Atherosclerosis, 83 (1990) 187-l 96 Elsevier Scientific Publishers Ireland,

ATHERO

187 Ltd.

04504

Plasma lipids, lipoproteins and apolipoproteins hypobetalipoproteinemia Jean-Franqois

in two kindreds of

I, Colette Serougne *, Denis Y. Dubois Lontie ‘, Claude L. Malmendier Christiane Dachet 3, Jacqueline Ferezou * and Denis Math6 3

‘,

’Fond&ion de Recherche sur I’Athe!roscl&ose, Brussels (Belgwm), ’ Laboratoire de Physiologie de la Nutrition, Univers& de Paris-Sud, Orsay (France), and ’ Unite de Recherche SW les DyslipidPmies et I’AthhrosciProse, INSERM

(1.32, CrPteil (France)

(Received 10 October, 1989) (Revised, received 13 February. 1990) (Accepted 4 April, 1990)

Summary Plasma lipids and apolipoproteins were quantified in two kindreds of hypobetalipoproteinemia. All affected members were asymptomatic but showed a decrease of 75% in apolipoprotein B and of 69% in LDL-cholesterol. There were no major changes in apo A-I and A-II but all affected family members had reduced levels of apo C-II (by 58%) and C-III (by 59%) without significant decrease in apo C-I and no specific decrease of apo C-III,. Apolipoprotein E is decreased in SDS-PAGE. The plasma level and phenotype of Lp(a) are not affected by HBL, suggesting that a catabolic rather than a synthetic mechanism is responsible for the disease. As shown by density gradient ultracentrifugation, HDL, particles that contain essentially apolipoprotein A-I, cholesterol and phospholipids represent in affected subjects the major part of HDL. Due to the net reduction of apolipoprotein B-containing particles (VLDL and LDL) as acceptors of lipids in HBL, there is an accumulation of large particles rich in cholesteryl esters.

Key words:

Hypobetalipoproteinemia; Lipoproteins; tion; Immunoaffinity chromatography; electrophoresis; Immunoblotting

Introduction Familial considered

hypobetalipoproteinemia a genetic disorder distinct

(HBL) is from classi-

Correspondence to: Prof. CL. Malmendier, Research Foundation on Atherosclerosis, Faculty of Medicine, Bat D, rue Evers 2. B-1000 Brussels, Belgium. 0021-9150/90/$03.50

0 1990 Elsevier Scientific

Publishers

Ireland,

Apolipoproteins; Density gradient ultracentrifugaFPLC gel filtration; Two-dimensional immuno-

cal abetalipoproteinemia (ABL) [1,2]. It is characterized by sharply reduced levels of plasma cholesterol, low density lipoprotein cholesterol (LDL-C), apolipoprotein B [l] and triglycerides [1,3-51. Whereas apolipoprotein B levels are reduced [6,7], the other apolipoproteins (A-I, A-II, C-II, C-III) are slightly or not affected [Alaupovic, personal communication]. The patients are generally asymptomatic and without physical findings. Ltd.

188 Different mechanisms have been proposed to define the cause of the reduced plasma low density lipoproteins in heterozygotes with familial HBL: reduced synthesis or secretion [6,8-lo], increased catabolism [ll], or genetic alterations of the apo B gene that cause truncated variants of apo B [1216]. The purpose of the current report is to provide a detailed characterization of plasma lipids and apolipoproteins in 2 kindreds with 3-generation vertical transmission of familial HBL associated with apolipoprotein C-II and C-III deficiencies. Material and methods Column chromatography

Total plasma lipoproteins were isolated from 4 ml plasma by ultracentrifugation at d = 1.225 g/ml. The supernatant (in 2 ml) was chromatographed without prior dialysis on Superose 6 Prep Grade column using an FPLC system (Pharmacia) as described in [17]. Density gradient ultracentrifugation

The density gradient ultracentrifugal procedure described by Chapman et al. [18] was followed. The gradient was prepared in 12 ml polyallomer tubes layering successively 1.4 ml distilled water, 1.5 ml d= 1.006, 1.5 ml d = 1.019, 2.5 ml d = 1.063, 3 ml d = 1.12, and 2 ml plasma at d = 1.21 g/ml. After a run at 40 000 rpm and at 15 o C for 24 h in an SW 41 swinging bucket (Beckman), 24 0.5-ml fractions were removed carefully with a syringe. In order to obtain the density profile realized after ultracentrifugation, a test tube containing 2 ml d = 1.21 instead of plasma was run and the refraction index (proportional to density) was measured in the 24 fractions. Lipid and apolipoprotein

determinations

Lipids (cholesterol, triglycerides and phospholipids) of plasma and lipoprotein fractions were analyzed using Lipid Research Clinics procedures [19]. HDL-cholesterol was measured after precipitating the apo B containing lipoproteins with phosphotungstate and magnesium chloride [20]. LDL-cholesterol was determined from the formula of Friedewald et al. [21]. Total proteins have been determined according to Lowry et al. [22]. Apoli-

poproteins A-I, A-II, B, C-I, C-II, C-III and Lp(a) were quantitated by enzyme immunoassay [23-281. Affinity chromatography

Rabbit antiserum was precipitated by 50% ammonium sulfate and dialyzed against 0.1 M borate, 0.1 M NaCl, 0.1% EDTA, pH 8.0. These Ig fractions were purified by passing through the specific antigen-coupled Sepharose 4B column. The purified antibodies were desorbed with 3 M sodium thiocyanate and immediately dialyzed against 50 mM NH,HCO,, freezed and lyophilized. These purified polyclonal Ig dissolved in 25 mM phosphate, 0.5 M NaCl, pH 6.5, were covalently bound to CNBr-activated Sepharose 4B as described by Cuatrecasas [29]. 250 ~1 fresh plasma was transferred to disposable plastic Econo-column [30] (BioRad) containing 1 ml of immunosorbent: anti apo A-I Sepharose, anti apo A-II or anti LDL-apo B Sepharose 4B. The sample was eluted with the equilibrium buffer 0.1 M borate, 0.1 M NaCl containing 0.1% EDTA (pH 8.0). After the unretained fraction was eluted, 3 M sodium thiocyanate was applied to desorb the retained lipoproteins before being re-equilibrated with the borate buffer [31]. The desorbed lipoproteins (retained fraction) were desalted on a Sephadex G25 column (PD-10, Pharmacia) equilibrated with 0.01 M PBS, pH 7.4 containing 0.1% EDTA. Apolipoproteins were determined in the eluates and in the desorbed lipoprotein particles. Two-dimensional

polyacrylamide

gel electrophoresis

Electrophoresis was carried out on whole plasma using the method of Sprecher et al. [32]. A capillary isoelectrofocusing was performed in the first dimension with as carrier Ampholine (pH 4.0-6.5). For the second dimension, electrophoresis was performed using 15% polyacrylamide gel with a stacking gel of 4% polyacrylamide. Isofocalisation

and immunoblotting

The focalization was carried out using LKB Multiphor II apparatus on lipoproteins isolated at d = 1.21 g/ml, dialyzed extensively in PBS 0.01 M, pH 7.4 and delipidated with acetone/ethanol (1 : 1) [33]. The procedure was strictly that described in the LKB manual [34]. Ultrathin plates (0.5 mm) of 4% polyacrylamide were focused in

Ampholine mixture (pH 3.5-5.0 and 4.0-6.5; 1 : 2). The transfer on nitrocellulose membranes was performed using the same apparatus and the apolipoproteins C-II and C-III were detected by the appropriate goat’s antibodies (Daishi, Japan) and revealed with peroxidase labeled anti-goat immunoglobulins (Biosis, France) and 4-chloronaphthol. For the determination of apoprotein E phenotypes, electrofocusing of apolipoproteins from heparin/Mg 2 + precipitated lipoproteins was achieved using the procedure of Utermann et al. ]351. SDS-gel electrophoresis SDS-PAGE was performed on plates covered with discontinuous gradient of polyacrylamide gels (5-10%) for total lipoproteins or VLDL [36]. SDS-PAGE followed by immunoblotting with Lp(a) antibodies was used for determination of Lp(a) phenotypes according to the method of Kraft et al. [37]. A

’

=Q&+a

LPDU

JMD

;

8

AMD

II

CD

III

Fig. 1. Pedigrees (three generations) of the D (upper) and C kindreds with familial HBL. Deceased parents are shown by an arrow. Subjects are designated by (0) males and (0) females; Heterozygous whose apo B concentrations are less than 33 mg/dl by t2 and 8. ? = no lipid or apoprotein determinations available, no mortality from CAD.

Subjects Four members of a Belgian family (GD, AMD, MD and CD) and 5 members of a Moroccan family (MZC, RC, MC, YC, PMC) with low cholesterol and apolipoprotein B plasma levels were diagnosed as heterozygous HBL. They were asymptomatic. The father (LPD) and the eldest child (JMD) of the first family, and the father and 4 children of the second family were normal and had normal or subnormal lipid and apolipoprotein values. The pedigrees (Fig. 1) establish an autosomal form of genetic transmission for the abnormality in these families. Results Lipid and apolipoprotein composition Plasma lipid and apolipoprotein levels of the family members are reported in Table 1. Three members of the D kindred (AMD, CD, and MD) and 4 members of the C kindred (RC, MMC, YC, and PMC) have low or very low levels of total cholesterol, LDL-C, phospholipids, triglycerides, apolipoproteins B, C-II and C-III, but normal values of apolipoproteins A-I, A-II and HDL-C for their age except RC. Apolipoproteins C-I are decreased less than C-II and C-III. The ratio C-I/C-III is increased in the affected members of the D and C families but apo C-II and C-III plasma levels are in the low range of normal values in the unaffected members of the Moroccan family. In the 4th affected member of the D family (GD) and in the mother of the C family (MZC) only LDL-C and apo B are decreased whereas apo A-I and HDL-C are higher than normal. The eldest son (JMD) and the father (LPD) have normal lipid and apolipoprotein values for their respective ages. The LDL-C/HDL-C ratio is low (0.64 + 0.25; range 0.38-1.20) in all affected members compared to normal (2.45 f. 0.94; range 1.76-4.86). The apo A-I/ape B ratio is much higher (8.24 f 2.76 vs 2.18 f 0.39) in all affected members compared to unaffected family members or normal subjects. Lp(a) plasma levels were low in the D family but elevated in the C family except for the youngest child (YC) and one male child (YMC) (Table 1). FPLC chromatography of plasma lipoproteins of the D family is shown in Fig. 2. Similar pat-

1

DATA,

MMC

n = 23

Normal

* HBL affected

M

3.9

10

25

30

39

41

42

43

80

75

46

58

12

66

75

69

members.

0.1

1.2

5

I

11

13

PMC

M

M

HMC

*

F

SC

YC

F

RC *

*

M

M

YMC

14

43

42

16

16

15

F

MZC

19

41

F

M

MC

*

F

MD*

12

Weight

(kg)

Age

46

AND

Height

3 6 2

201* 129+ 133+ 140+ 18Ok 2OOk 18Ok

172

165

175

164

167

152

157

8 6 3 3 2

154k 153k 126+_ 106* 97*

150

120

57

13

I10

206 f 33

6 2

149k

140

142 153+

5

9

12 9

209+11 25Ok12

165

PL

170

(cm) 8

93&37

69k

60+

235

56+

52k

23+

55+

68*

61k

131*15

28k

21+

29+

4

6

3

9

2

3

4

1

3

9

1

5

81 f 12

164k31

76+

TG

AND

9

2

4

6

4

8

6

8

2

4

5

3

6

7

13

199*36

57+

71*

105f

147*

148+

119&

146*

17Ok

164k

166+

102+

985

113f

194k

256&

156kll

Chol

2

5

6

5

1

5

4

3

4

132&30

2*1

195

2

32+_ 6

90f

9Ok

32+

86*

107*

695

94*10

36k

38+

59+

121*13

5 7

47+ 185+

LDL-C

48+11

41*2

40f

68+_

46+_

48k

83k

49*

49*

83*

40+

60+

56zk

49+

4X*

38+

94k

2

3

2

2

5

1

1

4

2

6

6

4

4

2

6

HDL-C

determinations.

APOLIPOPROTEIN

independent

LIPIDS,

and 3 (C family)

LIPOPROTEIN

1 SD for 6 (D family)

LIPID

(yrs)

HC

M

F

CD *

M

AMD

*

JMD

M

M

*

Sex

LPD

CD

Patient

Values are meansf

FAMILIES

CLINICAL

TABLE

7

161*

64+

102*

133+_

113+

114*

156+

123*

103+

203+

107+

2

2

7

6

5

4

6

1

4

2

33

146k12

4Ok8

17+1

30+2

25+1

32+1

31+1

29+1

30+1

31*1

31*2

21kl

28+2

30*1

37*2

139+

8

47*3 33*2 31+2

8

6

Apo A-II

EXPRESSED

137*11

147k

158*

187k

Apo A-I

LEVELS

2

7

5

3

4

6

6

9

3

4

4

8

6

ilk 79*19

12+

2 2

6.5&0.2

2.OkO.l

2.9kO.l

4.610.1

4.9kO.l

5.0*0.1

5.3kO.2

5.5*0.3

5.8kO.4

5.350.3

5.4kO.3

3.1 kO.2

3.2kO.2

3.1kO.l

5.5f0.2

7.2kO.3

6.550.2

Apo C-l

MG/DL

13+_ 3

49+

39+

13+

49*

48f

32+

59*

13zk

15+

33*

64+

lOO+

29*

Apo B

IN

OF

kO.1

*0.1

&1.7

+0.3

2.9

1.8

0.7

0.6

2.1

2.0

0.9

1.9

1.9

1.5

1.4

*1.2

+0.2

+O.l

+_O.l

+0.3

kO.3

+O.l

kO.1

kO.2

kO.2

kO.2

0.59kO.02

0.95+0.01

1.2

2.8

3.5

2.0

Apo C-II

PLASMA

THE

9.7+3.1

3.0 f 0.2

1.850.2

2.0+_0.2

3.9 + 0.2

4.0 + 0.3

3.5 kO.3

3.8 + 0.4

5.5 *0.1

6.7+0.2

5.OkO.l

2.8 k 0.4

2.9*0.4

3.1+0.5

8.8+1.3

12.5& 1.2

9.1 + 0.4

Apo C-Ill

OF

0

3kO.l

44+_3

33+2

61k2

25k2

7*0.5

57+4

33*3

2s*2

2kO.l

2kO.l

4*0.2

13*1

6+0.2

2kO.l

Lp(a)

D AND

C

5;

0

191 AM

LP

C

JM

M

10

08

t

30

50

70

90

110

ELUTION

VOLUME

(ML)

Fig. 2. Column elution profile of lipoproteins isolated by ultracentrifugation at d = 1.225 g/ml from 4 ml plasma. A final volume of 2 ml was injected on Superose 6 Prep Grad Pharmacia column (56 x 1.6 cm) chromatography using FPLC Pharmacia and eluted at 0.75 ml/mm with 0.15 M NaCl, 0.01% disodium EDTA and 0.02% NaN,, pH 7.2. LPD, AMD, JMD, CD and MD represent 5 members of the D family, V = VLDL; L = LDL; H = HDL. The peak without initials represents low molecular weight substances (less than 5000 daltons) and potassium bromide.

terns are observed for the C family (not shown). LDL peak is much reduced in AMD, CD, MD, RC, MMC, YC, PMC, low in JMD, MZC and normal in LPD. This method does not allow to discriminate between LDL-apo B and Lp(a) both eluted at the same elution volume. Density gradient ultracentrifugation profiles Figure 3 shows lipid and apolipoprotein profiles of the fractions separated by density gradient ultracentrifugal procedure in the D family. Cholesterol is low in VLDL and LDL of AMD, CD, MD, RC and MMC, and in all these patients cholesterol and phospholipids are essentially in the first HDL fractions (HDL,: fractions 13-16 of d = 1.063-1.115 g/ml). HDL, and HDL, constituting 9 and 25%, respectively, of total lipoprotein mass in normal subjects and unaffected members rose to 33% and 48% in HBL. Moreover, these HDL, were enriched in cholesterol esters (CE/CT ratio = 0.67 vs. 0.58 in normal subjects). Triglycerides are very low in all lipoprotein fractions of the 5 affected subjects. Apolipoprotein B is low in CD, MD, RC and MMC, and intermediate between these values and normal values in AMD. Apo A-I shows two peaks in JMD, LPD, MC and SC, but 3 peaks in AMD, CD, MD, RC

and MMC (first peak in fractions 13-16). Only one peak is observed for apo A-II corresponding to the second peak of Apo A-I (fractions 16-20). One peak (fractions 13-18) is visible for apo C-II (not shown) for patients AMD, CD, MD, RC and MMC, this peak being larger for JMD, LPD, MC and SC. The peak of apo C-III appears in less dense fractions and more reduced in AMD, CD and MD than in MC and SC. Peak of apo C-III (also apo C-II) is anterior to the major peak of apo A-I in JMD, LPD, MC and SC.

Immunoaffinity chromatography of lipoprotein particles Immunoaffinity chromatography showed as expected a reduction in the apo B containing particles but the distribution between LP-B and LPB : C is not affected. Ninety to 100% of apo C-II and C-III is distributed in the apo A-I containing particles in the affected members of both families compared to 68.9-90.2% in the unaffected members and 74.0~89.8% in 5 normal subjects. The percentage of apo A-I without apo A-II particles (LP-A-I) is increased significantly by HBL (44.1 + 7.3%) vs. 34.3 _+ 3.7% for unaffected members and 30.7 f 2.0% for normals.

192 CHOLESTEROL

PHOSPHOLIPIDS

APOPROTEIN Al

3*0UWrnl

260

300

A

0

APOPROTEIN All

100

1

w/ml

1

10

16

20

26

0

6

APOPROTEIN B

In\

10

20

1

APOPROTEIN Clll

200

80

160 00 100 40

20

20

0

0 0

6

20

26

0

10

II

Fractions

I

10

1s

20

Fractions

Fig. 3. Concentrations (pg/ml of fraction) of cholesterol, phospholipids, and apolipoproteins A-I, A-II, B, and C-III in 25 lipoprotein fractions isolated by density gradient ultracentrifugation in 5 members of the D family (2 unaffected: 0, LPD; and X , JMD, and 3 affected: 0, AMD; * , CD; and X , MD).

Polyaclylamide gel electrophoresis and isoelectric focusing SDS-PAGE of total lipoproteins (Fig. 4A) and of VLDL (Fig. 4B) confirmed the net decrease of apo C, and showed also a decrease of apo E in the affected members of both families (MD, AMD, CD, RC, YC, MMC). Two-dimensional electrophoresis of total plasma (Fig. 5) displayed a similar pattern of apo C-II and C-III for an unaffected member (LPD) and an affected member (AMD) of the D family despite a global reduction of apo C-II and C-III in the HBL patient. Isoelectric focusing followed by immunoblotting (Fig. 6A and B) showed normal apo C-II pattern and the presence of the three major isoforms of apo C-III

(0.1 and 2) in the 3 affected members (AMD, CD and MD) of the D family. The E-types of the D family were 3/2 FS for MD, CD and LPD and 3/3 F for AMD and JMD. The Lp(a) type was S3 for the whole D family. The phenotypes for the C family were MC S3, MZC S2, HC S2/S3, YMC S4, RC S3, SC S2/S3, HMC S2, MMC S2,‘S3. Discussion Most lipid and apolipoprotein values reported in the literature concerned ABL patients who have traces of apolipoprotein B in plasma. In our affected patients, the severe decrease of total cholesterol, LDL-cholesterol and triglycerides, and

2

,BlOO

>*

848

-

MW 45.000 E

:. 8,. .

Al

Fig. 4. SDS-gel electrophoresis in 5-108 polyacrylamide of: (A) Total lipoproteins of the D family and a pool of normolipidemic plasma (NP); VLDL of the same pool. (B) VLDL of the C family. See only traces of apo A-I and A-II compared to total lipoproteins. The band intermediate between albumin and apo E is of - 45000 daltons, corresponding to apo A-IV (in VLDL) and/or to apo E-A-II complex (in total lipoproteins).

the general increase in HDL-C corroborate previous findings [1,3-lo]. The low ratio of LDLC/HDL-C already observed by Glueck et al. [4] may be related to prolonged longevity and decreased morbidity for myocardial infarction in familial HBL. Apo B is always strongly reduced as in our families [6,7]. Whereas the concentrations of both apo A-I and apo A-II have been found lower than normal in HBL [6] or in ABL [38,39] most of members of our families D and C had normal apo A-I and A-II plasma levels for their age and ethnic group except GD, MZC and RC who had high apo A-I levels. Apo C-I, C-II and C-III were observed lower than normal in ABL (381. Despite the few apo C data available in the literature, two facts were noted: the reversal of the C-I/C-III ratio (P. Alaupovic, personal communication) and

Fig. 5. Two-dimensional electrophoresis of total plasma. A = normal pool; B = unaffected member (LPD), and C = affected member of the D family (AMD).

A

B

n”e

;i;,&:

..,_

:.,,,‘~

_’

GIIIG CIII, GIN2 OIlI,

Fig. 6. Isoelectric focusing followed by immunoblotting of (A) apolipoprotein C-II (D family) and (B) apo C-III (C family).

194 the reduction or absence of apo C-III, in VLDL [6] reported in one case of ABL [40]. In our affected patients, the apo C-I/C-III ratio and also the apo C-I/C-II ratio were increased due to the major decrease in apo C-III and apo C-II. In the Moroccan family the ratios C-I/C-III and C-I/CII are even higher in the unaffected members because of normally low apo C-II and C-III values in these populations (unpublished observations) while keeping apo C-I levels similar to those of the Caucasian population. This observation might be related to differences in dietary habits. 95% of apo C-II and C-III concentrations are recovered in the HDL range as observed in the density gradient fractions’ profile and in the affinity chromatography experiments. In GD and MZC apo C-II and C-III concentrations were normal likely due to the increase in HDL (high HDL-C and apolipoprotein A-I). The two-dimensional electrophoretic pattern of apo C-II and C-III was normal, observation not in favour of apo C structural abnormalities. In particular there was no absence of apo C-III,. In the D family (phenotype S3) Lp(a) levels are low as expected [37] but the ratio Lp(a)/apo B is much increased in the affected members. All members of the C family (except YMC) exhibit high levels of Lp(a) as expected in presence of their different phenotypes: S2, S3 and S2/S3. YMC, an unaffected member has a normal Lp(a) level but is of a different phenotype (S4) and comes from another father. It is interesting to point out that HBL does not seem to affect Lp(a) levels. As Lp(a) is constituted from apo B and ape(a) [41,42], this might imply that it is not the synthesis of apo B but more likely its catabolism that is at the origin of reduced plasma apo B levels in our cases of HBL. This also suggests independent catabolic mechanisms for apo B and Lp(a). In the 5 patients (AMD, CD, MD, RC and MMC) studied by density gradient ultracentrifugation a high percentage of HDL,, containing the major part of phospholipids, cholesterol, cholesterol ester, apo A-I, apo C-II and apo C-III was observed. A large increase of HDL, was already observed in ABL [43]. These particles may serve for transport and storage of excess cholesterol and phospholipids in the presence of very low amounts of VLDL and LDL. Apo A-I is implicated in the stabilization of these particles to the exclusion of

apo A-II which is included mainly in the second peak of A-I-containing particles. In conclusion, 4 major facts arose from the analysis of the two families: (a) besides the reduced level of apolipoprotein B, there was in all affected members an important decrease in apo C-II and C-III (ELISA, density gradient ultracentrifugation and SDS electrophoresis) and apo E plasma levels (SDS electrophoresis); (b) no specific reduction was observed in apo C-III,; (c) HBL does not affect phenotype and plasma concentrations of Lp(a) in the patients studied, which is in favour of a catabolic rather than a synthetic mechanism as etiology of the disease; (d) due to the imbalance of lipoproteins resulting from a dramatic reduction of VLDL, IDL and LDL much more HDL, particles were formed containing essentially apo A-I, cholesterol and phospholipids. Acknowledgments The authors wish to thank Dr. Hans-Jtirgen Menzel (University of Innsbruck, Austria) for having performed the identification of phenotypes of Lp(a) and E for the D family, and Dr. Pascal Mermillod (University of Louvain, Belgium) for performing Lp(a) phenotypes for the C family. References Herbert, P.N., Assmann, G., Gotto, A.M. and Fredrickson, D.S., Familial lipoprotein deficiency: abetalipoproteinemia, hypobetalipoproteinemia and Tangier disease. In: Stanbury, J.B., Wyngaarden, J.B., Fredrickson, D.S., (Eds.), The Metabolic Basis of Inherited Disease, Ch. 29, McGraw-Hill, New York, 1983. Ross, R.S., Gregg, R.E., Law, S.W., Monge, J.C., Grant, S.M., Higuchi, K., Triche, T.J., Jefferson, J. and Brewer, H.B., Jr., Homozygous hypobetalipoproteinemia: a disease distinct from abetalipoproteinemia at the molecular level, J. Clin. Invest., 81 (1988) 590. Cottrill, C., Glueck, C.J., Leuba, V., Millett, F., Puppione, D. and Brown, W.V., Familial homozygous hypobetalipoproteinemia, Metabolism, 23 (1979) 779. Glueck, C.J., Gartside, P., Fallat, R.W., Sielski, J. and Steiner, P.M., Longevity syndromes: familial hypobeta and familial hyperalphalipoproteinemia, J. Lab. Clin. Med., 88 (1976) 941. Mars, H., Lewis, L.A., Robertson, A.L., Butkus, A. and Williams, G.H., Jr., Familial hypo-/I-lipoproteinemia. A genetic disorder of lipid metabolism with nervous system involvement, Am. J. Med., 46 (1969) 886.

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