Amino Acid Stable Carbon Isotopes in Nail Keratin Illuminate Breastfeeding and Weaning Practices of Mother – Infant Dyads

doi:10.21203/rs.3.rs-4688041/v1

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Amino Acid Stable Carbon Isotopes in Nail Keratin Illuminate Breastfeeding and Weaning Practices of Mother – Infant Dyads

https://doi.org/10.21203/rs.3.rs-4688041/v1

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Compound-specific stable carbon isotope analysis of amino acids (CSIA-AA) is widely used in ecological studies to analyze food-webs and is gaining use in archaeology for investigating past diets. However, its use in reconstructing breastfeeding and weaning practices is not fully understood. This study evaluates the efficacy of stable carbon isotope analysis of amino acids in early life diet reconstruction by analyzing keratin from fingernail samples of three mother-infant pairs during late gestation and early postpartum periods. Our results show that stable carbon isotope ratios (δ¹³C) of glycine, and to a lesser extent glutamate, effectively trace the onset of exclusive breastfeeding and the end of weaning in infants. We propose that glycine’s ‘conditionally essential’ metabolic pathway during infancy allows it to reflect maternal glycine δ¹³C, indicating breastmilk consumption. Subtle changes in glutamate δ¹³C likely result from its ‘non-essential’ status. Additionally, δ¹³C values of glycine and glutamate indicate maternal physiological and pathological stress due to catabolic effects such as gluconeogenesis. These findings have significant implications for ecological and archaeological research using CSIA-AA for dietary reconstructions. They highlight the need to understand how metabolic pathways affecting δ¹³C of amino acids may change over an individual's lifespan or be altered due to various forms of stress.

Compound-specific

Isotope Analysis

Breastfeeding

Weaning

Stable isotope analysis (SIA) is a valuable tool for investigating past dietary practices, particularly between mothers and infants (Waters-Rist 2023). However, the interpretation of stable isotope data from bulk tissues (e.g., collagen from bones or teeth; keratin from hair or nails) can be complicated and uncertain due to the effect of factors like growth, disease, and physiological or nutritional stress (Fuller et al. 2004; Hobson et al. 1993; Waters-Rist & Katzenberg 2010). Unlike bulk tissue SIA, compound-specific isotope analysis (CSIA) examines individual amino acids (AA). Amino acids are the fundamental building blocks of proteins and their stable isotopic composition is influenced by the chemical nature of each individual AA and its unique metabolic pathway (Ambrose & Norr 1993; Choy et al. 2010). Some AAs are more indicative of the isotopic composition of diet, while others can provide insights into metabolic processes (McMahon & Newsome 2019), enabling researchers to distinguish between dietary and non-dietary isotopic changes. This study explores the effectiveness of compound-specific stable carbon isotopes (δ¹³C) for reconstructing early life diet via a longitudinal analysis of keratin from fingernail samples of three mother-infant dyads. We focus on specific AAs such as glycine, glutamate, and phenylalanine due to their distinct biosynthetic pathways, which provide unique insights into diet and physiological or pathological stress. Additionally, we investigate the application of a widely used spacing ratio between the δ¹³C of glycine (Gly) and phenylalanine (Phe) (Δ¹³C_Gly-Phe) to discern terrestrial and aquatic protein diets.

This study is among a few that focus on using CSIA-AA to exclusively examine maternal reproductive and infant feeding practices (e.g., Cheung et al. 2022; Harris 2020; Romek et al. 2013). To improve our understanding of CSIA-AA for reconstructing early childhood diets, this study delves into AA metabolic pathways and their influence on δ¹³C patterns during the late prenatal and early postnatal periods for mothers and fetuses/infants (i.e. 14 weeks in utero to 1.5 years of age). This investigation is vital because the body's AA sourcing may shift from dietary intake during childhood to endogenous synthesis later in life, impacting stable isotope patterns (Jim et al. 2006; Wu et al. 2013). These findings are significant for CSIA-AA ecological research, as researchers seek to understand diets and trophic positions across ecosystems, despite limited knowledge of AA routing changes over an individual's lifespan.

AA Routing & Metabolism

Amino acids are categorized as essential or non-essential based on their metabolic pathways. Essential amino acids (EAAs) like lysine, leucine, phenylalanine, and valine cannot be synthesized by the body and must be obtained from dietary protein sources. Since they are not significantly modified before incorporation into tissues, δ¹³C of EAAs (δ¹³C_EAA) closely reflect dietary δ¹³C_EAA. Non-essential amino acids (NEAAs), such as glutamic acid and alanine, can be synthesized from the body's carbon pool, leading to isotopic fractionation (Ambrose & Norr 1993; Newsome et al. 2014). Since NEAAs require significant energy for biosynthesis, they may also be derived from dietary sources (Hesse et al. 2022). The balance between dietary routing and de novo synthesis affects carbon fractionation from diet to tissue. Some NEAAs can act like EAAs under certain conditions. For instance, during infancy, the body may not produce enough glycine and proline, making these AAs conditionally essential (CEAA) during growth (Jim et al. 2006; Wang et al. 2014; Wu et al. 2013). This is crucial during breastfeeding and weaning periods, highlighting the need to understand how metabolic changes in CEAAs influence δ¹³C and the interpretations from CSIA data.

Glycine & Other NEAAs in utero

Glycine is an important AA in mammals and other animals as it plays a role in metabolic regulation, anti-oxidative reaction, and neurological function (Wang et al. 2013). It can be endogenously synthesized from other AAs, namely (1) serine (Arnstein & Keglevic 1956) and to lesser extents, (2) choline (Soloway & Stetten 1953) and (3) threonine (Dale 1978; Hartshorne & Greenberg 1964). Cetin’s (2003) study on transport of AAs from the placenta to the fetus in normal and growth-restricted pregnancies found that the transfer rate of glycine from mother to fetus was significantly lower than other AAs. Based on this finding, the author suggested that glycine can be newly synthesized in the feto-placental unit itself without significant input from the mother. Similarly, studies have shown that glutamic acid is not significantly taken up from the maternal placental side but is very efficiently synthesized within the placenta and transported to the fetus (Malek et al. 1993; Schneider et al. 1979). Hence, it is assumed that the most important pathway for fetal NEAAs is de novo placental production with minimal uptake from the mother, while EAAs are directly routed from the maternal to fetal units of the placenta with little or no fractionation. To shed light on the poorly understood topic of fetal routing of glycine in humans, we investigate in-utero glycine δ¹³C (δ¹³C_Gly) of mother-infant dyads.

Glycine in Diseased States & Neonates

Several studies have shown that the amount of glycine synthesized in vivo is insufficient to meet the metabolic demands of an organism in a diseased state (Jackson 1991; Melendez-Hevia et al. 2009; Wu 2010). Mild insufficiency of glycine is not life threatening; however, a chronic shortage may result in suboptimal growth, an impaired immune response, and other adverse metabolic and health effects (de Koning et al. 2003; Lewis et al. 2005). In birds, dietary glycine is essential for fetal (Jackson et al. 2002) and neonatal (Wu & Knabe 1994) development, since in early life they cannot synthesize sufficient glycine to meet requirements (Cetin et al. 1995; Paolini et al. 2001).Research on glycine routing in humans during the early stages of life remains limited, creating gaps in our knowledge concerning the use of δ¹³C_Gly. Our investigation explores glycine metabolism during early life, seeking to determine how its functionality as a NEAA or a CEAA may influence δ¹³C_Gly.

Applications of δ¹³C_Gly

Previous studies using CSIA-AA for dietary reconstruction have primarily utilized δ¹³C_Gly to differentiate between terrestrial and aquatic diets (Corr et al. 2005; Honch et al. 2012; Webb et al. 2018). Aquatic diets, both marine and freshwater, show higher δ¹³C_Gly, correlating with higher trophic levels due to greater isotopic fractionation in marine ecosystems (Larsen et al. 2013). As phenylalanine δ¹³C (δ¹³C_Phe) remains relatively unchanged between diet and consumer, Δ¹³C_Gly-Phe is used to distinguish aquatic from terrestrial protein diets. Corr et al. (2005) found that individuals consuming marine protein exhibited elevated Δ¹³C_Gly-Phe (12.0 ± 1.9‰) compared to those consuming C₃(5.1 ± 1.8‰) and C₄ terrestrial protein (4.0 ± 1.6‰). However, the study focused on adult human and faunal bone collagen, leaving the effectiveness of this metric for nonadults or diseased individuals unclear due to the conditionally essential nature of glycine. Here, we apply Δ¹³C_Gly-Phe to mother-infant dyads to investigate its effectiveness in distinguishing diets during breastfeeding and weaning, and under various pathological or physiological conditions.

Sample Collection

Fingernails were chosen for analysis because they grow incrementally and have the potential to provide longitudinal dietary information if collected over time (e.g., Buchardt et al. 2007; Fraser et al. 2006; O’Connell et al. 2001; Williams & Katzenberg 2012). A total of 43 fingernail samples were collected from three mother-infant dyads before and after birth, until the infants were at least six months of age (Online Resource 1, Table S1). Two of the dyads lived in Canada (MOM-CHIL 1 and 3), and the other in Western Europe (MOM-CHIL 2). Participants were asked to compete dietary and health surveys for themselves and their infant (see in Online Resource 2). All three dyads reported an omnivorous diet. All mothers exclusively breastfed (MOM 1 for ~ 4 months; MOM 2 for 9 months; MOM 3 for 4 months). MOM 1 began weaning at 4 months by providing formula and completed weaning by 9 months. By 1 year formula was completely removed from CHIL 1’s diet. MOM 2 gradually weaned her infant from 9 months to 1 year. MOM 3 provided formula to CHIL 3 for the first 8 weeks along with breastmilk, after which she switched to exclusive breastfeeding until 4 months of age.

CSIA-AA

Debris from fingernail samples was meticulously removed using a scalpel. After cleaning, the samples were degreased following the protocol established by Tea and colleagues (2013), involving an ethyl acetate wash followed by a hexane wash. The samples were dried and precisely weighed (6 mg). In preparation for CSIA-AA, a combination of protocols from Corr et al. (2007a,b), Styring et al. (2010), and Schwartz-Narbonne et al. (2015) were followed (see Online Resource 1 for details). For analysis, samples were dissolved in ethyl acetate (1 mL) and injected twice into the Thermo Trace GC 1310 gas chromatograph coupled to a Thermo Scientific Delta V Advantage IRMS via a GC Iso-Link II Combustion interface. The measurements are reported in per mil (‰) and calibrated to VPDB.

The δ¹³C values reported here are a mean of duplicate δ¹³C measurements. Amino acid δ¹³C measurements were adjusted using specific correction factors to account for additional carbon added during derivatization and for the kinetic isotope effect. The correction factors were calculated using Docherty et al.’s (2001) equation (presented in Online Resource 1, equation S1). As correction factors introduce an additional source of error, the total error generated for each AA was calculated using equation S2 in Online Resource 1, as presented by Docherty et al. (2001) and Soncin et al. (2021). Additionally, a standard AA mixture of alanine, valine, proline, glutamate, and phenylalanine (purchased through Sigma-Aldrich, UK, derivatized in-house) was analyzed after every 3 to 6 sample injections, and the averages from the standard mixture were used for the calibration of the samples. True δ¹³C of standards were measured by EA-IRMS: Val, –32.2 ± 0.2‰; Glu, –32.0 ± 0.4‰; Ala,–29.5 ± 0.0‰; Phe, –21.3 ± 0.6‰; Pro, –22.8 ± 0.5‰; Gly, –33.7 ± 0.1‰; Leu, –34.4 ± 0.7‰; Thr, –28.2 ± 0.6‰.

Stable carbon isotope profiles were generated for all participants using CSIA data. Below, we present the δ¹³C profiles of glutamate and glycine to illustrate both dietary and non-dietary changes experienced by the participants. Profiles and the δ¹³C of all 8 AAs for each participant can be found in Online Resource 1 (Table S2), along with the assessment of sample molecular preservation.

The δ¹³C data for glycine showed the highest intra-individual variation (average ± 2.1‰), followed by glutamate (average ± 1.1‰), making these amino acids the most effective for identifying dietary and physiological changes in infants and mothers. Fig. 1a indicates that CHIL 2's δ¹³C_Gly increased by 2.7‰ from 2 to 10 weeks, coinciding with exclusive breastfeeding. This increase aligns with the 1‰ rise observed in bulk tissue δ¹³C for breastfeeding infants (Fuller et al. 2003, 2006; Richards et al. 2002). Maternal δ¹³C_Glyalso rose by 1.3‰ from 12 weeks before birth to 9 weeks after birth, after it was stable until 61 weeks (1.2 years). The δ¹³C_Gly of mother and child were equivalent at ~ 9-10 weeks (–21.1‰ and –21.2‰). Between 10 to 41 weeks (9.4 months), CHIL 2’s δ¹³C_Gly remained stable, resembling maternal isotopic compositions, but increased by 4.7‰ by 57 weeks 1.1 years).

Starting at 9 months, CHIL 2 began consuming solid foods, gradually reducing breast milk intake. The elevated δ¹³C_Gly compared to maternal isotopic compositions during this period may indicate a shift in glycine sourcing from breast milk to a post-weaning diet with a different δ¹³C composition. It could also reflect a transition in the metabolic pathway for glycine from dietary routing to de novo synthesis. CHIL 2's δ¹³C of glutamate remained stable until 41 weeks (9.4 months), then declined by 4.6‰ with the introduction of solid foods.

The δ¹³C_Gly pattern observed in MOM-CHIL 1 is similar to that of MOM-CHIL 2, with both mother and infant showing comparable isotopic compositions during exclusive breastfeeding (Online Resource 1, Fig. S8). For instance, at around 4-5 weeks, δ¹³C_Gly for the mother and infant were –20.1‰ and –19.4‰, respectively. Additionally, their δ¹³C_Gly were similar from the start of weaning (4.4 months) until midway through weaning (7.4 months), at –17.3‰ and –17.7‰, respectively. In contrast, MOM and CHIL 3 exhibited a significant difference in δ¹³C_Gly, ranging from ~4-7‰, compared to a difference of 1-2‰ in the other MOM-CHIL pairs (Fig. 1b). This substantial disparity might be due to the multiple health challenges faced by MOM 3 during her pregnancy and the peripartum complications experienced by CHIL 3, as detailed below.

For CHIL 3, δ¹³C_Gly increased by 3.1‰ from 8 weeks before birth to the week of birth (Fig. 1b). This increase may be linked to the health issues MOM 3 encountered during pregnancy, which resulted in an emergency C-section 3 weeks early. At birth, CHIL 3 experienced hypoglycemia, labored breathing, and sepsis. Initially, formula was used to supplement breastmilk until ~8 weeks, after which MOM 3 transitioned to exclusive breastfeeding. As a result, δ¹³C_Gly remained stable until 4 weeks due to the heavy reliance on formula, then increased by 5.5‰ until 12 weeks (3 months) with the shift to exclusive breastfeeding. Solid foods were introduced around 4 months, correlating with a 1.5‰ decrease inδ¹³C_Glyby 19 weeks (4.4 months). Fluctuations in δ¹³C_Glx were more subtle during this period; δ¹³C_Glx increased by 1.4‰ between 9 and 13 weeks (3 months) and then decreased by 1.5‰ at 19 weeks (4.4 months).

Non-Dietary Changes – δ¹³C of Glycine & Glutamate

In addition to breastfeeding and weaning, glycine and glutamate δ¹³C profiles were effective in identifying non-dietary factors for the mothers. For instance, in Fig. 2a, MOM 1's δ¹³C for glycine and glutamate increased by 1.8‰ and 1.1‰, respectively, over a single week (20 to 19 weeks before birth), corresponding to the exact period MOM 1 had COVID-19. During the 3rd trimester, glycine and glutamate δ¹³C declined by 2.0‰ and 3.2‰, respectively. However, with only two data points at the start and end of the 3^rd trimester, it is difficult to draw definitive conclusions for this period. Notably, between 3 weeks before and the week of delivery, δ¹³C_Glx increased by 3.3‰, likely due to the catabolic effects of giving birth, similar to the increase observed during COVID-19. This significant increase was not observed in δ¹³C_Gly.

Fig. 2b presents the glycine and glutamate δ¹³C profile of MOM 3 from 25 weeks prior to and 6 weeks after delivery. MOM 3's δ¹³C_Gly declined by 4.3‰ from 25 to 15 weeks prior to delivery (during the second trimester). During this period, MOM 3 experienced leg and feet edema, increased migraines, and fatigue. Between 15 weeks before and 1 week after delivery, MOM 3's δ¹³C_Gly rose again, by 6.2‰. In the third trimester, MOM 3 faced multiple health issues, including respiratory syncytial virus (RSV), which progressed to pulmonary pneumonia. She also experienced preeclampsia, severe anemia, and an ear infection. Due to preeclampsia complications, MOM 3 was induced 3 weeks before her due date. In contrast, δ¹³C_Glx for MOM 3 were stable until 8 weeks prior to delivery, at which point they rose by only 0.8‰. Between 3 weeks before and 6 weeks after delivery, δ¹³C_Glx steadily declined by 1.4‰. Postpartum, MOM 3 underwent various medical treatments, including two different blood pressure medications, iron supplements, antibiotics for the ear infection, a puffer to alleviate pneumonia, and pain medications due to an emergency C-section.

Overall Diet of Mother-Infant Dyads

EAAs like phenylalanine, unlike NEAAs, are reliable diet composition indicators (Jim et al. 2006; Hare et al. 1991; Howland et al. 2003; McMahon et al. 2010). Table S3 in Online Resource 1 shows average δ¹³C_Phe and δ¹³C_bulk of keratin samples. Study participants' average δ¹³C_Phe ranged from –26.1 to –23.3‰, averaging –24.4 ± 1.3‰. MOM-CHIL 2's δ¹³C for bulk tissue and phenylalanine was 2–3‰ lower compared to the other pairs, likely due to residing in Western Europe during pregnancy and nursing, while the others were in Canada. Despite identical supermarket diets, North Americans and Europeans can have different tissue carbon isotopes, influenced by livestock feed variation, especially corn (Bataille et al. 2020; Nardoto et al. 2006; Schmidt et al. 2005). Alternatively, specific dietary choices, like MOM 2 avoiding processed foods and soft drinks, may also play a role.

As mentioned, researchers have used Δ¹³C_Gly-Phe to distinguish terrestrial and aquatic-based diets (Corr et al. 2005; Honch et al. 2012; Webb et al. 2018). Here, we found wide intra-individual variability of –3.0 to 10.6‰ (Fig. S9 in Online Resource 1). For example, MOM 1 showed Δ¹³C_Gly-Phe ranging from 3.4 to 10.6‰, while MOM 2 ranged from 3.4 to 9.6‰. Despite these values, dietary and health surveys indicate participants consumed a C₃-dominated, omnivorous diet with minimal aquatic food intake. Additionally, we noted that infants generally exhibit lower Δ¹³C_Gly-Phe compared to their mothers. (i.e. MOM 3 avg. 4.26 vs. CHIL 3 avg. 0.59).

Breastfeeding & Weaning

Our study demonstrates that δ¹³C_Gly effectively tracks breastfeeding and weaning practices. CHIL 1 and 2's profiles (Fig. S8 in Online Resource 1, and Fig. 1a) illustrate glycine's role in detecting the onset of exclusive breastfeeding. Specifically, during exclusive breastfeeding, δ¹³C_Gly for CHIL 1 and 2 (averaging –18.5‰ and –22.2‰) closely matched those of their mothers (–18.7‰ and –21.2‰), indicating maternal dietary influence. Postpartum, an increase in MOM 2's δ¹³C_Gly corresponded to a 2.7‰ rise in CHIL 2's δ¹³C_Gly between 2 and 10 weeks, while other AAs remained stable. However, from 9 to 13 months, CHIL 2's δ¹³C_Gly increased significantly by 4.7‰, diverging from MOM 2's stable δ¹³C_Gly. At 9 months, CHIL 2's diet comprised half breastmilk and half solid foods, reflecting maternal values. But by 13 months, breastmilk was completely removed from the diet and CHIL 2’s δ¹³C_Gly(–17.7‰) became distinct from the mother's (–21.4‰). Substantial changes in δ¹³C_Gly after the removal of breastmilk suggests that either this AA began to reflect a post-weaning diet with a different isotopic composition compared to the mother's diet, or it began to be biosynthesized. The former scenario seems probable as CHIL 2's δ¹³C_Gly become more akin to reported values for a C₃ diet consisting of soybean meal, barley, and alfalfa (–17.3‰) (Hare et al. 1991), or durum wheat (ranging from –17.3 to –15.0‰) (Paolini et al. 2015).

Unlike CHIL 2, MOM-CHIL 3 data present a distinct scenario. Theδ¹³C_Gly differences between MOM 3 and CHIL 3 range from 4 to 7‰, notably higher than other pairs in our study. While one interpretation could be endogenous glycine synthesis in the infant, the δ¹³C_Gly profile suggests an alternative explanation. From the week of birth to 4 weeks after, the δ¹³C of phenylalanine, glycine, and glutamate for CHIL 3 remained relatively stable (see Fig. 2b, and Fig. S7 in Online Resource 1), indicating that the overall diet was uniform during the first few weeks of life. This aligns with the fact that CHIL 3 was in neonatal intensive care unit (NICU) and was fed formula alongside breastmilk until approximately 8 weeks Subsequently, a 5.5‰ increase inδ¹³C_Gly occurred as CHIL 3 transitioned exclusively to breastmilk by 13 weeks. Additionally, δ¹³C_Gly became depleted of ¹³C at 19 weeks after birth (by 2.8‰), at approximately the time CHIL 3 was introduced to solid foods. This pattern affirms that glycine was incorporated and influenced by breastmilk consumption. Consequently, we propose that the disparity in δ¹³C_Gly between CHIL 3 and MOM 3 arises from a change in the biosynthetic pathway of glycine induced by pathological stress (see below).

Glutamate δ¹³C also provided insights into dietary changes for infants. For example, CHIL 2’s δ¹³C_Glx decreased by 4.6‰ after the cessation of breastmilk consumption. Similarly, CHIL 3’s δ¹³C_Glx increased by 1.4‰ during exclusive breastfeeding and then decreased by 1.5‰, 19 weeks after birth, following the introduction of solid foods. The subtle changes in δ¹³C_Glxin comparison to δ¹³C_Gly in CHIL 3 were surprising as NEAAs are expected to demonstrate a greater trophic level effect than EAAs. However, as the biosynthesis of NEAAs requires a large amount of energy, under conditions of high NEAA concentrations in the diet, these AAs can be routed from dietary sources (Ambrose & Norr 1993; Newsome et al. 2014). Considering that breastmilk contains high glutamate concentrations, infants may have directly sourced glutamate from their diet during certain infancy periods (Davis et al. 1994). The combined metabolic pathways (direct routing and biosynthesis) of glutamate may have led to variable changes in δ¹³C_Glx during infancy. In contrast, the distinctive CEAA role of glycine in infants resulted in patterns closely mirroring those of their mothers during exclusive breastfeeding, notably evidenced by the postpartum increases in δ¹³C_Gly. The postpartum increase in maternal δ¹³C_Glymay be associated with the physiological/caloric burden of breastfeeding as stress can lead to elevated δ¹³C (further explained below).

AA Biosynthetic Pathways & Stress

Before delving into how pathological or physiological conditions can change the δ¹³C of AAs, it is crucial to grasp the intricacies of AA biosynthetic pathways. Typically, NEAAs are synthesized during glycolysis, a process where glycolytic precursors (i.e., glucose arising from carbohydrates) are transformed to form lipids. Carbon in glycine is directly linked to carbohydrate digestion, while glutamic acid is synthesized from the pool of carbon from all macronutrients including protein (Soncin et al. 2021). Various stressors, whether nutritional, physiological, or pathological, can induce a reversal in metabolic pathways, prompting the breakdown of glycogen (the stored form of carbohydrates) and lipids to produce glucose for energy. This process, known as gluconeogenesis, also involves the simultaneous synthesis of NEAAs, which exhibit higher δ¹³C (Kaleta et al. 2011; Newsome et al. 2014). In addition to glycolysis and gluconeogenesis, normal mammalian metabolism includes extensive turnover and degradation of AAs (Adeniyi-Jones et al. 1981; Berg et al. 2012). Most AA catabolism occurs through transamination, a chemical reaction transferring an amino group from one AA (donor) to a ketoacid (acceptor) to form new AAs, and thus influencing δ¹⁵N (McMahon & McCarthy, 2016). Conversely, decarboxylation, which removes a carboxyl group and releases carbon dioxide, is associated with significant effects on δ¹³C (Fry & Carter, 2019). This process enriches the residual AA pool in ¹³C, leading to the enrichment of ¹³C in keratin formed from this pool (DeNiro et al. 1977).

Two mothers in our study experienced physiological or pathological stress. MOM 1, who had COVID-19, showed elevated δ¹³C for glycine and glutamate, likely due to catabolic breakdown. MOM 3 saw a significant decline in δ¹³C_Gly (by 4.3‰) during her second trimester, possibly linked to increased AA synthesis or glycolysis from maternal and fetal growth. In her third trimester, MOM 3's δ¹³C_Gly increased substantially (by 5.1‰) alongside multiple health issues. These trends suggest that breastfeeding-related stress may elevate postpartum δ¹³C_Gly in mothers and infants, given glycine's role as a CEAA during infancy.

These interpretations are supported by Fry and Carter’s (2019) investigation of δ¹³C of carboxyl groups (δ¹³C_CARBOXYL) in AAs of keratin samples from humpback whales, which proposes that AA uptake from diet and new AA synthesis lower δ¹³C_CARBOXYL while catabolic effects (gluconeogenesis or decarboxylation) increase δ¹³C_CARBOXYL. Carboxyl carbon represents one quarter of the carbon comprising an AA and is related to CSIA results that measure the average isotopic composition for non-carboxyl carbon (NCC) as well as carboxyl carbon. Fry and Carter’s (2019) results for humpback whales demonstrate ¹³C-enrichment in δ¹³C_CARBOXYL of both EAAs and NEAAs relative to bulk tissue δ¹³C due to extended fasting (>9 months). Fry and Carter (2019) further note a greater ¹³C-enrichment in EAAs than NEAAs. They suggest that these processes may exert a more pronounced influence on EAAs, as they are not replenished from the diet during fasting, leading to a decreasing supply as they become catabolized. In contrast, NEAAs are continuously synthesized, representing a partially replenished reservoir, and consequently exhibiting less overall ¹³C enrichment than EAAs (Fry & Carter 2019).

With this is mind, the substantial difference between mother and infant δ¹³C_Gly (4 – 7‰) in MOM-CHIL 3 is most likely caused by the combined effects of gluconeogenesis, catabolism, and increasing demand for AA synthesis. CHIL 3’s in utero δ¹³C_Gly(–26.8‰) was the most ¹³C depleted compared to all other AAs for the mother and child, except for valine (mother’s δ¹³C_Val: –26.8‰; child’s δ¹³C_Val: –28.9‰). Although CHIL 3’s δ¹³C_Gly eventually rose over time in response to breastmilk consumption, they remained depleted of ¹³C in comparison with the mother’s δ¹³C_Gly. Given the health complications of MOM 3 during pregnancy and CHIL 3 in the first 6 months of life, it is conceivable that, in addition to insufficient body reserves, new AA synthesis, and pathological stress, glycine metabolic pathways may have been altered to support the growing infant's needs.

In another study, Zignego et al. (2015) investigated human chondrocytes’ response to moderate compression. Based on changes in the concentrations of threonine, homoserine, and allothreonine, Zignego et al. (2015) suggested that rates of glycine, serine, and threonine metabolism were increased after mechanical loading. Similarly, Mickiewicz et al.’s (2015) analysis of metabolites in synovial fluid indicated that an anterior cruciate ligament reconstruction injury resulted in altered pathways for the metabolism firstly of glycine, serine, and threonine, then other AAs such as proline, alanine and glutamate.

Based on these studies, we can conclude that unexpected patterns may be observed, particularly for glycine, and not necessarily for other AAs. We emphasize that carbon for glycine is directly linked to carbohydrate digestion, and any factors affecting this glycolysis would directly affect δ¹³C_Gly. During periods of stress, such as extended fasting, glycogen, the stored form of glucose, is the first to be broken down by the body to obtain energy, followed by lipids and protein (Fry & Carter 2019). As such, δ¹³C_Gly would be one of the first AAs to be changed by these processes. In contrast, since glutamic acid is synthesized from all macronutrients, changes in δ¹³C_Glx may be less pronounced unless lipids and proteins are also being broken down.

Overall Diet

Phenylalanine is known for its minimal enrichment (1-2‰) from diet to tissue (Corr et al. 2005), making it a reliable indicator of the isotopic composition of terrestrial or marine plants at the base of the food web (e.g., Choy et al. 2010; Larsen et al. 2013; McMahon et al. 2010). The average δ¹³C_Phe for MOM 1, 2, and 3 (–24.0‰, –26.1‰, and –23.4‰, respectively) suggest a diet primarily composed of C₃plants and terrestrial livestock fed on a C₃ diet (McCullagh et al. 2005) (Table S3). This observation is supported by the bulk δ¹³C averages (–19.1‰, –21.4‰, and –18.7‰, respectively), which also indicate a C₃plant-dominated diet (Burt & Amin, 2014). Human collagen δ¹³C typically exhibits a positive offset of 3–5‰ from diet (Ambrose & Norr 1993). Subtracting 5‰ from the δ¹³C_bulk averages approximates theδ¹³C_Phe, providing further evidence of limited enrichment in δ¹³C of phenylalanine.

Application of Δ¹³C_Gly-Phe

Based on our findings, Δ¹³C_Gly-Pheproved unreliable for distinguishing diets heavily reliant on terrestrial versus aquatic resources due to significant intra-individual variability among participants. This challenge was also noted in Korean archaeological sites (Tongsamdong and Nukdo) by Choy et al. (2010), who found that Δ¹³C_Gly-Phewas not definitive without considering threonine δ¹³C. Threonine, an EAA, exhibited distinct δ¹³C between marine and terrestrial protein consumers (Choy et al. 2010). Their study indicated there is no universal Δ¹³C_Gly-Phethreshold for distinguishing C3, C4, or marine diets. Choy et al. (2010) examined bone samples from humans (n = 9) and animals (n = 27), and while the ages and sex of most samples were not provided, one human sample was noted as coming from a 7.5-month-old; this infant exhibited the lowest Δ¹³C_Gly-Pheamong the human samples. Cheung et al. (2022), studying weaning practices at two Middle Neolithic communities in the Paris Basin region, Balloy and Vignely, similarly found difficulty in interpreting Δ¹³C_Gly-Phe, particularly in cases where individuals consumed significant freshwater protein. They suggested low Δ¹³C_Gly-Phecould be due to unusually low baseline δ¹³C_Gly in the rivers or exploitation of lower trophic resources. Based on our study's δ¹³C_Gly results, we propose an alternative explanation: variability in glycine biosynthesis influenced by physiological factors rather than baseline conditions or specific dietary choices.

Our results also indicated that infants had smaller Δ¹³C_Gly-Phecompared to their mothers. Cheung et al. (2022) also observed that Δ¹³C_Gly-Phewere smaller for most infants (< 3 years, n = 6) compared to the adults (n = 4). In one group from Vignely in France, there were no overlapping Δ¹³C_Gly-Phebetween adults (n = 2, range 15.2 to 15.7‰) and infants (n = 4, range 10.2 to 13.8‰). To explain why adults had larger Δ¹³C_Gly-Phethan the nonadults, Cheung et al. (2022) proposed that foodstuffs with lower δ¹³C_Gly and δ¹³C_Phewere consumed by both adults and infants, with the latter consuming a higher portion of such foods. Our data do not support the idea that children consumed greater quantities of lower trophic level food or foods with lower δ¹³C_Gly and δ¹³C_Phe in comparison to mothers, given that smaller Δ¹³C_Gly-Phein children were also present during the prenatal period and exclusive breastfeeding, when they relied heavily on their mother's diet. In Cheung et al.’s (2022) study, the age range of Vignely infants spanned from 0 to 2.8 years, encompassing both the fetal stage and exclusive breastfeeding period as determined through their Bayesian statistical analysis (Tsutaya 2019). Therefore, we propose that metabolic processing of glycine during fetal and infant stages primarily contributed to the observed smaller Δ¹³C_Gly-Phe.

This study utilized CSIA-AA to reconstruct breastfeeding and weaning practices. Our findings highlight that δ¹³C of glycine, and to a lesser extent glutamate, effectively trace the onset of exclusive breastfeeding and the cessation of weaning. The pronounced fluctuations in δ¹³C_Gly, compared to other AAs, reflect glycine's unique role as a CEAA, sourced primarily from the diet during infancy and biosynthesized later in life. Elevated δ¹³C_Gly in infants, influenced by mothers' δ¹³C_Gly during breastfeeding, facilitated tracking of breastmilk consumption. Meanwhile, more subtle changes in δ¹³C_Glx can be attributed to glutamate's status as a NEAA, capable of switching between biosynthesis and dietary uptake, particularly evident during exclusive breastfeeding.

Stable carbon isotope compositions of glycine and glutamate also serve as indicators of physiological and pathological stress for mothers. Glycine's carbon is linked to carbohydrate digestion, while glutamic acid synthesis utilizes carbon from all macronutrients (carbohydrates, lipids, and proteins). Under stress, initial breakdown of glycogen affects glycine synthesis, with subsequent impacts on glutamic acid synthesis as lipid and protein breakdown occurs over longer periods. These catabolic stressors enrich δ¹³C of these AAs, increasing their δ¹³C values.

Furthermore, our study emphasizes the complexities of AA metabolic routing and its implications for dietary investigations using CSIA-AA data. The application of Δ¹³C_Gly-Phe to distinguish terrestrial from aquatic diets may lead to misleading interpretations due to glycine's CEAA functionality during infancy and the influence of anabolic or catabolic effects on δ¹³C_Gly (resulting in lower or higher values). Given our current limited understanding of AA metabolic mechanisms and their relationship to δ¹³C, future CSIA-AA research should explore these complexities more comprehensively. Long-term studies from childhood to adulthood are crucial to elucidate the transition from CEAA to NEAA status for glycine, thereby enhancing the interpretation of stable carbon isotope data for AAs in dietary reconstruction and assessing physiological and pathological stressors effectively.

The authors declare that there are no known financial interests or personal relationships that could have influenced the work reported in this article. All study participants gave their informed consent for inclusion before partaking in the study. This study is in accordance with the Tri-Council Policy Statement Ethical Conduct for Research Involving Humans (TCPS2) and the Ontario Personal Health Information Protection Act (PHIPA, 2004). The protocol used in this study was approved by the Non-Medical Research Ethics Board of Western University (Project ID 119889).

Acknowledgements

We are grateful to Li Huang, Kim Law, and Grace Yau for technical assistance in Western’s Laboratory for Stable Isotope Science (LSIS). FJL thanks NSERC (RGPIN 2019-05904) and the Canada Research Chairs Program (X1277C01) for financial support of this research, and the Canada Foundation for Innovation and the Ontario Research Fund, which funded the stable isotope infrastructure. HS is supported by funding from the Social Sciences and Humanities Research Council. This is LSIS Contribution #413.

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No competing interests reported.

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Amino Acid Stable Carbon Isotopes in Nail Keratin Illuminate Breastfeeding and Weaning Practices of Mother – Infant Dyads

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