In vitro optimization of antioxidants for total parenteral nutrition for newborns

Karthigesu, Kandeepan (2024) In vitro optimization of antioxidants for total parenteral nutrition for newborns. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Newborns with gastrointestinal anomalies or premature birth often require lifesaving nutritional support in the form of total parenteral nutrition (TPN). However, TPN administration is associated with several complications in newborns. Several studies have suggested that it could be due to oxidant generation in the TPN admixture due to various factors, including light exposure. Therefore, these studies aimed to assess the peroxidation of the commercially available TPN for newborns by increasing levels of antioxidants and decreasing levels of prooxidants and optimizing the environmental conditions, leading to the development of a new admixture to be tested in a cell culture model. Standard TPN, similar to that in use in the neonatal intensive care unit (NICU) of Janeway Children Hospital, St. John’s, Newfoundland, Canada (control diet) and the antioxidant-fortified all-in-one (AIO)-TPN (test diet), were prepared immediately before experiments commenced by optimizing each selected antioxidant. Both TPNs were light-exposed (LE) and light-protected (LP) for 24 hours. Standard and optimized AIO-TPN, both LE and LP, were treated to THP-1 human monocytes for further assessment of peroxidation, antioxidant status, and endoplasmic reticular (ER) stress. Peroxide levels in the AIO-TPN were gradually increased with increasing light intensity (at 0 Lux=1.020 (±0.07) vs. at 3000 Lux=1.588 (±0.088) mM H₂O₂ equivalents; p < 0.05). L-ascorbic acid at 60 mg/kg/day (2.27 mM), DL-α-tocopherol acetate at 8.8 mg/kg/day (124.1 μM), selenium at 4 μg/kg/day (0.24 μM), glutathione at 4 mg/kg/day (40 μM), copper at 40 μg/kg/day (4.2 M), and zinc at 300 μg/kg/day (30.6 μM) in TPN significantly decreased peroxide levels (p < 0.05). The peroxide levels of optimized LE AIO-TPN were significantly decreased compared to standard LE AIO-TPN (1.165 (±0.042) vs. 1.439 (±0.074) mM H₂O₂ equivalents, p < 0.05). L-ascorbic acid and DL-α-tocopherol acetate at 20 μM fortified SMOFlipid® treated to the THP-1 cells significantly decreased lipid peroxidation compared to the control (1.16 (±0.04) vs. 1.56 (±0.15) μM thiobarbituric acid reactive substances (TBARS), respectively; p < 0.001). Optimized AIO-TPN treated THP-1 cells generated less reactive oxygen species (ROS) than standard TPN. TBARS levels in LE-standard AIO-TPN (3.484 (±0.247) μM), for 24 hours treated THP-1 cells were significantly higher than LP 24 hour-AIO-TPN treated cells (1.257 (±0.153) μM); p < 0.05. The optimized LP AIO-TPN for 24 hours treated THP-1 cells had the highest cellular antioxidant activity (CAA) activity (36.97 (±9.63) %), followed by LE-optimized AIO-TPN for 24 hours (33.93 (±9.1) %), whereas the CAA of standard AIO-TPN was 13.37 (±5.069) %; p < 0.05. Though LP-optimized AIO-TPN for 24 hours treated THP-1 cells exhibited a higher ferric reducing antioxidant power (FRAP) activity (49.5 (±7.73) Trolox equivalents (TE) μg/mL), the FRAP activity of LE-optimized AIO-TPN treated cells was low (23.63 (±2.67) TE μg/mL). Even though the oxidized form of glutathione (GSSG) at 40 μM concentrations for TPN decreased the peroxide levels, the reduced form of glutathione (GSH)/GSSG ratio was low, indicating low redox potential in THP-1 cells. ER stress protein, GRP78 expression was high in THP-1 cells treated with LE AIO-TPN compared to LE standard TPN. However, it did not significantly differ. These studies concluded that peroxide levels were significantly increased when TPN was exposed to light. Optimizing TPN using antioxidants demonstrated lowering lipid peroxidation, better antioxidant capacity, and improving ER stress. Overall, these studies give novel insight into a promising strategy to develop a new TPN formula for newborns, particularly preterm babies, to decrease in vitro oxidants and eventually minimize the complications associated with oxidative stress in neonates.

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