Branched‐chain fatty acids in the vernix caseosa and meconium of infants born at different gestational ages

Abstract The functional lipid components found in breast milk, vernix caseosa, and meconium are Branched‐chain Fatty Acids (BCFA). The goal of this study was to establish the existence of BCFA in vernix and meconium in infants born at different gestational ages. TLC plates and GC‐MS were examined for the lipids in vernix caseosa and meconium. The results indicated that there were nine BCFA in vernix caseosa, including iso‐12:0, anteiso‐13:0, iso‐14:0, iso‐15:0, anteiso‐15:0, iso‐16:0, anteiso‐17:0, iso‐18:0, and iso‐20:0. Five BCFA (iso‐12:0, anteiso‐13:0, iso‐14:0, iso‐15:0, and anteiso‐15:0) were not contained in the meconium, suggesting that some of the BCFA may be digested and consumed by infants. In the vernix caseosa, the content of BCFA in triacylglycerol (TAG) and free fatty acid (FFA) was 15.59% and 11.82%, respectively. The vernix caseosa's wax ester fraction contained the highest content of BCFA, reaching up to 16.81%. The carbon chain length of fatty acids (FA) ranged from 12 to 24 in the vernix caseosa and 14 to 22 in meconium samples. The gestational age was likely to affect BCFA concentrations, with the vernix caseosa and meconium BCFA content being significantly higher in full‐term infants than in preterm infants (p < .001). Further research is required into the relationship between BCFA and gut microbiotas.


| Samples collection
The vernix caseosa was wiped from the newborn's skin, and the first meconium was collected according to Rissmann et al. (2006). All ex- Seventeen vernix and meconium samples were collected, including preterm infants (29-36 weeks, n = 11) and full-term infants (≥37 weeks, n = 6). The maternal age of the mother of preterm infants and the mother of term infants was 30.45 ± 2.98 years and 29.17 ± 4.22 years. With regard to the type of delivery, 36.36 percent of mothers of infants and 66.67 percent of mothers of infants chose eutocia. The gestational age of preterm infants and term infants was 237.55 ± 14.44 days and 272.50 ± 7.92 days, respectively.
The parity of preterm infants' mothers and term infants' mothers was 0.18 ± 0.40 and 0.50 ± 0.55, respectively. The proportion of male preterm infants and term infants was 81.82% and 66.67%. The birth weight of preterm infants and term infants was 2.15 ± 0.40 kg and 2.57 ± 0.23 kg, respectively.

| Lipid extraction
Lipids were extracted as described by Míková (Kaneda, 1977;Míková et al., 2014). Gauze and diapers containing the vernix caseosa and meconium were dissolved in 50 ml of chloroform/methanol (2:1, v/v) with 0.05% of BHT. The suspension was ultrasound for 40 min and filtered by SPE column (60-120 mm, 0.2 g). Trace amount of water was removed by adding anhydrous Na 2 SO 4 (approximately 5 g), and the suspension was filtered by SPE column. The solvent was removed by nitrogen. The lipids were stored at −20°C until further analysis.

| Lipid class analysis
The lipids (100 mg) were separated using hexane/diethyl ether (93:7, v/v) as a mobile phase on a 20 cm × 10 cm silicic acid TLC plates.
Each lipid band was scraped off from the TLC plate and added into the diethyl ester. After centrifugation, take the supernatant liquid to remove the solvents by nitrogen evaporated. By the weighing of each band, the lipid content was determined (Kärkkäinen et al., 1965). The lipids were stored −80°C until further analysis.

| Composition analysis of BCFA in sn-2 monoacylglycerols (sn-2 MAG)
The analysis of sn-2 MAG FA was based on the improved method described by Luddy et al. (1964). In brief, 7 ml of 1 M Tris-HCl buffer (PH = 8), 1.75 ml of 0.05% bile salts, 0.7 ml of 2.2% CaCl 2 solution, and 30 mg pancreatic lipase were applied to the extracted lipids. The mixture was incubated for 3 min at 37°C and shaken for 30 s. These steps were repeated three times, then 2 ml of ether was added, and the mixture was centrifuged (1800 g, 5 min). The ether layer was desiccated with anhydrous Na 2 SO 4, and evaporated to 200 μl under nitrogen. The ether layer hydrolysate was isolated on the TLC plate, to be unfolded using hexane, ether, and ethyl (50:50:1, v/v/v). The band corresponding to sn-2 MAG was scraped off, then methylated, and analyzed by GC-MS.

| Methylation of FA and GC analysis
The collected lipid samples were methylated, according to Míková et al. (2014). Samples were dissolved into chloroform/methanol (2:3, v/v) solution. Fatty acid methyl esters (FAMEs) were obtained via methylation with acetyl chloride (1 ml) at 70°C for 60 min. Trace amounts of water were removed from FAME extracts with anhydrous Na 2 SO 4 , and FAMEs were then dissolved in n-hexane.
Samples were analyzed using a DB-5 column-equipped

| Statistical analysis
Data treatment was performed using IBM SPSS Statistics 19 (Armonk, New York, USA). Statistical significance (p < .05) was analyzed by the Student's t test or analysis of variance (ANOVA).
Graphing was performed using GraphPad Prism 8.

| FA composition in vernix caseosa and meconium
A total of 23 FA were identified in the vernix caseosa of both preterm and term infant samples using GC-MS, and the FA carbon chain length ranged from 12 to 24.

| Distribution of BCFA in meconium
As shown in Table S1, 17 types of FA were measured in meconium samples, with a chain length variation between 14 and 22 carbons.
The composition of SFAs in preterm infants was almost the same as term infants'; 16:0 (~30%), 18:1 n-9 (~20%), and 18:0 (~10%) were most abundant in both preterm and term infants. Only five types of BCFA, including one anteiso-BCFA, were detected in the meconium, which accounted for 5.66 ± 0.22% and 8.11 ± 0.22% in preterm and term infants. Figure 1a shows the comparison of BCFA in the meconium of preterm and term infants. The concentration of BCFA with long carbon chains (iso-16:0, iso-18:0, and anteiso-17:0) in the meconium of preterm infants was significantly lower than in term infants (p < .01), which was in agreement with a previous report (Ran-

Ressler et al., 2008b).
The results indicated that there were significant differences between the FA composition in the vernix caseosa and meconium. In contrast to the vernix caseosa, the types and contents of most BCFA in the meconium of term infants were less than those in the vernix caseosa. The concentration of iso-16:0, iso-18:0, and Note: Data are represented as the mean ± SD (n = 3). *Significant differences from specific fatty acids in preterm infants: * p < .05; ** p < .01; *** p < .001.

| D ISCUSS I ON
In our last study, we reported the content of BCFA in breast milk and its influencing factors, showing eight BCFA with a 0.55% cumulative concentration, and most BCFA are in the sn-2 position. In this study, we studied and contrasted the FA composition of vernix caseosa and meconium in preterm infants and term infants. We further analyzed the impact of gestational age on BCFA in vernix caseosa and meconium compared to the Ran-Ressler team, which found that BCFA were the main components of the intestinal contents of healthy newborns and had been a natural constituent present in the human gastrointestinal tract since a very early age and played an extraordinary role in the life cycle of humans. In addition, vernix caseosa and meconium are more common in the forms and material of BCFA than they are in breast milk. It was proposed that breast milk should only be used as the supplement to BCFA after birth, but may not completely meet the requirements of infant BCFA. Moreover, the content of BCFA both vernix caseosa and meconium was significantly higher in term infants than in preterm infants (p < .001).
The main BCFA in vernix caseosa and meconium are iso-BCFA and anteiso-BCFA, but there was a significant difference of the BCFA component between vernix caseosa and meconium. The distribution of common BCFA in vernix caseosa and meconium is shown in Figure 1. In contrast to vernix caseosa, the types and contents of most BCFA in meconium of both preterm and term infants were somewhat less than those in vernix caseosa. There was only one kind of anteiso-BCFA (anteiso-17:0) was detected in meconium. It was speculated that chain elongation is likely metabolic transformation; short-chain BCFA may be transformed into long-chain BCFA during gastrointestinal digestion. Therefore, it can also explain the According to Friel et al. (1989), the meconium's wet weight in 27 term infants was about 8.9 g, and the average dry weight represents 32% of the wet weight, or 2.8 g.  et al., 2008b, 2013), implying that a large amount of BCFA may be absorbed by the neonatal intestines.
There are two possible reasons for this; first, the lack of BCFA in the vernix caseosa of preterm infants leads to a decrease in the amount of BCFA consumed, resulting in low detection of BCFA in meconium. The second explanation may be that preterm infants have a higher medium and long-chain BCFA absorption capacity than term infants, but are not prone to long-chain BCFA, such as iso-22:0, where the absorption capacity has decreased. It has been speculated that chain elongation is a likely metabolic transformation, and shortchain BCFA may be transformed into long-chain BCFA during gastrointestinal digestion (Mukherji et al., 2003;Terasaka et al., 1986).
The secretion of BCFA in amniotic fluid begins in the second trimester (>36 weeks), with a concentration of up to 17 mg/L (Egge et al., 1972). However, preterm infants (<37 weeks) rarely acquire BCFA at birth. Similarly, the reason for the low prevalence of breastfed infants is also associated with BCFA (Pisano et al., 2020; F I G U R E 1 Composition of BCFA (wt%) in the meconium of term and preterm infants (a), and in the vernix caseosa and meconium of term infants (b)