Precipitation of chylomicrons and very low density lipoproteins from human serum with sodium lauryl sulfate

Life Sciences Vol . 11, Part II, pp. 177-184, 1972 . Printed in Great Britain

Pergamon Preas

PRECIPITATION OF CHYLOMICRONS AND VERY LOW DENSITY LIPOPROTEINS FROM HUMAN SERUM WITH SODIUM LAURYL SULFATE

M. Bursteln and H. R. Scholnick Centre National

de Transfusion Sanguine, Paris, France

(Received in final form 3 January 1972) SUMMARY With appropriate concentration of sodium lauryl sulfate, VLDL from clear human sera and chylomicrons plus VLDL from ligemtc sera are specifically precipitated at 35 C . The precipitated lipoproteins float on centrifugation and a clear subnatant is obtained . This precipitation requires the presence of a non-dialyzable thermolabtle serun factor . Human serum lipoproteins are divided Into several groups : high density lipoproteins

(HDL) with density greater than 1 .063 g/ml and migrating as alpha

globulins ; low density lipoproteins

(LDL) with density range 1 .006 to 1 .063

Sf 0-20, and beta electrophoretic mobility and lipoproteins with density less than

1 .006 and Sf>20.

proteins

The last group Is subdivided Into very low density lipo-

(VLDL) with Sf 20-400 and chylomicrons with Sf~+00 .

origin) are found In low concentration in fasting normal in hyperlipemic states

VLDL (endogenous

sera and are Increased

(type IV and V) ; the Sf>400 particles (chylomicrons)

appear in serum of normal~subJects only after a fatty meal

and are found In

fasting sera from patients with types I and V hyperltpoprotelnemias .

LDL and

HDL are cholesterol ester and phosphollpld-rich, while VLDL and chylomicrons are triglycerlde-rich.

The protein content increases, and the triglyceride

content decreases as the density Increases from chylomicrons to HDL .

The sera

from patients with extrahepatlc biliary obstruction or blllary cirrhosis may contain abnormal

low density lipoproteins which have beta electrophoretic

Present address : Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, New York 10461, U.S .A .

177

178

Precipitation ad Serum Lipoproteins

mobility but do not react with anttsera against normal

Vol . il, No. 4

beta lipoproteins

Tha interaction of serum lipoproteins with anionic detergents known.

(1) .

is well

Burstein and Morfin (7) have shown that In the presence of divalent

cations or protamine,

anionic detergents such as sodium oleate, sodium lauryl

sulfate or bi nary salts (sodium dasoxycholate, sodium glycocholate and sodium cholate) precipitate LDL and HDL from human serum.

It will

be shown

In this paper that under certain conditions, chylomicrons and . VLDL are selectively precipitated by sodium lauryl sulfate, without the addition of divalent cations or protamine . Materials and Methods The following sera were used ; clear sera from normal sera prepared by ultracentrifugation (24 hours, triglycerida-rich sera

donors ; VLDL-free

100,000 g,

density 1 .006) ;

(post prandial, type IV, and type V) ; sera of patients

with obstructive Jaundice containing abnormal

lipoproteins .

The following

isolated lipoproteins ware used : chylomicrons and VLDL from lipemic sera chylomlcrons were

(the

isolated by centrifugation for 2 hours at 40,000 g and the

VLDL by ultracentrifugation of the chylomicron-free infranatant) ; VLDL from clear sera ; abnormal

lipoproteins Isolated from Jaundiced sera by precipitation

with heparin and MgC12 as previously described (2) . Triglycarides were determined by the method of Eggstein and Krenz (3) . Zonal electrophoresis was performed on agarose (4) and thin

layer chromato-

graphy (5) was run on sTlicic acid with a solvent system containing petroleum ether : diethyl ether: acetic acid,70 :25 :2 . Precipitation of Chylomicrons plus VLDL with S odium Laury l Sulfate. To one volume of fresh human serum was added 0.075 volumes of 10~ S .L .S . (final

concentration 0 .75°6) ; the mixture was incubated in a water bath for two

hours at 35°C.

Turbldity appeared in several minutes and increased with time .

After two hours, the serum was centrifuged for 10 min at 10,000 g ; the pracipitated lipoproteins floated and the clear subnatant was removed by aspiration and/or filtration .

Both the turbidity and the size of the creamy precipitate

Yol. 11, No . 4

Precïpitatioa ad Serum Lipoproteins

179

were visibly greater In triglycerlde-rich sera than in normal clear sera . The precipitation of chylomicrons plus VLDL Is selective ; there was no precipitation of LDL or HDL (no Increase of turbidity when S .L .S . was added to

a d 1 .006 infranate) and no precipitation of abnormal

lipoproteins (small

increase in turbidity with serum of obstructive Jaundice, no further increase in turbidity when Isolated abnormal lipoproteins were added to clear normal serum) . The optimal concentration of S .L .S . for both clear and lipemic sera is between 0.6 and 0 .75% ; with 1 .5 to 2 .OX there was no precipitation to either clear or lipemic sera .

The optimal concentration Is not related to the

amount of chylomicrons plus VLDL but is In fact related to the concentration of other serum proteins ; in a serum diluted 4-fold, with normal saline, the optimal final concentration was lower .

The precipitation is temperature

dependent, more complete at 35°C than at 20°C and is not affected by change of the pH of the serum in the range 6 .6 to 8 .8 . Table 1 demonstrates the decrease in triglycerlde concentrations of S .L .S . treated normal and triglycerlde-rich sera .

The drop In triglycerides

ranges from 20% In the normals to as much as 70~ In some of the hypertrtglyceridemic samples .

Precipitation ad Serum Lipoproteins

180

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TABLE 1 Trtglycerides (mg/100 ml serum) before (a) and after (b) S .L .S . precipitation In various normal and trtglycertde-rich sera (values after S .L .S . were corrected for dilution) . a

b

a

b

a

b

68

54

205

98

380

110

71

55

205

107

390

110

79

62

205

102

390

160

89

54

220

105

390

167

94

75

225

108

400

105

96

77

264

111

410

170

103

67

270

105

410

185

104

75

285

155

440

160

108

65

290

118

480

172

113

78

298

155

500

230

115

76

320

120

545

162

145

102

330

135

555

183

180

83

345

125

575

270

183

93

364

130

590

160

200

102

376

150

610

220

200

110

380

98

630

188

Thin layer chromatography (Fig . 1) reveals that in a type IV serum only the trtglyceridas decrease after S .L .S . precipitation ; there is no significant change to free and esteriftsd cholesterol or in phospholTpids .

Vol . ü, No. 4

Precipitation ad Serum Lipoproteins

181

Fig . l . Thin layer chromatogram of lipid extracts from Type IV serum before S .L .S . (left) and the same after S .L .S . (right) . From below, phospholipids, free cholesterol, trlglycerldns and esterified cholesterol . In highly lipemic sera there Is only partial precipitation, but after 2 or 4-fold dilution with clear normal serun, the precipitation is complete . Table 2 demonstrates that the pnrcentage reduction in triglycerides Is larger, after dilution of lipemic sera with normal serum .

This would seem to indicate

that a serum factor is involved In the precipitation of chylomicrons and VLDL wl th S . L . S .

182

Precipitation of Serum Lipoproteins

Vol . 11, No . 4

TABLE 2 Triglycertdes (mg/100 ml serum) before (a, al, a 2 ) and after (b, b l ,b2) S .L .S . a and b non-diluted lTpemic sera ; al, a2, b l , b2 ; the same diluted with normal serum ; a l and bl : 1 :2 dtlutton, a2 and b 2 1 :4 dtlutton . (Values after S .L .S . ware corrected for dilution with S . L .S .) . a

b

al

bl

a2

b2

960

390

515

155

980

375

530

150

1014

480

550

185

1140

375

610

140

1165

600

620

210

1430

575

750

200

1540

750

805

225

435

100

1560

615

450

120

1680

910

485

160

1970

868

550

155

2400

1515

660

260

Serum Factor The existence of a serum factor was confirmed by studies with isolated chylomlcrons and VLDL .

The isolated lipoproteins were suspended to buffered

(pH 7 .7) 1% sodlum chloride and varying amounts of the 10% S .L .S . solution (neutralized to pH 7 .7) were added .

No precipitate was found with any of the

final concentrations of S .L .S . tried ; 0 .01%, 0 .1%, 0 .25%, and 0 .75%.

However,

the same Isolated lipoproteins were precipitated by S .L .S . when they were suspended in serum instead of sodlum chloride . The factor is thennolabtle, destroyed by heating for 5 minutes at 65 °C . When normal or ltpemlc serum was heated for 5 minutes at 65 °C, and then cooled to 35°C, there was no longer precipitation of lipoproteins after the addition of S . L . S .

Th l s cannot be explained by modification of chylomicrons and VLDL

by heating, for when unheated isolated lipoproteins were added to heated serum there was also no precipitation with S .L .S .

On the other hand, there was pre-

cipitation with S .L .S . when these lipoproteins were added to a mixture (v/v) of

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Precipitation ad Serum Lipoproteins

189

heated and non-heated serum or when non-heated clear serum (or VLDL-free serum) was added to heated ltpemic serum .

These results are not compatible with the

hypothesis that an inhibitor is formed during the process of heating . Furthermore, the serum factor is not dialyzable and its presence can be demonstrated in a serun from which LDL and HDL have been removed by pracipitatton with dextran sulfate and manganese chloride (ti) . 2ona1 electroahorests Under the conditions described ; S .L .S . precipitates triglycertde-rich lipoproteins but not LDL and HDL .

However,

tt can be shown that S .L .S . forms

soluble complexes with LDL and HDL ; Fig . 2 demonstrates that with small amounts of detergent the eleetrophorettc mobility of LDL and HDL but not of the other serum proteins is increased .

On the other hand, LDL and HDL ara precipitated

by S .L.S . to the presence of protamine (7) .

FIg . 2 . Electrophoresis on agarose gel . 1, 2, and 3, protein stain ; 4, 5, and 6, lipid stain. 3 and 6, normal human serum ; 1, 2, 4, and 5, the same serum with S .L .S . added ; 2 and 5, 0 .1% ; 1 and 4, 0 .2%. Other anionic detergents such as sodium oleate and sodium desoxycholate also increase the electrophoretlc mobility of serum lipoproteins and precipitate

184

Precipitation ad Serum Lipoproteins

all serum lipoproteins In the presence of protamlne .

Vol. 11, No . 4 However,

the selective

precipitation of chylamicrons and VLDL does not occur with these detergents . Discussion " Selective precipitation can also be obtained by using heparin and MgCl 2 , but unlike S .L .S ., these reagents also precipitate the abnormal of Jaundiced sera abnormal

(2) .

lipoproteins

As In the case of VLDL, the protein/lipid ratio of the

lipoproteins is low, but the latter ara rich In phosphollpids rather

than in triglycerides .

Thus, with heparin-MgC1 2 the precipitation Is related

to the low protein/lipid ratio and is Independent of the composition of the lipid moiety

(2), but with S.L .S . only triglyceride-rich lipoproteins are

precipitated . The S.L .S . subnatant from lipemlc serum is completely clear but the trlglyceride content can be higher than in normal serum (tables 1 and 2) .

The

clarlficatlon of the serum indicates that all chylamicrons have been elimlnated .

It is known that VLDL (Sf

20-400)

are heterogeneous, having a wide

range of protein/triglyceride ratios and particle sizes ; with decreasing Sf, percentage protein Increases, and triglycerides decrease (8,9) .

It is possible

that VLDL with low Sf are not precipitated by S.L.S . REFERENCES 1.

H .A . Eder, E. M. Russ, R. A. Prltchett, M.M . Wilbert end D. P. Barr, J. Clin . Invest .

2.

~4, 1147 (1955) "

M. Burstein and J. Carols, Rev. Med. Chlr . Mal . Foie .

Rats Pancreas .

44,

125 (1969) . 3.

M. Eggsteln and F. H. Kreuz, Klin . Wschr.

4.

M. Burstein, P. Amouch and J.M . Fine, Nouv .Rev . Fr . Hematol .

5.

H. K. Mangold,

6.

M. Burstein, H. R. Scholnick and R . Morfin, J . Lipid Res .

7.

M. Burstein and R. Morfln, Nouv . Rev. Fr . Hematol . 11,

8.

A. Gustafson, P. Alaupovic and R. H. Furman, Biochemistry .

9.

W. J. Lossow, F . T. Lindgren, J.C . Murchlo, G. R. Stevens and L. C. Jensen, J. Lipid Res. ,

J . Am . Oil Chemists Soc. ,

s,

Ô8 (1969) "

44, 262 (1966) . ~8, 708 (1961) .

s,

8, 809 (1968) .

583 (1970) "

173 (1971) " 4, 596 (1965) .