Lp(a) lipoprotein: Relationship to sinking pre-β lipoprotein, hyperlipoproteinemia, and apolipoprotein B

Lp( a) Lipoprotein: Relationship to Sinking Pre-P Lipoprotein, Hyperlipoproteinemia, and Apolipoprotein B John J. Albers, Veneration G. Cabana, G. RussellWarnick, and William R. Hazzard To assess the relationship between the Lp(a) and the “sinking pre-6” (d > 1.006) lipoprotein, the concentration of Lp(a) was quantified by radial immunodiffusion and the presence or absence of sinking pre-8 was assessed by agarose electrophoresis in overnight fasting plasma samples from 485 adults, comprised of 320 with normal lipid levels, 48 with type Ila, 40 with type Ilb, and 77 with type IV lipoprotein phenotypes. The median Lp(a) level was 7.6 mg/lOO ml, 89% (433 of 485) having detectable Lp(a) levels. Twenty-two per cent (107 of 485) had detectable pre-/I lipoprotein in the d > 1.006 plasma fraction (sinking pre-/3). Of the sinking pre-8 positive plasma samples, 96% (102 of 107) exceeded the median Lp(a) level, and sinking pre-@ wos detected in all 44 samples with an Lp(a) concentration exceeding 40 mg/lOO ml. The relationship of Lp(a) and sinking p&3 to lipoprotein phenotype was assessed. Compared to the normolipidemic group, the type Ila group had higher Lp(a) percentile values ( p < 0.02), whereas the Ilb and type IV groups hod significantly lower Lp(a) values than the normolipi-

demic group. Ninety-two per cent (296 of 320) of the normolipidemic subiects had detectable levels of Lp(a) and 22% (70 of 320) had detectable sinking pre-/3 lipoprotein. Ninety-four per cent (45 of 48) of the type Ila plasmas had detectable Lp( a) levels ond 27% ( 13 of 48) had sinking pre-8 lipoproteins. Contrasted with the lla group, only 80% (32 of 40) of the Ilb plasmas had detectable Lp(o) levels and 18% (7 of 40) had sinking pre-8 lipoprotein. In the type IV plasmas, 78% (60 of 77) had detectable Lp(o) and 22% (17 of 77) had sinking pre-8 lipoprotein. Lp(a) or log Lp(a) levels were not correlated with opolipoprotein B levels (n = 485, I = 0.002 or 0.037, respectively). Furthermore, Lp(a) levels remained essentially constant in three subjects whose apoprotein B levels were altered in response to pharmacological and/or dietary manipulation. A fourth subject hod a 50% increase in Lp(a) but this change did not correlate with apoprotein B changes. Thus, these findings suggest that Lp( a) is metobolically independent of low density lipoprotein even though it shares the same structural protein, apoprotein B.

A

MONG IDENTIFIED LIPOPROTEINS, Lp(a) is particularly difficult to classify. It closely resembles low density lipoprotein (LDL) of d 1.0191.063 g/ml in lipid composition,’ with which it shares major antigenic determinants and overlaps in density (principally 1.050- 1.090 g/m1).2 Moreover, the major structural apoprotein of Lp(a) is identical to apolipoprotein B of LDL.“) However, the electrophoretic mobility of Lp(a) on paper4 or agarose’ is pre-/3

From the Departments of Medicine (Division of Metabolism and Gerontology) and Biochemistry, The Northwest Lipid Research Clinic, University of Washington School of Medicine, Seattle. Wash. Received for publication March I I, 1975. Supported in part by PHS contract NHLI-71-2157, PHS grant HL-15263. American Heart Association grant 72-639 (supported in part by the Idaho Heart Association and the Northwest Chapter of the Washington Heart Association); Dr. William R. Hazard is an Investigator of the Howard Hughes Medical Institute. Reprint requests should be addressed to John J. Albers. Northwest Lipid Research Clinic, Harborview Medical Center, 325 Ninth Avenue, Seattle, Wash. 98104. o 1975 by Grune & Stratton, Inc. Metabolism, Vol. 24, No. 9 (September), 1975

1047

ALBERS ET Al.

1048

whereas that of LDL is beta. Furthermore, Lp(a) is not significantly correlated with age, sex, total cholesterol, or glyceride concentration,‘*5 whereas LDL cholesterol or LDL apoprotein B (in females) shows a statistically significant increase with age and LDL apoprotein B levels are highly correlated with total cholesterol and positively correlated with triglyceride.‘j The distribution of Lp(a) in a healthy randomly selected population is highly skewed’ whereas the distribution of LDL cholesterol or apoprotein B is nearly normal.6 The observed quantitative variation of Lp(a) appears to be determined by a combination of polygenic and environmental factors but major gene effects could also be operative.5 An increased prevalence of Lp(a) reactivity in cases of angiographically proven coronary heart disease has been reported.’ Furthermore, Lp(a) has been demonstrated in atherosclerotic plaques.’ The characterization of Lp(a) is far from complete: many questions remain about its metabolic and genetic control, its relationship to normal and abnormal states of lipid metabolism, and its participation in the atherosclerotic process. The purpose of the present investigation was to: (1) compare the immunochemical assay of Lp(a) with an electrophoretic procedure, (2) determine the levels of Lp(a) in hyperlipoproteinemic states, and (3) assess the relationship of Lp(a) to apohpoprotein B. To assess the relationship between Lp(a) and the “sinking pre$” (d > 1.006) lipoprotein, the concentration of Lp(a) was quantified by radial immunodiffusion and the presence or absence of sinking pre-/3 was assessed by agarose electrophoresis in plasma samples from 485 adults, comprised of 320 with normal lipid levels, 48 with type IIa, 40 with type IIb, and 77 with type IV phenotypes. Apolipoprotein B was measured in all subjects by double antibody radioimmunoassay and, together with Lp(a) quantification, also in four subjects whose apoprotein B levels were altered in response to pharmacological and/or dietary manipulation. MATERIALS Subjects and plasma samples. from adult volunteers prevalence NWLRC

survey

after an overnight

being conducted

AND METHODS

samples were obtained (12-14

at the

consists of all employees

who work

containing

in King County,

dry disodium

centrifugation

of the Pacific

(I

mg/ml).

The

For estimation from

of time-to-time

variation

a hypertriglyceridemic at weekly

of Lp(a)

female

intervals

for

from a female and male subject at approximately

lmmunochemicalanalysis.

Lp(a) lipoprotein

those plasma samples which gave reactions method. Apolipoprotein

Elecrrophoresis. Noble”

Lipoprotein

using commercially

prepared

Electrophoresis

Bell Telephone

from

was promptly

study

Company,

aged

into Vacutainer

tubes

separated

by low-speed

wk at 4” in sealed Wheaton

and total

apoprotein

vials

B levels, plasma

2 hr for 24 hr. In addition,

IO wk from

four

healthy

females.

fasting

was

plasma

one male,

and

weekly intervals for 4 mo. was quantitated

B was quantified

by radial

diffusion

according

Laboratories

for 24 hr before use. Staining fraction

immunodiffusion.’

were considered

by a double antibody

was performed

slides from Bio-Rad

of the d > 1.006 plasma

or absence of sinking pre-/3 lipoprotein.

every

in double-gel

electrophoresis

were soaked in 0.05”/, bovine albumin 7B.”

(NWLRC).

analysis.

samples were obtained

quantitative

Clinic

blood was drawn

plasma

conditions’

of the hyperlipidemia

to the clinic. The prevalence

Northwest

Venous

standardized

a portion

Research

at 4°C and stored with O.OSo/;,sodium azide l-4

until immunochemical obtained

EDTA

Lipid

subjects referred

Washington.

under

hr) fast during

Northwest

employees, and from hyperlipidemic

population 20-65,

Plasma

radioimmunoassay. to the procedure

(Richmond,

was performed

was performed

Only

positive in the

to determine

Calif.).

of

Slides

with Fat Red the presence

1049

Lp (a) LIPOPROTEIN

Lipid and lipoprotein Hyperlipidemia

analysis.

was translated

Lipid and lipoprotein into specific lipoprotein

analysis was performed phenotypes

as specifically

as described.‘*6J outlined

by the

Lipid Research Clinic Program.’ Statistical Determination

methods.

Correlations

of statistical

were measured

significance

was carried

using the

Pearson’s

correlation

out using the Student’s

coefficient.

t test or Chi-square

test.

RESULTS

Lp(a) and Sinking Pre-@ Lipoprotein

The distribution of Lp(a) for 485 adult fasting subjects was highly skewed with a mean of 13.4 mg/ 100 ml and a median of 7.6 mg/ 100 ml (Fig. 1). Twentytwo per cent (107 of 485) of all subjects tested had detectable pre-8 lipoprotein in the d > 1.006 infranatant plasma fraction following ultracentrifugation. Fifty-two women and 55 men demonstrated sinking pre-/3 lipoprotein, indicating no effect of sex upon the presence of sinking pre-/3 lipoprotein. Ninetysix per cent (102 of 107) of sinking pre-/3 positive plasma samples had Lp(a) levels above the median level of 7.6 mg/lOO ml and 88% (84 of 107) above 20 mg/lOO ml. Moreover, all 44 subjects with Lp(a) greater than 40 mg/lOO ml had detectable sinking pre-/3 lipoprotein. Lp(a) Levels in Normolipidemic and Hyperlipidemic Subjects

The distributions of plasma Lp(a) levels as determined by radial immunodiffusion for the normolipidemic and hyperlipidemic groups were highly skewed (Fig. 2). For the normolipidemic group (n = 320), the median (50th percentile) was 7.8 mg/lOO ml and the 90th percentile was 38.8 mg/lOO ml (Table 1). Ninety-two per cent (296 of 320) of the normolipidemic subjects had detectable levels of Lp(a) in whole plasma and 22% (70 of 320) had detectable sinking pre-@ lipoprotein (Table 2). Compared to the normolipidemic group, the group

Fig. 1. Relationship of sinking pre-/3 lipoprotein, as detected by electrophoresir, to lp(a) lipoprotein levels quantitated by radial immunodiffusion.

Lp(0)

lipoprotein

(mg/lOOml)

ALBERS ET Al.

NORMOLIPIDEMIC

20 Lp(a)

lipoprotein

40

60

(mg/lOOml)

Fig. 2. Distribution of Lp(a) in normolipidemic and hyperlipidemic subjects.

Table I. Percentile Values of lp(a) (m&100

ml)

Percentile ”

Subject Group

Normolipidsmic

25th

50th

90th

95th

17.2

38.8

51.2

18.7

48.0

59.0

5.8

10.7

27.1

38.4

5.5

16.0

33.5

46.5

320

3.1

7.0

Type Ila

48

5.2

11.6

Type Ilb

40

2.2

Type IV

77

2.1

75th

with type IIa had higher Lp(a) values at all percentile cutoffs examined (Table l), statistically significant (p < 0.02) by the median test. Contrasted with the type IIa group, the IIb group had significantly lower Lp(a) percentile values than the normolipidemic group (p < 0.01) with a lower median (5.8 compared to 8.0) and a much lower 90th percentile cutoff (27.1 compared to 48.0) and only 80% (32 of 40) had detectable Lp(a) levels (Table 2). Similar to the IIb group, the type IV group had statistically significantly lower Lp(a) levels than the normolipidemic group (p < 0.01). Moreover, only 78% (60 of 77) had detectable Lp(a), significantly lower than the normolipidemic (93%) or the type IIa group (94%). Relationship of Lp(a) to Apolipoprotein B and Lipid Levels The apoprotein B levels of three subjects (A, B, and C) were altered sponse to pharmacological and/or dietary perturbation (Fig. 3). Subject

in reA is a

Table 2. Percentage of lp(a) and Sinking Pro-8 Positive Subiects Sinking

Subject

Total

LP(a) Positive

Per cent Lp(0)

Group

Subjects

Subjects

Positive

320

296

93%

70

22%

IIA

48

45

94%

13

27%

IIB

40

32

80%

IV

70

60

78%*

Normolipidemic

*Significantly

different

l

P&?

Per cent Sinking

Positive

Pre-8 Positive

7

18%

17

22%

from normolipidemic and IIA groups by Chi-square test (p < 0.01).

Lp (a) LIPOPROTEIN

1051

120 100 A

80 11 20

~_~~o’-~~~_,~~~o---~~~~~~~..~I”o---o~~P-~~-~~o

0

18

10

2

I 24

Hours

Months 80 60 40 20-

0@

oV’?“““‘V.

d-O_

o~~O__O’O~‘o-o~OIOI~O~~O~~O’~o---O ;....)~...:~.~..i: :;,:..,::.:.:.: .,.. ‘.‘.‘::..::(::..: .;,:.‘..‘1’.,.~., ‘:;:;.::..; :.,:.;:.... ,......,_,( ,.:,: ,..,.;.,:,:(‘;.. _,.,;;.,...,._._. ., ‘. 1

.>:.:.:. .....

1

2

4

Months3

80 60 40 20 ! 0

oI-_o~-~o--oII 2

4

8

10

Fig. 3. Relationship of Lp(a) lipoprotein to apoprotein 8. Solid line closed circles e---e, apoprotem 8; dotted line open circles C--------O, lp(a) lipoprotein. Subject A, hypertriglycendemic 359 old female maintained on 40% fat, 45% carbohydrate, and 15% protein liquid formula diet given in five equal feedings. Subject g is a healthy 23-yr old female who consumed an oral contraceptive during the interval indicated by . Subject C is a healthy 38-yr-old male maintained on an isocaloric liquid formula diet throughout with various dietary compositions and drug thempy as follows: diet: alcohol 20% (of calories), fat 20%. carbohydrate 45%, protein 15%; drug: clofibmto 2 g/day diet: fat 40%. carbohydmte 45%, protein 15%; drug: cloflbrate 2 g/day diet: fat 40%, carbohydmte 45%, protein 15%; drug: none diet: fat O%, carbohydmte g5%, protein 15%; drug: ethinyl estradiol 1 pg/kg/day diet: fat 40%, carbohydrate 45%, protein 15%; drug: ethinyl estradiol 1 pg/kg/day

ALBERS ET AL.

1052

35-year old hypertriglyceridemic female who underwent a study of diurnal variations in lipid and lipoprotein levels, for which she was maintained on 40% fat, 45% carbohydrate, and 15% protein liquid formula diet given in five equal feedings on the zero, third, sixth, ninth, and twelfth hours of the study. Plasma was obtained every 14 hr and apolipoprotein B and Lp(a) lipoprotein levels determined. Within a 24-hr period the apoprotein B level rose 44% (80- 115 mg/ 100 ml), whereas the Lp(a) level remained essentially constant (fluctuating < lo%, within the estimated coefficient of variation of the assay).’ Subject B is a healthy 23-year old female who began taking an oral contraceptive during the 4th wk of the study, continuing on this medication for 12 wk as indicated (Fig. 3). Immunochemical analysis of fasting plasma samples obtained at weekly intervals indicated that her apoprotein level rose approximately 30 mg/lOO ml (70-100 mg/lOO ml) between the 7th and 10th wk, whereas her Lp(a) levels remained constant. Subject C is a 38-year old male with tuberoeruptive xanthomas and known broad+ disease (type III hyperlipoproteinemia) of 3 yr duration, who was maintained on an isocaloric liquid formula throughout the 5 mo study with varying dietary composition and drug therapy as indicated in the legend to Fig. 3. His apoprotein B level dropped the first month (60-40 mg/ 100 ml) in response to clofibrate therapy (2 g/day) and then rose sharply (40-75 mg/ 100 ml), presumably in response to withdrawal of the drug. The apoprotein B levels then began to decline again but rose the latter portion of the 2nd mo from 50-75 mg/lOO ml while maintained on the 40% fat diet and estrogen therapy (ethinyl estradiol, 1 pg/kg/day). Weekly analysis during this 4-mo period showed that his Lp(a) levels remained essentially constant. Subject D is a healthy 32-year old female on no known medication and on a normal diet. Fasting plasma was obtained at weekly intervals for 10 wk. In contrast to subjects A, B, and C, subject D’s Lp(a) level increased by over 50% (40-64 mg/ 100 ml). Furthermore, the variation in apoprotein B, with an assay coefficient of variation of 8%, did not correlate with Lp(a) changes. In the population study (n = 485) Lp(a) levels were found to be independent of apoprotein B levels (Table 3). However, there was a low but statistically significant correlation between total cholesterol and Lp(a). Furthermore, the mean cholesterol levels of the 48 subjects with Lp(a) levels greater than the 90th percentile cutoff of 38.8 mg/lOO ml (defined herein as the hyper-Lp[a] lipoproteinemic group) was 227 mg/ 100 ml, statistically significantly greater (p < Table 3. Correlations

Between

Lp(o) and Apolipoprotein

B or Lipid Levels

Correlation coet%ent

Variables Lp(a)

vs. apolipoprotein

Log Lp(o) tp(o) tog

vs. cholesterol

*Total

0.9 < p < 1.0 0.2 < p < 0.3

0.108

0.02 < p < 0.05 0.01 < p < 0.002

vs. net cholesterol*

0.066

0.1
0.071

0.1

Lp(a) vs. net

Lp(o)

cholesterol

or log TG

vs. TG

cholesterol-tp(o)

tTriglyceride.

0.002 - 0.057 0.146

Lp(o) vs. TGt tog

B

Probability

vs. cholesterol

Lp(a)

Lp(a) tog

B

vs. opolipoprotein

(r)

or log TG cholesterol.

- 0.069; - 0.070 - 0.071;

-

0.073

<

p < 0.2

0.1 < p < 0.2 0.1 < p < 0.2

1053

Lp(a) LIPOPROTEIN

0.05) than the mean of 210 gm/lOO ml for the 437 subjects with Lp(a) levels below the 90th percentile Lp(a) values. However, the net cholesterol (total minus Lp[a] cholesterol, (0.281 x Lp[a] concentration, see’), was not significantly correlated with the Lp(a). Nor was the net cholesterol in the hyper-Lp(a) lipoproteinemic group (209 mg/lOO ml) significantly different than the net cholesterol of the nonhyper-Lp(a) lipoproteinemic group (207 mg/lOO ml). Moreover, there was no statistically significant correlation between Lp(a) and triglyceride (Table 3). DISCUSSION

The present results confirm the close association between elevated Lp(a) lipoprotein levels and the presence of sinking pre-/3 lipoprotein. Both sinking pre-@ and Lp(a) have similar pre-/3 mobilities on agarose. If we assume that the sinking pre-8 lipoprotein generally reflects the presence of Lp(a), then the average threshold for detection of Lp(a) by the electrophoretic method employed would be approximately the 78th percentile cutoff (22% of the population having detectable levels) or 21 mg/ 100 ml. Among 14 subjects with Lp(a) levels of 21 f 1 mg/lOO ml, seven had detectable sinking pre-/3 lipoprotein. When purified Lp(a) was electrophoresed under the identical conditions, 13 mg/lOO ml was the lowest level detectable. Nine per cent (ten of 107) of subjects with sinking pre-@ had Lp(a) levels below this minimum limit. Therefore, we can conclude that on most (91%) but not all occasions the sinking pre-8 lipoprotein may reflect the presence of the Lp(a) lipoprotein. The distribution of Lp(a) in the 320 normolipidemic fasting adults with a median of 7.8 mg/lOO ml and a 95th percentile upper cutoff of 51.2 mg/lOO ml was nearly identical to that reported on a different population of healthy fasting adults in which the median Lp(a) was 8.0 mg/lOO ml and the 95th percentile was 48 mg/lOO ml.’ Ninety-two per cent of the normolipidemic subjects had detectable levels of Lp(a) in unconcentrated plasma consistent with the concept that Lp(a) lipoprotein is a quantitative trait present in nearly all individuals.‘*’ Lp(a) levels appeared independent of apoprotein B levels (r = 0.002). Furthermore, changes in apoprotein B levels in response to dietary and/or pharmacological manipulations were not accompanied by changes in Lp(a). Lp(a) appeared refractory to dietary or pharmacological manipulation. Little physiological variation (total variation equal to or less than assay variation) was found in seven of eight subjects (subjects A, B, C, and three healthy females and one male examined at weekly intervals for 10 wk). These results, together with the findings that Lp(a) is not correlated with net cholesterol (total cholesterol minus Lp[a] cholesterol), triglyceride, or age,‘e5 suggest that Lp(a) is metabolically independent of LDL even though it shares the same structural protein, apoprotein B. The present finding that two apoprotein B-containing molecules may be metabolically independent may be important because other B protein subpopulations may also have different metabolic origins and/or fates than the bulk of the LDL molecules. Furthermore, these data raise the question as to the source and function(s) of the Lp(a) lipoprotein. The previous data suggest: (1) that Lp(a) [containing apoB and the Lp(a) antigen] and LDL [containing apoB but not the Lp(a) antigen] are produced from different VLDL precursor

1054

ALBERS

ET Al.

populations, or (2) that Lp(a) is not a byproduct of VLDL catabolism but is secreted “de novo.” Consistent with the former possibility, we have detected Lp(a) in VLDL by gel diffusion studies. Also, quantitative radial immunodiffusion studies indicated that Lp(a) levels in the d > 1.006 fraction from 35 normolipidemic subjects was 91 f 9”/, of the total plasma Lp(a) whereas type IV subjects (n = 16) had 94 + 6% of the total Lp(a) in the d > 1.006 fraction. Walton et a1.8 recently reported an overall increase in Lp(a) reactivity in hyperlipidemic subjects regardless of the type of hyperlipidemia. These findings are consistent with our findings of increased Lp(a) percentile values for the type IIa group, but contrary to our findings of decreased Lp(a) reactivity in the type IIb and type IV group. Differences in results could be a reflection of the different methodologies (“rocket” electrophoresis versus radial immunodiffusion) or perhaps due to the different populations examined. It should be noted that the “rocket” electrophoresis method has been reported to give a greater intensity of precipitation in sera containing high levels of triglycerides.’ The reasons why we found Lp(a) levels lower in hypertriglyceridemic subjects is not apparent. The type IIa classification of hyperlipoproteinemia is based upon the LDL cholesterol levels which we obtain by a combination of ultracentrifugation and heparin-manganese precipitation.’ Since the precipitation technique precipitates both LDL and Lp(a) lipoprotein (Albers, unpublished observations), Lp(a) cholesterol is included in the estimation of LDL cholesterol. Since the mean Lp(a) concentration of the type IIa group was 18 mg/lOO ml, the Lp(a) contributed on the average ca 5 mg/lOO ml of the total cholesterol and in many cases considerably more. Since a number of ongoing clinical trials select type IIa subjects using the heparin-manganese precipitation method, it is important to recognize this heterogeneity among the lipoproteins which contribute to the total LDL cholesterol in these subjects. REFERENCES I. Albers JJ, Hazzard WR: Immunochemical quantification of human plasma Lp(a) lipoprotein. Lipids 9: 15-26, 1974 2. Albers JJ, Chen CH, Aladjem F: Human serum lipoproteins. Evidence for three classes of lipoproteins in Sr O-2. Biochemistry 1 l:57-63, 1972 3. Ehnholm C, Garoff H, Renkonan 0, Simons K: Protein and carbohydrate composition of Lp(a) lipoprotein from human plasma. Biochemistry 11:3229-3232, 1972 4. Rider AK, Levy RI, Fredrickson DS: “Sinking” pre-beta lipoprotein and the Lp antigen. Circulation 42:(Suppl)III: IO, 1970 5. Albers JJ, Wahl P, Hazzard WR: Quantitative genetic studies of the human plasma Lp(a) lipoprotein. Biochem Gen I I:4755486, 1974 6. Albers J, Cabana V, Hazzard W: Radioimmunoassay of plasma and LDL apolipoprotein B levels. Circulation SO:(Suppl)III:20, 1974

7. Berg K, Dahlen G, Erickson C, Frick H, Furberg C, Wiljasalo M: Pre-&lipoprotein: Relationship to angiographically proven coronary heart disease and to Lp(a) lipoprotein, in Proceedings of the Third International Symposium on Atherosclerosis. Berlin, 1973. p 481 8. Walton KW, Hitchens J, Magnani HN, Khan M: A study of methods of identification and estimation of Lp(a) lipoprotein and its significance in health, hyperhpidaemia and atherosclerosis. Atherosclerosis 20:323-346, 1974 9. Lipid Research Clinics Program, Manual of Laboratory Operations, (vol I) 1974. U.S. Government Printing Office, Washington, D.C. 10. Noble RP: Electrophoretic separation of plasma lipoproteins in agarose gel. J Lipid Res 9:693-700, 1968 11. Hatch FT. Lindgren FT. Adamson CL, Jensen LC, Wong AW, Levy RI: Quantitative agarose gel electrophoresis of plasma lipoprotein: A simple technique and two methods for standardization. J Lab Clin Med 81:946960, 1973