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Myristic acid in amniotic fluid produces appetitive responses in human newborns
Early Human Development 115 (2017) 32–37
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Early Human Development journal homepage: www.elsevier.com/locate/earlhumdev
Myristic acid in amniotic fluid produces appetitive responses in human newborns
MARK
Ana G. Gutiérrez-Garcíaa,b,⁎, Carlos M. Contrerasb,c, Cynthia Díaz-Marted a
Facultad de Psicología, Universidad Veracruzana, Xalapa 91097, Veracruz, Mexico Laboratorio de Neurofarmacología, Instituto de Neuroetología, Universidad Veracruzana, Xalapa 91190, Veracruz, Mexico c Unidad Periférica Xalapa, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Xalapa 91190, Veracruz, Mexico d Hospital Civil de Perote, 91270, Veracruz, Mexico b
A R T I C L E I N F O
A B S T R A C T
Keywords: Appetitive behavior Fatty acids Myristic acid Newborn recognition Prenatal learning Olfactory stimulation
Background: A mixture of eight fatty acids (lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, elaidic acid, and linoleic acid) that are contained in human amniotic fluid, colostrum, and milk produces appetitive responses in newborns, suggesting the existence of a transition of sensorial cues that guide newborns to the maternal breast. Objective: To explore the ability of each of these eight fatty acids individually to produce appetitive responses in newborns. Methods: The study included 12 healthy human newborns < 24 h after birth. Using a longitudinal design, cotton swabs that were impregnated with each of the eight fatty acids and control substances (i.e., vehicle, saline, and vanilla) were placed approximately 1 cm from the newborns' nostrils for 30 s. Positive responses that were suggestive of acceptance included appetitive movements (i.e., suckling) and sniffing that were directed toward the cotton swab. Lateral movements of the head away from the swab were considered negative responses. Remaining stationary with no changes in facial expressions was considered indifference. Results: Compared with controls (i.e., vehicle, saline, and vanilla) and the other fatty acids tested, myristic acid produced the longest duration of positive facial responses (suckling and sniffing). No significant differences in negative facial responses were observed in response to the odoriferous stimuli. No reactions that were suggestive of disgust were observed. Conclusion: A complex combination of stimuli, including the odor of myristic acid, may integrate sensory cues that guide newborns to the maternal breast.
1. Introduction
mucosa is well developed [3] and contains ciliated olfactory receptors that have a mature appearance [4]. Olfactory marker proteins (an indicator of neuroreceptor functionality) [5] and connectivity with mitral cells in the main olfactory bulb [6] are present in epithelia by the 28th gestational week and in the main olfactory bulb between the 32nd and 35th gestational weeks [7]. Similarly, at approximately the 12th gestational week, the human vomeronasal organ is well developed [8] and visible in the developing fetus [3,9,10] and newborns [11]. From an anatomical perspective, human fetuses have a reasonably well developed olfactory system before birth. Some specific components of the interphase between mothers and newborns (e.g., amniotic fluid, colostrum, and milk) may serve as sensorial cues. Shortly after birth, human newborns and other mammals display movements of the head toward the maternal mammary gland [12,13], its own amniotic fluid odor [14–16], and maternal
Among the signaling systems, chemical cues that consist of pheromones [1] can cause notable behavioral responses, including anxiety [2], when perceived by other individuals in the group. The opposite is also true. Some pheromones can act as cues that indicate the existence of a safe environment [1] by informing other individuals of the same species about the absence of danger or presence of food through sensorial systems. Some chemical cues begin to achieve salience in the intrauterine milieu before birth under two conditions: a functional sensorial system and the presence of such cues in the intrauterine milieu (i.e., amniotic fluid). Such prenatal training may guide newborns to similar substances that are contained in colostrum and milk, acting as a guide to the maternal breast and nursing. In humans as early as the 24th gestational week, the olfactory
⁎
Correspondence to: A. Gutiérrez-García, Laboratorio de Neurofarmacología, Av. Dr. Luis Castelazo s/n, Col. Industrial Ánimas, Xalapa 91190, Veracruz, Mexico. E-mail address: [email protected] (A.G. Gutiérrez-García).
http://dx.doi.org/10.1016/j.earlhumdev.2017.08.009 Received 22 June 2017; Received in revised form 18 August 2017; Accepted 25 August 2017 0378-3782/ © 2017 Elsevier B.V. All rights reserved.
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axillary odor [17]. These observations suggest that prior exposure to sensorial cues, likely during intrauterine life, facilitates orientation toward the natural source of feeding (i.e., the mammary gland). Human amniotic fluid is a complex mixture of many substances. Eight fatty acids from amniotic fluid, colostrum, and maternal milk have consistently been identified, and an artificial fatty acid mixture that is based on the content and concentration of fatty acids in human amniotic fluid (i.e., lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, elaidic acid, and linoleic acid) produces appetitive responses in human newborns [18]. Therefore, at least in humans, the initial steps toward infant-mother interactions appear to occur after birth when the newborn seeks substances that are similar to those that it experienced during intrauterine life, such as those that emanate from the maternal breast [15,19], including fatty acids [19,20]. However, unknown is whether the entire fatty acid mixture produces these seeking behaviors or whether only some of the fatty acids do. Therefore, the present study evaluated human newborns a few hours after birth. We recorded appetitive responses to each of these eight fatty acids and compared the results with scents from vanilla, the vehicle, and neutral saline.
2.3. Olfactory stimuli
2. Materials and methods
2.4. Behavioral test and data analysis
2.1. Ethics
Detailed procedures have been published elsewhere [18]. Briefly, the tests were conducted in a nursery room at a temperature of 25 °C. The newborns were individually placed in a warming bed (Infant Warner IW 703, Fisher Paikel Heath Care) with controlled temperature (37 °C) and humidity (< 60%). Different disposable swabs were introduced in Vacutainer tubes, impregnated with the corresponding olfactory stimulus, and vertically placed 1 cm above the newborn's nose. Particular attention was paid to preventing the cotton swabs from touching the newborn's skin. All of the sessions were recorded with a digital videocamera (Sony, DCR-SR85, 25× optical zoom, Carl Zeiss lens) for further analysis. Two other independent observers who were blind to the sequence and nature of the stimuli separately scored the presence or absence of responses. The correlation between the observers' scores reached 0.886 (p < 0.001). The videorecordings were analyzed several times. The presence of responses that were suggestive of mucosal irritation (e.g., grimacing, crying, hiccupping, and sneezing) were first explored. For the evaluation of these behaviors, the presence and absence of each sensory stimulus presentation over 30 s was considered and marked on a binary scale (1 or 0). Subsequently, to classify the newborns' reactions to the stimuli, the data were analyzed based on categories of positive and negative facial responses according to previous reports [22], with slight modifications. Movements of the mouth and suckling behaviors were considered appetitive responses because they are directed toward the maternal breast or milk bottles [23]. Positive responses were scored when movements of the mouth (i.e., suckling) and nose (i.e., sniffing) were directed toward the swabs. Negative responses included lateral movements of the head away from the swab. Remaining stationary without any perceptible changes in facial expression was considered an indicator of indifference.
Each of the eight fatty acids that were previously identified in human amniotic fluid was prepared individually, strictly following the same concentration that was found in amniotic fluid in previous studies [18]. Pure fatty acids were individually dissolved in a volume of 100 mL of vehicle (96% propyl-n-glycol and 4% ethanol) at < 40 °C. All of the fatty acids were of analytical grade and purchased from SigmaAldrich (St. Louis, MO, USA). The final preparation of each of the fatty acids was based on previous reports [18,21] and the physiological concentrations of fatty acids that are detected in amniotic fluid: linoleic acid (4.4 mg/L), palmitoleic acid (7.1 mg/L), stearic acid (3.7 mg/L), myristic acid (3.0 mg/L), elaidic acid (1.5 mg/L), lauric acid (0.4 mg/ L), oleic acid (8.0 mg/L), and palmitic acid (15. 3 mg/L). We included three control odors: control-neutral (0.9% saline), control-aromatic (4% vanilla dissolved in distilled water), and control-vehicle (96% propyl-nglycol and 4% ethanol). The presentation of each sensorial stimulus lasted 30 s. Thus, a total of 11 olfactory stimuli were applied, with a 60 s interval between each stimulus presentation. The total duration of testing was < 17 min for each newborn. The sequence of presentation of the various stimuli varied between newborns according to a Latinsquare design. Afterward, each mother took care of her newborn.
The present study strictly followed the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments that involve humans. The local research ethics committee (Biomedical Research Institute of the National Autonomous University of Mexico and Hospital Civil de Perote, Veracruz, Mexico) approved the study. During the invitation sessions, all of the mothers received a detailed explanation of the purpose and risks of the study. Two physicians who did not participate in the experimental protocol gave the explanations to the mothers. We did not touch their newborns at any time during the study, with the exception of placing the newborn in the bed. The mothers were present near the bed throughout the behavioral tests.
2.2. Mother volunteers and newborns We obtained written consent from 12 mothers to include their babies in the study. Mothers who were previously diagnosed with any psychiatric or neurologic pathology were excluded from the study. All of the mothers were right-handed, and none were smokers. They were all in optimal health, confirmed by a complete clinical physical examination. The inclusion criteria included a minimum gestational age of 39 ± 0.23 weeks (range, 37–40 weeks), birth weight appropriate for gestational age (range, 2.7–3.7 kg), and minimal Apgar score of 9.0 immediately and 5 min after delivery. Well-being and the absence of hunger were ensured by feeding the newborns with colostrum from the maternal breast 15 min before the behavioral test. None of the newborns received any artificial milk formulation or bottle-feeding before the tests. In addition to the newborn and its mother, two researchers were in the examination room. One researcher applied the stimuli, and the other researcher videorecorded the session and coordinated the activities (i.e., the sequence of stimulus presentation) while explaining each step of the experimental procedure to the mother. Under these conditions, only one researcher was near the newborn, at a distance that was necessary to apply the stimuli while not touching the newborn. The other researcher and mother were at least 2 m away from the newborn, remaining as silent and stationary as possible.
2.5. Statistical analysis Possible gender differences were analyzed using the Mann-Whitney U test. Behaviors that were suggestive of mucosal irritation (e.g., grimacing, crying, hiccupping, and sneezing) were scored in binary form (1 or 0). The Cochran Q test was used to analyze the presence or absence of these behaviors. Because the data did not follow a normal distribution, we used nonparametric tests and Statistica 7.0 software. The duration of positive and negative responses to the stimuli was 33
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analyzed using Friedman's analysis of variance (ANOVA). The Wilcoxon test was used for comparisons of individual positive and negative responses to the eight fatty acids compared with the three controls (control-neutral, control-vehicle, and control-aromatic). We only considered responses to be significantly different when the difference reached statistical significance compared with the three control stimuli. Bonferroni correction was applied to adjust for multiple comparisons (adjusted p [padj] = p ≤ 0.0125). The results are expressed as mean ± standard error of the mean. 3. Results The mean age of the mothers was 22.8 ± 1.25 years (range, 14–32 years). All of the deliveries occurred by the vaginal route. No resuscitation or intensive care was needed in any of the cases. The Silverman score was zero. The newborns' average Capourro score was 39.5 ± 0.16. Twelve newborns were included in the behavioral study (five girls and seven boys). The newborns were tested approximately 22.0 ± 1.21 h after delivery (range, 18–24 h). 3.1. Mucosal irritation Of the 12 newborns who were included in the study, two sneezed, one hiccupped, one grimaced, and one cried at least once but less than three times without any clear accumulation in response to any of the stimuli. The Cochran Q test confirmed the absence of significant differences (Q = 7.727, df = 10, p = 0.655) between the different stimuli. 3.2. General aspects of behavior Notably, the behavior that was most often observed was indifference, followed by negative and then positive responses (Fig. 1A). Positive responses were observed only at the beginning of the test. We did not observe any effects of gender on positive responses (U = 8072.5, p = 0.516), negative responses (U = 2036.0, p = 0.708), or indifference (U = 1939.5, p = 0.412). 3.3. Positive responses Fig. 1. (A) Cumulative duration of positive responses (gray bars), negative responses (open bars), and indifference (hatched bars). All of the fatty acids, with the exception of myristic acid, produced similar reactions to the control stimuli. (B) Only myristic acid significantly increased the duration of positive responses (p < 0.0001, Wilcoxon test) compared with the three control stimuli and other fatty acids. V, control-vehicle (propyln-glycol); VA, control-aromatic (vanilla); Sal, control-neutral (saline); LA, lauric acid; MY, myristic acid; PA, palmitic acid; PO, palmitoleic acid; ST, stearic acid; OL, oleic acid; LI, linoleic acid.
Friedman's ANOVA indicated a significant effect of stimulus on positive responses (χ210,10 = 52.055, p < 0.0001). The Wilcoxon test indicated that the comparisons between the actions of myristic acid and the three control substances and all of the other fatty acids reached padj < 0.0001. Myristic acid produced the longest positive responses (Fig. 1B). No other significant differences were observed. 3.4. Negative responses
hippocampus and other deep temporal lobe structures, such as the amygdaloid complex [24], the mesolimbic system [25], and interactions among these structures and the prefrontal cortex [26,27], among other connections. A typical feature of vertebrates is the presence of connections between olfactory structures and cerebral nuclei that are responsible for processing emotional behavior, thus influencing adaptive behaviors that are essential for survival of the species [28]. Interestingly, in animal models, a single injection of amniotic fluid or a fatty acid mixture that contained myristic acid decreased the responsivity of the lateral septal nucleus-amygdala connection similarly to typical anxiolytic drugs [29]. This finding illustrates that fatty acids may exert actions on neuronal activity, and these actions occur through two forebrain structures that are well-known to be related to emotional processing and social behavior [30]. Additionally, this artificial mixture of fatty acids was shown to produce anxiolytic effects in animal models [21], and these actions appeared to occur through γ-aminobutyric acidA receptor neurotransmission [31]. Altogether, these neuronal and
Friedman's ANOVA revealed no significant effect of stimulus on negative responses (χ210,10 = 15.495, p = 0.115), but a trend toward myristic acid producing the lowest values was observed (Fig. 2A). 3.5. Indifference Friedman's ANOVA revealed no significant effect of stimulus on indifference (χ210,10 = 15.811, p = 0.105). Myristic acid shortened the duration of indifference, but this shorter duration did not reach statistical significance (Fig. 2B). 4. Discussion The present study found that myristic acid may act as an olfactory cue that guides newborns to the maternal breast. The behaviors that are elicited by chemical cues must be elaborated in brain regions that modulate emotional processing. Such processes involve the 34
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study, no differences in behavioral responses that were elicited by the control stimuli were observed, and only myristic acid produced significantly longer positive responses than the three control stimuli and other fatty acids, thus discarding the possible influence of diet on our results. After birth, the newborn is exposed to similar substances, and its movements are directed toward the maternal breast [15,41]. Fatty acids, mainly myristic acid, seemingly act as chemical cues to which the newborns were previously exposed during intrauterine development. Myristic acid produces some interesting actions, but it is not entirely devoid of toxicity. It is widely found in natural products, including bovine [42,43] and human [18,43] colostrum and milk, in addition to palm kernel oil and coconut oil. Myristic acid has been studied for its possible therapeutic uses. The moderate consumption of myristic acid, among other fatty acids, was shown to decrease total cholesterol, lowdensity lipoprotein cholesterol, and triglycerides but did not alter highdensity lipoprotein cholesterol [44]. Coconut oil is rich in saturated fatty acids. It has a high content of myristic acid and exerts supposedly renal protective effects in experimental animal models [45] and neuroprotective effects [46]. However, a maternal diet that consists of pure saturated fatty acids may produce persistent alterations in Na+,K+adenosine triphosphatase function in offspring, which may increase the risk of adulthood disease [47]. In the present study, we did not detect any relevant signs of mucosal irritation beyond those that were produced by the control stimuli. For the preparation of the artificial fatty acid mixture, we used fatty acid concentrations that were previously detected in amniotic fluid. Myristic acid was shown to exert anxiolytic effects at physiological concentrations that are found in amniotic fluid in animal models [48]. Therefore, although myristic acid may produce some toxic actions, its use at physiological concentrations may be considered safe for possible use. Coconut oil is one example of such use at physiological concentrations. The present study has at least three limitations. First, we only included a small sample of newborns. We did not observe any gender differences, but this observation may not be conclusive because of two aspects: the small sample size of the study and the young ages of the babies. The babies were tested within 24 h after delivery, which may be before possible gender differences would be expressed. Second, the newborns drank colostrum a few minutes before testing. We included newborns < 24 h after birth, and depriving them of feeding would be unethical. However, in the present study, the newborns were exposed to each of the eight fatty acids separately plus three controls (saline, vanilla, and vehicle) in a different sequence for each newborn. Likewise, neonatal oral and facial responses reflect namely behavioral attitudes directed toward feeding [49–51]. In the present study, the babies received some drops of colostrum approximately 15 min before the test. Considering that amniotic fluid, colostrum, and maternal milk contain similar amounts of the same fatty acids [18], the babies received three consecutive exposures to similar substances. The first exposure occurred during intrauterine life. The second exposure occurred when they received the drops of colostrum. The third exposure occurred during the test. Our results confirmed that prenatal learning appears to follow a transnatal olfactory continuum [52], in which the fetuses are exposed to similar substances that they will find after birth in a continuum of sensorial cues that leads to early learning and further recognition through olfactory stimulation [53]. Third, we did not directly record the sleep-wake state. In newborns, the sleep-wake cycle is evident within a few hours after birth [54], and feeding is a typical behavior during wakefulness, but both lip and tongue suckling movements are commonly observed during both rapideye-movement (REM) and non-REM sleep stages [55]. We used olfactory stimuli and avoided touching the baby's face or dropping any of the stimuli on the baby's face. Sniffing and suckling were presumably responses that were elicited by olfactory stimulation and not necessarily a primary gustatory response. An olfactory response may occur independently of the sleep/wake state, but a gustatory response may
Fig. 2. (A) Negative responses. A trend toward myristic acid producing the lowest negative response values was observed. (B) Indifference. A trend toward myristic acid producing the lower indifference values was observed. See Fig. 1 for abbreviations.
behavioral actions likely contribute to infant-mother recognition. Any odorant molecule that acts as a stimulus requires a binding carrier protein with high affinity for membrane olfactory receptor neurons [32]. At least three carrier proteins have been identified, including within the source of stimulation (i.e., amniotic fluid, colostrum, and milk) and in the receptor system (i.e., nasal mucosa and vomeronasal organ), and appear to mediate the fatty acid-induced initiation of perception [19,33,34]. These proteins have been identified in both human amniotic fluid [35] and colostrum [36], suggesting specific actions of these proteins even during intrauterine life. Importantly, the babies who were included in the study were tested between 18 and 24 h after delivery. Before the test, the babies were in contact with their mothers, the nursing staff, and pediatricians. The mothers received a bath before the test, and none of them used cosmetics during their hospitalization. Nonetheless, the babies were exposed to many relatively uncontrolled sensorial stimuli before the test, but all of the babies received similar sensorial stimulation before being tested. The ingestion of saturated and monounsaturated fatty acids is relatively constant across many countries [37]. Although we did not obtain information about the mothers' diets, the odoriferous content of amniotic fluid [38,39] and maternal milk [40] is closely related to the odoriferous content of the mother's diet. In an effort to control some possibly confounding aspects of the diet, we selected vanilla as one of the control stimuli (in addition to saline and propyl-n-glycol) because vanilla is commonly used as a flavoring in this country. In the present 35
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require a state of wakefulness to be expressed. In fact, olfaction is regulated by ancient brain structures that are common to all vertebrate species and have similar functions in all species [28]. Fatty acids are also widely distributed among the animal kingdom and exert similar functions [20], thus conferring relevance of the olfactory system in adaptive responses, such as orientation toward the feeding source independent of the sleep/wake state. 5. Conclusion The present findings are consistent with previous studies. Some components of amniotic fluid seemingly act as a primary intrauterine signal for the subsequent recognition of similar substances after birth. We found a significantly greater duration of appetitive responses when myristic acid was the stimulus, suggesting that it may be one of the main olfactory sensory cues that guide newborns to the maternal breast during the transition from intrauterine life. Our findings also suggest that there are endogenous phylogenetic mechanisms that allow the identification of essential nutrients. The possible applications of these findings require further research. These fatty acids at concentrations that are detected in amniotic fluid and combinations thereof are commonly found in many natural nutritional sources and some cosmetic products. This finding supports the use of baby cosmetics and skin care products that have a similar composition, such as coconut oil. Author contributions A.G. Gutiérrez-García had primary responsibility for protocol ecution, preliminary data analysis, and writing and revising manuscript. C.M. Contreras conceived the study and experimental sign and wrote and revised the manuscript. C. Díaz-Marte was sponsible for patient screening.
exthe dere-
Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements The authors thank Michael Arends for revising and editing the English of this paper. References [1] S. Jacob, M.K. McClintock, Psychological state and mood effects of steroidal chemosignals in women and men, Horm. Behav. 37 (1) (2000) 57–78. [2] A.G. Gutiérrez-García, C.M. Contreras, M.R. Mendoza-Lopez, S. Cruz-Sanchez, O. Garcia-Barradas, J.F. Rodriguez-Landa, B. Bernal-Morales, A single session of emotional stress produces anxiety in Wistar rats, Behav. Brain Res. 167 (1) (2006) 30–35. [3] T. Nakashima, C.P. Kimmelman, J.B. Snow, Vomeronasal organs and nerves of Jacobson in the human fetus, Acta Otolaryngol. 99 (3–4) (1985) 266–271. [4] G.A. Pyatkina, Development of the olfactory epithelium in man, Z. Mikrosk. Anat. Forsch. 96 (2) (1982) 361–372. [5] R.C. Gesteland, R.A. Yancey, A.I. Farbman, Development of olfactory receptor neuron selectivity in the rat fetus, Neuroscience 7 (12) (1982) 3127–3136. [6] P.C. Brunjes, L.L. Frazier, Maturation and plasticity in the olfactory system of vertebrates, Brain Res. 396 (1) (1986) 1–45. [7] M.I. Chuah, D.R. Zheng, Olfactory marker protein is present in olfactory receptor cells of human fetuses, Neuroscience 23 (1) (1987) 363–370. [8] N. Boehm, B. Gasser, Sensory receptor-like cells in the human foetal vomeronasal organ, Neuroreport 4 (7) (1993) 867–870. [9] E.W. Kreutzer, B.W. Jafek, The vomeronasal organ of Jacobson in the human embryo and fetus, Otolaryngol. Head Neck Surg. 88 (2) (1980) 119–123. [10] T.D. Smith, M.I. Siegel, M.P. Mooney, A.R. Burdi, A.M. Burrows, J.S. Todhunter, Prenatal growth of the human vomeronasal organ, Anat. Rec. 248 (3) (1997) 447–455. [11] T.D. Smith, K.P. Bhatnagar, The human vomeronasal organ, Part II: Prenatal development. Anat 197 (Pt 3) (2000) 421–436. [12] R.H. Porter, Olfaction and human kin recognition, Genetica 104 (3) (1998–1999) 259–263. [13] J. Winberg, R.H. Porter, Olfaction and human neonatal behaviour: clinical
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necrosis occurring in rats fed a methyl-deficient diet, Res. Exp. Med. 199 (4) (2000) 195–206. S.Y. Tsai, M.J. Pokrass, N.R. Klauer, H. Nohara, T.P. Su, Sigma-1 receptor regulates Tau phosphorylation and axon extension by shaping p35 turnover via myristic acid, Proc. Natl. Acad. Sci. U. S. A. 112 (21) (2015) 6742–6747. J.A. Armitage, S. Gupta, C. Wood, R.I. Jensen, A.M. Samuelsson, W. Fuller, M.J. Shattock, L. Poston, P.D. Taylor, Maternal dietary supplementation with saturated, but not monounsaturated or polyunsaturated fatty acids, leads to tissuespecific inhibition of offspring Na+,K +-ATPase, J. Physiol. 586 (20) (2008) 5013–5022. C.M. Contreras, J.F. Rodríguez-Landa, R.I. Garcia-Rios, J. Cueto-Escobedo, G. Guillen-Ruiz, B. Bernal-Morales, Myristic acid produces anxiolytic-like effects in Wistar rats in the elevated plus maze, Biomed. Res. Int. 2014 (2014) 492141. L. Marlier, B. Schaal, R. Soussignan, Orientation responses to biological odours in the human newborn. Initial pattern and postnatal plasticity, C. R. Acad. Sci. III 320 (12) (1997) 999–1005.
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Early Human Development journal homepage: www.elsevier.com/locate/earlhumdev
Myristic acid in amniotic fluid produces appetitive responses in human newborns
MARK
Ana G. Gutiérrez-Garcíaa,b,⁎, Carlos M. Contrerasb,c, Cynthia Díaz-Marted a
Facultad de Psicología, Universidad Veracruzana, Xalapa 91097, Veracruz, Mexico Laboratorio de Neurofarmacología, Instituto de Neuroetología, Universidad Veracruzana, Xalapa 91190, Veracruz, Mexico c Unidad Periférica Xalapa, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Xalapa 91190, Veracruz, Mexico d Hospital Civil de Perote, 91270, Veracruz, Mexico b
A R T I C L E I N F O
A B S T R A C T
Keywords: Appetitive behavior Fatty acids Myristic acid Newborn recognition Prenatal learning Olfactory stimulation
Background: A mixture of eight fatty acids (lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, elaidic acid, and linoleic acid) that are contained in human amniotic fluid, colostrum, and milk produces appetitive responses in newborns, suggesting the existence of a transition of sensorial cues that guide newborns to the maternal breast. Objective: To explore the ability of each of these eight fatty acids individually to produce appetitive responses in newborns. Methods: The study included 12 healthy human newborns < 24 h after birth. Using a longitudinal design, cotton swabs that were impregnated with each of the eight fatty acids and control substances (i.e., vehicle, saline, and vanilla) were placed approximately 1 cm from the newborns' nostrils for 30 s. Positive responses that were suggestive of acceptance included appetitive movements (i.e., suckling) and sniffing that were directed toward the cotton swab. Lateral movements of the head away from the swab were considered negative responses. Remaining stationary with no changes in facial expressions was considered indifference. Results: Compared with controls (i.e., vehicle, saline, and vanilla) and the other fatty acids tested, myristic acid produced the longest duration of positive facial responses (suckling and sniffing). No significant differences in negative facial responses were observed in response to the odoriferous stimuli. No reactions that were suggestive of disgust were observed. Conclusion: A complex combination of stimuli, including the odor of myristic acid, may integrate sensory cues that guide newborns to the maternal breast.
1. Introduction
mucosa is well developed [3] and contains ciliated olfactory receptors that have a mature appearance [4]. Olfactory marker proteins (an indicator of neuroreceptor functionality) [5] and connectivity with mitral cells in the main olfactory bulb [6] are present in epithelia by the 28th gestational week and in the main olfactory bulb between the 32nd and 35th gestational weeks [7]. Similarly, at approximately the 12th gestational week, the human vomeronasal organ is well developed [8] and visible in the developing fetus [3,9,10] and newborns [11]. From an anatomical perspective, human fetuses have a reasonably well developed olfactory system before birth. Some specific components of the interphase between mothers and newborns (e.g., amniotic fluid, colostrum, and milk) may serve as sensorial cues. Shortly after birth, human newborns and other mammals display movements of the head toward the maternal mammary gland [12,13], its own amniotic fluid odor [14–16], and maternal
Among the signaling systems, chemical cues that consist of pheromones [1] can cause notable behavioral responses, including anxiety [2], when perceived by other individuals in the group. The opposite is also true. Some pheromones can act as cues that indicate the existence of a safe environment [1] by informing other individuals of the same species about the absence of danger or presence of food through sensorial systems. Some chemical cues begin to achieve salience in the intrauterine milieu before birth under two conditions: a functional sensorial system and the presence of such cues in the intrauterine milieu (i.e., amniotic fluid). Such prenatal training may guide newborns to similar substances that are contained in colostrum and milk, acting as a guide to the maternal breast and nursing. In humans as early as the 24th gestational week, the olfactory
⁎
Correspondence to: A. Gutiérrez-García, Laboratorio de Neurofarmacología, Av. Dr. Luis Castelazo s/n, Col. Industrial Ánimas, Xalapa 91190, Veracruz, Mexico. E-mail address: [email protected] (A.G. Gutiérrez-García).
http://dx.doi.org/10.1016/j.earlhumdev.2017.08.009 Received 22 June 2017; Received in revised form 18 August 2017; Accepted 25 August 2017 0378-3782/ © 2017 Elsevier B.V. All rights reserved.
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axillary odor [17]. These observations suggest that prior exposure to sensorial cues, likely during intrauterine life, facilitates orientation toward the natural source of feeding (i.e., the mammary gland). Human amniotic fluid is a complex mixture of many substances. Eight fatty acids from amniotic fluid, colostrum, and maternal milk have consistently been identified, and an artificial fatty acid mixture that is based on the content and concentration of fatty acids in human amniotic fluid (i.e., lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, elaidic acid, and linoleic acid) produces appetitive responses in human newborns [18]. Therefore, at least in humans, the initial steps toward infant-mother interactions appear to occur after birth when the newborn seeks substances that are similar to those that it experienced during intrauterine life, such as those that emanate from the maternal breast [15,19], including fatty acids [19,20]. However, unknown is whether the entire fatty acid mixture produces these seeking behaviors or whether only some of the fatty acids do. Therefore, the present study evaluated human newborns a few hours after birth. We recorded appetitive responses to each of these eight fatty acids and compared the results with scents from vanilla, the vehicle, and neutral saline.
2.3. Olfactory stimuli
2. Materials and methods
2.4. Behavioral test and data analysis
2.1. Ethics
Detailed procedures have been published elsewhere [18]. Briefly, the tests were conducted in a nursery room at a temperature of 25 °C. The newborns were individually placed in a warming bed (Infant Warner IW 703, Fisher Paikel Heath Care) with controlled temperature (37 °C) and humidity (< 60%). Different disposable swabs were introduced in Vacutainer tubes, impregnated with the corresponding olfactory stimulus, and vertically placed 1 cm above the newborn's nose. Particular attention was paid to preventing the cotton swabs from touching the newborn's skin. All of the sessions were recorded with a digital videocamera (Sony, DCR-SR85, 25× optical zoom, Carl Zeiss lens) for further analysis. Two other independent observers who were blind to the sequence and nature of the stimuli separately scored the presence or absence of responses. The correlation between the observers' scores reached 0.886 (p < 0.001). The videorecordings were analyzed several times. The presence of responses that were suggestive of mucosal irritation (e.g., grimacing, crying, hiccupping, and sneezing) were first explored. For the evaluation of these behaviors, the presence and absence of each sensory stimulus presentation over 30 s was considered and marked on a binary scale (1 or 0). Subsequently, to classify the newborns' reactions to the stimuli, the data were analyzed based on categories of positive and negative facial responses according to previous reports [22], with slight modifications. Movements of the mouth and suckling behaviors were considered appetitive responses because they are directed toward the maternal breast or milk bottles [23]. Positive responses were scored when movements of the mouth (i.e., suckling) and nose (i.e., sniffing) were directed toward the swabs. Negative responses included lateral movements of the head away from the swab. Remaining stationary without any perceptible changes in facial expression was considered an indicator of indifference.
Each of the eight fatty acids that were previously identified in human amniotic fluid was prepared individually, strictly following the same concentration that was found in amniotic fluid in previous studies [18]. Pure fatty acids were individually dissolved in a volume of 100 mL of vehicle (96% propyl-n-glycol and 4% ethanol) at < 40 °C. All of the fatty acids were of analytical grade and purchased from SigmaAldrich (St. Louis, MO, USA). The final preparation of each of the fatty acids was based on previous reports [18,21] and the physiological concentrations of fatty acids that are detected in amniotic fluid: linoleic acid (4.4 mg/L), palmitoleic acid (7.1 mg/L), stearic acid (3.7 mg/L), myristic acid (3.0 mg/L), elaidic acid (1.5 mg/L), lauric acid (0.4 mg/ L), oleic acid (8.0 mg/L), and palmitic acid (15. 3 mg/L). We included three control odors: control-neutral (0.9% saline), control-aromatic (4% vanilla dissolved in distilled water), and control-vehicle (96% propyl-nglycol and 4% ethanol). The presentation of each sensorial stimulus lasted 30 s. Thus, a total of 11 olfactory stimuli were applied, with a 60 s interval between each stimulus presentation. The total duration of testing was < 17 min for each newborn. The sequence of presentation of the various stimuli varied between newborns according to a Latinsquare design. Afterward, each mother took care of her newborn.
The present study strictly followed the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments that involve humans. The local research ethics committee (Biomedical Research Institute of the National Autonomous University of Mexico and Hospital Civil de Perote, Veracruz, Mexico) approved the study. During the invitation sessions, all of the mothers received a detailed explanation of the purpose and risks of the study. Two physicians who did not participate in the experimental protocol gave the explanations to the mothers. We did not touch their newborns at any time during the study, with the exception of placing the newborn in the bed. The mothers were present near the bed throughout the behavioral tests.
2.2. Mother volunteers and newborns We obtained written consent from 12 mothers to include their babies in the study. Mothers who were previously diagnosed with any psychiatric or neurologic pathology were excluded from the study. All of the mothers were right-handed, and none were smokers. They were all in optimal health, confirmed by a complete clinical physical examination. The inclusion criteria included a minimum gestational age of 39 ± 0.23 weeks (range, 37–40 weeks), birth weight appropriate for gestational age (range, 2.7–3.7 kg), and minimal Apgar score of 9.0 immediately and 5 min after delivery. Well-being and the absence of hunger were ensured by feeding the newborns with colostrum from the maternal breast 15 min before the behavioral test. None of the newborns received any artificial milk formulation or bottle-feeding before the tests. In addition to the newborn and its mother, two researchers were in the examination room. One researcher applied the stimuli, and the other researcher videorecorded the session and coordinated the activities (i.e., the sequence of stimulus presentation) while explaining each step of the experimental procedure to the mother. Under these conditions, only one researcher was near the newborn, at a distance that was necessary to apply the stimuli while not touching the newborn. The other researcher and mother were at least 2 m away from the newborn, remaining as silent and stationary as possible.
2.5. Statistical analysis Possible gender differences were analyzed using the Mann-Whitney U test. Behaviors that were suggestive of mucosal irritation (e.g., grimacing, crying, hiccupping, and sneezing) were scored in binary form (1 or 0). The Cochran Q test was used to analyze the presence or absence of these behaviors. Because the data did not follow a normal distribution, we used nonparametric tests and Statistica 7.0 software. The duration of positive and negative responses to the stimuli was 33
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analyzed using Friedman's analysis of variance (ANOVA). The Wilcoxon test was used for comparisons of individual positive and negative responses to the eight fatty acids compared with the three controls (control-neutral, control-vehicle, and control-aromatic). We only considered responses to be significantly different when the difference reached statistical significance compared with the three control stimuli. Bonferroni correction was applied to adjust for multiple comparisons (adjusted p [padj] = p ≤ 0.0125). The results are expressed as mean ± standard error of the mean. 3. Results The mean age of the mothers was 22.8 ± 1.25 years (range, 14–32 years). All of the deliveries occurred by the vaginal route. No resuscitation or intensive care was needed in any of the cases. The Silverman score was zero. The newborns' average Capourro score was 39.5 ± 0.16. Twelve newborns were included in the behavioral study (five girls and seven boys). The newborns were tested approximately 22.0 ± 1.21 h after delivery (range, 18–24 h). 3.1. Mucosal irritation Of the 12 newborns who were included in the study, two sneezed, one hiccupped, one grimaced, and one cried at least once but less than three times without any clear accumulation in response to any of the stimuli. The Cochran Q test confirmed the absence of significant differences (Q = 7.727, df = 10, p = 0.655) between the different stimuli. 3.2. General aspects of behavior Notably, the behavior that was most often observed was indifference, followed by negative and then positive responses (Fig. 1A). Positive responses were observed only at the beginning of the test. We did not observe any effects of gender on positive responses (U = 8072.5, p = 0.516), negative responses (U = 2036.0, p = 0.708), or indifference (U = 1939.5, p = 0.412). 3.3. Positive responses Fig. 1. (A) Cumulative duration of positive responses (gray bars), negative responses (open bars), and indifference (hatched bars). All of the fatty acids, with the exception of myristic acid, produced similar reactions to the control stimuli. (B) Only myristic acid significantly increased the duration of positive responses (p < 0.0001, Wilcoxon test) compared with the three control stimuli and other fatty acids. V, control-vehicle (propyln-glycol); VA, control-aromatic (vanilla); Sal, control-neutral (saline); LA, lauric acid; MY, myristic acid; PA, palmitic acid; PO, palmitoleic acid; ST, stearic acid; OL, oleic acid; LI, linoleic acid.
Friedman's ANOVA indicated a significant effect of stimulus on positive responses (χ210,10 = 52.055, p < 0.0001). The Wilcoxon test indicated that the comparisons between the actions of myristic acid and the three control substances and all of the other fatty acids reached padj < 0.0001. Myristic acid produced the longest positive responses (Fig. 1B). No other significant differences were observed. 3.4. Negative responses
hippocampus and other deep temporal lobe structures, such as the amygdaloid complex [24], the mesolimbic system [25], and interactions among these structures and the prefrontal cortex [26,27], among other connections. A typical feature of vertebrates is the presence of connections between olfactory structures and cerebral nuclei that are responsible for processing emotional behavior, thus influencing adaptive behaviors that are essential for survival of the species [28]. Interestingly, in animal models, a single injection of amniotic fluid or a fatty acid mixture that contained myristic acid decreased the responsivity of the lateral septal nucleus-amygdala connection similarly to typical anxiolytic drugs [29]. This finding illustrates that fatty acids may exert actions on neuronal activity, and these actions occur through two forebrain structures that are well-known to be related to emotional processing and social behavior [30]. Additionally, this artificial mixture of fatty acids was shown to produce anxiolytic effects in animal models [21], and these actions appeared to occur through γ-aminobutyric acidA receptor neurotransmission [31]. Altogether, these neuronal and
Friedman's ANOVA revealed no significant effect of stimulus on negative responses (χ210,10 = 15.495, p = 0.115), but a trend toward myristic acid producing the lowest values was observed (Fig. 2A). 3.5. Indifference Friedman's ANOVA revealed no significant effect of stimulus on indifference (χ210,10 = 15.811, p = 0.105). Myristic acid shortened the duration of indifference, but this shorter duration did not reach statistical significance (Fig. 2B). 4. Discussion The present study found that myristic acid may act as an olfactory cue that guides newborns to the maternal breast. The behaviors that are elicited by chemical cues must be elaborated in brain regions that modulate emotional processing. Such processes involve the 34
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study, no differences in behavioral responses that were elicited by the control stimuli were observed, and only myristic acid produced significantly longer positive responses than the three control stimuli and other fatty acids, thus discarding the possible influence of diet on our results. After birth, the newborn is exposed to similar substances, and its movements are directed toward the maternal breast [15,41]. Fatty acids, mainly myristic acid, seemingly act as chemical cues to which the newborns were previously exposed during intrauterine development. Myristic acid produces some interesting actions, but it is not entirely devoid of toxicity. It is widely found in natural products, including bovine [42,43] and human [18,43] colostrum and milk, in addition to palm kernel oil and coconut oil. Myristic acid has been studied for its possible therapeutic uses. The moderate consumption of myristic acid, among other fatty acids, was shown to decrease total cholesterol, lowdensity lipoprotein cholesterol, and triglycerides but did not alter highdensity lipoprotein cholesterol [44]. Coconut oil is rich in saturated fatty acids. It has a high content of myristic acid and exerts supposedly renal protective effects in experimental animal models [45] and neuroprotective effects [46]. However, a maternal diet that consists of pure saturated fatty acids may produce persistent alterations in Na+,K+adenosine triphosphatase function in offspring, which may increase the risk of adulthood disease [47]. In the present study, we did not detect any relevant signs of mucosal irritation beyond those that were produced by the control stimuli. For the preparation of the artificial fatty acid mixture, we used fatty acid concentrations that were previously detected in amniotic fluid. Myristic acid was shown to exert anxiolytic effects at physiological concentrations that are found in amniotic fluid in animal models [48]. Therefore, although myristic acid may produce some toxic actions, its use at physiological concentrations may be considered safe for possible use. Coconut oil is one example of such use at physiological concentrations. The present study has at least three limitations. First, we only included a small sample of newborns. We did not observe any gender differences, but this observation may not be conclusive because of two aspects: the small sample size of the study and the young ages of the babies. The babies were tested within 24 h after delivery, which may be before possible gender differences would be expressed. Second, the newborns drank colostrum a few minutes before testing. We included newborns < 24 h after birth, and depriving them of feeding would be unethical. However, in the present study, the newborns were exposed to each of the eight fatty acids separately plus three controls (saline, vanilla, and vehicle) in a different sequence for each newborn. Likewise, neonatal oral and facial responses reflect namely behavioral attitudes directed toward feeding [49–51]. In the present study, the babies received some drops of colostrum approximately 15 min before the test. Considering that amniotic fluid, colostrum, and maternal milk contain similar amounts of the same fatty acids [18], the babies received three consecutive exposures to similar substances. The first exposure occurred during intrauterine life. The second exposure occurred when they received the drops of colostrum. The third exposure occurred during the test. Our results confirmed that prenatal learning appears to follow a transnatal olfactory continuum [52], in which the fetuses are exposed to similar substances that they will find after birth in a continuum of sensorial cues that leads to early learning and further recognition through olfactory stimulation [53]. Third, we did not directly record the sleep-wake state. In newborns, the sleep-wake cycle is evident within a few hours after birth [54], and feeding is a typical behavior during wakefulness, but both lip and tongue suckling movements are commonly observed during both rapideye-movement (REM) and non-REM sleep stages [55]. We used olfactory stimuli and avoided touching the baby's face or dropping any of the stimuli on the baby's face. Sniffing and suckling were presumably responses that were elicited by olfactory stimulation and not necessarily a primary gustatory response. An olfactory response may occur independently of the sleep/wake state, but a gustatory response may
Fig. 2. (A) Negative responses. A trend toward myristic acid producing the lowest negative response values was observed. (B) Indifference. A trend toward myristic acid producing the lower indifference values was observed. See Fig. 1 for abbreviations.
behavioral actions likely contribute to infant-mother recognition. Any odorant molecule that acts as a stimulus requires a binding carrier protein with high affinity for membrane olfactory receptor neurons [32]. At least three carrier proteins have been identified, including within the source of stimulation (i.e., amniotic fluid, colostrum, and milk) and in the receptor system (i.e., nasal mucosa and vomeronasal organ), and appear to mediate the fatty acid-induced initiation of perception [19,33,34]. These proteins have been identified in both human amniotic fluid [35] and colostrum [36], suggesting specific actions of these proteins even during intrauterine life. Importantly, the babies who were included in the study were tested between 18 and 24 h after delivery. Before the test, the babies were in contact with their mothers, the nursing staff, and pediatricians. The mothers received a bath before the test, and none of them used cosmetics during their hospitalization. Nonetheless, the babies were exposed to many relatively uncontrolled sensorial stimuli before the test, but all of the babies received similar sensorial stimulation before being tested. The ingestion of saturated and monounsaturated fatty acids is relatively constant across many countries [37]. Although we did not obtain information about the mothers' diets, the odoriferous content of amniotic fluid [38,39] and maternal milk [40] is closely related to the odoriferous content of the mother's diet. In an effort to control some possibly confounding aspects of the diet, we selected vanilla as one of the control stimuli (in addition to saline and propyl-n-glycol) because vanilla is commonly used as a flavoring in this country. In the present 35
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