311
Clinica Chimica Acta, 78 (1977) 311-314 0 Elsevier/North-Holland Biomedical Press
CCA 8646
SERUM LECITHIN-CHOLESTEROL ACYLTRANSFERASE ACTIVITY CHILDREN WITH FAMILIAL HYPERBETALIPOPROTEINAEMIA
D.C.K. ROBERTS *, JOAN M. ROUND AUDREY S. FOSBROOKE Department
(Received
of Child Health,
February
Institute
**, JUNE K. LLOYD
of Child Health,
IN
*** and
Guildford
Street,
London
(U.K.)
3rd, 1977)
Summary Serum (non-fasting) was obtained from ‘71 healthy school children (12-14 years) and from 16 children with the heterozygous form of familial hyperbetalipoproteinaemia being treated by diet or ion-exchange resin. The activity of lecithin-cholesterol acyltransferase (EC 2.3.1.43) expressed as nmol cholesterol esterified/hour/ml serum did not change with increasing concentrations of unesterified cholesterol in the healthy children. In children with familial hyperbetalipoproteinaemia, lecithin-cholesterol acyltransferase activity was higher than in the healthy children and this activity increased with increasing concentrations of unesterified cholesterol.
Introduction Primary hyperlipoproteinaemias are characterized by increased concentration of one or more of the major lipoprotein classes or by the presence of an abnormal lipoprotein [ 11. One of the roles ascribed to the serum enzyme, lecithin-cholesterol acyltransferase (LCAT) is the maintenance of lipoprotein structure [2]. This enzyme catalyses the formation of cholesteryl esters by the transfer of fatty acids from the 2 position of phosphatidyl choline (lecithin) to the 3-hydroxyl of cholesterol. The activity of LCAT, that is the rate of esterification of cholesterol, has been reported to be increased in adult patients with familial hyperbetalipoproteinaemia (FH) [3]. The present study reports the LCAT activity observed in children with familial hyperbetalipoproteinaemia in comparison with that found in a group of healthy school children. * To
whom correspondence should be addressed at the following address: Department of Biochemistry, University of Western Ontario, London, Ontario, Canada. NfiA 5Cl. ** Present address: Department of Human Metabolism, University College Hospital. London. U.K. *** Present address: Department of Child Health, St. George’s Hospital Medical School, Blackshaw Road, London, S.W. 17, U.K.
312
Materials and methods Patients and collection
of samples
Venous blood (non-fasting) was collected by one of us (J.M.R.) between 10 and 11 a.m. from a total of 71 healthy children, 34 boys and 37 girls, aged 12-14 years who attended a London school. Blood was already being obtained for an unrelated study for which both the children and their parents had given full consent and the project had been approved by the Hospital (University College Hospital) Ethical Committee. The blood was stored and transported in an icebox at 4°C until the serum was separated by centrifugation (600 X g for 10 min) always by 3 p.m. of the same day. For all children, LCAT activity was assayed on the same day as the blood was collected. Sixteen children with 11 boys and 5 girls aged 5-15 years, familial hyperbetalipoproteinaemia, attending the Hospital for Sick Children, Great Ormond Street, and being treated with diet or ion exchange resin (J.K.L.), had blood taken between 9.30 a.m. and 12 noon during the course of a routine clinic visit. Assay of lecithin-cholesterol
acyltransferase
Serum LCAT was assayed by the method of Soloff et al. [4]. The endogenous lipoproteins were radioactively labeled with [ 4-14C] cholesterol (Radiochemical Centre Ltd., Amersham, Bucks., U.K.) during which time the enzyme was inhibited by the addition of 5,5-dithio-bis-2-nitrobenzoic acid (British Drug Houses Ltd., Poole, Dorset, U.K.). At the start of the assay, the inhibition was reversed by the addition of 2-mercaptoethanol (B.D.H.). After the addition of 2-mercaptoethanol, samples (100 ~1) were taken at 0, 10, 20 and 40 minutes after which time the rate of reaction was no longer linear. The lipids from the serum samples were extracted with chloroform/methanol (2 : 1, v/v) overnight, filtered and the extract dried. The lipid-containing residue was then dissolved in petroleum ether (60-80” C) and applied to a thin layer (500 pm) of silica gel G and chromatographed to separate unesterified cholesterol from cholesteryl ester in a solvent system consisting of petroleum ether (60~-8O”C)/diethyl ether/glacial acetic acid (70 : 30 : 2, by vol.). The lipid bands were visualised under ultra violet light with an aqueous spray of Rhodamine 6G. The silica gel containing the individual lipids was scraped into scintillation vials, scintillant added (10.0 ml; 0.5% PPO, 0.025% POPOP (w/v) in toluene) and the radioactivity counted in a Phillips scintillation counter. The appropriate quench correction curves were constructed and the sample counts corrected for any quenching. Assay of serum
lipids
Lipids were extracted from serum (0.2 ml) and chromatographed as already described to separate unesterified cholesterol and cholesteryl ester. The silica gel containing the individual components was scraped into screw-capped glass vials (5 ml) and isopropanol (2.0 ml) was added. Elution of the cholesterol and cholesteryl esters from the gel was aided by placing the vials with the caps screwed on tightly in an incubator at 37°C for 1 h. After centrifugation (600 X g for 10 min) to precipitate the gel, the cholesterol content of the supernatant was assayed using a FeC13 autoanalyser method [ 51.
313 TABLE I SERUM CHOLESTEROL CHILDREN
CONCENTRATION
WITH FAMILIAL
AND LCAT ACTIVITY
BOYS
Cholesterol concentration (rnrnOl/l, Mean +_S.D.) (a) Total (b) Free rc) Ester LCAT activity (nmol unesterified cholesterol esterified/h/ml serum) Mean r S.D.
CHILDREN
AND
Girls
Healthy (34)
~~.
IN HEALTHY
HYPERBETALIPOPROTEINAEMIA
Patients (11)
Healthy (37)
._~.
4.1 f 1.1 + 2.7 f
0.62 0.23 0.46
7.6 f 1.5 1.8 * 0.49 5.3 +_ 0.85
31.6 ? 10
4.6 + 1.2 + 3.2 f.
45.3 f 16
0.85 0.23 0.83
30.3 + 13
Patients (5)
6.2 f 1.8 k 4.3 k
0.95 0.28 0.83
42.8 f 12
Results and discussion The mean serum cholesterol concentration was higher in the healthy girls than in the healthy boys and the difference was statistically significant for both unesterified (P < 0.05) and esterified (P < 0.01) fractions (Table I). No differ-
. l
Healthy Chit&en r= 0.22 PBO.05
.
Childra wilh FH r=O.70 PO.01
A A
6 A
I
0.5
1.0
A
A
5%
0
1
2.0 2.5 UNEsroRlFlED cxusr& WNcENTnATlw(mmd/l) Fig. 1. Regression of the rate of esterification of cholesterol by the enzyme lecithin-cholesterol acyltransferase on the unesterified cholesterol concentration in serum of healthy children, and children with familial hyperbetalipoproteinaemia (FH).
314
ence in serum cholesterol concentration was found between the boys and girls with FH. The LCAT activity did not differ significantly between the sexes in either group of children but was higher in the children with FH than in the healthy children (P < 0.05, Table I). Fabien et al. [6] in a study of adults with FH found the LCAT activity to be significantly increased. However, the magnitude of the rate of esterification for the adults is twice that reported here for the children with FH. Since they comment that their method (gas-liquid chromatography) gives higher LCAT activity (plus 74%) than a radioassay procedure, it seems possible that the differences in absolute levels of activity between their study of adults and our study of children are due to the use of different methods. The relationship between the quantity of cholesterol esterified (LCAT activity) and the concentration of unesterified cholesterol is shown in Fig. 1. Whereas there is no significant correlation between LCAT activity and unesterified cholesterol concentrations in the healthy children, in the children with FH there is a significant positive correlation (r = 0.70, P < 0.01). Thus, in the children with FH, LCAT activity increases with increasing unesterified cholesterol concentration and occurs at a higher rate than in the healthy children. This may be due to increased enzyme quantity or increased enzyme saturation. Acknowledgements D.R. gratefully acknowledges receipt of a Wellcome Research Fellowship and wishes to thank Professor O.H. Wolff of the Department of Child Health for the provision of the facilities to undertake this research. References 1
Fredrickson,
D.S.
Wyngaarden,
J.B.
Levy,
R.I.
Fredrickson,
and
Adams,
in The
D.S.
eds.).
edn.,
Basis
Rutenberg,
H.L.
and
Lacko,
A.G.
(1972)
Circulation
4
Soloff.
L.A..
Rutenberg,
H.L.
and
Lacko.
A.G.
(1973)
Am.
G.A.
(1970)
Clin.
Chim.
AS.
and
Pringle,
Davignon,
J. and
Marcel,
Y.L.
(1973)
Biochem.
Acta Can.
of
Inherited
545-614,
L.A.,
H.D.,
Rev.
pp.
Soloff,
Fosbrooke,
Annu.
Metabolic
Schumaker,
Fabien.
(1969)
3rd
2
5
G.H.
(1972)
3
6
V.N.
and and
Disease Hill,
38,113-136 Suppl.
Heart 30,
McGraw
J. 85,
II, 45, 153-161
47-52
J. Biochem.
51,
550-555
224
(Stanbury,
New
York
J.B..
Clinica Chimica Acta, 78 (1977) 311-314 0 Elsevier/North-Holland Biomedical Press
CCA 8646
SERUM LECITHIN-CHOLESTEROL ACYLTRANSFERASE ACTIVITY CHILDREN WITH FAMILIAL HYPERBETALIPOPROTEINAEMIA
D.C.K. ROBERTS *, JOAN M. ROUND AUDREY S. FOSBROOKE Department
(Received
of Child Health,
February
Institute
**, JUNE K. LLOYD
of Child Health,
IN
*** and
Guildford
Street,
London
(U.K.)
3rd, 1977)
Summary Serum (non-fasting) was obtained from ‘71 healthy school children (12-14 years) and from 16 children with the heterozygous form of familial hyperbetalipoproteinaemia being treated by diet or ion-exchange resin. The activity of lecithin-cholesterol acyltransferase (EC 2.3.1.43) expressed as nmol cholesterol esterified/hour/ml serum did not change with increasing concentrations of unesterified cholesterol in the healthy children. In children with familial hyperbetalipoproteinaemia, lecithin-cholesterol acyltransferase activity was higher than in the healthy children and this activity increased with increasing concentrations of unesterified cholesterol.
Introduction Primary hyperlipoproteinaemias are characterized by increased concentration of one or more of the major lipoprotein classes or by the presence of an abnormal lipoprotein [ 11. One of the roles ascribed to the serum enzyme, lecithin-cholesterol acyltransferase (LCAT) is the maintenance of lipoprotein structure [2]. This enzyme catalyses the formation of cholesteryl esters by the transfer of fatty acids from the 2 position of phosphatidyl choline (lecithin) to the 3-hydroxyl of cholesterol. The activity of LCAT, that is the rate of esterification of cholesterol, has been reported to be increased in adult patients with familial hyperbetalipoproteinaemia (FH) [3]. The present study reports the LCAT activity observed in children with familial hyperbetalipoproteinaemia in comparison with that found in a group of healthy school children. * To
whom correspondence should be addressed at the following address: Department of Biochemistry, University of Western Ontario, London, Ontario, Canada. NfiA 5Cl. ** Present address: Department of Human Metabolism, University College Hospital. London. U.K. *** Present address: Department of Child Health, St. George’s Hospital Medical School, Blackshaw Road, London, S.W. 17, U.K.
312
Materials and methods Patients and collection
of samples
Venous blood (non-fasting) was collected by one of us (J.M.R.) between 10 and 11 a.m. from a total of 71 healthy children, 34 boys and 37 girls, aged 12-14 years who attended a London school. Blood was already being obtained for an unrelated study for which both the children and their parents had given full consent and the project had been approved by the Hospital (University College Hospital) Ethical Committee. The blood was stored and transported in an icebox at 4°C until the serum was separated by centrifugation (600 X g for 10 min) always by 3 p.m. of the same day. For all children, LCAT activity was assayed on the same day as the blood was collected. Sixteen children with 11 boys and 5 girls aged 5-15 years, familial hyperbetalipoproteinaemia, attending the Hospital for Sick Children, Great Ormond Street, and being treated with diet or ion exchange resin (J.K.L.), had blood taken between 9.30 a.m. and 12 noon during the course of a routine clinic visit. Assay of lecithin-cholesterol
acyltransferase
Serum LCAT was assayed by the method of Soloff et al. [4]. The endogenous lipoproteins were radioactively labeled with [ 4-14C] cholesterol (Radiochemical Centre Ltd., Amersham, Bucks., U.K.) during which time the enzyme was inhibited by the addition of 5,5-dithio-bis-2-nitrobenzoic acid (British Drug Houses Ltd., Poole, Dorset, U.K.). At the start of the assay, the inhibition was reversed by the addition of 2-mercaptoethanol (B.D.H.). After the addition of 2-mercaptoethanol, samples (100 ~1) were taken at 0, 10, 20 and 40 minutes after which time the rate of reaction was no longer linear. The lipids from the serum samples were extracted with chloroform/methanol (2 : 1, v/v) overnight, filtered and the extract dried. The lipid-containing residue was then dissolved in petroleum ether (60-80” C) and applied to a thin layer (500 pm) of silica gel G and chromatographed to separate unesterified cholesterol from cholesteryl ester in a solvent system consisting of petroleum ether (60~-8O”C)/diethyl ether/glacial acetic acid (70 : 30 : 2, by vol.). The lipid bands were visualised under ultra violet light with an aqueous spray of Rhodamine 6G. The silica gel containing the individual lipids was scraped into scintillation vials, scintillant added (10.0 ml; 0.5% PPO, 0.025% POPOP (w/v) in toluene) and the radioactivity counted in a Phillips scintillation counter. The appropriate quench correction curves were constructed and the sample counts corrected for any quenching. Assay of serum
lipids
Lipids were extracted from serum (0.2 ml) and chromatographed as already described to separate unesterified cholesterol and cholesteryl ester. The silica gel containing the individual components was scraped into screw-capped glass vials (5 ml) and isopropanol (2.0 ml) was added. Elution of the cholesterol and cholesteryl esters from the gel was aided by placing the vials with the caps screwed on tightly in an incubator at 37°C for 1 h. After centrifugation (600 X g for 10 min) to precipitate the gel, the cholesterol content of the supernatant was assayed using a FeC13 autoanalyser method [ 51.
313 TABLE I SERUM CHOLESTEROL CHILDREN
CONCENTRATION
WITH FAMILIAL
AND LCAT ACTIVITY
BOYS
Cholesterol concentration (rnrnOl/l, Mean +_S.D.) (a) Total (b) Free rc) Ester LCAT activity (nmol unesterified cholesterol esterified/h/ml serum) Mean r S.D.
CHILDREN
AND
Girls
Healthy (34)
~~.
IN HEALTHY
HYPERBETALIPOPROTEINAEMIA
Patients (11)
Healthy (37)
._~.
4.1 f 1.1 + 2.7 f
0.62 0.23 0.46
7.6 f 1.5 1.8 * 0.49 5.3 +_ 0.85
31.6 ? 10
4.6 + 1.2 + 3.2 f.
45.3 f 16
0.85 0.23 0.83
30.3 + 13
Patients (5)
6.2 f 1.8 k 4.3 k
0.95 0.28 0.83
42.8 f 12
Results and discussion The mean serum cholesterol concentration was higher in the healthy girls than in the healthy boys and the difference was statistically significant for both unesterified (P < 0.05) and esterified (P < 0.01) fractions (Table I). No differ-
. l
Healthy Chit&en r= 0.22 PBO.05
.
Childra wilh FH r=O.70 PO.01
A A
6 A
I
0.5
1.0
A
A
5%
0
1
2.0 2.5 UNEsroRlFlED cxusr& WNcENTnATlw(mmd/l) Fig. 1. Regression of the rate of esterification of cholesterol by the enzyme lecithin-cholesterol acyltransferase on the unesterified cholesterol concentration in serum of healthy children, and children with familial hyperbetalipoproteinaemia (FH).
314
ence in serum cholesterol concentration was found between the boys and girls with FH. The LCAT activity did not differ significantly between the sexes in either group of children but was higher in the children with FH than in the healthy children (P < 0.05, Table I). Fabien et al. [6] in a study of adults with FH found the LCAT activity to be significantly increased. However, the magnitude of the rate of esterification for the adults is twice that reported here for the children with FH. Since they comment that their method (gas-liquid chromatography) gives higher LCAT activity (plus 74%) than a radioassay procedure, it seems possible that the differences in absolute levels of activity between their study of adults and our study of children are due to the use of different methods. The relationship between the quantity of cholesterol esterified (LCAT activity) and the concentration of unesterified cholesterol is shown in Fig. 1. Whereas there is no significant correlation between LCAT activity and unesterified cholesterol concentrations in the healthy children, in the children with FH there is a significant positive correlation (r = 0.70, P < 0.01). Thus, in the children with FH, LCAT activity increases with increasing unesterified cholesterol concentration and occurs at a higher rate than in the healthy children. This may be due to increased enzyme quantity or increased enzyme saturation. Acknowledgements D.R. gratefully acknowledges receipt of a Wellcome Research Fellowship and wishes to thank Professor O.H. Wolff of the Department of Child Health for the provision of the facilities to undertake this research. References 1
Fredrickson,
D.S.
Wyngaarden,
J.B.
Levy,
R.I.
Fredrickson,
and
Adams,
in The
D.S.
eds.).
edn.,
Basis
Rutenberg,
H.L.
and
Lacko,
A.G.
(1972)
Circulation
4
Soloff.
L.A..
Rutenberg,
H.L.
and
Lacko.
A.G.
(1973)
Am.
G.A.
(1970)
Clin.
Chim.
AS.
and
Pringle,
Davignon,
J. and
Marcel,
Y.L.
(1973)
Biochem.
Acta Can.
of
Inherited
545-614,
L.A.,
H.D.,
Rev.
pp.
Soloff,
Fosbrooke,
Annu.
Metabolic
Schumaker,
Fabien.
(1969)
3rd
2
5
G.H.
(1972)
3
6
V.N.
and and
Disease Hill,
38,113-136 Suppl.
Heart 30,
McGraw
J. 85,
II, 45, 153-161
47-52
J. Biochem.
51,
550-555
224
(Stanbury,
New
York
J.B..
