Differences in the Triacylglycerol and Fatty Acid Compositions of


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Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Differences in the Triacylglycerol and Fatty Acid Compositions of Human Colostrum and Mature Milk Pu Zhao,† Shuwen Zhang,† Lu Liu, Xiaoyang Pang, Yang Yang, Jing Lu,* and Jiaping Lv* Key Laboratory of Agro-Food Processing and Quality Control, Institute of Food Science and Technology, Chinese Academy of Agricultural Science, Beijing 100193, People’s Republic of China S Supporting Information *

ABSTRACT: Human colostrum is important for immune system development and plays a protective role for infants. However, the comprehensive exploration of lipids, which account for 3−5% of milk, and their biological functions in human colostrum was limited. In present study, the triacylglycerol (TAG) and fatty acid (FA) compositions of human colostrum and mature milk were analyzed and compared. Variations were observed in both the TAG and FA compositions. The concentrations of 18:1/18:1/16:0 TAG, high-molecular-weight and unsaturated TAGs were significantly higher in colostrum, whereas mature milk contained more low/medium-molecular-weight TAGs and medium-chain FAs. Furthermore, there were also specific TAGs in both colostrum and mature milk. Our data highlighted targets for further investigation to elucidate the biological function of lipids in colostrum milk. In addition, the comprehensive analysis of TAGs in Chinese colostrum might help in designing infant formula for Chinese babies, especially the preterm ones. KEYWORDS: human colostrum, mature milk, triacylglycerols, fatty acids, UPLC-ESI-MS

1. INTRODUCTION Human milk, which contains essential nutrients and other physiological substances, is regarded as the optimal food for neonates.1 Among these substances, a fat content of only 3−5% in milk can provide approximately 50% of the energy required by infants.2 Ninety-eight percent of the milk fat consists of triacylglycerols (TAGs), which are molecules of glycerol esterified to three fatty acids (FAs), with the side-chains at the Sn-1, Sn-2, and Sn-3 positions. The different FAs at these three positions result in unique TAGs that play important roles in improving digestion and absorption,3 and determine the applications of TAGs. For example, TAG containing palmitic acid (C16:0), a FA with a higher melting point than the human body temperature (37 °C), in the Sn-1/Sn-3 position can combine with Ca2+ or Mg2+ to form insoluble solids that may be excreted in hard stool.4 More than 20% of all fatty acids in human milk fat (HMF) are C16:0, with 60% of C16:0 in the Sn-2 position to reduce calcium loss and improve FA absorption.5,6 Globally, only 35% of newborn to 6-month-old babies are breastfed. Therefore, developing infant formula (IF) with a composition similar to that of breast milk will help ensure suitable nutrition for all infants. Based on previous studies of TAGs in human milk,7,8 the addition of 18:1/16:0/ 18:1 TAG (OPO) is common when formulating IF. However, adding only OPO does not simulate the overall structure and composition of HMF. Therefore, investigating the changes in the TAGs over the lactation periods is important for accurately mimicking HMF for IF. Colostrum is the milk produced during the first several days after parturition. Colostrum is important for the development of the immune system and protects infants against allergies and chronic diseases. Infants could gain long-term metabolic benefits by consuming colostrum during the first several days of life.9 To the best of our knowledge, information about the © XXXX American Chemical Society

TAGs in human colostrum is scarce. Martin and co-workers analyzed the TAGs in human colostrum and mature milk by using enzymatic degradation,10 but information on individual molecular species was absent. Benefiting from the development of analytical techniques in recent years, 24 TAGs were determined in Danish human colostrum, transitional, and mature milk.11 Thirty TAGs were compared in Spanish human colostrum and mature milk.12 Most recently, Chinese human transitional and mature milk were analyzed by using supercritical fluid chromatography (SFC) coupled with quadruple time-of-flight mass spectrometry (Q-TOF-MS).13 The lipid composition of breast milk is influenced by diet, age, parity, and stage of lactation.14 Large variations exist between the Western and Asian diets, and the TAG data for Western colostrum is thus not representative of Chinese data. The TAGs with specific FA chain composition in Chinese colostrum could aid in the determination of the nutritional value and health benefits of colostrum and the exploration of the effect of diet on TAGs. For TAG analysis, most studies focus on developing suitable analytical methods, including traditional methods, such as enzymatic degradation using thin layer chromatography (TLC) and gas chromatography (GC),15 as well as new methods, such as high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with atmospheric pressure chemical ionization (APCI)16 for plant oil and electrospray ionization (ESI) for milk fat.17 The methods used for sample preparation and TAG separation for human milk were developed by Ten-Doménech and co-workers. In their study, 42 TAGs were characterized and quantified in human milk by Received: Revised: Accepted: Published: A

February 14, 2018 April 9, 2018 April 10, 2018 April 16, 2018 DOI: 10.1021/acs.jafc.8b00868 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 1. TAG with Specific FA Chain and Composition of TAGs in Colostrum and Mature Milka (mol %) colostrum (n = 40) compound

composition

34-0 TAG 34-0 TAG 36-0 TAG

12:0/12:0/10:0 14:0/12:0/8:0 12:0/12:0/12:0 + 14:0/12:0/10:0 14:0/12:0/12:0 16:0/12:0/10:0 18:1/16:0/4:0 18:1/10:0/10:0 + /16:1/12:0/10:0 16:0/12:0/12:0 + 18:0/12:0/10:0 16:0/14:0/10:0 18:1/12:0/10:0 18:2/12:0/10:0 16:0/14:0/12:0 16:0/16:0/10:0 18:0/12:0/12:0 18:1/12:0/12:0 18:2/12:0/12:0 18:2/14:0/10:0 18:0/14:0/12:0 18:0/16:0/10:0 18:1/16:0/10:0 18:1/14:0/12:0 18:2/16:0/10:0 18:2/14:0/12:0 16:0/16:0/14:0 + 18:0/16:0/12:0

La/La/C M/La/Cy La/La/La + M/La/C M/La/La P/La/C O/P/B O/C/C + Po/La/C P/La/La + S/La/C P/M/C O/La/C L/La/C P/M/La P/P/C S/La/La O/La/La L/La/La L/M/C S/M/La S/P/C O/P/C O/M/La L/P/C L/M/La P/P/M + S/P/La

18:0/14:0/14:0 18:1/16:0/12:0 18:1/14:0/14:0 18:1/18:1/10:0 + 18:1/16:1/12:0 18:2/18:1/10:0 16:0/16:0/16:0 + 18:0/16:0/14:0 18:1/16:0/14:0 18:1/16:1/14:0 18:1/18:1/12:0 18:2/16:0/14:0 18:2/18:1/12:0 18:2/18:2/12:0 18:1/16:0/15:0 18:0/16:0/16:0 18:1/16:0/16:0 18:1/16:0/16:1 18:1/18:1/14:0 18:2/16:0/16:0 18:2/18:1/14:0 18:2/16:0/16:1 18:2/18:2/14:0 18:1/17:0/16:0 18:1/17:1/16:0 18:1/18:1/15:0 18:0/18:0/16:0 18:0/18:1/16:0 18:1/18:1/16:0 18:2/18:1/16:0 18:2/18:2/16:0 18:2/18:1/16:1 18:2/18:2/16:1

S/M/M O/P/La O/M/M O/O/C + O/Po/La L/O/C P/P/P + S/P/M O/P/M O/Po/M O/O/La L/P/M L/O/La L/L/La O/P/Pe S/P/P OP/P O/P/Po O/O/M L/P/P L/O/M L/P/Po L/L/M O/Ma/P O/Mo/P O/O/Pe S/S/P S/O/P O/O/P L/O/P L/L/P L/O/Po L/L/Po

38-0 38-0 38-1 38-1

TAG TAG TAG TAG

40-0 TAG 40-0 40-1 40-2 42-0 42-0 42-0 42-1 42-2 42-2 44-0 44-0 44-1 44-1 44-2 44-2 46-0

TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG

46-0 46-1 46-1 46-2

TAG TAG TAG TAG

46-3 TAG 48-0 TAG 48-1 48-2 48-2 48-2 48-3 48-4 49-1 50-0 50-1 50-2 50-2 50-2 50-3 50-3 50-4 51-1 51-2 51-2 52-0 52-1 52-2 52-3 52-4 52-4 52-5

TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG

abbreviation

mature (n = 26)

precursor ion

product ion

RT (min)

average

range

average

range

628.8 628.8 656.7

439.3 383.2 439.3

4.065 4.065 5.022

0.01 ± 0.013 0.003 ± 0.003 0.09 ± 0.107

0.001−0.052 0.001−0.011 0.009−0.479

0.053 ± 0.045a ND 0.255 ± 0.22a

0.01−0.273 ND 0.034−1.371

684.6 684.6 682.7 682.7

439.4 495.3 577.2 493.3

6.141 6.141 5.44 5.19

0.121 ± 0.139 0.041 ± 0.046 0.035 ± 0.03 ND

0.014−0.643 0.005−0.193 0.011−0.181 ND

0.338 ± 0.288a 0.107 ± 0.073a ND 0.043 ± 0.022

0.047−1.772 0.02−0.458 ND 0.008−0.112

712.8

495.5

7.398

0.216 ± 0.208

0.043−0.926

0.516 ± 0.349a

0.11−2.133

712.8 710.8 708.8 740.9 740.9 740.9 738.8 736.6 736.6 768.9 768.9 766.9 766.9 764.9 764.9 796.6

467.4 411.4 411.4 495.5 551.3 439.2 521.3 519.4 547.3 551.4 495.4 577.3 549.4 575.4 547.4 523.4

7.398 6.173 5.169 8.958 8.958 8.958 7.47 6.315 6.48 10.625 10.625 9.056 9.056 7.68 7.68 12.478

0.108 ± 0.114 0.111 ± 0.103 0.071 ± 0.101 0.13 ± 0.098 0.052 ± 0.042 0.056 ± 0.053 0.286 ± 0.249 0.189 ± 0.258 ND 0.208 ± 0.117 0.396 ± 0.197 0.287 ± 0.2 0.295 ± 0.184 0.171 ± 0.173 0.136 ± 0.153 0.847 ± 0.311

0.016−0.542 0.022−0.405 0.006−0.371 0.036−0.455 0.015−0.162 0.009−0.183 0.054−1.096 0.017−1.087 ND 0.094−0.559 0.177−0.987 0.072−0.688 0.13−0.863 0.024−0.591 0.029−0.711 0.477−1.542

0.281 ± 0.252a 0.278 ± 0.14a 0.136 ± 0.069a 0.279 ± 0.208a ND ND 0.665 ± 0.397a 0.324 ± 0.158a 0.088 ± 0.042 0.331 ± 0.168a 0.606 ± 0.305a 0.59 ± 0.181a 0.571 ± 0.348a 0.28 ± 0.086a 0.229 ± 0.124a 1.05 ± 0.501a

0.027−1.535 0.07−0.773 0.032−0.338 0.036−1.187 ND ND 0.181−2.405 0.074−0.882 0.024−0.241 0.074−0.834 0.154−1.541 0.239−1.133 0.102−2.119 0.145−0.469 0.053−0.707 0.275−2.434

796.6 794.9 794.9 792.8

495.4 577.3 549.3 493.4

12.478 10.689 10.689 9.249

0.251 1.077 0.322 1.158

± ± ± ±

0.136 0.436 0.164 0.696

0.087−0.672 0.564−2.225 0.103−0.812 0.412−3.173

0.241 ± 0.131 2.526 ± 0.994a ND 1.926 ± 0.499a

0.035−0.558 0.929−5.852 ND 0.945−3.034

790.7 824.8

601.4 551.4

7.71 14.5

0.135 ± 0.132 1.496 ± 0.626

0.017−0.487 0.811−3.856

0.267 ± 0.101a 1.172 ± 0.503

0.081−0.537 0.31−2.548

822.8 820.9 820.9 820.9 818.9 816.9 836.7 852.8 850.8 848.7 848.7 848.7 846.9 846.9 844.8 864.9 862.7 862.7 880.7 878.9 876.7 874.8 872.8 872.8 870.8

523.4 549.4 603.3 523.4 519.4 519.4 537.6 579.4 577.4 577.3 603.5 551.4 547.3 575.5 547.3 565.3 589.4 603.4 579.4 577.4 577.4 577.3 599.4 601.3 599.2

12.541 10.657 10.657 10.657 9.28 7.878 13.405 16.53 14.462 12.573 12.573 12.573 10.882 10.882 9.477 15.368 13.505 13.505 18.557 16.499 14.431 12.763 11.23 11.23 9.863

2.009 ± 0.617 0.199 ± 0.062 1.65 ± 0.584 0.867 ± 0.363 0.753 ± 0.554 0.432 ± 0.537 0.057 ± 0.02 0.854 ± 0.385a 6.851 ± 1.865a 1.298 ± 0.354 4.456 ± 1.267 4.315 ± 0.78a 1.264 ± 0.344 0.542 ± 0.156 0.557 ± 0.422 0.099 ± 0.024 0.072 ± 0.015 0.161 ± 0.036 0.336 ± 0.175 3.612 ± 1.259 17.589 ± 2.793a 11.279 ± 1.785 2.331 ± 1.304 0.544 ± 0.141 0.462 ± 0.342

0.995−3.67 0.088−0.32 0.692−3.037 0.453−1.934 0.178−2.347 0.05−1.892 0.032−0.129 0.385−2.306 2.936−10.878 0.576−2.231 1.977−7.058 2.929−5.951 0.802−2.052 0.324−0.822 0.18−1.892 0.055−0.172 0.055−0.11 0.088−0.227 0.154−0.811 1.535−6.465 9.837−21.576 7.199−15.151 0.805−5.407 0.291−0.871 0.138−1.463

2.048 ± 0.733 0.205 ± 0.096 2.577 ± 0.966a 0.986 ± 0.375 1.153 ± 0.372a 0.586 ± 0.393 0.059 ± 0.033 0.659 ± 0.29 5.133 ± 1.461 1.151 ± 0.466 4.013 ± 1.271 3.495 ± 1.027 1.341 ± 0.368 0.515 ± 0.19 0.63 ± 0.382 0.091 ± 0.037 0.065 ± 0.021 0.158 ± 0.063 0.301 ± 0.159 3.187 ± 1.095 14.575 ± 1.962 11.132 ± 1.452 5.366 ± 2.479a ND 0.493 ± 0.265

0.597−3.781 0.074−0.438 0.969−5.134 0.333−2.149 0.391−2.129 0.14−1.733 0.019−0.193 0.225−1.35 2.406−9.098 0.513−2.32 1.907−7.474 1.472−5.601 0.737−2.371 0.218−0.897 0.154−1.986 0.039−0.219 0.035−0.132 0.064−0.321 0.087−0.765 1.303−5.17 9.753−20.173 7.003−14.391 1.34−9.429 ND 0.103−1.366

B

DOI: 10.1021/acs.jafc.8b00868 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 1. continued colostrum (n = 40) compound 52-5 54-1 54-1 54-2 54-2 54-3 54-3 54-4 54-5 54-6

TAG TAG TAG TAG TAG TAG TAG TAG TAG TAG

composition

abbreviation

precursor ion

18:3/18:2/16:0 18:0/18:1/18:0 20:0/18:1/16:0 18:1/18:0/18:1 20:1/18:1/16:0 18:1/18:1/18:1 18:1/18:0/18:2 18:1/18:2/18:1 18:2/18:1/18:2 18:2/18:2/18:2

Ln/L/P S/O/S A/O/P O/S/O E/O/P O/O/O O/S/L O/L/O L/O/L L/L/L

870.8 906.9 906.9 904.9 904.9 902.8 902.8 900.8 898.8 896.8

mature (n = 26)

product ion

RT (min)

average

range

average

range

575.3 605.3 577.4 603.4 631.4 603.4 605.5 601.4 601.3 599.4

9.863 18.411 18.411 16.435 16.435 14.494 15.342 12.794 11.199 9.736

0.331 ± 0.285 0.241 ± 0.13 0.067 ± 0.026 2.958 ± 1.38a 0.553 ± 0.322a 10.768 ± 3.258a ND 8.944 ± 1.352a 3.829 ± 1.884 1.419 ± 1.392

0.1−1.169 0.074−0.551 0.029−0.129 0.824−6.051 0.101−1.345 4.673−14.756 ND 5.913−11.757 1.3−8.45 0.286−6.175

0.332 ± 0.194 0.198 ± 0.106 0.062 ± 0.025 2.283 ± 1.073 0.306 ± 0.206 8.509 ± 2.557 1.553 ± 0.41 7.84 ± 1.943 4.093 ± 2.346a 1.75 ± 1.485a

0.066−0.894 0.07−0.47 0.031−0.147 0.748−5.287 0.101−1.083 4.135−16.976 0.586−2.563 4.049−12.445 0.792−9.36 0.177−6.843

The values represent the means ± SD. The “a” in an entry denotes significant differences (P < 0.05) between colostrum and mature milk groups. ND is not detected. Abbreviations used for acyl chains in the triacylglycerols (TAGs): B, butyric; Cy, caprylic; C, capric; La, lauric; M, myristic; Pe, pentadecanoic; P, palmitic; Po, plamitoleic; Ma, margaric; Mo, margaroleic; S, stearic; O, oleic; L, linoleic; Ln, linolenic; A, arachic; E, eicosaenoic. RT, retention time.

a

mixture was oscillated again for 15 min and centrifuged at 2250g for 30 min. The organic fraction was filtered through filter paper containing 2 g of Na2SO4, dried under a nitrogen stream, and stored at −80 °C before analysis. 2.4. TAG Composition of HMF Detected by UPLC-MS/MS. Referring to the method of Guan,19 the extracted lipids were dissolved in CHCl3/ MeOH (1:2, v/v) at 5 mg/mL and then underwent a 50fold dilution using MeOH with an internal standard (d5-(17:0/17:1/ 17:0) TAG). Samples were loaded through a UPLC system (I-class Acquity UPLC, Waters) with an Autosampler. The lipids were separated on a BEH C18 reversed-phase column (1.7 μm, 2.1 mm i.d. × 100 mm, Waters Corporation). The column was maintained at 60 °C during the whole process. Mobile phase A was acetonitrile/water (1:1, v/v), and mobile phase B was IPA/acetonitrile (9:1, v/v), both of which contained 10 mM ammonium formate and 0.1% formic acid. The flow rate was 0.3 mL/min, with a linear gradient of solvent B from 70% to 85% over 28 min, and then to 70% at 28.1 min, followed by maintenance at 70% for 1.9 min, and 5 μL samples were injected for analysis by MS (API 4500 Q-Trap, AB SCIEX) which was equipped with an ESI source. Samples were detected in the positive ionization mode and both the nebulizer and desolvation gases were nitrogen. The typical operating parameters were set as follows: curtain gas (CUR) 25; collision gas (CAD) medium; ion source gas 1 (GS1) 45; ion source gas 2 (GS2) 50; electrospray voltage 5500 V; and a temperature of 550 °C. The detected method was the same with Guan. Briefly, first the parent ions were obtained using the scan mode, with the declustering potential (DP) set to 100 V, the mass range set to 400−1200m/z, and the scan rate set to 1000 Da/s. Parent ion of TAGs in colostrum and mature milk can be obtained using the scan mode; then an instrument method in MRM (Q1 = Q3)-information dependent acquisition (IDA)-enhanced product ion (EPI) mode was established for the qualification of TAGs with specific FA chain compositions; finally, for the quantification of TAGs with specific FA chain compositions, MRM (Q1 ≠ Q3) mode was applied. Specific ion pairs of each TAG isomers were selected and used in MRM survey channels, which was shown in Table 1. The isomeric species of TAGs were determined by referring to the mass-to- charge ratio (m/z) and abundance. The peak areas of TAGs isomers were collected and compared with the peak areas of d5−17:0/17:1/17:0 TAG to calculate the concentrations of TAGs in colostrum and mature samples. 2.5. FAs Detected by GC. The sample pretreatment method followed the Chinese national standard 541327-2010. Sample (0.5 g) was weighed, and then it was placed in a screw-cap test tube. Then, 5 mL of PhMe and 6 mL of acetyl chloride-MeOH (1:9, v/v) were added to the tube. After the tube was shaken, it was filled with a nitrogen stream for 60 s. The mixture was placed in an 80 °C water bath and shaken six times every 20 min. After the mixture was cooled to room temperature, it was transferred to a 50 mL centrifuge tube and

HPLC with evaporative light scattering detection and tandem mass spectrometry (HPLC-ELSD MS/MS).18 TAGs in milk of different species have also been analyzed by HPLC-APCI-MS.11 To the best of our knowledge, in most of the works related to TAG analysis in milk, the detailed TAGs in Chinese human milk at different lactation stages have not been well characterized. Thus, in the present study, we analyzed the TAGs in colostrum and mature Chinese human milk by the advanced ultraperformance liquid chromatography-tandem mass spectrometry equipped with an electrospray ionization source (UPLC-ESI-MS) method reported by M Guan.19 In such a method, one sample can be analyzed in 30 min, and a small sample amount can yield highly accurate and precise results. More than 66 different TAGs were characterized and quantified. Additionally, there were also significant differences between human colostrum and mature milk; for example, colostrum milk had more UUU (three unsaturated fatty acids in Sn-1, 2, 3 positions) and LLL (three long chain fatty acids in Sn-1, 2, 3 positions) types of TAGs, whereas mature milk had more low- and medium-molecular-weight TAGs. The variations and patterns of the TAGs observed in the present study can form the basis for IF production.

2. MATERIALS AND METHODS 2.1. Chemicals. Internal standard TAG, 1,3(d5)-diheptadecanoyl2-heptadecenoyl-glycerol (d5-(17:0/17:1/17:0) TAG), was purchased from Avanti Polar Lipids (Birmingham, AL, U.S.A.). Standard FAs (Supelco 37-component FAME mix) were obtained from SigmaAldrich Co. LLC. HPLC-grade acetonitrile, methanol (MeOH), isopropanol (IPA), chloroform (CHCl3), formic acid, ammonium formate, and toluene (PhMe) were purchased from Fisher Scientific (Pittsburgh, PA, U.S.A.). Analytical-grade anhydrous sodium carbonate, anhydrous sodium sulfate, and acetyl chloride were purchased from Sinopharm Chemical Reagent Co., Ltd. 2.2. Sample Collection. Twenty-six colostrum samples (collected on the fourth day of lactation) and 40 mature milk samples (collected on the 30th day of lactation) were obtained from healthy volunteer mothers in the morning in the hospital in Beijing and were transported to the laboratory on ice. All the participating mothers were informed of the project in advance. The samples were stored at −80 °C before analysis. 2.3. Extraction of Human Milk Lipids. According to the Bligh and Dyer method,20 one human milk sample was mixed with CHCl3, MeOH, and Milli-Q water (1:1:2:0.8, v/v/v/v), and the mixture was oscillated for 30 min. CHCl3 and Na2SO4 (2%, w/v) were added for a final volume ratio of 2:2:1.8 (MeOH/CHCl3/water, v/v/v). The C

DOI: 10.1021/acs.jafc.8b00868 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Total ion chromatograms (TIC) of human colostrum (A) and mature milk (B), the extracted ion chromatograms (XICs) of 40-2 TAG (m/z: 708.8, RT: 5.16 min) (a), 40-1 TAG (m/z: 710.8, RT: 6.16 min) (b), 40-0 TAG (m/z: 712.8, RT: 7.43 min) (c) in colostrum milk and their corresponding MS/MS spectra of ammonium adducts (d−f) obtained by UPLC-ESI-MRM (Q1 = Q3)-IDA-EPI mode. The detection method was performed according to Chavarri et al.21 with a slight modification of the capillary column and temperature program. Samples were analyzed by GC (7890B Agilent) with a

washed three times with 3 mL of 6% Na2CO3. The mixture was centrifuged at 5000 rpm for 5 min, and then the upper phase was transferred to a gas vial with a 0.2 μm organic filter. D

DOI: 10.1021/acs.jafc.8b00868 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 2. Characteristics of TAGs in Colostrum and Mature Milka

a b

characteristics

colostrum milk

mature milk

molecular weight of TAGs molecular weight/carbon number/mol % 498−610/26−34/mol % (low) 624−694/35−40/mol % (medium) 708−918/41−56/mol % (high) number of FAs in TAGs chain length of FAsb LLL/mol % L2M/mol % LM2/mol % MMM/mol % L2S/mol % un/saturation of FAsc SSS S2U SU2 UUU

610−888

610−888

0.013 ± 0.015 0.794 ± 0.798 99.193 ± 0.813a 16 4−20 91.601 ± 4.935a 7.169 ± 3.718 1.117 ± 1.146 0.078 ± 0.094 0.035 ± 0.030a

0.053 ± 0.045a 1.955 ± 1.305a 97.992 ± 1.347 14 4−20 84.295 ± 5.320 12.782 ± 3.775a 2.470 ± 1.388a 0.246 ± 0.213a 0

5.215 ± 1.719 21.101 ± 2.761 47.717 ± 1.939 25.967 ± 3.814a

6.189 ± 2.743 20.99 ± 4.278 50.134 ± 2.097a 22.686 ± 6.095

The values represent the means ± SD. The “a” in an entry denotes significant differences (P < 0.05) between colostrum and mature milk groups. L, long-chain FA; M, medium-chain FA; S, short-chain FA. cS, saturated FA; U, unsaturated FA.

capillary column (Agilent HP-88 100 m × 0.25 mm × 0.2 μm). The carrier gas (nitrogen) flow rate was 1 mL/min, and the oven temperature was increased from 80 °C (1 min) to 200 °C at 2 °C/ min, held for 2 min, increased to 230 °C at 5 °C/min, and held at 230 °C for 15 min. The temperature of the flame ionization detector was 300 °C. The qualitative and quantitative methods used an external standard (Supelco 37 component FAME mix) and area normalization. 2.6. Statistical Analysis. All measurements were made in duplicate. IBM SPSS 22.0 was used to determine significant differences of the data.

In total, 68 and 66 TAGs were characterized in colostrum and mature human milk, respectively. 3.2. Quantitative Analysis of TAGs in Colostrum and Mature Milk. After the characterization of each TAG isomer, the ion pairs, which were shown in Table 1, were carefully chosen, and UPLC-ESI-MRM (Q1 ≠ Q3) mode was applied for the quantification of TAGs with specific FA chain (Table 1). The TAGs with contents exceeding 5 mol % were O/O/P, 18:2/18:1/16:0 TAG (L/O/P), 18:1/18:1/18:1 TAG (O/O/ O), 18:2/18:1/18:1 TAG (L/O/L), 18:1/16:0/16:0 TAG (O/ P/P), and 18:2/18:2/16:0 TAG (L/L/P) (Table 1). Among these TAGs, the L/L/P content was less than 5 mol % in colostrum milk. Considering the major TAGs that accounted for more than 1 mol % of the total content, colostrum milk contained more O/O/P, O/O/O, O/L/O, O/P/P, 18:2/16:0/ 16:0 TAG (L/P/P), 18:1/18:0/18:1 TAG (O/S/O), and 16:0/ 16:0/16:0 TAG (P/P/P) than mature milk (p < 0.05), whereas mature milk contained more 18:2/18:1/18:2 TAG (L/O/L), LPL, 18:1/18:1/12:0 TAG (O/O/La), 18:2/18:2/18:2 TAG (L/L/L), 18:1/16:0/12:0 TAG (O/P/La), 18:1/18:0/18:2 TAG (O/S/L), 18:2/18:1/12:0 TAG (L/O/La), and 18:1/ 16:1/12:0 TAG (O/Po/La) than colostrum milk (Table 1). Six TAGs were detected only in colostrum: 18:2/18:1/16:1 TAG (L/O/Po), 18:1/14:0/14:0 TAG (O/M/M), 18:0/12:0/12:0 TAG (S/La/La), 16:0/16:0/10:0 TAG (P/P/C), 18:1/16:0/ 4:0 TAG (O/P/B), and 14:0/12:0/8:0 TAG (M/La/Cy). The total content of these TAGs was 1.012 mol %, and all of these TAGs were less than 1 mol %. Four TAGs were detected only in mature milk: 18:1/18:0/18:2 TAG (O/S/L), 18:2/14:0/ 10:0 TAG (L/M/C), 18:1/10:0/10:0 TAG (O/C/C), and 16:1/12:0/10:0 TAG (Po/La/C). The total content of these TAGs was only 1.684 mol %. Among these TAGs, the main structure was O/S/L, with a content of 1.553 mol % (Table 1). 3.3. Characteristics of the TAGs in Colostrum and Mature Milk. The types of TAGs in colostrum and mature milk were similar but with variations. The range of TAGs with different molecular weights in the colostrum and mature milk samples was between 610 and 888. Most of the TAGs in human milk had high molecular weights (Table 2 and Figure 2). The concentrations of low- and medium- molecular-weight

3. RESULTS 3.1. Characterization of TAGs Based on UPLC-ESIMRM (Q1 = Q3)-IDA-EPI Mode. The UPLC-ESI-MRM (Q1 = Q3)-IDA-EPI mode19 was applied for the determining TAGs. In this mode, Q1 was set as the same as Q3 to lock the parent ions. The locked parent ions will be fragmented in EPI experiment if the intensity of parent ions exceeds the threshold of IDA. From the real-time fragments of certain parent ions, the compositions of TAGs can be deduced. The total ion chromatograms (TICs) of human colostrum and mature milk were showed in Figure 1A,B respectively. Figure 1a−f shows the extracted ion chromatograms (XICs) of 40-2−40-0 TAGs in colostrum and their corresponding MS/ MS spectra of ammonium adducts. The TAGs were separated according to the combined effects of the number of carbon atoms in FA moieties and the number of double bonds. To be specific, the retention times of 40-2, 40-1, and 40-0 TAGs were 5.16, 6.16, and 7.43 min, respectively, after eliminating the isobaric interferences.22 Figure 1d−f shows the MS/MS spectra of the ammonium adducts of 40-2, 40-1, and 40-0 TAGs detected in MRM (Q1 = Q3)-IDA-EPI mode. For example, in Figure 1d, product ions at m/z 411.4, 519.5, and 491.3 were corresponding to the neutral loss (NL) of NH3 and 18:2, 10:0, and 12:0 FAs of ammonium adduct of 40-2 TAG, respectively, and 40-2 TAG can thus be deduced as 18:2/12:0/10:0 TAG. Similarly, 40-1 TAG eluted at 6.16 min was deduced as 18:1/12:0/10:0 TAG shown in Figure 1e. However, 40-0 TAG (RT: 7.43 min), a more complex TAG, was deduced as a mixture of 16:0/12:0/12:0 TAG, 18:0/12:0/ 10:0 TAG, and 16:0/14:0/10:0 TAG (Table 1). E

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Figure 2. Relative contents of TAGs analyzed by ACN-DB (acyl carbon number−number of double bonds) (mol %) .The “★” in an entry denotes significant differences (P < 0.05) between colostrum and mature milk groups.

4. DISCUSSION The number of characterized TAGs in the present study was comparable to that in previous studies. Ten-Domenech and coworkers characterized 50 TAGs in human milk.18 Zou characterized 24 TAGs in human milk at different lactation stages.11 Linderborg compared 60 TAGs in milk from normal and overweight women.23 Haddad et al. characterized 98 TAGs in milk, but two C18 columns connected in series were used in their study, which resulted in a very long analysis time.17 In the present study, the analytical methods applied were relatively simple, the amount of sample required was small, and the analysis time was only 30 min. These features could indicate advances in the present methods for studying human milk TAGs. Furthermore, the TAG concentrations determined in the present study are comparable to those in previous studies. The content of O/O/P, one of the most abundant TAGs in human milk, was approximately 14−17 mol % in the present study and ranged from 7.25 mol % to 24.68 mol % in previous studies.13,18,23 The variations among different studies could be due to the variations in human milk samples, as they are from humans in various lactation stages and from different areas of the world. In addition, the concentrations of the other abundant TAGs, including L/O/P and O/P/P, were consistent with those in other studies. In our present work, acyl carbon number (ACN) of TAGs ranged from 34−54, DBs ranged from 0−6 (Figure 2), which were similar to Tu’s work,13 with ACN 38−56, and DBs 0−5 in human transitional and mature milk. Furthermore, we also characterized some TAGs which were reported the first time, such as M/La/Cy, P/M/C, S/M/La. Table S1 shows types and contents of TAGs (wt %) in colostrum and mature milk in the present work compared with data of Zou11 and Pons.12 We could see that in the three studies, O/O/P and L/O/P were the first two abundant TAGs in human milk, although the concentrations were different, which could be explained by the fewer kinds of TAGs characterized in their studies leading to higher relative contents of these two TAGs. In Spanish human milk, there was no TAGs with ACN 34−46, and there were only 7 TAGs within this ACN range in Danish human milk. In addition, there was a similar change of TAGs as the lactation prolonged; for example, more S/P/P, O/P/P, O/O/ P, O/S/O, O/O/O, and O/L/O were in colostrum than in mature milk, but more O/La/La, O/M/La, O/P/La and L/O/ La were in mature milk than in colostrum. Furthermore, there

TAGs were significantly higher in mature milk than in colostrum, whereas colostrum contained more TAGs with high molecular weights (Table 2 and Figure 2). Sixteen and 14 FAs were observed in colostrum and mature milk, respectively. C4:0 and C8:0 were determined exclusively in TAGs of human colostrum, but with a relatively low concentration of 0.035 mol % (18:1/16:0/4:0 TAG (O/P/B)) and 0.003 mol % (14:0/ 12:0/8:0 TAG (M/La/Cy)), respectively. Human milk TAGs consisted of more long-chain fatty acids (LCFAs) than medium-chain fatty acids (MCFAs) and short-chain fatty acids (SCFAs). More TAGs with LCFAs in the Sn-1, Sn-2, and Sn-3 positions (LLL) were observed in human colostrum than in mature milk. FAs can also be classified as saturated fatty acids (SFAs) and unsaturated fatty acids (UFAs), and TAGs can thereby be classified as SSS, S2U, SU2, and UUU. The concentrations of SU2 TAGs were much higher in mature milk than in colostrum, whereas the concentrations of TAGs with three UFAs (UUU type of TAG) were significantly higher in colostrum than in mature milk (Table 2). 3.4. FA Compositions in Colostrum and Mature Milk Detected by GC. The results of the FA compositions and contents in colostrum and mature milk are shown in Table 3. The major FAs found in both types of human milk were C18:1n9c (∼30%), C18:2n6c (∼20%), C16:0 (∼15%), C18:0 (∼5%), C14:0 (∼4%), and C12:0 (∼3%). Compared with those in colostrum, the contents of SFAs, including C6:0, C8:0, C13:0, C21:0, and C22:0, were significantly decreased, and the contents of C10:0 and C12:0 were significantly increased in mature milk. The contents of C16:0 and C18:0 were not significantly different. Regarding monounsaturated fatty acids (MUFAs), the contents of C15:1 and C24:1n9 were significantly decreased in mature milk, but the total MUFA content was not significantly different, possibly because C18:1n9c, which accounted for more than 30% of the content, did not change notably as the lactation stage was prolonged. In addition, C18:1n9c was an important MUFA, which relatively accounted for more than 86%. Regarding polyunsaturated fatty acids (PUFAs), the contents of C20:2, C22:2n6, and C20:5n3 significantly decreased as the lactation stage increased. However, the content of C18:2n6c, which accounted for more than 84% of the PUFAs, was not significantly different between colostrum and mature milk. ARA and DHA, which accounted for approximately 0.3% of the PUFAs, were detected by GC but not by UPLC-MS/MS. F

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Journal of Agricultural and Food Chemistry Table 3. Content and Composition of FAs in Colostrum and Mature Milka (wt%) FA

colostrum (n = 20)

range

mature (n = 30)

range

C4:0 C6:0 C8:0 C10:0 C12:0 C13:0 C14:0 C15:0 C16:0 C17:0 C18:0 C20:0 C21:0 C22:0 C23:0 C24:0 SFA SC-SFA MC-SFA LC-SFA C14:1 C15:1 C16:1 C17:1 C18:1n9t C18:1n9c C20:1 C22:1n9 C24:1n9 MUFA C18:2n6t C18:2n6c C18:3n6 C18:3n3 C20:2 C20:3n6 C20:4n6 C20:3n3 C22:2n6 C20:5n3 C22:6n3 PUFA PUFA ω-3 PUFA ω-6

0.042 ± 0.062 0.262 ± 0.140a 0.550 ± 0.351a 0.838 ± 0.602 2.966 ± 1.681 0.262 ± 0.154a 3.075 ± 0.923 0.115 ± 0.035 18.681 ± 1.87 0.241 ± 0.056 5.466 ± 0.785 0.173 ± 0.073 0.996 ± 0.542a 2.841 ± 1.006a 0.334 ± 0.280 0.195 ± 0.139 37.036 ± 3.548 0.042 ± 0.062 4.878 ± 2.016 32.116 ± 3.725 0.092 ± 0.079 0.180 ± 0.116a 1.608 ± 0.505 0.152 ± 0.039 0.446 ± 0.725 31.082 ± 4.689 0.897 ± 0.568 1.013 ± 0.472 0.537 ± 0.361a 36.007 ± 5.494 1.77 ± 2.499 21.175 ± 6.154 1.862 ± 1.182 0.077 ± 0.051 0.200 ± 0.179a 0.763 ± 0.723 0.387 ± 0.401 0.040 ± 0.065 0.177 ± 0.098a 0.105 ± 0.067a 0.385 ± 0.158 26.942 ± 6.228 0.607 ± 0.182 26.135 ± 6.373

0.000−0.209 0.101−0.596 0.112−1.350 0.133−1.992 1.375−7.297 0.095−0.622 1.572−5.254 0.064−0.190 15.183−21.652 0.166−0.389 4.285−6.952 0.000−0.320 0.406−2.058 1.338−4.813 0.000−1.058 0.000−0.553 30.549−42.958 0.000−0.209 2.572−9.939 25.193−37.679 0.000−0.353 0.074−0.503 0.962−2.539 0.086−0.215 0.070−2.467 22.413−38.722 0.118−2.050 0.152−2.147 0.048−1.128 25.297−45.086 0.000−10.88 14.056−32.832 0.623−4.120 0.000−0.176 0.000−0.657 0.000−2.47 0.000−1.46 0.000−0.209 0.000−0.365 0.000−0.282 0.151−0.749 17.456−38.743 0.297−1.031 16.548−38.062

0.054 ± 0.075 0.173 ± 0.079 0.279 ± 0.141 1.458 ± 0.293a 4.857 ± 1.51a 0.161 ± 0.077 3.725 ± 1.605 0.106 ± 0.036 17.939 ± 2.658 0.231 ± 0.052 5.277 ± 0.809 0.209 ± 0.063 0.478 ± 0.149 2.095 ± 0.776 0.211 ± 0.09 0.143 ± 0.08 37.396 ± 5.077 0.054 ± 0.075 6.928 ± 1.824a 30.414 ± 4.142 0.077 ± 0.044 0.117 ± 0.062 1.814 ± 0.583 0.148 ± 0.048 0.251 ± 0.486 32.231 ± 5.232 0.61 ± 0.447 0.843 ± 0.614 0.25 ± 0.157 36.341 ± 5.68 0.877 ± 1.151 22.173 ± 6.66 1.762 ± 1.071 0.077 ± 0.057 0.106 ± 0.082 0.458 ± 0.438 0.274 ± 0.214 0.068 ± 0.077 0.075 ± 0.041 0.071 ± 0.061 0.324 ± 0.178 26.264 ± 6.703 0.539 ± 0.234 25.618 ± 6.735

0.000−0.359 0.086−0.391 0.089−0.668 0.908−2.417 2.056−9.928 0.000−0.303 1.764−8.293 0.06−0.209 14.638−20.878 0.166−0.337 3.445−6.658 0.113−0.374 0.315−0.922 0.776−3.819 0.099−0.368 0.041−0.396 29.557−48.495 0.000−0.359 3.441−13.155 24.042−38.414 0.022−0.145 0.059−0.249 1.176−3.404 0.062−0.221 0.062−0.223 24.623−41.489 0.101−2.06 0.279−3.061 0.114−0.657 27.527−44.797 0.029−2.888 10.645−33.65 0.000−3.383 0.000−0.282 0.000−0.301 0.000−2.165 0.000−0.756 0.000−0.294 0.000−0.138 0.020−0.136 0.143−1.154 15.367−38.835 0.202−1.571 13.795−39.961

The values represent the means ± SD. The “a” in an entry denotes significant differences (P < 0.05) between colostrum and mature milk groups. Abbreviations: SC-SFA, short-chain saturated FA; MC-SFA, medium-chain saturated FA; LC-SFA, long-chain saturated FA; MUFA, monounsaturated FA; PUFA, polyunsaturated FA.

a

was also a TAG, O/O/La that showed a different trend in Chinese and Danish human milk. In a short, there were variations in the concentrations of TAGs in human milk of three countries; however, similar changes of TAGs were observed in colostrum and mature milk, which could indicate the specific functions of TAGs in colostrum for infants and provide clues for further investigation. In the present study, more UUU and LLL types of TAGs were observed in colostrum. As known, UFAs have lower melting points than the human body temperature (37 °C) and are easily digested and absorbed by infants.24 A higher amount of UUU types of TAGs in colostrum might mitigate the inferior

digestive capacity of infants, who might require nutrients to be easily digested during their first several days of life. Furthermore, during the first several days of lactation, the mammary gland might not be fully matured for fat synthesis and secretion, resulting in a low capability of synthesizing MCFAs and a high need of circulating FAs from blood. Thus, LLL types of TAGs were significantly higher in colostrum because of the input from circulating LCFAs from blood.25 In the present study, the content of TAGs with low and medium molecular weights increased as the lactation stage increased, which is consistent with previous results (Tu and coworkers13). It is speculated that as lactation is prolonged, more G

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energy is needed for infant growth. The direct source of energy from human milk would thus be desired. This could also be explained by the higher amount of SSS type of TAGs in mature human milk, as the saturated SCFAs and MCFAs could be a good, rapidly utilized source of energy.25 In the analysis of FA composition, the concentration of MCFAs was increased in mature human milk compared with colostrum. Even though large variations in the change in FA composition with prolonged lactation could be observed among different studies,26−28 MCFAs in mature milk increased across all samples despite the region, diet, and race of the mothers. This could indicate that the MCFAs concentrations were not significantly influenced by these factors. This increase is also consistent with our TAG analysis showing a higher amount of low- and medium-molecular-weight TAGs in mature milk. The mammary gland can synthesize FAs with 10−14 carbons.27 The maturation of the mammary gland during lactation could explain the increase in MCFAs in mature milk. PUFAs also changed in a similar pattern in human milk samples from various mothers. Colostrum contained more PUFAs, however, with different species among studies.27−29 Jiang et al. found that the concentrations of C22:6 n-3 and C22:5 n-3 were lowest in mature milk,27 while Molto et al. found that the amounts of C20:2 n-6, C20:3 n-6, C20:4 n-6, C22:2 n-6, and C22:5 n-6 decreased over the lactation period.29 In our study, the concentrations of C20:2, C22:2 n-3, and C20:5 n-3 were decreased in mature milk compared with colostrum. The variations among studies could result from the milk samples being from different areas of the world and the different diets of the mothers, since PUFAs are influenced by diet.30 Numerous TAGs were characterized in Chinese human colostrum and mature milk. Human milk contained high contents of high-molecular-weight TAGs and LLL type of TAGs. C16:0, C18:1, and C18:2 were the dominant FAs in human milk. Variations were observed in both the TAGs and FA composition between colostrum and mature milk. In colostrum milk, the concentrations of O/O/P, LLL, and UUU types of TAGs were significantly higher than those in mature milk, indicating that these TAGs might be easier to digest and play important roles in infant development during the first several days of life. In contrast, mature milk contained more low- and medium-molecular-weight TAGs and MCFAs than colostrum, which could be due to the high energy demand of infants and the maturation of the mammary gland as lactation progresses. To the best of our knowledge, few studies have been done on TAGs in colostrum, and our work might be the first focused on human colostrum from Chinese mothers. This comprehensive analysis and comparison of Chinese human colostrum and mature milk might provide targets for further study in human lipids.



Pu Zhao: 0000-0002-4306-0762 Xiaoyang Pang: 0000-0003-1813-2039 Author Contributions †

P.Z and S.Z contributed equally to this work.

Funding

This research was funded by the National Natural Science Foundation of China (No. 31371808), Special Research Grant for Nonprofit Public Service (No. 201303085), and Beijing Innovation Team of Technology System in Dairy Industry. Notes

The authors declare no competing financial interest.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b00868. Types and contents of TAGs in colostrum and mature milk compared with other countries (wt %) (PDF)



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AUTHOR INFORMATION

Corresponding Authors

*E-mail for J.L.: [email protected] *E-mail for J.-p.L.: [email protected] H

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DOI: 10.1021/acs.jafc.8b00868 J. Agric. Food Chem. XXXX, XXX, XXX−XXX