Hd4 platform

Non-targeted mass spectrometry analysis was performed at Metabolon, and sample preparation was carried out as published by Schlosser et al.^{5 (link)}. Automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries (>4,500 purified standards) was used for metabolite identification. Known metabolites reported in this study conformed to confidence level 1 (the highest confidence level of identification) of the Metabolomics Standards Initiative^{58 (link),59 (link)}, unless otherwise denoted with an asterisk. Additional mass spectral entries have been created for compounds of unknown structural identity (unnamed biochemicals; >2,750 in the Metabolon library), which have been identified by virtue of their recurrent nature (both chromatographic and mass spectral). Peaks were quantified using the area under the curve and normalized to correct for variation resulting from instrument interday tuning differences by the median value for each run day. Likewise, metabolites in the ARIC replication sample were also quantified with the Metabolon HD4 platform.

Untargeted ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) plasma metabolomics profiling of the pregnant mothers at 24 week and 1 week postpartum and of the children at age 6 months, 18 months and 6 years was performed using untargeted plasma metabolomic profiling including relative abundances of PFOS and PFOA from the HD4 platform Metabolon, Inc. (NC, USA) as described previously.¹¹ (link)

Metabolic measurements were conducted at ADLQ by deploying implemented Metabolon Inc. HD4 platform as previously described (34 (link)). Briefly, the samples extracts, obtained from methanol-based solvent extraction with recovery standards, were divided into equal parts and evaporated under nitrogen stream [TurboVap (Zymark)]. The samples were further reconstituted in four different solvents compatible with each analytical methods as previously described (38 (link)). The metabolic measurements were conducted with Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution (44 (link)). The raw data were analyzed at Metabolon Inc. (Durham, NC, United States) using Metabolon’s hardware and software for compound identification. The obtained peaks were compared to library entries of purified, and the components were identified based on standard retention index, their accurate mass match to the library ±10 ppm, and MS/MS forward and reverse scores (45 (link)). Each obtained compound was corrected in a run day and all the metabolic data were normalized to correct for variations resulting from inter-day tuning differences in the instrument.

Metabolon (Morrisville, North Carolina, USA) performed the metabolite analysis using the HD4 Platform. Lyophilized faecal samples were extracted with methanol to precipitate protein and dissociate small molecules bound to protein or trapped in the precipitated protein matrix, followed by centrifugation to recover chemically diverse metabolites. The resulting extract was divided into fractions. These were used for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods using positive ion mode electrospray ionization (ESI), for analysis by RP/UPLC-MS/MS using negative ion mode ESI and for analysis by HILIC/UPLC-MS/MS using negative ion mode ESI. Metabolon’s peak identification software was used to match ions to a library of standards for metabolite identification and for metabolite quantitation by peak area integration.

During enrollment of the study at gestation week 24, plasma samples were collected from the mothers. Metabolic profiling on these plasma samples was carried out by Metabolon, Inc. (Durham, NC, USA) using its HD4 platform. A detailed description of the metabolomics protocol is provided in the Supplementary Materials Section S1 and has also been described previously [23 (link)]. In brief, metabolites with missingness ≥ 30% as well as unannotated metabolites were excluded. This resulted in a metabolic profile consisting of levels of 753 metabolites used for analyses.

Analysis of plasma metabolites composition was performed using non-targeted MS by Metabolon Inc (North Carolina, US) in HD4 platform (Sperk et al., 2021 (link)). Levels of glucose, glutamine, glutamate, and lactate were compared for differential plasma levels between the groups. Statistical significance was determined using Mann-Whitney U-test in R (significance level, p<0.05).

Plasma un-targeted metabolomics was performed using the HD4 Platform (Metabolon, Morrisville, North Carolina, USA) in a cohort including HC (n = 22), PWH_ART (n = 29), and PWH_VP (n = 11). The samples were selected randomly. The method was performed as previously described [11 (link)]. Plasma targeted metabolomics was performed towards central carbon metabolism (CCM) and sugars using gas chromatography–mass spectrometry (GC–MS) and amino acids by liquid chromatography (LC)–MS/MS in a cohort of HC (n = 37), PWH_ART (n = 55), and PWH_VP (n = 24) at the Swedish Metabolomics Centre (Umeå, Sweden).