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Quantitative plasma lipoprotein estimation by agarose gel electrophoresis
203
CLINICA CHIMICA ACTA
QUANTITATIVE
PLASMA LIPOPROTEIN
ESTIMATION
BY AGAROSE GEL
ELECTROPHORESIS
J. DYERBERG
Central
Laboratory,
(Received
AND N. HJ0RNE
Aalborg
September
City Hospital,
Aalborg
(Denmark)
roth, 1969)
SUMMARY
Agarose gel electrophoresis as a quantitative method for plasma lipoprotein estimation is examined. The technique is found reliable and fit for clinical use. Linearity between plasma lipoprotein content and dye uptake is demonstrated for all fractions. This seems to justify the relating of dye uptake to total plasma lipid concentration. 31 young healthy males and 30 young healthy females were examined with the method. No significant sex difference between plasma concentrations of chylomicrons, p- and pre-&lipoproteins was found. Detectable amounts of pre+?-lipoproteins were found in nearly all of the young persons. Two fractions of cr-lipoproteins were seen, and a significantly higher concentration of the fastest-moving fraction was found among the males.
INTRODUCTION
During recent years plasma lipoprotein estimations have become still more important in the investigation of disorders of lipid metabolism. Lipoprotein electrophoresis on paper has, due to the works of Fredrickson and Lees’, received great clinical attention. However, certain disadvantages of paper lipoprotein electrophoresis, in our opinion, limit its general use. One of the major problems has been to obtain good separation between p- and pre-/?-lipoproteins; the lack of good separation causes difficulties in the exact interpretation and the quantitative evaluation of the results. Techniques which allow quantitative evaluation of the results are in our opinion preferable to qualitative ones, especially when the aims are to follow the effect of treatment of hyperlipidemia and when population studies, e.g. of the epidemiology of atherosclerotic diseases, are carried out. Medias other than paper have been tried for better separation of the lipoproteins; the two most suitable seem to be cellulose acetate2-4 and agarose6-7. The purpose of the present paper has been to study the value of lipoprotein electrophoresis on agarose gel used as a quantitative method for clinical purposes. Secondarily we have studied the results obtained by this method on 61 young healthy persons. C&n.Chim.Acta,
28 (1970)
203-208
DYERBERG,
204
HJBRNE
METHODS
Plasma total lipid concentration was determined gravimetrically after extraction by the method of Folch et al.*. Plasma total cholesterol concentration was determined according to Rundee. Plasma triglyceride was estimated with an enzymatic glycerol determination lo. Phospholipids were determined as lipid phosphoruslr. Plasma lipoprotein electrophoresis (mainly adopted from Nobles but with own modifications) was carried out as follows: Plasma was separated immediately after sampling, and electrophoresis was carried out without delay. Agarose gel (12 (g/l) 1’Industrie biologique franc;aise !?A.) in 0.05 M barbital buffer (pH 8.6) enriched with human albumin (2.5 g/l) (~albiochem; Human Albumin, Fraction V) was used as supporting media. 25 ml of agarose gel, boiled and then cooled to 60”, were spread on a 15 cm x 20 cm Cronar Clear Base film, spec. goz, subbed on one side. After I min, an acryl templet with twelve 13 mm x 1.5 mm prongs, all 3 mm apart, was passed into the gel layer 2 cm from the catodal side of the plate. After the gel had solidified, the templet was removed, and the slits were filled with IO ~1 plasma mixed with IO ,ul of agarose gel (24 g/l) by means of a preheated micropipette. A serum agarose mixture containing 25% (v/v) of 1% bromophenol blue solution, placed in the first and the last slit, was used as an indicator for the electrophoretic run. The electrophoresis was carried out in a closed horizontal chamber, cooled with tap water (IO”). The chamber was built according to directions given by C. B. Laurel& Allmanna Sjukhuset, Malmo. Electrical connection was achieved by agarose gel wicks (18 g of agarose in I 1 of barbital buffer). A constant voltage of 12 V/cm for approx. 2.5 h was found suitable for good separation. The electrophoresis was stopped when the bromophenol blue-colored albumin fraction had moved g-10 cm. The plate was fixed in 50% (v/v) aqueous methanol for 15 min. After this the plate was dried in a 60” hot oven overnight and stained in Sudan Black staining solution (Sudanschwarz B, Merck 1387, 0.4 g; ethanol, 120 ml; zinc acetate, 4 g; water, 80 ml) for z h. The plate was then rinsed under running tap water and gently rubbed with cotton wool. No further destaining was necessary. After drying, the different lipoprotein fractions were cut out and eluted in glacial acetic acid-ethanol-water (II :5 :4, by vol.), 3 ml per fraction, and read photometrically at 590 nm against a blank, cut catodically to the starting point. Before cutting the strips were also scanned directly at 578 nm with a modified Bender and Hobein densitometer connected with a logarithmical recorder with electronic integrator. Nornzat material. 31 young healthy males and 30 young healthy females, mean age 23 years, range 18-40 years, were used as normaI material. All assays were performed after overnight fasting. RESULTS
Good separation of the various lipoprotein fractions was obtained (Fig. I). Usually five fractions could be seen. The two fastest-moving fractions for routine use were grouped and called cx-lipoproteins. After this a pre-~-lipoprotein fraction was nearly always seen, well separated from the ~-lipoprotein band. Trace amounts of chylomicrons at the starting point were often found. Quantitation of the lipoprotein content was obtained by expressing the dye Clin. Claim. Acta,
28 (1970)
203-208
QUANTITATIVE
PLASMA LIPOPROTEIN
ESTIMATION
20.5
Fig. I. (a) One plate with ten applications of the same plasma. In positions I and samples. (b) I, normal plasma; 2, chylomicronemia (Type I) ; 3. hyper-~-lipoproteinemia 4, “Broad p band” hyperlipoproteinemia (Type III) ; 5, hyper-pre+lipoproteinemia 6, chylomicronemia and hyper-pre-/$-lipoproteinemia (Type V).
indicator (Type II) (Type IV)
12
; ;
uptaking of the individual fractions as the percent of the total dye uptake, and this was related to the plasma total lipid concentration evaluated gravimetrically. This seems justified by the fact that linearity between lipoprotein amount and dye uptake could be demonstrated for all fractions (Fig. z), contrary to the results found by Jencks and Durrumx2 using paper electrophoresis and Oil-Red-O staining. The precision of the method was found satisfactory with a standard deviation of 10-25 mg/roo ml on the five fractions, calculated from 60 pairs of double estimations and 40 single estimations on a pooled serum. C&z. Chim. A&a,
28
(1970) 203-208
DYERBERG,
206
Fig. 2. Dilution proteins; 3---crons. TABLE
of a plasma preparation with physiological saline soIution. A,-0, a-lipoprotein complex; O-----O, pre-&lipoproteins; O---C,
HJBRNE
A, /?-lipochylomi-
I
PLASMA LIPID AND LIPOPROTEIN CONCENTRATIONS(elating method) 61 young persons. Values are means -+ S.D.
Total lipid Cholesterol Triglyceride Phospholipid Chylomicrons #?-Lipoprotein Pre-B-lipoprotein x-Lipoprotein complex _ * No normal distribution.
20
Sex difjeerence
Concn. (mglroo ml) -__ MllltS All
Compound
554 -i: 209 zt 86 i 168 f 15 f 278 f88 & 171 *
83 39 3o 31 15 67 41 39
553 i 199 f 93 f 153 zt ‘3 * 263 zt 95 i 179 i
Females 82 34 29 25 IO* 56 33 34
555 220 80 184
+ & f &
86 42 30 25
17
*
I$*
293 _t 74 81 & 47 163 I 43
+ (P
(P
._-_I
Chyfomicrons
f
n
mg/lOOml
Fig. 3. Frequency of distribution of lipoprotein results.
The results of lipid and lipoprotein estimations young persons are given in Table I and Fig. 3. Clin. Chim. Acta, 28 (1970) 203-208
(obtained
by elution)
in 61
QUANTITATIVE PLASMA LIPOPROTEIN ESTIMATION TABLE
207
II
COMPARISOX
OF RESULTS
OBTAINED
BY
ELUTING
AND
DIRECT
SCANNING
Values are means j, S.D. Compound
Concn. Euting
Chylomicrons /?-Lipoproteins We-p-lipoproteins a-Lipoprotein complex
I5 278 88 ‘7’
f f f. k
(mgg/Ioo ml --
plasma)
Densitomstric scanning
15 67 4’ 39
II $L If 259 + 61 81 & 40 203 rt 45
A comparison of the results obtained by elution and by direct scanning is given in Table II. DISCUSSION
The described method is found reliable and highly suitable for clinical use. The quantitative results expressed as lipoprotein lipid are sufficient for the great majority of situations where information on the plasma ~poprotein content is wanted. However, two facts must be kept in mind when using a method like this: firstly that only the lipid moiety of the lipoprotein complex is measured, secondly that the optical absorption of the stain-lipid complex need not be the same for the various lipid types, weight to weight. These problems are similar to those of the generally accepted electrophoretic estimation of plasma protein fractions. Several facts of interest were found when examining the 61 young healthy persons: Chylomicrons were found in detectable amounts in 77% of the persons after an overnight fasting. The amounts were, however, very small, and the results were distributed as a “one-tail distribution” with maximum at zero. The distribution of the /3- and pre-#I-lipoprotein values were slightly skew. Compared with the results of Postma and Stroes4 from cellulose acetate electrophoresis, our estimations were found substantialiy smaller. The main reason for this is the difference between the plasma total lipid concentration in their material (av, 700 mg/Ioo ml) and in our’s (av. 550 mg/roo ml). Pre-B-lipoprotein in detectable amount was found in all the young persons in contrast to results obtained by paper electrophoresisl~ls and to those of Chin and Blankenhorn3 on cellulose acetate who found pre-#?-lipoprotein to constitute less than jy$ of the total lipoprotein content in “several fasting young individuals”. A pre-@-lipoprotein content of less than 5% of the total lipoproteins was in our material only found in four persons, all female. This can be due either to differences in sensitivity of the two methods or to geographical population difference. The cr-lipoproteins were ordinarily found separated into two bands. Great confusion concerning the nomenclature of these and other lipoprotein fractions exists in the literature. Widely accepted nomenclature for the ‘~~-lipoprotein complex” is “/?- and pre-/%lipoproteins”, and we therefore suggest the same system for the “alipoprotein complex”, namely ‘ICI-and pre-a-lipoproteins”. Whereas no sex difference could be demonstrated in the plasma concentrations of chylomicrons, #&, pre-& and a-lipoproteins as a whole, a highly significant sex difference (P < 0.005) existed in the plasma concentration of the “pre-or-lipoproC&z. Claim. Acta, 28 (1970) 203-208
DYERBERG,
208 TABLE
HJ0RNE
III
PLASMACONCENTRATI~PI‘S OF THE VARIOUS K-LIPOPROTEINSRELATED TO SEX (ELUTING METHOD) Values are means & S.D.
u-lipoprotein
a-Lipoprotein complex Pre-a-lipoproteins cc-lipoproteins
Concn. (mglIo0
ml)
Males
Females
‘79 I-t 34 82 * I7 97 * 21
163 i 43 64 xt 27 98 + 29
Sex differewe
+ (P
teins” with the highest concentration among the men (Table III). Neither the meaning of this sex difference nor the clinical significance of changes in the individual a-lipoprotein fractions is known. Further investigations need to be carried out in order to elucidate these problems of lipid metabolism. ACKNOWLEDGMENT
This work was supported
by a grant from MIFU-Fonden
and Hjerteforeningen.
REFERENCES I 2 3 4 5
6 7 8 g IO II 12
13
D. S. FREDERICKSON AND R. S. LEES, Circulation, 35 (1965) 321. B. COLFS AND J. VERHEYDEN, Clin. Chim. Acta, 18 (1967) 325. H. P. CHIN AND D. H. BLANKENHORN, Clin. Chim. Acta, 20 (1968) 305. T. POSTMA AND J. A. P. STROES, Clin. Chim. Acta, 22 (1968) 569. W. RAPP AND W. KAHLKE, Clin. Chim. Acta. Ig (1968) 493. R. P. NOBLE, J. Lipid Res., g (1968) 693. D. A. K. MCGLASHAN AND T. R. E. PILKINGTON, Clin. Chim. Acta, 22 (1968) 646. J. FOLCH, M. LEES AND S. G. H. SLOANE, J. Biol. Chem., 226 (1957) 497. I. RUNDE, &and. J. Clin. Lab. Invest., 18 (1966) 461. M. EGCSTEIN AND F. H. KREUTZ, Klin. Wochschr., 44 (1966) 262. Official and Tentative Methods of the American Oil Chemists’ Society, Ca 12. American Oil Chemists’ Society, Chicago, Ill., 1968. W. P. JENCKS AND E. L. DURRUM, J. Clin. Invest., 34 (1955) 1437. J. DYERBERG, H. 0. BANG AND J. A. NIELSEN, Stand. J. Clin. Lab. Invest. Suppl., 110(1969) 54.
Clin. Chim. Acta, 28 (1970) 203-208
CLINICA CHIMICA ACTA
QUANTITATIVE
PLASMA LIPOPROTEIN
ESTIMATION
BY AGAROSE GEL
ELECTROPHORESIS
J. DYERBERG
Central
Laboratory,
(Received
AND N. HJ0RNE
Aalborg
September
City Hospital,
Aalborg
(Denmark)
roth, 1969)
SUMMARY
Agarose gel electrophoresis as a quantitative method for plasma lipoprotein estimation is examined. The technique is found reliable and fit for clinical use. Linearity between plasma lipoprotein content and dye uptake is demonstrated for all fractions. This seems to justify the relating of dye uptake to total plasma lipid concentration. 31 young healthy males and 30 young healthy females were examined with the method. No significant sex difference between plasma concentrations of chylomicrons, p- and pre-&lipoproteins was found. Detectable amounts of pre+?-lipoproteins were found in nearly all of the young persons. Two fractions of cr-lipoproteins were seen, and a significantly higher concentration of the fastest-moving fraction was found among the males.
INTRODUCTION
During recent years plasma lipoprotein estimations have become still more important in the investigation of disorders of lipid metabolism. Lipoprotein electrophoresis on paper has, due to the works of Fredrickson and Lees’, received great clinical attention. However, certain disadvantages of paper lipoprotein electrophoresis, in our opinion, limit its general use. One of the major problems has been to obtain good separation between p- and pre-/?-lipoproteins; the lack of good separation causes difficulties in the exact interpretation and the quantitative evaluation of the results. Techniques which allow quantitative evaluation of the results are in our opinion preferable to qualitative ones, especially when the aims are to follow the effect of treatment of hyperlipidemia and when population studies, e.g. of the epidemiology of atherosclerotic diseases, are carried out. Medias other than paper have been tried for better separation of the lipoproteins; the two most suitable seem to be cellulose acetate2-4 and agarose6-7. The purpose of the present paper has been to study the value of lipoprotein electrophoresis on agarose gel used as a quantitative method for clinical purposes. Secondarily we have studied the results obtained by this method on 61 young healthy persons. C&n.Chim.Acta,
28 (1970)
203-208
DYERBERG,
204
HJBRNE
METHODS
Plasma total lipid concentration was determined gravimetrically after extraction by the method of Folch et al.*. Plasma total cholesterol concentration was determined according to Rundee. Plasma triglyceride was estimated with an enzymatic glycerol determination lo. Phospholipids were determined as lipid phosphoruslr. Plasma lipoprotein electrophoresis (mainly adopted from Nobles but with own modifications) was carried out as follows: Plasma was separated immediately after sampling, and electrophoresis was carried out without delay. Agarose gel (12 (g/l) 1’Industrie biologique franc;aise !?A.) in 0.05 M barbital buffer (pH 8.6) enriched with human albumin (2.5 g/l) (~albiochem; Human Albumin, Fraction V) was used as supporting media. 25 ml of agarose gel, boiled and then cooled to 60”, were spread on a 15 cm x 20 cm Cronar Clear Base film, spec. goz, subbed on one side. After I min, an acryl templet with twelve 13 mm x 1.5 mm prongs, all 3 mm apart, was passed into the gel layer 2 cm from the catodal side of the plate. After the gel had solidified, the templet was removed, and the slits were filled with IO ~1 plasma mixed with IO ,ul of agarose gel (24 g/l) by means of a preheated micropipette. A serum agarose mixture containing 25% (v/v) of 1% bromophenol blue solution, placed in the first and the last slit, was used as an indicator for the electrophoretic run. The electrophoresis was carried out in a closed horizontal chamber, cooled with tap water (IO”). The chamber was built according to directions given by C. B. Laurel& Allmanna Sjukhuset, Malmo. Electrical connection was achieved by agarose gel wicks (18 g of agarose in I 1 of barbital buffer). A constant voltage of 12 V/cm for approx. 2.5 h was found suitable for good separation. The electrophoresis was stopped when the bromophenol blue-colored albumin fraction had moved g-10 cm. The plate was fixed in 50% (v/v) aqueous methanol for 15 min. After this the plate was dried in a 60” hot oven overnight and stained in Sudan Black staining solution (Sudanschwarz B, Merck 1387, 0.4 g; ethanol, 120 ml; zinc acetate, 4 g; water, 80 ml) for z h. The plate was then rinsed under running tap water and gently rubbed with cotton wool. No further destaining was necessary. After drying, the different lipoprotein fractions were cut out and eluted in glacial acetic acid-ethanol-water (II :5 :4, by vol.), 3 ml per fraction, and read photometrically at 590 nm against a blank, cut catodically to the starting point. Before cutting the strips were also scanned directly at 578 nm with a modified Bender and Hobein densitometer connected with a logarithmical recorder with electronic integrator. Nornzat material. 31 young healthy males and 30 young healthy females, mean age 23 years, range 18-40 years, were used as normaI material. All assays were performed after overnight fasting. RESULTS
Good separation of the various lipoprotein fractions was obtained (Fig. I). Usually five fractions could be seen. The two fastest-moving fractions for routine use were grouped and called cx-lipoproteins. After this a pre-~-lipoprotein fraction was nearly always seen, well separated from the ~-lipoprotein band. Trace amounts of chylomicrons at the starting point were often found. Quantitation of the lipoprotein content was obtained by expressing the dye Clin. Claim. Acta,
28 (1970)
203-208
QUANTITATIVE
PLASMA LIPOPROTEIN
ESTIMATION
20.5
Fig. I. (a) One plate with ten applications of the same plasma. In positions I and samples. (b) I, normal plasma; 2, chylomicronemia (Type I) ; 3. hyper-~-lipoproteinemia 4, “Broad p band” hyperlipoproteinemia (Type III) ; 5, hyper-pre+lipoproteinemia 6, chylomicronemia and hyper-pre-/$-lipoproteinemia (Type V).
indicator (Type II) (Type IV)
12
; ;
uptaking of the individual fractions as the percent of the total dye uptake, and this was related to the plasma total lipid concentration evaluated gravimetrically. This seems justified by the fact that linearity between lipoprotein amount and dye uptake could be demonstrated for all fractions (Fig. z), contrary to the results found by Jencks and Durrumx2 using paper electrophoresis and Oil-Red-O staining. The precision of the method was found satisfactory with a standard deviation of 10-25 mg/roo ml on the five fractions, calculated from 60 pairs of double estimations and 40 single estimations on a pooled serum. C&z. Chim. A&a,
28
(1970) 203-208
DYERBERG,
206
Fig. 2. Dilution proteins; 3---crons. TABLE
of a plasma preparation with physiological saline soIution. A,-0, a-lipoprotein complex; O-----O, pre-&lipoproteins; O---C,
HJBRNE
A, /?-lipochylomi-
I
PLASMA LIPID AND LIPOPROTEIN CONCENTRATIONS(elating method) 61 young persons. Values are means -+ S.D.
Total lipid Cholesterol Triglyceride Phospholipid Chylomicrons #?-Lipoprotein Pre-B-lipoprotein x-Lipoprotein complex _ * No normal distribution.
20
Sex difjeerence
Concn. (mglroo ml) -__ MllltS All
Compound
554 -i: 209 zt 86 i 168 f 15 f 278 f88 & 171 *
83 39 3o 31 15 67 41 39
553 i 199 f 93 f 153 zt ‘3 * 263 zt 95 i 179 i
Females 82 34 29 25 IO* 56 33 34
555 220 80 184
+ & f &
86 42 30 25
17
*
I$*
293 _t 74 81 & 47 163 I 43
+ (P
(P
._-_I
Chyfomicrons
f
n
mg/lOOml
Fig. 3. Frequency of distribution of lipoprotein results.
The results of lipid and lipoprotein estimations young persons are given in Table I and Fig. 3. Clin. Chim. Acta, 28 (1970) 203-208
(obtained
by elution)
in 61
QUANTITATIVE PLASMA LIPOPROTEIN ESTIMATION TABLE
207
II
COMPARISOX
OF RESULTS
OBTAINED
BY
ELUTING
AND
DIRECT
SCANNING
Values are means j, S.D. Compound
Concn. Euting
Chylomicrons /?-Lipoproteins We-p-lipoproteins a-Lipoprotein complex
I5 278 88 ‘7’
f f f. k
(mgg/Ioo ml --
plasma)
Densitomstric scanning
15 67 4’ 39
II $L If 259 + 61 81 & 40 203 rt 45
A comparison of the results obtained by elution and by direct scanning is given in Table II. DISCUSSION
The described method is found reliable and highly suitable for clinical use. The quantitative results expressed as lipoprotein lipid are sufficient for the great majority of situations where information on the plasma ~poprotein content is wanted. However, two facts must be kept in mind when using a method like this: firstly that only the lipid moiety of the lipoprotein complex is measured, secondly that the optical absorption of the stain-lipid complex need not be the same for the various lipid types, weight to weight. These problems are similar to those of the generally accepted electrophoretic estimation of plasma protein fractions. Several facts of interest were found when examining the 61 young healthy persons: Chylomicrons were found in detectable amounts in 77% of the persons after an overnight fasting. The amounts were, however, very small, and the results were distributed as a “one-tail distribution” with maximum at zero. The distribution of the /3- and pre-#I-lipoprotein values were slightly skew. Compared with the results of Postma and Stroes4 from cellulose acetate electrophoresis, our estimations were found substantialiy smaller. The main reason for this is the difference between the plasma total lipid concentration in their material (av, 700 mg/Ioo ml) and in our’s (av. 550 mg/roo ml). Pre-B-lipoprotein in detectable amount was found in all the young persons in contrast to results obtained by paper electrophoresisl~ls and to those of Chin and Blankenhorn3 on cellulose acetate who found pre-#?-lipoprotein to constitute less than jy$ of the total lipoprotein content in “several fasting young individuals”. A pre-@-lipoprotein content of less than 5% of the total lipoproteins was in our material only found in four persons, all female. This can be due either to differences in sensitivity of the two methods or to geographical population difference. The cr-lipoproteins were ordinarily found separated into two bands. Great confusion concerning the nomenclature of these and other lipoprotein fractions exists in the literature. Widely accepted nomenclature for the ‘~~-lipoprotein complex” is “/?- and pre-/%lipoproteins”, and we therefore suggest the same system for the “alipoprotein complex”, namely ‘ICI-and pre-a-lipoproteins”. Whereas no sex difference could be demonstrated in the plasma concentrations of chylomicrons, #&, pre-& and a-lipoproteins as a whole, a highly significant sex difference (P < 0.005) existed in the plasma concentration of the “pre-or-lipoproC&z. Claim. Acta, 28 (1970) 203-208
DYERBERG,
208 TABLE
HJ0RNE
III
PLASMACONCENTRATI~PI‘S OF THE VARIOUS K-LIPOPROTEINSRELATED TO SEX (ELUTING METHOD) Values are means & S.D.
u-lipoprotein
a-Lipoprotein complex Pre-a-lipoproteins cc-lipoproteins
Concn. (mglIo0
ml)
Males
Females
‘79 I-t 34 82 * I7 97 * 21
163 i 43 64 xt 27 98 + 29
Sex differewe
+ (P
teins” with the highest concentration among the men (Table III). Neither the meaning of this sex difference nor the clinical significance of changes in the individual a-lipoprotein fractions is known. Further investigations need to be carried out in order to elucidate these problems of lipid metabolism. ACKNOWLEDGMENT
This work was supported
by a grant from MIFU-Fonden
and Hjerteforeningen.
REFERENCES I 2 3 4 5
6 7 8 g IO II 12
13
D. S. FREDERICKSON AND R. S. LEES, Circulation, 35 (1965) 321. B. COLFS AND J. VERHEYDEN, Clin. Chim. Acta, 18 (1967) 325. H. P. CHIN AND D. H. BLANKENHORN, Clin. Chim. Acta, 20 (1968) 305. T. POSTMA AND J. A. P. STROES, Clin. Chim. Acta, 22 (1968) 569. W. RAPP AND W. KAHLKE, Clin. Chim. Acta. Ig (1968) 493. R. P. NOBLE, J. Lipid Res., g (1968) 693. D. A. K. MCGLASHAN AND T. R. E. PILKINGTON, Clin. Chim. Acta, 22 (1968) 646. J. FOLCH, M. LEES AND S. G. H. SLOANE, J. Biol. Chem., 226 (1957) 497. I. RUNDE, &and. J. Clin. Lab. Invest., 18 (1966) 461. M. EGCSTEIN AND F. H. KREUTZ, Klin. Wochschr., 44 (1966) 262. Official and Tentative Methods of the American Oil Chemists’ Society, Ca 12. American Oil Chemists’ Society, Chicago, Ill., 1968. W. P. JENCKS AND E. L. DURRUM, J. Clin. Invest., 34 (1955) 1437. J. DYERBERG, H. 0. BANG AND J. A. NIELSEN, Stand. J. Clin. Lab. Invest. Suppl., 110(1969) 54.
Clin. Chim. Acta, 28 (1970) 203-208