Breast Milk from Frequent Trans Fatty Acid Consumers Shows High Triglyceride and Glucose Levels,but Low Cholesterol and Apolipoprotein A-I levels,with Resulting Impaired In Vitro Zebrafish Embryo Growth and Survival

Abstract

Background:

It is well known that breast milk is the best nutritional source for infant growth. However, there has been no information about the quality of breast milk from individuals who daily consume a trans fatty acid (TFA)-enriched diet.

Subjects and Methods:

We performed compositional and functional analyses with breast milk from lactating mothers, in terms of lipid content and zebrafish embryo survivability, among individuals who daily consumed TFA-enriched food (n = 5), normal diet as control (n = 5), and powder formula (n = 5).

Results:

In lipid content of breast milk, the control group showed 2.5- and 4.5-fold higher cholesterol content than the TFA group and infant formula, respectively. The TFA group and infant formula showed 1.8- and 2.0-fold higher triglyceride (TG) than the control group. Moreover, the TFA group and formula showed 1.4- and 4.8-fold higher glucose levels compared with control. The TFA group also showed 25% lower protein content than control. Microinjection with breast milk (50 nL) from the TFA group showed significantly lower zebrafish embryo survivability (50% ± 4%) compared with the control (66% ± 5%), whereas microinjection with formula showed the lowest survivability (39% ± 5%) with the slowest developmental speed. Immunodetection revealed that breast milk from the TFA group showed smaller-sized apoA-I (25.5 ± 0.6 kDa) than that from the control group (27.5 ± 1.5 kDa), whereas formula did not contain apoA-I. Larger apoA-I size in breast milk was directly associated with higher embryo survivability.

Conclusions:

Breast milk from the TFA group showed increased TG and loss of cholesterol, lactalbumin (14 kDa), and apoA-I proteins, resulting in functional impairment of development and growth.

Introduction

Human milk is a good nutritional source for infant growth and development due to its bioactive factors and immune components. Regarding lipid ingredients, breast milk contains oleic acid and a significant amount of cholesterol, whereas infant formula contains only plant oil-derived fatty acids¹ and very low cholesterol content.^2,3 However, no report has examined the physiological effect of lipid content in breast milk on infant development. Infant formula is produced mainly from corn oil and sunflower oil, which lack w-3 fatty acid (linolenic acid) and cholesterol, but contain ω-6 fatty acid.¹ Our research group recently reported that linolenic acid (ω-3 fatty acid) displays enhanced cholesterol efflux and antiatherosclerotic activities.⁴ However, linoleic acid (ω-6 fatty acid) is known to increase proatherogenic activity in human macrophages, whereas ω-3-rHDL treatment inhibits the atherogenic process.

Trans fatty acid (TFA) is an artificial and hydrogenated unsaturated fatty acid with deleterious effects on various aspects of human health,⁵ including cardiovascular toxicity,⁶ neurotoxicity,⁷ and embryonic toxicity.⁸ High TFA content is also associated with increased risk of cancers,^9,10 such as human breast and colorectal cancers, and teratogenicity.¹¹ A Canadian study observed that the total TFA content of women's breast milk was reduced in response to mandatory TFA labeling regulations and recommendations.¹² The study suggests that TFA consumption by lactating mothers is directly related to increased TFA content in breast milk. However, change of specific nutritional composition and bioactive factors have not been characterized in breast milk from those who consumed TFA frequently.

Apolipoprotein A-I (apoA-I) is the principal protein behind the beneficial functions of high-density lipoprotein (HDL), which has potent antioxidant, anti-inflammatory, and antiatherosclerotic activities in blood.¹³ ApoA-I is found in human breast milk,¹⁴ although the reason why is still unknown. Since apoA-I is a major protein of HDL, which participates in reverse cholesterol transport in blood, it is possible that apoA-I is a cholesterol carrier in breast milk and provides antioxidant ability.

Although many studies have analyzed the ingredients of breast milk, there has been no report on embryo growth in the context of nutrition. This study investigated differences in the quality of breast milk based on nutritional composition and pollutant exposure, especially in breast milk from trans fat-consuming women. We compared breast milk in terms of macronutrient composition, enzyme activity, embryo survivability, and embryo development.

Subjects and Methods

Recruiting of breast milk donor

Control breast milk was obtained from healthy lactating women (average age 31 ± 6 years, n = 5) volunteers in the Seoul Metropolitan area, South Korea. The TFA group (average age 32 ± 3 years, n = 5), who lived in Seoul Metropolitan area, consumed TFA-enriched food daily, at least two-serving size, such as French fries and processed foods, before and during pregnancy and lactation period.

All lactating women consumed a typical Korean diet, which is enriched with rice and vegetables, and no changes in dietary patterns were observed in the past 3 years based on the survey. The questionnaire was a survey for the food frequency per week. The TFA group consumed at least three food items containing trans fat per day and five times per week. As shown in the Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/bfm), average consumption of trans fat was 8.3 ± 0.9 g per week in the TFA group. TFA consumption per week was maintained for 6 months before the survey. The questionnaire was a survey for the food frequency per week. Informed consent was obtained from all participants before enrollment in the study, and the Institutional Review Board at Yeungnam University (Gyeongsan, South Korea) approved the protocol (IRB #7002016-A-2015-043).

Quantification of lipids, glucose, and proteins in breast milk

Breast milk was obtained and stored at −70°C without further dilution or concentration until analysis. Breast milk (around 100 mL) was collected from both breasts from each mother and then mixed well and aliquoted into 1 mL aliquots. We did not homogenize breast milk. Aliquots were stored in a −80°C freezer until analysis. Once thawed, aliquots were not refrozen.

Commercially available protein powder formula was dissolved in water as recommended by the manufacturer. Total cholesterol (TC) and triglycerides (TGs) were determined in breast milk and formula without further dilution using commercially available assay kits (cholesterol, T-CHO, and triglycerides, Cleantech TS-S; Wako Pure Chemical). Glucose level was measured using a commercially available assay kit (Asan Pharmaceutical), as in our previous report.¹⁵

SDS-PAGE and western blot

Protein compositions of breast milk were compared by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under the same dilution of phosphate-buffered saline, and the bands were visualized by Coomassie blue staining.

Total protein compositions and apolipoprotein A-I (apoA-I) contents were compared through SDS-PAGE with identical protein loading quantities (5 μg of total protein per lane) from breast milk, and expression levels of apoA-I were analyzed through immunodetection. Anti-human apoA-I antibody (ab7613; Abcam) was used to detect the expression level. Relative molecular weight size and band intensity (BI) were compared through band scanning with Chemi Doc^® XR (Bio-Rad) using Quantity One software (version 4.5.2). The BI was compared by Student's t-test using the SPSS program (version 14.0; SPSS, Inc.) from three independent blots.

Zebrafish and embryos

Wild-type zebrafish and embryos were maintained according to standard protocols.¹⁶ Zebrafish maintenance and experimental procedures were approved by the Committee of Animal Care and Use of Yeungnam University (Gyeongsan, Korea). Zebrafish and embryos were maintained in a system cage (3-L volume acrylic tank) and six-well plates, respectively, at 28°C during treatment under a 14-hour light:10-hour dark cycle.

Microinjection of zebrafish embryos

To compare antioxidant and anti-inflammatory activities between the Transfat group and control group, breast milk was injected into zebrafish embryos, as in our previous report.¹⁷ Embryos at 1 day postfertilization (dpf) were individually microinjected using a pneumatic picopump (PV820; World Precision Instruments) equipped with a magnetic manipulator (MM33; Kantec) and a pulled microcapillary pipette-using device (PC-10; Narishigen). To minimize bias, injections were performed at the same position on each yolk. Filter-sterilized solution of each breast milk sample (50 nL) was injected into flasks of embryos as in our previous report.

We repeated injection experiments five times with each individual breast milk sample for more than 100 eggs for control and Transfat group to get reliable results. Following injection, live embryos were observed under a stereomicroscope (Motic SMZ 168, Hong Kong) and photographed using a Motic cam2300 CCD camera. Correlations of embryo survival between apoA-I size, cholesterol, and TG content were analyzed by regression analysis using Sigma Plot (Systat Software, Inc.) through polynomial equation linear regression.

Imaging of reactive oxygen species

After injection with breast milk, changes in reactive oxygen species (ROS) levels in larvae were imaged by dihydroethidium (DHE; cat #37291; BioChemika) staining, as previously described.¹⁸ Images were obtained by fluorescence microscopy (Ex = 588 nm and Em = 605 nm) on a Nikon Eclipse TE2000 Instrument. To avoid bias, red fluorescence was measured in the trunk area away from the injection site. Quantification of the stained area was carried out through computer-assisted morphometry using Image Proplus software (version 4.5.1.22; Media Cybernetics).

Statistical analysis

All data are expressed as the mean ± standard deviation from at least three independent experiments with duplicate samples. In comparison with three groups, data in the same group were evaluated through one-way analysis of variance using SPSS (version 14.0;), and differences between the means were assessed using Duncan's multiple-range test. Data comparisons for band size and intensity from western blots were assessed by Student's t-test using the SPSS program (version 14.0; SPSS, Inc.) between two groups. Statistical significance was defined as p < 0.05.

Results

Composition analysis

Breast milk from the control group showed a total cholesterol concentration of 55 ± 25 mg/dL, whereas breast milk from the TFA group and formula showed total cholesterol concentrations of 22 ± 9 and 11 ± 6 mg/dL, respectively, as shown in Figure 1A. Infant formula showed the highest triglyceride level around 3,212 ± 463 mg/dL, whereas breast milk from the control group showed a triglyceride level of 1,564 ± 786 mg/dL. Breast milk from the TFA group showed a 1.8-fold higher TG level around 2,841 ± 1107 mg/dL (Fig. 1B), suggesting that TFA consumption was directly associated with higher secretion of TGs into breast milk.

FIG. 1.

Compositional analysis of breast milk from control and TFA groups and formula. Total cholesterol (A), triglyceride (B), glucose (C), and protein (D) contents were determined as described in the text. TFA, trans fatty acid.

Glucose content of breast milk was similar between the control and TFA groups, around 39–53 mg/dL. However, infant formula showed around fivefold higher glucose content, 190 ± 113 mg/dL (Fig. 1C). Breast milk from the TFA group showed the lowest content of total proteins, around 9 mg/mL, whereas formula showed twofold higher protein content (Fig. 1D). As formula is made from plant-based oil and whey protein, it is enriched in ω-6 fatty acid and milk proteins such as casein. Shortly, commercially available formula was found to contain higher triglyceride and glucose contents, but a remarkably lower cholesterol level than breast milk due to its vegetable oil-derived production.

Embryo survivability and developmental speed

As shown in Figure 2A, at 8 hours postinjection, the control breast milk group showed 84% survivability, whereas the TFA group and formula caused 77% and 62% survivability, respectively. At 48 hours postinjection, the control breast milk group showed 66% survivability, whereas the TFA group and formula caused 50% and 39% survivability, respectively. However, the noninjected group and H₂O-injected group showed 88% and 79% of survival, respectively, at 48 hours postinjection. This result suggests that the difference in embryonic cell death occurring in early phase of postinjection depends on the breast milk and formula.

FIG. 2.

Embryo survivability after microinjection with breast milk from control and TFA groups and formula. DHE, dihydroethidium.

DHE staining revealed that control group breast milk-injected embryos showed the fastest developmental speed with the lowest ROS production, whereas the TFA group and infant formula showed slower developmental speeds and 1.5- and 1.6-fold higher ROS production than H₂O-injected embryos (Fig. 2). Especially, the infant formula-injected group showed the highest mortality, lowest eye pigmentation, and slowest skeletal development as indicated by the arrowhead (Fig. 3).

FIG. 3.

Extent of ROS production by microinjection with breast milk from control and TFA groups and formula. ROS, reactive oxygen species.

Electrophoretic analysis of protein composition

SDS-PAGE analysis detected totally different protein compositions between breast milk and infant formula, as shown in Figure 4. Control breast milk showed a stronger lactalbumin band (14 kDa) indicated by the arrowhead, while infant formula showed a strong casein band (20 kDa), which was not detected in breast milk. Especially, lactalbumin, which is enriched with essential amino acids for growth, showed no band for TFA breast milk and infant formula.

FIG. 4.

Electrophoretic profiles of breast milk from control and TFA groups and formula (15% SDS-PAGE). SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Expression level of apoA-I

Western blotting with apoA-I antibody, which can recognize full-length apoA-I (28 kDa), detected a distinct band in 1/4-diluted breast milk from both the control and TFA groups, as shown in Figure 5A. However, formula did not show any apoA-I band as formula protein contains mostly whey protein. Densitometric analysis revealed that apoA-I from the TFA group had a band size of 26.4 ± 1.2 kDa, whereas that from the control group showed a band size of 28.7 ± 1.5 kDa, suggesting that putative modification or cleavage occurred in apoA-I from breast milk of the TFA group. More interestingly, embryo survivability was positively correlated with higher molecular weight of apoA-I, as shown in Figure 5B; larger apoA-I in breast milk correlated well with higher survivability.

FIG. 5.

Immunodetection of apoA-I for size comparison between control and TFA groups (A). Correlations of apoA-I size and zebrafish embryo survivability (B).

Influential factors for embryo survivability

Higher cholesterol content in breast milk was well associated with higher embryo survivability and faster developmental speed, as shown in Figure 6A. However, embryo survivability was negatively correlated with TG level in breast milk, as shown in Figure 6B. Although powder formula showed the highest contents of TG, glucose, and proteins, formula showed the lowest survivability. These results suggest that cholesterol and apoA-I in breast milk are essential for neonate growth and development since they are directly associated with embryo survivability (Figs. 1 and 2).

FIG. 6.

Correlations of cholesterol (A) and triglyceride contents (B) in breast milk with zebrafish embryo survivability. TC, total cholesterol; TG, triglyceride.

Discussion

It has been well established that breast milk is important to optimize not only infant growth and development but also immunization and anti-infectious activity. Although breast milk compositions were roughly identified,¹⁹ it is still unknown what kind of macronutrients are important for growth and defense activity. In addition, the quality of breast milk might differ depending on the mother's lifestyle, such as smoking status, dietary habits such as trans fat consumption,²⁰ and exposure to environmental pollutants, such as particulate matter, phthalate, and bisphenol. Therefore, it is critical to monitor the quality of breast milk using an appropriate evaluation system.

In the current study, breast milk from the TFA group showed remarkably lower cholesterol and protein contents than that from the control (Fig. 1) and resulted in lower survivability of zebrafish embryos with higher ROS production (Figs. 2 and 3). Breast milk from the TFA group showed increased TG levels and loss of lactalbumin (Fig. 4). Reduced cholesterol in breast milk from the TFA group has been reported previously,²¹ in which human subjects showed reduced serum cholesterol upon TFA consumption for 4 weeks. Increased TG levels in breast milk upon TFA consumption is in good agreement with our previous report, which observed that TFA consumption for 20 weeks increased serum TG levels and fatty liver changes with adverse effects on lipoprotein metabolism. The TFA group also showed less and smaller-sized apoA-I in breast milk (Fig. 5) compared with the control.

ApoA-I is the major protein of HDL and important for maintaining antioxidant and anti-inflammatory properties in serum.²² Lactalbumin consists of many peptides having antibacterial and antihypertensive effects.²³ Zebrafish embryo survivability was directly correlated with apoA-I size (Fig. 5), and breast milk from the TFA group containing smaller-sized apoA-I resulted in greater embryo death. This result is in good agreement with our previous report, which found that elderly subjects (71 ± 4 years old) showed functionally impaired and truncated apoA-I, as well as lower cholesterol levels than young and healthy control subjects (22 ± 2 years old).²⁴ These reports suggest that the functionality and properties of apoA-I in serum are reproduced in breast milk in terms of lipid and lipoprotein levels. We reported that smoker HDL is functionally and structurally modified, resulting in atherogenesis and diabetes. We also reported that apoA-I modified by oxidation and glycation through smoking causes inflammatory death of zebrafish embryos and more rapid atherogenesis.²⁵ Glycated apoA-I is more aggregated and fragmented with altered electromobility.²⁶

Interestingly, a proteomic approach revealed a considerable amount of apoA-I in breast milk, although its precise role remains unknown.¹⁴ In addition to cholesterol delivery, apoA-I has antiviral and antibacterial activities in blood.^27,28 In the current study, higher apoA-I content and larger apoA-I size were directly associated with embryo survivability. To the best of our knowledge, this is the first report showing that TFA consumption can lower cholesterol and apoA-I size in breast milk.

Breast milk contains considerable amounts of cholesterol, oleic acid, and essential fatty acids, whereas formula does not since its lipids are made from plant oil lacking cholesterol and long-chain polyunsaturated fatty acids.¹ Higher cholesterol in breast milk is associated with a normal level of serum cholesterol in adults since breast milk-fed infants show a threefold lower cholesterol synthesis rate than formula-fed infants.²⁹ A longitudinal study revealed that breast feeding is associated with 10% risk reduction for cardiovascular disease in adulthood.³⁰

High content of TFA is also associated with increased risk of cancer^9,10 and teratogenicity.¹¹ The current study demonstrated an association between TFA consumption and slower developmental speed and lower embryo survivability (Figs. 2 and 3) through reduced cholesterol and apoA-I contents. Modification of apoA-I is directly related to production of dysfunctional HDL, which has more atherogenic and inflammatory properties that exacerbate cellular senescence.³¹

In conclusion, the current findings show that dietary habits such as TFA consumption can influence the quality of breast milk by lowering cholesterol and protein contents while raising TG levels. Loss of lactalbumin and smaller-sized apoA-I is well correlated with lower embryo survivability. Further studies are necessary to investigate the molecular mechanism by which TFA consumption contributes to loss of cholesterol and apoA-I secretion in breast milk.

Footnotes

Acknowledgments

This work was supported by a grant from the Mid-Carrier Researcher Program (2014-11049455) and Medical Research Center Program (2015R1A5A2009124) through the National Research Foundation (NRF), funded by the Ministry of Science, ICT, and Future Planning of Korea.

Disclosure Statement

No competing financial interests exist.

References

Giovannini

, Agostoni

, Riva

. Fat needs of term infants and fat content of milk formulae. Acta Paediatr Suppl, 1994; 402:59–62.

Delplanque

, Gibson

, Koletzko

, et al. Lipid quality in infant nutrition: Current knowledge and future opportunities. J Pediatr Gastroenterol Nutr, 2015; 61:8–17.

Bayley

, Alasmi

, Thorkelson

, et al. Influence of formula versus breast milk on cholesterol synthesis rates in four-month-old infants. Pediatr Res, 1998; 44:60–67.

Park

, Kim

, Choi

, et al. ω-6 (18:2) and ω-3 (18:3) fatty acids in reconstituted high-density lipoproteins show different functionality of anti-atherosclerotic properties and embryo toxicity. J Nutr Biochem, 2015; 26:1613–1621.

Calder

. Functional roles of fatty acids and their effects on human health. JPEN J Parenter Enteral Nutr, 2015; 39:18S–32S.

Mozaffarian

, Katan

, Ascherio

, et al. Trans fatty acids and cardiovascular disease. N Engl J Med, 2006; 354:1601–1613.

Zhang

, Chen

, Huang

, et al. Elaidic acid enhanced the simultaneous neurotoxicity attributable to the cerebral pathological lesion resulted from oxidative damages induced by acrylamide and benzo(a)pyrene. Toxicol Ind Health, 2011; 27:661–672.

Park

, Kim

, Cho

. Elaidic acid (EA) generates dysfunctional high-density lipoproteins and consumption of EA exacerbates hyperlipidemia and fatty liver change in zebrafish. Mol Nutr Food Res, 2014; 58:1537–1545.

Thompson

, Shaw

, Minihane

, et al. Trans-fatty acids and cancer: The evidence reviewed. Nutr Res Rev, 2008; 21:174–188.

10.

, La Vecchia

, de Groh

, et al. Dietary transfatty acids and cancer risk. Eur J Cancer Prev, 2011; 20:530–538.

11.

Carlson

, Clandinin

, Cook

, et al. trans fatty acids: Infant and fetal development. Am J Clin Nutr, 1997; 66:715S–736S.

12.

Ratnayake

, Swist

, Zoka

, et al. Mandatory trans fat labeling regulations and nationwide product reformulations to reduce trans fatty acid content in foods contributed to lowered concentrations of trans fat in Canadian women's breast milk samples collected in 2009–2011. Am J Clin Nutr, 2014; 100:1036–1040.

13.

Cho

. Biomedicinal implications of high-density lipoprotein: Its composition, structure, functions, and clinical applications. BMB Rep, 2009; 42:393–400.

14.

Roncada

, Stipetic

, Bonizzi

, et al. Proteomics as a tool to explore human milk in health and disease. J Proteomics, 2013; 88:47–57.

15.

Kim

, Lim

, Yoo

, et al. Consumption of high-dose vitamin C (1250 mg per day) enhances functional and structural properties of serum lipoprotein to improve anti-oxidant, anti-atherosclerotic, and anti-aging effects via regulation of anti-inflammatory microRNA. Food Funct, 2015; 6:3604–3612.

16.

Nusslein-Volhard

, Dahm

. Zebrafish: A Practical Approach. New York, Oxford University Press, Inc., 2002.

17.

Park

, Cho

. A zebrafish model for the rapid evaluation of pro-oxidative and inflammatory death by lipopolysaccharide, oxidized low-density lipoproteins, and glycated high-density lipoproteins. Fish Shellfish Immunol, 2011; 31:904–910.

18.

Owusu-Ansah

, Yavari

, Mandal

, et al. Distinct mitochondrial retrograde signals control the G1-S cell cycle checkpoint. Nat Genet, 2008; 40:356–361.

19.

Ballard

, Morrow

. Human milk composition: Nutrients and bioactive factors. Pediatr Clin North Am, 2013; 60:49–74.

20.

Sheila

. Trans fatty intakes during pregnancy, infancy and early childhood. Atheroscler Suppl, 2006; 7:17–20.

21.

de Roos

, Schouten

, Katan

. Consumption of a solid fat rich in lauric acid results in a more favorable serum lipid profile in healthy men and women than consumption of a solid fat rich in trans-fatty acids. J Nutr, 2001; 131:242–245.

22.

Tabet

, Lambert

, Cuesta Torres

, et al. Lipid-free apolipoprotein A-I and discoidal reconstituted high-density lipoproteins differentially inhibit glucose-induced oxidative stress in human macrophages. Arterioscler Thromb Vasc Biol, 2011; 31:1192–1200.

23.

Wada

, Lönnerdal

. Bioactive peptides derived from human milk proteins—Mechanisms of action. J Nutr Biochem, 2014; 25:503–514.

24.

Park

, Shin

, Kim

, et al. Senescence-related truncation and multimerization of apolipoprotein A-I in high-density lipoprotein with an elevated level of advanced glycated end products and cholesteryl ester transfer activity. J Gerontol A Biol Sci Med Sci, 2010; 65:600–610.

25.

Park

, Shin

, Cho

. Dysfunctional lipoproteins from young smokers exacerbate cellular senescence and atherogenesis with smaller particle size and severe oxidation and glycation. Toxicol Sci, 2014; 140:16–25.

26.

Jang

, Shim

, Lee

, et al. Rapid detection of dysfunctional high-density lipoproteins using isoelectric focusing-based microfluidic device to diagnose senescence-related disease. Electrophoresis, 2011; 32:3415–3423.

27.

Srinivas

, Birkedal

, Owens

, et al. Antiviral effects of apolipoprotein A-I and its synthetic amphipathic peptide analogs. Virology, 1990; 176:48–57.

28.

Beck

, Adams

, Biglang-Awa

, et al. Apolipoprotein A-I binding to anionic vesicles and lipopolysaccharides: Role for lysine residues in antimicrobial properties. Biochim Biophys Acta, 2013; 1828:1503–1510.

29.

Wong

, Hachey

, Insull

, et al. Effect of dietary cholesterol on cholesterol synthesis in breast-fed and formula-fed infants. J Lipid Res, 1993; 34:1403–1411.

30.

Rich-Edwards

, Stampfer

, Manson

, et al. Breastfeeding during infancy and the risk of cardiovascular disease in adulthood. Epidemiology, 2004; 15:550–556.

31.

Park

, Cho

. High-density lipoprotein (HDL) from elderly and reconstituted HDL containing glycated apolipoproteins A-I share proatherosclerotic and prosenescent properties with increased cholesterol influx. J Gerontol A Biol Sci Med Sci, 2011; 66:511–520.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.02 MB