Immune system stimulation increases the plasma cysteine flux and whole-body glutathione synthesis rate in starter pigs1

Abstract

Glutathione (GSH) is the major intracellular thiol that plays a role in numerous detoxification, bio-reduction, and conjugation reactions. The availability of Cys is thought to be the rate-limiting factor for the synthesis of GSH. The effects of immune system stimulation (ISS) on GSH levels and the GSH synthesis rate in various tissues, as well as the plasma flux of Cys, were measured in starter pigs fed a sulfur AA (SAA; Met + Cys) limiting diet. Ten feed-restricted gilts with initial body weight (BW) of 7.0 ± 0.12 kg were injected i.m. twice at 48-h intervals with either sterile saline (n = 4; ISS-) or increasing amounts of Escherichia coli lipopolysaccharide (n = 6; ISS+). The day after the second injection, pigs received a primed constant infusion of 35S-Cys (9,300 kBq/pig/h) for 5 h via a jugular catheter. Blood and tissue free Cys and reduced GSH were isolated and quantified as the monobromobimane derivatives by HPLC. The rate of GSH synthesis was determined by measurement of the specific radioactivity of GSH and tissue free Cys at the end of the infusion period. Plasma Cys and total SAA levels were reduced (16% and 21%, respectively), but plasma Cys flux was increased (26%) by ISS (P < 0.05). Immune system stimulation increased GSH levels in the plasma (48%; P < 0.05), but had no effect on GSH levels in the liver, small and large intestines, heart, muscle, spleen, kidney, lung, and erythrocytes. The fractional synthesis rate (FSR) of GSH was higher (P < 0.05) in the liver (34%), small intestine (78%), large intestine (72%), heart (129%), muscle (37%), and erythrocytes (47%) of ISS+ pigs compared to ISS- pigs. The FSR of GSH tended (P = 0.08) to be higher in the lungs (45%) of ISS+ pigs than in ISS- pigs. The absolute rate of GSH synthesis was increased by ISS (mmol/kg wet tissue/d ± SE, ISS- vs. ISS+; P < 0.05) in the liver (5.22 ± 0.22 vs. 7.20 ± 0.59), small intestine (2.54 ± 0.25 vs. 4.52 ± 0.56), large intestine (0.61 ± 0.06 vs. 1.06 ± 0.16), heart (0.21 ± 0.03 vs. 0.48 ± 0.08), lungs (1.50 ± 0.10 vs. 2.90 ± 0.21), and muscle (0.21 ± 0.03 vs. 0.34 ± 0.04), but it remained unchanged in erythrocytes, the kidney, and the spleen (P > 0.80). The current findings suggest that GSH synthesis is increased during ISS, contributing to enhanced maintenance sulfur amino acid requirements in starter pigs during ISS.

Keywords: cysteine; glutathione; lipopolysaccharide; pig; synthesis rate.

Figure 1.

Impact of immune system stimulation (ISS) on plasma free cysteine (Cys) specific radioactivity in pigs. Pigs with an initial BW of 7.0 ± 0.12 kg were injected (i.m.) every 48 h (total of 2 injections) with either increasing amounts of lipopolysaccharide (ISS+; n = 6; white circles) or a sterile saline solution (ISS−; n = 4; black circles). Two days after the start of ISS, pigs were given a primed constant i.v. infusion of ³⁵S-Cys. Plasma specific radioactivity of Cys (dpm/nmol) was determined as, Cys radioactivity (dpm)/Cys concentration (nmol).

Figure 2.

Impacts of immune system stimulation (ISS) on plasma levels of free Met + Cys and Cys, as well as the plasma Cys infusion rate, specific radioactivity (SRA) and flux. Data are least square mean values ± SE and were observed at the end of a 5-h continuous infusion of ³⁵S-Cys. Pigs with an initial body weight of 7.0 ± 0.12 kg were injected (i.m.) every 48 h (total of 2 injections) with either increasing amounts of lipopolysaccharide (ISS+; n = 6) or sterile saline solution (ISS−; n = 4). Two days after the start of ISS, pigs were given a primed constant i.v. infusion of ³⁵S-cysteine. Plasma SRA data are the average of the last 3 h of infusion when ³⁵S-Cys SRA was at steady state. Plasma SRA of Cys was measured as, Cys radioactivity (dpm)/metabolite concentration (nmol). Plasma Cys flux was determined as, ³⁵S-Cys infusion rate (dpm/kg BW/h)/SRA of ³⁵S-Cys at steady state (dpm/nmol). *P < 0.05, **P < 0.01.

Figure 3.

Impacts of immune system stimulation (ISS) on the specific radioactivity (SRA) of ³⁵S-Cys in selected tissues of starter pigs. Data are the least square means ± SE. Measurements was taken after 5 h of infusion. Six out of 10 pigs were injected (i.m.) every 48 h (total of 2 injections) with increasing amounts of lipopolysaccharide (LPS) (ISS+). The remaining pigs (n = 4) were injected with a sterile saline solution (ISS−). In the morning of the day after the second injection with LPS, pigs were given a primed constant i.v. infusion of ³⁵S-Cys for 5 h. Tissue SRA of Cys was measured as, Cys radioactivity (dpm)/metabolite concentration (nmol). Small Int. = small intestine; Large Int. = large intestine; Erythro. = erythrocyte. *P < 0.05, **P < 0.01.

Figure 4.

Impacts of immune system stimulation (ISS) on the specific radioactivity of glutathione (GSH) in various tissues of pigs. Data are the least square means ± SE and were observed at the end of a 5-h continuous isotope tracer infusion. Pigs were injected i.m. 2 times with either increasing amounts of lipopolysaccharide (LPS) (ISS+; n = 6) or a sterile saline solution (ISS−; n = 4). The 2 injections occurred 48 h apart. In the morning of the day after the second injection with LPS, pigs were given a primed constant i.v. infusion of ³⁵S-Cys. Glutathione was isolated and quantified as monobromobimane derivatives by HPLC. Specific radioactivity of GSH was determined as, GSH radioactivity (dpm)/GSH concentration in the tissue (nmol). Small Int. = small intestine; Large Int. = large intestine; Erythro. = erythrocyte. *P < 0.05, **P < 0.01.