Comparison of Catabolic Rates of sn-1, sn-2, and sn-3 Fatty Acids in Triacylglycerols Using 13 CO 2 Breath Test in Mice

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1 Journal of Oleo Science Copyright 2017 by Japan Oil Chemists Society doi : /jos.ess16124 Comparison of Catabolic Rates of sn-1, sn-2, and sn-3 Fatty Acids in Triacylglycerols Using 13 CO 2 Breath Test in Mice Fumiaki Beppu 1, Takashi Kawamatsu 1, Yoshio Yamatani 1, Toshiharu Nagai 2, Kazuaki Yoshinaga 2, Hoyo Mizobe 2, Akihiko Yoshida 2, Atsushi Kubo 3, Jota Kanda 3 and Naohiro Gotoh 1* 1 Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan, Minato-ku, Tokyo , JAPAN 2 Tsukishima Foods Industry Co. Ltd., Higashi Kasai, Edogawa-ku, Tokyo , JAPAN 3 Department of Ocean Science, Tokyo University of Marine Science and Technology, Konan, Minato-ku, Tokyo , JAPAN Abstract: Fatty acids in triacylglycerols (TAGs) are catabolized after digestion. However, the catabolic rates of the fatty acids at the sn-1, sn-2, and sn-3 positions of TAGs have not been compared. To elucidate the differences, we studied the catabolic rates of 13 C-labeled palmitic acid, oleic acid, and capric acid at the sn-1, sn-2, or sn-3 position of TAGs using isotope-ratio mass spectrometry. Specifically, we measured the 13 C-to- 12 C ratio in CO 2 (Δ 13 C ( )) exhaled by mice. For all analyzed fatty acids, we observed significant differences between sn-2 and other binding positions. In contrast, no significant difference was detected between the sn-1 and sn-3 positions. These results indicated that the catabolic rates of fatty acids are strongly influenced by their positions in TAGs. Key words: binding position, catabolism, 13 C-labeled fatty acid, lipase, triacylglycerol 1 INTRODUCTION Lipids are one of the three major nutrients in our diet and triacylglycerols TAGs are the main component of lipids. TAGs consist of one glycerol molecule esterified to three fatty acids 1. In a Fischer projection 2, the glycerol backbone of the TAG shows three binding positions: sn-1 α, primary alcohol group of glycerol, sn-2 β, secondary alcohol group of glycerol, and sn-3 α, primary alcohol group of glycerol Fig. 1. Therefore, three kinds of isomers, called TAG molecular species TAG-MS, TAG positional isomer TAG-PI, and TAG enantiomer TAG-E, must be considered when the TAG is esterified by three different fatty acids 3 5. For example, consider a TAG with the fatty acids A, B, and C. The TAG structure considering just the A, B, and C combination would be TAG-MS and would be indicated by ABC. In case two binding positions, α primary alcohol group of the glycerol backbone and β secondary alcohol group of the glycerol backbone, can be distinguished in the TAG, it would provide three isomers: β-abc, β-acb, and β-bac the β prefix indicates that the fatty acid at the β position is fixed 2. These isomers would be called TAG-PI. Furthermore, the sn-1, sn-2, and sn-3 positions can be distinguished when the central glycerol carbon is a chiral center. In this case, the TAG consisting of A, B, and C would have six possible isomers: sn-abc, sn-cba, sn-acb, sn-bca, sn-bac, and sn-cab. These isomers would be called TAG-E. Nature can distinguish these binding positions. For example, pancreatic lipase hydrolyzes bile acid-bound fatty acids at the α po- Abbreviations: CCK, cholecystokinin; IRMS, isotope-ratio mass spectrometry; LPL, lipoprotein lipase; 2-MAG, 2-monoacylglycerol; TAG, triacylglycerol; sn-*c 8 OO, 1-[1-13 C]-capryl-2,3-dioleoyl-sn-glycerol; sn-o*c 8 O, 1,3-dioleoyl-2-[1-13 C]-capryl-sn-glycerol; sn-oo*c 8, 1,2-dioleoyl-3-[1-13 C]-capryl-sn-glycerol; sn-oo*o, 1,2-dioleoyl-3-[1-13 C]-oleoyl-sn-glycerol; sn-o*oo, 1,3-dioleoyl-2-[1-13 C]-oleoyl-sn-glycerol; sn-*ooo, 1-[1-13 C]- oleoyl-2,3-dioleoyl-sn-glycerol; sn-o*po, 1,3-dioleoyl-2-[1-13 C]-palmitoyl-sn-glycerol; sn-oo*p, 1,2-dioleoyl-3-[1-13 C]- palmitoyl-sn-glycerol; sn-*poo, 1-[1-13 C]-palmitoyl-2,3- dioleoyl-sn-glycerol; TAG-E, TAG enantiomer; TAG-MS, TAG molecular species; TAG-PI, TAG positional isomer; VPDB, Vienna Pee Dee Belemnite * Correspondence to: Naohiro Gotoh, Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan, Minato-ku, Tokyo , JAPAN ngotoh@kaiyodai.ac.jp Accepted August 22, 2016 (received for review June 28, 2016) Journal of Oleo Science ISSN print / ISSN online

2 F. Beppu, T. Kawamatsu and Y. Yamatani et al. Fig. 1 TAG isomers. sition in the small intestine to yield two fatty acids and 2-monoacylglycerol 2-MAG 6. The relationship between the fatty acid position in TAGs and the function or catabolic rate of fatty acids has been studied. For example, the catabolic rate of stable isotope-labeled palmitic acid *P bound at α or β position was compared using a CO 2 breath test in mice 7. The results suggested that the catabolic rate was different between the α and β positions and that in the first stage, the fatty acid at α position was catabolized faster than that at the β position. However, the rates gradually changed, and at 4-6 hours after administration of labeled TAG, the catabolic rate of the fatty acid at β position became faster than that of the fatty acid at the α position. This difference could be due to the characteristics of pancreatic lipase. This specificity of pancreatic lipase results in differential absorption and function of the fatty acids in TAGs. Other types of lipases, namely, lingual lipase and gastric lipase, also participate in TAG digestion. These socalled acid lipases can function at low ph 8, 9 and do not need bile acid for their activity. These lipases also have stereo-specificity and characteristically hydrolyze fatty acids at the sn-3 α position For example, lingual lipase hydrolyzes the fatty acids at sn-3 position twice as fast as those at the sn-1 position 10, 11. Gastric lipase hydrolyzes of TAGs to free fatty acids during digestion. Therefore, the role of lipases that selectively hydrolyze the fatty acid at the sn-3 position might also be important in digestion in humans. It is known that 1,2-diacylglycerol is formed after digestion by gastric lipase; however, the effect of the sn-1, sn-2, and sn-3 positions on the catabolic rate of fatty acids in TAGs has not been examined. The fatty acids in TAGs are beta-oxidized to acetyl-coa in the mitochondrial matrix after digestion. The acetyl-coa is then converted to CO 2 in the tricarboxylic acid cycle, which generates high-energy compounds such as nicotinamide adenine dinucleotide 12. In the present study, we compared the catabolic rates of 13 C-labeled sn-1, sn-2, or sn-3 fatty acids of TAGs by measuring the 13 C-labeled CO 2 expired by ddy mice that were administered emulsions of the labeled TAGs and to understand the effect of gastric lipase which favorites to hydrolyze fatty acid bound to sn-3 position of TAG for digestion. 2 MATERIALS AND METHODS 2.1 Chemicals and materials All the TAG samples C -capryl-2,3-dioleoyl-snglycerol sn-*c 8 OO, 1,3-dioleoyl C -capryl-sn-glycerol sn-o*c 8 O, 1,2-dioleoyl C -capryl-sn-glycerol 86

3 Comparison of fatty acid catabolism rate among 3 binding positions in TAG sn-oo*c 8, C -palmitoyl-2,3-dioleoyl-sn-glycerol sn-*poo, 1,3-dioleoyl C -palmitoyl-sn-glycerol sn-o*po, 1,2-dioleoyl C -palmitoyl-sn-glycerol sn-oo*p, C -oleoyl-2,3-dioleoyl-sn-glycerol sn- *OOO, 1,3-dioleoyl C -oleoyl-sn-glycerol sn- O*OO, and 1,2-dioleoyl C -oleoyl-sn-glycerol sn- OO*O were in-house products Fig. 2. Other reagents were purchased from Wako Pure Chemical Industries, Ltd. Osaka, Japan. 2.2 Preparation of TAG emulsions Each species of 13 C-labeled TAG mol was placed in a 10-mL screw-cap vessel. An emulsifying agent Triton X-100, 5 of TAG and distilled water were added to the vessel to obtain a TAG concentration of mol/l. The vessel was closed, and the contents were thoroughly mixed using a tube mixer. This crude mixture was further emulsified using an ultrasonic homogenizer Ultrasonic Disruptor UD-200, Tomy Seiko Co. Ltd., Tokyo, Japan. 2.3 Animal species, emulsion dosing, and measurement of expired gas All experiments involving animals were performed in accordance with the Guidelines for Animal Use and Care set forth by the Tokyo University of Marine Science and Technology. Four-week-old male ddy mice were obtained from Japan SLC, Inc. Hamamatsu, Japan. The mice were housed in stainless-steel wire-mesh cages under a controlled 12-h light and 12-h dark cycle at 22 2 and relative humidity. The mice had free access to water and were fed rodent chow MF Oriental Yeast Co., Ltd., Tokyo, Japan. Forty-five mice were divided equally into nine groups: i sn-*c 8 OO group, ii sn-o*c 8 O group, iii sn-oo*c 8 group, iv sn-*poo group, v sn-o*po group, vi sn-oo*p group, vii sn-*ooo group, viii sn-o*oo group, and ix sn-oo*o group. The emulsion was orally administered 0.10 ml/20 g body weight to the mice using a feeding needle. The expired gas was sampled using a 450-mL plastic vessel. The mice were placed in the vessel for 5 min, and the air 50 ml in the vessel was sampled using a syringe. The sampled air was divided into three evacuated 12-mL vials Exetainer, Labco Ltd., High Wycombe, UK. Sampling was performed at 0 before administration, 15, 30, 45, 60, 75, 90, 105, 120, 240, and 360 min after administration of the emulsion. The mice were not fed between samplings. 2.4 Determination of the ratio of 13 CO 2 to 12 CO 2 in sampled gas The natural abundances of 13 C and 12 C in G1-grade CO 2 purchased from Taiyo Nippon Sanso Corporation Tokyo, Japan were analyzed by SI Science Co. Ltd., Sugito, Japan, δ 13 C-Vienna Pee Dee Belemnite VPDB The CO 2 was diluted 10 fold with nitrogen gas, and the mixed gas was packed into a 12-mL evacuated vial. The mixed gas was used as a standard to adjust the value measured using isotope-ratio mass spectrometry IRMS ANCA-GSL, SerCon Ltd., Cheshire, UK. The ratio of 13 C to 12 C in the Fig. 2 Structures of TAG isomers used in this study. 87

4 F. Beppu, T. Kawamatsu and Y. Yamatani et al. CO 2 expired by the mice was analyzed by IRMS. The following parameters were used: ionization method, electron ionization; gas chromatography column temperature, 100 ; carrier gas, helium; carrier gas flow rate, 60 ml/min; and injection time, 12 s. The δ 13 C values in each sample of the expired gas were calculated using the following equation 13 : δ 13 C 13 C/ 12 C sample / 13 C/ 12 C standard Equation 1 where 13 C/ 12 C sample denotes the ratio of 13 C to 12 C in the expired CO 2, and 13 C/ 12 C standard represents the ratio of 13 C to 12 C in VPDB The δ 13 C values at each sampling time were adjusted by subtracting the δ 13 C at 0 h using the following equation, and the resulting values were reported as Δ 13 C. Δ 13 C δ 13 Ct δ 13 C 0 Equation 2 where δ 13 Ct represents the δ 13 C value at time t after sample administration, and δ 13 C 0 represents the δ 13 C value before sample administration. 2.5 Statistical analysis Differences among the groups were analyzed by one-way ANOVA and all individual differences were further analyzed using a Steel-Dwass test. Differences were considered significant when p was less than RESULTS AND DISCUSSION 3.1 Comparison of TAGs containing *C 8 and O The time course of 13 C-labeled CO 2 expiration from the sn-*c 8 OO, sn-o*c 8 O, and sn-oo*c 8 groups was compared Fig. 3. C 8 is a medium-chain fatty acid that is rapidly catabolized to CO 2 gas after absorption. This rapid catabolism is possible because medium-chain fatty acids pass through the inner mitochondrial membrane without binding to carnitine and are quickly beta-oxidized to acetyl-coa. Therefore, the increments in Δ 13 C occurred more rapidly for this TAG group than for the others. The sn-*c 8 OO and sn- OO*C 8 groups exhibited maximum Δ 13 C at 15 min after administration of the sample emulsion. Thereafter, the Δ 13 C decreased gradually over time and returned to the original value at 360 min. In contrast, the Δ 13 C of the sn-o*c 8 O group peaked at 60 min. These results suggest that the fate of the fatty acid at the sn-2 β position is different from that of the fatty acids at the other two positions. Significant difference was observed between the sn- O*C 8 O group and the sn-*c 8 OO and sn-oo*c 8 groups at min, but not between the sn-*c 8 OO and sn-oo*c 8 groups. The difference between the sn-o*c 8 O group and the sn-*c 8 OO and sn-oo*c 8 groups at 15 min could be due Fig. 3 Comparison of Δ 13 C in CO 2 expired from mice among sn-*c 8 OO, sn-o*c 8 O, and sn-oo*c 8 groups. Values represent mean SE. Different letters indicate significant difference at p 0.05 at the respective sampling time. to the preference of pancreatic lipase for the fatty acid at α position sn-1 and sn-3 positions. Owing to this preferential hydrolysis, the fatty acid at β position sn-2 position is not released from the glycerol backbone, resulting in the formation of 2-monoacyl glycerol 2-MAG, which is absorbed by the epithelial cells of the small intestine, where the TAG gets resynthesized. The catabolic rate of mediumchain fatty acids at the sn-2 position is lower than that of fatty acids at the sn-1 and sn-3 positions 7. This is consistent with our results. In contrast, the effect of gastric lipase was not confirmed in this study because no significant difference was observed between the sn-*c 8 OO and sn- OO*C 8 groups. Gastric lipase displays a preference for the fatty acid at the sn-3 position. Therefore, at earlier time points, the Δ 13 C increment of the sn-oo*c 8 group was faster than that of the sn-*c 8 OO groups. This could imply that unlike pancreatic lipase, gastric lipase does not directly affect the digestion of TAGs and that it might have a different function. Furthermore, the products of gastric lipolysis are known to facilitate subsequent rapid TAG digestion by pancreatic lipase 12, 14. Bile acids are essential for the hydrolysis of TAGs by pancreatic lipase in the digestive tract. Pancreatic lipase and bile acids are released simultaneously from the pancreas and gallbladder, respectively 16 18, following stimulation by cholecystokinin CCK, a peptide hormone secreted by enteroendocrine cells in the duodenum 15. The secretion of CCK is triggered by the presence of free fatty acids, peptides, and amino acids in the duodenum 19, 20. These findings suggest that the release of fatty acids from TAGs in the stomach by the action of gastric lipase is linked with CCK secretion in the duodenum and that TAG digestion is not completed in the stomach. This hypothesis is supported by the lack of significant differences in Δ 13 C between the sn-*c 8 OO and sn-oo*c 8 88

5 Comparison of fatty acid catabolism rate among 3 binding positions in TAG groups. 3.2 Comparison of TAGs containing *P and O The Δ 13 C values of the sn-*poo, sn-o*po, and sn-oo*p groups are shown in Fig. 4. Both *P and *C 8 are saturated fatty acids with different carbon chain lengths; *C 8 has an 8-carbon chain, while *P has an 18-carbon chain. Unlike in Fig. 3, the Δ 13 C of all the groups reached the maximum at 75 min. This delay in Δ 13 C increment may be due to the low preference of lipase for long-chain fatty acids. This result is similar to that reported by the study comparing the catabolic rates of 13 C-labeled palmitic acid at the α and β positions 7. Significant difference among the three groups was detected at 15 min, and the Δ 13 C values of the sn-*poo and sn-oo*p groups were higher than that of the sn-o*po group. However, no significant difference was detected between the sn-*poo and sn-oo*p groups. This is comparable to the result obtained for sn-*c 8 OO, sn-o*c 8 O, and sn-oo*c 8. In particular, the lack of a significant difference in the Δ 13 C values for the sn-*poo and sn-oo*p groups suggests that unlike pancreatic lipase, gastric lipase does not strongly affect the hydrolysis of TAGs in digestive tract. 3.3 Comparison of TAGs containing *O and O The comparison of Δ 13 C among sn-*ooo, sn-o*oo, and sn-oo*o groups is shown in Fig. 5. All the groups reached the maximum Δ 13 C at min. Significant difference among the three groups was detected at 15 and 30 min. The Δ 13 C values of the sn-*ooo and sn-oo*o groups were higher than that of the sn-o*oo group. However, the difference between the Δ 13 C of the sn-*ooo and sn-oo*o groups was not significant. These trends were the same as those observed for sn-*poo, sn- O*PO, and sn-oo*p Fig. 4. Hence, it can be said that the structural difference between the long-chain fatty acids, namely, monounsaturated fatty acid *O and saturated fatty acid *P, does not critically influence their fate. In contrast, the length of the fatty acid chains in TAGs strongly affects their hydrolytic and catabolic rates Figs. 3, 4 and 5. There was no difference between the stable isotope-labeled fatty acids at sn-1 and sn-3 positions even in the case of long-chain monounsaturated fatty acid. This result suggests that the low activity of gastric lipase might not be due to the structure of the fatty acids, but might be naturally very low. Interestingly, significant difference among the three groups was detected at 360 min; however, the Δ 13 C values of the sn-*ooo and sn-oo*o groups were lower than that of the sn-o*oo group in this case. A similar trend was observed in Fig. 3 and our previous study using *P 7. We previously thought that the difference is attributable to the difference in the characteristics of the two lipases 7. As mentioned above, the fatty acid at the sn-2 position is absorbed by the epithelial cells of the small intestine in the form of 2-MAG, which is then resynthesized to TAG. This indicates that the position of the sn-2 fatty acid in the original TAG is retained in the resynthesized TAG. The resynthesized TAGs are incorporated into chylomicrons in the epithelial cells of the small intestine and circulate through the bloodstream. The TAGs in the chylomicrons are hydrolyzed by lipoprotein lipase LpL located on the capillary walls. LpL is expressed in the adipose tissue, heart, spleen, and lung. However, it is not active in the liver 21. Instead of LpL, the liver expresses hepatic lipase, which has different characteristics from those of LpL. Specifically, LpL hydrolyzes sn-1 and 3 positions of TAGs to 2-MAG and free fatty acids. In contrast, hepatic lipase hydrolyzes sn-2 fatty acids 22, 23. The 2-MAG formed in this re- Fig. 4 Comparison of Δ 13 C in CO 2 expired from mice among sn-*poo, sn-o*po, and sn-oo*p groups. Values represent mean SE. Different letters indicate significant difference at p 0.05 at the respective sampling time. Fig. 5 Comparison of Δ 13 C in CO 2 expired from mice among sn-*ooo, sn-o*oo, and sn-oo*o groups. Values represent mean SE. Different letters indicate significant difference at p 0.05 at the respective sampling time. 89

6 F. Beppu, T. Kawamatsu and Y. Yamatani et al. action is transferred to albumin or high-density lipoprotein and is hydrolyzed to glycerol and free fatty acids by hepatic lipase in the liver 22, 24. The free fatty acids that were originally at the sn-2 position of the resynthesized TAGs are ultimately beta-oxidized in the liver. This metabolic pathway requires a long time for completion and might be responsible for the significant differences observed at 360 min in Fig CONCLUSIONS We compared Δ 13 C of 13 C-labeled sn-1, sn-2, and sn-3 fatty acids of TAGs using mouse breath test. The differences between the α sn-1 and sn-3 position and β sn-2 positions were observed in all the experiments and the fatty acids at the α position were catabolized faster than those at the β position. This result was attributed to the substrate preference of pancreatic lipase. In contrast, the characteristic of gastric lipase did not express as the different pattern of the Δ 13 C and no significant difference was observed between the sn-1 and sn-3 positions. These results suggest that unlike pancreatic lipase, gastric lipase does not directly participate in the digestion of TAGs and may have a different role. We propose that the gastric lipase-mediated release of fatty acids from TAGs in the stomach stimulates CCK secretion in the duodenum. This is because free fatty acids are known to trigger CCK secretion in the duodenum. However, further studies are needed to confirm our hypothesis. Conflict of interest The authors declare no conflicts of interest. References 1 Botham, K.M.; Mayes, P.A. Lipids of physiologic significance. in Harper s Illustrated Biochemistry Murray, R.K.; Granner, D.K.; Rodwell, V.W. eds., 27th ed. Mc- Graw-Hill Companies, New York USA, pp Owen R.F. Food chemistry. 3rd ed. Marcel Dekker Inc., New York USA, pp Kuroda, I.; Nagai, T.; Mizobe, H.; Yoshimura, N.; Gotoh, N.; Wada, S. HPLC separation of triacylglycerol positional isomers on a polymeric ODS column. Anal. Sci. 24, Nagai, T.; Gotoh, N.; Mizobe, H.; Yoshinaga, K.; Kojima, K.; Matsumoto, Y.; Wada, S. Rapid separation of triacylglycerol positional isomers binding two saturated fatty acids using octacocyl silylation column. J. Oleo Sci. 60, Nagai, T.; Mizobe, H.; Otake, I.; Ichioka, K.; Kojima, K.; Matsumoto, Y.; Gotoh, N.; Kuroda, I.; Wada, S. Enantiomeric separation of asymmetric triacylglycerol by recycle high-performance liquid chromatography with chiral column. J. Chromatogr. A 1218, Brockerhoff, H.; Yurkowski, M. Stereospecific analysis of several vegetable fats. J. Lipid Res. 7, Beppu, F.; Konno, K.; Kawamatsu, T.; Nagai, T.; Yoshinaga, K.; Mizobe, H.; Kojima, K.; Watanabe, H.; Gotoh, N. Comparison of catabolic rates of 13 C-labeled palmitic acid bound to the alpha and beta positions of triacylglycerol using CO 2 expired from mice. Eur. J. Lipid Sci. Technol. 117, Hamosh, M.; Scow, R.O. Lingual lipase and its role in the digestion of dietary lipid. J. Clin. Invest. 52, Roussel, A.; Canaan, S.; Egloff, M.P.; Rivière, M.; Dupuis, L.; Verger, R.; Cambillau, C. Crystal Structure of human gastric Lipase and model of lysosomal acid lipase, two lipolytic enzymes of medical interest. J. Biol. Chem. 274, Staggers, J.E.; Fernando-Warnakulasuriya, G.J.P.; Wells, M.A. Studies on fat digestion, absorption, and transport in the suckling rat. II. Triacylglycerols: molecular species, stereospecific analysis, and specificity of hydrolysis by lingual lipase. J. Lipid Res. 22, Fink, C.S.; Hamosh, P.; Hamosh, M. Fat digestion in the stomach: Stability of lingual lipase in the gastric environment. Pediatr. Res. 18, Rogalska, E.; Ransac, S.; Verger, R. Stereoselectivity of lipases. II. Stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases. J. Biol. Chem. 265, Shibata, R.; Gotoh, N.; Kubo, A.; Kanda, J.; Nagai, T.; Mizobe, H.; Yoshinaga, K.; Kojima, K.; Watanabe, H.; Wada, S. Comparison of catabolism rate of fatty acids to carbon dioxide in mice. Eur. J. Lipid Sci. Technol. 114, Gargouri, Y.; Moreau, H.; Verger, R. Gastric lipases: biochemical and physiological studies. Biochim. Biophys. Acta 1006, Liddle, R.A. Cholecystokinin cells. Annu. Rev. Physiol. 59, Hopman, W.P.M.; Kerstens, P.J.S.M.; Jansen, J.B.M.J.; Rosenbusch, G.; Lamers, C.B.H.W. Effect of graded physiologic doses of cholecystokinin on gallbladder contraction measured by ultrasonography. Gastroenterology 89, Liddle, R.A.; Morita, E.T.; Conrad, C.K.; Williams, J.A. Regulation of gastric emptying in humans by cholecystokinin. J. Clin. Invest. 72,

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