91
Clinica Chimica Acta, 62 (1975) 97-101 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CCA 7097
PLASMA APOLIPOPROTEIN
A LEVELS
IN HEALTHY
HUMAN ADULTS
ELIZABETH FARISHa, J. SHEPHERDb, T.D.V. LAWRIEa and H.G. MORGANb technical assistance from SANDRA WIGHTMAN ‘University Department of Medical Cardiology, Royal Infirmary, Glasgow and bUniversity Department of Pathological Biochemistry, Royal Infirmary,
Glasgow (U.K.)
(Received December 24, 1974)
Summary The apolipoprotein A concentration in the plasma of ten healthy human subjects was measured immunologically on three occasions at monthly intervals and the following results recorded. 1. There was no difference between fasting and non-fasting apoprotein A concentrations. 2. Apolipoprotein A levels were higher in females than in males (p < 0.001). 3. The correlation coefficient calculated for high density lipoprotein cholesterol and apolipoprotein A was 0.62. 4. The apoprotein values varied widely between subjects and there were considerable concentration differences from month to month in the plasma of each individual.
Introduction Many studies have been made of the lipoproteins of normal and diseased human plasma. Most of this work has involved the measurement of total lipoproteins or their lipid moiety despite the fact that the apolipoproteins have important physiological functions (e.g. Apolipoprotein AI activates the enzyme 1ecithin:cholesterol acyltransferase [l] ; Apolipoprotein CII is required to activate lipoprotein lipase [ 2-4]), and are considered by many authorities to be the key components involved in lipoprotein metabolism. The lipoproteins are normally classified according to their density or electrophoretic mobility, the three major classes being as follows: high density lipoproteins (HDL) or o1 lipoproteins, low density lipoproteins (LDL) or /3 lipoproteins, and, very low density lipoproteins (VLDL) or cr2 lipoproteins. Since each of these classes is heterogeneous with respect to the protein moiety,
98
Alaupovic et al. [ 53, suggested that a better system of classification would be based on the apoproteins present. He indicated that the lipoproteins could be divided into three groups, thus: LP-A group, characterised by the A apoproteins; LP-B group, by apoprotein B; LP-C group, by the C apoproteins. Since the apoproteins are immunologically distinct, their concentrations in whole plasma may be readily determined by immunoelectrophoresis into specific antisera using the “rocket” technique described by Laurel1 [6]. The technique has been used in this study to measure the concentration of A apoproteins in healthy young adult subjects not receiving drug or oral contraceptive therapy. Methods Blood was taken from five male and five female healthy young adults on three occasions at monthly intervals. On each occasion, two samples were obtained, the first after an overnight fast and the second one hour after eating. The blood was collected in 0.1% lithium sequestrene and analysis and fractionation of the plasma samples were begun on the day of collection. fractionation of lipoproteins Each plasma sample was separated into three fractions (d < 1.006 g/ml, d = 1.006--1.063 g/ml, d > 1.063 g/ml) by sequential ultracentrifugation at 114 000 X g in the 40.3 rotor of the Beckman L2-65B ultracentrifuge. The procedure followed was essentially the same as that described by Lees and Hatch [7]. Analysis Cholesterol concentrations were measured in total plasmas and in the fractions obtained by ultracentrifugation [ 81. Apolipoprotein A concentrations were determined by the Laurel1 “rocket” technique [6]. Specific antiserum to human al lipoprotein produced in rabbits, was supplied by Hoechst Pharmaceuticals. This antise~m had been previously characterised by Lee and Alaupovic [9], and found to contain antibodies to A apoproteins only, In this study immunoelectrophoresis showed that the antiserum (Batch No. 2348) did not cross-react with VLDL, LDL, albumin, fibrinogen or y-globulin. Crossed immunoelectrophoresis demonstrated monospecificity of the antiserum against our HDL3 standard and so quantitative measurement of A apoproteins could be obtained. Duplicate electrophoresis was performed for sixteen hours at 2.5 V/cm on approp~ately diluted plasma using a 1% agarose gel in 0.075 M barbitone buffer (pH 8.6), containing 2.5% anti ffl lipoprotein antibody. The plates were washed, dried, stained with amido black (0.1% in methanolic acetic acid) and resulting peaks measured. On each plate four HDL standards (prepared in the laborato~) were run alongside the samples being assayed. A graph of g protein/litre versus peak height was plotted and the eoncentrations of A apoproteins in the samples under investigation were obtained
WI * Preparation of standard The standard chosen
in this study was an HDL3 fraction
(d = 1.10-1.21
99
g/ml),
since it contains only small amounts (less than 5%) of C apoproteins [ll] , is completely free from apoprotein B [2], and may be prepared very simply in the laboratory, rendering it suitable for use in routine estimation. Centrifuge tubes were filled 2/3 full with plasma whose density had been adjusted to 1.1 g/ml using solid KBr, and overlayered with a solution of KBr in 0.01 M Tris buffer (pH 7.4, d = 1.1 g/ml) containing 0.05% disodium EDTA. After centrifugation for 22 hours at 5°C and 114 000 X g the tubes were sliced and the top l/3 discarded. This process was repeated twice. The density was then increased to 1.21 g/ml by a further addition of KBr and the plasma over-layered with buffer at 1.21 g/ml density. The crude HDL3 which collected at the top of the tube after a 22 hour spin was removed by tube slicing and washed three times by re-centrifugation at d = 1.21 g/ml. The resultant purified HDLJ was dialysed exhaustively against 0.15 M NaCl containing 0.05% disodium EDTA. The standard was tested for purity by two dimensional immunoelectrophoresis [12] into 3% rabbit anti human serum (Hoechst Pharmaceuticals). The protein concentration was estimated by the method of Lowry et al. [13], and dilutions were made to obtain a set of four suitable standards. Solutions were stored at 4°C in the presence of sodium azide (1 mg/ml). TABLE
Subject
F.B.
J.McA.
F.D.
A.F.
J.S.
S.W.
A.T.
J.C.
L.B.
M.McD.
I
S‘ZX
M
M
M
M
M
F
F
F
F
F
Month
A apoproteins
HDL
(g/l)
(mmol/l)
cholesterol
Whole
plasma
cholesterol
(mmol/l)
Fasting
Non-fasting
Fasting
Non-fasting
Fasting
Non-fasting
1
2.05
2.14
1.34
1.44
5.68
5.50
2
2.15
2.20
1.42
1.36
5.19
5.19
3
2.19
2.48
1.34
1.16
5.16
5.16
1
1.70
1.90
1.13
1.44
5.37
5.16
2
1.70
1.70
1.52
1.44
5.42
5.29
3
2.36
2.36
1.42
1.34
5.42
5.29
1
2.30
1.80
1.73
1.88
4.26
4.03
2
2.53
2.77
1.88
2.22
4.39
4.90
3
2.75
2.59
1.83
1.83
4.26
4.39
1
1.20
1.00
1.44
1.42
5.24
4.65
2
1.87
1.87
1.60
1.65
5.32
4.90
3
2.15
1.71
1.57
1.36
5.29
4.85
1
1.93
1.85
1.57
1.49
5.34
5.42
2
1.65
1.65
1.36
1.39
4.75
4.85
3
1.98
2.20
1.36
1.34
5.16
5.16
1
2.35
2.34
1.83
1.65
5.94
5.76
2
2.39
2.45
1.80
1.73
5.32
4.98
3
2.09
2.31
1.65
1.73
5.81
5.89
1
2.50
2.70
1.62
1.60
4.88
5.06
2
2.31
2.37
1.52
1.52
4.00
4.49
3
2.46
2.56
1.65
1.42
4.78
4.54
1
2.34
2.46
1.91
1.86
5.09
5.16
2
2.32
2.46
1.93
2.01
4.59
4.52
3
2.73
2.86
2.37
2.42
5.29
5.29
1
2.46
2.46
1.57
1.52
5.03
5.16
2
2.64
2.62
1.65
1.44
5.27
4.90
3
2.75
2.64
1.57
1.65
4.90
4.90
1
2.40
2.30
2.09
2.06
5.14
5.09
2
2.70
2.56
1.70
1.80
4.70
4.57
3
2.53
2.48
1.60
1.80
4.39
4.65
100
Results The values obtained for whole plasma cholesterol, HDL cholesterol and A apolipopro~ins, are shown in Table I. Statistical analyses of these results revealed no significant difference between the apoprotein or HDL cholesterol values in the fasting and non-fasting state. However, a significant difference Cp < 0.001) was found to exist between male and female values of the apoproteins and HDL cholesterol. The values in females were consistently higher than in males. The fasting apoprotein values showed a within-patient variation of up to 54% from month to month. The HDL cholesterol/A apoprotein ratios varied considerably from subject to subject, the correlation coefficient for the whole group being 0.62 (the correlation coefficient calculated for whole plasma cholesterol/A apoproteins was 0.32). On the first monthly collection, the A apoprotein concentrations were measured in the HDL fractions (obtained by ultracentrifugation) as well as in whole plasma. All the values obtained using HDL were less than those in the whole plasma, being on average 20% lower. Since cholesterol concentrations in total plasma always agreed with the summation of the concentrations in the individual fractions to within 10% the difference in apoprotein measurements could not be accounted for by loss of material during fractionation. Laurel1 rocket electrophoresis demonstrated that the protein deficit in the HDL fraction did not result from its transference into LDL or VLDL. It therefore appeared that the protein moiety of the HDL was being degraded to some extent during ultracentrifugation and that the values obtained using total plasma were in fact the true values. Discussion There is considerable discrepancy in literature values for human plasma A apolipoproteins. Using ultracentrifugal isolation followed by protein estimation, Have1 et al. [14] obtained a value of 1.7 g/l protein for the d 1.063-1.21 g/ml fraction of normal plasma. This is lower than our estimation (2.62 it 0.41 g/l for men; 2.48 & 0.17 g/l for women). The discrepancy could be accounted for by loss of some HDL into the d > 1.21 g/ml fraction during ultracentrifugation [ 151, or by loss of material during the necessary “washing” procedure required to obtain an albumin-free HDL sample by centrifugation. Jones and Ways [16] measured HDL protein concentrations in seven normal individuals by the technique of Have1 et al. [14] and also obtained lower values than ours. It should be remembered that the values found using our standard will be 5% higher than the true values of apolipoprotein A concentration, However, correction for the presence of C apoproteins in both sets of data will increase the disparity between them since total HDL apoprotein contains lo-15% C proteins. When our results are compared with values for HDL protein calculated from measurements of total lipoprotein determined by Donner ultracentrifugation [17], they are again high, but are in agreement with those obtained by Cohn ~actionation [lS]. Finally, a radioimmuno~say has recently been described for the measurement of apolipoprotein AI [ 191. The values given for healthy men (1.00 + 0.35 g/l) and women (1.04 f 0.35 g/l) which the authors
101
estimate as representing munological estimations
51% of the total apo HDL agree well with our imof A apolipoproteins.
References 1 C.J. Fielding, Biochem. Biophys. Res. Commun.. 46 (19’72) 1493 2 R.J. Havel, V.G. Shore, B. Shore and D.M. Bier, Circ. Res.. 27 (1970) 595 3 J.C. La Rosa, R.I. Levy, P. Herbert, S.E. Lux and D.S. Fredrickson, Biochem. Biophys. Res. Commun., 41(1970) 57 4 R.M. Krauss, P.N. Herbert, R.I. Levy and D.S. Fredrickson, Circ. Res., 33 (1973) 403 5 P. Alaupovic, D.M. Lee and W.J. McConathy. Biochim. Biophys. Acta, 260 (1972) 689 6 C.B. Laurel& Anal. Biochem., 15 (1966) 45 7 R.S. Lees and F.T. Hatch, Adv. Lipid Res., 6 (1968) 33 8 W. Annan and D.M. Isherwood, J. Med. Lab. Technol.. 26 (1969) 202 9 D.M. Lee and P. Alaupovic. Biochemistry, 9 (1970) 2244 10 B. Weeke in N.H. Ax&on, J. KrolI and B. Weeke (eds). Quantitative Immunoelectrophoresis. Universitet Sforlaget, Oslo, 1973, p. 37 11 A.M. Scanu and C. Wisdom, Ann. Rev. Biochem., 41 (1972) 709 12 M.H.G. Clarke and T. Freeman, Clin. Sci., 35 (1968) 403 13 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem.. 193 (1951) 265 14 R.J. Havel, H.A. Eder and J.H. Bragdon, J. Clin. Invest., 34 (1955) 1345 15 R.I. Levy and D.S. Fredrickson, J. Clin. Invest., 44 (1965) 426 16 J.W. Jones and P. Ways. J. Clin. Invest.. 46 (1967) 1151 17 D.S. Fredrickson, R.I. Levy and F.T. Lindgren, J. Clin. Invest., 47 (1968) 2446 18 W.F. Lever, F.R.N. Gurd, E. Uroma, R.K. Brown. B.A. Barnes, K. Schmidt and E.L. Schultz, J. Chin. Invest., 30 (1951) 99 19 G. Schonfeld and B. Pfleger. J. Clin. Invest., 54 (1974) 236
Clinica Chimica Acta, 62 (1975) 97-101 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CCA 7097
PLASMA APOLIPOPROTEIN
A LEVELS
IN HEALTHY
HUMAN ADULTS
ELIZABETH FARISHa, J. SHEPHERDb, T.D.V. LAWRIEa and H.G. MORGANb technical assistance from SANDRA WIGHTMAN ‘University Department of Medical Cardiology, Royal Infirmary, Glasgow and bUniversity Department of Pathological Biochemistry, Royal Infirmary,
Glasgow (U.K.)
(Received December 24, 1974)
Summary The apolipoprotein A concentration in the plasma of ten healthy human subjects was measured immunologically on three occasions at monthly intervals and the following results recorded. 1. There was no difference between fasting and non-fasting apoprotein A concentrations. 2. Apolipoprotein A levels were higher in females than in males (p < 0.001). 3. The correlation coefficient calculated for high density lipoprotein cholesterol and apolipoprotein A was 0.62. 4. The apoprotein values varied widely between subjects and there were considerable concentration differences from month to month in the plasma of each individual.
Introduction Many studies have been made of the lipoproteins of normal and diseased human plasma. Most of this work has involved the measurement of total lipoproteins or their lipid moiety despite the fact that the apolipoproteins have important physiological functions (e.g. Apolipoprotein AI activates the enzyme 1ecithin:cholesterol acyltransferase [l] ; Apolipoprotein CII is required to activate lipoprotein lipase [ 2-4]), and are considered by many authorities to be the key components involved in lipoprotein metabolism. The lipoproteins are normally classified according to their density or electrophoretic mobility, the three major classes being as follows: high density lipoproteins (HDL) or o1 lipoproteins, low density lipoproteins (LDL) or /3 lipoproteins, and, very low density lipoproteins (VLDL) or cr2 lipoproteins. Since each of these classes is heterogeneous with respect to the protein moiety,
98
Alaupovic et al. [ 53, suggested that a better system of classification would be based on the apoproteins present. He indicated that the lipoproteins could be divided into three groups, thus: LP-A group, characterised by the A apoproteins; LP-B group, by apoprotein B; LP-C group, by the C apoproteins. Since the apoproteins are immunologically distinct, their concentrations in whole plasma may be readily determined by immunoelectrophoresis into specific antisera using the “rocket” technique described by Laurel1 [6]. The technique has been used in this study to measure the concentration of A apoproteins in healthy young adult subjects not receiving drug or oral contraceptive therapy. Methods Blood was taken from five male and five female healthy young adults on three occasions at monthly intervals. On each occasion, two samples were obtained, the first after an overnight fast and the second one hour after eating. The blood was collected in 0.1% lithium sequestrene and analysis and fractionation of the plasma samples were begun on the day of collection. fractionation of lipoproteins Each plasma sample was separated into three fractions (d < 1.006 g/ml, d = 1.006--1.063 g/ml, d > 1.063 g/ml) by sequential ultracentrifugation at 114 000 X g in the 40.3 rotor of the Beckman L2-65B ultracentrifuge. The procedure followed was essentially the same as that described by Lees and Hatch [7]. Analysis Cholesterol concentrations were measured in total plasmas and in the fractions obtained by ultracentrifugation [ 81. Apolipoprotein A concentrations were determined by the Laurel1 “rocket” technique [6]. Specific antiserum to human al lipoprotein produced in rabbits, was supplied by Hoechst Pharmaceuticals. This antise~m had been previously characterised by Lee and Alaupovic [9], and found to contain antibodies to A apoproteins only, In this study immunoelectrophoresis showed that the antiserum (Batch No. 2348) did not cross-react with VLDL, LDL, albumin, fibrinogen or y-globulin. Crossed immunoelectrophoresis demonstrated monospecificity of the antiserum against our HDL3 standard and so quantitative measurement of A apoproteins could be obtained. Duplicate electrophoresis was performed for sixteen hours at 2.5 V/cm on approp~ately diluted plasma using a 1% agarose gel in 0.075 M barbitone buffer (pH 8.6), containing 2.5% anti ffl lipoprotein antibody. The plates were washed, dried, stained with amido black (0.1% in methanolic acetic acid) and resulting peaks measured. On each plate four HDL standards (prepared in the laborato~) were run alongside the samples being assayed. A graph of g protein/litre versus peak height was plotted and the eoncentrations of A apoproteins in the samples under investigation were obtained
WI * Preparation of standard The standard chosen
in this study was an HDL3 fraction
(d = 1.10-1.21
99
g/ml),
since it contains only small amounts (less than 5%) of C apoproteins [ll] , is completely free from apoprotein B [2], and may be prepared very simply in the laboratory, rendering it suitable for use in routine estimation. Centrifuge tubes were filled 2/3 full with plasma whose density had been adjusted to 1.1 g/ml using solid KBr, and overlayered with a solution of KBr in 0.01 M Tris buffer (pH 7.4, d = 1.1 g/ml) containing 0.05% disodium EDTA. After centrifugation for 22 hours at 5°C and 114 000 X g the tubes were sliced and the top l/3 discarded. This process was repeated twice. The density was then increased to 1.21 g/ml by a further addition of KBr and the plasma over-layered with buffer at 1.21 g/ml density. The crude HDL3 which collected at the top of the tube after a 22 hour spin was removed by tube slicing and washed three times by re-centrifugation at d = 1.21 g/ml. The resultant purified HDLJ was dialysed exhaustively against 0.15 M NaCl containing 0.05% disodium EDTA. The standard was tested for purity by two dimensional immunoelectrophoresis [12] into 3% rabbit anti human serum (Hoechst Pharmaceuticals). The protein concentration was estimated by the method of Lowry et al. [13], and dilutions were made to obtain a set of four suitable standards. Solutions were stored at 4°C in the presence of sodium azide (1 mg/ml). TABLE
Subject
F.B.
J.McA.
F.D.
A.F.
J.S.
S.W.
A.T.
J.C.
L.B.
M.McD.
I
S‘ZX
M
M
M
M
M
F
F
F
F
F
Month
A apoproteins
HDL
(g/l)
(mmol/l)
cholesterol
Whole
plasma
cholesterol
(mmol/l)
Fasting
Non-fasting
Fasting
Non-fasting
Fasting
Non-fasting
1
2.05
2.14
1.34
1.44
5.68
5.50
2
2.15
2.20
1.42
1.36
5.19
5.19
3
2.19
2.48
1.34
1.16
5.16
5.16
1
1.70
1.90
1.13
1.44
5.37
5.16
2
1.70
1.70
1.52
1.44
5.42
5.29
3
2.36
2.36
1.42
1.34
5.42
5.29
1
2.30
1.80
1.73
1.88
4.26
4.03
2
2.53
2.77
1.88
2.22
4.39
4.90
3
2.75
2.59
1.83
1.83
4.26
4.39
1
1.20
1.00
1.44
1.42
5.24
4.65
2
1.87
1.87
1.60
1.65
5.32
4.90
3
2.15
1.71
1.57
1.36
5.29
4.85
1
1.93
1.85
1.57
1.49
5.34
5.42
2
1.65
1.65
1.36
1.39
4.75
4.85
3
1.98
2.20
1.36
1.34
5.16
5.16
1
2.35
2.34
1.83
1.65
5.94
5.76
2
2.39
2.45
1.80
1.73
5.32
4.98
3
2.09
2.31
1.65
1.73
5.81
5.89
1
2.50
2.70
1.62
1.60
4.88
5.06
2
2.31
2.37
1.52
1.52
4.00
4.49
3
2.46
2.56
1.65
1.42
4.78
4.54
1
2.34
2.46
1.91
1.86
5.09
5.16
2
2.32
2.46
1.93
2.01
4.59
4.52
3
2.73
2.86
2.37
2.42
5.29
5.29
1
2.46
2.46
1.57
1.52
5.03
5.16
2
2.64
2.62
1.65
1.44
5.27
4.90
3
2.75
2.64
1.57
1.65
4.90
4.90
1
2.40
2.30
2.09
2.06
5.14
5.09
2
2.70
2.56
1.70
1.80
4.70
4.57
3
2.53
2.48
1.60
1.80
4.39
4.65
100
Results The values obtained for whole plasma cholesterol, HDL cholesterol and A apolipopro~ins, are shown in Table I. Statistical analyses of these results revealed no significant difference between the apoprotein or HDL cholesterol values in the fasting and non-fasting state. However, a significant difference Cp < 0.001) was found to exist between male and female values of the apoproteins and HDL cholesterol. The values in females were consistently higher than in males. The fasting apoprotein values showed a within-patient variation of up to 54% from month to month. The HDL cholesterol/A apoprotein ratios varied considerably from subject to subject, the correlation coefficient for the whole group being 0.62 (the correlation coefficient calculated for whole plasma cholesterol/A apoproteins was 0.32). On the first monthly collection, the A apoprotein concentrations were measured in the HDL fractions (obtained by ultracentrifugation) as well as in whole plasma. All the values obtained using HDL were less than those in the whole plasma, being on average 20% lower. Since cholesterol concentrations in total plasma always agreed with the summation of the concentrations in the individual fractions to within 10% the difference in apoprotein measurements could not be accounted for by loss of material during fractionation. Laurel1 rocket electrophoresis demonstrated that the protein deficit in the HDL fraction did not result from its transference into LDL or VLDL. It therefore appeared that the protein moiety of the HDL was being degraded to some extent during ultracentrifugation and that the values obtained using total plasma were in fact the true values. Discussion There is considerable discrepancy in literature values for human plasma A apolipoproteins. Using ultracentrifugal isolation followed by protein estimation, Have1 et al. [14] obtained a value of 1.7 g/l protein for the d 1.063-1.21 g/ml fraction of normal plasma. This is lower than our estimation (2.62 it 0.41 g/l for men; 2.48 & 0.17 g/l for women). The discrepancy could be accounted for by loss of some HDL into the d > 1.21 g/ml fraction during ultracentrifugation [ 151, or by loss of material during the necessary “washing” procedure required to obtain an albumin-free HDL sample by centrifugation. Jones and Ways [16] measured HDL protein concentrations in seven normal individuals by the technique of Have1 et al. [14] and also obtained lower values than ours. It should be remembered that the values found using our standard will be 5% higher than the true values of apolipoprotein A concentration, However, correction for the presence of C apoproteins in both sets of data will increase the disparity between them since total HDL apoprotein contains lo-15% C proteins. When our results are compared with values for HDL protein calculated from measurements of total lipoprotein determined by Donner ultracentrifugation [17], they are again high, but are in agreement with those obtained by Cohn ~actionation [lS]. Finally, a radioimmuno~say has recently been described for the measurement of apolipoprotein AI [ 191. The values given for healthy men (1.00 + 0.35 g/l) and women (1.04 f 0.35 g/l) which the authors
101
estimate as representing munological estimations
51% of the total apo HDL agree well with our imof A apolipoproteins.
References 1 C.J. Fielding, Biochem. Biophys. Res. Commun.. 46 (19’72) 1493 2 R.J. Havel, V.G. Shore, B. Shore and D.M. Bier, Circ. Res.. 27 (1970) 595 3 J.C. La Rosa, R.I. Levy, P. Herbert, S.E. Lux and D.S. Fredrickson, Biochem. Biophys. Res. Commun., 41(1970) 57 4 R.M. Krauss, P.N. Herbert, R.I. Levy and D.S. Fredrickson, Circ. Res., 33 (1973) 403 5 P. Alaupovic, D.M. Lee and W.J. McConathy. Biochim. Biophys. Acta, 260 (1972) 689 6 C.B. Laurel& Anal. Biochem., 15 (1966) 45 7 R.S. Lees and F.T. Hatch, Adv. Lipid Res., 6 (1968) 33 8 W. Annan and D.M. Isherwood, J. Med. Lab. Technol.. 26 (1969) 202 9 D.M. Lee and P. Alaupovic. Biochemistry, 9 (1970) 2244 10 B. Weeke in N.H. Ax&on, J. KrolI and B. Weeke (eds). Quantitative Immunoelectrophoresis. Universitet Sforlaget, Oslo, 1973, p. 37 11 A.M. Scanu and C. Wisdom, Ann. Rev. Biochem., 41 (1972) 709 12 M.H.G. Clarke and T. Freeman, Clin. Sci., 35 (1968) 403 13 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem.. 193 (1951) 265 14 R.J. Havel, H.A. Eder and J.H. Bragdon, J. Clin. Invest., 34 (1955) 1345 15 R.I. Levy and D.S. Fredrickson, J. Clin. Invest., 44 (1965) 426 16 J.W. Jones and P. Ways. J. Clin. Invest.. 46 (1967) 1151 17 D.S. Fredrickson, R.I. Levy and F.T. Lindgren, J. Clin. Invest., 47 (1968) 2446 18 W.F. Lever, F.R.N. Gurd, E. Uroma, R.K. Brown. B.A. Barnes, K. Schmidt and E.L. Schultz, J. Chin. Invest., 30 (1951) 99 19 G. Schonfeld and B. Pfleger. J. Clin. Invest., 54 (1974) 236
