The lipid ratios that were associated with the desaturases activities in animal study were also examined in lipidomics data from an infant cohort, in order to verify the associations between lipid ratios associated with desaturase enzyme activity and weight gain in humans.

The experiment built upon the methodology employed in our previous studies [6,15,16]. The Cambridge Baby Growth Cohort Study (CBGS) is a UK prospective observational cohort, with longitudinal infancy anthropometric measures and collection of detailed demographics and infant feeding [9]. Mother-infant pairs were recruited from a single maternity unit in Cambridge, UK. Inclusion criteria included singleton, term, and healthy infants with normal birth weight and no significant comorbidities during pregnancy. Infants with a genetic or syndromal disease were excluded from the analyses. Studies were approved by National Research Ethics Service Cambridgeshire 2 Research Ethics Committee (REC) with REC reference 00/325, and all mothers gave informed written consent. This study involved a subgroup of CBGS (total n=201) with available dried capillary blood spots samples. Subjects were recruited between 02/10/2003 and 29/08/2009.

Birth weight was obtained from hospital records. Other birth and subsequent (3, 12, and 24 months) anthropometry measures were performed by three trained paediatric research nurses. Weight was measured to the nearest 1 g using a Seca 757 electronic baby scale and length was measured to the nearest 0.1 cm using an Infantometer (SECA 416). Skinfold thickness was measured in triplicate at four sites (triceps, subscapular, flank, and quadriceps) on the left side of the body using a Holtain Tanner/Whitehouse Skinfold Caliper (Holtain Ltd).

Blood samples from capillary heel-prick sampling were collected at 3 months of age. Blood was dropped onto filtered paper cards, air-dried at ambient room temperature overnight, and stored in Ziploc bags at -80°C. A single spot with diameter 3.2 mm was punched from the card for analysis.

Blood spots samples were extracted by adapting a protocol described previously [6]. Bloods/analytes were placed in the wells of glass coated 2.4 ml deep well plate and added with 100 μl of MilliQ H2O, 250 μl of methanol, and 500 μl of methyl tertiary butyl ether to partition the lipids. The plates were then centrifuged for 10 minutes at 6,000 rpm after being shaken for 10 minutes at 600 rpm. The organic layer on top of the aqueous phase was transferred, dried down, reconstituted, and used for lipid analysis by direct infusion high-resolution untargeted mass spectrometry (HRMS), as previously described [6,15]. Additional lipid identification was done by LC-MS/MS. Selected masses were isolated, and all spectra were recorded [6]. Only the signals of (CE(16:1), CE(16:0), PC(32:1), PC(32:0) PC(38:4), PC(38:3) TG(54:4), TG(54:3), PC(36:3), PC(36:2), TG(50:3) and TG(50:2) were used to calculate the desaturase activities yielding 6 values of lipid ratios (Table 3).

Regression models associating lipid ratios at 3 months and subsequent growth

In the mouse models the natural variation in response to HFD feeding was used to establish correlations between plasma lipids and specified obesity endpoints as was reported in Wopereis et al [14]. Correlations and corresponding p-values were calculated in R version 3.6.3 using Spearman's tests. The resulting p-values were corrected for multiple testing by calculating the false discovery rate (FDR) using the Benjamini Hochberg method. Graphs were produced using GraphPad Prism version 8.4.2.

Infancy age- and sex-appropriate standard deviation scores (SDS) were calculated for weight and length measurements, (with adjustment for gestational age at birth and 3 months), by comparison to the UK 1990 growth reference using LMSgrowth software [17]. Internally-derived SDS were calculated for each individual skinfolds, adjusted for infant sex, GA, and exact age at visit. Growth gains were derived from delta weight, height, and mean skinfolds SDS between 3-12 and 12-24 months. Maternal BMI was derived from self-reported pre-pregnancy weight divided by the square of measured height (kg/m2).

Unless otherwise stated, all descriptive data are presented as means±standard deviations (SD) for continuous variables or as a percentage (%) for categorical variables. Multiple linear regression was used to investigate associations between lipid ratios separately at 3 months of age with weight, height, and skinfolds (reflecting adiposity) gains during both 3-12 and 12-24 months. For weight and height SDS, models were adjusted for maternal parity, maternal pre-pregnancy BMI, and infant feeding history. For mean skinfold, models were adjusted for infant sex, gestational age, postnatal age at measurement, and feeding history, as well as maternal pre-pregnancy BMI and parity. For linear regression models, assumptions of normality, linearity, homoscedasticity, and absence of multicollinearity had been evaluated and none were violated. Since there were only 6 variables/lipid ratios in the regression models, statistical significance was achieved if p values<0.05. Statistical analyses were carried out using SPSS version 25.0 (IBM) and R version 1.0.136.