Detection of subtle abnormalities of serum β- and pre-β-lipoproteins in “normal” individuals by turbidimetric and electrophoretic methods

CLINICA CHIMICA ACTA

DETECTION

OF SUBTLE

/!&LIPOPROTEINS

ABNORMALITIES

IN “NORMAL”

AND ELECTROPHORETIC

ALFRED0

LOPEZ-S.,

KRISHNAMURTHY

SATHANUR AND

Departments of Medicine Orleans, La. (U.S.A.) (Received

GERALD

OF SERUM

INDIVIDUALS

,5- AND PRE-

BY TURBIDIMETRIC

METHODS*

R. SRINIVASAN,

FORTUNE

A. DUGAN,

B. RADHA-

S. BERENSON

and Biochemistry,

Louisiana

State

University

School of Medicine,

New

March 10, 1970)

SUMMARY

A simple method is described for use in screening “normal” individuals for lipid abnormalities. The method is reproducible and consists of turbidimetric measurement of cholesterol of serum /?-lipoproteins (p and pre-/?) by use of heparin, CaZ+, without elaborate equipment. When this method is combined with estimation of the proportion of b- and pre-p-lipoproteins (by agar gel electrophoresis), a very good and practical assessment of serum lipids can be made. Subtle lipid abnormalities in presumably healthy individuals can be detected by these methods. A relationship between the pre-/?-lipoproteins and p-lipoproteins was made which can easily be related to the phenotyping of hyperlipoproteinemias, as described by Fredrickson and Lees. An evaluation of serum lipids in 69 clinically “normal” individuals is presented, showing that these lipid studies are very useful in the screening of “presumably normal” individuals and should be used for routine clinical observations. The procedures are adaptable for clinical purposes and should offer better evaluations than by total cholesterol and/or triglyceride determinations alone.

INTRODUCTION

Epidemiological and experimental evidence indicates an important relationship between abnormalities in serum lipoproteins and atherosclerosis. The earlier studies by Gofman and co-workers’ served to emphasize differences in the serum lipoproteins which could be characterized by ultracentrifugation, and numerous investigators using other physical or chemical means have related changes of lipoproteins with susceptibility to coronary artery disease. With the introduction in 1965 by Fredrickson and Lees* of a system for phenotyping hyperlipoproteinemias, quantitation of * Supported bv funds from the National Heart Association.

Heart

Institute

of USPHS

(HE 02942)

Ctin. Chim. Acta,

and Louisiana

31 (1971)

123-132

12-t

LOPEZ-S.

L’t al.

pre-/3- and p-1il)oproteins gained much significance, and, hence, the development of simple, reproducible methods for studying serum lipids and lipoproteins is needed. Altllougll ultracentrifugation offers an accurate method for assay of different serum lipoproteins, it has limitation for use in many clinical laboratories because the equipment is expensive and the procedures are cumbersome. In the past, several methods II:I\Y been proposed for the estimation of serum $-lipoproteins, based on the ability of these protein5 to form complexes with pal\.anionic macromolecule+ !‘. Sulfated polysaccharides (acid mucopolvsaccllarides (XI’S) have often been used) react to form insoluble complexes with pre-@- and plipoproteins in the presence of certain cations, particularly- Ca*’ ¢ studies from this laboratory ~~1 indicate that the nature and concentration of LIPS, lipoproteins in serum, and (‘as- are all critical for quantitative precipitation of these specific complexes. As the primary interest in those investigations was to study the nature of the complexing of p-lipoproteins, no attempt was made to individuall!. assay pre--/$and p-lipoproteins. Further observations Jrielded a simple and reproducible turbidimetric method for lipoprotein analysis and allowed approximation of serum pre-@lipoproteins when combined with electrophoretic studies of tile lipoproteins. The mctllods employed are particularly useful in screening “normal” individuals for subtle lipoprotein abnormalities such as an ultracentrifuge.

Fasting from patients

and can be performed

serum was obtained admitted to Charity

Reagents The reagents

consisted

of

without elaborate

from medical students, Hospital, New Orleans,

0.25%

aqueous

solution

equipment,

house staff, faculty, La.

of sodium heparinate

and

(Up-

john Co.) containing approximately 140 units/mg; 0.5 M solution of CaCl, (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, and 0.05 ionic strength; Oil red 0 stain** (stock solution: g5 mg in IOO ml isopropyl alcohol; working solution: 3.0 ml of stock solution + 50 ml of 950/b ethanol

+ 30 ml water).

EquiPwent A Coleman Jr. spectrophotometer was used to read absorbance at 600 nm with cuvettes (15x 125 mm). An International clinical centrifuge, table model, and an electrophoresis chamber, made in the laboratory to accommodate three 8 x IO-cm glass (slide) plates, and D.C. power supply were used. For densitometric scanning of stained electropherograms, a Photovolt densitometer (Model 52-C) with a yellow filter (Y-3 (K3) Ednalite Optical Co., Inc., Peekskill, N.Y.), attached to a Photomultiplier microphotometer (American Instrument Co., Inc., Silver Springs, Md.) and Sargent recorder (Model SR), was used.

Clin. Chim. Acta,

31 (1971)

X23-132

~~olest~r~~ alad t~i~l~~~?Yi~i? analyses

Cholesterol was determined by the method of Pearson, 1stal.*” using the VniKit (Cole Technical Laboratory) or was determined simultaneously with triglycerides using a Techniron autoanalyzer’*.

~~~~~j~~irnet~i~ ~YoGed~~reforestjn~atio~~ofGholesteyolin sewm

/3’-and pve-S-li~o~rotfins” The following were mixed well, in the order given, in a cuvette or centrifuge tube: serum 0.2 ml; distilled water, 3.2 ml; heparin (0.2574, 2 140 units/m&, 0.1 ml; CaCl, (0.5 XI), 0.5 ml; and a similar mixture omitting heparin, water being suhstituted, was prepared simultaneously as a blank. The turbidity obtained after 15 min at room temperature was read against the blank at fjoo nm in the spectrophotometcr and values for /?- plus pre-&lipoprotein cholesterol were calculated from a standard curve. The standard curve was obtained as follows: the absorbance (turbidity) was measured after 15 min of incubating increasing amounts of serum (0.~15-0.5 ml) with 0.1 ml of o.z50/ heparin solution, plus 0.5 ml of 0.5 M CaCl, and enough distilled water to make a total volume of 4 ml. In order to relate the cholesterol content to turbidimetric measurements, a parallel set of experiments, exactly as described above, was carried out using centrifuge tubes. The tubes after incubation for 15 min were centrifuged for IO min, the supernatant discarded, and the centrifuge tubes filled with cold water (4’). These were allowed to stand for z-3 min and then decanted without loss of precipitate. The washing was repeated once more and tubes were then alfowed to stand inverted on a filter paper for IO min. The precipitate was dissolved in 0.2 ml of 1.7% sodium chloride solution and cholesterol content determined. The amount of cholesterol found in the precipitate was plotted against the turbidity produced by the same volume of serum (Fig. I). From this standard curve, the p-

P 8

0.60.40.2-

0.2

t 0.4 0.6 0.8 mg Cholesterol

Fig. I. Relationship between turbidity and cholesterol of serum b- plus pre-~-lipoproteins. The cholesterol of & plus pre-@-lipoproteins of a given serum can be determined from the curve by measuring the turbidity alone, e.g. to express as mg”,b @- and pre-&lipoprotein cholesterol: 100

x mg

cholesterol

(turbidity

curve)

ml serum or for 0.2 ml serum, multiply cholesterol

value

x 500 mg%.

plus pre-@lipoprotein choIest&roI value of any given serum can be calculated by measurement of the turbidity alone. For sera giving high turbidity values (above 0.6 A) less than 0.2 ml of serum should be used. More detailed studies of serum lipids can be c!i?$. Chim. Act@, 3X (1971)

123-132

126

LOPEZ-S. et nl.

conducted below.

using the @- and pre-#-lipoprotein

cholesterol

determinations

as described

Agarose-agav gel electrophoresis Electrophoretic studies of serum lipoproteins were performed according to the technique described by Nobler5 with some modification, Gel electrophoresis of 10-30 ~1 serum sample was performed on glass slides instead of Cronar polyester film. A Verona1 buffer was used and 22 mA current per slide achieved adequate lipoprotein separation in 2 h. Usually, three slides were used and 18 to 21 samples could be run simultaneously. 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 distilled water, The piates were dried in an oven at 60-70”. This staining technique is less time-consuming and gives more uniform staining of the plates than the method adopted by NobleIs. In the present investigation, a concentrated stock solution of Oil red 0 was conveniently prepared by dissolving the dye in isopropanol to maintain maximum concentration, instead of refluxing the dye in aqueous ethanol for several hours and keeping the prepared solution at 37-40’. The stock solution of Oil red 0 in isopropanol is stable for several weeks even at room temperature and can be diluted with ethanol-water (5:3 v/v) to any required concentration prior to use. The proportion of alcohol to water appears to be critical for rapid and uniform staining as well as destaining the background. The serum lipoprotein bands obtained were scanned with a densitometer and the curves integrated to estimate proportion of z, @, and pre-p-lipoproteins. Indirect estimation f$ serztm @e-/J- and /Mipo$uatein content Based on ratios of pre-p- to ,&lipoprotein obtained from densitometric scanning, by the turbidimetric procedure, JI- and pre-fi-l’pI op ro t ein cholesterol determinations and from the reported average values* of cholesterol present in /?- (46.9%) and pre-j?lipoproteins (22.27") (ref. r6), the approximate serum content of pre-/I- and #?-lipoproteins can be calculated indirectly as follows: 100 x 2 _____~.. -.-46.9 X + 22.2 y

:

rng% @-lipoprotein

rng% pre+lipoprotein

:

rooyz ___.46.9 X --j- 22.2 y

where : X = y. &lipoprotein (densitometric ratio, /I+pre-b = 100%) y = o/o pre-/3-lipoprotein (densitometric ratio) Z = rng% /?- and pre$-lipoprotein cholesterol (turbidimetric

method)

RESULTS

After extensive experiments, it was found that several factors (concentration of heparin, Cazf, and different ions, etc.) critically affect the nature and amount of * Variations of the cholesterol content of 5 to IO~/~ will only change B-lipoprotein estimations the same magnitude and do not appreciably change pre-/?-lipoprotein estimations. Cfin. Chim. A&Z, 31 (1971)

123-132

to

ABNORMALITIES

IN SERUM LIPOPROTEIN

127

b-lipoproteins precipitated. Electrophoretic and immuno-electrophoretic techniques further illustrated that only the p-lipoproteins (pre-p-+,&lipoproteins) were precipitated by this methodlOpll and that the fi- plus pre-/!&lipoprotein cholesterol could be measured turbidimetrically. a-Lipoprotein was not precipitated. The turbidimetric method was applied particularly to study of “normal” individuals, although a number of patients with different diseases as exploratory to its general use were included. The reproducibility of the turbidimetric method was determined by replicate analyses of several individual serum samples using reagents prepared in different batches. For example, a quintuplicate analysis of IO serum samples showed a maximum deviation of 54% from the average value obtained for the individual sample. Similarly, a 4% variation was observed when the analysis of the same serum sample was performed using reagents prepared in three different batches. Table I shows a very good correlation between the values of cholesterol of the fi- plus pre-p-l’p1 oproteins estimated by the turbidimetric procedure and the actual TABLE

I

COMPARISOY AND _-

CHEMICAL -..

OF fi-

AND

PRE-~-LIPOPROTEIN

ANALYSESl’

CHOLESTEROL

OF PRECIPITATED Absorbance*

VALUES

* --

Cholesterol T~~ba~ity * * (rng~r~o ml)

“Normals”

Diabetes

(69)

0.24

(.x4-.33)

(211

M.I.

(14)

Liver disease

(16)

Renal disease

(‘4)

Xanthoma

l

(41

0.32

(.22--.53) 0.28 (.r7-.45) 0.23 (.r8-,491 0.34 (.IO-.57) o-39 (.23-.62)

OBTAINED

RY

TURBIDIMETRIC

LIPOPROTEINS

127.1 (z-55) (115-280) 148.9 (90-240) 124.9 (z?’ (55-305) 207.5 (120-330)

t

~Precifiitate * * ~rng~~oo ml) 130.6 (75-175) 159.4 (79-270) 139.5 (72-218) ‘24.9 (9o--301) 146.5 Wzd35)

- 1.76t 2.85 2.77 1.oot 3.74

(129-350) --_*-Number of subjects. * * --Mean and ranges. t-Test of significance for the difference between the two methods {calculated from individual values)i7. t-The difference is not significant (p > o.o5), i.e. there is good agreement between turbidity and analyses of precipitate.

values obtained by analyzing the precipitate. In the group of “normal” individuals, the difference between the two methods (3.5 mg) is less than 2%. (Statistically, the difference is not significant, P > 0.05.) A somewhat greater difference between the two methods was observed in patients with diabetes, myocardial infarction, and renal disease, as it might be expected. This greater variability is probably due to the increase in pre-&lipoproteins, which contain relatively less cholesterol. From a study of apparently “normal” individuals, values by this technique above an A of 0.24 or @- plus pre-~-lipoprotein chotesterol over 130 rng% are considered above “normal” limits. Such individuals should be studied in more detail for total serum cholesterol, triglyceride levels, and lipoprotein patterns. Obviously, C&Z. ChZ,B.ACta, 31 (1971)

123-132

128

LCJPEZ-S. CL 01.

there is an overlap in the A estimation between “normals” and those for individual patients within disease groups. The overlap furthcar illustrates the presence of lipid abnormalities in a number of persons otherwise considered ncltmal. Ideally, the turbidimetric screening technique should be used in combination with total cholesterol, triglyceride and electrophoretic- lip~)protei~l ~leter~ninat~ot-ts. Agar-agarose gel electrophoresis has been shown to be an improved method over paper in respect to lipoprotein evaluation, since suitably distinct ,zparations between ,5- and pre-#?-lipoprotein can be obtained even in normal individuals (Fig. 2). {Other methods which achieve sel~aration and d~~nsitonletri~ study of lil)(~~r(~teins

Fig. 2. Agar-agarase gel electroph~rogram of 7 different human sera, showing different proportions of GYZ, j?, and pre-@lipoproteins, Oil red 0 stain. For details of staining technique, please refer to text.

would be as satisfactory.) An example of agarose gel separation and densitometric scanning used to determine the relative proportion of each of the lipoprotein fractions (r, pre-/? and p) is shown in Fig. 3. From ratios of the pre-/3 to p-fraction (from densitometry) and the turbidimetric assay of the cholesterol of the /?- plus pre-/?lipoproteins, pre-p- and b-lipoprotein concentrations in serum were calculated for a group of 69 “normal” individuals. (A much larger sampling of serum from individuals with various diseases is in progress.) Using the reported values for content of cholesterol in pre-@- and @lipoproteins, indirect estimations (approximate values) for these lipoproteins can be obtained. As an example, /3- and pre-~-l~~protein determinations of 4 serum samples are shown in Table II. Duplicate analyses of several samples showed a good reproducibility of these determinations. A maximum variation of 6.2% for @-lipoproteins and 13,7% for pre-&lipoproteins was observed in duplicate C&n. Chim.

a‘icta, 31 (1971)

123-132

AB~~R~fALlTIES

IN SERUM

LIPOPROTEINS

129

Fig. 3. Densitometric tracing of the serum electropherogram shown at the bottom of the figure. Th? relative proportions of the three lipoprotein fractions can be calculated from peak areas.

ESTIMATION

Sample*

OF @- AND

Turbidity (A)

PRE-$-LIPOPROTEINS

BY

TURBIDIMETRIC

p- + Pre-/Llipoprotein cholesterol * *

AND

B 9/,

pqe-P

140 140

0.3”

160

93 93 6x

0.31

165

3.

0.27

4.

0.2g 0.34 0.34

I45 15.5 180 ISO

64 85 83 83 83

7 7 39 36 I5 ‘7 ‘7 ‘7

I.

2.

0.26

0.26

* -Duplicate **-Calculated ***-Calculated

ELECTROPHORETIC

Densitometric ratio (/3-+-pve-/I = 100)

METHODS

Lipoproteins**

$

288

22

288

22

261

‘67 156 50

278 28.1 301 348 348

62

7’ 7’

analyses. from turbidity vaiues. from the formula given in the text. Clin.

C&m.

Acta,

31 (1971)

123-137

I>OPEZ-S.

r3o

ct 01.

determinations. Altl~ougl~ it has been shown I8 that the proportion of cholesterol in the lipoproteins can vary, Smithrg indicates little change in Sfo 121ipoprotein. From a clinical standpoint the approximation of lipoprotein content can be obtained bv this method to yield useful inforn~ation, as shown in l:ig. 4. Lipoprotein abn~~rn~aliti~s are of a much greater magnitude than the repr(~d~~cibil~t~ of the method or the estimations based on the above formulas.

l:ic h. ,+, Iiclationship between the serum & and prc-B-lipoprotein concentrations in 6g “normal” individuals. The lipoprotein concentrations were calculated indirectly by assuming average cholesterol contents of the two types of lipoproteinsiB and from the determinations of cholesterol in the ,% plus pre-@-lipoprotein fractions (turbidity) and of proportions of pre-&- and /&lipoprotein (electrophoresis). The values were grouped according to percent of pre-~-lipoprotein and arbitrary iimits assigned.--“Ideal” lipid values are contained in the lower left quadrant within the broken iincs, less rigid criteria, the solid lines. From the scattergram, it can be seen that half or more of the “healthy” individuals have high values accordin g to the arbitrary divisions. The graph can serve as a quick reference to the nature of abnormalities, for example, abnormally high values falling in the upper left quadrant, high pre-B-lipoprotein, suggest diabetic and/or carbohydrateinduced lipid abnormalities with high triglycerides and correspond to Fredrickson and Lees Type IS. Thelower right corresponds to Type Ii with high cholesterol and high &lipoproteins, and the upper right to the mixture of high pre-fi- and ~-lipoproteills, Type III, with both high cholesterol and triglycerides.

DISCUSSION

The availability of uncomplicated methodology to estimate the serum lipids, including ~-lipoproteins (low density) and pre-~-lipoproteins (very low density), is of great practical and clinical importance. In the present study a reproducible method to study cholesterol of the fl-lipoprotein fractions (including pre-/$lipoproteins) has been described, Similar techniques have been described earlier but without chemically characterizing the nature of the material precipitated. For example, the use of an excess of heparin” or the substitution of Mn for Ca at levels which have been of other serum proteins used for lipoprotein isolations*o results in the co-precipitation in addition to the @-lipoproteins It. Other polyanions, such as dextran sulfate or carrageenin, have been used for similar studies and may be equally good. In our studies heparin seemed to have an advantage of specificity and reproducibility (there seems to be a relationship between the anticoagulant ability of different MPS, sulfate content and their compiexing abilitv with /3-lipoproteins). The reliability of the turb~dimetric procedure for the estimation of the cholesterol of the pre-#?- plus j3-lipoprotein fractions was demonstrated by the excellent correlation in individuals considered clinically normal between the values obtained Clin. Chim. ACta, jr

(1971)

123-132

ABNORMALITIES

IN SERUM LIPOPROTEINS

r3r

by turbidity and those measuring cholesterol in precipitated pre$- and t!%lipoproteins (Table I). Jordan et ~1.~ (although using more heparin than proposed in these studies) have found very good agreement in normal individuals between the cholesterol precipitated by calcium and heparin and the cholesterol content of the low density lipoproteins isolated by ultracentrifugation from the same serum. Unfortunately, they did not attempt to correlate these values with turbidimetric estimates of the cholesterol of the low density lipoproteins. The turbidimetric procedure as described allows a rapid and simple estimation of this lipoprotein fraction and when used in colnbination with electrophoretic data allows a useful characterization of lipid abnormalities (Fig. 4). The turbidimetric technique is rather easy to develop and can be performed as an office procedure if a calorimeter is available. For screening purposes this technique might yield results more valuable than the mere determination of cholesterol or triglycerides alone. But, individuals with abnormal values by this technique also need determinations of total cholesterol, triglycerides, and the electrophoretic lipoprotein pattern since abnormal values by the turbidimetric method do not indicate the abnormality due to changes in either pre-@- or #?-lipoproteins or both. In the course of the studies it was found that the presence of chylomicrons or fibrinogen interferes with the method. For this reason, fasting serum is used instead of plasma, and if the serum is already grossly turbid, the complete determination of cholesterol, triglycerides, and lipoprotein pattern is obviously indicated. For characterization of lipoprotein patterns, el~~trophoresis in agar-agarose gel plates offers several advantages over the paper electrophoretic technique which permitted Fredrickson and Lees* to classify the hyperlipoproteins into phenotypes. Among these advantages are a greater sensitivity and resolution of the very low density lipoproteins, which are evident even in normal sera. The method is also timesaving and the glass plates are permanent. Staining of the serum lipoproteins prior to electrophoresis has been suggested by some investigators 21-*3but this procedure may cause denaturation of lipoproteins and consequently alter electrophoretic patterns. The staining technique adapted in the present work was found to be sensitive and uniform. Objections to densitometric determination of lipoprotein by electrophoresis have been expressed. These objections are based on the non-linear relationship between concentration and density of bound dye*a, due mainly to the variable dye-binding capacity of different lipids**. Nowever, a good correlation between densitometric scan of electrophoretic patterns of lipoproteins and ultracentrifugal analyses has been observed by Hatch el al.26 and also by Noble ct aLz7 and in preliminary studies by ourselves. For clinical purposes, the degree of agreement appears quite adequate. Significant abnormalities of lipoprotein metabolism are of much greater magnitude than the comparatively minor deviations observed among the different methods. Combining the turbidimetric procedure with the agarose gel electrophoretic patterns, it was observed, based upon arbitrary divisions from mean turbidity values and suggested normal limits for serum lipoprotein concentrations**, that about half of our “normal” group according to such rigid criteria had some lipid values which are considered in excess of ideal levels. It is interesting to note that our “normal” group was composed mainly of medical students, interns, residents, and individuals associated with medical activities. Noble** has reported similar findings in 18% of a Cli%. ChiW. A&,

31

(1971)

123-132

132

LOPEZ-S. et al.

group of I& teenagers, and Brown and L)oyle30 in 3oyb of a group of students. abnormal lipid values were also found31J2 in 14% of a large group of school children. More recently, Blankenhorn et a1.33 found that in IO young patients with coronary heart disease, 50% had Type IV hyperlipidemia. All these findings point out the importance of early detection of abnormal serum lipids which can guide modification of environmental factors to restore these values to an “ideal” range. Strong evidence points to serum lipids as useful markers to detect coronary artery disease proneness in The need for simple and effective screening young, “normal, healthy” individuals. procedure which can be applied routinely or for clinical evaluation without elaborate equipment is apparent.

The authors wish to acknowledge the assistance of Dr. C. A. McMahan, man, Department cf Biometry, for statistical analyses.

8 9 I”

Chair-

1. 11'.GOFRIAN, I‘.T. LISDGREN AXU H. A. ELLIOTT, I.Viol.Chem.. 170 (IcM~! 971 b. S. FREDRICKSON AND R. S. LEES, Circulation.3I (;965) 32r. '- _ '-' '. hl. RURSTEIN AKD J. SAMAILLE, J. Physiol. (Pauis), 49 (1957) 83. P. BERNFELD AND J. S. NISSELBAUM. Fedevalion Pvoc., I5 (1956) 220. P. BERNPELD, M. E. BERKOIYITZ AND V. M. DONAHUE, J. Clin. Invest., 36 (1957) 1363. A. SCAR’U, L. A. LEWIS ANI) I. H. PAGE, J. Lab. C/in. Med., 51 (1958) 325. H.N. ANTONIADES, J. L. TULLIS, L. H. SARGEANT. R.B. PENNELI. AXE J.L. ONCLEY, J. Lab. Clin. Med., 5’ (1958) 630. W. G. DANCERFIELD AKL) G. FAULKNER, Clin.CAim. Acta, IO (1951) 123. W. J. JORDAN, JR., A. G. FAULKNER AND E. C. KX~BLOC~C, .4nal. B&hem.. ‘4 (1966) 91. A. LOPEZ-S., S. R. SRIVI\~~SAN,B. RADHAKRISHNAMURTHY AND G. S. BERENSON, Federation

Pi%, 28 (1969) 5’5. S. R. SRIXIVASAN, A. LOPEZ-S;.,B.RADI~AKRISHKAMURTHY AP~DG.S. BSRENSOX, J.Atkevoscier. lies., 12 (‘97~‘) 321. 12 P. C. ARQL~EMBOURG, J, E. SALV.~(;GIOASD J. N. BICKERS, Primtfv of Immunorlectrophorc,sls. S. Karger, 1~1~1, 1970. I .3 S. PEARSON, S. STERX AND T. II. hfcCi.tv.4CK. Anal. Chrm., 25 (1953) 813. ‘4 W. A. KREXL, A. LOPEZ-S. ASD E. I. GOOD, Amer. J. Clin. Nuts., 20 (1967) 968. 15 R. P. NOBLE, J. Lipid Res., 9 (1968) 693. I6 R. STR~U~ AND M. WURM. in F. W. SUNDERMAX AND F. W. SUNDERMAN, JR. (Eds.), Lipids a:hd the Steroid Hormones in Clinical Medicine, Lippincott, Philadelphia, 1960, p. 47. I7 C. A. MCMAHAN, Rudimepzts ojRiometv,y, Edwards Brothers, Inc., Ann Arbx, Mich., 1967. 18 R. S. LEES, Clin. Res., 17 (1969) 388. 19 E. B. SMITH, Lawcet, ii (1962) 530. 20 M. BURST&IN AND J. SAMAILLE. Cliu. Chim. Acta, 5 (1960) 609. 21 H. S. MCDONALD AND L. P. RIBEIRO, Ckn. Chim. Ada, 4 (1959) ~58. 22 K. A. NARAYAN, S. NARAYAN AND F. A. KUMMEROW, Nature. 205 (1965) 246. 23 S. RAYMOND, J. L. MILES AND J. C. J. LEE, Science, 151 (1966) 3~6.

24 W. P. JENCKS AND E. L. DURRUM, J. 2.5 B. S:VAHN, &and. J. Clin. Lab. Invest.,

CEin. IwesI., 3.1 (1955) 1437. 5 (Suppl. 9) (1953) 7. zb F. T. HATCH, J. L. MOORE, F. T. LINDGREN, N. K. JENSEN, N. K. FREEDMAN AND R. D. WILLS, Civculalion, 36 (1969) I I. 27 R. P. NOBLE, F. T. HATCH, J. A. MAZRIMAS, R. T. LIX~GREN, L. C. JENSEN AND G. L. ADAM-

SON, Lzpids, j (1969) 55. 28 D. S. FREDRICKSON, R. I. LEVY AND R. S. LEES, New En&. J. Med., 276 (1967) 148. 29 R. P. NOBLE, Circulation, 37, 38 (Suppl. 6) (1968) 18. 30 D. F. BROWN AND J. T. DOYLE, Amer. J. Clin. Nutr., 20 (1967) 324. 31 R. E. HODGES AND W. A. KREHL, Amw. J.Clin. Nutr., 17 (1965) 200. 32 A. LOPEZ-S., W. A. KREHL AND R. E. HODGES, Amer. J. Clin. Nuts., 20 (1967) 808. 33 D. H. BLANKENHORN, H. P. CHIN AND F. Y. K. LAW, Ann. Internal fifed., 69 (1968) 21. Clin. Chim. Acta.

31 (1971) 123-132