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Page 4 of 18 Monaco et al. J Environ Expo Assess 2024;3:18 https://dx.doi.org/10.20517/jeea.2024.10

Disaccharidase activity
Intestinal lactase and sucrase activities were measured as previously described [24,25] . Small intestine mucosal
homogenates were prepared and incubated with 0.6 M lactose (ThermoFisher Scientific, Waltham, MA) or
0.3 M sucrose (ThermoFisher Scientific) buffer (dissolved in 0.0625 M maleate buffer) for 60 min at 37 °C.
The reaction was stopped by the addition of 2.0% zinc sulfate (ThermoFisher Scientific) and 1.8% barium
hydroxide (ThermoFisher Scientific), and the amount of glucose released was detected with a glucose
oxidase reagent (ThermoFisher Scientific). The Bradford method measured protein in intestinal
homogenates (Bio-Rad Laboratories, Inc, Hercules, CA). Enzyme activity was expressed as μmoles of
glucose per minute per gram of protein.

Urinary DEHP metabolites
Urinary DEHP metabolites were assessed as described by Warner et al. . Mono-(2-ethyl-5-hydroxyhexyl)
[26]
phthalate (MEHHP), mono-(2-ethyl-5-hexyl) phthalate (MEHP), mono-(2-ethyl-5-oxohexyl) phthalate
(MEOHP) and mono-(2-ethyl-5-carboxypentyl) phthalate (MECPP) were analyzed in the 5500 QTRAP LC/
MS/MS system (Sciex, Framingham, Massachusetts) at the Metabolomics Lab of the Roy J. Carver
Biotechnology Center (UIUC) and Analyst 1.6.2 software was used for data acquisition.

Statistical analyses
Analysis of variance (ANOVA) was conducted using the MIXED procedure of SAS 9.4 (SAS Institute Inc.,
Cary, NC, USA) to differentiate dose effects on young pigs. Depending on the outcome, one of two
statistical models was used: (1) data collected at a single time point were analyzed by 1-way ANOVA; and
(2) data collected from the same animal on more than one occasion (i.e., daily BW and formula intake) were
analyzed by ANOVA with repeated measures, in which dietary treatment, time, and treatment by time
interaction were added to the statistical model. Normality was checked by the Shapiro-Wilk test and data
were log-transformed if distribution did not follow normal distribution (urinary metabolite data). Outliers
were identified by a studentized residual procedure where values of > 3 or < -3 were removed from the
statistical analysis. Data were expressed as mean ± standard error of the means (SEM) and statistical
significance was defined at P < 0.05. Microbial statistical analyses are described below.

Microbiota analysis
DNA extraction
DNA was extracted from AC and RC contents with the QIAamp DNA Stool Mini Kit (Qiagen,
Germantown, MD) in combination with the FastPrep-24 System (MP Biomedicals, Solon, OH) as
previously described . DNA concentration was determined with a NanoDrop 1000 spectrophotometer
[27]
(NanoDrop Technologies, Wilmington, DE).

Polymerase chain reaction amplification, library preparation, and sequencing of 16S rRNA genes
Polymerase chain reaction (PCR) amplification, library preparation, and sequencing of 16S rRNA genes
were performed at Roy J. Carver Biotechnology Center, UIUC. The full-length 16S rRNA genes were
amplified from the extracted DNA using universal primers 27F: AGRGTTYGATYMTGGCTCAG and
[28]
1492R: RGYTACCTTGTTACGACTT . For each sample, both the forward and reverse primers were tailed
with sample-specific PacBio barcode sequences to allow for multiplexed sequencing. The PCR amplification
mixture contained 0.3 µM of both primers, 2.5 ng of genomic DNA, 12.5 µL Kappa Hotstart Ready Mix
(Roche Sequencing and Life Science, Wilmington, MA) in a final volume of 25 µL. PCR temperature profile
included 1 cycle at 95 °C for 3 min, followed by 25 cycles at 95 °C for 30 s, 57 °C for 30 s, and 72 °C for
1 min. The concentrations of PCR products were measured on a Qubit 3.0 Fluorometer (Thermo Fisher
Scientific), and amplicon size (~1.5 kb) was checked by running on an Agilent Fragment Analyzer. Samples
were pooled in equimolar concentrations and purified with AMPure PB beads according to the