Acute effects of the pattern of fat ingestion on plasma high density lipoprotein components in man

567

Atherosclerosis, 36 (1980) 567-573 0 Elsevier/North-Holland Scientific Publishers, Ltd.

ACUTE EFFECTS OF THE PATTERN OF FAT INGESTION HIGH DENSITY LIPOPROTEIN COMPONENTS IN MAN

RUTH M. KAY, SURESH RAO, CAROLE ARNOTT, LEWIS

ON PLASMA

NORMAN E. MILLER and BARRY

Department of Surgery, Toronto Western Hospital and Department of Nutrition, University of Toronto, Toronto M5T 2S8 (Canada) and Department of Chemical Pathology and Metabolic Diseases, St. Thomas' Hospital Medical School, London SE1 7EH (Great Britain) (Received 7 December, 1979) (Revised, received 12 March, 1980) (Accepted 13 March, 1980)

Summary Since Apoprotein A-I (apo A-I) is partially synthesized in the intestine and is secreted into plasma via chylomicrons, we have tested the effects of the daily distribution of fat intake on the concentration and composition of plasma high density lipoprotein (HDL). Ten normal subjects ingested 136 g fat either as a single load (SL) at 0 h or in 6 divided doses from 0 to 10 h (DL). Serial blood samples were obtained over a 24-h period. Studies were performed 7 days apart using a double crossover design and paired comparisons. HDL apo A-I increased during DL (+ll% at 9 h, P < 0.01) but was not significantly altered after SL. The HDL Apo A-II concentration did not change. HDL cholesterol decreased significantly (-4 to -7%) during postprandial lipaemia in both phases of the study. Apo A-I and A-II were detected in lipoproteins of d < 1.006 during alimentary lipaemia but not in fasting plasma. These results indicate that HDL apo A-I increases when fat intake is distributed throughout the day (DL), perhaps due to intestinal production of particles with a higher protein/lipid ratio during DL than after a large bolus of oral lipid (SL). Alimentary lipaemia is associated with acute alterations in HDL components including a transient reduction in HDL cholesterol and an increase in the apo A-I/A-II ratio during DL. Key words:

Apoprotein A - Cholesterol Lipolysis -Lipoprotein

Financial SUPPoh

WBS

-

Chylomicron

- High density lipoprotein

-

Provided by DmtS Tom the Medical Research Council of Canada and Uaever

(U.K.) Ltd.

Address co=espondence to: Dr. Street, Toronto, MST 258 Canada.

Ruth

M.

Kay,

Lab 1223,

Toronto

Western Hospital, 399 Rathurst

568

Introduction Apolipoproteins A-I and A-II (apo A-I, A-II) comprise the major protein fraction of plasma high density lipoprotein (HDL). These apoproteins are partially synthesized in the intestine and are secreted into plasma as surface components of nascent chylomicrons [ 1,2]. This suggests that intestinal synthesis of apo A-I and A-II may be influenced by factors regulating chylomicron formation and size (surface/lipid ratio). Lewis and colleagues demonstrated that after large oral fat loads, the rise in plasma triglyceride (TG) was largely accounted for by an increase in chylomicrons (S, > 400). Smaller fat meals resulted in a proportionately greater increase in very low density lipoprotein (VLDL) (S, 20-400) [3]. In the present study we have investigated the acute effects of the pattern of fat ingestion on plasma levels of apo A-I, A-II and HDL cholesterol (HDL-C) in normal volunteers. Subjects

and Methods

The subjects were healthy volunteers aged 15-64 years (9 males, 1 postmenopausal female) within 10% of normal body weight (Table 1). They were non-smokers and were receiving no medication; habitual alcohol intake was less than 15 g/day. Informed consent was obtained from all. Each subject ingested a weighed diet containing the same amount of butterfat (136.2 f 2.3 (SEM) g for different individuals) on 2 occasions 7 days apart using a double crossover design and paired comparison. Lipid, mainly in the form of cream was consumed either as a single bolus at the first meal (SL) or in 6 equally-divided doses at 2-h intervals throughout the day (DL). The cream containers were rinsed with warm water to ensure quantitative ingestion of lipid. During both SL and DL, meals identical in protein and carbohydrate content and composition were given at the same 2-h intervals. During SL, except for the first meal, these contained less than 1.5 g of fat (Table 2). Subjects fasted for 12-14 h before the start of the study and no additional food or beverage except water, clear tea or coffee was permitted during each 24-h study period. Subjects abstained from alcohol during and for 36 h prior to TABLE 1 DESCRIPTION Subjects

RI RM HM EL RL MP SE GE MS KM

OF SUBJECTS Sex

M M M M M M M M M

F

Age (Yr)

Weight

Plasma Chol.

Plasma TG

(4)

(mM P)

(mMP)

31 25 32 24 23 24 17 15 36 64

67 68 59 64 57 66 71 61 73 58

5.50 5.65 3.75 5.25 4.28 4.75 3.23 2.98 5.05 5.35

1.23 3.23 0.68 1.48 0.83 0.75 1.48 0.58 1.38 0.68

569 TABLE 2 DISTRIBUTION

OF NUTRIENT Time

INTAKE Energy

Protein

Carbohydrate

Fat

Cholesterol

&cd)

(g)

w

w

(ma

1386 230 2450

9.8 9.8 58.8

45.0 45.0 270.0

129.7 1.3 136.2

360 2 370

423 2540

9.8 58.8

45.0 270.0

22.7 136.2

61 370

Single fat load 1st meal 5 meals (6 X 2-h) Daily total Distributed fat load 6 meals (6 X 2-h) Daily total

Oh 2-10h

O-10

h

each study, physical exercise was curtailed during each 24-h study period. Blood samples were obtained before and at 3, 6, 9 and 24 h after the start of the first meal. Subjects were seated for 5 min prior to venepuncture to standardize blood volume. Blood was drawn without stasis into sterile “vacutainer” tubes containing EDTA (1 mg/ml). Plasma was immediately separated and stored at 2-4°C prior to analysis. Triglyceride-rich lipoproteins were isolated in a MSE preparative ultracentrifuge. The rotor geometry corresponded to the Beckman 40.3 rotor. Plasma samples in Beckman 6.5ml tubes were spun as follows: chylomicrons, 20,000 X g for 30 min; VLDL, 105,000 X g for 16 h. HDL cholesterol and apoproteins were measured in the supernatant after precipitation of apoBassociated lipoprotein by heparin (1.3 mg/dl) and MnCl, (92 mM) at 4” C [ 41. Any turbid supernatants were treated by centrifuging at 12,000 X g for 10 min. Cholesterol was then determined in the clear subnatant solution. HDL cholesterol was assayed manually by the cholesterol esterase-cholesterol oxidase method [5] after addition of EDTA (pH 7.4, 400 mM, 20 I.rl/ml supernatant) and chylomicron and VLDL triglyceride by the lipase-glycerol kinase technique [ 61. Cholesterol and triglycerides in whole plasma were determined by modified Auto Analyzer AA-II techniques. Apoproteins A-I and A-II were measured by electroimmunoassay, using a modification of the method of Curry et al. [ 71. Monospecific antibodies to A-I and A-II were produced in rabbits as described by Albers et al. [8]. Antibody was incorporated into clear gels consisting of 1.5% electrophoresis grade agarose in 50 mM sodium barbitone buffer (pH 8.6) containing 0.01 g/l sodium azide. Standards and diluted samples were assayed in batch lots. Electrophoresis was carried out at 8 V/cm for 16 h. The within-batch coefficients of variation for the above analyses were 2.5% for cholesterol, 2.7% for HDL cholesterol and 3% for triglycerides, apo A-I and A-II. Statistical analyses utilized Student’s t-test for paired comparison and linear regression analysis. Results The diets were consumed completely and were well tolerated. Plasma total lipids - Plasma cholesterol increased by

5% (P<

0.01)

Apoprotein

Ok++---?4

-q-

..__ ,. . 14h FastI Ime (nj

1--

-12L

-

A-I

Distributed fat load Single fat load

Fig. 1. Post-prandial changes in plasma total Iipids (mean. SEMI. Si$niflcance of change from baseline value: * P < 0.05, ** P < 0.01. n = 10. Fig. 2. Changes in apoprotein A-I concentrations in HDL fraction after single fat load and during distributed fat load, mean, SEM. n = 10. ** Significant increase. P < 0.01.

following ingestion of the single load of fat (SL) but no significant changes occurred during DL. Plasma triglyceride levels increased by 138% after SL and by 68-128% during DL (P < 0.01). Fasting plasma triglyceride concentrations

TABLE 3 ACUTE EFFECTS OF FAT INGESTION Mean SEM

ON HDL COMPOSITION

Time (h) from start of first meal 3

0 Single fat load HDL-C (mM/l)

1.24

6

1.18 *

24

APO A-I (mg/dl)

(0.11) 102.7

(0.10) 97.5

(0.11) 104.7

1.17 (0.12) 107.7

1.24 (0.10) 100.0

APO A-II (mg/dI)

(6.0) 26.1

(5.7) 24.7

(4.5) 27.3

(6.7) 28.4

(6.0) 25.5

Apo A-I/ape A-II

(1.1) 3.94

(2.7) 3.69

HDL-C/ape

(0.18) 1.20

(0.17) 1.18

(1.3) 3.86 (0.10) 1.05

(0.09)

(0.06)

(0.09)

(1.8) 3.84 (0.22) 1.07 (0.06)

(1.7) 3.98 (0.17) 1.27 (0.06)

Distributed

A3 (X 102)

1.09

9

fat load

HDGC (mM/l)

1.24

1.19 *

1.16 ** (0.09) 106.4

1.16 * (0.10) 109.5 b

1.22 (0.09) 103.7

APO A-I (mg/dl)

(0.09) 98.5

(0.09) 101.3

APO A-II (mg/dl)

(7.5) 27.2

(9.3) 27.1

(7.7) 25.8

(7.5) 27.5

(7.1) 25.9

APO A-I/ape

(2.1) 3.68

(2.8) 3.88

(0.21) 1.27

(0.30) 1.21 (0.07)

(2.4) 4.21 * (0.15) 1.11 (0.07)

(2.0) 4.01 (0.15) 1.06 *** (0.04)

(2.6) 4.16 (0.25) 1.18 (0.06)

HDGC/apo

A-II A-I (X 10’)

(0.06)

Significantly different from baseline value (t = 0 h): * P < 0.05,

** P < 0.01. *** P < 0.001.

571

HDL Cholesterol

1.4r

:IIjl~~~ l

z

3

6

9

24

Time (h) Fig. 3. Alterations in plasma HDL cholesterol concentration of change from baseline value: * P < 0.05.

during alimentary

lipaemia,

mean,

SEM.

Significance

on the morning following each of the SL and DL studies were significantly greater than baseline levels (Fig. 1). HDL apoproteins - Plasma HDL apo A-I increased by 11% during DL (P < 0.01). In contrast, after SL, changes in apo A-I were smaller and less consistent (Fig. 2). Apo A-II levels were not significantly altered after SL or during DL. The ratio of apo A-I to apo A-II in the HDL fraction increased during DL (P < 0.05) (Table 3). Neither Apo A-I or Apo A-II were present in the VLDL fraction of fasting plasma. Apo A-I and apo A-II were detectable in both VLDL and chylomicron fractions of plasma obtained during postprandial lipaemia although amounts were too small to be quantified. HDL cholesterol - Plasma HDL cholesterol concentrations decreased by 4.0-7.5s (P < 0.05) during postprandial lipaemic phases of both the SL and DL studies (Fig. 3). Linear regression analyses were performed for percentage change in HDL cholesterol versus percentage change in plasma triglycerides at 3, 6 and 9 h. Initially, the 2 phases of the experiment were considered separately. In each of SL and DL, there were significant negative correlations between these variables. As there was no significant difference between the slopes or y-axis intercepts for the SL and DL correlations, the data were combined. The regression coefficient (rs) was -0.408 (n = 56, P < 0.01). The equation for the regression line was y = 0.025x - 1.62. The y-axis intercept was not significantly different from 0. The ratio of cholesterol to apo A-I in the HDL fraction decreased during DL (P < 0.001) (Table 3). Discussion Apoprotein A-I is the major structural protein of HDL and is believed to derive in part from the surface components of chylomicrons released into lymph during fat absorption. Fat ingestion increases the mucosal synthesis of A peptides [9] and the formation of chylomicrons and intestinal VLDL [lo]. Hydrolysis of the triglyceride of these particles is accompanied by rapid transfer of apoproteins into the plasma HDL fraction [1,11,12] and a rise in plasma apoprotein A-I concentration [2,10]. The observation in the present study that apoprotein A-I and A-II were detectable in chylomicrons and postprandially sampled VLDL but not in VLDL during the fasting state is compatible with an intestinal role in apoprotein A synthesis. Little is known about factors regulating intestinal synthesis and secretion of

A peptides, but particles within the density range of chylomicrons and VLDL are known to become relatively richer in protein as they become smaller and more dense [13,14]. Previous studies in both man [3] and animals [15] have clearly demonstrated that the size of the triglyceride-bearing lipoprotein is in part dependent on the size of the fat load presented to the intestinal mucosa. In the present study, the distributed fat load resulted in a significant postprandial increase in the apo A-I content of the HDL fraction whereas the single lipid bolus did not. This is consistent with the hypothesis that intestinal synthesis of apo A-I is enhanced when conditions are conducive to the formation of small rather than large chylomicrons. Unlike apo A-I, plasma levels of apo A-II were not significantly altered by lipid feeding. This was not unexpected, since studies in chyluric subjects have suggested that the intestine may make a less important overall contribution to apo A-II synthesis than to apo A-I synthesis [2]. During the period of prolonged fat ingestion, the apo A-I/ape A-II ratio increased moderately. This suggests that the distributed fat load increased the concentration of HDLz (d 1.063-1.125 g/ml) relative to HDL3 (1.125-1.21 g/ml), since the former has been shown to have a higher apo A-I/ape A-II ratio [ 151 and thus lends support to the suggestion that chylomicron catabolism leads to the conversion of HDL3 to HDLl [17,18]. Alternatively, the increase in HDL apo A-I could reflect a selective increase in the protein concentration of the HDLz subfraction as has been reported previously following a lipid meal [ 191. Other compositional changes in HDL during alimentary lipaemia were evidenced by a decrease in HDL cholesterol in both phases of the present experiment. Transfer of cholesteryl ester from HDL to triglyceride-rich lipoproteins has been documented under in vivo [ 191 and in vitro conditions [ 20-231. This may be due to nonenzymatic exchange of cholesteryl ester for triglyceride [ 221 or may be mediated by a specific exchange protein [ 241. It is also possible that the observed loss of cholesterol from HDL during lipolysis could represent transfer into the LDL fraction [ 121. The increase in plasma total cholesterol (7.9 + 2.2 mg/dl) which was observed 3 h after ingestion of the single bolus of fat could be fully accounted for by the cholesterol content (360 mg) of the lipid vehicle. We also noted a substantial rise in plasma triglycerides 14 h after the last meal irrespective of the pattern of butterfat ingestion. Such an effect may have been due to competition between endogenous and exogenous particles for triglyceride clearing mechanisms or to enhanced splanchnic secretion of VLDL. By contrast, subjects given a similar amount of fat as safflower oil had lower fasting triglycerides on the morning following a lipid-loading study [ 251. Dietary modifications which increase the amount of A apoproteins and of other chylomicron surface components may lead to enhanced levels of circulating HDL2. Studies are now underway to determine the long-term effects of the pattern of fat ingestion on HDL concentration and composition in man. Acknowledgements The authors study,

would like to thank the volunteers

for their participation

in this

573

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