Main

Glutathione plays a central role as antioxidant in the organism(1) because of its capacity to regenerate other antioxidants, its antiperoxide activity as well as radical scavenging properties, its recycling ability, and elevated concentrations(2). Decreased tissue GSH concentrations have been reported in several diseases involving reactive oxygen metabolites(3). Depleting pulmonary concentrations of this cysteine-containing tripeptide by chemical means accelerates the development of oxygen-induced lung injury in neonatal animal models(4,5). Similarly, dietary deficiency of sulfur-containing amino acids induces a low tissue GSH concentration, which exacerbates pulmonary oxygen toxicity(6). In contrast, glutathione supplementation protects preterm rabbits from oxidative lung injury(7). These experimental observations support the contention that low tissue GSH precedes toxicity and disease(8).

In newborn rats, hyperoxia induces a compensatory mechanism stimulating the synthesis of antioxidant enzymes as well as lung glutathione(9). Yet, in ventilated newborn infants, there is a positive umbilical arterio-venous gradient of GSSG(10), suggesting a loss of GSSG by the lungs to maintain the cellular redox ratio. Exposure of premature infants to hyperoxia is associated with lower plasma cysteine levels(11) and blood GSH concentrations(12), suggesting an increased demand for glutathione(11). The low level of blood glutathione may reflect a low hepatic production as the liver is a net glutathione producer from sulfur amino acids(13), although blood GSH originating from the liver is a main source of substrate for peripheral organs such as lungs. In premature infants exposed to oxygen, we have documented that the levels of glutathione in cells isolated from tracheal aspirates remain low(14) despite a stimulated synthetic activity(15), suggesting a poor substrate availability.

Even if early introduction of amino acids has been proposed(16), newborn infants are often restricted, in the first days of life, to a dextrose infusion during the acute phase of their clinical problems. Malnourished subjects such as sick newborn infants receiving a poor nutrient support during the first days of life could be more susceptible to oxygen toxicity because of a lack of substrate for glutathione synthesis. To test this hypothesis a study was designed to verify if optimizing the feeding regimen had an effect on survival in oxygen in relation to glutathione status in neonates from a species susceptible to oxygen toxicity.

METHODS

Experimental protocols. In newborn guinea pig pups, the developmental expression of antioxidant enzymes in lung and other tissues(17), as well as the mortality in oxygen is well characterized(5,18–20).

Newborn Hartley guinea pigs provided by Charles River (St-Constant, Québec, Canada) as 1-day old pups were randomly assigned to room air or oxygen for 3 d before sampling of lung [net consumer of circulating glutathione(21)] and liver [net producer of glutathione(13)]. To sample tissues from a significant number of surviving animals, the study duration of 3 d was chosen based on a report documenting a survival rate of less than 20% by 5 d in term newborn guinea pigs kept in a hyperoxic environment(20).

To ensure that the effects of hyperoxia on glutathione were related to substrate availability rather than feeding problems related to dyspnea, the animals were fed i.v. with dextrose alone or fat-free TPN or they were nursed by lactating dams. As sick infants are often started on i.v. glucose alone or TPN, we elected to perform these experiments in a neonatal animal model that we could feed parenterally. Animals receiving glucose or nursed by lactating dams were considered to receive a suboptimal nutrient intake. Indeed, previous experience with this animal model had shown that in nursing pups, surgery performed under the same conditions as in this study was associated with a 10% weight loss after 4, although nursing pups who did not undergo surgery had a 10% weight gain over the same interval(22). The day the animals were received, pups were operated under katamine/xylazine. The i.v.-fed animals were fitted with a 0.4-mm polyurethane catheter (Luther Medical Products, Tustin, CA) positioned in the superior vena cava; it was tunneled s.c. and exteriorized in the scapular region and connected to a flow-through swivel permitting mobility of the animals fitted with a harness. Enterally fed animals were returned to the mother after undergoing the same surgical procedures, except for the placement of the catheter. All surgical procedures were performed in room air. The animals dying within the first 24 h were excluded, as the cause of death was assumed to be related to surgery.

Pups nursed by lacting dams were housed three to a mother (lactating surrogate mother or own mother), with free access to mothers' food (water and sham: Pro-lab RMH 4018). Because the survival rate of adult guinea pigs is only 10% after 3 d in oxygen(20), the mothers were separated from the pups and put in room air 1 h twice daily to retard the toxic effects of oxygen. Pups fed i.v. received a continuous infusion of 5% (w/v) dextrose + 0.45% (w/v) NaCl solution at approximately 1 mL/h100 g body weight, providing the recommended fluid intake of 200-250 mL/kg/d(23–24).

To isolate the effect of substrate availability on glutathione status, a further group of pups fed i.v. received fat-free TPN. The TPN regimen consisted of fluids 1 mL/h/100 g body weight, 7.5% dextrose, 1.8% amino acids (Travasol 10% Blend B, Clintec-Baxter, Mississauga, Ontario, Canada) containing 26.8 mM methionine, 1% multivitamin (MVI Pediatric, Rhône Poulenc Rorer, Montreal, Quebec, Canada), electrolytes, minerals, and micronutrients, at concentrations commonly prescribed in neonatology. To isolate the effect of amino acids from the other components of TPN on glutathione production, an alternate source of substrate was infused in a limited number of animals. Under the same experimental conditions, animals cared for in oxygen received i.v. 5% dextrose + 0.45% NaCl supplemented only with 20 mM of a stable precursor of cysteine, OTC (Transcend Therapeutics, Cambridge, MA).

After the surgical procedure, the pups were housed in plastic boxes with wire mesh bottoms in a temperature-controlled room (22-24°C) with a 12-h light to 12-h obscurity cycle. The closed boxes were exposed to a flow (3 to 5 L/min) of room air or 100% oxygen sufficient to maintain a constant normoxic (O2 = 21%) or hyperoxic (O2 > 95%) environment. Under hyperoxic conditions, environmental oxygen concentration was verified at least twice daily with a TED 60-T portable oxygen analyzer (Teledyne Brown Engineering, City of Industry, CA). The cages were changed daily, at which time the oxygen concentration dropped momentarily less than 95%. At the end of each protocol the surviving animals were killed by decapitation. This study was approved by the "Institutional Animal Care and Use Committee."

Sample preparation and analytical procedures. The animals were weighted at induction of anesthesia and before they were killed. Upon decapitation, lungs and liver were immediately sampled, weighed, minced, and aliquoted on ice. Samples were frozen at -70°C until the later biochemical determinations.

An aliquot of tissue was homogenized for 30 s on ice in a 10-fold volume of buffer (100 mM Tris, 0.1 mM EDTA, 5 mM L-serine, 10 mM sodium borate, pH 7.6). After centrifugation at 5500 × g the supernatant was used for the determination of total glutathione and activities of glutathione-reductase, -peroxidase, and synthesis. The pellete of an other homogenate without serine-borate served to measure γ-GT activity.

Total glutathione was measured using the method described by Griffith(14,25). The results were reported as GSH/mg protein. The level of detection was 20 pmol. GSSG-R activity was determined according to the method of Becker et al.(14,26). The level of detection was 50 pmol/10 min. The activity of glutathione peroxidase was determined using a variant of the method for measuring GSSG-R activity. The sample was mixed with 1 mM GSH, 1 mM tert-butyl hydroperoxide (Aldrich Chemical Co, Milwaukee, WI), 0.1 mM NADPH and 5 µg of GSSG-R in a buffer (250 mM Tris, 0.1 mM EDTA, pH 7.6, 30°C). The disapperance of NADPH was recorded at 340 nm. The level of detection was 50 pmol/10 min. The determination of γ-GT activity was performed as described previously(27). The threshold of the assay was 50 pmol/10 min. Glutathione synthetic activity was determined using a technique previously developed to measure the overall synthesis resulting from the combined activities of γ-glutamylcysteine synthetase and glutathione synthetase(15). The threshold of the assay was 70 pmol of GSH per hour. The level of protein was determined by the method of Bradford (Bio-Rad, Mississauga, Ontario, Canada).

Statistics. Results are expressed as mean ± SEM. Nonparametric data on viability were compared by one-tailed Fisher's exact test, whereas parametric biologic data were compared by analysis of variance. All comparisons were performed orthogonally and homoscedasticity was tested by Bartlett. The level of significance was set at p < 0.05. Tissue glutathione was compared using a 2 × 3 factorial analysis of oxidative stress over the source of feeding regimen (normoxia, hyperoxia x enteral, i.v. dextrose, TPN). For each feeding regimen, correlations were sought between tissue glutathione levels and enzyme activities.

RESULTS

Study population. Of the 61 animal preparations 3 enterally fed pups were excluded (2 in oxygen and 1 in air) as they died within 24 h of surgery. Twenty pups were nursed by lactating dams, 17 pups were fed i.v. with dextrose 5%, and 21 pups received TPN. Of the 58 animals included in the study, 25 were randomized to be placed in oxygen. The description of the study population is presented in Table 1. The initial body weight of the animals in the enteral group was higher than those in the i.v. 5% dextrose group [F(1,52) = 5.8, p < 0.05]. There was no statistically significant difference in initial body weight between animals exposed or not exposed to hyperoxia [F(1,52) = 0.3]. The animals receiving TPN had no weight loss as opposed to the other two groups (without TPN), leading to a significant difference in change in body weight [F(1,44) = 7.1, p < 0.02] between those receiving TPN or not. Therefore, animals were separated between those with or without TPN.

Table 1 Description of the study population
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Without TPN, the overall death rate after 3 days of study was 1/23 for the animals placed in room air compared with 6/14 for those placed in oxygen (p = 0.024). The relative lung weight was significantly increased [F(1,28) = 17.0, p < 0.001] in the animals cared for in the oxygen enriched environment (Table 1).

With TPN, the pups were protected against the lethal effects of hyperoxia, because the death rate was 0/11 for the animals placed in oxygen. The relative lung weight was not significantly modified [F(1,18) = 0.05] in the animals cared for in oxygen when compared with room air (Table 1).

Glutathione. In lungs, hyperoxia induced overall a significant increase in total glutathione concentration (Fig. 1) normalized for lung protein content [F(1,44) = 13.6, p < 0.002]. The possible artifact caused by the lower lung protein content in animals cared for in hyperoxia [90.6 ± 4.4 versus 108.8 ± 3.1 mg/g, F(1,44) = 12.6, p < 0.002] was dealt with by further analyzing the effect of hyperoxia on glutathione expressed per unit of organ weight. The effect was found to be similar [F(1,44) = 14.5, p < 0.001]. Without TPN hyperoxia induced a 33% increase in total glutathione, and with TPN a 67% increase. Oxidized glutathione (% of total glutathione) was measured in a limited number of subjects in the enteral group [normoxia 5 ± 1%, n = 7 versus hyperoxia: 8 ± 2%, n = 4; F(1,10) = 4.41, NS]. The feeding regimens had no detectable effect on glutathione [F(2,44) = 0.7].

Figure 1
figure 1

Effect of hyperoxia and nutritional support on tissue glutathione. Newborn guinea pig pups fed either by mothers (enteral) or i.v. (i.v. dextrose or TPN) were randomly assigned to room air (air) or a fraction of inspired oxygen of more than 0.95 (oxygen). After 3 days, the surviving animals were killed and lung as well as liver total glutathione contents (as GSH equivalents) were compared by analysis of variance. In the lungs, hyperoxia was associated with a significantly (**p < 0.002) higher GSH content compared with room air, all regimens confounded. In the liver, hyperoxia had no detectable effect on GSH content, but the TPN regimen was associated with a significantly higher GSH level compared with regimens without TPN. Bars represent mean ± SEM. Sample sizes (n) are shown for each group of animals.

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In liver, hyperoxia had overall no detectable effect [F(1,44) = 0.3] on total glutathione concentration (Fig. 1). However, with TPN, glutathione was significantly higher compared with feeding regimens without TPN, when glutathione is normalized for liver protein content [F(1,44) = 27.3, p < 0.001]. The possible artifact caused by the lower liver protein content in animals receiving TPN [112.8 ± 3.4 versus 133.9 ± 2.9 mg/g tissue, F(1,44) = 11.3, p < 0.002] was dealt with by further analyzing the effect of feeding regimen on glutathione expressed per unit of organ weight. The effect was found to be similar [F(1,44) = 22.5, p < 0.001].

Glutathione-related enzymes. The activities of the enzymes involved in maintaining intracellular glutathione levels in the lungs and the liver are presented in Table 2 for each treatment group. Because no effect of feeding regimens was observed on lung glutathione, the effects of normoxia and hyperoxia on activities of peroxidase, reductase, γ-GT, and synthesis are presented in Figure 2 for all feeding regimens combined. Hyperoxia induced a significant [F(1,44) = 5.3, p < 0.05] rise of 32%, only in synthetic activity. Synthesis was significantly correlated with total lung glutathione, overall (r2 = 0.35, p < 0.01) as well as for each feeding regimen (enteral: r2 = 0.47, p < 0.01; i.v. dextrose: r2 = 0.46, p < 0.05; TPN: r2 = 0.36, p < 0.01).

Table 2 Activities of glutathione related enzymes
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Figure 2
figure 2

Effect of hyperoxia on the activities of glutathione-related enzymes in lung tissue. Because no effect of feeding regimens was observed on lung glutathione, the effects of normoxia (air) and hyperoxia (oxygen) on activities of peroxidase, reductase, γ-GT, and synthesis are shown for all feeding regimens combined. Hyperoxia induced a significantly (*p < 0.05) higher synthetic activity. Bars represent mean ± SEM. Sample sizes are 31 for air and 19 for oxygen.

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Because no difference between normoxia and hyperoxia was observed on liver glutathione, the effects of feeding regimens (with and without TPN) on activities of peroxidase, reductase, γ-GT, and synthesis are presented in Figure 3 for all concentrations of oxygen combined (21 and 95%). The TPN regimen had a significant effect on glutathione synthesis [+24%, F(1,44) = 5.2, p < 0.05], GSSG-R [+10%, F(1,44) = 5.0, p < 0.05] and γ-GT [-18%, F(1,44) = 5.2, p < 0.05]. Within the feeding regimens there was no correlation between enzymatic activities and glutathione content.

Figure 3
figure 3

Effect of the nutritional support on the activities of glutathione-related enzymes in liver. Because no difference between normoxia and hyperoxia was observed on liver glutathione, the effects of feeding regimens (with TPN, without TPN) on activities of peroxidase, reductase, γ-GT, and synthesis are shown for both concentrations of oxygen combined (21 and 95%). TPN was associated with a significantly (*p < 0.05) higher activity of glutathione reductase and synthesis, although γ-GT activity was significantly lower. Bars represent mean ± SEM. Sample sizes are 30 for those without TPN and 20 for the TPN regimen.

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Alternate source of glutathione substrate. In a separate group of animals (weight 108 ± 5 g, n = 4) cared for in oxygen under the same experimental conditions as the study population, and receiving i.v. 5% dextrose + 0.45% NaCl supplemented only with 20 mM OTC (stable cysteine precursor), glutathione concentrations were measured in lung (38.5 ± 5.1 nmol/mg protein) and liver (72.5 ± 11.1 nmol/mg protein).

DISCUSSION

In room air, the three feeding regimens had no effect on lung glutathione status, whereas in the liver only TPN induced an increase in glutathione. This observation combined with the 10% loss in body weight in animals without TPN (Table 1) underlines that these animals were not nourished optimally. The nutritional status plays an important role in maintaining glutathione homeostasis, as protein malnutrition exacerbates glutathione deficiency(6). Consequently, after comparing between enterally and dextrose-fed animals, results were analyzed by comparing with or without TPN. The postoperative state accounts for the poor weight gain in the enterally fed animals kept in room air(22); in contrast to the i.v.-fed pups, and although they are precocious and able to feed on their own, these animals had no nutrient intake until they were able to resume feeding after surgery. Furthermore, the oxygen-induced lung disease might have kept dyspneic pups from suckling. Therefore, under the present experimental conditions, enteral feeding was associated with an undernourished state similarly to the dextrose group that did not receive amino acids and multivitamins. Only in the i.v. dextrose group was the hepatic activity of γ-GT increased (Table 2), as observed in fasted guinea pigs(25).

In response to oxygen exposure, pulmonary glutathione levels rose by 33% in the groups without TPN. This confirms the findings of others, showing that neonatal guinea pigs can maintain tissue glutathione status during periods of nutrient stress(28). Depleting pulmonary glutathione accelerates the development of oxygen toxicity in neonatal animal models(4,5). However, the data from this animal study show that the lethal effect of the hyperoxic environment was not caused by the depletion of lung total glutathione. Among the surviving guinea pig pups kept in oxygen, two animals receiving i.v. dextrose were killed in a premortal state. Their glutathione concentrations were not outside the range of values found in other animals that survived in oxygen. This suggests that the glutathione levels of the deceased animals were not different from those analyzed. The concentrations of glutathione found in our study are in the same range as those reported by others(5).

Although the accumulation of oxidized glutathione is proposed as a marker of oxidant-induced lung injury(10), we found in a limited number of enterally fed animals, that the level of oxidized glutathione in the lungs (<10%) could not account for the oxygen-induced glutathione stimulation (>30%). This confirms observations in rats showing that glutathione stimulation was primarily due to an increase in reduced glutathione(9). An oxidant environment stimulates the synthesis of γ-glutamylcysteine synthetase, the rate-limiting enzyme responsible for the synthesis of glutathione(29). The higher glutathione synthetic activity measured in lungs from oxygen-exposed animals enhanced pulmonary glutathione production. This is supported by the fact that only the synthetic activity correlated with glutathione levels in the lungs.

Despite an increased lung glutathione content, the animals without TPN cared for in the oxygen-enriched environment developed some characteristics of hyperoxic lung injury. Indeed, the higher mortality rate was associated with an increased relative lung weight(30). In contrast, this was not observed in animals receiving parenteral nutrition containing precursors of glutathione. Because liver is the main site of glutathione production(13) that is delivered to the lungs via the circulation, the 67% increase in lungs observed with TPN suggests that the pulmonary demand for glutathione was met during hyperoxia by a greater liver production (Fig. 1). The beneficial effect of TPN might be explained by the provision of amino acids as substrates for glutathione metabolism as documented by the glutathione levels observed in hyperoxic animals receiving i.v. 5% dextrose supplemented with OTC, a stable cysteine precursor for glutathione production.

Because no correlation was found in liver between enzyme activities and glutathione levels, the higher liver glutathione content induced by TPN could be related to substrate availability as this regimen contained methionine, a precursor of cysteine, a rate-limiting step in glutathione production(1). Indeed, the enzymatic activity required to transform methionine into cysteine is present in guinea pigs(31), and the results of our study suggest that it is functional in newborn pups as glutathione rose by 67% despite the absence of cysteine in the infused amino acid solution.

A better nutritional status could lead to an anabolic state and account for the effect of TPN on the statistical differences observed in the liver glutathione-related enzymes (Fig. 3). However, the absence of correlation with glutathione suggests that the effect of the regimens on enzyme activities was a statistical finding with little biologic significance. The 40% higher relative liver weight of animals on TPN was associated with a significant drop in protein content. The absolute amount of protein by organ was not modified suggesting a dilutional effect caused by some form of hepatic storage, a recognized complication of TPN.

The pivotal findings of this study are first, that oxygen toxicity was not related with a decrease in glutathione concentrations; and second as underlined by Meister(1), the ability to maintain an adequate level of GSH in the face of an oxidant stimulus is more important than the initial quantity of GSH. Newborn and particularly premature infants are subjected to sources of oxidative stress associated with therapeutic strategies such as oxygen supplementation and TPN(32). Combined with an immature antioxidant activity, this accounts for a significant source of morbidity(33). As newborn human infants have an immature transsulfuration pathway, this may limit cysteine availability for glutathione synthesis. Indeed, in premature neonates we found a low level of total glutathione in cells from tracheal aspirates(14), confirming similar findings in bronchoalveolar fluid(24). Therefore, under hyperoxic conditions early nutritional support could be of vital importance to support an optimal glutathione production. However, before extrapolating observations from the newborn guinea pig model to humans, it is important to keep in mind that, in the days after birth, conditions inducing changes in lung and liver glutathione might have interspecies differences.