Lactate, Sodium

MRS was performed at 37°C on a Bruker 11.7 T spectrometer. Hyperpolarized ¹³C in vitro: 18 mg [1-¹³C]pyruvic acid (99% isotopically enriched, Sigma-Aldrich, UK containing 15 mM trityl free radical OX63, Oxford Instruments, UK) was polarized in a HyperSense® DNP polarizer (Oxford Instruments Molecular Biotools Ltd, UK) for 1 hour. The polarized sample was dissolved in 4 ml aqueous buffer (50 mM sodium lactate, 50 mM NaOH, 1 mM EDTA) resulting in a 50 mM pyruvate solution at pH 7, 37°C. A solution of 100 µl, 50 mM hyperpolarized [1-¹³C]pyruvate was mixed with 500 µl cell suspension, and ¹³C spectra were acquired every 2 s using a single scan and a 10° flip angle.

Cauda epididymides were collected from both groups, cut using micro-spring scissors, squeezed, and transferred to 1 mL of human tubal fluid (HTF) medium [25 (link)]. The medium consisted of 101.6 mM NaCl, 4.7 mM KCl, 0.37 mM K₂PO₄, 0.2 mM MgSO₄∙7H₂O, 2 mM CaCl₂, 25 mM NaHCO₃, 2.78 mM glucose, 0.33 mM sodium pyruvate, 21.4 mM, sodium lactate, 286 mg/L penicillin G, 228 mg/L streptomycin, and 5 mg/mL bovine serum albumin. After incubation for 60 min at 37.5°C in 5% CO₂ in humidified air, the upper layer of the medium containing motile and capacitated sperm was collected. Sperm from 10 animals in each group were used for subsequent IVF experiments, and sperm from 3 animals in each group were used for DNA extraction.

Sodium lactate (lactate), 2,2′-azobis(2-methylpropionitrile)
(AIBN), methacrylic acid (MAA), ethylene glycol dimethacrylate (EGDMA),
phosphate-buffered saline (PBS) tablets (pH 8.0 ± 0.02, 0.1 M),
anhydrous dichloromethane >99.8% purity containing 40–150
ppm
amylene as a stabilizer, and anhydrous methanol of 99.8% purity were
all obtained from Sigma-Aldrich and used in the synthesis of both
MIP and NIP powders. All chemicals were used as received with the
exception of MAA and EGDMA, which had inhibitors removed by gravity
filtration via prepacked aluminum oxide (activated, basic) columns
designed for the removal of hydroquinone and monomethyl ether hydroquinone.
Deionized (DI) water and ultrapure water (resistivity of 18 MΩ
cm^–1) were produced using a Purelab Chorus 1 Complete
water purification system (Elga LabWater, Veolia Water Systems Ltd.).
Polymer powders were synthesized by first decanting aluminum oxide
into two syringes filled with glass fibers. MAA as a functional monomer
and EGDMA as a cross-linker were filtered (separately) through the
columns under gravity. Once the inhibitors were removed, MAA (0.24
mL, 2.4 mmol) and EGDMA (2.26 mL, 12.0 mmol) were combined in a reaction
vessel with the addition of 4 mL of dichloromethane. Lactate (44.8
mg, 0.4 mmol) as the template molecule was then added and solubilized
with the addition of 0.4 mL of anhydrous methanol. After the reaction
vessel was concealed from light, AIBN (16.4 mg, 0.1 mmol) was added
as the initiator. The mixture was degassed with nitrogen for 25 min
and then photochemically polymerized for 30 h using a standard laboratory
UV reactor (Cole-Parmer 4-W UV lamp) at a wavelength of 365 nm and
kept at 4 °C using a recirculatory bath (Isotemp). The obtained
colorless and translucent polymer was manually ground using a pestle
and mortar, mechanically milled (Retsch MM 400) at a frequency of
25 Hz for 25 min, and sieved using a 0.25 mm mesh.

Jugular vein catheters (Instech Labs) were implanted in the right jugular vein of 10-wk-old mice (n = 6 per group) under aseptic conditions. The catheter was connected to a vascular access button (Instech Labs) into which the tracer was infused. After 1 wk of the recovery period, mice were fasted for 6 h, and then infused for 2.5 h with U-¹³C-glucose (0.2 M, CLM-1396), U-¹³C-sodium lactate (0.49 M, CLM-1579), 0.2 M ¹³C-alanine (0.2 M, CLM-2184-H), and U-¹³C-glycerol (0.1M, CLM15101), respectively, at 2 to 3-d interval. The infusion rate was 0.1 µL g⁻¹ min⁻¹, and mice moved freely in a cage during the intravenous infusions. Blood (~ 10 µL) was collected from the tail into microvettes with coagulating activator (Starstedt Inc, 16.440.100). Blood samples were kept on ice, and serum was separated by centrifugation at 3,000 g for 10 min at 4 °C. 4 µL serum was added to 60 µL ice-cold extraction solvent (methanol: acetonitrile: water at 40:40:20), vortexed vigorously and incubated on ice for at least 5 min. The samples were centrifuged at 16,000 g for 10 min at 4 °C, and the supernatant was transferred to LC–MS tubes for analyses.