395

Clinica Chimica Acta, 87 (1978) @ Elsevier/North-Holland

395-404 Biomedical Press

CCA 9538

EFFECTS OF DIETARY SATURATED AND POLYUNSATURATED ON THE METABOLISM OF APOLIPOPROTEINS A-I AND B STUDY OF A PATIENT

FAT

WITH TYPE IIb HYPERLIPOPROTEINAEMIA

JAMES SHEPHERD *, CHRISTOPHER 0. DAVID TAUNTON

J. PACKARD,

ANTONIO

M. GOTTO

Jr. and

Division of Atherosclerosis and Lipoprotein Research, Department of Medicine, Baylor College of Medicine and The Methodist Hospital, Houston, Texas 77030 (U.S.A.) (Received

February

28th,

1978)

Summary The effects of dietary saturated and polyunsaturated fat on the metabolism of apolipoprotein A-I (apoA-I) and apolipoprotein B (apoB) were studied in a patient with type IIb hyperlipoproteinaemia. On the saturated fat diet, the rate of synthesis of very low density lipoprotein apoprotein B (VLDL-apoB) was approximately twice normal, accounting for the increased plasma VLDL pool in this subject. However, 54% of the synthesized VLDL-apoB was catabolized by a pathway independent of low density lipoproteins (LDL). The metabolic conversion rate of VLDL-apoB to LDL-apoB was normal in this subject and his expanded plasma LDL-apoB pool resulted, not from increased input of the apoprotein from VLDL, but from a decrease in its fractional clearance rate. On the polyunsaturated diet, there was a significant fall in the plasma cholesterol and triglyceride concentrations and a change in the fatty acid composition of all plasma lipoprotein fractions. These changes were accompanied by a decrease in the plasma concentrations of apoA-I and apoB which resulted from a reduction of apoprotein synthetic rate.

Introduction The management of primary hyperlipoproteinaemia institution of an appropriate dietary regimen [l]. which is characterized by an increase in circulating

* All

correspondence

Royal

Infirmary,

should Glasgow

be G4

sent OSF,

to U.K.

Dr.

James

Shepherd,

always begins with the The type IIb phenotype, very low density lipopro-

Department of

Pathological

Biochemistry.

396

teins (VLDL) and low density lipoproteins (LDL responds best, in terms of diet, to cholesterol restriction and substitution of polyunsaturated for saturated fats. The effectiveness of such dietary intervention is now well established, but its mechanism of action is not yet understood. Since the question of how polyunsaturated fats lower plasma cholesterol may be related to their long term safety and ultimate benefits, numerous studies have been directed towards elucidating the mechanism [ 21. Most of these have attempted to relate the hypocholesterolaemic actions of polyunsaturated fats to a direct effect on cholesterol metabolism. The results of many of these studies have not been consistent from one laboratory to another and have generated considerable controversy regarding the actions of polyunsaturated fats. In this report we examine the effect of dietary fat saturation level, not on cholesterol metabolism directly, but on the metabolic handling of two proteins associated with cholesterol in the plasma (apolipoprotein A-I (apoA-I) and apolipoprotein B (apoB)). The study was performed on a patient with type IIb hyperlipoproteinaemia. Patient and methods The subject A 66-year-old male with type IIb hyperlipoproteinaemia as defined by the Lipid Research Clinics’ criteria [3] gave informed consent to this study. His plasma lipid and lipoprotein profiles on the saturated and polyunsaturated fat diets are shown in Table I. Routine clinical, haematological and biochemical analysis revealed no evidence of hepatic, renal or endocrine disease. However, asymptomatic myocardial ischaemia, without impairment of exercise tolerance, was demonstrated during a treadmill electrocardiographic study. Thyroidal sequestration of radioiodide was prevented by administration of potassium

PL.AN

OF

METABOLIC VS

INVESTIGATION POLYUNSATURATED

OF

EFFECTS

FAT

OF

SATURATED

DIETS

Time (weeks) 0

I t Admit + start diet

I

2 I t

3

I

4

I

Inject

STUDY Saturated Cholesterol P/S ratlo isocaloric

1 t Discharge

‘25I VLDL 13’1

5

HDL I

A

0

I t

I 1

2

3

I t-

Admit

inject

t start

‘25I

diet

VLDL

13’1 STUDY

Diet = 400mg/day = 0.25

HDL II

Polyunsaturated Cholesterol P/S ratio

Diet

= 400 = 4.0

Isocalorlc

Fig. 1. Plan of metabolic study of the type IIb hyperlipoproteinaemic

4

patient.

mg /day

5

397

iodide (300 mg twice daily, by mouth) during the period of investigation. No other medications were given for four weeks prior to and throughout the study. Pro toco1

The study protocol is shown in Fig. 1. The subject was investigated in the General Clinical Research Center of the Methodist Hospital and received only food prepared in the Research Center Metabolic Kitchen. Both diet regimens were isocaloric and were designed to maintain constant body weight. Cholesterol intake was 400 mg/day and the calorie distribution was 40%, 40% and 20% as carbohydrate, fat and protein respectively. In the first phase of the study (diet I) the polyunsaturated/saturated (P/S) fat ratio was 0.25, while in the second phase (diet II), the ratio was adjusted to 4.0. Methods

All metabolic studies were performed after two weeks of dietary equilibration (Fig. l), by which time diet-induced changes in lipoprotein composition were complete [ 41. Plasma cholesterol and triglyceride were measured on alternate days and P-quantification was performed twice weekly throughout the study, using the Lipid Research Clinics’ methodology [ 31. (a) ApoA-I

metabolism

ApoA-I was purified from high density lipoproteins (HDL, d = 1.063-1.21 kg/l) isolated from normal postabsorptive plasma, radiolabelled with 1311 (Amersham-Searle, Arlington Heights, Ill. 60005), and incorporated into the subject’s HDL (d = 1.063-1.21 kg/l) using an in vitro incubation procedure described previously [5,6]. The labeled lipoprotein was then reisolated by ultracentrifugal flotation at d = 1.21 kg/l [7] sterilized by filtration through 0.22 pm cellulose membranes (Millipore Corp., Bedford, Mass. 01730) and 25 PCi of this material (approximately 1.0 mg protein) injected intravenously into the patient. A blood sample was removed 10 min after the injection and subsequently at daily intervals for the next 14 days, and the rate of clearance of plasma radioactivity determined on a Packard Autogamma Spectrometer (Packard Instruments Inc., Downers Grove, Ill.). From this data, the fractional catabolic rate (FCR) of the 1311-apoA-I/HDL (i.e. the fraction of the intravascular 1311-apo-A-I/HDL pool catabolized per day) was calculated using a mathematical procedure described elsewhere [ 8,9]. Measurement of the plasma apoA-I concentration performed at daily intervals throughout the study by electroimmunoassay [6], permitted calculation of the absolute rate of catabolism (ACR) of apoA-I in the plasma (ACR of apoA-I = plasma apoA-I pool X FCR). (b) ApoB

metabolism

Postabsorptive VLDL was isolated from the patient’s plasma [7] two weeks after commencement of each diet, labeled with “‘1 [lo], sterilized by filtration through 0.45 pm cellulose membranes (Millipore Corp., Bedford, Mass. 01730) and a 25 &i aliquot (approximately 0.25 mg protein) reinjected into the donor simultaneously with the 1311-apoA-I/HDL. Blood samples were collected at fre-

398

quent intervals over the first 72 h and thereafter every 24 h for 14 days. After removal of chylomicra (30 min ultracentrifugation at lo4 rpm, lO”C, in a 40,3 Beckman anglehead rotor), VLDL (d < 1,006 kg/l), LDL (d = 1.006-1.063 kg/l) and HDL (d = 1.063-1.21 kg/l) were prepared by sequential ultracentrifugation [7] of 4-ml aliquots of plasma, density adjustment being made by addition of concentrated NaBr solutions. ApoB in VLDL and LDL was precipitated with tetramethylurea [ 111 freed of contaminant lipids by chloroform/ methanol (1 : I, v/v) extraction, washed with ether, and dried under nitrogen. The protein exhibited the amino acid composition and sodium dodecyl sulphate acrylamide gel electrophoretic mobility of apoB. The dried apoprotein pellet was solubilized in 1 .O ml of 0.5 M NaOH and its radioactivity and protein content [ 121 determined. From this was calculated the specific activity of the apoprotein in both lipoprotein fractions at each plasma sampling time. Also, since the VLDL-apoB had been isolated from 4 ml of plasma, it was possible to calculate the plasma concentration (in mg/lOO ml) of apoB in each lipoprotein fraction. The resultant data were analyzed by the SAAM 27 program [13] adapted for use in a Univac 1110 computer. The metabolic studies described above were performed twice, first on diet I (P/S ratio = 0.25), then on diet II (P/S ratio = 4.0). The interval between both studies was five weeks. Results The effects of dietary fat saturation level on the plasma lipids and lipoproteins of the type IIb subject are shown in Tables I and II. Compared to saturated fat, ingestion of polyunsaturated fats caused a significant (P < 0.01) reduction in plasma cholesterol (4 10%) and triglyceride (J 14%) and a 12% fall (P < 0.01) in the concentration of circulating LDL-cholesterol. No significant difference in plasma VLDL- or HDL-cholesterol was observed as a result of polyunsaturated fat ingestion. These changes in absolute concentrations of plasma lipids and lipoproteins, which did not reduce the subject’s values to

TABLE I EFFECTS OF DIETARY FAT SATURATION LIPOPROTEINAEMIC SUBJECT

LEVEL

ON PLASMA

LIPIDS OF A TYPE IIb HYPER-

Values in parentheses indicate the number of samples analysed. Diet

Saturated Polyunsaturated

% Change on polyunsaturated diet ---*

t

Plasm a cholesterol (mmol/l)

Plasma triglyceride (mmol/l)

VLDL cholesterol (mmol/l)

LDL cholesterol (mmol/l)

HDL cholesterol (mmol/l)

8.73 f 0.34 (11)

2.11 r 0.14 (11)

0.74 i 0.11 (7)

6.90

0.98 ? 0.21

7.94 ? 0.42 (12)

1.82 k 0.19 (12)

0.82 i 0.26 (8)

6.11

410% P < 0.01 *

414% P < 0.01

N.S.

test on unpaired samples.

0.37

(7)

(7) 0.29

1.03 + 0.19

(8)

(8)

3-12% P < 0.01

N.S.

399

TABLE

II

CHANGES

IN

LIPOPROTEIN

TION

IN A TYPE

Fatty

acids

FATTY

ACIDS

IIb HYPERLIPOPROTEINAEMIC

INDUCED

BY

ALTERED

DIETARY

FAST

SATURA-

SUBJECT

Saturated

Polyunsaturated

(%I VLDL

14:o

2.1

16:O

LDL 1.8

27.0

19.9

16:l

4.3

3.2

18:0

4.9

5.7

HDL 3.7

VLDL

LDL

0.9

0.4

16.2

14.5

1.8

3.0

1.6

11.8

3.9

5.3

24.2

HDL 2.7 23.1 2.8 7.5

18:l

31.6

18.2

27.0

21.1

12.1

16.5

18:2

22.7

40.0

24.7

48.1

54.6

34.0