Naphthylethylenediamine

Soy phosphatidylcholine (Lipoid S100, SPC) was generously provided by Lipoid GmbH (Ludwigshafen, Germany). The 1-palmitoyl-2-(dipyrrometheneboron difluoride)undecanoyl-sn-glycero-3-phosphocholine (TopFluor^®-PC, T) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5) (18:0 Cy5-PE, C) were purchased from Avanti Polar Lipids, AL, USA. Methanol, naphthylethylenediamine, potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic dodecahydrate, sulfanilamide, phosphoric acid (H₃PO₄), RPMI-1640 medium, Dulbecco’s phosphate buffer, lipopolysaccharide (LPS, Escherichia coli, 055:B5) and propidium iodide (PI) were obtained from Sigma-Aldrich, Steinheim, Germany.

NO was measured by determining the total quantity of nitrite (NO²⁻), which is the stable product of NO metabolism in plasma. The Griess reagent, an aqueous solution of 1% sulfanilamide (Sigma-Aldrich^®, Darmstadt, Germany) and 0.1% naphthylethylenediamine (Sigma-Aldrich^®, Darmstadt, Germany) in 2.5% H3PO4 (JT Baker^®, Philadelphia, PA, USA), forms a stable chromophore with NO²⁻. The reaction’s absorbance was determined at 546 nm in a xMark Microplate Absorbance Spectrophotometer (BIORAD, Berkeley, CA, USA). The calibration curve was constructed using different concentrations of sodium nitrite dissolved in 0.9% NaCl.
The tertiles of NO were constructed based on concentrations in mothers and newborns. The tertiles of NO for the mothers were as follows: from 1.0 to 6.5 µmol/L, tertile 1; from 6.8 to 22.8 µmol/L, tertile 2; and from 23.0 to 384.4 µmol/L, tertile 3. The tertiles of NO for the newborns were as follows: from 0 to 9.6 µmol/L, tertile 1; from 10.2 to 29.1 µmol/L, tertile 2; and from 29.2 to 231.9 µmol/L, tertile 3.

NO was measured by determining the total quantity of nitrite (NO₂⁻), which is the stable product of NO metabolism in plasma. Griess reagent was used (an aqueous solution of 1% sulfanilamide (Sigma-Aldrich, St. Louis, MO, USA) with 0.1% naphthylethylenediamine (Sigma-Aldrich, St. Louis, MO, USA) in 2.5% H₃PO₄ (2.5%, JT Baker, Xalostoc, Mexico), which forms a stable chromophore with NO₂⁻ and absorbs light at 546 nm (Green LC 1982). The calibration curve was constructed using different concentrations of sodium nitrite dissolved in 0.9% NaCl. The NO level was expressed in µmol/L.

For comparisons of microaerobic growth, cells from overnight starter cultures in the MACS cabinet were inoculated in 200 ml MHS in 500 ml conical flasks with appropriate antibiotics to a final OD (600 nm) of 0.1 and grown at 42 °C until an OD 600 nm of approximately 0.4–0.5. They were then back diluted to an OD 600 nm of 0.1 in fresh 100 ml MHS in shaken 250 ml conical flasks and the OD 600 nm monitored every hour using Jenway 6705 UV spectrophotometer. All Growth curves were done in triplicates. For growth and supernatant sample collection under oxygen-limited conditions, cells grown microaerobically as above, were back diluted to an OD of 0.1 in fresh 200 ml MHS in 250 ml conical flasks, with and without TMAO, nitrate or fumarate, and OD 600 nm monitored every hour. Cells in 1 ml samples were separated from the media supernatant by centrifugation (13,800 × g, 5 min) and the supernatants removed and stored frozen at −20 °C until ready for analysis. For nitrite determination, diluted culture supernatants (50 μl) from oxygen-limited growth experiments were added to 850 μl of 1% (w/v) sulphanilamide (Sigma) dissolved in 1 M HCl and 100 μl of 0.02% (w/v) naphthylethylenediamine (Sigma). After 15 min, the absorbance at 540 nm was measured and nitrite concentrations were determined by reference to a standard curve.