set against the fact that plasma lecithin values were independent of the blood urea.
225
I
I
100
0
Blood
we0
203
I 300
(mg/Ul)
Fig. 1. The ‘lysolccithin index’ (the ratio of log plasma lysolecithin phosphorus to total lipid phosphorus times ten) has been plotted against blood urea for uraemic patients (triangles), acute renal failure cases after recovery (open circles) and controls (black dots).
The likely explanation is that in advanced uraemia there is a defect of the enzyme lecithin-cholesterol acyltransferase (LCAT). This enzyme normally engages in a reaction whereby LDL or HDL cholesterol is esterified at the expense of lecithin, which in turn is converted to lysolecithin. In previous reports all the profound reductions of LCAT have been found in patients with liver disease. Studies of this enzyme in uraemia now seem worth while. Acknowledgements Our thanks are due to Mr G.A. Cheyne of the MRC Growth and Reproduction unit, Princess Mary Hospital for help with the technique and to Dr A. Cassels-Smith of Newcastle General Hospital for the facilities. References 1 2 3 4 5 6 7
E.M.M. Besterman and M.P.T. Gillett, Atherosclerosis. 14 (1971) 323 E.M.M. Besterman and M.P.T. Gill&t, Nature New Biology, 241 (1973) 223 HI. Horowitz, B.D. Cohen, P. Martinez and M.F. Papogoanou. Blood, 30 (1967) 331 HI. Horowitz. I.M. Stein, B.D. Cohen and .J.G. White, Am. J. Med., 49 (1970) 336 S.F. Rabiner and F. Molinas. Am. J. Med., 49 (1970) 346 E.N. Wardle. W. Schardt and P.R. Uldall. Post-grad. Med. J., 50 (1974) 737 E.N. Wardle, IS. Menon and J. Anderson. Q. J. Med.. 41 (1971) I5
