405
Clinica Chimica Acta, 60 (1975) 405-408 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed in The Netherlands
SHORT COMMUNICATION ~_. CGA 7030
PLASMA AND RED BLOOD CELL PHOSPHOLIPIDS IN CHRONIC LIVER DISEASES
L. CANTONI,
S.B. CURRIa, P. ANDREUZZI
and P. ROCCHETTI
Ospedale Maggiore Ca’ Granda, Milano, Divisione Medica “Crespi” Centro Ambulatoriale e di Laboratorio per la Prevenzione, Diagnosi e Terapia delle Malattie Epato-Spleniche and aCentro di Biologia Molecolare Milan0 (Italy)
(Received
November
23, 1974)
Summary
The total phospholipid content and the individual phospholipids, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, and sphingomyelin were determined in the plasma and red blood cells of 58 patients with chronic liver disease and 12 control subjects by quantitative extraction and thin-layer chromatography. The total phospholipids were significantly reduced in both the plasma and red blood cells of the patients with liver disease. For the individual phospholipids, plasma sphingomyelin was significantly decreased, in the red blood cells there was an increase in lysophosphatidylcholine and a decrease in phosphatidylethanolamine and phosphatidylserine compared to the control subjects. It is suggested that in chronic liver disease the red blood cell lipid disturbances follow more closely those of the hepatocyte than those in plasma.
Introduction
Abnormalities of plasma phospholipids have been described in chronic liver disease [l--4], and it has been suggested that these changes might reflect the disruption of hepatocyte lipids. Plasma phospholipids are known to exchange with those in the red blood cell, particularly phosphatidylcholine, lysophosphatidylcholine and sphingomyelin [ 5-81 and such an exchange may be a selective process [9,10]. It is possible that erythrocyte lipids, being membrane components, might more closely reflect liver cell phospholipids than those found in plasma. However, although changes in the phospholipid pattern have been found in the red blood cells of patients with chronic liver disease
406
[ll-131 no systematic study has been made to compare them with plasma phospholipids. In this paper we report the preliminary results of such a study. Methods A total of 70 subjects was investigated. 12 were normal controls of the same age range as the 58 patients with chronic liver disease. The liver disease in 12 of these with cirrhosis was compensated, in 41 decompensated, the remainder having chronic aggressive hepatitis. Diagnosis was made by liver biopsy, laparoscopy (in some cases), liver scan and serum enzymes. Blood samples were taken, after fasting, for the phospholipid estimations and the plasma was immediately separated from the red blood cells. The quantitative extraction of plasma phospholipids was performed mainly after Parker and Peterson [14] and phospholipid phosphorous was estimated by the method of Wagner et al. [15] as modified by Gonzato [16]. The individual phospholipids were separated by thin-layer chromatography on silica gel plates (F-254 Merck, Darmstadt) using the solvent systems chloroform/methanol/water (65 : 25 : 4, by vol.) and chloroform/methanol/acetic acid/water (65 : 25 : 8 : 4, by vol; see Angelelli et al. [18] ). The RF values were compared with those of authentic reference compounds. The phosphorous contents of the individual fractions were determined as published. [ 19-211 . Results The total phospholipid levels (see Fig. 1) were significantly (P < 0.01) reduced in both the plasma and red blood cells of subjects with liver disease as compared with the normal values.
Fig. 1. Total liver disease.
phospholipids
in plasma
and red blood
cells in normal
subjects
and patients
with chronic
407
Control s”b,ect
Liver disease ,,g phospholnpld phosphorus/ml Standard ermr
Phosphat~dylcholone
Fig. 2.
Lysophosphabdyi choline
Phosphofipidfractionsin plasma
Phosphattldylethanolamlne + phosphatidylsetine
Sphmgomyelln
from normal subjects and Patients
with
chronic
liver
disease.
Plasma. Sphingomyelin was significantly decreased in chronic liver disease (see Fig. 2). There were no significant differences between the control and liver disease values for phosphatidylcholine, lysophosphatidylcholine and phosphatidylethanolamine and phosphatidyl serine. Red cell. There was a marked increase (P < 0.01) in lysophosphatidylchoiine and also a significant decrease in phosphatidylethanolamine and phosphatidylserine (see Fig. 3). There were no differences in phosphatidylcholine and sphingomyelin. Discussion The reduction
60
50
pg phopsholipid phosphorus/ml CellS
in the total
plasma and red blood
r-l
I
m I
cell phospholipid
is in
Control subject Liver dwase
Standard error
40
L
Phosphatidylcholme
Fig. 3. Phosphoapid fractions disease.
in
LysophosphatidyL Cil0li”e
P‘MS, phatidylethanolamme + ph(mphatidylserine
Sphingomyehn
red blood cells from normal subjects and Patients
with chronic liver
408
agreement with previous studies [l .3] and this could have implications in the reduced incidence of atherosclerosis found in cirrhosis. The results summarised in Figs 2 and 3 indicate clear differences in the patterns of individual phospholipids in plasma and the red blood cell. In chronic liver disease different changes take place, in plasma the level of sphingomyelin decreases while in the red blood cells lysophosphatidylcholine increases and the combined phosphatidylcholine and phosphatidylserine decrease. It is known that the phospholipid synthesis in mature erythrocytes [ 221 is very limited hence other factors must account for these differences. The plasma enzyme lecithin acyltransferase (LCAT) might be implicated particularly as it is thought to be synthesised by the liver and has been implicated in changes in red cell lipid [ 131. In patients recovering from acute hepatitis the red blood cell lipid composition paralleled the return to normal of the liver function more closely than the plasma phospholipids [ 121. This suggests that liver cell phospholipids correlate better with those in the red blood cells than with those in the plasma. It is also possible that the biosynthesis of the phospholipids is reduced in liver disease, or else the transfer of the phospholipid from plasma to the red blood cell could be altered. The latter could be caused by a direct effect of the altered plasma environment of the red blood cell as a result of the increased bile salt concentration in liver disease, as suggested by Gjone and Norum [6] . In the red blood cell altered lipid composition can give rise to structural changes, and this could also be occurring in the liver. An inability to synthesise phospholipids for regenerating hepatocytes would obviously exacerbate the chronicity of liver disease. References 1
Phillips,
2
Gjone,
G.B.
3
Wolfram,
4
Kunz,
5
Phillips,
6
Gjone.
(1960)
E. and
G. and F. and
and
E. and
7
Shohot, Rowe,
9
Sakagami,
K.R.
(1970)
T..
Minari,
P. and
Invest.
40,
J. Clin.
Reed,
0.
J. 76,
and
Ways,
11
Neerhout.
R.C.
(1968)
J. Lab.
12
Neerhout,
R.C.
(1968)
J. Pediatr.
13
Simon,
J.B.
14
Parker.
F. and
15
Wagner,
16
Gonzato,
(1971)
H.,
17
Rose,
18
AngeleIIi.
19
Mussini,
20 21 22
Marks,
C.F.
J. Lab.
Peterson.
and L.,
Oklander, G.,
M.
Faggionato,
M.,
Manzini,
E..
Curri,
Mussini,
ZiIiotto,
G.R. P.A.,
and
S.B., Curri,
GeIIhorn.
Med.
A. and
Med.
109,
360-364
153-161
71,
77,
Acta
98.
356-384
891-900
11,
J. Lipid
G. and
Res.
6, 455460 Biochem.
Z. 175.
334
334 Res.
6. 428-431
Siniscalchi,
E. and
C. and
Kidson,
Biophys. 34
438447
P. (1961)
Sper.
(1968)
10,
364-373
Wolff,
Manzini.
S.B.
Biol. 187,
Biochim.
J. Lipid
(1965)
C.,
209
1668-1678
Med.
73.
Biol.
Moretti.
Exp.
&and.
Haematol.
(1965)
Biochim.
Carina,
CIin.
L. and
18,
199
27,185
Sm.
Med.
T. (1965)
Ser.
C&n.
N.F.
HGrhammer,
P. (1963)
H.G.
(1965)
Invest. 49,
471-475
Orii,
10
Acta
Proc.
Acta
Lab.
Wochenschr.
Chim.
(1962)
(1970)
Biochem.
J. CIin.
KIin.
CIin.
N.S.
1639-1650
Sand.
N. (1971)
D. (1970)
(1960)
39,
(1966)
Roome.
Norum,
S.B. C.E.
Invest.
O.M.
ZiiIIner.
Kosin,
G.B.
8
J. CIin.
Orning.
Cwri,
Neviani,
P. (1966) S.B.
V.
(1967)
(1968)
G. Gerontol.
16,
C. (1960)
J. Biol.
I1 Farm.
Ed.
Pratt.
Haematologica
Biochim.
Biol.
Sper.
1 Chem.
235.
2579-2583
21.
52,
851
7. 61
493-507
Clinica Chimica Acta, 60 (1975) 405-408 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed in The Netherlands
SHORT COMMUNICATION ~_. CGA 7030
PLASMA AND RED BLOOD CELL PHOSPHOLIPIDS IN CHRONIC LIVER DISEASES
L. CANTONI,
S.B. CURRIa, P. ANDREUZZI
and P. ROCCHETTI
Ospedale Maggiore Ca’ Granda, Milano, Divisione Medica “Crespi” Centro Ambulatoriale e di Laboratorio per la Prevenzione, Diagnosi e Terapia delle Malattie Epato-Spleniche and aCentro di Biologia Molecolare Milan0 (Italy)
(Received
November
23, 1974)
Summary
The total phospholipid content and the individual phospholipids, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, and sphingomyelin were determined in the plasma and red blood cells of 58 patients with chronic liver disease and 12 control subjects by quantitative extraction and thin-layer chromatography. The total phospholipids were significantly reduced in both the plasma and red blood cells of the patients with liver disease. For the individual phospholipids, plasma sphingomyelin was significantly decreased, in the red blood cells there was an increase in lysophosphatidylcholine and a decrease in phosphatidylethanolamine and phosphatidylserine compared to the control subjects. It is suggested that in chronic liver disease the red blood cell lipid disturbances follow more closely those of the hepatocyte than those in plasma.
Introduction
Abnormalities of plasma phospholipids have been described in chronic liver disease [l--4], and it has been suggested that these changes might reflect the disruption of hepatocyte lipids. Plasma phospholipids are known to exchange with those in the red blood cell, particularly phosphatidylcholine, lysophosphatidylcholine and sphingomyelin [ 5-81 and such an exchange may be a selective process [9,10]. It is possible that erythrocyte lipids, being membrane components, might more closely reflect liver cell phospholipids than those found in plasma. However, although changes in the phospholipid pattern have been found in the red blood cells of patients with chronic liver disease
406
[ll-131 no systematic study has been made to compare them with plasma phospholipids. In this paper we report the preliminary results of such a study. Methods A total of 70 subjects was investigated. 12 were normal controls of the same age range as the 58 patients with chronic liver disease. The liver disease in 12 of these with cirrhosis was compensated, in 41 decompensated, the remainder having chronic aggressive hepatitis. Diagnosis was made by liver biopsy, laparoscopy (in some cases), liver scan and serum enzymes. Blood samples were taken, after fasting, for the phospholipid estimations and the plasma was immediately separated from the red blood cells. The quantitative extraction of plasma phospholipids was performed mainly after Parker and Peterson [14] and phospholipid phosphorous was estimated by the method of Wagner et al. [15] as modified by Gonzato [16]. The individual phospholipids were separated by thin-layer chromatography on silica gel plates (F-254 Merck, Darmstadt) using the solvent systems chloroform/methanol/water (65 : 25 : 4, by vol.) and chloroform/methanol/acetic acid/water (65 : 25 : 8 : 4, by vol; see Angelelli et al. [18] ). The RF values were compared with those of authentic reference compounds. The phosphorous contents of the individual fractions were determined as published. [ 19-211 . Results The total phospholipid levels (see Fig. 1) were significantly (P < 0.01) reduced in both the plasma and red blood cells of subjects with liver disease as compared with the normal values.
Fig. 1. Total liver disease.
phospholipids
in plasma
and red blood
cells in normal
subjects
and patients
with chronic
407
Control s”b,ect
Liver disease ,,g phospholnpld phosphorus/ml Standard ermr
Phosphat~dylcholone
Fig. 2.
Lysophosphabdyi choline
Phosphofipidfractionsin plasma
Phosphattldylethanolamlne + phosphatidylsetine
Sphmgomyelln
from normal subjects and Patients
with
chronic
liver
disease.
Plasma. Sphingomyelin was significantly decreased in chronic liver disease (see Fig. 2). There were no significant differences between the control and liver disease values for phosphatidylcholine, lysophosphatidylcholine and phosphatidylethanolamine and phosphatidyl serine. Red cell. There was a marked increase (P < 0.01) in lysophosphatidylchoiine and also a significant decrease in phosphatidylethanolamine and phosphatidylserine (see Fig. 3). There were no differences in phosphatidylcholine and sphingomyelin. Discussion The reduction
60
50
pg phopsholipid phosphorus/ml CellS
in the total
plasma and red blood
r-l
I
m I
cell phospholipid
is in
Control subject Liver dwase
Standard error
40
L
Phosphatidylcholme
Fig. 3. Phosphoapid fractions disease.
in
LysophosphatidyL Cil0li”e
P‘MS, phatidylethanolamme + ph(mphatidylserine
Sphingomyehn
red blood cells from normal subjects and Patients
with chronic liver
408
agreement with previous studies [l .3] and this could have implications in the reduced incidence of atherosclerosis found in cirrhosis. The results summarised in Figs 2 and 3 indicate clear differences in the patterns of individual phospholipids in plasma and the red blood cell. In chronic liver disease different changes take place, in plasma the level of sphingomyelin decreases while in the red blood cells lysophosphatidylcholine increases and the combined phosphatidylcholine and phosphatidylserine decrease. It is known that the phospholipid synthesis in mature erythrocytes [ 221 is very limited hence other factors must account for these differences. The plasma enzyme lecithin acyltransferase (LCAT) might be implicated particularly as it is thought to be synthesised by the liver and has been implicated in changes in red cell lipid [ 131. In patients recovering from acute hepatitis the red blood cell lipid composition paralleled the return to normal of the liver function more closely than the plasma phospholipids [ 121. This suggests that liver cell phospholipids correlate better with those in the red blood cells than with those in the plasma. It is also possible that the biosynthesis of the phospholipids is reduced in liver disease, or else the transfer of the phospholipid from plasma to the red blood cell could be altered. The latter could be caused by a direct effect of the altered plasma environment of the red blood cell as a result of the increased bile salt concentration in liver disease, as suggested by Gjone and Norum [6] . In the red blood cell altered lipid composition can give rise to structural changes, and this could also be occurring in the liver. An inability to synthesise phospholipids for regenerating hepatocytes would obviously exacerbate the chronicity of liver disease. References 1
Phillips,
2
Gjone,
G.B.
3
Wolfram,
4
Kunz,
5
Phillips,
6
Gjone.
(1960)
E. and
G. and F. and
and
E. and
7
Shohot, Rowe,
9
Sakagami,
K.R.
(1970)
T..
Minari,
P. and
Invest.
40,
J. Clin.
Reed,
0.
J. 76,
and
Ways,
11
Neerhout.
R.C.
(1968)
J. Lab.
12
Neerhout,
R.C.
(1968)
J. Pediatr.
13
Simon,
J.B.
14
Parker.
F. and
15
Wagner,
16
Gonzato,
(1971)
H.,
17
Rose,
18
AngeleIIi.
19
Mussini,
20 21 22
Marks,
C.F.
J. Lab.
Peterson.
and L.,
Oklander, G.,
M.
Faggionato,
M.,
Manzini,
E..
Curri,
Mussini,
ZiIiotto,
G.R. P.A.,
and
S.B., Curri,
GeIIhorn.
Med.
A. and
Med.
109,
360-364
153-161
71,
77,
Acta
98.
356-384
891-900
11,
J. Lipid
G. and
Res.
6, 455460 Biochem.
Z. 175.
334
334 Res.
6. 428-431
Siniscalchi,
E. and
C. and
Kidson,
Biophys. 34
438447
P. (1961)
Sper.
(1968)
10,
364-373
Wolff,
Manzini.
S.B.
Biol. 187,
Biochim.
J. Lipid
(1965)
C.,
209
1668-1678
Med.
73.
Biol.
Moretti.
Exp.
&and.
Haematol.
(1965)
Biochim.
Carina,
CIin.
L. and
18,
199
27,185
Sm.
Med.
T. (1965)
Ser.
C&n.
N.F.
HGrhammer,
P. (1963)
H.G.
(1965)
Invest. 49,
471-475
Orii,
10
Acta
Proc.
Acta
Lab.
Wochenschr.
Chim.
(1962)
(1970)
Biochem.
J. CIin.
KIin.
CIin.
N.S.
1639-1650
Sand.
N. (1971)
D. (1970)
(1960)
39,
(1966)
Roome.
Norum,
S.B. C.E.
Invest.
O.M.
ZiiIIner.
Kosin,
G.B.
8
J. CIin.
Orning.
Cwri,
Neviani,
P. (1966) S.B.
V.
(1967)
(1968)
G. Gerontol.
16,
C. (1960)
J. Biol.
I1 Farm.
Ed.
Pratt.
Haematologica
Biochim.
Biol.
Sper.
1 Chem.
235.
2579-2583
21.
52,
851
7. 61
493-507
