Ribitol

Sample preparation was carried out as described previously (32 (link)). C. carassius cultured at 20 or 30°C were euthanized in ice slush (5 parts ice/1 part water, 0–4°C) for at least 10 min following cessation of gill movement, and left in the ice water for a total of 20 min after cessation of all movement to ensure death by hypoxia following the guidelines of NIH. C. carassius were rinsed with distilled water and then wiped thoroughly with sterilized gauze. Spleens were removed ascetically, where 25 mg of spleens were cut and immensed immediately in 1 mL cold methanol. Then, the samples were sonicated for 5 min at 10W-power setting at Ultrasonic Processor (JY92-IIDN, SCIENTZ), followed by centrifugation at 12,000 × g in 4°C for 10 min. The supernatant was collected and 10 μl 0.1 mg/ml ribitol (Sigma) was added as the internal standard. The supernatant was concentrated in a rotary vacuum centrifuge device (LABCONCO). The dried polar extracts were incubated with 80 μl methoxy amination hydrochloride (20 mg/ml pyridine) for 90 min at 37°C, followed by an addition of 80 μl N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) (Sigma) and incubated for another 30 min at 37°C. Finally, the resulted samples were cooled down to room temperature prior to mass spectrometry analysis.

Ribitol was purchased from Sigma (A5502). Ribitol-5-phosphate and CDP-Ribitol were synthesized by Z Biotech (Aurora, CO). MCF7 cells were incubated with or without10 mM Ribitol in growth medium for 3 days, harvested by trypsinization, and washed with PBS three times. Total cells of 10⁵ and 10⁶ each were analyzed for three metabolites: Ribitol, Ribitol 5 phosphate (rib-5P) and CDP-Ribitol. Samples were blinded and then subjected to the following procedures. Cells were homogenized with 400 μl of MeOH:Acetonitrile (ACN) (1:1) and then centrifugated for 5 min at 10,000 rpm. The supernatants were removed, transferred to individual wells of 96-well plate and analyzed by LC/MS-MS performed by Z Biotech. An Applied Biosystems Sciex 4000 (Applied Biosystems; Foster City, CA) equipped with a Shimadzu HPLC (Shimadzu Scientific Instruments, Inc. Columbia, MD) and Auto-sampler (LEAP Technologies; Carrboro, NC) were used to detect Ribitol, Ribitol-5P and CDP-Ribitol. The analysis of metabolites was performed by Z Biotech, LLC (Aurora, CO) and described previously (33).

All chemicals including nutrient broth, methanol, ribitol, methoxamine hydrochloride, N‐methyl‐N‐(trimethylsilyl) trifluoroacetamide (MSTFA), trimethylchlorosilane (TMCS), Folin Ciocalteu's reagent, 20% sodium carbonate, 1‐1,‐diphenyl‐2‐picryl‐hydrazil (DPPH), ethanol, 6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid (Trolox), and gallic acid were purchase from Sigma‐Aldrich. Fresh okara samples were kindly provided by Vitasoy International Singapore Pte Ltd, Singapore. Okara were separated into aliquots and sealed in airtight polyethylene bags and stored at −20°C in the dark.

Methanol of HPLC grade was purchased from Fisher Scientific (Fair lawn, NJ, USA). Standard of n-paraffins mixture (C7-C40) were purchased from ANPEL Laboratory Technologies (Shanghai) Inc. (Shanghai, China). Sodium chloride was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Methyl nonanoate, Ribitol, N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) and methoxamine hydrochloride were purchased from Sigma Aldrich (Saint Louis, MO, USA). All the chemicals and reagents were of analytical grade.

Zebrafish body fluid was collected as previously described with a few modifications [14 (link)]. In brief, zebrafish were rinsed with distilled water and then wiped thoroughly with sterilized gauze. These animals were cut into five pieces on ice and then weighted. The appropriate volume of saline (100 μL/100 mg) was added according to the weight. After centrifugation at 3000 × g, 4 ⁰C, 100 μL fluid was isolated for the further study of metabolites. Metabolites were extracted with 0.2 mL cold methanol (Sigma) containing 10 μL 0.1 mg/mL ribitol (Sigma) as an analytical internal standard. After centrifugation at 12,000 × g for 10 min, the supernatant was concentrated in a rotary vacuum centrifuge device (LABCONCO). The dried polar extracts were used for GC-MS analysis.

Korean perilla and sesame cultivars were grown at the National Institute of Crop Science, Rural Development Administration, Wanju-gun, Korea, during the 2018 growing season (June to November). Chinese perilla and sesame samples were procured from a local market in Xinzhou and JiangXia district (Wuhan city), China. The Chinese samples including perilla and sesame were from the recent harvests of November 2017 and 2016, respectively. Three biologic replicates were prepared for each sample. 5α-Cholestane, ribitol, pentadecanoic acid, fatty acid methyl ester (FAME) mixture, N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA) and pyridine were purchased from Sigma-Aldrich (St. Louis, Mo, USA). All other chemicals used in this study were reagent grade unless stated otherwise.