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Carbohydrate content of human VLDL, IDL, LDL and HDL plasma apoproteins from fasting normal and hyperlipemic patients
91
Clinica
Chimica
0 Elsevier
Acta,
Scientific
64 (1975)
Publishing
91-93 Company,
Amsterdam
SHORT COMMUNICATION
-Printed
in The Netherlands
_
CCA 7306
CARBOHYDRATE CONTENT OF HUMAN VLDL, IDL, LDL AND HDL PLASMA APOPROTEINS FROM FASTING NORMAL AND HYPERLIPEMIC PATIENTS
M. FONTAINE
and C.L. MALMENDIER
Laboratory (Belgium)
of Clinical
Chemistry,
(Received
May 14, 1975)
St-Pierre
University
Hospital,
Free
University
of Brussels
Few data are available in the literature on the carbohydrate content of apolipoproteins when compared to the huge number of papers dealing with their protein composition. The object of this study was to examine the carbohydrate composition of major apolipoproteins in normal subjects and in hyperlipoproteinemic patients of types III, IV and V [l], treated or untreated. Blood was collected with disodium EDTA (1 mg/ml) as anticoagulant. Plasma was separated by centrifugation at 4°C. Lipoprotein fractions were isolated as described by Lee and Alaupovic [ 21 by ultracentrifugation at 10°C in a Beckman L2-65B ultracentrifuge. Each fraction, removed by the tube-slicing technique, was dialyzed for 24 h against 0.01% EDTA and then lyophilized. Samples containing l-4 mg of protein were delipidated with 10 ml of chloroform/methanol (1 : 4, v/v) [ 31, followed by 5 ml of diethyl ether and then dried under nitrogen at 35°C. Proteins were determined by a modification of the method of Lowry et al. [4], with bovine serum albumin as standard. Delipidated lipoproteins were dissolved in water with 20 mg of cetyltrimethylammonium bromide [3]. Hexoses were determined by the method of Hewitt [ 51 using mannose/galactose (1 : 1, w/w) as standard. Hexosamines were analyzed by the method of Gatt and Berman [6], with galactosamine/glucosamine (1 : 1, w/w) as standard. Sialic acid was assayed after hydrolysis and elution through Dowex l-X8 [ 71 by the resorcinol reaction [ 81 using N-acetylneuraminic acid as standard. VLDL and IDL contain a significant amount of hexose (Table I). No data are available in the literature for these lipoproteins in humans. The carbohydrate
Abbreviations: VLDL, very low density lipoproteins (d < 1.006 g/ml); IDL, intermediate low density lipoproteins (1.006 < d < 1.019); LDL. low density lipoproteins (1.019 < d < 1.063); HDL. high density lipoproteins (1.063 < d < 1.21).
92
TABLE
1
CARBOHYDRATE Values
CONTENT
are means
S.D.
OF
(fig/mg
AI’OLlPKOTEINS
of protein)
HeX0W
for
IN NORMAL
9 normal
PLASM,\
volunteers.
Hexosamine
Sialic
13.3
acid
VLDL
57.5
i 12.5
22.1
i 3.9
IDL
85.5
123.2
23.2
i 4.7
5.7
LDL
42.9
i
6.7
20.0
t 3.0
6.3
’
3.0
HDL
16.8
+
2.8
9.9
i 1.7
5.5
’
1.1
!
3.2
r 0.7
content of LDL-apolipoprotein is compatible with the value reported by Ehnholm et al. [3]. The HDL hexose value is slightly higher than that given by Scanu [9]. The hexose content of IDL and HDL is increased by a factor of two in type V untreated hyperlipemic patients while it decreases in type III hyperlipoproteinemia. An improvement of the hyperlipemic state (reduction in the serum lipid levels) causes a normalization of the hexose content both in type V and type III patients (Table II). While hexoses are submitted to obvious variations, hexosamines only increase in HDL-apolipoprotein of type V (27.2 pg/mg of protein) and remain practically constant for the other apolipoproteins (Table I). Hexosamine content for LDL is compatible with that described by Lee and Alaupovic [lo] and Ehnholm et al. [ 31, slightly higher than the values of Shore and Shore [ 111. HDL values are similar to those found by Scanu [9]. Sialic acid contents in HDL and LDL confirm the reported values [9,11,12] (Table I). In a normal or “normalized” fasting subject, IDL are, among lipoproteins, those which contain the highest percentage of hexose (Table II). As both VLDL and LDL (derived essentially from VLDL through LDL) contain less hexose, the high hexose content of IDL may only be explained by three hypotheses: (a) an addition of carbohydrate when the IDL particle is formed, (b) a false evaluation of the amount of hexose per mg of protein due to the fact
TABLE HEXOSE Values
I1 CONTENT are means
OF
? S.D.
PLASMA (pg/mg
APOLIPOPROTEINS of protein).
Number
IN HYPERLIPEMIC of patients
analyzed
PATIENTS is given
in parentheses.
~~~
~_~ Type
Type
III
IV
Type
Untreated
Treated*
Untreated
Treated*
(1)
(1)
(2)
(4)
43.9
40.4
48.7
IDL
30.8
66.4
102.0
LDL
50.7
41.8
HDL
14.8
16.8
* Types **
Type alcohol
III and VI
IV
received restriction.
were
treated
2
*
1
2
4.5
60.1
* 37.5
61.8
67.1
58.4
41.0
* 14.3
81.0
f 32.6
180.0
182.3
74.1
65.3
39.7
?
0.7
40.9
f
2.2
61.7
85.5
57.3
35.9
15.1
i-
1.2
17.1
i
2.7
12.9
45.8
15.2
13.2
with
an hypocaloric
t
Treated*
Untreated 1
VLDL
V
oxandrolone diet
(1000
(Lonavar, Cal/day)
Searle).
and type
V2
was maintained
under
complete
93
that apoproteins B and C, which are the normal constituents of both VLDL and IDL, contain radically different amounts of hexose (preliminary results show that this is not the case), (c) less likely, a qualitative change in the hexose linked to IDL (e.g. an increase in glucose or fucose, to which the anthrone reaction is more sensitive at the expense of galactose and mannose, used as standard in our procedure). The increase in hexose content of IDL-apolipoprotein from type V patients, compared to normal, could possibly result from the presence of two distinct lipoproteins in the class of density 1.006-1.019, one of which may be increased in this pathologicalstate. Evidence for two different lipoprotein populations accumulating 6 h post prandially in this density range has been previously reported by Fellin et al. [13] . References 1 D.S. Fredrickson and RI. Levy, in J.B. Stanbury, J.B. Wyngaarden and D.S. Fredrickson feds), The Metabolic Basis of Inherited Disease, McGraw-Hill, New York, 1972, pp. 545-614 2 D.M. Lee and P. Alaupovic, Atherosclerosis. 19 (1974) 501-520 3 C. Ehnholm, H. Garoff. 0. Renkonen and K. Simons. Biochemistry. 11 (1972) 3229-3232 4 G.R. Schachterle and R.L. Pollack, Anal. Biochem., 51 (1973) 654-655 5 B.R. Hewitt, Nature, 182 (1958) 246-247 6 R. Gatt and E.R. Berman, Anal. Biochem., I5 (1966) 167-171 7 R.G. Spiro, Methods Enzymoi., 8 (1966) 3-52 8 L. Svennerholm, Biochim. Biophys. Acta, 24 (1957) 604-611 3 A. Scanu, J. Lipid Res., 7 (1966) 295-306 10 D.M. Lee and P. Alaupovic, Biochemistry, 9 (1970) 2244-2252 11 V.G. Shore and B, Shore, in Nelson, G.J. (ed.), Blood I&ids and ~poproteins: quantitation, composition and metabolism, Wiley Interscience, New York. 1972. pp. 78B--824 12 S. Margolis and R.G. Langdon, J. Biol. Chem., 241 (1966) 469476 13 R. Fellin, B. Agostini, W. Rost and D, Seidel, Clin. Chim. Acta. 54 (1974) 325-333
Clinica
Chimica
0 Elsevier
Acta,
Scientific
64 (1975)
Publishing
91-93 Company,
Amsterdam
SHORT COMMUNICATION
-Printed
in The Netherlands
_
CCA 7306
CARBOHYDRATE CONTENT OF HUMAN VLDL, IDL, LDL AND HDL PLASMA APOPROTEINS FROM FASTING NORMAL AND HYPERLIPEMIC PATIENTS
M. FONTAINE
and C.L. MALMENDIER
Laboratory (Belgium)
of Clinical
Chemistry,
(Received
May 14, 1975)
St-Pierre
University
Hospital,
Free
University
of Brussels
Few data are available in the literature on the carbohydrate content of apolipoproteins when compared to the huge number of papers dealing with their protein composition. The object of this study was to examine the carbohydrate composition of major apolipoproteins in normal subjects and in hyperlipoproteinemic patients of types III, IV and V [l], treated or untreated. Blood was collected with disodium EDTA (1 mg/ml) as anticoagulant. Plasma was separated by centrifugation at 4°C. Lipoprotein fractions were isolated as described by Lee and Alaupovic [ 21 by ultracentrifugation at 10°C in a Beckman L2-65B ultracentrifuge. Each fraction, removed by the tube-slicing technique, was dialyzed for 24 h against 0.01% EDTA and then lyophilized. Samples containing l-4 mg of protein were delipidated with 10 ml of chloroform/methanol (1 : 4, v/v) [ 31, followed by 5 ml of diethyl ether and then dried under nitrogen at 35°C. Proteins were determined by a modification of the method of Lowry et al. [4], with bovine serum albumin as standard. Delipidated lipoproteins were dissolved in water with 20 mg of cetyltrimethylammonium bromide [3]. Hexoses were determined by the method of Hewitt [ 51 using mannose/galactose (1 : 1, w/w) as standard. Hexosamines were analyzed by the method of Gatt and Berman [6], with galactosamine/glucosamine (1 : 1, w/w) as standard. Sialic acid was assayed after hydrolysis and elution through Dowex l-X8 [ 71 by the resorcinol reaction [ 81 using N-acetylneuraminic acid as standard. VLDL and IDL contain a significant amount of hexose (Table I). No data are available in the literature for these lipoproteins in humans. The carbohydrate
Abbreviations: VLDL, very low density lipoproteins (d < 1.006 g/ml); IDL, intermediate low density lipoproteins (1.006 < d < 1.019); LDL. low density lipoproteins (1.019 < d < 1.063); HDL. high density lipoproteins (1.063 < d < 1.21).
92
TABLE
1
CARBOHYDRATE Values
CONTENT
are means
S.D.
OF
(fig/mg
AI’OLlPKOTEINS
of protein)
HeX0W
for
IN NORMAL
9 normal
PLASM,\
volunteers.
Hexosamine
Sialic
13.3
acid
VLDL
57.5
i 12.5
22.1
i 3.9
IDL
85.5
123.2
23.2
i 4.7
5.7
LDL
42.9
i
6.7
20.0
t 3.0
6.3
’
3.0
HDL
16.8
+
2.8
9.9
i 1.7
5.5
’
1.1
!
3.2
r 0.7
content of LDL-apolipoprotein is compatible with the value reported by Ehnholm et al. [3]. The HDL hexose value is slightly higher than that given by Scanu [9]. The hexose content of IDL and HDL is increased by a factor of two in type V untreated hyperlipemic patients while it decreases in type III hyperlipoproteinemia. An improvement of the hyperlipemic state (reduction in the serum lipid levels) causes a normalization of the hexose content both in type V and type III patients (Table II). While hexoses are submitted to obvious variations, hexosamines only increase in HDL-apolipoprotein of type V (27.2 pg/mg of protein) and remain practically constant for the other apolipoproteins (Table I). Hexosamine content for LDL is compatible with that described by Lee and Alaupovic [lo] and Ehnholm et al. [ 31, slightly higher than the values of Shore and Shore [ 111. HDL values are similar to those found by Scanu [9]. Sialic acid contents in HDL and LDL confirm the reported values [9,11,12] (Table I). In a normal or “normalized” fasting subject, IDL are, among lipoproteins, those which contain the highest percentage of hexose (Table II). As both VLDL and LDL (derived essentially from VLDL through LDL) contain less hexose, the high hexose content of IDL may only be explained by three hypotheses: (a) an addition of carbohydrate when the IDL particle is formed, (b) a false evaluation of the amount of hexose per mg of protein due to the fact
TABLE HEXOSE Values
I1 CONTENT are means
OF
? S.D.
PLASMA (pg/mg
APOLIPOPROTEINS of protein).
Number
IN HYPERLIPEMIC of patients
analyzed
PATIENTS is given
in parentheses.
~~~
~_~ Type
Type
III
IV
Type
Untreated
Treated*
Untreated
Treated*
(1)
(1)
(2)
(4)
43.9
40.4
48.7
IDL
30.8
66.4
102.0
LDL
50.7
41.8
HDL
14.8
16.8
* Types **
Type alcohol
III and VI
IV
received restriction.
were
treated
2
*
1
2
4.5
60.1
* 37.5
61.8
67.1
58.4
41.0
* 14.3
81.0
f 32.6
180.0
182.3
74.1
65.3
39.7
?
0.7
40.9
f
2.2
61.7
85.5
57.3
35.9
15.1
i-
1.2
17.1
i
2.7
12.9
45.8
15.2
13.2
with
an hypocaloric
t
Treated*
Untreated 1
VLDL
V
oxandrolone diet
(1000
(Lonavar, Cal/day)
Searle).
and type
V2
was maintained
under
complete
93
that apoproteins B and C, which are the normal constituents of both VLDL and IDL, contain radically different amounts of hexose (preliminary results show that this is not the case), (c) less likely, a qualitative change in the hexose linked to IDL (e.g. an increase in glucose or fucose, to which the anthrone reaction is more sensitive at the expense of galactose and mannose, used as standard in our procedure). The increase in hexose content of IDL-apolipoprotein from type V patients, compared to normal, could possibly result from the presence of two distinct lipoproteins in the class of density 1.006-1.019, one of which may be increased in this pathologicalstate. Evidence for two different lipoprotein populations accumulating 6 h post prandially in this density range has been previously reported by Fellin et al. [13] . References 1 D.S. Fredrickson and RI. Levy, in J.B. Stanbury, J.B. Wyngaarden and D.S. Fredrickson feds), The Metabolic Basis of Inherited Disease, McGraw-Hill, New York, 1972, pp. 545-614 2 D.M. Lee and P. Alaupovic, Atherosclerosis. 19 (1974) 501-520 3 C. Ehnholm, H. Garoff. 0. Renkonen and K. Simons. Biochemistry. 11 (1972) 3229-3232 4 G.R. Schachterle and R.L. Pollack, Anal. Biochem., 51 (1973) 654-655 5 B.R. Hewitt, Nature, 182 (1958) 246-247 6 R. Gatt and E.R. Berman, Anal. Biochem., I5 (1966) 167-171 7 R.G. Spiro, Methods Enzymoi., 8 (1966) 3-52 8 L. Svennerholm, Biochim. Biophys. Acta, 24 (1957) 604-611 3 A. Scanu, J. Lipid Res., 7 (1966) 295-306 10 D.M. Lee and P. Alaupovic, Biochemistry, 9 (1970) 2244-2252 11 V.G. Shore and B, Shore, in Nelson, G.J. (ed.), Blood I&ids and ~poproteins: quantitation, composition and metabolism, Wiley Interscience, New York. 1972. pp. 78B--824 12 S. Margolis and R.G. Langdon, J. Biol. Chem., 241 (1966) 469476 13 R. Fellin, B. Agostini, W. Rost and D, Seidel, Clin. Chim. Acta. 54 (1974) 325-333