Effects

of Estrogen Therapy on Apolipoprotein in Type III Hyperlipoproteinemia

James M. Falko, Gustav Schonfeld,

Joseph L. Witztum,

Type III hyperlipoproteinemia (HLPj is characterized by the accumulation in plasma of an abnormal cholesterol-enriched lipoprotein WLDL-Chol/VLDLTG ratio > 0.42, VLDL-Chol/total TG ratio > 0.30) that floats in the S, 12-100 range and migrates as /?-VLDL on lipoprotein electrophoresis. Type Ill patients have increased levels of apolipoprotein E and the apoEsubspecies is deficient. while apoEand apoEare increased. The role of the apoE abnormalities in the production of abnormal lipoprotein compositions is unknown. Since estrogens exert a hypolipidemic effect, correct the VLDL composition, and improve rates of remnant removal in type Ill HLP, we investigated whether the administration of estrogen would also correct the apoEdeficiency. Three type Ill subjects (two females. one male) were given ethinyl estradiol 1 pg/kg/day for 6-6 wk and fasting plasmas were tested at 2-wk intervals. Lipoprotein lipids were determined chemically, apoA-I, apoC-II, and apoC-Ill by radioimmunoasaay, and the subspecies of apoC and apoE in VLDL by analytic gel isoelectric focusing (IEFj. In the women total TG, total Chol, VLDL-Chol, and LDLChol decreased whereas HDL-Chol and apoA-I increased. The abnormal lipid ratios reverted to normal and the broad beta lipoprotein decreased or disappeared. Furthermore, the apoE/apoC area ratio (by IEF) in apoVLDL decreased in response to estrogen. This occurred because of a decrease in apoE levels in VLDL. Despite these changes, the apoEdeficiency remained. In contrast to the females, the male type Ill exhibited a progressive hyperlipidemia. By the 8th wk. total-TG had risen to 2387 mg/dl. His apoE/apoC area ratio in apoVLDL increased because of an increase in apoE levels in VLDL. Despite the greet increases in lipid levels, the relative proportions of the apoE subspecies and apoE deficiency remained unaltered. The dissociation between apoE composition and lipoprotein physiology suggests either that apoE plays no important role in the pathophysiology of the dysbetalipoproteinemia and hyperlipoproteinemia, or if apoE does play an important role in the untreated patient, treatment bypasses any “metabolic blocks” due to the apoE abnormalities.

III hyperlipoproteinemia (HLP), T YPE broad-beta disease, is a familial disorder of lipoprotein metabolism associated with cardiovascular disease, tuberoeruptive xanthomatosis, elevations of both plasma cholesterol (Chol) and triglyceride (TG) levels and the accumulation in plasma of an abnormal Chol-enriched lipoprotein found in the Sr 12-100 flotation range’“, which migrates as ,&VLDL on lipoprotein elecMefabdism, Vol.

28, No. 11, (November), 1979

E

Janet Kolar, and Stuart W. Weidman

trophoresis. The presence of this abnormal lipoprotein defines dysbetalipoproteinemia. It is possible to have dysbetalipoproteinemia and normal total TG and Chol levels in plasma, and it is probably useful to distinguish between dysbetalipoproteinemia alone and dysbetalipoproteinemia in the presence of abnormal levels of TG and Chol (hyperlipidemia). The laboratory diagnosis of type III HLP has evolved from its initial definition, i.e., the finding of P-VLDL on electrophoresis and hyperlipidemia, to the quantitation of the abnormality in VLDL composition (VLDL-Chol/VLDL-TG ratio > 0.42, or VLDL-Chol/total-TG ratio > 0.307*8),and more recently, to abnormalities in apolipoprotein E (arginine-rich protein or apoE).9s’o ApoE is composed of several subspecies, which are detectable by gel isoelectric focusing (IEF). Some people have three major subspecies and others have four.“-‘4 In type III HLP, the levels of total apoE in plasma are elevated.” but one of the apoE subspecies, apoEis deficient, whereas apoE- and apoEare increased.“*” Utermann et alI3 have suggested that the apoEIII deficiency is a true qualitative marker for the disease, and work in other laboratories, including ours, has confirmed this.‘&17However, the apoEIII deficiency, as is true for dysbetalipoproteinemia, may occur without hyperlipidemia. Thus, at the present time, the laboratory diagnosis of type III HLP is most firmly made by demon-

From the Lipid Research Center, Departments of Preventive Medicine and Medicine, Washington University School of Medicine, St. Louis, MO. Receivedfor publication February 13. 1979. Supporied in part by NIH Contract NOI-HV-2-2916-L (Lipid Research Clinics Program). NIH Grant HL-15308. and National Science Foundation Grant PCM 74-21871. Dr. Witztum was supported in part by the American Heart Association, Missouri Afiliate. Dr. Falko was supported by U.S. Public Health Service Training Grant lF32HL05566-OI. Address reprint requests to Gustav Schonfeld, M.D., Lipid Research Center, Washington University School of Medicine, Box 8046. 4566 Scott Avenue, St. Louis, MO. 63110. o I979 by Grune & Stratton, Inc. 0026~495/79/2811~015$01.00/0

1171

1172

FALKO ET AL.

strating hyperlipidemia and both the lipoprotein lipid and apoE abnormalities. In addition to the compositional, structural, and apoE abnormalities, there are also abnormalities in lipoprotein kinetics in type III HLP. Following their secretion by intestinal and hepatic cells, chylomicrons (from gut) and VLDL (from gut and liver) enter plasma, where they are degraded to smaller particles, called chylomicron remnants and intermediate density lipoproteins (IDL) (sf 12-l00).‘8-2’ Remnants, particularly those derived from chylomicrons, are taken up by liver, whereas IDL is further metabolized to LDL, and the latter is taken up by peripheral tissues. The uptake of lipoproteins serves to regulate endogenous Chol synthesis in tissues. The accumulation of the SI 12-100 particles in plasma in type III HLP results from delays in their clearance from plasma.63’8-*’This may be due to delays in the conversion of Sf 12-100 particles to LDL, or to the defective uptake of these particles by tissue (e.g., liver), or both. The role of the apoE abnormalities in the production of the abnormal lipoprotein kinetics and compositions is unknown. One way to establish a connection would be to normalize lipoprotein kinetics and compositions by therapy and then to assess whether the apoE abnormalities have also been normalized. Since estrogen therapy reduces lipid levels and normalizes lipoproin type III HLP, we sought tein compositions 22m2g to ascertain whether it also corrects the abnormality in the distributions of the apoE subspe-

ties. For comparative purposes we also examined the behaviors of the subspecies of apoC. MATERIALS AND METHODS

Subjects and Sample Collections Pertinent initial clinical findings of the study subjects are given in Table I. All type III subjects had unequivocal laboratory manifestations of broad-beta disease. None of the patients had clinical or laboratory evidence of an aggravating cause for their hyperlipidemia such as hypothyroidism, renal disease, or clinically manifest diabetes mellitus, and none received any medication for at least 2 mo before and/or during the study period. Patients were followed as outpatients in the Lipid Research Center’s clinic, where both dietary adherence and compliance with medication regimen were monitored by specially trained dietitians and physicians. Informed consent was obtained and the study was carried out by procedures and according to a protocol approved by the Human Studies Committee of our institution. Two females, M. E. and D. D., and a male, J. F., were given ethinyl estradiol I rg/kg/day for 6-8 wk. Blood samples were taken after 14 hr of fasting at baseline, and at 2-wk intervals on medication. During the estrogen treatment all subjects were weight stable on an isocaloric diet (40% protein, 40% carbohydrate, 20% fat, < 300 mg Chol/day, P/S ratio > 1.O).

Analytic Techniques Lipoprotein and lipid analyses. Ultracentrifugations were begun on the same days that bloods were drawn. Plasmas were spun in a 40.3 rotor, and VLDL-Chol and VLDL-TG were measured directly on the ultracentrifugally isolated d -c 1.006 g/ml fractions. HDL-Chol and HDL-TG were determined on the 1.006 g/ml infranates from which LDL had been removed by precipitation with heparin and MnCl,.*’ LDL-Chol was obtained by difference. TG and Chol were assayed by Autoanalyzer II (Technicon Instruments, Tarrytown, N.Y.) in the Core Laboratory of the Washington University Lipid Research Center. Electrophor-

Table 1. Clinical and Chemical Characteristics

of Study Subjects Diagnostic Criteria

Subject

Age

Sex

M.E.

47

F

Ideal

Initial

Initial

Body

Cholesterol

Triglyceride

(mg/dl)

Img/dl)

Weight*

1.37

370

540

Initial Clinical Status

Palmar xanthoma,

ApoE-lll

VLDL-Chd ___ Tofal TG

-VLDL-Chol VLDL-TG

Yes

0.45

0.60

Yes

Yes

0.52

0.67

Yes

Yes

0.45

0.56

Yes

Floating Betat

Deficiency$

peripheral vascular and coronary heart disease D.D.

58

F

1.26

590

712

Tuberoeruptive xanthoma

J.F.

45

M

1.24

820

1396

Palmar, tuberoeruptive xanthoma

*Ideal bodY weight based on Metropolitan Life Insurance Tables. tFloating beta lipoprotein in agarose gel electrophoresis.25 SApoiipoprotein E-III subspecies deletion was quantified by photometric scanning of the stained analytic isoelectric gels. ApoEIll/apoE-II dYe uptake ratios for normals, heterozygotes, and homozygotes are > 1.1, 0.2- 1.1, and 4 0.2, respectively.

ESTBOGEN EFFECT ON apoE SUBSPECIES

csis in agarose

was performed on whole plasma and on d < I .006 g/ml fractions to detect beta-migrating VLDL.2S Apoprotein assays. ApoA-I,% apoC-II, and apoC-III” were measured by previously described radioimmunoassays (RIA). The apoC-III assay measures apoC-III,, apoC-III,, and apoC-III,, which together make up > 95% of apoC-III in plasma VLDL and HDL.” Coefficients of variation of triplicate points for standard curves range from 3% to 7% and average 4%. Intra- and interassay coefficients of variation for plasma samples ranged from 6% to 11% and from 9% to 13%. Gel iswlecfricfbcusing. We have reported details of this technique before.‘6*‘7 Briefly, it consists of the following

steps: (1) isolation of the d < 1.006 or VLDL subfractions by ultraccntrifugation, (2) gel filtration of the fractions through a 0.7 x 20 cm Sepharose 2B column to separate lipoproteins from other plasma proteins (e.g., albumin), (3) lyophilization and delipidation of the lipoprotein (400 pg protein). (4) solubilization of the apoproteins in 0.01 M Tris, pH 8.2, 8 M urea, 0.01 M dithiothreitol, (5) isoelectric focusing in 7.5% polyacrylamide gels containing 8 M urea and 2.0% ampoline (LKB), pH 3.5-10 at -Y’C, (6) staining of the protein bands with Coomassie blue using the method of Malik and Berrie,**

1173

(7) scanning of the gels at 550 nm in a Gilford Model 2400s spectrophotometer equipped with a linear transport accessory, (8) integration of the areas under the peaks on the gel scans with a Hewlett-Packard Model IO calculator equipped with a digitizer, and (9) calculation of area ratios. The linearity of dye uptake with increasing loads of protein has been demonstrated in this system” and loads were always kept within the linear range (IO&400 fig). In -70% of volunteer populations, apoE focuses into four main bands designated apoE-I’, apoE-I, apoE-II, and apoEIII, in order of increasing pi (Fig. 1). In approximately 28% of volunteers without type III HLP, a fifth band (apoE-IV) is present. ApoE-III/apoE-II ratios for type III homozygotes, heterozygotes, and normals are < 0.2, 0.2-1.1, and > 1.1, respectively.” ApoC-II and apoC-III,, are also well separated. ApoB does not enter the gels. RESULTS

Ethinyl estradiol, I pg/kg body approximately equivalent to that contraceptives (SO-100 pg/day). subjects, M. E. and D. D., total

weight/day, is given in oral In the female TG and total

0+

Fig. 1. Effect of estrogen treatment in subject Cl. D. Gels are: (1) apoVLDL before estrogen, (2) apoVLDL after 2 wk of estrogen therapy. (31 apoVLDL after 4 wk. (4) apoVLDL after 6 wk. and (6) apoVLDL in a normal subject.

1174

FALKO ET AL.

Table 2.

Effect of Ethinyl Estradiol on Plasma Lipoproteins Triglycerides (mg/dl)

Time Subject

(wk)

Total

VLDL

M.E.

0

255

179

2

289

174

4

228

145

6

234

160

D.D.

J.F.

and ApoA-I Levels in Type Ill Subjects

Cholesterol (mg/dll

Plasm8 HDL

-VLDL-Chol Total TG

-VLDL-Chol VLDL-TG

61

0.36

0.52

+

101

86

78

0.17

0.28

-

221

47

65

82

0.21

0.32

Yk

152

31

72

90

0.13

0.19

_

216

LDL

HDL

Total

VLDL

LDL

49

20

303

93

151

54

35

220

48

45

52

190

46

38

187

712

558

84

22

590

373

159

31

0.52

0.67

+

103

442

96

34

422

243

122

45

0.40

0.55

+

186

4

542

387

75

48

318

160

103

49

0.30

0.41

+

191

6

413

280

76

40

278

110

111

51

0.27

0.39

_

231

8

515

361

71

59

235

116

74

46

0.23

0.32

_

187

0

614

509

57

18

328

212

85

29

0.34

0.42

+

88

2

842

705

84

30

370

255

76

35

0.30

0.36

+

95 125

4

1045

913

56

38

440

342

59

32

0.33

0.38

+

6

1426

1330

77

31

556

468

66

27

0.33

0.35

+*

73

8

2387

2136

67

35

612

501

65

24

0.21

0.25

+*

120

were also present.

1.006 g/ml fraction by electrophoresis, decreased in intensity or disappeared. There were also estradiol-induced changes in VLDL apoproteins (Table 3, Fig. 1). The dye uptake areas produced in IEF gels by apoE and apoC were measured by planimetry. ApoE areas fell, while apoC areas remained relatively constant. Thus, apoE/apoC area ratios decreased in the VLDL of the women, i.e., apoE fell relative to other VLDL apoproteins. The relative proportions of the apoE subspecies was typical of type III HLP in the basal state, i.e., apoEwas nearly absent, and apoE-III/apoEin VLDL Percent Total ApoC Areas

Percent Total ApoE Areas ApoE-III/ApoE-II

Time

J.F.

(mg/dl)

611

Table 3. The Effect of Estradiol on the ApoE Subspecies

D.D.

Beta

0

Chol both fell during the 6-8 wk of therapy (Table 2). The falls in TG were due to VLDLTG, whereas LDL-TG remained relatively constant and HDL-TG rose. The falls in Chol were due to both VLDL- and LDL-Chol, whereas HDL-Chol rose. ApoA-I levels increased, too. Thus, VLDL and LDL mass fell and HDL mass rose. These results resemble those we have reported for diet and clofibrate therapy in type III HLP.29 The characteristically elevated VLDL-Chol/VLDL-TG ratios of type III reverted to normal. Simultaneously, the broad beta lipoprotein band, detected in the d c

M.E.

ApoA-I

2

lChylomicrons

Subject

Floating

(wk)

E-l’

E-l

E-II

E-III

Area Ratio

ApoW Area Ratio

C-III,

C-II

C-III

1

C-III,

C-III,

0

12.5

25.4

56.9

5.1

0.09

1.07

7.4

15.6

48.8

25.4

2.8

2

17.3

25.2

57.6

0.0

0.00

0.65

9.0

19.9

44.9

24.2

2.4

4

12.2

35.6

52.2

0.0

0.00

0.81

9.0

16.2

50.0

25.4

2.8

6

16.2

28.8

55.0

0.0

0.00

0.49

8.5

11.2

47.8

28.8

3.7

0

9.7

25.8

60.4

4.1

0.07

0.71

15.6

21.0

34.2

26.0

3.2

2

9.2

23.5

66.1

1.2

0.02

0.54

12.8

11.4

42.9

29.9

2.0

4

11.2

28.6

56.0

4.3

0.08

0.44

14.2

11.4

45.6

27.5

1.6

6

10.4

27.5

54.2

7.9

0.15

0.29

11.9

8.6

48.9

27.5

2.7

8

15.1

30.5

48.5

6.0

0.12

0.30

14.2

9.3

46.3

26.9

3.3

0

14.0

25.4

53.8

6.8

0.13

0.48

11.1

16.8

47.6

22.0

2.6

2

12.9

28.7

56.2

2.2

0.04

0.89

10.4

18.0

43.1

26.1

2.4

4

12.3

23.6

53.2

10.8

0.20

0.92

10.6

10.7

52.5

24.3

1.9

6

12.8

25.8

52.6

8.7

0.17

0.83

13.3

10.6

51.2

22.7

2.3

8

12.0

24.2

60.2

3.6

0.06

1.38

11.6

11.4

52.0

22.6

2.5

Urea-soluble VLDL apoproteins were separated by isoelectric focusing, stained, and photometrically scanned. Total apoE areas represent sums of the areas apoE-V-III. Total apoC areas represent sums of ApoC-II + ApoC-Ill (see Table 4). Area units are arbitrary.

ESTROGEN EFFECT ON apoE SUBSPECIES

Table 4.

1175

Effect of Estradiol on Levels of ADoproteins ApoC-II

C-II and C-Ill in Type III Subjects

ApoGlll d>

A&-II

+ ApoC-Ill

subject

(wk)

M.E.

0

7.5

3.0

4.6

33.4

12.6

21.3

0.22

0.19

2

6.0

3.0

2.9

26.6

6.5

21.0

0.23

0.32

4

4.3

1.7

2.9

30.6

10.2

20.5

0.14

0.14

6

4.5

2.4

2.3

25.7

10.2

19.1

0.18

0.19

0

15.5

9.7

2.7

65.3

44.2

17.9

0.24

0.18

2

11.2

8.5

3.3

63.1

39.5

22.2

0.18

0.18

4

8.3

6.0

2.5

54.3

38.8

16.2

0.15

0.13

6

7.9

5.0

2.0

53.2

32.0

18.2

0.15

0.14

8

6.6

5.3

2.0

58.7

29.2

28.8

0.11

0.15

D.D.

J.F.

wp

ApoC-ll

ApoC-Ill

ApoC-Ill

Time d