Tandem Mass Spectrometric Analysis of Regio-Isomerism of

Chapter 21

Tandem Mass Spectrometric Analysis of RegioIsomerism of Triacylglycerols in Human Milk and Infant Formulae

Downloaded by UNIV OF ARIZONA on April 29, 2017 | http://pubs.acs.org Publication Date: August 21, 1998 | doi: 10.1021/bk-1998-0702.ch021

H. Kallio, J.-P. Kurvinen, O. Sjövall, and A. Johansson Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland Human milk lipids are generally recognized as an optimal source of fatty acids for both energy production and synthesis of functional lipids for infants. In addition to the special composition of fatty acids, combinations of fatty acids and their distribution within triacylglycerol molecules are also specific. Negative ion chemical ionization mass spectrometry was used to determine the molecular weight pattern and collision induced dissociation tandem mass spectrometry applied to identify the sn-2 vs.sn-1/3positions of fatty acids in human milk and infant formula triacylglycerols. Significant differences were found between the products. Mammal and marsupial dams exude milk for their neonates, as does a mother for her baby. The composition of the milk of various genera and species are analogous, though distinct differences do exist. Adaptation to different environments can be seen in milk composition. Whale calves swim in the ocean, kangaroo joeys are hidden in pouches whereas human children and their mothers try to survive in very variable climates and weather on dry land. The nutrient content of milk is also in balance with the speciesspecific speed of growth which is reflected by the proportions of proteins, lactose, lipids and minerals. We may assume that the long evolution of mammals guarantees the milk of healthy, well-fed mothers to be optimal nourishment for their descendants. A man in modern society is no longer totally under the control of the natural selection of evolutive forces as in ancient times. Despite the rapidly changed and somewhat unnatural environment of infants, the composition of human milk remains the best food known for a child. Detailed evidence to the contrary has not been forthcoming. Human milk is an excellent example of a functional food, even though the importance and detailed effects of its individual components are not yet fully understood. For the food industry, human milk is, however, the clear model for infant formulae. Milk fat is a natural source of essential fatty acids and their precursors for cell

©1998 American Chemical Society Shibamoto et al.; Functional Foods for Disease Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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membranes, eicosanoids and other functional lipids of infants. Fat is also needed for energy production and functions as an antimicrobial agent. In addition to the fat composition its digestion, absorption, resynthesis, transport, metabolism and placing in various tissues define the final physiological effect. The less developed lipase system of infants, high pH in the stomach, lipolytic enzymes in human milk and highly specific fatty acid positioning in the milk triacylglycerols make the fat metabolism of infants clearly distinct from that of adults. The aim of the study was to compare the lipids of commercial infant formulae with mothers milk. Fatty acids, molecular weight distribution of triacylglycerols (TAG) and the regioisomeric structures of the major TAG were analysed by chromatographic and mass spectrometric methods. Materials and Methods Human Milk and Formulae. Milk was received from three Finnish mothers (28-34 years), about three months after delivery. The milk was pooled and frozen at -70 °C. Commercial brands of human milk formulae were either donations from producers or bought from retailers. The products were prepared according to the instructions on the packages. Lipids were isolated by a modified Folch extraction procedure with chloroform/methanol (2:1, v/v) (7) and triacylglycerols purified by a Florisil column, with 10 mL n-hexane/diethylether, (4:1, v/v). Results of the human milk sample, three infant formulae and one infant formula ingredient are shown and discussed as examples. Fatty acid Analysis. Methyl esters of the TAG fatty acids were prepared by sodium methoxide transesterification (2). Gas chromatographic analysis was performed by a Perkin Elmer Auto Systems gas chromatograph (Perkin Elmer, Norwalk, Conn.), equipped with a split injector (split ratio 1:40, 230 °C) and a flame ionization detector (250 °C). An NB-351 column (25 m, 0.32 mm i.d., d 0.20 urn, HNU-Nordion Instruments Ltd, Helsinki, Finland) was used with temperature programming from 50 °C to 240 °C at a rate of 4 °C/min and held at 240 °C for 10 min. The flow rate of the He carrier was 31 cm/s measured at 120 °C. Relative responses of various fatty acid methyl esters for the GC analysis were defined, using commercial mixtures GLC-60 and 68 D (Nu Chek Prep, Inc., Elysian, MN) as quantitative standards. Correction factors from methyl butanoate up to methyl docosahexaenoate varied between 1.15 and 1.00, where methyl hexadecanoate was used as the reference compound. f

Mass Spectrometry of Triacylglycerols. The molecular weight pattern of TAG was determined by ammonia negative ion chemical ionization with a triple quadrupol tandem mass spectrometer (TSQ-700, Finnigan MAT, San Jose, CA) (3). Optimized parameters for relative abundances of [M - HJ ions were used as published earlier (4). Each sample was analyzed four times and the averaged spectra displayed. Tandem mass spectrometric analysis (TSQ-700) based on negative ion chemical ionization / collision induced dissociation was applied to the regioisomer analysis of TAG as described earlier (5,6). Triacylglycerols 16:0-18:1-16:0, rac-16:0-16:0-18:1,

Shibamoto et al.; Functional Foods for Disease Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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18:1-16:0-18:1, rac-18:1-18:1-16:0 (Larodan Ab, Malmo, Sweden), rac-16:0-18:l18:0,rac-16:0-18:1-18:2,rac-18:l-18:l-18:2andrac-18:2-18:2-16:0(Sigma Chemical Co, St Louis, MO) were used as calibration standards. Each of the standard TAG was analyzed four times and averaged results of both the correction factors of RCOO ions. The discrimination between the fatty acids in sn-2 and sn-l/3 of [M - H - RCOOH 100] ions were determined for the default values in TAGS-100 programme based on the Simplex method (6). The correction factors were: stearic acid (18:0) 1.0; palmitic acid (16:0) 1.1; oleic acid (18:ln-9) 1.1 and linoleic acid (18:2n-6) 1.2. The averaged ratio of the abundances of [M - H - RCOOH™ - 100] / [M - H - R C O O I W - 100] was 0.10 showing a typical discrimination, when using a rhenium wire loop introduction of the sample. Downloaded by UNIV OF ARIZONA on April 29, 2017 | http://pubs.acs.org Publication Date: August 21, 1998 | doi: 10.1021/bk-1998-0702.ch021

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Results and Discussion Fatty Acids. A gas chromatogram of fatty acid methyl esters of the pooled human milk sample, is shown as an example in Figure 1. Standard deviations of the ten major peaks typically varied between 1 and 4 %. Separation was sufficient, the only significant problem being the overlapping of fatty acids 22:6n-3 and 24:ln-9. Fatty acid profiles of the five samples analyzed are shown in Table I. The pattern of human milk fatty acids corresponds well to the earlier investigations (1,7,8). The contents of fatty acids 4:0, 6:0 and 8:0 were higher than typically reported in the literature. This may be due to the special GC technique required for the proper analysis of short-chain fatty acids. However, Baldwin and Longenecker (1944) (9) have already correctly reported the existence of these compounds in human milk using fractional distillation techniques. The wide compositional variation of the commercial baby milk formulae became clear in our investigations. The fatty acid profiles shown in Table I indicate the use of e.g. cow milk, vegetable oils and synthetic mixtures as ingredients. Formula I has a clear profile of cow milk fatty acids (#); Formula II is mainly based on cow milk. According to the fatty acid pattern, the formula ingredient oil could be related to the Formula III. Molecular Weights of Triacylglycerols. Molecular weight patterns of triacylglycerols of the products are displayed in Figure 2. In addition to being fast, the MS method applied gives a more accurate molecular weight distribution than chromatographic methods. Comparison of the samples is easy and reliable. Figure 2a shows the typical pattern of human milk TAG, being in accordance with the results of e.g. Breckenridge et al. (10). Formula I (Figure 2b) is clearly based on bovine milk triacylglycerols, but Formula II (Fig 2c) may be composed of several milk TAG fractions, possibly supplemented with some vegetable oils. The molecular weight pattern of Formula III (Fig 2d) and the formula ingredient (Fig 2e) are practically identical. The only distinct difference being the high content of the ACN:DB species 52:2 of the ingredient. The molecular weight profiles of formulae II and III are similar to each other, but are clearly built up from different fat ingredients.



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HUMAN MILK FATTY ACIDS


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Table I. Relative proportions of TAG fatty acids of pooled human milk, formula ingredient fat and three commercial infant formulae. Fatty acid (CN:DB)

Human Formula I Formula II Formula III Fat Milk Ingredient 0.2 9.2 4.9 0.6 0.2 0.3 4.3 1.9 10.1 0.4 1.9 2.9 0.9 5.2 2.0 1.8 2.9 1.5 15.4 6.6 13.0 2.8 1.9 5.3 6.9 5.3 8.2 4.8 0.3 0.8 0.3 0.3 0.6 17.2 24.0 21.6 29.3 20.7 2.3 0.1 0.1 0.9 0.6 0.3 0.3 4.0 10.3 2.3 7.7 5.5 24.7 31.0 38.8 17.2 29.8

4:0 6:0 8:0 10:0 12:0 14:0 14:l(n-5) 15:0 16:0 16:l(n-7) 17:0 18:0 18:l(n-9) 18:l(n-7)/ 1.7 (n-9)trans 18:2(n-6) 8.0 1.2 18:3(n-3) 20:0 0.3 0.4 20:l(n-9) 0.2 20:2(n-6) 0.2 20:3(n-6) 0.3 20:4(n-6) 20:3(n-3) 0.1 20:5(n-3) 22:0 22:l(n-9) 24:0 22:6(n-3)+ 0.3 24:l(n-9) 2.4 others total (mol %) 100.0

0.8 11.7 1.2 0.2 0.4 0.1 0.1 0.1

1.0 14.5 2.3 0.2 0.1 0.1 0.1

1.4 12.8 1.4 0.3 0.3 0.1 0.1

0.6 14.3 1.3 0.2 0.1 0.2 0.0

0.4 100.0

3.3 100.0

2.3 100.0

0.8 100.0

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Tandem Mass Spectrometric Analysis of Regio-Isomerism of

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