Pinolenic Acid in Structured Triacylglycerols Exhibits Superior

Pinolenic Acid in Structured Triacylglycerols Exhibits Superior Intestinal .... A–C, Uncommon Bis-sesquiterpenoids from the Chinese Red Alga Laurenc...
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Pinolenic Acid in Structured Triacylglycerols Exhibits Superior Intestinal Lymphatic Absorption As Compared to Pinolenic Acid in Natural Pine Nut Oil Min-Yu Chung,† Hyunjoon Woo,‡ Juyeon Kim,§ Daecheol Kong,§ Hee-Don Choi,† In-Wook Choi,† In-Hwan Kim,∥ Sang K. Noh,*,§ and Byung Hee Kim*,⊥ †

Korea Food Research Institute, Seongnam 13539, Korea Department of Food Science and Technology, Chung-Ang University, Anseong 17546, Korea § Department of Food and Nutrition, Changwon National University, Changwon 51140, Korea ∥ Department of Food and Nutrition, Korea University, Seoul 02841, Korea ⊥ Department of Food and Nutrition, Sookmyung Women’s University, Seoul 04310, Korea ‡

S Supporting Information *

ABSTRACT: The positional distribution pattern of fatty acids (FAs) in the triacylglycerols (TAGs) affects intestinal absorption of these FAs. The aim of this study was to compare lymphatic absorption of pinolenic acid (PLA) present in structured pinolenic TAG (SPT) where PLA was evenly distributed on the glycerol backbone, with absorption of pine nut oil (PNO) where PLA was predominantly positioned at the sn-3 position. SPT was prepared via the nonspecific lipase-catalyzed esterification of glycerol with free FA obtained from PNO. Lymphatic absorption of PLA from PNO and from SPT was compared in a rat model of lymphatic cannulation. Significantly (P < 0.05) greater amounts of PLA were detected in lymph collected for 8 h from an emulsion containing SPT (28.5 ± 0.7% dose) than from an emulsion containing PNO (26.2 ± 0.6% dose), thereby indicating that PLA present in SPT has a greater capacity for lymphatic absorption than PLA from PNO. KEYWORDS: lymphatic absorption, Novozym 435, pinolenic acid, stereospecific numbering position, structured triacylglycerols

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INTRODUCTION Oil from the Korean pine nut (Pinus koraiensis) is a major food source of pinolenic acid (PLA; c5,c9,c12-18:3), a type of Δ5unsaturated polymethylene-interrupted fatty acid (FA). Pine nut oil has been shown to confer beneficial effects on human health, including blood lipid-lowering and hypocholesterolemic activity in animal models. It can also suppress appetite by enhancing the effect of gut satiety hormones including cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1) in humans.1−3 In the triacylglycerol (TAG) structure, a stereospecific numbering (sn) system is often used to differentiate the three carbon atoms present in the glycerol backbone, because the middle carbon becomes a chiral pivot if different FAs are esterified on the primary top and bottom hydroxyl groups. The fatty acyl moiety positions on the glycerol backbone are labeled sequentially as sn-1, sn-2, and sn-3 (from top to bottom; in a planar Fischer projection of TAG) by convention. Absorption of FA in the intestine from TAG’s sn-2 position differs from that of FA esterified at the sn-1 and sn-3 positions. Pancreatic lipase preferentially hydrolyzes ester bonds located at the sn-1 and sn-3 positions of TAG within the duodenum, causing the release of free FA (FFA) from those positions and the formation of sn-2-monoacylglycerols (sn-2-MAGs), which are major factors responsible for the carriage of FA through the intestinal wall.4 Thus, FAs located at the sn-2 position are more effectively absorbed through the intestinal wall into the body as compared to FAs present in the sn-1 and sn-3 positions.5−7 © XXXX American Chemical Society

Published reports supporting this hypothesis have been covered by several studies.8,9 The PLA content of Korean pine nut oil is approximately 14 mol % of the total FA present. However, according to a study conducted by Choi et al.,10 the PLA content at the sn-3 position of Korean pine nut oil is ∼39 mol %, while at the sn-1 and sn-2 positions, it is less than 1 mol %, indicating that ∼95% of the total PLA found in the oil is esterified at the sn-3 position. This unusual positional distribution of PLA in pine nut oil may be causing a low absorption efficiency of PLA from oil in the diet. Structured TAGs refer to those for which the composition and/ or positional distribution of FA are chemically or enzymatically modified from the naturally occurring form. Such structural alterations provide structured TAGs with specific and desirable nutritional properties, which are often significantly different from the corresponding natural form.11 For this reason, structured TAGs have attracted interest as a class of functional foods and nutraceuticals that may address consumer needs. Recently, Choi et al.10 attempted to prepare structured pinolenic TAG (SPT), in which PLA was evenly distributed across the glycerol backbone (with total PLA content the same as in pine nut oil) employing a two-step nonspecific lipasecatalyzed esterification of the fatty acyl moieties in pine nut oil. Received: Revised: Accepted: Published: A

November 20, 2016 February 15, 2017 February 15, 2017 February 15, 2017 DOI: 10.1021/acs.jafc.6b05216 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Preparation of SPT through Novozym 435-catalyzed esterification of glycerol with FFA obtained from pine nut oil. The total PLA content (∼13 mol %) was not significantly different between pine nut oil and SPT. However, PLA was predominantly positioned at the sn-3 positions in pine nut oil, whereas it was evenly distributed at all sn positions in SPT. 600 rpm using a magnetic stirrer at 400 Pa. The reaction proceeded for 18 h. Analysis of TAG Content in Pine Nut Oil and SPT. The relative amounts of TAG, DAG, MAG, and FFA in pine nut oil and SPT were analyzed by GC following the method of Woo et al.12 A gas chromatograph (Agilent Technologies 7890A; Palo Alto, CA), equipped with a flame ionization detector (FID) and a fused silica capillary column (DB-17HT, 15 m × 0.25 mm i.d. × 0.15 μm film thickness, J&W Scientific, Folsom, CA), was used for the analysis. The oil samples (20 mg) were dissolved in n-hexane (1 mL), and 1 μL of the solution was injected in split mode with a split ratio of 50:1. Helium was used as the carrier gas at a flow rate of 2.0 mL/min. The temperature of both the injector and the detector was maintained at 360 °C. The column was initially held at 120 °C for 1 min and increased to 360 °C at a rate of 3 °C/min. It was then held constant at 360 °C for a further 5 min. The TAG content in the reaction products was calculated from the GC profile using the following eq 1:

The study used Novozym 435 (lipase B from Candida antarctica immobilized on a macroporous acrylic resin) as the biocatalyst for the reaction. However, hydrolysis occurring during the process results in the formation of diacylglycerol (DAG), MAG, and FFA, thereby diminishing the relative amount of TAG (∼92 wt %) in the reaction products. In the present study, we considered a new approach to improve the TAG content. SPT was prepared using a Novozym 435catalyzed esterification of glycerol with FFA obtained from pine nut oil under a vacuum. The TAG content in the SPT was thereby increased up to 97 wt %. We hypothesize that reallocation of the PLA from the sn-3 position in pine nut oil to the sn-2 position in SPT enhances the lymphatic absorption capacity of PLA. The aim of the present study was to determine whether the lymphatic absorption capacity of PLA from SPT was greater than that of PLA from natural pine nut oil. Emulsions containing known amounts of SPT or pine nut oil were infused into rat duodenum, and the lymph was collected using microsurgery techniques through lymph duct cannulation. Total PLA content was tracked by gas chromatography (GC) analysis.

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TAG content (wt%) =

PA TAG × 100 PA TAG + PADAG + PAMAG + PAFFA (1)

where PATAG, PADAG, PAMAG, and PAFFA are the total peak areas of TAG, DAG, MAG, and FFA, respectively. Analysis of FA Composition of Pine Nut Oil and SPT. The total FA composition of the pine nut oil and SPT was determined in accordance using the method described by Kang et al.,13 with a minor modification. Twenty milligrams of the oil samples was saponified with 0.5 N methanolic NaOH (3 mL) at 85 °C for 10 min, and then allowed to cool to room temperature. Following methylation with 3 mL of 14% BF3 in methanol at 85 °C for 10 min, the mixture was again cooled to room temperature before isooctane (3 mL) and saturated NaCl solution (5 mL) were added, and the solution was vortexed. FAME (the upper isooctane layer) was then collected and passed through an anhydrous Na2SO4 column. The FAME was then analyzed by GC using an Agilent Technologies 7890A gas chromatograph, equipped with an FID and a fused silica capillary column (SP2560, 100 m × 0.25 mm i.d. × 0.2 μm film thickness, Supelco). The sample (1 μL) was injected in split mode with a split ratio of 200:1, with helium as the carrier gas (flow rate of 1.0 mL/min). The temperature of the injector and detector was held at 225 and 285 °C, respectively. The column was initially heated to 100 °C for 4 min before increasing to 240 °C at a rate of 3 °C/min. The temperature was then held constant for 17 min. The FAMEs were identified using a comparison of their retention times with those of the standards, before relative quantities were calculated and expressed as mol %. Pancreatic hydrolysis was conducted to determine the positional distribution of the FA residues in pine nut oil and SPT.14 Twenty milligrams of the oil samples was added to 1 M Tris-HCl buffer (2 mL; pH 7.6), 0.05% sodium cholate solution (0.5 mL), and 2.2% calcium chloride solution (0.2 mL) and mixed thoroughly for emulsion. Pancreatic lipase (20 mg) was then added to the mixture, before incubation in a water bath at 40 °C for 2 min, and vigorous vortexing for 30 s. Next, 5 mL of 6 N hydrochloric acid was added to stop the reaction, and 15 mL of anhydrous diethyl ether was added twice and

MATERIALS AND METHODS

Chemicals. Pine nut oil containing 13.3 mol % PLA was obtained from Komega Co. (Eumseong, Korea). Novozym 435 was purchased from Novozymes A/S (Bagsvaerd, Denmark). Pancreatic lipase and glycerol (purity >99%) were purchased from Sigma Chemical Co. (St. Louis, MO). Silica gel thin layer chromatography (TLC) plates were supplied by Merck (Darmstadt, Germany). The FA methyl ester (FAME) standards were obtained from Supelco (Bellefonte, PA). Preparation of FFA from Pine Nut Oil. One hundred milligrams of pine nut oil was saponified with 400 mL of 10 wt % NaOH in 75 vol % ethanol. The saponified mixture then had 200 mL of distilled water added, and the emergent aqueous layer was acidified by adding 250 mL of 6 N HCl. After the addition of 500 mL of n-hexane and 300 mL of distilled water, the aqueous layer was removed using a separating funnel, and the n-hexane layer containing FFA was washed five times with 100 mL of distilled water. The n-hexane layer was dried over anhydrous sodium sulfate before the n-hexane was removed using a rotary vacuum evaporator at 40 °C. Preparation of SPT. SPT with PLA evenly distributed on the glycerol backbone was prepared via esterification of glycerol with the pine nut oil FFA fraction in a vacuum stirred-batch reactor with Novozym 435 as the biocatalyst (Figure 1). A flat-bottom glass vessel [80 mm × 35 mm internal diameter (i.d.)] equipped with a water jacket for temperature control served as the reactor. The vessel was heated to 60 °C using a water circulator (model RW-0252G, Jeio Tech, Seoul, Korea) prior to initiation of the reaction. The substrates in the reaction were glycerol and the FFA fraction at a molar ratio of 1:3. The substrates (4 g) and Novozym 435 (0.4 g) (i.e., 10 wt % based on total substrates) were placed into the vessel and agitated at B

DOI: 10.1021/acs.jafc.6b05216 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

samples were then reconstituted in petroleum ether before injection into a gas chromatograph (Agilent Technologies 7890A) equipped with an FID and DB-225 column (30 m × 0.25 mm i.d. × 0.25 μm film thickness; Agilent J&W Scientific) to quantify the FAMEs. One microliter of the sample was injected in split mode at a split ratio of 30:1. Helium was used as the carrier gas at a flow rate of 1.0 mL/min. The temperature of the injector and detector was set to 250 and 280 °C, respectively, and the column was initially held at 180 °C before the temperature was increased to 220 °C at a rate of 7 °C/min. It was then held at that temperature for a further 9 min. The FAMEs were identified via comparison of their retention times with those of the standards, and the FA content was determined using an internal standard (heptadecanoic acid; 17:0). Statistical Analysis. All data are presented as mean ± standard error of the mean (SEM). Student’s t-test was used to compare the TAG content and total FA composition between pine nut oil and SPT, as well as lymph volume and PLA content between the pine nut oil and SPT in the rat model of lymph duct cannulation. One-way analysis of variance (ANOVA) was used to examine the differences between total, sn-2, and sn-1,3 FA compositions of the pine nut oil and SPT. ANOVA was followed by Bonferroni’s multiple-range test.

the mixture was vortexed. The upper diethyl ether layer containing pancreatic lipase hydrolysates was collected and passed through an anhydrous sodium sulfate column. The hydrolysates were then spotted on TLC plates and developed with diethyl ether/hexane/acetic acid (50:50:1, v/v/v). The sn-2 MAG band was scraped from the plates, directly methylated, and analyzed by GC as described above, to determine the FA composition at the sn-2 position. The FA composition at the sn-1,3 positions was acquired using the following eq 2:15

FAsn ‐ 1,3(mol %) =

3 × FA total (mol %) − FAsn ‐ 2(mol %) 2

(2)

where FAsn‑1,3 is the content of a particular FA at the sn-1,3 positions of the TAG, FAtotal is the total content of the particular FA in the TAG, and FAsn‑2 is the content of the particular FA at the sn-2 position of the TAG. Animals and Diet. Eight-week-old male Sprague−Dawley rats (n = 12; 295.2 g) were purchased from Japan SLC (Shizuoka, Japan). Rats were housed individually under controlled temperature (22 °C) and lighting (12-h light-dark cycle) conditions. All animal procedures were approved by the Changwon National University Institutional Animal Care and Use Committee. The diet was supplied by Dyets (Bethlehem, PA) in accordance with the AIN-93 recommendations. Dried egg whites and tocopherol-free soybean oil were used as the protein and fat sources, respectively,16 with food and water provided ad libitum. Surgical Cannulation of the Mesenteric Lymph Duct. Following a fasting period of 16 h, the animals were anesthetized with isoflurane (2.0% isoflurane with O2 at 2.0 L/min), before the duodenum and mesenteric lymph ducts were cannulated.17,18 Polyethylene tubing (SV31; Dural Plastics & Engineering, Auburn, Australia) was inserted into the superior mesenteric lymph duct to collect lymph. An intraduodenal catheter (Silastic Medical grade Tubing; Dow Corning, Midland, MI) was then inserted into the duodenum approximately 2 cm below the pylorus to infuse the TAG emulsion and saline. After cannulation, the rats were individually placed into cages in a chamber with the temperature maintained at 30 °C for 20 h. During this recovery period, a maintenance solution (277.0 mM glucose in phosphate buffered saline (PBS) containing 6.8 mM Na2HPO4, 16.5 mM NaH2PO4, 115 mM NaCl, and 5 mM KCl, pH 6.4) was infused into the intraduodenal catheter at 3.0 mL/h with an infusion pump (NE-1600; New Era Pump Systems, New York, NY). Preparation of TAG Emulsions and Lymph Collection. A TAG emulsion containing either pine nut oil or SPT was prepared prior to infusion using ultrasonication. Emulsion has been prepared according to our previous studies with slight modification.16,18 The emulsion consisted of pine nut oil or SPT (452.0 μmol), Nataurocholate (396.0 μmol), cholesterol (20.7 μmol), α-tocopherol (3.1 μmol), retinol (75.4 μmol), phosphatidylcholine (10.0 μmol), and PBS (pH 6.4, 24.0 mL). Cholesterol was used as a representative dietary lipid, and the concentration was estimated on the basis of the daily intake of each lipid. α-Tocopherol and retinol were selected as they are representative fat-soluble vitamins. The emulsion was then infused through the intraduodenal catheter for 8 h (3.0 mL/h; 24 mL total) with an infusion pump. Lymph samples were simultaneously collected every hour for a total of 8 h into conical tubes containing 25 mM EDTA as an anticoagulant. Measurement of PLA Absorption. Total FAs, including PLA, were extracted from the collected lymph samples and quantified using the methods described by Folch,19 and Slover and Lanza20 with modifications. The lymph samples (100 μL) were dissolved in a chloroform:methanol mixture (2:1, v/v; 2 mL), saponified with 0.5 N methanolic NaOH (2 mL), and methylated with 14% BF3 in methanol (2 mL). At room temperature, the samples were then mixed with petroleum ether (2 mL) and saturated NaCl solution (2 mL) prior to centrifugation. The upper petroleum ether layer was then transferred to a clean tube containing 20 mg of anhydrous Na2SO4. Following incubation at room temperature, the collected petroleum ether was evaporated under a stream of N2 to remove residual water. The

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RESULTS Comparison of Chemical Compositions between Natural Pine Nut Oil and SPT. Table 1 shows the TAG, Table 1. TAG, DAG, MAG, and FFA Content of Pine Nut Oil and SPT Prepared by Novozym 435-Catalyzed Esterificationa of Glycerol with FFA Obtained from Pine Nut Oil (wt %)b TAG DAG MAG FFA

pine nut oil

SPT

98.9 ± 0.1 0.363 ± 0.015

97.0 ± 0.1c 1.85 ± 0.01c

0.681 ± 0.147

1.10 ± 0.07

Performed at 60 °C with a reaction time of 18 h, substrate molar ratio of 1:3 (glycerol-to-FFA), enzyme loading of 10 wt % (based on total substrates), and 600 rpm agitation in a stirred-batch reactor at 0.4 kPa. b Mean ± SEM (n = 2). cSignificantly different from pine nut oil (P < 0.05). a

DAG, MAG, and FFA contents in natural pine nut oil and SPT prepared by Novozym 435-catalyzed esterification of the glycerol with the FFA obtained from the pine nut oil. The TAG content was significantly greater in pine nut oil (98.9 wt %) than in SPT (97.0 wt %), while SPT (1.85 wt %) had greater DAG content than did pine nut oil (0.363 wt %). MAG was not found in either pine nut oil or and SPT. The FFA content was not significantly different between the oils (0.681 mol % for pine nut oil and 1.10 mol % for SPT). Table 2 compares total FA compositions of pine nut oil and SPT. The major FA residues in both oils were linoleic acid, 18:2n-6 (∼46 mol %); oleic acid, 18:1n-9 (∼26 mol %); and PLA (∼13 mol %), which constituted ∼85% of the total FA. The total PLA contents of pine nut oil and SPT were 13.7 and 13.3 mol %, respectively. Taxoleic acid, c5,c9-18:2 (∼2 mol %), and sciadonic acid, c5,c11,c14-20:3 (∼1 mol %) were also found as minor forms of Δ5-unsaturated polymethylene-interrupted FA in both oils. The total content of all FA including PLA in SPT was not significantly different from that of pine nut oil. However, the positional distribution of FA in SPT was markedly different from FA positioning in pine nut oil. In pine nut oil, most of the PLA was located at sn-1,3 positions (19.7 mol %) rather than the sn-2 position (1.6 mol %), C

DOI: 10.1021/acs.jafc.6b05216 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Table 2. Total and Positional FA Composition of Pine Nut Oil and SPT Prepared by Novozym 435-Catalyzed Esterificationa of Glycerol with FFA Obtained from Pine Nut Oil (mol %)b pine nut oil FA 16:0 16:1n-7 18:0 18:1n-9 18:1n-7 c5,c9-18:2 18:2n-6 c5,c9,c12-18:3 (PLA) 18:3n-3 20:0 20:1 20:2 c5,c11,c14-20:3 unidentified

sn-2

total 5.3 0.1 2.1 25.7 0.5 2.2 46.0 13.7 0.6 0.3 1.1 0.6 1.1 0.7

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.0 0.0 0.0 0.1 0.0 0.0 0.1 0.2 0.0 0.0 0.0 0.0 0.0 0.1

SPT

B A B A B B B B B B B B B AB

0.9 ± 0.0 C

25.4 ± 0.3 A

72.1 ± 0.3 A 1.6 ± 0.0 C

sn-1,3 7.5 0.1 3.2 25.8 0.8 3.3 32.9 19.7 1.0 0.5 1.7 0.9 1.6 1.0

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.0 0.0 0.1 0.1 0.0 0.0 0.1 0.3 0.0 0.0 0.0 0.0 0.0 0.2

total A A A A A A C A A A A A A A

5.3 0.1 2.2 26.0 0.5 2.2 45.6 13.3 0.6 0.3 1.2 0.6 1.1 1.0

± ± ± ± ± ± ± ± ± ± ± ± ± ±

c

0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

sn-2 A B AB A A A A B A A A A A B

sn-1,3

6.2 ± 0.5 A 2.3 25.4 0.6 2.3 45.3 14.6 0.3 0.3 1.0 0.6 1.1

± ± ± ± ± ± ± ± ± ± ±

0.1 0.4 0.1 0.0 0.6 0.2 0.1 0.1 0.1 0.1 0.0

A A A A A A B A A A A

4.9 0.1 2.1 26.3 0.5 2.2 45.7 12.7 0.8 0.3 1.3 0.6 1.1 1.4

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 0.0 0.0 0.1 0.0 0.0 0.3 0.1 0.0 0.0 0.0 0.0 0.0 0.0

A A B A A A A B A A A A A A

Performed at 60 °C with a reaction time of 18 h, substrate molar ratio of 1:3 (glycerol-to-FFA), enzyme loading of 10 wt % (based on total substrates), and 600 rpm agitation in a stirred-batch reactor at 0.4 kPa. bMean ± SEM (n = 2); means with the same letter in the same row indicate no significant difference between the total, sn-2, and sn-1,3 position FA compositions (P > 0.05). cTotal content of all FA in the SPT was not significantly different from that in the pine nut oil (P > 0.05). a

whereas PLA tended to evenly exist between the sn-2 (14.6 mol %) and sn-1,3 positions (12.7 mol %) in SPT, and the difference was statistically significant. In addition, the PLA content at the sn-1,3 positions of the SPTs was not significantly different from the total PLA content of the oil. Linoleic acid, which was the most abundant FA in both pine nut oil and SPT, was predominantly located at the sn-2 position (72.1 mol %) as compared to the sn-1,3 positions (32.9 mol %) in pine nut oil. In SPT, no significant difference was found in linoleic acid content at the sn-2 (45.3 mol %) and sn-1,3 positions (45.7 mol %). However, neither oil had a significant difference in the oleic acid positioning (25.4 mol % at sn-2 and 25.8 mol % at sn-1,3 for pine nut oil; 25.4 mol % at sn-2 and 26.3 mol % at sn-1,3 for SPT). SPT also did not have significant differences in the positioning of other FA, such as palmitic (16:0), taxoleic, and sciadonic acids between the sn-2 and sn-1,3 positions. Therefore, these results showed that in SPT, all FAs including PLA were almost evenly distributed through the glycerol backbone of the oil. Comparison of PLA Lymphatic Absorption from Pine Nut Oil and SPT. To compare the capacity for lymphatic absorption of PLA between pine nut oil and SPT, hourly and cumulative concentrations of PLA in lymph were measured during 8 h of the continuous infusion of an emulsion containing equimolar concentrations of either pine nut oil or SPT into the duodenum (Figure 2). There was no significant difference in total lymph volume collected over 8 h from the rats infused with the emulsion containing either pine nut oil (22.5 mL) or SPT (23.3 mL) (Table 3). In both pine nut oil and SPT groups, hourly lymphatic absorption of PLA gradually increased during the first 4 h of the infusion, reached a maximum after 4 h of the infusion, and then dropped. Although hourly lymphatic absorption of PLA tended to be greater in the SPT group than in the pine nut oil group after 4, 5, 6, 7, and 8 h of infusion, this difference was not statistically significant (Figure 1A). However, cumulative lymphatic absorption of PLA was significantly greater in the SPT group versus the pine nut oil group at all time points after 6 h of the infusion (Figure 1B). Thus, these results suggest that

Figure 2. Hourly (A) and cumulative (B) lymphatic absorption of PLA in rats infused with an emulsion containing either equimolar concentrations of pine nut oil or SPT prepared by Novozym 435catalyzed esterification of glycerol with FFA obtained from pine nut oil. Data are presented as mean ± SEM (n = 7). *Denotes significant difference (P < 0.05).

PLA from SPT of which the PLA was evenly distributed on the glycerol backbone underwent greater lymphatic absorption than did PLA from pine nut oil where PLA was predominantly D

DOI: 10.1021/acs.jafc.6b05216 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

A major factor contributing to the retention of FA at the sn-2 position is the regiospecificity of pancreatic lipase for FA located at the sn-1 and sn-3 positions and the chain length of FA.8 Long-chain FA (LCFA) including PLA located at the sn-1 and sn-3 positions is absorbed via different pathways as compared to sn-2-MAG, in that sn-2-MAG isabsorbed via passive diffusion, while LCFA requires a protein-mediated process.28,29 After absorption, sn-2-MAG is either utilized for synthesizing gut or liver phospholipids or incorporated into lymph chylomicrons after fast esterification into TAG, which then enters the circulation and is delivered to target organs (liver, heart) or tissues (adipose, skeletal) via the thoracic duct.30 Meanwhile, LCFA released from the sn-1 and sn-3 positions of TAG is reassembled into new TAG via the phosphatidic acid pathway31 and monoacylglycerol pathway,32 and these FAs are slowly absorbed into the circulation. This study is important, because several recently published studies emphasize the health benefits of the pine nut oil, particularly in terms of obesity and metabolic disorders. For instance, potential antiobesity and antidiabetic effects of pine nut oil have been suggested by Le et al.30 They found that pine nut oil replacement causes less weight gain that was associated with increased adenosine monophosphate-activated protein kinase, and increased uncoupling protein-1 gene expression in brown adipose tissue in high-fat diet-induced obese mice. Christiansen et al.33 also proposed that PLA acts as a dual FFA1/FFA4 agonist (FFA1 free fatty acid receptor 1; FFA4, free fatty acid receptor 4), indicating that PLA enhances glucose-stimulated insulin secretion and has insulin-sensitizing and anti-inflammatory effects. Park et al.34 demonstrated that 10% pine nut oil replacement significantly reduced liver weight and liver TAG levels in high-fat diet-induced obese mice, which was likely related to increased sirtuin-3 (SIRT3) gene expression. A study by Lee et al.35 further supported the antiobesity effect of pine nut oil by providing evidence that PLA attenuated lipid anabolism pathway by reducing gene expression of sterol regulatory element-binding protein 1c (SREBP1c), fatty acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD1), and acetyl-CoA carboxylase 1 (ACC1) in the HepG2 cell line. In conclusion, pine nut oil has received great attention due to its content of PLA, which suppresses appetite and thereby aids in weight loss. Pine nut oil is the sole commercial form of PLA and is the only known major natural source of this FA. The importance of FA stereospecificity during digestion and absorption raises the hypothesis that TAG with an even distribution of PLA on the glycerol backbone would have greater lymphatic PLA absorption than natural pine nut oil. To test this hypothesis, SPTs were prepared, and the lymphatic absorption rate of pine nut oil and SPT was compared using mesenteric lymph duct cannulated rat model. The current study demonstrated that consumption of synthesized SPT results in a higher lymphatic PLA absorption than that of natural pine nut oil. However, further investigations on the PLA content at the sn-2 position of TAG recovered from lymph would be required to provide direct evidence that enriching PLA at the sn-2 position of SPT leads to increases in absorption capacity. Given that pine nut oil has potential antiobesity and antidiabetic effects, SPT with greater lymphatic PLA absorption can be used as an alternative to the natural pine nut oil in obesity prevention.

Table 3. Total Lymph Volume and Lymph PLA Concentrationsa pine nut oil total lymph volume (mL/8 h) μmol per 8 h % dose per 8 h FFA

22.5 118 26.2 0.7

± ± ± ±

1.6 3 0.6 0.1

SPT 23.3 129 28.5 1.1

± ± ± ±

0.7 3b 0.7b 0.1

a Mean ± SEM (n = 7). bSignificantly different from pine nut oil (P < 0.05).

located at the sn-3 position. After 8 h of infusion, the amounts of all FA including 16:0, stearic acid (18:0), 18:1n-9, 18:2n-6, and arachidonic acid (20:4n-6) as well as PLA in lymph collected from an emulsion containing SPT were significantly greater than those in lymph collected from an emulsion containing PNO (data available in Table S1).

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DISCUSSION Since the late 2000s, natural pine nut oil has received much attention for use as an appetite suppressant because an unusual polyunsaturated FA called PLA in this oil increases CCK and GLP-1 secretion in humans.2,3 However, PLA is predominantly esterified at the sn-3 position of the oil, resulting in the relatively low absorption efficiency of PLA. Unlike natural pine nut oil, SPT prepared in this study did show an even distribution of PLA on the glycerol backbone by enriching PLA at the sn-2 position, leading to increases in absorption capacity in a rat model of lymphatic cannulation. Our research group has employed the Novozym 435catalyzed esterification of glycerol with FFA, including conjugated linoleic acid and/or PLA with antiobesity properties to prepare structured TAG with an even distribution of the FA on the glycerol backbone.12,21 In the present study, SPT was prepared under the optimal conditions for esterification, which were established in previously published studies. As with the natural pine nut oil, SPT was comprised almost exclusively of TAG with very low levels of DAG and FFA. Although there was a statistically significant difference in TAG content between the oils, the difference arose from the very high precision (i.e., the SEM value was 0.1) of our analysis technique. The FA analysis proved that SPT contained equal amounts of PLA as pine nut oil, but the PLA was evenly distributed at all sn positions in SPT. The metabolic fate of TAG during digestion and absorption is determined by FA stereospecificity and chain length.22,23 Gastric and pancreatic lipases hydrolyze TAG to produce FFA (from the sn-1 and sn-3 positions of TAG) and sn-2-MAG during digestion. Indeed, due to their high affinity for ester bonds in the sn-1 and sn-3 positions of TAG, lipases readily hydrolyze FA at these positions, but hydrolyze only ∼22% of FA at the sn-2 position.24,25 This suggests that FA present at the sn-2 position remains intact in the glycerol backbone (∼75% conservation rate).8 Much evidence indicates that polyunsaturated fatty acid (PUFA) present at sn-2 position is preferentially absorbed. Linoleic acid in sn-2 position is correlated with plasma concentration of arachidonic acid, indicating that 18:2 was maintained in the sn-2 position during absorption.26 In addition, while saturated fatty acids from cocoa butter, present in the sn-1 and -3 positions, were lost in feces, 18:2 presented in sn-2 position was found in the epididymal fat tissue.27 These collectively suggest that PUFA located in sn-2 position plays an important role in both metabolism and biological effects. E

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(9) Karupaiah, T.; Sundram, K. Effects of stereospecific positioning of fatty acids in triacylglycerol structures in native and randomized fats: a review of their nutritional implications. Nutr. Metab. 2007, 4, 16. (10) Choi, J. H.; Kim, B. H.; Hong, S. I.; Kim, C. T.; Kim, C. J.; Kim, Y.; Kim, I. H. Lipase-catalysed production of triacylglycerols enriched in pinolenic acid at the sn-2 position from pine nut oil. J. Sci. Food Agric. 2012, 92, 870−876. (11) Kim, B. H.; Akoh, C. C. Recent research trends on the enzymatic synthesis of structured lipids. J. Food Sci. 2015, 80, C1713− C1724. (12) Woo, H.; Kim, J.; Kim, I. H.; Choi, H. D.; Choi, I. W.; Kim, B. H. Substrate selectivity of Novozym 435 in the esterification of glycerol with an equimolar mixture of linoleic, conjugated linoleic, and pinolenic acids. Eur. J. Lipid Sci. Technol. 2016, 118, 928−937. (13) Kang, K. K.; Kim, S.; Kim, I. H.; Lee, C.; Kim, B. H. Selective enrichment of symmetric monounsaturated triacylglycerols from palm stearin by double solvent fractionation. LWT - Food Sci. Technol. 2013, 51, 242−252. (14) Luddy, F. E.; Barford, R. A.; Herb, S. F.; Magidman, P.; Riemenschneider, R. W. Pancreatic lipase hydrolysis of triglycerides by a semimicro technique. J. Am. Oil Chem. Soc. 1964, 41, 693−696. (15) Kim, B. H.; Lumor, S. E.; Akoh, C. C. trans-Free margarines prepared with canola oil/palm stearin/palm kernel oil-based structured lipids. J. Agric. Food Chem. 2008, 56, 8195−8205. (16) Kim, J.; Koo, S. I.; Noh, S. K. Green tea extract markedly lowers the lymphatic absorption and increases the biliary secretion of 14Cbenzo[a]pyrene in rats. J. Nutr. Biochem. 2012, 23, 1007−1011. (17) Koo, S. I.; Noh, S. K. Phosphatidylcholine inhibits and lysophosphatidylcholine enhances the lymphatic absorption of alphatocopherol in adult rats. J. Nutr. 2001, 131, 717−722. (18) Noh, S. K.; Koo, S. I. Milk sphingomyelin is more effective than egg sphingomyelin in inhibiting intestinal absorption of cholesterol and fat in rats. J. Nutr. 2004, 134, 2611−2616. (19) Folch, J.; Lees, M.; Sloane Stanley, G. H. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 1957, 226, 497−509. (20) Slover, H. T.; Lanza, E. Quantitative analysis of food fatty acids by capillary gas chromatography. J. Am. Oil Chem. Soc. 1979, 56, 933− 943. (21) Kang, I.; Bang, H. J.; Kim, I. H.; Choi, H. D.; Kim, B. H. Synthesis of trans-10,cis-12 conjugated linoleic acid-enriched triacylglycerols via two-step lipase-catalyzed esterification. LWT - Food Sci. Technol. 2015, 62, 249−256. (22) Small, D. M. The effects of glyceride structure on absorption and metabolism. Annu. Rev. Nutr. 1991, 11, 413−434. (23) Decker, E. A. The role of stereospecific saturated fatty acid positions on lipid nutrition. Nutr. Rev. 1996, 54, 108−110. (24) Rogalska, E.; Ransac, S.; Verger, R. Stereoselectivity of lipases. II. Stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases. J. Biol. Chem. 1990, 265, 20271−20276. (25) Mattson, F. H.; Volpenhein, R. A. The digestion and absorption of triglycerides. J. Biol. Chem. 1964, 239, 2772−2777. (26) Renaud, S. C.; Ruf, J. C.; Petithory, D. The positional distribution of fatty acids in palm oil and lard influences their biologic effects in rats. J. Nutr. 1995, 125, 229−237. (27) Apgar, J. L.; Shively, C. A.; Tarka, S. M., Jr. Digestibility of cocoa butter and corn oil and their influence on fatty acid distribution in rats. J. Nutr. 1987, 117, 660−665. (28) Yang, L. Y.; Kuksis, A. Apparent convergence (at 2monoacylglycerol level) of phosphatidic acid and 2-monoacylglycerol pathways of synthesis of chylomicron triacylglycerols. J. Lipid Res. 1991, 32, 1173−1186. (29) Schulthess, G.; Lipka, G.; Compassi, S.; Boffelli, D.; Weber, F. E.; Paltauf, F.; Hauser, H. Absorption of monoacylglycerols by small intestinal brush border membrane. Biochemistry 1994, 33, 4500−4508. (30) Le, N. H.; Shin, S.; Tu, T. H.; Kim, C. S.; Kang, J. H.; Tsuyoshi, G.; Teruo, K.; Han, S. N.; Yu, R. Diet enriched with korean pine nut oil improves mitochondrial oxidative metabolism in skeletal muscle

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05216. Table S1 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +82-55-213-3516. Fax: +82-55-281-7480. E-mail: [email protected]. *Tel.: +82-2-2077-7241. Fax: +82-2-710-9479. E-mail: bhkim@ sookmyung.ac.kr. ORCID

Byung Hee Kim: 0000-0002-4599-6775 Funding

This research was supported by the Main Research Program (E0124200-03) of the Korea Food Research Institute (KFRI) funded by the Ministry of Science, ICT & Future Planning, and was also supported by the Sookmyung Women’s University Research Grants (1-1603-2008). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ACC1, acetyl-CoA carboxylase 1; CCK, cholecystokinin; DAG, diacylglycerol; FA, fatty acid; FAME, fatty acid methyl ester; FAS, fatty acid synthase; FFA, free fatty acid; FFA1, free fatty acid receptor 1; FFA4, free fatty acid receptor 4; GC, gas chromatography; GLP-1, glucagon-like peptide-1; LCFA, longchain fatty acid; MAG, monoacylglycerol; PLA, pinolenic acid; SCD1, stearoyl-CoA desaturase 1; SIRT3, sirtuin-3; SPT, structured pinolenic triacylglycerol; SREBP1c, sterol regulatory element-binding protein 1c; TAG, triacylglycerol; TLC, thin layer chromatography

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REFERENCES

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