The Journal of Nutrition Vol. 128 No. 3 March 1998, pp. 570-576

Composition of Human VLDL Triacylglycerols after Ingestion of Olive Oil and High Oleic Sunflower Oil^1,2

Valentina Ruiz-Gutiérrez³, Nora Morgado^*, José Luis Prada, Francisco Pérez-Jiménez^**, and Francisco J. G. Muriana

Instituto de la Grasa (C.S.I.C.), Seville, Spain; ^* Instituto de Nutrición y Tecnología de los Alimentos, Santiago de Chile, Chile;  Hospital Costa del Sol, Marbella, Spain; and ^** Departamento de Medicina, Facultad de Medicina, Córdoba, Spain

	ABSTRACT

Abstract Introduction Methods Results Discussion References

This work was undertaken to determine the effect of diets enriched with olive oil or high oleic sunflower oil on very low density lipoprotein (VLDL) triacylglycerol composition of healthy human subjects. Both oils contain a similar proportion of monounsaturated fatty acids (MUFA) but differ in their triacylglycerol composition. All 22 human subjects initially consumed a low fat, high carbohydrate diet as recommended by the National Cholesterol Education Program (NCEP-I). They then consumed the two experimental oils (40% dietary energy) in a crossover design. The olive oil and high oleic sunflower oil diets resulted in significant increases in palmitoleic (55%, P < 0.05), oleic (27%, P < 0.01) and eicosenoic (>100%, P < 0.001) acids of VLDL triacylglycerols, whereas there was a significant decrease in linoleic acid (38%, P < 0.001). In addition, the high oleic sunflower oil diet increased the content of stearic acid (60%, P < 0.05) and total saturated fatty acids (14%, P < 0.05). Both MUFA-rich diets significantly (P < 0.01) decreased the content of sn-glycerol-palmitate-linoleate-oleate, sn-glycerol-palmitoleate-dioleate and sn-glycerol-palmitate-dilinoleate in VLDL with regard to the NCEP-I diet, whereas they increased the content of sn-glycerol-trioleate (>100%, P < 0.001 after the olive oil diet; 80%, P < 0.05 after the high oleic sunflower oil diet). Intake of olive oil, in particular, significantly decreased the content of sn-glycerol-tripalmitate (36%, P < 0.01) and increased the content of dioleoyl-containing triacylglycerols. MUFA (P < 0.01) and arachidonic acid (P < 0.001) tended to be rich in the sn-2 position of VLDL triacylglycerols during the periods of consuming the olive oil or high oleic sunflower oil diets. In addition, olive oil, but not high oleic sunflower oil, further contributed to VLDL triacylglycerols that contained -linolenic and docosahexaenoic acids acylated in the sn-2 position. These data suggest that differences in the composition of VLDL triacylglycerols may be of major importance in explaining the beneficial effects of dietary olive oil in reducing the atherogenic risk profile in healthy subjects.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Very low density lipoproteins (VLDL)⁴ make up a family of particles with a high degree of variability in size, density and chemical composition (Thompson 1994). VLDL are secreted mainly by the liver; their chief function is the transport of endogenously synthesized triacylglycerols. These lipoproteins receive additional cholesteryl esters from high density lipoprotein (HDL) by the action of cholesteryl ester transfer protein, which also transfers cholesteryl ester to low density lipoprotein (LDL). Formation of remnant particles and conversion of VLDL to LDL are dependent upon removal of triacylglycerol core molecules by lipolytic pathways, such as those mediated by hepatic lipase and lipoprotein lipase, the rate-limiting enzyme in providing cells with fatty acids for either energy or storage (Deckelbaum et al. 1992). Lipoprotein lipase also enhances the binding of non-HDL to the extracellular matrix of endothelial cells and the uptake of these proteins by cell-specific receptors through independent hydrolytic mechanisms (Olivecrona and Olivecrona 1995).

The intake of polyunsaturated and monounsaturated fatty acids (PUFA and MUFA) may reduce plasma triacylglycerol-rich lipoproteins by two complementary mechanisms, changes in the composition of VLDL and changes in the expressed activities of the enzymes and proteins involved in intravascular processing and catabolism of VLDL (McNamara 1995). The fatty acid pattern of triacylglycerols in VLDL is influenced by the fatty acid composition of dietary fats so that the metabolic fates of VLDL and clearance of triacylglycerols may be altered profoundly (Campos et al. 1995, Montalto and Bensadoun 1993). However, there are virtually no reports investigating the effects of dietary triacylglycerols on the composition of VLDL.

The positional specificity of lipolytic enzymes during digestion affects the absorption rate of monoacylglycerols (with ~75% of fatty acids conserved at sn-2 position), providing the basic structure for the resynthesis of triacylglycerol molecules (Bracco 1994). Preliminary studies (Zampelas et al. 1994) indicate that the positional distribution of fatty acids on dietary triacylglycerols does not constitute an important determinant of postprandial lipemia; however, the contribution of triacylglycerol composition to the understanding of endogenous triacylglycerol origin and fasting hiperlipidemia remains to be elucidated. First, an appropriate analysis of triacylglycerols in VLDL seems of major interest, especially when emerging evidence suggests a link between fat quality and removal of triacylglycerol-rich lipoproteins in atherogenesis (Benlian et al. 1996, Ginsberg et al. 1995, Reznik et al. 1996).

Among dietary fats, olive oil intake has been associated with a low incidence of coronary heart disease (Keys et al. 1986) by reducing the atherogenic risk profile in both healthy subjects (Mata et al. 1992, Ruiz-Gutiérrez et al. 1996) and hyperlipidemic patients (Nydahl et al. 1994, Ruiz-Gutiérrez et al. 1996). High oleic sunflower oil (HOSO) is a new vegetable source that contains a proportion of oleic acid similar to that of olive oil, but the oils differ in their triacylglycerol composition and distribution of other minor fatty acids (Carelli and Cert 1993, Pérez-Jiménez et al. 1995). Interestingly, dietary olive oil (but not HOSO) has important benefits for membrane homeostasis of healthy subjects and patients with different cardiovascular risk factors (Ruiz-Gutiérrez et al. 1996). In this study, olive oil and HOSO were incorporated into the diets of healthy subjects to establish effects of dietary triacylglycerols on triacylglycerol composition of VLDL. In addition, fatty acid composition and sn-2 positional distribution have been studied to obtain as much data as possible on the effects of the two monounsaturated-enriched oils, which differ in triacylglycerol composition, on human triacylglycerol metabolism. This paper presents further analysis of data obtained in a previous study (Pérez-Jiménez et al. 1995).

	SUBJECTS AND METHODS

Abstract Introduction Methods Results Discussion References

Subjects.  Twenty-two healthy volunteers (23 ± 0.4 y) were recruited from among medical students at the University of Cordoba (Spain). All subjects had a comprehensive medical history, physical examination and clinical chemistry analysis before enrollment (Pérez-Jiménez et al. 1995). All subjects gave their informed consent before participating in the study.

Experimental design and diets.  The study design included an initial 25-d period during which all participants consumed a modified diet as recommended by the National Cholesterol Education Program (NCEP-I), calculated to contain 15% of energy as protein, 55% as carbohydrate and 30% as fat [10% saturated fatty acids (SFA), 12% MUFA and 8% PUFA]. After this period, two groups of 11 subjects each were assigned to eat olive oil or high oleic sunflower oil (HOSO) diets over two 28-d periods in a crossover design. Both diets were calculated to contain 15% of energy as protein, 45% as carbohydrate and 40% as fat (10% SFA, 22% MUFA and 8% PUFA). The daily energy consumption of participants was ~10.2 MJ. Dietary cholesterol intake was maintained during the three diet periods at 285 mg/d. All diet trials were performed between Christmas and Easter to ensure temporal consistency. To calculate nutrient composition and to design the menus for each diet period, a computerized database was created (Pérez-Jiménez et al. 1995). Virgin olive oil (Olea europaea, Extra Sublime, Koype, Andujar, Jaen) or HOSO (Helianthus annuus, Vipaceite, Koype) was used for cooking and salad dressing and was occasionally spread on bread slices. To avoid any isomerization, only oils obtained after the first frying were used. The stability index (AOCS 1994) of the oils was 72.3 h for the olive oil and 20.8 h for the HOSO. The concentration of total polar compounds in the meals was never > 5% (Jorge et al. 1996). Fatty acid composition, triacylglycerols and fatty acid sn-2 positional distribution of the oils are depicted in Tables 1, 2 and 3, respectively. The design of this study was approved by the Human Investigation Review Committee at the Hospital Universitario Reina Sofia, Cordoba.

Blood sampling and biochemical determinations.  Blood samples were obtained from fasting subjects by venous puncture into EDTA-containing (1 g/L) tubes. Plasma was separated by centrifugation at 1500 × g at 4°C for 30 min. Lipoprotein fractions were isolated from fresh plasma samples by centrifugation at 105,000 × g at 4°C for 20 h with the use of sequential flotation (Schumaker and Puppione 1986). VLDL were harvested at density 1.006 kg/L (Havel et al. 1955). Compliance with the diets was determined by evaluation of daily food questionnaires and by analysis of fatty acid composition of the cholesterol ester fraction in LDL (Sarkkinen et al. 1994).

Separation of triacylglycerols from VLDL.  Neutral lipids from VLDL were extracted with chloroform/methanol (2:1, v/v) and separated by TLC on silica gel 60 plates using a solvent system of hexane/diethylether/acetic acid (80:20:1, v/v/v). The band corresponding to triacylglycerols was scraped off and eluted with hexane/chloroform (9:1, v/v).

Analysis of fatty acid methyl esters.  Triacylglycerols were saponified by heating for 10 min with 0.2 mol/L sodium methylate at 80°C. Fatty acid methyl esters were formed by heating again for 5 min with 60 g/L H₂SO₄ in anhydrous methanol (Ruiz-Gutiérrez et al. 1996). After extraction with hexane, fatty acid methyl esters were analyzed in a Hewlett-Packard 5890 series II gas chromatograph (Hewlett-Packard, Avondale, PA) equipped with a flame ionization detector and using an Omegawax 320 fused-silica capillary column (30 m × 0.32 mm i.d., 0.25 µm film). The initial column temperature was 200°C, which was held for 10 min, then programmed to change from 200 to 230°C at 2°C/min. The injection and detector temperatures were 250 and 260°C, respectively. The flow rate of helium was 2 mL/min. Peak areas were calculated by a Hewlett-Packard 3390A recording integrator. Individual fatty acid methyl esters were identified on isothermal analyses by comparison of their retention times with those of reference standards. Fatty acid methyl esters for which no standard was available were quantified using calibration tables of relative response ratios constructed according to carbon number with the use of gas chromatography-mass spectrometry, performed on a Konik KNK-2000 chromatograph (Konik, Barcelona, Spain) interfaced directly to an AEJ MS30/70 VG mass spectrometer, using the electron impact ionization mode. The ion source temperature was maintained at 200°C, the multiplier voltage was 4.0 kV, the emission current was 100 µA and the electron energy was 70 eV.

Fatty acids in the sn-2 position of triacylglycerols.  Triacylglycerols were partially hydrolyzed by pancreatic lipase from pigs (EC 3.1.1.3; Sigma Chemical, St. Louis, MO) and then separated by TLC on silica gel 60 plates by using a solvent system of hexane diethylether acetic acid (60:40:2, v/v/v) (Ruiz-Gutiérrez and Mazuelos 1985). The band corresponding to monoacylglycerols was scraped off, eluted with hexane and treated as above for analysis of fatty acid methyl esters.

Analysis of triacylglycerols.  Triacylglycerols were analyzed in a Chrompack (Middleburg, The Netherlands) CP9000 gas chromatograph fitted with a flame ionization detector and a split injection system (splitting ratio 1:30) (Carelli and Cert 1993, Ruiz-Gutiérrez and Barron 1995). Separation was conducted on a high temperature aluminium-clad fused-silica capillary column (25 m × 0.25 mm i.d.) coated with methyl 65% phenylsilicone (0.1 µm film). The initial column temperature was 350°C, which was held for 1 min, then programmed to change from 350 to 360°C at 0.5°C/min and remaining at 360°C for 8 min. The injection and detector temperatures were 360 and 365°C, respectively. Helium was used as the carrier gas at a column head pressure of 130 kPa. Trilinolein, trimyristin, trinonadecanoin, triolein, sn-glycerol-palmitate-dioleate, tripalmitin, tripalmitolein and tristearin were used as reference standards (Nu-Chek-Prep, Elysian, MN). Tripentadecanoin was used as an internal standard (Sigma Chemical). Triacylglycerols were then identified by their elution times, because retention is affected not only by the number of carbon atoms but also by the number of double bonds (Ruiz-Gutiérrez et al. 1992a).

Statistical analyses.  The significance of the differences between the groups was assessed by one-way (repeated measures) ANOVA with Tukey's post-hoc comparison of the means (Neter et al. 1985). Data were transformed reciprocally before statistical analysis. All comparisons were considered statistically significant at P < 0.05. The analyses were done with the GraphPAD InStat (GraphPAD Software, San Diego, CA) and CoStat (CoHort Software, Berkeley, CA) statistical packages.

	RESULTS

Abstract Introduction Methods Results Discussion References

Diets.  The fatty acid composition of olive oil and HOSO was characterized by a high content of MUFA [mainly oleic acid (77-78% of total fats)] (Table 1). Palmitic acid [(16:0) 68% of total SFA] and -linolenic acid [18:3(n-3) 9% of total PUFA] were more abundant in the olive oil than in the HOSO, whereas the content of linoleic acid [18:2(n-6)] was greater in the HOSO (11.4% of total fats) than in the olive oil (8% of total fats). sn-Glycerol-trioleate (OOO) was the major triacylglycerol found in olive oil (49.8% of the total) and HOSO (65.1% of the total) (Table 2). sn-Glycerol-palmitate-dioleate (POO) was also abundant in olive oil, making up 30% of the total. Oleic acid was the predominant fatty acid located at the sn-2 position of triacylglycerols from either olive oil or HOSO (Table 3).

All of the participants responded in a similar manner to the diets and completed the study according to schedule, except one subject who developed diarrhea and anorexia and was excluded from the study. Oleic acid content was significantly enhanced (16%, P < 0.05) in the cholesterol ester fraction of plasma LDL during consumption of MUFA-rich diets (Pérez-Jiménez et al. 1995), indicating good adherence to the diets. Order of diet consumption did not have a significant effect.

Fatty acid composition of VLDL triacylglycerols.  Compared with the NCEP-I diet, the olive oil and HOSO diets resulted in significant increases in palmitoleic acid [16:1(n-9) 55%, P < 0.05], oleic acid (28%, P < 0.01) and eicosenoic acid [20:1(n-9) >100%, P < 0.001] of VLDL triacylglycerols (Table 4) whereas there was a significant decrease in linoleic acid (38%, P < 0.001). Both oil diets increased total MUFA (29%, P < 0.01) and decreased total PUFA (32%, P < 0.001); the ingestion of HOSO, in particular, increased the content of stearic acid (18:0 60%, P < 0.01) and total SFA (14%, P < 0.05).

Triacylglycerol composition of VLDL.  sn-Glycerol-tripalmitate (PPP, 36%, P < 0.01) was significantly decreased and dioleoyl-containing triacylglycerols [sn-glycerol-myristate-dioleate (MOO, 63%, P < 0.05; POO, 30%, P < 0.01); sn-glycerol-stearate-dioleate (SOO, 94%, P < 0.01); and sn-glycerol-linoleate-dioleate (OLO, >100%, P < 0.001)] were significantly increased in VLDL of healthy subjects consuming the olive oil diet (Table 5). Compared with the period of consuming the NCEP-I diet, both MUFA diets decreased the content of sn-glycerol-palmitate-linoleate-oleate (PLO), sn-glycerol-palmitoleate-dioleate (PoOO) (P < 0.001) and sn-glycerol-palmitate-dilinoleate (PLL) (P < 0.01), and increased the content of OOO (>100%, P < 0.001 after ingestion of olive oil; 80%, P < 0.05 after ingestion of HOSO). OLO was also significantly increased during the period in which the HOSO diet was consumed.

Fatty acids at the sn-2 position of VLDL triacylglycerols.  In contrast to linoleic acid, MUFA [16:1(n-9), 18:1(n-9) and 20:1(n-9)] (P < 0.01) and arachidonic acid [20:4(n-6)] (P < 0.001) were rich in the sn-2 position of VLDL triacylglycerols during both the olive oil and HOSO diet periods (Table 6). Olive oil, but not HOSO, further contributed to a well-defined intramolecular structure of VLDL triacylglycerols having mainly -linolenic acid [18:3(n-3)], dihomo--linolenic acid [20:3(n-6)] and docosahexaenoic acid [22:6(n-3)] acylated in the sn-2 position and SFA (including 14:0, 16:0 and 18:0) acylated in the sn-1 and sn-3 positions with regard to the NCEP-I diet. Consequently, there was an increase of total MUFA (21%, P < 0.01) and a decrease of total SFA (17%, P < 0.01) after ingestion of olive oil, whereas there was an increase of total MUFA (10%, P < 0.05) and a decrease of total PUFA (18%, P < 0.05) after ingestion of HOSO.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Colipase-dependent pancreatic lipase and pancreatic lipase-related proteins hydrolyze fatty acids presented at the sn-1 and sn-3 positions of triacylglycerols, which releases free fatty acids and 2-monoacylglycerols (Lehner and Kuksis 1996). Therefore, fatty acids at the sn-2 position of dietary triacylglycerols are largely retained during absorption processes. In the liver, VLDL are assembled, they contain triacylglycerols derived metabolically from both the diet and de novo synthesis of fatty acids.

Recently, we showed that two MUFA-rich (olive oil and HOSO) diets led to a similar modification of plasma lipid and lipoprotein profiles in healthy subjects (Pérez-Jiménez et al. 1995) and hypertensive patients with or without hyperlipidemia (Ruiz-Gutiérrez et al. 1996). However, olive oil (but not HOSO) was particularly effective in normalizing the activity of a membrane-bound enzyme responsible for sodium transport, and the distribution and transbilayer movement of erythrocyte membrane cholesterol (Muriana et al. 1997a and 1997b). These studies suggest that factors other than oleic acid content in the oils may explain diet-induced metabolic responses. Certainly, olive oil and HOSO differ in their concentrations of minor fatty acids (Table 1), triacylglycerol composition (Table 2) and sn-2 positional distribution of fatty acids (Table 3).

We found that the fatty acid composition of VLDL triacylglycerols did not differ during the periods of consuming the olive oil and HOSO diets (Table 4). It was interesting to note that both MUFA-rich diets significantly decreased the ability of VLDL to transport linoleic acid [18:2(n-6)] and thereby the substrate for arachidonic acid [20:4(n-6)] formation, thus probably affecting their incorporation into the peripheral tissues. Fatty acids are released from circulating triacylglycerols by the action of lipoprotein lipase (LPL), which is localized and attached on the surface of cells via heparan sulfate proteoglycans (Wang et al. 1992). The enzyme is tightly regulated by the composition of dietary fats, because linoleic acid and fatty acids of the (n-3) family markedly decrease LPL synthesis and secretion (Montalto and Bensadoun 1993). In addition, LPL can differentiate between substrates (Calder et al. 1994) and exhibits specificity with respect to the position of fatty acid chains in the triacylglycerol molecule (Wang et al. 1982); the ester bond at the sn-1 position is attacked in preference to that at position 2. Therefore, the composition of VLDL triacylglycerols is a determinant for the conversion of VLDL into other lipoproteins and the metabolism of triacylglycerols by cells.

Our data show that OOO was the main triacylglycerol found in olive oil (50% of the total) and HOSO (65% of the total), whereas POO was the main triacylglycerol found in VLDL after the MUFA diets (Table 5). The incorporation into VLDL of OOO from olive oil was more efficient than that from HOSO, suggesting differences in the absorption, metabolism or clearance of dietary OOO. The same was true for SOO and OLO. The positional distribution of fatty acids on dietary triacylglycerols can affect their digestion and absorption (Bracco 1994), but oleic acid is mainly at the sn-2 position in the two oils. The content and positional distribution of other minor fatty acids (palmitic, stearic and -linolenic acids) could partially explain the preference for long-chain fatty acids in the hepatocyte triacylglycerol-synthesizing pathways after olive oil intake. It has been calculated that a minimum of 60% of VLDL triacylglycerols are derived via hydrolysis to diacylglycerols and reesterification (monoacylglycerol pathway), and a maximum of 40% originate via the conventional phosphatidic acid pathway (Lehner and Kuksis 1996). The contribution of another pathway involving a transacylation of diacylglycerols (diacylglycerol transacylase pathway) is presently not known, but the high content of POO in olive oil would probably affect the incorporation of OOO into VLDL by the transacylation of sn-glycerol-dioleate with oleoyl-CoA. In addition, about 70% of liver triacylglycerols are subjected to a lipolysis/reesterification cycle before secretion as VLDL (Wiggins and Gibbons 1992). The influence of olive oil and HOSO ingestion on the activity of enzymes that may participate in this cycle should therefore be analyzed. Secondary source of fatty acids for triacylglycerol synthesis are the fatty acids released from body stores. Interestingly, fatty acid and triacylglycerol compositions of adipose tissue in healthy subjects are highly conserved with regard to fatty acid and triacylglycerol compositions of VLDL (Ruiz-Gutiérrez et al. 1992b and 1993).

The regiospecific analysis of VLDL triacylglycerols revealed a significant decrease of linoleic acid (compensated by an increase in arachidonic acid) at the sn-2 position after the MUFA diets (Table 6). Olive oil in particular, (but not HOSO) promoted the presence of -linolenic and docosahexaenoic [22:6(n-3)] acids in the sn-2 position of VLDL triacylglycerols. Olive oil and HOSO diets induced a significant decrease of SFA and a significant decrease of PUFA, respectively, at the sn-2 position. These unprecedented findings suggest that the availability of (n-3) PUFA in the liver may be increased by consumption of olive oil. Fatty acids of the (n-3) family suppress hepatic fatty acid synthesis and formation of VLDL (Surette et al. 1992), whereas oleic acid has been shown to be a potent stimulator of triacylglycerol synthesis and secretion of triacylglycerol-rich lipoproteins (Field et al. 1988, Montalto and Bensadoun 1993). Olive oil may help to establish an interactive mechanism whereby MUFA and PUFA are functionally interchangeable.

The amount of fat (40% of total energy) was similar in both MUFA-rich diets and before and after the dietary supplementation; as determined by food-frequency questionnaires, we found no significant changes in the intake of MUFA, SFA or PUFA after consumption of olive oil or HOSO. Therefore, it is unlikely that the explanation is based on uncontrolled dietary alterations.

Our study demonstrates that dietary fatty acids are esterified at different positions within the triacylglycerols of human VLDL after consumption of two MUFA-rich oils (olive oil and HOSO). Importantly, a decrease of (n-6) PUFA and an increase of (n-3) PUFA at the sn-2 position of VLDL triacylglycerols were specific effects of olive oil. This is of major metabolic importance, given the evidence that PUFA of the (n-3) family competitively inhibit the utilization of arachidonic acid by cyclooxygenase pathway (output of eicosanoids) (James 1996, Kinsella 1994) and the evidence that there is a link between fat quality and metabolism of triacylglycerol-rich lipoproteins in atherogenesis (Benlian et al. 1996, Ginsberg et al. 1995, Reznik et al. 1996).

	FOOTNOTES

Manuscript received 14 May 1997. Initial reviews completed 11 July 1997. Revision accepted 21 October 1997.

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