![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
The Journal of Nutrition Vol. 128 No. 4 April 1998,
pp. 671-676
Department of Human Nutrition, The Ohio State University, Columbus, OH 43210-1295 and * Department of Nutritional Sciences, University of Guelph, Guelph, ON, Canada, N1G 2W1
 |
ABSTRACT |
|---|
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
It has become recognized that enhancing the antioxidant defense system during the early phase of rehabilitation is important to the survival of wasting protein-energy malnourished (PEM) patients. In this study, we compared the efficacy of dietary protein replenishment and supplementation with L-2-oxothiazolidine-4-carboxylate (OTC, 3.5 mg/d), a cysteine precursor, to protect against hyperoxia-induced lung damage in PEM rats. The PEM rats were produced by feeding weanling rats a protein-deficient diet (0.5% protein) for 14 d. PEM rats were then divided in three dietary treatment groups, 0.5% protein (
Pr), 0.5% protein plus the OTC supplement (+OTC), or 15% protein (+Pr) during 4 d of either hyperoxia (85% O2) or air exposure. Increased lung-to-body weight ratios, indicative of oxidative tissue damage, were observed following exposure to hyperoxia in Pr and +Pr rats, but not in +OTC rats, even though the OTC supplement and the 15% protein diet contained a comparable amount of cysteine. Tissue reduced glutathione (GSH) status, GSH-dependent enzyme activity and antioxidant defense enzyme activities were monitored in the lung, liver and blood during 4 d of hyperoxia exposure. OTC supplementation enhanced GSH levels significantly in the lung of PEM rats, whereas protein repletion significantly elevated blood GSH concentrations. The protective effect of OTC was not a function of changes in activity of GSH-dependent enzymes or oxygen defense enzymes in the lung. These results indicate that a short-term strategy that selectively elevates GSH levels in the lung is more effective than protein repletion in protecting against hyperoxia-induced oxidative lung damage in PEM rats.
Protein-energy malnutrition (PEM)6 is a wasting malnutrition that manifests itself in various clinical forms and affects millions of people worldwide. In children, kwashiorkor and marasmus are the two most recognized and devastating forms of PEM. In adults, PEM may occur secondarily to many diseases including cancer, AIDS and chronic digestive disorders (Bistrian et al. 1974 In addition to an impaired antioxidant defense system, free radical production is often increased in PEM patients. For example, PEM patients are susceptible to opportunistic lung infections that often require oxygen or drug therapy (Bray and Taylor 1994 GSH, a thiol-containing tripeptide that acts as a substrate for GSH-Px and GSH-Tr, plays a central role in cellular defense against free radicals and xenobiotic compounds (Meister and Anderson 1983 It has become recognized that enhancing the antioxidant defense system during the early phase of rehabilitation is important to the survival of wasted PEM patients. However, nutritional support for growth and development is also of paramount importance in PEM patients, especially in premature infants and children with inadequate nutrient stores (Solomons 1985 Animals and diets.
Male Wistar weanling rats (Charles River Canada, St. Constant, Canada) with initial weights of 55-65 g were housed individually in suspended stainless steel mesh cages in a temperature- and humidity-controlled room with light from 0800 to 2000 h. Animals were fed a casein-based purified diet (Table 1) containing 0.5% protein for 14 d to produce wasting malnutrition (Taylor et al. 1992
Assessment of lung damage due to hyperoxia exposure.
In the first experiment, the effect of OTC supplementation and protein repletion on oxygen toxicity was studied. Rats from each of the three diet treatment groups were exposed to either hyperoxia (85% oxygen) or a normal oxygen atmosphere (air) for 4 d, n = 6 rats for each treatment. Hyperoxia was produced by mixing compressed air and oxygen with a Bird 3800 Microblender (Bird Product, Palm Spring, CA) in a Plexiglas chamber with a flow rate of 8-10 chamber changes/h. The oxygen concentration was monitored continuously using a Servomex Oxygen Analyzer OA570 (Servomex, Crowborough, Sussex, UK). The chamber was opened once daily to maintain feeding regimens and administer OTC. After the third day of hyperoxia, the lung was removed and weighed immediately after CO2 intoxication. To assess the severity of hyperoxia-induced lung damage, lung-to-body weight ratios were measured. The lung-to-body weight ratio in rats remains constant during normal growth and development; lung damage detected by changes in the ratio of lung to body weight has been described and corroborated with damage detected by MRI and histopathologic analysis (Taylor et al. 1992 Assessment of GSH status and antioxidant enzymes.
In the second experiment, the roles of GSH status, GSH-dependent enzyme activity and antioxidant defense enzyme activity in the development of lung damage during hyperoxia exposure were assessed. After a 14-d feeding period of 0.5% protein, PEM rats were placed into one of the three dietary treatment groups as described in Experiment 1. Rats in all groups were exposed to 85% oxygen. OTC supplementation, protein repletion and hyperoxia exposure started at the beginning of the dark period to accommodate the nocturnal nature of the rats. Rats were killed by CO2 intoxication at the beginning of the light period of d 0, 2 and 4. Lung and liver were collected and frozen immediately in liquid N2 and stored at Measurement of GSH and GSSG.
Total GSH and oxidized glutathione (GSSG) were measured by the method of Tietze (1969) Evaluation of enzymatic defense systems.
To determine enzyme activities, lung or liver tissue was homogenized in ice-cold 0.01 mol/L phosphate, 0.15 mol/L KCl buffer for 10 s (Ultra-Turrax disperser with stainless steel shaft, Terochem Laboratories, Mississauga, Canada). The homogenate was centrifuged for 1 h at 100,000 × g at 4°C. The supernatant was used as the source of the cytosolic enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (EC 1.11.1.6), GSH-Px (EC 1.11.1.9) and GSH-Tr (EC 2.5.1.18). SOD activity was determined by the method of Crapo et al. (1978) Statistical analysis.
Data were analyzed by the General Linear Models procedure of SAS (SAS Institute 1985) to determine significant (P < 0.05) main effects due to atmosphere, diet or day of hyperoxia exposure and their interactions. When the probability of obtaining a larger P-value was <0.05, the differences were considered significant. Values were expressed as means ± SEM of at least 6 rats in each treatment.
A model of PEM was previously established by feeding weanling rats a diet containing 0.5% protein for 14 d (Taylor et al. 1992
This study reveals that a strategy that selectively elevates GSH in the lung is more effective than repletion of protein in protecting the lung against hyperoxia-induced oxidative tissue damage. Nutritional rehabilitation with the 15% protein diet, significantly elevated blood GSH concentrations (Fig. 4) but failed to elevate lung GSH levels (Fig. 2) or protect the lung against oxygen toxicity in wasting rats (Fig. 1). However, short-term OTC supplementation without nutritional rehabilitation successfully elevated GSH concentrations in the lung (Fig. 2), the target organ of oxygen toxicity, and protected it against oxidative injury during hyperoxia in PEM rats (Fig. 1). The protective effect of OTC was not a function of changes in activity of GSH-dependent enzymes or oxygen defense enzymes in the lung. GSH-Px and GSH-Tr activity generally were equal and unchanged in the three diet groups throughout the hyperoxia period (Fig. 5). Although lung SOD and catalase activity increased in response to hyperoxia exposure, the increase was similar among all treatment groups. Thus, the protective effect of OTC supplementation may have been due to its ability to elevate GSH concentrations in the organ in which the oxidative stress occurred.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
and 1976, Woodward and Filteau 1990). It has been suggested that many of the clinical and pathological manifestations of PEM result from an imbalance between free radical defenses and free radical production (Golden 1995, Ramdath and Golden 1993). Often the free radical defense system appears to be depressed in PEM patients. For example, low circulating levels of antioxidant nutrients such as vitamin E, vitamin C and 
-carotene have been found in children with kwashiorkor (Golden and Ramdath 1987). Alterations in the activity of free radical defense enzymes such as reduced glutathione (GSH) peroxidase (GSH-Px) and GSH transferases (GSH-Tr) have also been observed in PEM patients (Golden and Ramdath 1987).
, Tupasi et al. 1990). Unfortunately, both the immune response and the necessary treatment to infection result in increased free radical production and therefore an increased potential for free radical-mediated tissue damage. Thus enhancing the antioxidant defense as well as the detoxification systems during the early phase of rehabilitation may be crucial to the survival of the most debilitated PEM patients.
). GSH depletion, which often occurs in the blood and tissue of malnourished patients, has been associated with an increased toxicity of drug and oxygen therapy (Meister and Anderson 1983), an effect that is reduced by enhanced tissue GSH levels (Stevens and Anders 1981, Taylor et al. 1992). Although various approaches have been used to increase tissue GSH concentrations, the results are often negative. For example, attempts to elevate tissue GSH levels through supplementation of GSH itself have not proven effective (Bray and Taylor 1994). Dietary supplementation with cysteine, the limiting amino acid of GSH synthesis, has some limitations because it can be rapidly oxidized and is toxic at high levels (Harper et al. 1970, Olney et al. 1971). In addition, the concentration of intracellular cysteine is tightly controlled, i.e., a rapid increase in dietary cysteine induces the activity of cysteine dioxygenase, the first enzyme in cysteine degradation (Bauman et al. 1988, Kohashi et al. 1978). However, a novel strategy of using L-2-oxothiazolidine-4-carboxylate (OTC) has proven effective in elevating tissue GSH levels (Hazelton et al. 1986, Rose 1984) and reducing oxidative lung damage (Taylor et al. 1992). OTC has a low toxicity and is readily transported into the cell where it is slowly converted to cysteine by 5-oxoprolinase, an enzyme of the GSH synthesis cycle (Rose 1984). It therefore acts as an effective cysteine delivery system for sustainable tissue GSH synthesis.
). In this study, we compared the effectiveness of dietary protein replenishment and OTC supplementation to protect against hyperoxia-induced lung damage in PEM rats. We hypothesize that if each treatment contains a comparable amount of cysteine or cysteine precursors for GSH synthesis, repletion with dietary protein will be as effective as OTC supplementation in protecting against oxygen toxicity. It is also proposed that dietary protein will provide amino acids and energy to facilitate GSH synthesis during rehabilitation. Thus the specific objectives of this study are as follows: 1) to compare the protective effects of dietary protein repletion and oral supplementation of OTC against hyperoxia induced lung injury, and 2) to evaluate whether tissue GSH status, GSH-dependent enzyme activity or antioxidant enzyme activity play a role in the protection against hyperoxia-induced lung injury in PEM rats.
METHODS AND MATERIALS
Abstract
Introduction
Methods
Results
Discussion
References
). On d 15, rats were divided into three groups and fed one of three diets during the 4 d of exposure to either 85% oxygen or air. The first group, the nonsupplemented rats (Pr), continued to consume the 0.5% protein diet. The second group, the OTC supplemented group (+OTC), also continued to consume the 0.5% protein diet but were given a daily oral supplement of OTC (3.5 mg in 0.25 mL water). The dose of OTC was chosen such that rats would receive an amount equivalent to the amount of sulfur amino acid available to rats fed a 15% protein diet. The third group of rats, the 15% protein-repletion group (+Pr) were switched from the 0.5% protein diet to a casein-based diet containing 15% protein (Table 1). All experiments and procedures were reviewed and approved by the Animal Care Committee of the University of Guelph.
View this table:
Table 1.
Diet composition
and 1997).
80°C until analyzed. Trunk blood was collected in heparinized tubes and 50 µL was immediately fractionated into 1 mL 5% tricholroacetic acid (TCA), centrifuged and the supernatant fraction stored frozen at 80°C until analysis for total GSH.
. Tissue was homogenized in 5% TCA for 10 s and centrifuged at 10,000 × g for 15 min at 4°C. The supernatant was mixed with 0.6 mmol/L 5,5
-dithiobis-(2-nitrobenzoic acid) (DTNB) and GSH reductase. The formation of a GSH-DTNB conjugate was then measured at 412 nm. GSSG was similarly measured, except that the sample was first incubated for 20 min in 0.1 mol/L phosphate buffer containing 10 mmol/L N-ethylmaleimide (NEM). The NEM forms a complex with GSH, which is then removed by column chromatography (SEP-PAK C18 Column, Waters, Framingham, MA). The remaining GSSG was incubated with GSH reductase and DTNB and the subsequent product was assayed at 412 nm.
. Inhibition of cytochrome C reduction by superoxide produced by the reaction of xanthine-xanthine oxidase was measured at 418 nm. Catalase was determined by the method of Aebi (1983), which measures the decomposition of H2O2 at 240 nm. GSH-Px activity was determined by the method of Paglia and Valentine (1967) as modified by Prohaska and Gutsch (1983). tert-Butyl hydroperoxide was used as the substrate during the GSH-Px-catalyzed oxidation of GSH to GSSG. GSSG is converted to GSH by GSH reductase, consuming NADPH. GSH-Px activity was therefore determined by measuring the decrease in absorbance of NADPH at 314 nm and expressed as µmol NADPH oxidized/(min·mg protein). GSH-Tr activity was determined by the method of Baars et al. (1978), which measures the formation of a GSH-1-chloro-2,4-dinitrobenzene (CDNB) conjugate at 340 nm. The reaction mixture (1.5 mL volume) contained 1 mmol/L GSH, 0.5 mmol/L CDNB, and 10-20 µg cytosolic protein in 0.01 mol/L phosphate buffer with 0.1 mmol/L EDTA at pH 6.5.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
). In this experiment, the appetite of rats fed 0.5% protein was suppressed as indicated by an ~50% reduction in total food intake for 2 wk (114.2 ± 2.4 g of food ingested) compared with previous data of rats fed an adequate protein diet (213.7 ± 4.8 g of food ingested) (Taylor et al. 1992). Rats fed the 0.5% protein diet lost 27% (15.3 ± 0.5 g) of their original body weight during the 2-wk feeding period.
Pr (control) rats by 56%. Similarly, 4 d of repletion with the 15% protein diet was not effective in preventing hyperoxia-induced lung damage in PEM rats. The lung/body weight ratio of this group was significantly greater than that of air-exposed rats fed the same diet. However, no difference in lung/body weight ratio was observed in the +OTC groups exposed to hyperoxia or air.
View larger version (12K):
[in a new window]
Fig 1.
Effect of L-2-oxothiazolidine-4-carboxylate (OTC) supplementation or protein repletion on the lung-to-body weight ratio of PEM rats after 4 d of hyperoxia exposure. Values are expressed as means ± SEM, n = 6. Values marked with an asterisk are significantly different (P < 0.05) from their respective control group exposed to air.
Pr and +Pr groups had significantly increased GSSG/GSH ratios at d 2 and 4. Overall, the GSH status in the lung of the Pr group and the +Pr group was similar during the 4 d of hyperoxia exposure. An elevated GSH concentration and constant GSSG/GSH ratio in the lung of OTC supplemented rats correlated with a low lung/body weight ratio, indicative of little or no lung damage after hyperoxia.
View larger version (30K):
[in a new window]
Fig 2.
Effect of L-2-oxothiazolidine-4-carboxylate (OTC) supplementation or protein repletion on lung reduced glutathione (GSH), oxidized glutathione (GSSG) and the ratio of GSSG/GSH in PEM rats during 4 d of hyperoxia exposure. Values are expressed as means ± SEM, n = 6. Values not sharing a letter are significantly different (P < 0.05).
Pr and +Pr rats. Repletion with the 15% protein diet caused only a slight increase in GSH level on d 2, with a return to the control value by d 4. Liver GSSG levels remained relatively low and constant regardless of dietary treatment or hyperoxia exposure. The pattern of the ratio of GSSG/GSH in the liver in response to hyperoxia exposure was also similar to that observed in the lung. However, the ratios were much lower in all groups; for example, when lung damage was observed after hyperoxia exposure in Pr and +Pr rats, the ratio of GSSG/GSH in the liver was 1.25 and 1.35%, respectively, compared with ~10 and 14% in the lung.
View larger version (23K):
[in a new window]
Fig 3.
Effect of L-2-oxothiazolidine-4-carboxylate (OTC) supplementation or protein repletion on liver reduced glutathione (GSH), oxidized glutathione (GSSG) and the ratio of GSSG/GSH in PEM rats during 4 d of hyperoxia exposure. Values are expressed as means ± SEM, n = 6. Values not sharing a letter are significantly different (P < 0.05).
View larger version (34K):
[in a new window]
Fig 4.
Effect of L-2-oxothiazolidine-4-carboxylate (OTC) supplementation or protein repletion on blood reduced glutathione (GSH), oxidized glutathione (GSSG) and the ratio of GSSG/GSH in PEM rats during 4 d of hyperoxia exposure. Values are expressed as means ± SEM, n = 6. Values not sharing a letter are significantly different (P < 0.05).
View larger version (45K):
[in a new window]
Fig 5.
Effect of L-2-oxothiazolidine-4-carboxylate (OTC) supplementation or protein repletion on lung superoxide dismutase, catalase, reduced glutathione (GSH) peroxidase and GSH transferase activity in PEM rats during 4 d of hyperoxia exposure. Values are expressed as means ± SEM, n = 6. Values not sharing a letter are significantly different (P < 0.05).
View larger version (45K):
[in a new window]
Fig 6.
Effect of L-2-oxothiazolidine-4-carboxylate (OTC) supplementation or protein repletion on liver superoxide dismutase, catalase, reduced glutathione (GSH) peroxidase and GSH transferase activity in PEM rats during 4 d of hyperoxia exposure. Values are expressed as means ± SEM, n = 6. Values not sharing a letter are significantly different (P < 0.05).
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). Thus OTC bypassed the erythrocytes and was shunted directly to lung and liver for GSH synthesis.
Pr and +Pr rats, in which lung damage was observed, but remained relatively constant in the +OTC rats in which lung damage was not observed. In liver, hyperoxia caused very little fluctuation in GSH, GSSG or GSSG/GSH in Pr or +Pr rats. Interestingly, blood GSH levels were influenced mainly by nutritional status and did not always reflect changes of GSH status in other tissues. However, circulating GSSG concentrations were increased after prolonged exposure to hyperoxia in all treatment groups. Unfortunately, blood GSSG/GSH ratios showed inconsistent patterns among the different treatment groups. Taken together, these results indicate that the ratio of GSSG/GSH is a good indicator of oxidative damage in tissues directly exposed to oxidative stress, but is much less reliable in other tissues. Therefore, although the concentration of blood GSH and GSSG is a convenient measurement for human studies, judicious interpretation is required when it is used as a marker for total antioxidant status or oxidative stress.
). Treatment strategies that counteract the effects of free radical-mediated tissue damage may be very beneficial. This may be particularly relevant to PEM patients who may be exposed to additional oxidative stress during rehabilitation. GSH is derived from protein; as a key component of cellular defense systems that protect against xenobiotics and free radicals, it may be particularly critical during the rehabilitation of PEM patients. In this study, we have demonstrated in an animal model of PEM that GSH levels can be rapidly elevated and maintained in a target organ through OTC supplementation, a procedure that may prove very effective in aiding the recovery of PEM patients. Although nutritional rehabilitation with protein repletion failed to protect against hyperoxia-induced lung damage in PEM rats, it did provide insight into tissue priority for GSH synthesis. In the future, a short-term therapy that selectively elevates antioxidant status in target organ combined with a long-term treatment of nutritional support may significantly increase the chances of survival and recovery in wasting PEM patients.
| |
FOOTNOTES |
|---|
-dithiobis-(2-nitrobenzoic acid); GSH, reduced glutathione; GSH-Px, glutathione peroxidase; GSH-Tr, glutathione transferase; GSSG, oxidized glutathione; NEN, N-ethylmaleimide; OTC, L-2-oxothiazolidine-4-carboxylate; +OTC, L-2-oxothiazolidine-4-carboxylate supplemented group; PEM, protein-energy malnutrition; +Pr, 15% protein repletion group; Pr, nonsupplemented group; SOD, superoxide dismutase; TCA, trichloroacetic acid.
Manuscript received 18 April 1997. Initial reviews completed 18 August 1997. Revision accepted 8 December 1997.
| |
LITERATURE CITED |
|---|
|
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
selective alteration of glutathione metabolism.
Nutr. Rev.
1984;
42:397-410
[Medline]
This article has been cited by other articles:
|
|
 |
|
  P. J. Fraker, L. E. King, T. Laakko, and T. L. Vollmer The Dynamic Link between the Integrity of the Immune System and Zinc Status J. Nutr., May 1, 2000; 130(5): 1399S - 1406. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
