- Email: [email protected]
Changes in the concentration of lipoprotein-X during incubation of postheparin plasma from patients with familial lecithin:Cholesterol acyltransferase (LCAT) deficiency
63
Ctinica Ch~rn~caActa, 67 (1976) 63-69 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CCA 7556
CHANGES IN THE CONCENTRATION OF LIPOPROTEIN-X DURING INCUBATION OF POSTHEPARIN PLASMA FROM PATIENTS WITH FAMILIAL L~CITHIN:CHOLESTEROL ACYLTRANSFERASE (LCAT) DEFICIENCY STALE RITLAND,
KJELL TORGEIR STOKKE and EGIL GJONE
Medical Department A and Institute of Clinical Biochemistry, The National Hospital of Norway, Rikshospitalet, Oslo 1 (Norway) (Received September 10, 1975)
Summary The concentration of lipoprotein-X (LP-X) has been studied in six patients with familial 1ecithin:cholesterol acyltransferase (LCAT) deficiency before and after the administ~tion of heparin. In preheparin plasma, LP-X was present in all the patients, the concentrations ranging from 40 to 251 mg/lOO ml. When postheparin plasma samples were incubated at 37°C for 4 hours, LP-X could not be detected, whereas it was still present before incubation. LCAT activity was absent in the preheparin as well as in the postheparin samples. Accordingly, the changes in the concentration of LP-X could not be attributed to alterations in plasma LCAT activity. An increase in lysolecithin and a decrease in lecithin was observed during incubation of postheparin plasma samples. It is suggested that the changes in the concentration of LP-X during incubation of postheparin samples are due to phospholipase activity released by heparin. Patients with familial LCAT deficiency thus seem to have normal postheparin phospholipolytic activity.
Introduction Familial 1ecithin:choIesterol acyltransferase (LCAT) deficiency is an inborn error of metabolism with complete lack of plasma LCAT activity leading to a variety of abnormalities in the composition and metabolism of plasma lipoproteins [l].The presence of the abnormal lipoprotein lipoprotein-X (LP-X) has been demonstrated previously in patients with this disorder [ 21; the concentrations ranging from 40 mg/lOO ml to 251 mgflO0 ml ]3]. Intravenous administration of heparin leads to release into the plasma of lipoprotein lipases either of peripheral or hepatic origin [4,5,6] .
Normal activity of both peripheral and hepatic triglyceride lipases has been demonstrated in the postheparin plasma from patients with this disease 171. The present investigation reports changes in the concentration of LP-X after injection of heparin in patients with familial LCAT deficiency. Material and methods Patients Seven patients with familial LCAT deficiency were studied. Five of them were in good general health without any special treatment, whereas one was being treated with immunosuppressive agents (Prednison and Azathioprine) after uncomplicated renal transplantation. In these six patients the plasma concentration of LP-X was studied after an overnight fast before and at different intervals after intravenous injection of heparin (50 I.U./kg body weight). In a seventh patient, changes in the composition of phospholipids were studied while the patient was on haemodialysis due to renal failure. Prior to haemodialysis this patient received 3000 I.U. of heparin as injection and during the haemodialysis 1200 I.U./h were administered as a continuous infusion. At different intervals samples were drawn and the phospholipids were examined both before and after incubation at 37°C for five hours. Blood sampling Blood was immediately cooled on ice, and separation took place at 4°C. Plasma (K,-EDTA, 2 mg/ml) was kept at -15°C for later determination of lipids and LCAT activity. The concentration of LP-X was measured in unfrozen aliquots the same day. Lipoprotein-X The presence of LP-X was demonstrated by precipitation with polyanion, using the method of Seidel and coworkers [8] with slight modifications as earlier described [9]. The concentration of LP-X was measured by the method of Ritland [lo]. By this method endogenous free cholesterol of the plasma lipoproteins was equilibrated with exogenous labelled cholesterol during an incubation at 37°C for four hours. After separation of LP-X from the other lipoproteins by electrophoresis in agar, the percentage of the label found in the LP-X fraction was measured by liquid scintillation. Based on this percentage, the plasma concentration of free cholesterol and the known composition of LP-X, the concentration of LP-X was calculated. Isolation 0 f lipopro teias Low density lipoproteins (LDL) (density 1.019-1.063) were isolated in one patient from preheparin as well as plasma taken 10 minutes after the injection of heparin. After incubation of the plasma samples at 3’7°C for four hours, sequential ultracentrifugation was performed at 5°C for 22 hours in a preparative ultracentrifuge and recentrifuged once under identical conditions. Densities were adjusted with solid KBr.
65
Other laboratory tests LCAT activity
was estimated according to the assay of Stokke and Norum cholesterol were measured by gas-liquid chromatography [12] and phospholipids (total and fractionated) as described by Gjone and Orning [13], triglycerides by the method of Laurel1 [14], and proteins by the method of Lowry et al. [ 151. [ 111. Free and total
Results
Lipoprotein-X LP-X was present in the preheparin sample in all patients in quantities ranging from 40 mg/lOO ml to 251 mg/lOO ml (Table I). When plasma obtained 10 minutes after the injection of heparin was incubated at 37°C for 4 hours, LP-X could not be detected by the qualitative method in any of the patients (Fig. 1, A and B). By the quantitative method only traces were found in three of the patients. After incubation of samples drawn 3, 30 and 60 minutes after the heparin injection in three of the patients, LP-X was not found. When samples taken 90 minutes (one patient) and two hours (two patients) after the injection of heparin had been incubated at 37°C for four hours, the concentration of LP-X had increased, reaching approximately the preheparin level. When electrophoresis was performed immediately after the samples had been drawn 10 minutes after the injection of heparin (i.e. without incubation) LP-X was found to be present. Quantitative determinations in these unincubated samples could not be undertaken because the method for quantitation of LP-X involves incubation. Incubation of the postheparin samples at 4°C demonstrates a slower fall in LP-X. After incubation for 90 minutes only small changes were observed. After 24 hours, however, no LP-X could be detected (Fig. 2). The effect of heparin on plasma during incubation was also studied. To preheparin plasma (250 ~1) was added 50 ~1 of heparin, either 50 I.U./ml or 5000 U.I./ml, and to controls 50 ~1 of physiological saline. The samples were incubated at 37°C for 4 hours. On electrophoresis in agar no change in the cathodal mi-
TABLE LP-X
I (m~/lOOml)
AFTER
INCUBATION
Patients
Minutes
0
OF after
PRE-
AND
injection
POSTHEPARIN
PLASMA
of heparin
10
30
60
90
120
-
AA
20.01.74
49
3
6
_*
76
LG
19.02.74
72
0
0
4
-
7.03.74
251
0
-
-
-
IS
85
KA
29.04.74
71
9
-
-
-
MRM
19.11.74
128
0
-
0
-
111
ML
15.04.74
40
3
-
-
-
-
* -denotes:
no sample
taken.
-
1
2 3 Fig. 1. LP-X in pre- and postheparin samples before and after incubation. Elertrophoresis in 1% agar. 1. preheparin plasma; 2. plasma obtained 10 minutes after injection of heparin; 3. plasma taken 30 minutes after injection of heparin. A, electrophoresis before incubation. B. electrophorcsis after incubation at 37°C for 4 hours. Cathode to thr left.
g-ration of LP-X was found. The concentrations of LP-X in the samples added with heparin were the same as in those added with saline. LCA T activity In two patients the plasma LCAT activity was studied in the preheparin as well as in the postheparin samples. No activity was discovered before or after the injection of heparin.
LP-X LP-x mg/lCKlml
IN POSTHEPARIN
PLASMA
A A Fam LCAT Def lncubatlon at 4°C
60_
...‘..
::........ ...’ ”
Incub. for 90mln lncub for 24 h
,:’ ..
0 30 90 min Fig. 2. Changes in the concentration of LP-X during incubation at 4’C. LP-X (mg/lOO ml) in preheparin plasma and plasma obtained 10, 30, fi0, and 90 minutes after the injection of heparin after incubation at 4°C for 90 minutes and 24 hours, respectively.
67 TABLE II THE COMPOSITION AND POSTHEPARIN
.~_
OF LDL (DENSITY 1.019-1.063) AFTER INCUBATION OF PREHEPARIN PLASMA FROM A PATIENT WITH FAMILIAL LCAT DEFICIENCY (1.S.)
---.
--.__-_-__I
Before heparin
10 minutes after heparin --.--____
Total cholesterol
%
20
23 22
Free cholesterol
o/o
20
Triglycerides
%
28
27
Phospholipids
%
35
32
Protein
%
LP-X
mgjlOOm1
16
18
300
30
” -
LDL (density 1.019-1.063) Before separation by ultracentrifugation preheparin plasma as well as plasma taken 10 minutes after the injection of heparin were incubated at 37°C for 4 hours. In the isolated LDL fraction from preheparin plasma the concentration of LP-X was 300 mg/lOO ml, whereas in that of the postheparin plasma the level of LP-X was 30 mg/lOO ml. The concentration of total and free cholesterol, triglycerides, total phospholipids and proteins of the LDL fractions of preheparin and postheparin plasma were equal (Table II).
Prior to haemodi~ysis and hep~inization in the patient A.R. the plasma concentration of phospholipids was 3.3 mmoles 1-‘, the relative composition being: lecithin 86%, lysolecithin 1.2%, phosphatidyl ethanolamine 3.4% phosPhospholipids in postheparin -----Changes duringmcubation Fom
plasma.
LCAT de‘
b
C
30 min
120 min
d 6 days
Fig. 3. Changes (9’0) in the composition of phospholipids during incubation at 37’C for 5 hours. a, before heparin; b. 30 min; c, 120 min and d, 6 days after heparinization. The patient (A.R.) was given 3000 I.U. of heparln as a rapid intravenous injection followed by a continuous infusion of 1200 I.U. h-’ for 4 hours.
phatidyl serine 1.4% and sphingomyelin 8.3%.. During incubation at 3’7’C for 5 hours no change in the total phospholipids took place in any sample. The relative composition of the phospholipids taken before or 6 days after haemodialysis and heparinization was also unchanged. In samples taken 30 and 60 minutes after heparinization, however, alterations were observed during incubation. The relative amount of Iysolecithin increased, whereas that of lecithin and phosphatidyf ethanolamine decreased (Fig. 3). Discussion The present investigation has demonstrated reduction in the concentration of LP-X after incubation of postheparin plasma from patients with familial LCAT deficiency. Addition of heparin to preheparin plasma did not affect the level of LP-X. It therefore seems likely that the injection of heparin releases a factor acting on LP-X. The in vivo changes in the concentration of LP-X seem to be small. Most of the decrease in the level of LP-X occurs during incubation. Ras et al. [ 161 have reported disappearance of LP-X after injection of heparin in patients with cholestatic liver disorders. Incubation of the postheparin plasma did not seem to be necessary as there was no specific information about this. Moreover, no data on the interval from the blood sampling until the LP-X test was performed were reported. They found similar changes in the composition of the phospholipids as we did. In a previous report from our laboratory, changes in the concentration of LP-X were found after incubation of postheparin plasma in most of the patients with cholestatic liver disorders [ 171. Also in patients with liver disorders, incubation was necessary to make LP-X disappear. The presence of LP-X in plasma seems to be related to either absolute or reIative LCAT deficiency [ 181, and LP-X is consumed when cholesterol esterification takes place ]3]. Ras and coworkers [ f6] suggest that the clearance of LPX might be caused by a LCAT-like effect of heparin. Our observations do not support this hypothesis as no plasma LCAT activity was induced in our patients. In addition an agent blocking any LCAT activity which might be present, was added during incubation when the decrease in LP-X took place. We think it is more reasonable to believe that the change in LP-X is caused by lipolytic activity reIeased by heparin. LP-X is rich in phospholipids [ 191, The reduction in ZIP-X might accordingly be due to phospholipolytic activity released by heparin. During incubation of posthep~in plasma an increase in lysolecithin and a decrease in lecithin were observed. These findings are compatible with this hypothesis. According to our observations the effect of heparin lasts for about two hours. This also favours the view that lipolytic activity is responsible for the degradation of LP-X as other studies have reported that the duration of the postheparin lipolytic activity is about two hours. In patients with liver disorders the changes in the concentration of LP-X during incubation of posthepa~n plasma seem in some way or other to be related to the function of the liver [17 J , This makes it tempting to suggest that the alterations in LP-X during incubation of postheparin plasma are caused by a hepatic phospholipase. Patients with familial LCAT deficiency have normal liver function [l] as
69
judged by the usual liver function tests. Recently, Greten et al. [7] reported normal postheparin hepatic and peripheral triglyceride lipase activity in plasma from patients with this disorder. As the clearance of LP-X in postheparin plasma probably is caused by a phospholipase, patients with familial LCAT deficiency seem to have normal postheparin activity of hepatic phospholipases. This is in accordance with the view that the lipase of hepatic origin also has phospholipolytic activity [6]. Acknowledgements Excellent technical assistance has been given us by Mrs. Hilde Letnes, Mrs. Ingrid Wiencke and Mr. Eystein Jensen. This investigation has been supported by grants from Tom Wilhelmsens Foundation and the Norwegian Research Council for Science and the Humanities. References 1 Gjone, E. and Norum, K. (1968) Acta Med. Stand. 283, 107 2 Torsvik, H.. Berg, K., Magnani, H.N., McConathy, W.J., Alaupovic, P. and Gjone, E. (1972) FEBS Lett. 24,165 3 Ritland, S. and Gjone. E. (1975) CIin. Chim. Acta 59, 109 4 Greten, H., Sniderman, A.D., Chandler. J.G., Steinberg, D. and Brown, W.V. (1974) FEBS Lett. 42, 157 5 Krauss, R.M., Levy, R.I. and Fredrickson, D.S. (1974) J. CIin. Invest. 54.1107 6 Ehnhohn. C.. Shaw, W., Greten. H.. Langfelder. W. and Brown. W.V. (1974) in Atherosclerosis III (Schettler, G. and Weizel, A., eds.). p. 557. Springer-Verlag, Berlin 7 Greten. H., DeGreIIa. R.. Klose, G.. Rascher. W.. de Gennes, J.-L. and Gjone, E. (1975). in press 8 Seidel. D.. Wieland, H. and Ruppert, C. (1973) CIin. Chem. 19, 737 9 Ritland, S. (1974) Stand. J. Gastroenterol. 9. 507 10 Ritland, S. (1974) CIin. Chim. Acta 55. 359 11 Stokke, K.T. and Norum. K. (1971) Stand. J. CIin. Lab. Invest. 27. 21 12 Blomhoff, J.P. (1973) CIin. Chim. Acta 43. 257 13 Gjone. E. and Oming, O.M. (1966) Stand. J. Clin. Lab. Invest. 18, 209 14 LaureII, S. (1966) Stand. J. Clin. Lab. Invest. 18,668 15 Lowry, O.H.. Rosebrough, N.J.. Farr. A. and Randall, R.J. (1953) J. Biol. Chem. 193, 265 16 Ras, M.R., Frison, J.6.. Rubies-Prat. J., Masdeu. S. and Bacardi, R. (1975) CIin. Chim. Acta 61. 91 17 Ritland. S. (1975) Eur. J. CIin. Invest. 5, Abstr. 120 18 Ritland. S.. Blomhoff. J.P. and Gjone, E. (1973) CIin. Chim. Acta 49, 251 19 Seidel, D.. Alaupovic. P. and Furman. R.H. (1970) J. CIin. Invest. 19.1211
Ctinica Ch~rn~caActa, 67 (1976) 63-69 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CCA 7556
CHANGES IN THE CONCENTRATION OF LIPOPROTEIN-X DURING INCUBATION OF POSTHEPARIN PLASMA FROM PATIENTS WITH FAMILIAL L~CITHIN:CHOLESTEROL ACYLTRANSFERASE (LCAT) DEFICIENCY STALE RITLAND,
KJELL TORGEIR STOKKE and EGIL GJONE
Medical Department A and Institute of Clinical Biochemistry, The National Hospital of Norway, Rikshospitalet, Oslo 1 (Norway) (Received September 10, 1975)
Summary The concentration of lipoprotein-X (LP-X) has been studied in six patients with familial 1ecithin:cholesterol acyltransferase (LCAT) deficiency before and after the administ~tion of heparin. In preheparin plasma, LP-X was present in all the patients, the concentrations ranging from 40 to 251 mg/lOO ml. When postheparin plasma samples were incubated at 37°C for 4 hours, LP-X could not be detected, whereas it was still present before incubation. LCAT activity was absent in the preheparin as well as in the postheparin samples. Accordingly, the changes in the concentration of LP-X could not be attributed to alterations in plasma LCAT activity. An increase in lysolecithin and a decrease in lecithin was observed during incubation of postheparin plasma samples. It is suggested that the changes in the concentration of LP-X during incubation of postheparin samples are due to phospholipase activity released by heparin. Patients with familial LCAT deficiency thus seem to have normal postheparin phospholipolytic activity.
Introduction Familial 1ecithin:choIesterol acyltransferase (LCAT) deficiency is an inborn error of metabolism with complete lack of plasma LCAT activity leading to a variety of abnormalities in the composition and metabolism of plasma lipoproteins [l].The presence of the abnormal lipoprotein lipoprotein-X (LP-X) has been demonstrated previously in patients with this disorder [ 21; the concentrations ranging from 40 mg/lOO ml to 251 mgflO0 ml ]3]. Intravenous administration of heparin leads to release into the plasma of lipoprotein lipases either of peripheral or hepatic origin [4,5,6] .
Normal activity of both peripheral and hepatic triglyceride lipases has been demonstrated in the postheparin plasma from patients with this disease 171. The present investigation reports changes in the concentration of LP-X after injection of heparin in patients with familial LCAT deficiency. Material and methods Patients Seven patients with familial LCAT deficiency were studied. Five of them were in good general health without any special treatment, whereas one was being treated with immunosuppressive agents (Prednison and Azathioprine) after uncomplicated renal transplantation. In these six patients the plasma concentration of LP-X was studied after an overnight fast before and at different intervals after intravenous injection of heparin (50 I.U./kg body weight). In a seventh patient, changes in the composition of phospholipids were studied while the patient was on haemodialysis due to renal failure. Prior to haemodialysis this patient received 3000 I.U. of heparin as injection and during the haemodialysis 1200 I.U./h were administered as a continuous infusion. At different intervals samples were drawn and the phospholipids were examined both before and after incubation at 37°C for five hours. Blood sampling Blood was immediately cooled on ice, and separation took place at 4°C. Plasma (K,-EDTA, 2 mg/ml) was kept at -15°C for later determination of lipids and LCAT activity. The concentration of LP-X was measured in unfrozen aliquots the same day. Lipoprotein-X The presence of LP-X was demonstrated by precipitation with polyanion, using the method of Seidel and coworkers [8] with slight modifications as earlier described [9]. The concentration of LP-X was measured by the method of Ritland [lo]. By this method endogenous free cholesterol of the plasma lipoproteins was equilibrated with exogenous labelled cholesterol during an incubation at 37°C for four hours. After separation of LP-X from the other lipoproteins by electrophoresis in agar, the percentage of the label found in the LP-X fraction was measured by liquid scintillation. Based on this percentage, the plasma concentration of free cholesterol and the known composition of LP-X, the concentration of LP-X was calculated. Isolation 0 f lipopro teias Low density lipoproteins (LDL) (density 1.019-1.063) were isolated in one patient from preheparin as well as plasma taken 10 minutes after the injection of heparin. After incubation of the plasma samples at 3’7°C for four hours, sequential ultracentrifugation was performed at 5°C for 22 hours in a preparative ultracentrifuge and recentrifuged once under identical conditions. Densities were adjusted with solid KBr.
65
Other laboratory tests LCAT activity
was estimated according to the assay of Stokke and Norum cholesterol were measured by gas-liquid chromatography [12] and phospholipids (total and fractionated) as described by Gjone and Orning [13], triglycerides by the method of Laurel1 [14], and proteins by the method of Lowry et al. [ 151. [ 111. Free and total
Results
Lipoprotein-X LP-X was present in the preheparin sample in all patients in quantities ranging from 40 mg/lOO ml to 251 mg/lOO ml (Table I). When plasma obtained 10 minutes after the injection of heparin was incubated at 37°C for 4 hours, LP-X could not be detected by the qualitative method in any of the patients (Fig. 1, A and B). By the quantitative method only traces were found in three of the patients. After incubation of samples drawn 3, 30 and 60 minutes after the heparin injection in three of the patients, LP-X was not found. When samples taken 90 minutes (one patient) and two hours (two patients) after the injection of heparin had been incubated at 37°C for four hours, the concentration of LP-X had increased, reaching approximately the preheparin level. When electrophoresis was performed immediately after the samples had been drawn 10 minutes after the injection of heparin (i.e. without incubation) LP-X was found to be present. Quantitative determinations in these unincubated samples could not be undertaken because the method for quantitation of LP-X involves incubation. Incubation of the postheparin samples at 4°C demonstrates a slower fall in LP-X. After incubation for 90 minutes only small changes were observed. After 24 hours, however, no LP-X could be detected (Fig. 2). The effect of heparin on plasma during incubation was also studied. To preheparin plasma (250 ~1) was added 50 ~1 of heparin, either 50 I.U./ml or 5000 U.I./ml, and to controls 50 ~1 of physiological saline. The samples were incubated at 37°C for 4 hours. On electrophoresis in agar no change in the cathodal mi-
TABLE LP-X
I (m~/lOOml)
AFTER
INCUBATION
Patients
Minutes
0
OF after
PRE-
AND
injection
POSTHEPARIN
PLASMA
of heparin
10
30
60
90
120
-
AA
20.01.74
49
3
6
_*
76
LG
19.02.74
72
0
0
4
-
7.03.74
251
0
-
-
-
IS
85
KA
29.04.74
71
9
-
-
-
MRM
19.11.74
128
0
-
0
-
111
ML
15.04.74
40
3
-
-
-
-
* -denotes:
no sample
taken.
-
1
2 3 Fig. 1. LP-X in pre- and postheparin samples before and after incubation. Elertrophoresis in 1% agar. 1. preheparin plasma; 2. plasma obtained 10 minutes after injection of heparin; 3. plasma taken 30 minutes after injection of heparin. A, electrophoresis before incubation. B. electrophorcsis after incubation at 37°C for 4 hours. Cathode to thr left.
g-ration of LP-X was found. The concentrations of LP-X in the samples added with heparin were the same as in those added with saline. LCA T activity In two patients the plasma LCAT activity was studied in the preheparin as well as in the postheparin samples. No activity was discovered before or after the injection of heparin.
LP-X LP-x mg/lCKlml
IN POSTHEPARIN
PLASMA
A A Fam LCAT Def lncubatlon at 4°C
60_
...‘..
::........ ...’ ”
Incub. for 90mln lncub for 24 h
,:’ ..
0 30 90 min Fig. 2. Changes in the concentration of LP-X during incubation at 4’C. LP-X (mg/lOO ml) in preheparin plasma and plasma obtained 10, 30, fi0, and 90 minutes after the injection of heparin after incubation at 4°C for 90 minutes and 24 hours, respectively.
67 TABLE II THE COMPOSITION AND POSTHEPARIN
.~_
OF LDL (DENSITY 1.019-1.063) AFTER INCUBATION OF PREHEPARIN PLASMA FROM A PATIENT WITH FAMILIAL LCAT DEFICIENCY (1.S.)
---.
--.__-_-__I
Before heparin
10 minutes after heparin --.--____
Total cholesterol
%
20
23 22
Free cholesterol
o/o
20
Triglycerides
%
28
27
Phospholipids
%
35
32
Protein
%
LP-X
mgjlOOm1
16
18
300
30
” -
LDL (density 1.019-1.063) Before separation by ultracentrifugation preheparin plasma as well as plasma taken 10 minutes after the injection of heparin were incubated at 37°C for 4 hours. In the isolated LDL fraction from preheparin plasma the concentration of LP-X was 300 mg/lOO ml, whereas in that of the postheparin plasma the level of LP-X was 30 mg/lOO ml. The concentration of total and free cholesterol, triglycerides, total phospholipids and proteins of the LDL fractions of preheparin and postheparin plasma were equal (Table II).
Prior to haemodi~ysis and hep~inization in the patient A.R. the plasma concentration of phospholipids was 3.3 mmoles 1-‘, the relative composition being: lecithin 86%, lysolecithin 1.2%, phosphatidyl ethanolamine 3.4% phosPhospholipids in postheparin -----Changes duringmcubation Fom
plasma.
LCAT de‘
b
C
30 min
120 min
d 6 days
Fig. 3. Changes (9’0) in the composition of phospholipids during incubation at 37’C for 5 hours. a, before heparin; b. 30 min; c, 120 min and d, 6 days after heparinization. The patient (A.R.) was given 3000 I.U. of heparln as a rapid intravenous injection followed by a continuous infusion of 1200 I.U. h-’ for 4 hours.
phatidyl serine 1.4% and sphingomyelin 8.3%.. During incubation at 3’7’C for 5 hours no change in the total phospholipids took place in any sample. The relative composition of the phospholipids taken before or 6 days after haemodialysis and heparinization was also unchanged. In samples taken 30 and 60 minutes after heparinization, however, alterations were observed during incubation. The relative amount of Iysolecithin increased, whereas that of lecithin and phosphatidyf ethanolamine decreased (Fig. 3). Discussion The present investigation has demonstrated reduction in the concentration of LP-X after incubation of postheparin plasma from patients with familial LCAT deficiency. Addition of heparin to preheparin plasma did not affect the level of LP-X. It therefore seems likely that the injection of heparin releases a factor acting on LP-X. The in vivo changes in the concentration of LP-X seem to be small. Most of the decrease in the level of LP-X occurs during incubation. Ras et al. [ 161 have reported disappearance of LP-X after injection of heparin in patients with cholestatic liver disorders. Incubation of the postheparin plasma did not seem to be necessary as there was no specific information about this. Moreover, no data on the interval from the blood sampling until the LP-X test was performed were reported. They found similar changes in the composition of the phospholipids as we did. In a previous report from our laboratory, changes in the concentration of LP-X were found after incubation of postheparin plasma in most of the patients with cholestatic liver disorders [ 171. Also in patients with liver disorders, incubation was necessary to make LP-X disappear. The presence of LP-X in plasma seems to be related to either absolute or reIative LCAT deficiency [ 181, and LP-X is consumed when cholesterol esterification takes place ]3]. Ras and coworkers [ f6] suggest that the clearance of LPX might be caused by a LCAT-like effect of heparin. Our observations do not support this hypothesis as no plasma LCAT activity was induced in our patients. In addition an agent blocking any LCAT activity which might be present, was added during incubation when the decrease in LP-X took place. We think it is more reasonable to believe that the change in LP-X is caused by lipolytic activity reIeased by heparin. LP-X is rich in phospholipids [ 191, The reduction in ZIP-X might accordingly be due to phospholipolytic activity released by heparin. During incubation of posthep~in plasma an increase in lysolecithin and a decrease in lecithin were observed. These findings are compatible with this hypothesis. According to our observations the effect of heparin lasts for about two hours. This also favours the view that lipolytic activity is responsible for the degradation of LP-X as other studies have reported that the duration of the postheparin lipolytic activity is about two hours. In patients with liver disorders the changes in the concentration of LP-X during incubation of posthepa~n plasma seem in some way or other to be related to the function of the liver [17 J , This makes it tempting to suggest that the alterations in LP-X during incubation of postheparin plasma are caused by a hepatic phospholipase. Patients with familial LCAT deficiency have normal liver function [l] as
69
judged by the usual liver function tests. Recently, Greten et al. [7] reported normal postheparin hepatic and peripheral triglyceride lipase activity in plasma from patients with this disorder. As the clearance of LP-X in postheparin plasma probably is caused by a phospholipase, patients with familial LCAT deficiency seem to have normal postheparin activity of hepatic phospholipases. This is in accordance with the view that the lipase of hepatic origin also has phospholipolytic activity [6]. Acknowledgements Excellent technical assistance has been given us by Mrs. Hilde Letnes, Mrs. Ingrid Wiencke and Mr. Eystein Jensen. This investigation has been supported by grants from Tom Wilhelmsens Foundation and the Norwegian Research Council for Science and the Humanities. References 1 Gjone, E. and Norum, K. (1968) Acta Med. Stand. 283, 107 2 Torsvik, H.. Berg, K., Magnani, H.N., McConathy, W.J., Alaupovic, P. and Gjone, E. (1972) FEBS Lett. 24,165 3 Ritland, S. and Gjone. E. (1975) CIin. Chim. Acta 59, 109 4 Greten, H., Sniderman, A.D., Chandler. J.G., Steinberg, D. and Brown, W.V. (1974) FEBS Lett. 42, 157 5 Krauss, R.M., Levy, R.I. and Fredrickson, D.S. (1974) J. CIin. Invest. 54.1107 6 Ehnhohn. C.. Shaw, W., Greten. H.. Langfelder. W. and Brown. W.V. (1974) in Atherosclerosis III (Schettler, G. and Weizel, A., eds.). p. 557. Springer-Verlag, Berlin 7 Greten. H., DeGreIIa. R.. Klose, G.. Rascher. W.. de Gennes, J.-L. and Gjone, E. (1975). in press 8 Seidel. D.. Wieland, H. and Ruppert, C. (1973) CIin. Chem. 19, 737 9 Ritland, S. (1974) Stand. J. Gastroenterol. 9. 507 10 Ritland, S. (1974) CIin. Chim. Acta 55. 359 11 Stokke, K.T. and Norum. K. (1971) Stand. J. CIin. Lab. Invest. 27. 21 12 Blomhoff, J.P. (1973) CIin. Chim. Acta 43. 257 13 Gjone. E. and Oming, O.M. (1966) Stand. J. Clin. Lab. Invest. 18, 209 14 LaureII, S. (1966) Stand. J. Clin. Lab. Invest. 18,668 15 Lowry, O.H.. Rosebrough, N.J.. Farr. A. and Randall, R.J. (1953) J. Biol. Chem. 193, 265 16 Ras, M.R., Frison, J.6.. Rubies-Prat. J., Masdeu. S. and Bacardi, R. (1975) CIin. Chim. Acta 61. 91 17 Ritland. S. (1975) Eur. J. CIin. Invest. 5, Abstr. 120 18 Ritland. S.. Blomhoff. J.P. and Gjone, E. (1973) CIin. Chim. Acta 49, 251 19 Seidel, D.. Alaupovic. P. and Furman. R.H. (1970) J. CIin. Invest. 19.1211