Orotic acid

The polymer PLA2002D was obtained from NatureWorks^® Ingeo™ (Minnetonka, MN, USA); its weight average molecular weight (MW) was about 120 kg·mol⁻¹. Orotic acid (Figure 1) was from Sigma-Aldrich (St. Louis, MO, USA). It was in the form of fine powder, the grains were of uneven shape, where the majority of particle sizes was in the interval from 7 to 30 µm. The distribution of the particle cross-section arreas was evaluated with optical microscopy using ImageJ 1.5 software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) (Figure S1 in Supplementary Materials). OA has melting point of about 345 °C so it was still solid at the processing temperature. First, PLA masterbatch with 5% (w/w) of OA was prepared by melting and mixing the components in the two-screw laboratory mini-extruder DSM Xplore 15 (Xplore Instruments BV, Sittard, The Netherlands) at 190 °C for 5 min. Then materials containing 0.1, 0.3 and 0.5% (w/w) of OA were prepared under the same conditions by mixing the masterbatch and neat PLA. Finally, the materials were dried in the vacuum oven at 60 °C for 24 h. Sample films about 100 µm thick were prepared by compression molding at 180 °C and 20 MPa for 1 min. Then the hot plates were removed from the press and let to cool down slowly on air for about 2 h.

The assay comprised enzymatic reaction of DHODH with dihydroOrotic acid (DHO) substrate, followed by fluorescence detection specific for Orotic acid using the 4-trifluoromethyl-benzamidoxime (4-TFMBAO) fluorogenic reagent^{55 (link)}. Briefly, 3 × 10⁵ lymphocytes were lysed by sonication for 10 min at 4 °C. Lysates were incubated with 1.0 ml aqueous solution containing 500 μM DHO, 200 mM K₂CO₃-HCl (pH 8.0), 0.2% Triton X-100 and 100 μM coenzyme Q10 at 37 °C for 1 h. Hundred microliter aliquot was mixed with 150 μL H₂O, 250 μL of 4.0 mM 4-TFMBAO, 250 μL of 8.0 mM K₃[Fe(CN)₆] and 250 μL of 40 mM K₂CO₃ (pH 11) and then heated at 80 °C for 4.0 min. The reaction was stopped by cooling in an ice-water bath and the intensity was measured with a spectrofluorometer. Excitation and emission wavelengths were 340 nm and 460 nm, respectively. For the calculation of the production, the preexisting level of Orotic acid at non-incubation time was subtracted from total amount of Orotic acid observed at the end of the hour incubation. Orotic acid, coenzyme Q10, 4-TFMBAO and DHO were purchased from Sigma-Aldrich.

Firstly, α-d-Lactose monohydrate (98%, Acros Organics, Fair Lawn, NJ, USA), lactulose (99%, Alfa Aesar, Haverhill, MA, USA), lactobionic acid (97%, Sigma-Aldrich, St. Louis, MO, USA), D-(+)-glucose (99%, Sigma-Aldrich), D-(+)-galactose (99%, Sigma-Aldrich), D-gluconic acid sodium salt (99%, Sigma-Aldrich), pyruvic acid (98%, Sigma-Aldrich), L-(+)-lactic acid (85%, Sigma-Aldrich), formic acid (99%, Fisher Scientific, Waltham, MA, USA), citric acid (99%, Sigma-Aldrich), and orotic acid (98%, Sigma-Aldrich) were purchased from commercial suppliers. Reduced ruthenium supported on activated carbon (5% Ru/C, Alfa Aesar) was purchased from commercial suppliers, and it was used without further preparation. Sweet whey permeate (SWP) was obtained from a regional cheese factory (Valley Queen Co., Milbank, SD, USA), while the acid whey permeate (AWP) was obtained from a Greek yogurt plant (Chobani Co., Twin Falls, ID, USA).

The standard orotic acid, dihydroorotic acid (DHO), cytidine, uridine, uracil, aspartic acid, glutamine, carbamoyl aspartic acid, uridine diphosphate glucose (UDP-Glu), uridine monophosphate (UMP), cytidine triphosphate (CTP), uridine triphosphate (UTP), adenosine triphosphate (ATP), and guanidine triphosphate (GTP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Table S1 summarizes the characteristics of each compound. The HPLC grade reagents ammonium acetate, ammonium formate, acetic acid, formic acid, and ammonium hydroxide were purchased from Sigma-Aldrich. The HPLC grade solvents, acetonitrile, and methanol, were purchased from Honeywell International, Inc. (Morris Plains, NJ, USA).

Orotate-supplemented chow (1% w/w) was prepared by adding orotic acid (Sigma-Aldrich, #O8402) to moistened chow followed by pressing the chow into pellets. These pellets were dried at room temperature and stored at −20 °C until use. The treated mice received this chow ad libitum from weaning (P19-22) until sample collection at P29.
The ketogenic diet intervention has been described in detail previously^{27 (link)}. Briefly, we utilized here samples from mice fed control chow or ketogenic diet (TD.96355, Harlan) from weaning until approximately P45.

A 20 mM teriflunomide (Sigma-Aldrich) stock solution was prepared in DMSO and further diluted in the appropriate cell culture medium. Diluted teriflunomide or DMSO corresponding to 1% of the supernatant volume was added to cells. A 25 mM orotic acid (Sigma-Aldrich) stock solution was prepared in 0.1 M NaOH and further diluted in 0.1 M NaOH. Diluted orotic acid or 0.1 M NaOH corresponding to 4% of the supernatant volume was added to cells. Inhibitors were added at the time of transfection/infection, and in the case of medium changes, fresh inhibitors were added at the time of the medium change. All concentrations indicated in the figures are final concentrations.