European Journal of Clinical Investigation (1978) 8, 115-120

Metabolism of apolipoproteins A-I and A-ll and its influence on the high density lipoprotein subfraction distribution in males and females

JAMES SHEPHERD, CHRISTOPHER J. PACKARD, JOSEF R. PATSCH, ANTONIO M. GOTTO, Jr & 0. DAVID TAUNTON, Division of Atherosclerosis and Lipoprotein Research, Department of Medicine, Baylor College of Medicine and The Methodist Hospital, Houston, Texas, U.S.A.

Received 14 October 1977 and in revised form 28 December 1977

Abstract. Rate zonal ultracentrifugation of plasma samples from ten healthy age-matched volunteers (five males, five females) indicated that the high density lipo- protein subfraction ratio (HDb:HDL3) in females was significantly higher than in males. The cause of this phenomenon was investigated by simultaneous examina- tion of the metabolism of the major HDL apoproteins (apoA-I and apoA-11) in both groups. The results show that there is no significant sex-related difference in the plasma pool size, fractional catabolic rate, or synthetic rate of either apoprotein. We conclude that the increased HDL:HDL3 ratio in females versus males does not derive from measurable differences in the metabolic handling of either apoprotein.

Key words. Rate zonal ultracentrifugation, HDL2 and HDL3, fractional catabolic rate ; absolute catabolic rate.

Introduction

Human plasma high density lipoproteins (HDL) are separable into two major subspecies, HDLz (d = 1.063- 1.125 kg/l) and HDL3 (d= 1.125-1.210 kg/l) by rate zonal ultracentrifugation [ I ] . Although the significance of these subspecies is not yet apparent, a number of physiological and pharmacological stimuli are known to influence their respective concentrations in the plasma. For example, others have observed [2] and their study confirmsthat the plasma level of HDL, in premenopausal women is higher than in men, suggesting a relationship between HDL and oestrogen levels [3, 41, and presum- ably accounting for the increase in cholesterol circulat- ing in the HDL fraction of female plasma [5]. A similar increase in HDL2 with respect t o HDLJ is also seen in the plasma of subjects receiving nicotinic acid [6, 71 or clofibrate drug therapy [ 6 ] .

All of the above changes in HDL subfraction distri- bution may be associated with an alteration in the meta-

Correspondence: Dr James Shepherd, Department of Patho- logical Biochemistry, Royal Infirmary, Glasgow G4 OSF, Scotland.

01978 Blackwell Scientific Publications 0014-2972/78/0600-O115$02.00

bolism of one or both of the major apoprotein compo- nents of HDL, apolipoproteins A-1 and A-I1 (apoA-1 and apoA-11). Despite the apparent importance of these two proteins, little is known of their metabolism, although Blum et al. [7] have found that their plasma clearance rates are identical even when HDL subfraction metabolism is perturbed by dietary or therapeutic means. In the present study, we have measured simultaneously the kinetic parameters of apoA-I and apoA-I1 in ten subjects (five male, five female) in order to determine whether the sex-related difference in HDLsubfraction distribution is associated with changes in the metabolism of either or both apoproteins.

Methods

This study was approved by the Human Research Com- mittee of Baylor College of Medicine and The Methodist Hospital.

Subjects Ten healthy normolipaemic young adults (five male,

five female) gave informed consent to the study. None demonstrated clinical, biochemical or haematological evidence of cardiovascular, hepatic, renal or endocrine disease. The subjects ate a normal diet and were examined as out-patients, an arrangement w h c h minimized pertur- bation of their life style and which has been shown [8] t o provide steady-state conditions suitable for turnover studies. For 3 days before and throughout the investi- gation, they received 300 mg of potassium iodide three times daily to prevent thyroidal sequestration of radio- iodide. No other medications (including the contraceptive pill) were given. The subjects were weighed, while fasting, at 08.00 hours daily. Blood was also withdrawn at this time thrice weekly for plasma cholesterol and triglyceride estimation [ 9 ] , and twice weekly for measurement of VLDL, LDL and HDL cholesterol [9] .

Rate zonal ultracen trifugation The ratio of HDL2:HDL3 in the plasma of each sub-

115

1 16 JAMES SHEPHERD et al.

ject was determined by rate zonal ultracentrifugation [ I ] . The HDL subfractions in 15 ml portions of fasting plasma were separated and their relative amounts mea- sured from the areas which they subtended on the zonal elution profile. These profiles showed a typical male/ female distribution difference [ l ] .

Purification and iodination of apolipoproteins A-I and A-11

HDL (d = 1.063-1.210 kg/l) was isolated from pooled normal plasma by ultracentrifugation, delipidated, and the released apoproteins separated by gel filtration as described previously [ l o ] . The purity of the apopro- teins was established by amino acid analysis [ l l ] , acrylamide gel electrophoresis [12], and crossed im- munoelectrophoresis [ 131 . After dialysis against NH4 HC03 (100 mmol/l, pH 8.6), they were lyophilized and stored at -70°C. Specific antibodies were raised in rab- bits against the purified apoproteins 1141.

ApoA-I and apoA-I1 were labelled with 13'1 and '''I respectively by the IC1 procedure described elsewhere [ lo ] . Labelling efficiency for both proteins was approxi- mately 50% and the I/protein ratio in the products was 1/1. The labelled apoproteins were dialysed against bar- bital buffer (50 mmol/l, pH 8.6) prior to incorporation into HDL.

HDL labelling

HDL (d = 1.063-1.210 kg/l), isolated from the plasma of each volunteer by ultracentrifugation 1151, was labelled with [lJII] apoA-I and apoA-I1 by in vifro exchange [ l o ] . ApoA-I1 associated with the HDL in a manner similar t o that of apoA-I.

Turnover study protocol 25 pCi of ['311]apoA-I/HDL and ["'I] apoA-II/HDL

were mixed together, sterilized by membrane filtration, and a weighed volume injected into the bloodstream of the respective donor. The subsequent turnover of the apoproteins was determined as described before for LDL [S], using the mathematical procedures of Matt- hews [I61 and Berson & Yalow [I71 for data analysis. Plasma concentrations of apoA-I and apoA-I1 in each subject were measured by electroimmunoassay [18] at daily intervals throughout the study.

A polipoprotein A-I and A-11 electroimmunoassay A previously described electroimmunoassay procedure

for apoA-I [ 101 was adapted to measure apoA-I in whole plasma samples. 2 ml of an 8.0 mol/l urea/50 mmol/l sodium barbital buffer solution, pH 8.6, were added to 0.1 ml of each plasma sample to be assayed and the apoA-I content of 5 pl of this mixture measured by electrophoresis into a 1% agarose gel containing 2% anti- apoA-I antibody. ApoA-I standards, dissolved in same buffer, were also applied t o each plate. The assay was

linear over the range 25-130 pg apoA-I/ml (r> 0.995) and gave results comparable to those obtained with de- lipidated plasma. The within and between batch coeffi- cients of variation were 3.6% (n = 15) and 4.8% (n = 19) respectively.

ApoA-I1 was quantified in whole plasma by a method similar to that for apoA-I. However, in this case the plasma samples to be assayed were diluted with two volumes of sodium barbital buffer (50 mmol/l, pH 8.6) and heated to 52°C for 3 h as described in an apoA- I radioimmunoassay by Karlin et al. [ 191 . This was found to result in maximum estimation of apoA-I1 immuno- reactivity (the values were approximately 10% higher than untreated plasma and comparable to those measured in delipidated specimens). Standards of apoA-I1 in the same barbital buffer were routinely applied to each plate. The standard curves were linear ( r > 0.990) over the range 30-200 pg/ml. In this assay, the within and between batch coefficients of variation were 4.5% (n = 15) and 3.6% (n = 15).

Results

Plasma lipoprotein values in subjects The clinical and biochemical variables of the ten sub-

jects investigated in this study are presented in Table 1. There was no age difference between the male and female groups, and with the exception of subject B.S. who was obese, their weights were within normal limits with res- pect t o age, height and sex. All had normal lipid profiles according to the Lipid Research Clinics Program criteria [9]. Although there was a significant increase (42%, P < 0.05) in plasma triglyceride in the male group, no difference was observed in total plasma cholesterol. HDL- cholesterol, however, showed the well-established [20, 2 11 plasma concentration differential, being higher (34%, P < 0.02) in the females.

Rate zonal ultracentrifugation of fasting plasma from each subject demonstrated a significant increase in the plasma HDLz:HDL3 ratio (Table 1) in the females (mean increase = 240%, P < 0.001).

Validation of methodology Throughout the study there was n o significant varia-

tion in the body weight, fasting plasma lipid levels, and plasma apoA-I and apoA-I1 concentrations in all subjects (Table 1). Consequently, application of Matthews's procedure [16] was justified. The constancy of the sub- jects' daily urine/plasma radioactivity ratios [ 171 sup- ported this conclusion. Ultracentrifugation of plasma from each subject on day 6 of the study confirmed (Fig. 1A) that both labelled apoproteins retained their associa- tion with a plasma component of d < 1.225 kg/l (i.e. with a plasma lipoprotein); and subsequent gel filtration [22] of the total plasma lipoprotein fraction through 6% agarose (Fig. 1 B) demonstrated that the [ 13'1] apoA- I and ['''I]apoA-II coeluted with HDL. We therefore concluded that the injected radiolabelled HDL probes

METABOLISM OF APOA-I AND APOA-I1 117

Table 1. Lipid values of male and female subjects

Plasma Plasma HDL cholesterol trigly cerid e cholesterol HDL,:HDL, Subject Weight (kg)

(Sex) Age (yr) (n = 15) (mmol/l) (mm ol/l) (mmol/l) ratio

B.S. (m) 27 116.2 t 0.5 4.00 1.48 0.96 0.0503 F.G. (m) 29 82.5 i 0.7 4.75 1.12 0.98 0.0941 G.B. (m) 25 69.7 i 0.4 4.52 0.80 1.29 0.1947 J.B. (m) 23 80.5 i 0.5 4.16 0.90 1.11 0.1076 K.W. (m) 24 68.4 i 0.6 4.42 0.73 1.34 0.1298 Mean i 1SD 25 k 2 83.5 i 19.4 C.C. (f) 29 58.2 i 0.4 4.50 0.68 1.63 0.3891 I.J. (f) 25 70.1 i 0.7 3.93 0.62 1.32 0.4682 M.W. (f) 27 70.0 f 0.2 4.08 0.64 1.32 0.2665 S.S. (f) 25 56.3 i 0.5 4.24 0.73 1.68 0.4781 S.W. (f) 24 57.4 i 0.5 4.26 0.68 1.71 0.3425 Mean f 1SD 26 f 2 62.4 i 7.0 4.20 f 0.21 0.67 f 0.04 1.53 f 0.20 0.391 i 0.091 Significance

4.37 f 0.30 0.95 i 0.20 1.14 f 0.17 0.115 f 0.053

( f test) NS* P < 0.05 NS P < 0.05 P < 0.02 P < 0.001

FRACTION FROM TOP

0 1 2 3 4 5 6 7 0 9 1 0

FRACTION

7 9 2 .o r(

W 1 5 2 f P 'or 3

5 1 - 1

z

V

*Not significant.

maintained their integrity in vivo, a conclusion substan- tiated by the finding that the radioactivity was distri- buted in the body in a manner consistent with its con- tinued associated with a high molecular weight species such as HDL.

Kinetic parameters of /'3'I/apoA-I/HDL and /'"I/ apoA-IIiHDL metabolism in males and females

Typical plasma clearance curves of [ 1311] apoA-I/HDL and [12'1] apoA-II/HDL are shown in Fig. 2 and the kinetic parameters of their metabolism, derived by Matthews's procedure, are presented in Tables 2 and 3 respectively. Both decay curves could be resolved into twoexponential components, indicative of a two-compartment system.

(a) ApoA-1 metabolism (Table 2 ) The half-life of apoA-I and its intercompartmental

(3 1.0 z z a z w rx W m 0

o A - I 0 A - I I

-

n n + 0.1 w u

-

5 f,

L U LL :: 001

Figure 1. Location of [13'I]apoA-I and ['2SI]apoA-11 in the plasma on day 6 after injection. 6 ml of plasma from subject I.J. were ultracentrifuged a t d = 1.225 kg/l t o isolate the buoyant lipoprotein fraction by flotation. 1 ml portions were removed progressively from the top of the centrifuge tube and counted in a scintillation spectrometer (A). The isolated lipoproteins were then concentrated and fractionated by gel filtration on 6% agarose [22]. The elution profile with associated radioactivity is shown (B).

0 2 4 6 8 1 0 1 2 1 4 DAYS

Figure 2. Typical plasma clearance curves of apoA-I and apoA-11.

1 18 JAMES SHEPHERD et al.

Table 2. Kinetic parameters: apolipoprotein A-I turnover

Fractional catabolic rate Plasma apoA-I concentration ApoA-I (b) Urine/plasma Absolute catabolic rate?

half-life Per cent of tracer ratio (f 1SD) (g/l f ISD) Subject (n = 15) (days) intravascular (a) Calculated (n = 15) mg/kg/day nmol/kg/day

B.S. F.G. G.B. J.B. K.W. Mean +_ 1SD C.C. I.J. M.W. S . S . S.W. Mean f 1SD Male/female Comparison (t test)

1.22 f 0.06 3.08 64.6 1.18 f 0.01 3.39 59.9 1.36 f 0.07 4.38 64.8 1.27 f 0.03 4.82 64.9 1.30 f 0.13 4.13 64.4 1.27 f 0.07 3.96 f 0.71 63.7 f 2.14 1.46 f 0.06 4.92 65.6 1.30 f 0.04 4.81 56.9 1.11 k 0.08 4.05 60.4 1.42 i: 0.01 4.80 67.2 1.50 f 0.07 3.96 64.5 1.36 i: 0.16 4.51 * 0.46 62.9 i: 4.2

0.352 0.411 0.277 0.242 0.288 0.314 f 0.067 0.239 0.287 0.3 11 0.230 0.300 0.273 f 0.037

0.331 f 0.04 0.358 f 0.04 0.217 f 0.03 0.194 f 0.03 0.228 f 0.02 0.266 f 0.074 0.254 f 0.03 0.260 i 0.04 0.267 f 0.03 0.264 f 0.03 0.252 f 0.02 0.259 f 0.006

17.2 607 19.4 685 15.1 533 12.3 434 14.9 526 15.8 f 2.7 14.0 494 14.9 5 26 13.8 487 13.1 462 18.0 635 14.8 * 1.9

557 i: 94

521 f 68

NS* NS NS NS NS NS NS

*Not significant. ?Derived from calculated FCR.

distribution were similar in the male and female groups (T+ k 1 SD in males = 3 9 6 ? 0.7 1 days, and in females = 4.51 f 0.46 days; per cent apoA-I intravascular in males = 63.7 k 2.1, in females = 62.9 ? 4.2). Furthermore, no difference was detected in the calculated fractional clearance rate (FCR) of apoA-I from the intravascular compartment of the male and female subjects (31.4 5 6.7% and 27.3 f 3.7% of the intravascular apoA-I was catabolized per day in the respective groups). Nor was the FCR, as determined from the daily urine/plasma

radioactivity ratio, significantly different in both groups. However, when the male and female data were analysed together, the FCRs derived from the urine/plasma ratios were statistically lower (12%, P < 0.05) than the values obtained by the mathematical procedure of Matthews

Although the plasma apoA-I concentration in the female group, as measured by electroimmunoassay, was 7% hgher than in the males (136 ? 16 mg/100 ml versus 127 _+ 7 mg/100 ml), the difference was not significant.

[161.

Table 3. Kinetic parameters: apolipoprotein A-11 turnover ~ ~ ~ ~~~

Fractional catabolic rate Plasma apoA-I1 concentration ApoA-I1 (b) Urine/Plasma Absolute catabolic rate? (g/l f 1SD) half-life Per cent of tracer ratio (* 1SD)

Subject (n = 15) (days) intravascular (a) Calculated (n = 15) mg/kg/day nmol/kg/day

B.S. 0.258 i 0.015 4.10 62.5 0.300 0.289 f 0.02 3.10 178 F.G. 0.273 f 0.016 4.18 60.4 0.313 0.294 f 0.04 3.42 196 G.B. 0.286 f 0.016 5.66 57.0 0.240 0.180 f 0.02 2.75 158 J.B. 0.256 f 0.016 6.44 59.8 0.196 0.169 f 0.03 2.00 115 K.W. 0.300 f 0.017 5.41 62.3 0.223 0.186 f 0.02 2.68 154 Mean f 1SD 0.275 f 0.02 5.16 i: 1.0 60.4 * 2 0.254 * 0.050 0.234 f 0.062 2.79 f 0.53 160 f 30 C.C. 0.292 f 0.014 5.93 61.8 0.206 0.226 i: 0.02 2.41 138 I.J. 0.261 f 0.015 5.94 56.1 0.231 0.216 i 0.03 2.4 1 138 M.W. 0.275 f 0.012 5.25 55.7 0.258 0.216 f 0.02 2.84 163 S . S . 0.256 f 0.014 5.84 65.3 0.203 0.230 f 0.02 2.08 119 S.W. 0.336 i 0.019 4.89 62.7 0.246 0.219 f 0.02 3.31 190 Mean i 1SD 0.284 f 0.032 5.57 i: 0.48 59.9 i 3.7 0.229 f 0.024 0.224 f 0.062 2.61 f 0.48 149 * 27 Male/female comparison ( t test) NS* NS NS NS NS NS NS

*Not significant. ?Derived from calculated FCR.

METABOLISM OF APOA-I AND APOA-I1 119

Consequently, the absolute rate of catabolism of apoA-I in both groups (the product of FCR and apoA-I pool size) was the same. This value (ACR in males = 15.8 * 2.7 mg/kg/day = 557 f 9 4 nmoles/kg/day, and in females = 14.8 f 1.9 mg/kg/day = 521 f 68 nmoles/kg/day) is equivalent to the rate of synthesis of the apoprotein under the steady-state conditions of the study.

(b) ApoA-ll metabolism (Table 3) As was found for apoA-I, the kinetic parameters of

apoA-I1 metabolism in the males and females were not statistically different. The mean plasma apoA-I1 concen- tration in the females were higher than in the males (3.2%), but not significantly so; nor were statistically significant differences seen in the half-life, the inter- compartmental distribution, the fractional catabolic rate (whether determined by calculation from the plasma decay curves or from the urine/plasma radioactivity ratios), or the absolute catabolic rate of the apoprotein. Again, however, the urine/plasma ratios of both groups taken together were lower (by 9%) than the calculated FCRs, although only significant at the 10% level.

Discussion

Although there is not complete unanimity [14, 231, it is now widely accepted, from various studies [19,20, 24-26] that the plasma concentration of apoA-I in fe- males is significantly higher than in males. In the present investion, we have also noted this sex-related difference in apoA-I concentration, although, because the com- plexity of the procedure limited the number of subjects, the difference did not reach the Ievel of statistical signi- ficance. With respect to the concentration of apoA-I1 in the plasma, no such difference has been noted between the sexes [26,27]. Our results support this finding.

Since it is known that the molar ratio of apoA-I: apoA-I1 is higher in HDLz than in HDL3 [27, 281, it is reasonable to propose that the difference in the plasma HDL,:HDL3 ratio between males and females may arise from sex-related differences in the metabolism of apoA-I and apoA-11. However, kinetic analysis of the [1311] apoaA-I/HDL and [12sI] apoA-II/HDL turnover data indi- cated that metabolic handling of each protein was similar in both sexes (Tables 2 and 3). Neither protein demon- strated a difference with respect to its plasma concentra- tion, half-life, intercompartmental distribution, or the fractional or absolute rate of catabolism. Incidentally, we noted that the fractional rates of catabolism of apoA-I and apoA-I1 determined by Matthew's procedure [16] were higher (12% and 9% respectively) than the values obtained from the urine/plasma radioactivity ratios, a finding described by others in relation to apolipopro- tein B metabolism [29-321. We have no ready explana- tion for this finding.

When the kinetic parameters of [ '311]apoA-I decay are compared with those of ['251]apoaA-II, two points of interest emerge. First, the clearance rates of the apoproteins are highly correlated (r = 0.967), suggesting

that they may be removed from the plasma by the same catabolic process as, for example, would occur were each HDL subfraction handled metabolically as a unit. Secondly, the mean FCR of apoA-I (determined either by calculation or from U P ratios), when compared with that of apoA-I1 is increased by approximately 20% over the latter (P < 0.0 1). This is in accord with our unpub- lished observations that apoA-11, when incorporated into HDL in vitro, is metabolically indistinguishable from the same protein labelled in situ in the lipoprotein. Conversely, apoA-I incorporated in vitro, which represents only two-thirds of the total HDL apoA-I complement, is cleared from the plasma approximately 20% faster than its endogenous counterpart [ 101 . When this is taken into account, the fractional clearance rates of the apo- proteins are not measurably different. Consequently, the ratio of their synthetic rates (the product of pool size and FCR), becomes equivalent to that of their molar concentration in the plasma (molar ratio of apoA-I/ apoA-I1 in plasma = 2.9/1).

From these observations that there is no measurable difference in the metabolic handling of apoA-I and apoA- I1 in males and females, we conclude that the sex-related difference in HDL2:HDL3 ratio does not occur by the mechanism proposed above. In the light of our finding [33] that there is a dynamic relationship between the protein components of HDL, and HDL3, we speculate that protein redistribution between the HDL subfractions may make a more important contribution to the sex- related fluctuations in their plasma levels.

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