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Clinical application of an indirect method for quantitating serum lipoproteins
CLINICA
CHIMICA ACTA
175
CCA 47’34
CLINICAL
APPLICATION
QUANTITATING
OF AN INDIRECT
G. S. BERENSON, S. R. SRINIVASAN, A. LOPEZ-S., P. S. PARGAONKAR AND R. H. DEUPREE The Departments of Medicine, Biochemistry, Medicine, New Orleans, La, (U.S.A.) Received
METHOD FOR
SERUM LIPOPROTEINS
B. RADHAKRISHNAMURTHY,
and Biometry, Louisiana
State University School of
July 13. 1971)
SUMMARY
Application of an indirect method for quantitating serum ,!I- and pre-glipoproteins in screening individuals for lipid abnormalities is described. The method consists of turbidimetric measurement of cholesterol of serum ,9- plus pre+lipoproteins by use of heparin and Caa+. When this method is combined with estimation of the proportion of /?-and pre-/?-lipoproteins by agar-agarose gel electrophoresis, very good quantitation of these two classes of lipoproteins can be made. The validity of this method was evaluated by comparing the results with those obtained with the analytical ultracentrifuge; the results were found to be in good agreement, as well as more reproducible. /I- and pre-/?-lipoproteins determined by this method correlated well with the total serum cholesterol and triglyceride values respectively. Good reproducibility and simplicity of the turbidimetric method in measuring j3- plus pre/5lipoproteins make this ideal as a rapid initial screening procedure to select individuals with serum lipid elevations for further study. The observations on patients with various diseases usually associated with lipid changes suggested that this method to quantitate lipoproteins is useful in diagnosis of serum lipoprotein abnormalities.
INTRODUCTION
The relationship of abnormal lipid parameters to a susceptibility for coronary artery disease is now well recognized. Unfortunately, a lack of simplified techniques to quantitate serum lipoproteins has inhibited study of specific lipoprotein concentrations for large numbers of individuals, and has limited understanding of their role in disease. Since Fredrickson and Lees1 introduced a system in 1956 for characterizing hyperlipoproteinemias, the need for further study of /I- and pre-/?-lipoproteins has become more obvious. The earlier work of Gofman et al.2 had emphasized differences in serum lipoproteins which could be characterized byultracentrifugation, and although ultracentrifugation offered an accurate method for assay of the different serum lipoproteins, the technique could not be used in many clinical laboratories because of expense and the cumbersome procedure. Clin. Chim. Acta, 36 (1972) 175-183
I76
BERENSON
et al.
An important aspect of the present work is to present a reproducible procedure for quantitating the serum lipoproteins without requiring elaborate equipment and one which is useful for clinical purposes. Several methods have been proposed for the estimation of serum lipoproteins based on the ability of these proteins to form complexes with polyanionic macromolecules 3- 6. Sulfated polysaccharides (acid mucopolysaccharides (MPS)) have often been used, since these materials react to form insoluble complexes with the pre-,8- and @lipoproteins in the presence of certain cations, particularly Ca2+. In routine diagnostic purposes, currently, one or all of the following are being used to observe serum lipid abnormalities: total serum cholesterol, serum triglycerides, and visualization of electrophoretic pattern of lipoproteins. Paper electrophoresis is imprecise, and other electrophoretic techniques using different support systems have been introduced and seem to be better. However, efforts should be made to determine the concentrations of serum lipoproteins accurately and reproducibly, since information on lipoproteins is valuable for diagnostic and therapeutic measures. Studies from this laboratory indicate that under appropriate concentrations of heparin and Cal+ only serum /?- and pre$-lipoproteins can be precipitated and that the cholesterol in these serum proteins can be measured very simply by turbidimetry’. When the turbidimetric assay is combined with electrophoretic studies of the lipoproteins, serum concentrations of these two classes of lipoproteins can be madea. Evahation of “healthy” subjects and patients with a variety of diseases has shown the usefulness of this method @*lo.In order to further test its validity, an attempt has been made to compare results to those obtained by the analytical ultracentrifuge, to evaluate the relationship between the turbidity values and /I-plus pre$-lipoprotein concentrations, to observe the relationship between /I- and pre-p-lipoproteins and the concentrations of total serum cholesterol and serum triglycerides respectively, and to observe the lipoprotein abnormalities in patients with several common diseases usually associated with lipid abnormalities, e.g., diabetes, myocardial infarction, and renal disease. MATERIALS
AND
METHODS
Serum. Fasting sera were obtained from medical students, house staff, faculty of L.S.U. School of Medicine, and patients admitted to Charity Hospital of Louisiana in New Orleans. Reagents, The reagents consisted of: 0.25% aqueous solution of sodium heparin (Upjohn Co.) containing approximately 140units/mg; 0.5 M solution of CaCI, (ACS reagent grade) ; agar, Ionagar No. 2 (Consolidated Laboratory, Inc., Chicago Heights, Ill.) ; agarose, electrophoresis grade (General Biochemicals, Chagrin Falls, Ohio) ; Verona1 buffer, pH 8.6, 0.05 ionic strength; oil red 0 stanll (stock solution: 95 mg in IOO ml isopropyl alcohol; working solution: 3.0 ml of stock solution + 50 ml of 95% ethanol + 30 ml water). Cholesterol and triglyceride analyses. Cholesterol and triglycerides were determined using a Technicon autoanalyze+. Ultracentrifugal analysis of serum lipoproteins. Serum samples were sent to Bio-Science Laboratory, Van Nuys, California, in blind duplicates for quantitative determination of lipoproteins. Clin. Chim. Ada,
36 (1972) 175-183
SERUM LIPOPROTEIN
QUANTITATION
177
Turbidimetric method for determination of cholesterol in serum p- plus @e-/Ilipoproteins. This has been described in detail previously7-0. Briefly, the method consisted of mixing serum (0.2 ml), distilled water (3.2 ml), heparin (0.25%, 0.1 ml) and CaCl, (0.5 M, 0.5 ml) in the order given and measuring the turbidity obtained after 15 min at 600 rnp against a blank containing a similar mixture but omitting heparin. The cholesterol content in B- plus pre-/&lipoproteins was related to turbidimetric measurements by constructing a standard curve. This curve was obtained by measuring turbidity with increasing amounts of serum (0.05-0.5 ml), the addition of distilled water to make the total volume 4 ml, and analyzing the corresponding cholesterol content of the precipitate after centrifugation and washing. From this standard curve, the /I- plus pre-/I-lipoprotein cholesterol value of any given serum could also be calculated by turbidimetry alone. In the analysis of individual serum samples, for those giving high values (above 0.6 O.D.), less than 0.2 ml of serum was used. Agarose-agar gel electrophoresis. Electrophoretic studies of serum lipoproteins were performed according to the technique described by Cawley13 and Noble’* with some modification. Gel electrophoresis of 10-20 ,ul samples was performed on glass slides (8.3 x IO cm) instead of cronar polyester film, using a Verona1 buffer, pH 8.6, 0.05 M and 22 mA current per slide. Staining was done with oil red 0 for 6 h or overnight, followed by washing successively with alcohol-water (5:3, v/v) for 5 min and then distilled water. The lipoprotein bands obtained were scanned with a densitometer and the curves integrated to estimate the proportion of #I- and pre-p-lipoproteins. Estimation of sevum /I- and pre-/I-lipoprotein content. Based on ratios of pre-/Ito ,!I-lipoprotein obtained from densitometric scanning, /I- and pre-p-lipoprotein cholesterol determinations by the turbidimetric procedure and from the reported average (22.2%)15, the values* of cholesterol present in @- (46.9%) and pre-Slipoproteins approximate serum content of pre- /I- and @-lipoproteins can be calculated indirectly as follows : rng% p-lipoprotein rng% pre-Slipropotein Where:
: :
100 x
2
46.9 X +22.2 iod Y z
Y
46.9 X $22.2
Y
X = y. p-lipoprotein (densitometric ratio, @ + pre-b = 100%) Y = y. pre-/I-lipoprotein (densitometric ratio) Z = mg% /I- and pre-/?-lipoprotein cholesterol (turbidimetric method)
RESULTS
Previous studies indicated that the turbidity produced by addition of an appropriate concentration of heparin and Ca 8+ is a measure of serum #I- and pre-&lipoproteinsV-a, and this method coupled with electrophoretic studies can achieve a measure of each individually. A comparison of lipoprotein analysis by the analytical ultracentrifuge with the present method was made by analyzing eight sera (5 “normal” * Variations of the cholesterol content of =Jto 10% will only change @-lipoprotein estimations the same magnitude and do not appreciably change pre-Slipoprotein estimations.
to
CZin. Chim. Acta, 36 (1972) 175-183
BERENSON
178
et al.
individuals and 3 patients with diabetes) in blind duplicates. Ultracentrifugal analyses of low density lipoprotein (Sfo-IZ) and very low density lipoproteni (S~IZ-400), which corresponds to j3- and pre-b-lipoprotein, respectivelyle, were performed commercially according to the method of deLalla and Gofmanl’. Table I shows results with good reproducibility using the present method as compared to data obtained by use of the ultracentrifuge. Duplicate analyses by the present method gave percent mean variations of f 1.3 for ,!?-and & 3.7 for pre-p-lipoproteins, compared to f 5.4 (Sfo-12) and f 9.3 (Sf12-400) with the ultracentrifugal analyses. The values obtained for p-lipoproteins compared reasonably well with ultracentrifugal low density (&o-12) fraction TABLE
I
COMPARISON
OF LIPOPROTEIN
Sample *
ANALYSES
BY
ULTRACENTRIFUGE
AND
Serum triglyceride (mg%)
rng% lipoprotein Ultracentrifuge
Turbidimetry
and electrophoresis
I
TURBIDIMETRIC
METHODS
Serum
cholesterol Img%j
B
Prfd
sfo-I2 ** B
SfI2_400 (Pre-81* *
376
220
359
209
390
250
359
231
235
310
81
210
275 268
49 55
266 318
44 49
318 3’2
I63 I69
370
I96
281
194
280
I85
320 306
I25 II9
423 453
99 140
I63
215
296 300
61 62
308 338
I32 I71
110
I95
6
215 219
I38 I23
223 234
91 102
I40
I72
7
273 -
I35
34’ 3’6
I23 87
120
210
9I
246
118
218
2
3 4 5
8
273 -
Mean percent difference (Jy) between duplicates
I.3
3.7
(:.63-2.2) * ** (0.8-6.3) -~ * Duplicate analysis ** Hatch, F. T. and Lees, R. S. I8 *** Range
5.4 (0-14.0)
43 9.3 (0.51-17.6)
-
and pre-,!?-lipoprotein with very low density (Sfrz-400) fraction. Significant differences were observed in some samples, e.g., Number 5, where the pre-&lipoprotein value obtained by the present method was low compared to the SfIz-4oo fraction (the low serum triglyceride value correlated better with the lower pre-b-lipoprotein value obtained by the indirect method). These comparisons are in adequate ranges for clinical use. In order to evaluate the relationship between turbidity and the values obtained for serum p- plus pre+lipoprotein concentrations in different individuals, a series of IOO serum samplks, mostly from “healthy” individuals (85 “normals ” 15 diabetics) Ck.
Chim. Acta, 36 (1972)
175-183
SERUM LIPOPROTEIN QUANTITATIOK
I79
were studied. Diabetic sera were introduced in this series to extend the range of values. Fig. I illustrates an excellent relationship (correlation coefficient r = 0.98, standard deviation from regression, 27.6) between turbidity values and ,!?-plus pre-j3-lipoprotein concentrations. It should be mentioned that the total /I-plus pre$-lipoprotein concentration is the summation of individual lipoprotein concentrations calculated Relationship Between Turbidity and Serum fi plus preg- Lipoprotein Concenlrotions
.
4
. .:.*
Ida 2b
l*
.
t
3bo 4bo 5i30 &I
fit pre /?- lipoprotein
7bo ebo
mg %
Fig. I. Relationship between turbidity (X) and serum /?-plus pre-B-lipoprotein (Y) concentrations in sera from IOO individuals (85 “normal” and 15 diabetic patients). Regression equation: Y = - rr.6+ 1342.8 X. Correlation coefficient: r = 0.98; standard deviation from regression, 27.6.
separately. It was obvious that two samples of sera with different proportions of /?or pre-/&lipoprotein could give similar turbidity values. Since /3- and pre$-lipoproteins are the major carriers of serum cholesterol and triglycerides respectively, correlation coefficients were also determined for these two classes of lipoproteins and serum lipids (total cholesterol, triglycerides). The results are presented in Figs. 2 and 3. A very good correlation (r = 0.96, standard deviation from regression, 23.6) was Relationship Between Serum Triglyceride and preb-Lipoprotein Concentroiiam
pre B -lipoprotein
mg%
Fig. 2. Correlation between serum triglyceride (X) and pre-7%lipoprotein (Y) content as determined by the turbidimetric and electrophoretic methods. 85 “normal” and 15 diabetic subjects were studied, as shown in Fig. I. Regression equation: Y = -32.3+ 1.27 X. Correlation coefficient: r = 0.96. Standard deviation from regression, 23.‘6. Clin. Chim. Acta. 36 (1972) 175-183
180
BERENSON
et d.
Relationship BetweenTotal Serum Cholesierol ond ~-li~prot~in Co~entroti~s
350 1
Fig. 3. Correlation of serum cholesterol values (X) with /?-lipoprotein (Y) concentrations in the same group of individuals shown in Figs. I and 2. Regression equation: Y = -71.8+ 1.73 X. Correlation coefkient : r = 0.92. Standard deviation from regression, 27.1.
obtained between pre-&lipoprotein determined by the present method and level of serum triglycerides. A good correlation (r = 0.92, standard deviation from regression, 27.x) for @-lipoprotein measurements and total serum cholesterol was also observed. These results indicate turbidity to be a good measure of the total of the two lipoproteins, exclusive of a-lipoprotein. Application of the present method to evaluate serum ,6- and pre-b-lipoprotein concentrations in a group of “normal” individuals and in patients with different diseases is shown by the several scattergrams in Fig. 4. Quite arbitrary divisions of “normal” values were made for p- and pre-p-lipoprotein determinations. These divisions were based on earlier studies from this laboratory819 and the suggested normal limits for serum lipoprotein concentrations18 as “ideal” values are contained within the lower left quadrant; high values of pre-~-~poprotein occur in the upper left quadrant; high p-lipoproteins are in the lower right quadrant; and, mixed elevations are in the upper right quadrant. The graph serves as a quick reference to the nature of serum lipoprotein abnormalities. For example, a significant number of the supposedly “normal” individuals have serum /?-and pre-&lipoprotein valties which can be considered above ideal values. It is interesting to note that the mean values for /$-and pre-/?-lipoprotein (B, 240 mg%; pre-/?, go mg%) in the case of “normals” are still contained in the lower left (ideal) quadrant, whereas mean values for diabetics (&, 288 mg% ; pre$, 180mg%), kidney disease (8, 275 mg% ; pre-l), 145mg%f and myocardial infarction (/?,260 mg*h; pre-B, 130 mg%) are contained in the upper right quadrant. Patients with the above mentioned diseases lie predominantly in the upper right quadrant (representing mixed h~r~poproteine~a) while a few of the values fall in upper left quadrant (elevated pre-@-lipoproteins). In the case of patients with liver disease (mostly advanced cirrhosis) the serum profile of j3-and pre-@-lipoproteins showed significantly low pre-&lipoprotein values. DISC’JSSION
These observations illustrate the usefulness of the technique, to quantltate Clir.
Chim. A&J, 36 (1972)
175-183
the
181
SERUM LIPOPROTEIN QUANTITATION Relationship Between,8 and Prep -Lipoproteins in’Ncmals”and
in Different Diseases
Fig. 4. Serum @- and pre-/?-lipoprotein profiles in “healthy” individuals and patients with several diseases. It can be seen from the scattergram that mean values (0) for p- and pre$-lipoproteins in the case of ‘%rormals” are contained in the lower left quadrant, whereas mean values for patients with diabetes, kidney disease and myocardial infarction are contained in the upper right quadrant and show a mixture of elevated /I- and pre-/&lipoproteins. Patients with advanced liver disease show significantly lower values of pre-/?-lipoproteins. Please refer to text for further details.
serum lipoproteins in a rather simple manner and without elaborate equipment. Although several methods have been proposed earlier to estimate serum /?-lipoproteins (,!I- plus pre-p-lipoproteins) based on their ability to form complexes with polyanionic macromolecules, unfortunately these methods did not achieve quantitation of the individual lipoproteins. Objections to densitometric determination of lipoproteins by electrophoresis alone have been expressed based on the nonlinear relationship between concentration and density of bound dyer@, although other9 have demonstrated a linearity. A reasonably good correlation of analyses by agarose gel, paper electrophoresis, and the analytical ultracentrifugation has also been reported by Hatch et aLa and by Noble et al.en. The results obtained by this combination of turbidimetry and electrophoresis also show an accuracy of levels for lipoproteins to be in a range similar to that obtained by the ultracentrifuge while achieving more reproducible determinations. The correlations of serum triglyceride analyses with the levels of pre+lipoproteins in respective sera and that of total serum cholesterol to the ,!I-lipoproteins were found to be unusually close. These observations indicate that simultaneous determination of both serum cholesterol and triglycerides can be adequate for survey of individuals for coronary heart disease risk factors. In addition, the very high correlation and simplicity of the turbidimetric method in determining pre-/3- plus @lipoproteins indicate another procedure which could be used clinically. The turbidity Clin. Chim. Acta, 36 (1972) 175-183
BERENSON
182
et a/.
measurement is easier to perform than serum cholesterol or triglyceride determinations and is more reproducible. It could aid as a rapid initial screening procedure in small laboratories to select individuals requiring further study of serum lipoproteins. Over the past two decades there have been conflicting claims over which is the most useful means to study serum lipids. It is now clear that serum cholesterol alone is inadequate to make such an evaluation. Only more recently have serum triglycerides been introduced, but this determination alone is also inadequate. Gofman and co-workersz3 much earlier advocated ultracentrifugal lipoprotein analyses by weighting the lipoprotein (LDL and VLDL) classes and the use of the atherogenic index. The ultracentrifuge studies, however, cannot be applied to mass survey, and such an index does not adequately categorize specific lipoprotein abnormalities. With the development of techniques that can be applied to small laboratories without the need for expensive equipment, more widespread attention can now be given to individual serum lipoprotein concentrations on a wide scale. Our studies and those of others24925 indicate that the definition of lipid abnormalities should be made by quantitation of lipoproteins rather than the integral substances, as cholesterol and/or triglycerides. This information is important for therapeutic purposes and would be helpful in observing metabolic changes at a macromolecular level instead of substances common to all of the lipoproteins. In our survey a large number of lipoprotein abnormalities seem to fall into a class of mixed hyperlipoproteinemia, with elevated pre-b-lipoproteins occurring as frequently if not more so than the @-lipoproteins. This observation is in the term “mixed hyperlipemia”. The agreement with Brownz5, who has introduced significance of this common occurrence of mixed hyperlipoproteinemias needs to be evaluated further in its relationship to the high incidence of coronary artery disease in our population. ACKNOWLEDGEMENT
Supported Louisiana Heart
by funds from National Association.
Heart
Institute
USPHS
HE
02942
and
REFERENCES I 2 3 ,+
D. J. M. P. 5 W. 6 E.
S. FREDRICKSON AND R. 5. LEES, Circulation, 31 (1965) 321. W. GOFMAN, F. T. LINDGREN AND H. A. ELLIOTT, J. Viol. Chem., BURSTEIN AND J. SAMAILLE, J. Physiol. (Paris), 49 (1957) 83. BERNFELD. M. E. BERKOWITZ AND V. M. DONAHUE, J. C&z. Invest., J. JORDAN, JR., A. G. FAULKNER AND E. C. KNOBLOCK, Analyt. BOYLE AND R. V. MOORE, J. Lab. Clin. Med., 53 (1959) 272. 7 S. R. SRINIVASAN. A. LOPEZ-S., B. RADHAKRISHNAMURTHY AND G. S. I2 (1970) 321. 8 S. R. SRINIVASAN, A. LOPEZ-S., B. RADHAKRISHNAMURTHY AND G. S.
7 (1970) 344.
179 (1949) 973.
36 (1957) 1363. Biochem., 14 (1966) 91. BERENSON. Atheroscler., BERENSON, Angiologica,
9 A. LOPEZ-S., S. R. SRINIVASAN, F. A. DUGAN, B. RADHAKRISHNAMURTHY C&z. Chim. Acta, 31 (1971) 123.
AND G. S. BERENSON,
IO G. S. BERENSON, S. R. SRINIVASAN, P. S. PARGAONKAR, S. LINDSEY, F. PLAVIDAL, B. RADHAKRISHNAMURTHY, E. R. DALFERES, JR. AND A. LOPEZ-S., J. La. State Med. sot., 123 (1971) 49. II P. C. ARQUEMBOURG, J. E. SALVAGGIO AND J. N. BICKERS, Primer of Immunoelectrophoresis, S. Karger, Basel, 1970. I2 W. A. KREHL, A. LOPEZ-S. AND E. I. GOOD, Amer. J. Clin. Nutr., 20 (1967) 968. 13 L. P. CAWLEY, Electrophovesis and Immunoelectrophovesis, Little Brown, Boston, 1969. I4 R. P. NOBLE, J. Lipid Res., 9 (1968) 693. Clin. Chim. Acta,
36 (1972) 175-183
SERUM LIPOPROTEIN
15
QUANTITATION
183
STRAUSANDM.WURM,~~F.W.SUNDERMANANDF.W.SUNDERMAN,JR.(E~.), Lipidsand in Cliz.-Rical Medicine, Lippincott, Philadelphia, 1960,p. 47. F. T, HATCH AND R. S.LEHS, Advan.L~~~d~es.,b(lg68) I. 0. DE LALLA AND J. W. GOFMAN, Methods Biochem. Anal., I (1954) 459. D. S. FREDRICKSON,R. I.LEVY AND R. S. LEES,NewEng.J. Med., 276(1967) 34. W. P. JENCKS AND E. L.DuRRuM,J. Clin.Invest., 34 (1~55)1437. J. DYERBERG AND N. HJORNE,C&. Chim. Acta, 28 (1970)203. F.T. HATCH,J.L.MOORE,F.T.LINDGREN,L.C.JENSEN,N.K.FREEMANAND R.D.WILLS, Circulation, 36 ([email protected])(x967)16. R.P. NOBLE,F.T.HATCH, J. A.MAZRIMAS,R.T.LINDGREN,L.C. JENSEN AND G.L. ADAMSON,Lipids, 4 (1969) 55. J. W. GOFMAN, B. STRISOWER,0. DE LALLA, A.TAMPLIN, H. B. JONES AND F. LINDGREN, Mod. Med., ZI (1953) 119. L. A. LEWIS.Ann. N.Y. Acad. Sci., 94 (1961)320. D. J. BROWN, Amer.J.Med., 46 (1969) 691. R.
Steroid Hormones
16 17
18 19
20 21
22
23 24 25
C&n. Chim. Acta, 36 (1972)175-183
CHIMICA ACTA
175
CCA 47’34
CLINICAL
APPLICATION
QUANTITATING
OF AN INDIRECT
G. S. BERENSON, S. R. SRINIVASAN, A. LOPEZ-S., P. S. PARGAONKAR AND R. H. DEUPREE The Departments of Medicine, Biochemistry, Medicine, New Orleans, La, (U.S.A.) Received
METHOD FOR
SERUM LIPOPROTEINS
B. RADHAKRISHNAMURTHY,
and Biometry, Louisiana
State University School of
July 13. 1971)
SUMMARY
Application of an indirect method for quantitating serum ,!I- and pre-glipoproteins in screening individuals for lipid abnormalities is described. The method consists of turbidimetric measurement of cholesterol of serum ,9- plus pre+lipoproteins by use of heparin and Caa+. When this method is combined with estimation of the proportion of /?-and pre-/?-lipoproteins by agar-agarose gel electrophoresis, very good quantitation of these two classes of lipoproteins can be made. The validity of this method was evaluated by comparing the results with those obtained with the analytical ultracentrifuge; the results were found to be in good agreement, as well as more reproducible. /I- and pre-/?-lipoproteins determined by this method correlated well with the total serum cholesterol and triglyceride values respectively. Good reproducibility and simplicity of the turbidimetric method in measuring j3- plus pre/5lipoproteins make this ideal as a rapid initial screening procedure to select individuals with serum lipid elevations for further study. The observations on patients with various diseases usually associated with lipid changes suggested that this method to quantitate lipoproteins is useful in diagnosis of serum lipoprotein abnormalities.
INTRODUCTION
The relationship of abnormal lipid parameters to a susceptibility for coronary artery disease is now well recognized. Unfortunately, a lack of simplified techniques to quantitate serum lipoproteins has inhibited study of specific lipoprotein concentrations for large numbers of individuals, and has limited understanding of their role in disease. Since Fredrickson and Lees1 introduced a system in 1956 for characterizing hyperlipoproteinemias, the need for further study of /I- and pre-/?-lipoproteins has become more obvious. The earlier work of Gofman et al.2 had emphasized differences in serum lipoproteins which could be characterized byultracentrifugation, and although ultracentrifugation offered an accurate method for assay of the different serum lipoproteins, the technique could not be used in many clinical laboratories because of expense and the cumbersome procedure. Clin. Chim. Acta, 36 (1972) 175-183
I76
BERENSON
et al.
An important aspect of the present work is to present a reproducible procedure for quantitating the serum lipoproteins without requiring elaborate equipment and one which is useful for clinical purposes. Several methods have been proposed for the estimation of serum lipoproteins based on the ability of these proteins to form complexes with polyanionic macromolecules 3- 6. Sulfated polysaccharides (acid mucopolysaccharides (MPS)) have often been used, since these materials react to form insoluble complexes with the pre-,8- and @lipoproteins in the presence of certain cations, particularly Ca2+. In routine diagnostic purposes, currently, one or all of the following are being used to observe serum lipid abnormalities: total serum cholesterol, serum triglycerides, and visualization of electrophoretic pattern of lipoproteins. Paper electrophoresis is imprecise, and other electrophoretic techniques using different support systems have been introduced and seem to be better. However, efforts should be made to determine the concentrations of serum lipoproteins accurately and reproducibly, since information on lipoproteins is valuable for diagnostic and therapeutic measures. Studies from this laboratory indicate that under appropriate concentrations of heparin and Cal+ only serum /?- and pre$-lipoproteins can be precipitated and that the cholesterol in these serum proteins can be measured very simply by turbidimetry’. When the turbidimetric assay is combined with electrophoretic studies of the lipoproteins, serum concentrations of these two classes of lipoproteins can be madea. Evahation of “healthy” subjects and patients with a variety of diseases has shown the usefulness of this method @*lo.In order to further test its validity, an attempt has been made to compare results to those obtained by the analytical ultracentrifuge, to evaluate the relationship between the turbidity values and /I-plus pre$-lipoprotein concentrations, to observe the relationship between /I- and pre-p-lipoproteins and the concentrations of total serum cholesterol and serum triglycerides respectively, and to observe the lipoprotein abnormalities in patients with several common diseases usually associated with lipid abnormalities, e.g., diabetes, myocardial infarction, and renal disease. MATERIALS
AND
METHODS
Serum. Fasting sera were obtained from medical students, house staff, faculty of L.S.U. School of Medicine, and patients admitted to Charity Hospital of Louisiana in New Orleans. Reagents, The reagents consisted of: 0.25% aqueous solution of sodium heparin (Upjohn Co.) containing approximately 140units/mg; 0.5 M solution of CaCI, (ACS reagent grade) ; agar, Ionagar No. 2 (Consolidated Laboratory, Inc., Chicago Heights, Ill.) ; agarose, electrophoresis grade (General Biochemicals, Chagrin Falls, Ohio) ; Verona1 buffer, pH 8.6, 0.05 ionic strength; oil red 0 stanll (stock solution: 95 mg in IOO ml isopropyl alcohol; working solution: 3.0 ml of stock solution + 50 ml of 95% ethanol + 30 ml water). Cholesterol and triglyceride analyses. Cholesterol and triglycerides were determined using a Technicon autoanalyze+. Ultracentrifugal analysis of serum lipoproteins. Serum samples were sent to Bio-Science Laboratory, Van Nuys, California, in blind duplicates for quantitative determination of lipoproteins. Clin. Chim. Ada,
36 (1972) 175-183
SERUM LIPOPROTEIN
QUANTITATION
177
Turbidimetric method for determination of cholesterol in serum p- plus @e-/Ilipoproteins. This has been described in detail previously7-0. Briefly, the method consisted of mixing serum (0.2 ml), distilled water (3.2 ml), heparin (0.25%, 0.1 ml) and CaCl, (0.5 M, 0.5 ml) in the order given and measuring the turbidity obtained after 15 min at 600 rnp against a blank containing a similar mixture but omitting heparin. The cholesterol content in B- plus pre-/&lipoproteins was related to turbidimetric measurements by constructing a standard curve. This curve was obtained by measuring turbidity with increasing amounts of serum (0.05-0.5 ml), the addition of distilled water to make the total volume 4 ml, and analyzing the corresponding cholesterol content of the precipitate after centrifugation and washing. From this standard curve, the /I- plus pre-/I-lipoprotein cholesterol value of any given serum could also be calculated by turbidimetry alone. In the analysis of individual serum samples, for those giving high values (above 0.6 O.D.), less than 0.2 ml of serum was used. Agarose-agar gel electrophoresis. Electrophoretic studies of serum lipoproteins were performed according to the technique described by Cawley13 and Noble’* with some modification. Gel electrophoresis of 10-20 ,ul samples was performed on glass slides (8.3 x IO cm) instead of cronar polyester film, using a Verona1 buffer, pH 8.6, 0.05 M and 22 mA current per slide. Staining was done with oil red 0 for 6 h or overnight, followed by washing successively with alcohol-water (5:3, v/v) for 5 min and then distilled water. The lipoprotein bands obtained were scanned with a densitometer and the curves integrated to estimate the proportion of #I- and pre-p-lipoproteins. Estimation of sevum /I- and pre-/I-lipoprotein content. Based on ratios of pre-/Ito ,!I-lipoprotein obtained from densitometric scanning, /I- and pre-p-lipoprotein cholesterol determinations by the turbidimetric procedure and from the reported average (22.2%)15, the values* of cholesterol present in @- (46.9%) and pre-Slipoproteins approximate serum content of pre- /I- and @-lipoproteins can be calculated indirectly as follows : rng% p-lipoprotein rng% pre-Slipropotein Where:
: :
100 x
2
46.9 X +22.2 iod Y z
Y
46.9 X $22.2
Y
X = y. p-lipoprotein (densitometric ratio, @ + pre-b = 100%) Y = y. pre-/I-lipoprotein (densitometric ratio) Z = mg% /I- and pre-/?-lipoprotein cholesterol (turbidimetric method)
RESULTS
Previous studies indicated that the turbidity produced by addition of an appropriate concentration of heparin and Ca 8+ is a measure of serum #I- and pre-&lipoproteinsV-a, and this method coupled with electrophoretic studies can achieve a measure of each individually. A comparison of lipoprotein analysis by the analytical ultracentrifuge with the present method was made by analyzing eight sera (5 “normal” * Variations of the cholesterol content of =Jto 10% will only change @-lipoprotein estimations the same magnitude and do not appreciably change pre-Slipoprotein estimations.
to
CZin. Chim. Acta, 36 (1972) 175-183
BERENSON
178
et al.
individuals and 3 patients with diabetes) in blind duplicates. Ultracentrifugal analyses of low density lipoprotein (Sfo-IZ) and very low density lipoproteni (S~IZ-400), which corresponds to j3- and pre-b-lipoprotein, respectivelyle, were performed commercially according to the method of deLalla and Gofmanl’. Table I shows results with good reproducibility using the present method as compared to data obtained by use of the ultracentrifuge. Duplicate analyses by the present method gave percent mean variations of f 1.3 for ,!?-and & 3.7 for pre-p-lipoproteins, compared to f 5.4 (Sfo-12) and f 9.3 (Sf12-400) with the ultracentrifugal analyses. The values obtained for p-lipoproteins compared reasonably well with ultracentrifugal low density (&o-12) fraction TABLE
I
COMPARISON
OF LIPOPROTEIN
Sample *
ANALYSES
BY
ULTRACENTRIFUGE
AND
Serum triglyceride (mg%)
rng% lipoprotein Ultracentrifuge
Turbidimetry
and electrophoresis
I
TURBIDIMETRIC
METHODS
Serum
cholesterol Img%j
B
Prfd
sfo-I2 ** B
SfI2_400 (Pre-81* *
376
220
359
209
390
250
359
231
235
310
81
210
275 268
49 55
266 318
44 49
318 3’2
I63 I69
370
I96
281
194
280
I85
320 306
I25 II9
423 453
99 140
I63
215
296 300
61 62
308 338
I32 I71
110
I95
6
215 219
I38 I23
223 234
91 102
I40
I72
7
273 -
I35
34’ 3’6
I23 87
120
210
9I
246
118
218
2
3 4 5
8
273 -
Mean percent difference (Jy) between duplicates
I.3
3.7
(:.63-2.2) * ** (0.8-6.3) -~ * Duplicate analysis ** Hatch, F. T. and Lees, R. S. I8 *** Range
5.4 (0-14.0)
43 9.3 (0.51-17.6)
-
and pre-,!?-lipoprotein with very low density (Sfrz-400) fraction. Significant differences were observed in some samples, e.g., Number 5, where the pre-&lipoprotein value obtained by the present method was low compared to the SfIz-4oo fraction (the low serum triglyceride value correlated better with the lower pre-b-lipoprotein value obtained by the indirect method). These comparisons are in adequate ranges for clinical use. In order to evaluate the relationship between turbidity and the values obtained for serum p- plus pre+lipoprotein concentrations in different individuals, a series of IOO serum samplks, mostly from “healthy” individuals (85 “normals ” 15 diabetics) Ck.
Chim. Acta, 36 (1972)
175-183
SERUM LIPOPROTEIN QUANTITATIOK
I79
were studied. Diabetic sera were introduced in this series to extend the range of values. Fig. I illustrates an excellent relationship (correlation coefficient r = 0.98, standard deviation from regression, 27.6) between turbidity values and ,!?-plus pre-j3-lipoprotein concentrations. It should be mentioned that the total /I-plus pre$-lipoprotein concentration is the summation of individual lipoprotein concentrations calculated Relationship Between Turbidity and Serum fi plus preg- Lipoprotein Concenlrotions
.
4
. .:.*
Ida 2b
l*
.
t
3bo 4bo 5i30 &I
fit pre /?- lipoprotein
7bo ebo
mg %
Fig. I. Relationship between turbidity (X) and serum /?-plus pre-B-lipoprotein (Y) concentrations in sera from IOO individuals (85 “normal” and 15 diabetic patients). Regression equation: Y = - rr.6+ 1342.8 X. Correlation coefficient: r = 0.98; standard deviation from regression, 27.6.
separately. It was obvious that two samples of sera with different proportions of /?or pre-/&lipoprotein could give similar turbidity values. Since /3- and pre$-lipoproteins are the major carriers of serum cholesterol and triglycerides respectively, correlation coefficients were also determined for these two classes of lipoproteins and serum lipids (total cholesterol, triglycerides). The results are presented in Figs. 2 and 3. A very good correlation (r = 0.96, standard deviation from regression, 23.6) was Relationship Between Serum Triglyceride and preb-Lipoprotein Concentroiiam
pre B -lipoprotein
mg%
Fig. 2. Correlation between serum triglyceride (X) and pre-7%lipoprotein (Y) content as determined by the turbidimetric and electrophoretic methods. 85 “normal” and 15 diabetic subjects were studied, as shown in Fig. I. Regression equation: Y = -32.3+ 1.27 X. Correlation coefficient: r = 0.96. Standard deviation from regression, 23.‘6. Clin. Chim. Acta. 36 (1972) 175-183
180
BERENSON
et d.
Relationship BetweenTotal Serum Cholesierol ond ~-li~prot~in Co~entroti~s
350 1
Fig. 3. Correlation of serum cholesterol values (X) with /?-lipoprotein (Y) concentrations in the same group of individuals shown in Figs. I and 2. Regression equation: Y = -71.8+ 1.73 X. Correlation coefkient : r = 0.92. Standard deviation from regression, 27.1.
obtained between pre-&lipoprotein determined by the present method and level of serum triglycerides. A good correlation (r = 0.92, standard deviation from regression, 27.x) for @-lipoprotein measurements and total serum cholesterol was also observed. These results indicate turbidity to be a good measure of the total of the two lipoproteins, exclusive of a-lipoprotein. Application of the present method to evaluate serum ,6- and pre-b-lipoprotein concentrations in a group of “normal” individuals and in patients with different diseases is shown by the several scattergrams in Fig. 4. Quite arbitrary divisions of “normal” values were made for p- and pre-p-lipoprotein determinations. These divisions were based on earlier studies from this laboratory819 and the suggested normal limits for serum lipoprotein concentrations18 as “ideal” values are contained within the lower left quadrant; high values of pre-~-~poprotein occur in the upper left quadrant; high p-lipoproteins are in the lower right quadrant; and, mixed elevations are in the upper right quadrant. The graph serves as a quick reference to the nature of serum lipoprotein abnormalities. For example, a significant number of the supposedly “normal” individuals have serum /?-and pre-&lipoprotein valties which can be considered above ideal values. It is interesting to note that the mean values for /$-and pre-/?-lipoprotein (B, 240 mg%; pre-/?, go mg%) in the case of “normals” are still contained in the lower left (ideal) quadrant, whereas mean values for diabetics (&, 288 mg% ; pre$, 180mg%), kidney disease (8, 275 mg% ; pre-l), 145mg%f and myocardial infarction (/?,260 mg*h; pre-B, 130 mg%) are contained in the upper right quadrant. Patients with the above mentioned diseases lie predominantly in the upper right quadrant (representing mixed h~r~poproteine~a) while a few of the values fall in upper left quadrant (elevated pre-@-lipoproteins). In the case of patients with liver disease (mostly advanced cirrhosis) the serum profile of j3-and pre-@-lipoproteins showed significantly low pre-&lipoprotein values. DISC’JSSION
These observations illustrate the usefulness of the technique, to quantltate Clir.
Chim. A&J, 36 (1972)
175-183
the
181
SERUM LIPOPROTEIN QUANTITATION Relationship Between,8 and Prep -Lipoproteins in’Ncmals”and
in Different Diseases
Fig. 4. Serum @- and pre-/?-lipoprotein profiles in “healthy” individuals and patients with several diseases. It can be seen from the scattergram that mean values (0) for p- and pre$-lipoproteins in the case of ‘%rormals” are contained in the lower left quadrant, whereas mean values for patients with diabetes, kidney disease and myocardial infarction are contained in the upper right quadrant and show a mixture of elevated /I- and pre-/&lipoproteins. Patients with advanced liver disease show significantly lower values of pre-/?-lipoproteins. Please refer to text for further details.
serum lipoproteins in a rather simple manner and without elaborate equipment. Although several methods have been proposed earlier to estimate serum /?-lipoproteins (,!I- plus pre-p-lipoproteins) based on their ability to form complexes with polyanionic macromolecules, unfortunately these methods did not achieve quantitation of the individual lipoproteins. Objections to densitometric determination of lipoproteins by electrophoresis alone have been expressed based on the nonlinear relationship between concentration and density of bound dyer@, although other9 have demonstrated a linearity. A reasonably good correlation of analyses by agarose gel, paper electrophoresis, and the analytical ultracentrifugation has also been reported by Hatch et aLa and by Noble et al.en. The results obtained by this combination of turbidimetry and electrophoresis also show an accuracy of levels for lipoproteins to be in a range similar to that obtained by the ultracentrifuge while achieving more reproducible determinations. The correlations of serum triglyceride analyses with the levels of pre+lipoproteins in respective sera and that of total serum cholesterol to the ,!I-lipoproteins were found to be unusually close. These observations indicate that simultaneous determination of both serum cholesterol and triglycerides can be adequate for survey of individuals for coronary heart disease risk factors. In addition, the very high correlation and simplicity of the turbidimetric method in determining pre-/3- plus @lipoproteins indicate another procedure which could be used clinically. The turbidity Clin. Chim. Acta, 36 (1972) 175-183
BERENSON
182
et a/.
measurement is easier to perform than serum cholesterol or triglyceride determinations and is more reproducible. It could aid as a rapid initial screening procedure in small laboratories to select individuals requiring further study of serum lipoproteins. Over the past two decades there have been conflicting claims over which is the most useful means to study serum lipids. It is now clear that serum cholesterol alone is inadequate to make such an evaluation. Only more recently have serum triglycerides been introduced, but this determination alone is also inadequate. Gofman and co-workersz3 much earlier advocated ultracentrifugal lipoprotein analyses by weighting the lipoprotein (LDL and VLDL) classes and the use of the atherogenic index. The ultracentrifuge studies, however, cannot be applied to mass survey, and such an index does not adequately categorize specific lipoprotein abnormalities. With the development of techniques that can be applied to small laboratories without the need for expensive equipment, more widespread attention can now be given to individual serum lipoprotein concentrations on a wide scale. Our studies and those of others24925 indicate that the definition of lipid abnormalities should be made by quantitation of lipoproteins rather than the integral substances, as cholesterol and/or triglycerides. This information is important for therapeutic purposes and would be helpful in observing metabolic changes at a macromolecular level instead of substances common to all of the lipoproteins. In our survey a large number of lipoprotein abnormalities seem to fall into a class of mixed hyperlipoproteinemia, with elevated pre-b-lipoproteins occurring as frequently if not more so than the @-lipoproteins. This observation is in the term “mixed hyperlipemia”. The agreement with Brownz5, who has introduced significance of this common occurrence of mixed hyperlipoproteinemias needs to be evaluated further in its relationship to the high incidence of coronary artery disease in our population. ACKNOWLEDGEMENT
Supported Louisiana Heart
by funds from National Association.
Heart
Institute
USPHS
HE
02942
and
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SERUM LIPOPROTEIN
15
QUANTITATION
183
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Steroid Hormones
16 17
18 19
20 21
22
23 24 25
C&n. Chim. Acta, 36 (1972)175-183