5-Aminolevulinic acid-induced alterations of oxidative metabolism in sedentary and exercise-trained rats B. PEREIRA,
R. CURI,
E. KOKUBUN,
AND
E. J. H. BECHARA
Department of Biochemistry, Institute de Q&mica, Universidude de Sa”o Pa&, 01498 Sao Puulo; Department of Physiology and Biophysics, Institute de Citkcias Biome’dicus, Universidude de S&o Pa&, 05508 Sao Pa&; and Department of Physical Education, Institute de Biocieizcias, Universidade Estudual Puulista, 13500 Rio Clara, Brazil R. CURI, E. KUKWWN, AND E. J. H. BECHARA. acid-induced alterations of oxidatiue ‘metabolism in sedentary and exercise-trained rats. J. Appl. Physiol. 72( 1): 226-230, l992.-5Aminolevulinic acid (ALA), a heme precursor that accumulates in acute intermittent porphyria patients and lead-exposed individuals, has previously been shown to autoxidize with generation of reactive oxygen species and to cause in vitro oxidative damage to rat liver mitochondria. We now demonstrate that chronically ALA-treated rats (40 mg/kg body wt every 2 days fur 15 days) exhibit decreased mitochondrial enzymatic activities (superoxide dismutase, citrate synthase) in liver and soleus (type I, red) and gastrocnemius (type IIb, white) muscle fibers. Previous adaptation of rats to endurance exercise, indicated by augmented (cytosolic) CuZn-superoxide dismutase (SOD) and (mitochondrial) Mn-SOD activities in several organs, does not protect the animals against liver and soleus mitochondrial damage promoted by intraperitoneal injections of ALA. This is suggested by loss of citrate synthase and Mn-SOD activities and elevation of serum lactate levels, concomitant to decreased glycogen content in soleus and the red portion of gastrocnemius (type IIa) fibers of both sedentary and swimming-trained ALA-treated rats. In parallel, the type IIb gastrocnemius fibers, which are known to obtain energy mainly by glycolysis, do not undergo these biochemical changes. Consistently, ALA-treated rats under swimming training reach fatigue significantly earlier than the control group. These results indicate that ALA may be an important prooxidant in vivo. PEREIRA,
B.,
5-Amideuulinic
porphyria; lead poisoning; reactive oxygen species; physical exercise; mitochondrial damage &AMINOLEVULINIC ACID (ALA), a heme precursor accumulated in chemical (e.g., plumbism) and inborn [e.g., acute intermittent porphyria- (AIP)] porphyrias, rapidly undergoes enolization and subsequently aerobic oxidation at pH levels above 7.0 (22-24). Superoxide species and carbon-centered radicals were proved to be intermediates in the chain reaction of ALA oxidation, and ammonium ions, hydrogen peroxide, and hydroxyl radicals were proved to be its products (Fig. 1). These properties make ALA a possible endogenous prooxidant. On addition of hemoglobin, myoglobin, and EDTA- or ADP-iron complexes, the oxygen-consuming reaction of ALA is accelerated (21,24). Analogous behavior, i.e., aerobic oxidation with generation of reactive oxygen species (ROS), has been demonstrated for a number of endogenous enolizable and hydroquinone derivative metabo226
0161-7567/92
$2.00
lites, such as dihydroxyacetone (18) and other carbohydrates (26), homogentisic acid (2), coenzyme Q (3), and 6-hydroxydopamine (1,7). In all cases, the autoxidationgenerated ROS have been implicated in the clinical manifestations of various diseases. Because 1) AIP patients (20), lead-exposed workers (22), and rats (28) exhibit elevated erythrocytic superoxide dismutase (SOD) activities, 2) histopathological studies of liver biopsy samples of AIP cases revealed extensive mitochondrial damage, such as fat, lipofucsin, and ferritin deposits, in addition to ultrastructural changes (4), 3) renal mitochondrial damage has been reported in cases of plumbism (6), and 4) incubation of rat liver mitochondrial preparations with succinate in the presence of ALA leads to the impairment of vital functions, such as respiratory rate, calcium fluxes, and membrane potential, which are abolished by ROS scavengers and ortho-phenanthroline, an iron chelator (15), we have suggested that ROS generated by autoxidization of ALA may underline the syndromes of both plumbism and AIP. Alternative biochemical mechanisms have been suggested by other authors on the basis of the deleterious effects of ROS in plumbism; stimulation by lead (and aluminum) of iron-dependent lipoperoxidation of membranes (25) is an example of these. The aim of this work is to examine whether intraperitoneal injection of ALA in rats would cause oxidative stress and promote mitochondrial injury. Inasmuch as physical exercise alters the biological redox balance, in which oxyradicals might have a role in mitochondrial lesions (ll), a possible synergistic effect of ALA treatment and exercise training was also investigated. These questions were approached by comparing the CuZn- and Mn-SOD activities in several tissues of sedentary and swimming-trained rats untreated or treated with ALA. In parallel, other parameters closely related to the aerobic (citrate synthase activity, free fatty acid serum level) and glycolytic (lactate serum level and liver and muscle glycogen content) metabolism, as well as the exercise performance under ALA treatment, were also monitored. MATERIALS
AND
METHODS
Reagents and equipment. Reagents of the purest quality available were used as purchased from commercial sources: bovine erythrocytic SOD, catalase, 2-amino-2methyl-1-propanol, 5,5’-dithiobis(2-nitrobenzoic acid), oxalacetic acid, acetyl CoA, Triton X-100, and 5-amino-
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Society
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5-ALA-PROMOTED 0
METABOLIC
CHANGES
IN RATS
227
OH 02
CC+-
+
/ +
=
NH2
ccy
complexes
NH2
0
0~ ,H202,H0
iron
FIG. 1. Chain reaction of 5-aminolevulinic acid oxidation.
0
NH4+
levulinic acid from Sigma Chemical; diethylenetriaminepentaacetic acid from Jensen; ethylenediaminetetraacetic acid and tris(hydroxymethyl)aminomethane (Tris) from Reagen. All other reagents were of analytic grade. All solutions were prepared with bidistilled deionized water obtained with Millipore (Milli Q) equipment. All spectrophotometric measurements were performed with a DMR-10 Zeiss apparatus. Animak Female Wistar albino rats (3-mo-old and +?50 g) were maintained in a room at 25OC under a 12:12-h light-dark cycle and fed a commercial diet ad libitum. Rats were killed by decapitation 24 h after the last exercise session without anesthesia. Blood was immediately collected and centrifuged at 2,000 g, and the separated plasma was kept at -2OOC for further assay of lactate and free fatty acids. Fragments of -250 mg of predominantly type IIb (white) and type IIa (red) fibers from the gastrocnemius muscle, type I soleus muscle (red) fibers, and two liver fragments (chopped to -500 mg) for measuring the glycogen content were also obtained. For hepatic and muscle glycogen determination, the tissues were digested for 30 min in a 30% KOH boiling solution, and extraction was performed in two steps of precipitation by 70% ethanol under heat. Glycogen was finally assayed by the anthrone method at low pH (16). Free fatty acids were calorimetrically determined according to Falholt (13) after extraction with chloroformheptane-methanol (28:21:1). Serum lactate was assayed as described by Engel and Jones (12). The extraction medium for SOD measurements contained 140 mM KC1 and 10 mM sodium phosphate at a final pH 7.0. The assay was performed on aliquots of the supernatant after centrifugation at 10,000 g for 30 min. SOD activities are expressed as units per gram of fresh weight and were measured as described by Marklund (17). The extraction medium for the measurement of citrate synthase activity contained 50 mM Tris-HCl and 1 mM EDTA, pH 7.4. The assays were performed as described by Cooney (8), and the activities were expressed as prnol. min-’ g-l fresh tissue wt. Endurance exercise training. A special swimmingtraining model described elsewhere (9,10) was used here. The rats were conditioned to swim for 90 min at 30°C daily, 5 days/wk for 2 mo, with an extra weight (5% of body wt) fixed on the tail. Sedentary group. As a control for the exercising group, groups of rats were housed in cages until the end of the training session of the experimental group. ALA treatment. A group of female Wistar rats was inl
jetted with ALA solution (40 mg/kg body wt ip) at a final pH adjusted to between 6 and 7 with sodium bicarbonate as described by McGillion (19). In this acute treatment, the treated rats were killed 3 h after injection, and the tissues were dissected for enzyme and glycogen determination. The control group received the same volume of saline as that of the ALA injection (0.5 ml). In the chronic treatment of rats with ALA (40 mg/kg body wt), sedentary rats received the ALA solution every 2 days for 15 days and were killed 24 h after the last injection. After 2 mo of swimming training, a group of exercised rats received the ALA solution after exercise sessions. The exercised rats were trained until the end of ALA treatment, every 2 days for 15 days, and then were killed 24 h after the last injection. The volume of ALA solution injected in the chronic treatment was the same as that injected for acute treatment. Statistical treatment. Two-way analysis of variance (ANOVA) with post hoc contrasts was used to compare the groups. The level of significance wa .s set at P < 0.01. RESULTS
The effect of acute and chronic ALA treatment on the aerobic meta .bolism o f swimming-train .ed and sedentary rats was evaluated. Under acute treatment, we did not observe any alteration in the metabolite parameters studied (Table 1). On the other hand, after chronic treatment (Table 1) we found elevated serum lactate (141%) and free fatty acid (33%) levels, whereas glycogen decreased in liver (50%), soleus muscle (83%), and the type IIa fibers of the gastrocnemius muscle (43%). Previous endurance swimming training of the animals did not reverse the ALA effects mentioned above (Table 1); that is, lactate increased 139% and glycogen decreased 81% in soleus muscle, 44% in the type IIa gastrocnemius fibers, and 27% in liver. No significant alteration was observed in the glycogen content of the type IIb gastrocnemius fibers of either sedentary or trained ALA-treated rats (Table 1). With regard to the serum fatty acid levels, there is a trend toward higher values in ALA-treated rats. The effect of ALA treatment on the CuZn-SOD and Mn-SOD activity of sedentary and swimming-trained rats is described in Tables 2 and 3. The results obtained for CuZn-SOD and Mn-SOD activities with the sedentary group chronically treated with ALA vs. the control (saline) group are described in Table 2. The CuZn-SOD activity (Table 2) increased in the ALA-treated rat groups in the following tissues: brain (61%), type IIa gas-
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228
5-ALA-PROMOTED
TABLE 1. Effect
of acute
and chronic treatment Acute
Blood lactate, mmol/l Serum FFA, meq/l Glycogen, mg/IOO mg Soleus (type I) Gastrocnemius Type IIa Type IIb Liver
METABOLIC
CHANGES
zuith ALA on metabolic parameters
Sedentary
Chronic
Chronic
Sedentary
Exercised
ALA
Saline
ALA
Saline
ALA
1.40t0.02 l.llt0.02
1.4ltO.02 0.91&0.05*
1.43kO.03 l.lkkO.02
3.46*0.07* 1.48*0.04*
1.49t0.02 1.02t0.05
3.56t0.16* 1.23t0.04*
0.41t0.02
0.40~0.01
0.44kO.02
0.24kO.02'
0.49t0.02
0.27*0.03*
0.40t0.02 0.44t0.02 5.86t0.06
0.48t0.01* 0.44t0.02 6.01tO.10*
0.43kO.02 0.50t0.03
0.3Ot0.02" 0.50t0.02 3.86?0,12*
0.49t0.02 0.49+0*03 5.96rtO.08
0.34t0.02* 0.47t0.02 4.66t0.13*
5.81t0.11
trocnemius fibers (57%), and liver (148%); decreased CuZn-SOD levels were observed in the (red) soleus fibers (184%). On the other hand, the Mn-SOD activity (Table 3) decreased in the type IIa gastrocnemius fibers (51%) and in the soleus muscle (78%), as well as in the liver (250%). When the groups of swimming-trained rats either untreated or treated with ALA were compared with regard to SOD activities (Tables 2 and 3), the same trends of CuZn-SOD and Mn-SOD alterations as those obtained with the sedentary group were observed. That is, the CuZn-SOD activity increased in brain and liver but decreased in the soleus muscle, whereas the Mn-SOD activity decreased in liver and the type IIa fibers of the gastrocnemius and soleus muscles. Citrate synthuse uctiuity. The activity of citrate synthase was measured in the soleus muscle of the rats to indicate the effect of the exercise training and ALA treatment on the aerobic metabolism. The sedentary and swimming-trained groups showed maximal enzyme activity of 25.4 t 6 and 49.0 t 15 pmol mine1 g fresh tissue-l, respectively. The soleus citrate synthase activity of ALA-treated swimming-trained rats (17.5 t 1.4 pm01 min-l g tissue-l, n = 10) decreased 45% (P < 0.01) relative to the sedentary group and 180% relative to the swimming-trained nontreated rats. The endurance of the rats in swimming (a mean value for 15 days of ALA treatment) before and after chronic treatment with ALA was found to be 90 t 12 and 40 t 6 min, respectively. l
l
l
DISCUSSION
The possible involvement of ALA as an endogenous source of reactive intermediates that may promote oxidative stress in plumbism and AIP patients is suggested by several lines of evidence. First, spectrophotometric TABLE 2. CuZn-SOD
of sedentary and exercised ruts
Saline
Values are means t SD for 10 rats in each group. See MATERIALS AND METHODS levulinic acid; FFA, free fatty acids. * P < 0.01 vs. saline-treated rats.
l
IN RATS
activity in tissues
of sedentary
for discussion of treatments and parameters. ALA, &amino-
and electron spin resonance techniques demonstrated rapid formation of oxygen- and carbon-centered radicals during ALA aerobic oxidation in slightly alkaline buffers (23). Second, incubation of succinate-sustained mitochondrial preparations with 5-15 mM ALA leads to acceleration of the state 4 respiration, release of intramitochondrial calcium ions, collapse of the membrane potential (15), and, at 50-100 PM ALA, calcium-dependent mitochondrial swelling (unpublished results). Third, several authors have reported extensive mitochondrial damage in liver biopsy samples of AIP patients (4), as well as kidney insufficiency in lead poisoning (6). Fourth, the neurological manifestations typical of both plumbism and AIP have been attributed to competition between ALA and y-aminobutyric acid for synaptic sites of nerve cells (5) but may also be linked to membrane lipoperoxidation (25). Finally, indication of oxidative stress in lead-exposed workers (22) and AIP patients (20) is suggested by the observed increased erythrocyte levels of the antioxidant enzymes SOD and glutathione peroxidase in these individuals. In one AIP patient, Gorschein (14) determined the ALA concentration in the serum as 8 PM, which is roughly one order of magnitude above the normal level. Inasmuch as studies of rats injected intraperitoneally with ALA (40 mg/kg body wt) revealed that ALA is distributed among and accumulated in several organs (X lo3 in liver, 3 X 10’ in heart, and X10 in brain after 3 h of injection) and that it slowly decays to the normal levels within 2-3 days (19), one could predict that either continuous overproduction of ALA in AIP and lead-poisoning patients or chronic ALA treatment of rats would expose their biomolecules and supramolecular cell structures to a potentially higher risk of damage by ALA-generated
and endurance-truined
ruts chronically
Sedentary
treated with ALA
Endurance
Trained
Tissues
Saline
ALA
Saline
ALA
Brain
12.28t0.76
19.83*0.45*
53.93t2.11
l&94* 1.59*
11.23H.46 55.6313.91
19.74*0.51*
Soleus muscle Gastrocnemius muscle Type Ifa Type IIb Liver
22.1621.36 11 l98t0.28 120.28t1.24
34.99t1.91* 11.95kO.40 299.30&6.46*
20.77+1.02*
28.18t1.29 9.55t0.82 117.96t1.91
Values are means (X10w3) k SD for 10 rats in each group. SOD, superoxide dismutase. *P < 0.01 vs. saline-treated
29.94k1.18 12.11st0.79” 213.56*4.0* rats.
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5-ALA-PROMOTED
3. Mn-SOD actiuity in tissues
TABLE
METABOLIC
of sedentary
CHANGES
and endurance-trained
229
IN RATS
rats chronically treated with ALA
Sedentary Tissues
Saline
Brain Soleus muscle Gastrocnemius muscle Type Ifa Type IIb Liver Values
are means
Endurance ALA
Saline
ALA
0.647tO.019
0.672tO.022
0.620t0.025
0.515t0.020
0.289+0.019*
0.96l-tO.039
0.336t0.012 0.142t0.028 3.764kO.lOO
0.223t0.01 0.115t0.009* 1.075t0.089”
0.525t0.02 0.110-+0.010 3.590~0.102
l*
(X10B3) t SD for 10 rats in each group, *P -C 0.01 vs. saline-treated
ROS and carbon-centered radicals. In this study the hypothesis that ALA might represent an endogenous prooxidant, an important consideration in elucidating the biochemical basis of the clinical manifestations of acquired and inherited porphyrias, is tested with ALAtreated sedentary and swimming-trained rats. The comparison between these two groups is justified by the evidence obtained by other authors (11) and ourselves (unpublished results) that endurance training triggers adaptations to an essentially aerobic muscle metabolism. In fact, we found that rats that have been swimming exercised for 2 mo present elevated Mn-SOD activity in the type I fiber of soleus muscle and type IIa fibers of gastrocnemius muscle concomitantly with twofold increased citrate synthase activity, compared with a control group (unpublished results). This result is consistent with the proliferation of mitochondria already described as a result of endurance training (11). That is, any difference on the deleterious effects of ALA at abnormally high concentrations on, for example, mitochondrial functions could eventually be better verified by comparing the sedentary and trained groups of rats submitted to ALA treatment. Table 1 shows that just one intraperitoneal injection of ALA in sedentary rats causes no significant differences in the rat metabolism, as indicated by measurement of the level of serum lactate and free fatty acids and that of liver and muscle (soleus and gastrocnemius) glycogen. However, chronic treatment of sedentary rats with ALA at the same dose (40 mg/kg body wt every 2 days for 15 days) that, according to McGillion (19), should maintain ALA at high concentration in the plasma and several organs, culminated with an apparent shift of an essentially aerobic to a more anaerobic metabolism (Table 1). This shift is suggested by significant increase of lactate and free fatty acids in the serum, which could be attributed to decreased consumption of fuels in oxidative tissues as a consequence of impairment of the mitochondrial functions, and the decrease of both hepatic and muscle glycogen. These sa.me effects were also observed with ALA-treated rats submitted to the endurance training: higher mobilization of glycogen and production of lactate (Table 1). In parallel, we also observed a loss of citrate synthase activity, and half of the swimming endurance (see RESULTS). This result is in line with observations in the literature (4, 6, 15) that in vitro and in vivo data point to mitochondria as main targets for ALA-generated ROS. The in vivo prooxidant properties of ALA are also suggested in this study by the observed increase of CuZn-
Trained
0.721t0.017* 0.296-tO.O49* 1
0.277,to.o19*
0.113rt0.008 1.559tO.087*
rats.
SOD in brain, muscle (type IIa fibers of gastrocnemius), and liver of untrained rats submitted to a chronic treatment with ALA (Table 2). This increase had also been observed with AIP patients and lead-poisoned workers by measurement of the erythrocyte SOD activity (20,22). The observed decrease of CuZn-SOD activity in the soleus muscle, richer in mitochondria than the type IIb and type IIa fibers of gastrocnemius muscle, may be linked to the expected augmented oxidative mitochondrial damage that may also affect other cytosolic enzymes and organelles. With regard to the swimming-trained rats chronically treated with ALA, one can also verify augmented CuZn-SOD levels in brain and liver and decreased levels in the soleus muscle (Table 2). Of striking interest, however, are the Mn-SOD data for both trained and sedentary rats chronically treated with ALA compared with the untreated control rats. Tables 2 and 3 show a dramatic loss of mitoehondrial SOD activity in the muscle and liver tissues of both trained and sedentary ALA-treated groups. It can also be verified that the higher mitochondrial SOD levels in red fibers of soleus and gastrocnemius muscles reached by swimming training (Table 3) were brought down to the same levels in both sedentary and trained rats by ALA treatment. That is, the ALA effect in lowering the Mn-SOD is more pronounced in trained rats than in the sedentary group. In conclusion, ALA treatment of rats results in elevated total SOD in several tissues (except red muscle fibers), which, like the previously reported augmented total SOD and glutathione peroxidase in the blood of AIP (20) and plumbism patients (22), is interpreted as a protective response against the deleterious oxidative effects of ROS-generated ALA. One can speculate that mitochondria are in fact main targets of autoxidizing ALA, as suggested by 1) the data described here for the mobilization of glycogen as major fuel and consequent increase of lactate in serum under ALA treatment, 2) loss of mitochondrial enzymatic activities especially in liver and red muscle fibers, such as those of citrate synthase and MnSOD, and finally, 3) 40% reduction in swimming endurance. These results are consistent with our previous in vitro studies on mitochondrial damage promoted by ALA (15). If, indeed, the heme precursor ALA, synthesized in the mitochondria and distributed and accumulated in several organs, represents a dangerous source of ROS in AIP and plumbism, it is important to recall that this role requires that ALA be first enolized to the autoxidizable form (Fig. 1). That is, enolic ALA cannot be stocked or circulated
among the organs without
causing damage. We hypothe-
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230
5-ALA-PROMOTED
METABOLIC
size that ALA may be maintained somehow in the keto, nonautoxidizable form, for example by association with plasma proteins. That the position of a keto-enol equilibrium is influenced by the solvent polarity and the two form s under equilibrium are partitioned between the aqueous and lipid phases of interfaces was demonstrated recently in studies by Ueno et al. (27) with use of a-benzoylacetanilide-liposomes. Therefore, one could predict that, in the lipid phase of membranes, which is less polar than water and lo-fold richer in 0, than the aqueous phase, the oxidation of ALA should be more important because it could initiate lipoperoxidation. Perhaps this could explain the demyelinization (6) and inhibition of K+-stimulated release of y-aminobutyric acid from preloaded synaptosomes (5) promoted by ALA and then could be related to the typical neurological manifestation of AIP and plumbism. We thank Prof. G. Cilento (Instituto de Quimica, University of Sao Paulo) for reading the manuscript. The study was supported by the FundaeBo de Amparo & Pesquisa do Estado de 5350 Paulo, the Conselho National de Desenvolvimento Cientifico e Tecnologico, and the Financiadora de Estudos e Projetos. Address for reprint requests: E. J. H. Bechara, Dept. of Biochemistry, Instituto de Q&mica, Universidade de Sao Paulo, CP20780,01498 Sao Paulo, SP, Brazil.
CHANGES
cal effort 2. Metabolic changes induced by acute exercise. Physiol. Behav.
11. DAVIES, 12.
13. 14. 15.
16. 17. 18. 19.
Received 26 December 1990; accepted in final form 4 September 1991.
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l
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R. CURI,
E. KOKUBUN,
AND
E. J. H. BECHARA
Department of Biochemistry, Institute de Q&mica, Universidude de Sa”o Pa&, 01498 Sao Puulo; Department of Physiology and Biophysics, Institute de Citkcias Biome’dicus, Universidude de S&o Pa&, 05508 Sao Pa&; and Department of Physical Education, Institute de Biocieizcias, Universidade Estudual Puulista, 13500 Rio Clara, Brazil R. CURI, E. KUKWWN, AND E. J. H. BECHARA. acid-induced alterations of oxidatiue ‘metabolism in sedentary and exercise-trained rats. J. Appl. Physiol. 72( 1): 226-230, l992.-5Aminolevulinic acid (ALA), a heme precursor that accumulates in acute intermittent porphyria patients and lead-exposed individuals, has previously been shown to autoxidize with generation of reactive oxygen species and to cause in vitro oxidative damage to rat liver mitochondria. We now demonstrate that chronically ALA-treated rats (40 mg/kg body wt every 2 days fur 15 days) exhibit decreased mitochondrial enzymatic activities (superoxide dismutase, citrate synthase) in liver and soleus (type I, red) and gastrocnemius (type IIb, white) muscle fibers. Previous adaptation of rats to endurance exercise, indicated by augmented (cytosolic) CuZn-superoxide dismutase (SOD) and (mitochondrial) Mn-SOD activities in several organs, does not protect the animals against liver and soleus mitochondrial damage promoted by intraperitoneal injections of ALA. This is suggested by loss of citrate synthase and Mn-SOD activities and elevation of serum lactate levels, concomitant to decreased glycogen content in soleus and the red portion of gastrocnemius (type IIa) fibers of both sedentary and swimming-trained ALA-treated rats. In parallel, the type IIb gastrocnemius fibers, which are known to obtain energy mainly by glycolysis, do not undergo these biochemical changes. Consistently, ALA-treated rats under swimming training reach fatigue significantly earlier than the control group. These results indicate that ALA may be an important prooxidant in vivo. PEREIRA,
B.,
5-Amideuulinic
porphyria; lead poisoning; reactive oxygen species; physical exercise; mitochondrial damage &AMINOLEVULINIC ACID (ALA), a heme precursor accumulated in chemical (e.g., plumbism) and inborn [e.g., acute intermittent porphyria- (AIP)] porphyrias, rapidly undergoes enolization and subsequently aerobic oxidation at pH levels above 7.0 (22-24). Superoxide species and carbon-centered radicals were proved to be intermediates in the chain reaction of ALA oxidation, and ammonium ions, hydrogen peroxide, and hydroxyl radicals were proved to be its products (Fig. 1). These properties make ALA a possible endogenous prooxidant. On addition of hemoglobin, myoglobin, and EDTA- or ADP-iron complexes, the oxygen-consuming reaction of ALA is accelerated (21,24). Analogous behavior, i.e., aerobic oxidation with generation of reactive oxygen species (ROS), has been demonstrated for a number of endogenous enolizable and hydroquinone derivative metabo226
0161-7567/92
$2.00
lites, such as dihydroxyacetone (18) and other carbohydrates (26), homogentisic acid (2), coenzyme Q (3), and 6-hydroxydopamine (1,7). In all cases, the autoxidationgenerated ROS have been implicated in the clinical manifestations of various diseases. Because 1) AIP patients (20), lead-exposed workers (22), and rats (28) exhibit elevated erythrocytic superoxide dismutase (SOD) activities, 2) histopathological studies of liver biopsy samples of AIP cases revealed extensive mitochondrial damage, such as fat, lipofucsin, and ferritin deposits, in addition to ultrastructural changes (4), 3) renal mitochondrial damage has been reported in cases of plumbism (6), and 4) incubation of rat liver mitochondrial preparations with succinate in the presence of ALA leads to the impairment of vital functions, such as respiratory rate, calcium fluxes, and membrane potential, which are abolished by ROS scavengers and ortho-phenanthroline, an iron chelator (15), we have suggested that ROS generated by autoxidization of ALA may underline the syndromes of both plumbism and AIP. Alternative biochemical mechanisms have been suggested by other authors on the basis of the deleterious effects of ROS in plumbism; stimulation by lead (and aluminum) of iron-dependent lipoperoxidation of membranes (25) is an example of these. The aim of this work is to examine whether intraperitoneal injection of ALA in rats would cause oxidative stress and promote mitochondrial injury. Inasmuch as physical exercise alters the biological redox balance, in which oxyradicals might have a role in mitochondrial lesions (ll), a possible synergistic effect of ALA treatment and exercise training was also investigated. These questions were approached by comparing the CuZn- and Mn-SOD activities in several tissues of sedentary and swimming-trained rats untreated or treated with ALA. In parallel, other parameters closely related to the aerobic (citrate synthase activity, free fatty acid serum level) and glycolytic (lactate serum level and liver and muscle glycogen content) metabolism, as well as the exercise performance under ALA treatment, were also monitored. MATERIALS
AND
METHODS
Reagents and equipment. Reagents of the purest quality available were used as purchased from commercial sources: bovine erythrocytic SOD, catalase, 2-amino-2methyl-1-propanol, 5,5’-dithiobis(2-nitrobenzoic acid), oxalacetic acid, acetyl CoA, Triton X-100, and 5-amino-
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Society
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5-ALA-PROMOTED 0
METABOLIC
CHANGES
IN RATS
227
OH 02
CC+-
+
/ +
=
NH2
ccy
complexes
NH2
0
0~ ,H202,H0
iron
FIG. 1. Chain reaction of 5-aminolevulinic acid oxidation.
0
NH4+
levulinic acid from Sigma Chemical; diethylenetriaminepentaacetic acid from Jensen; ethylenediaminetetraacetic acid and tris(hydroxymethyl)aminomethane (Tris) from Reagen. All other reagents were of analytic grade. All solutions were prepared with bidistilled deionized water obtained with Millipore (Milli Q) equipment. All spectrophotometric measurements were performed with a DMR-10 Zeiss apparatus. Animak Female Wistar albino rats (3-mo-old and +?50 g) were maintained in a room at 25OC under a 12:12-h light-dark cycle and fed a commercial diet ad libitum. Rats were killed by decapitation 24 h after the last exercise session without anesthesia. Blood was immediately collected and centrifuged at 2,000 g, and the separated plasma was kept at -2OOC for further assay of lactate and free fatty acids. Fragments of -250 mg of predominantly type IIb (white) and type IIa (red) fibers from the gastrocnemius muscle, type I soleus muscle (red) fibers, and two liver fragments (chopped to -500 mg) for measuring the glycogen content were also obtained. For hepatic and muscle glycogen determination, the tissues were digested for 30 min in a 30% KOH boiling solution, and extraction was performed in two steps of precipitation by 70% ethanol under heat. Glycogen was finally assayed by the anthrone method at low pH (16). Free fatty acids were calorimetrically determined according to Falholt (13) after extraction with chloroformheptane-methanol (28:21:1). Serum lactate was assayed as described by Engel and Jones (12). The extraction medium for SOD measurements contained 140 mM KC1 and 10 mM sodium phosphate at a final pH 7.0. The assay was performed on aliquots of the supernatant after centrifugation at 10,000 g for 30 min. SOD activities are expressed as units per gram of fresh weight and were measured as described by Marklund (17). The extraction medium for the measurement of citrate synthase activity contained 50 mM Tris-HCl and 1 mM EDTA, pH 7.4. The assays were performed as described by Cooney (8), and the activities were expressed as prnol. min-’ g-l fresh tissue wt. Endurance exercise training. A special swimmingtraining model described elsewhere (9,10) was used here. The rats were conditioned to swim for 90 min at 30°C daily, 5 days/wk for 2 mo, with an extra weight (5% of body wt) fixed on the tail. Sedentary group. As a control for the exercising group, groups of rats were housed in cages until the end of the training session of the experimental group. ALA treatment. A group of female Wistar rats was inl
jetted with ALA solution (40 mg/kg body wt ip) at a final pH adjusted to between 6 and 7 with sodium bicarbonate as described by McGillion (19). In this acute treatment, the treated rats were killed 3 h after injection, and the tissues were dissected for enzyme and glycogen determination. The control group received the same volume of saline as that of the ALA injection (0.5 ml). In the chronic treatment of rats with ALA (40 mg/kg body wt), sedentary rats received the ALA solution every 2 days for 15 days and were killed 24 h after the last injection. After 2 mo of swimming training, a group of exercised rats received the ALA solution after exercise sessions. The exercised rats were trained until the end of ALA treatment, every 2 days for 15 days, and then were killed 24 h after the last injection. The volume of ALA solution injected in the chronic treatment was the same as that injected for acute treatment. Statistical treatment. Two-way analysis of variance (ANOVA) with post hoc contrasts was used to compare the groups. The level of significance wa .s set at P < 0.01. RESULTS
The effect of acute and chronic ALA treatment on the aerobic meta .bolism o f swimming-train .ed and sedentary rats was evaluated. Under acute treatment, we did not observe any alteration in the metabolite parameters studied (Table 1). On the other hand, after chronic treatment (Table 1) we found elevated serum lactate (141%) and free fatty acid (33%) levels, whereas glycogen decreased in liver (50%), soleus muscle (83%), and the type IIa fibers of the gastrocnemius muscle (43%). Previous endurance swimming training of the animals did not reverse the ALA effects mentioned above (Table 1); that is, lactate increased 139% and glycogen decreased 81% in soleus muscle, 44% in the type IIa gastrocnemius fibers, and 27% in liver. No significant alteration was observed in the glycogen content of the type IIb gastrocnemius fibers of either sedentary or trained ALA-treated rats (Table 1). With regard to the serum fatty acid levels, there is a trend toward higher values in ALA-treated rats. The effect of ALA treatment on the CuZn-SOD and Mn-SOD activity of sedentary and swimming-trained rats is described in Tables 2 and 3. The results obtained for CuZn-SOD and Mn-SOD activities with the sedentary group chronically treated with ALA vs. the control (saline) group are described in Table 2. The CuZn-SOD activity (Table 2) increased in the ALA-treated rat groups in the following tissues: brain (61%), type IIa gas-
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228
5-ALA-PROMOTED
TABLE 1. Effect
of acute
and chronic treatment Acute
Blood lactate, mmol/l Serum FFA, meq/l Glycogen, mg/IOO mg Soleus (type I) Gastrocnemius Type IIa Type IIb Liver
METABOLIC
CHANGES
zuith ALA on metabolic parameters
Sedentary
Chronic
Chronic
Sedentary
Exercised
ALA
Saline
ALA
Saline
ALA
1.40t0.02 l.llt0.02
1.4ltO.02 0.91&0.05*
1.43kO.03 l.lkkO.02
3.46*0.07* 1.48*0.04*
1.49t0.02 1.02t0.05
3.56t0.16* 1.23t0.04*
0.41t0.02
0.40~0.01
0.44kO.02
0.24kO.02'
0.49t0.02
0.27*0.03*
0.40t0.02 0.44t0.02 5.86t0.06
0.48t0.01* 0.44t0.02 6.01tO.10*
0.43kO.02 0.50t0.03
0.3Ot0.02" 0.50t0.02 3.86?0,12*
0.49t0.02 0.49+0*03 5.96rtO.08
0.34t0.02* 0.47t0.02 4.66t0.13*
5.81t0.11
trocnemius fibers (57%), and liver (148%); decreased CuZn-SOD levels were observed in the (red) soleus fibers (184%). On the other hand, the Mn-SOD activity (Table 3) decreased in the type IIa gastrocnemius fibers (51%) and in the soleus muscle (78%), as well as in the liver (250%). When the groups of swimming-trained rats either untreated or treated with ALA were compared with regard to SOD activities (Tables 2 and 3), the same trends of CuZn-SOD and Mn-SOD alterations as those obtained with the sedentary group were observed. That is, the CuZn-SOD activity increased in brain and liver but decreased in the soleus muscle, whereas the Mn-SOD activity decreased in liver and the type IIa fibers of the gastrocnemius and soleus muscles. Citrate synthuse uctiuity. The activity of citrate synthase was measured in the soleus muscle of the rats to indicate the effect of the exercise training and ALA treatment on the aerobic metabolism. The sedentary and swimming-trained groups showed maximal enzyme activity of 25.4 t 6 and 49.0 t 15 pmol mine1 g fresh tissue-l, respectively. The soleus citrate synthase activity of ALA-treated swimming-trained rats (17.5 t 1.4 pm01 min-l g tissue-l, n = 10) decreased 45% (P < 0.01) relative to the sedentary group and 180% relative to the swimming-trained nontreated rats. The endurance of the rats in swimming (a mean value for 15 days of ALA treatment) before and after chronic treatment with ALA was found to be 90 t 12 and 40 t 6 min, respectively. l
l
l
DISCUSSION
The possible involvement of ALA as an endogenous source of reactive intermediates that may promote oxidative stress in plumbism and AIP patients is suggested by several lines of evidence. First, spectrophotometric TABLE 2. CuZn-SOD
of sedentary and exercised ruts
Saline
Values are means t SD for 10 rats in each group. See MATERIALS AND METHODS levulinic acid; FFA, free fatty acids. * P < 0.01 vs. saline-treated rats.
l
IN RATS
activity in tissues
of sedentary
for discussion of treatments and parameters. ALA, &amino-
and electron spin resonance techniques demonstrated rapid formation of oxygen- and carbon-centered radicals during ALA aerobic oxidation in slightly alkaline buffers (23). Second, incubation of succinate-sustained mitochondrial preparations with 5-15 mM ALA leads to acceleration of the state 4 respiration, release of intramitochondrial calcium ions, collapse of the membrane potential (15), and, at 50-100 PM ALA, calcium-dependent mitochondrial swelling (unpublished results). Third, several authors have reported extensive mitochondrial damage in liver biopsy samples of AIP patients (4), as well as kidney insufficiency in lead poisoning (6). Fourth, the neurological manifestations typical of both plumbism and AIP have been attributed to competition between ALA and y-aminobutyric acid for synaptic sites of nerve cells (5) but may also be linked to membrane lipoperoxidation (25). Finally, indication of oxidative stress in lead-exposed workers (22) and AIP patients (20) is suggested by the observed increased erythrocyte levels of the antioxidant enzymes SOD and glutathione peroxidase in these individuals. In one AIP patient, Gorschein (14) determined the ALA concentration in the serum as 8 PM, which is roughly one order of magnitude above the normal level. Inasmuch as studies of rats injected intraperitoneally with ALA (40 mg/kg body wt) revealed that ALA is distributed among and accumulated in several organs (X lo3 in liver, 3 X 10’ in heart, and X10 in brain after 3 h of injection) and that it slowly decays to the normal levels within 2-3 days (19), one could predict that either continuous overproduction of ALA in AIP and lead-poisoning patients or chronic ALA treatment of rats would expose their biomolecules and supramolecular cell structures to a potentially higher risk of damage by ALA-generated
and endurance-truined
ruts chronically
Sedentary
treated with ALA
Endurance
Trained
Tissues
Saline
ALA
Saline
ALA
Brain
12.28t0.76
19.83*0.45*
53.93t2.11
l&94* 1.59*
11.23H.46 55.6313.91
19.74*0.51*
Soleus muscle Gastrocnemius muscle Type Ifa Type IIb Liver
22.1621.36 11 l98t0.28 120.28t1.24
34.99t1.91* 11.95kO.40 299.30&6.46*
20.77+1.02*
28.18t1.29 9.55t0.82 117.96t1.91
Values are means (X10w3) k SD for 10 rats in each group. SOD, superoxide dismutase. *P < 0.01 vs. saline-treated
29.94k1.18 12.11st0.79” 213.56*4.0* rats.
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5-ALA-PROMOTED
3. Mn-SOD actiuity in tissues
TABLE
METABOLIC
of sedentary
CHANGES
and endurance-trained
229
IN RATS
rats chronically treated with ALA
Sedentary Tissues
Saline
Brain Soleus muscle Gastrocnemius muscle Type Ifa Type IIb Liver Values
are means
Endurance ALA
Saline
ALA
0.647tO.019
0.672tO.022
0.620t0.025
0.515t0.020
0.289+0.019*
0.96l-tO.039
0.336t0.012 0.142t0.028 3.764kO.lOO
0.223t0.01 0.115t0.009* 1.075t0.089”
0.525t0.02 0.110-+0.010 3.590~0.102
l*
(X10B3) t SD for 10 rats in each group, *P -C 0.01 vs. saline-treated
ROS and carbon-centered radicals. In this study the hypothesis that ALA might represent an endogenous prooxidant, an important consideration in elucidating the biochemical basis of the clinical manifestations of acquired and inherited porphyrias, is tested with ALAtreated sedentary and swimming-trained rats. The comparison between these two groups is justified by the evidence obtained by other authors (11) and ourselves (unpublished results) that endurance training triggers adaptations to an essentially aerobic muscle metabolism. In fact, we found that rats that have been swimming exercised for 2 mo present elevated Mn-SOD activity in the type I fiber of soleus muscle and type IIa fibers of gastrocnemius muscle concomitantly with twofold increased citrate synthase activity, compared with a control group (unpublished results). This result is consistent with the proliferation of mitochondria already described as a result of endurance training (11). That is, any difference on the deleterious effects of ALA at abnormally high concentrations on, for example, mitochondrial functions could eventually be better verified by comparing the sedentary and trained groups of rats submitted to ALA treatment. Table 1 shows that just one intraperitoneal injection of ALA in sedentary rats causes no significant differences in the rat metabolism, as indicated by measurement of the level of serum lactate and free fatty acids and that of liver and muscle (soleus and gastrocnemius) glycogen. However, chronic treatment of sedentary rats with ALA at the same dose (40 mg/kg body wt every 2 days for 15 days) that, according to McGillion (19), should maintain ALA at high concentration in the plasma and several organs, culminated with an apparent shift of an essentially aerobic to a more anaerobic metabolism (Table 1). This shift is suggested by significant increase of lactate and free fatty acids in the serum, which could be attributed to decreased consumption of fuels in oxidative tissues as a consequence of impairment of the mitochondrial functions, and the decrease of both hepatic and muscle glycogen. These sa.me effects were also observed with ALA-treated rats submitted to the endurance training: higher mobilization of glycogen and production of lactate (Table 1). In parallel, we also observed a loss of citrate synthase activity, and half of the swimming endurance (see RESULTS). This result is in line with observations in the literature (4, 6, 15) that in vitro and in vivo data point to mitochondria as main targets for ALA-generated ROS. The in vivo prooxidant properties of ALA are also suggested in this study by the observed increase of CuZn-
Trained
0.721t0.017* 0.296-tO.O49* 1
0.277,to.o19*
0.113rt0.008 1.559tO.087*
rats.
SOD in brain, muscle (type IIa fibers of gastrocnemius), and liver of untrained rats submitted to a chronic treatment with ALA (Table 2). This increase had also been observed with AIP patients and lead-poisoned workers by measurement of the erythrocyte SOD activity (20,22). The observed decrease of CuZn-SOD activity in the soleus muscle, richer in mitochondria than the type IIb and type IIa fibers of gastrocnemius muscle, may be linked to the expected augmented oxidative mitochondrial damage that may also affect other cytosolic enzymes and organelles. With regard to the swimming-trained rats chronically treated with ALA, one can also verify augmented CuZn-SOD levels in brain and liver and decreased levels in the soleus muscle (Table 2). Of striking interest, however, are the Mn-SOD data for both trained and sedentary rats chronically treated with ALA compared with the untreated control rats. Tables 2 and 3 show a dramatic loss of mitoehondrial SOD activity in the muscle and liver tissues of both trained and sedentary ALA-treated groups. It can also be verified that the higher mitochondrial SOD levels in red fibers of soleus and gastrocnemius muscles reached by swimming training (Table 3) were brought down to the same levels in both sedentary and trained rats by ALA treatment. That is, the ALA effect in lowering the Mn-SOD is more pronounced in trained rats than in the sedentary group. In conclusion, ALA treatment of rats results in elevated total SOD in several tissues (except red muscle fibers), which, like the previously reported augmented total SOD and glutathione peroxidase in the blood of AIP (20) and plumbism patients (22), is interpreted as a protective response against the deleterious oxidative effects of ROS-generated ALA. One can speculate that mitochondria are in fact main targets of autoxidizing ALA, as suggested by 1) the data described here for the mobilization of glycogen as major fuel and consequent increase of lactate in serum under ALA treatment, 2) loss of mitochondrial enzymatic activities especially in liver and red muscle fibers, such as those of citrate synthase and MnSOD, and finally, 3) 40% reduction in swimming endurance. These results are consistent with our previous in vitro studies on mitochondrial damage promoted by ALA (15). If, indeed, the heme precursor ALA, synthesized in the mitochondria and distributed and accumulated in several organs, represents a dangerous source of ROS in AIP and plumbism, it is important to recall that this role requires that ALA be first enolized to the autoxidizable form (Fig. 1). That is, enolic ALA cannot be stocked or circulated
among the organs without
causing damage. We hypothe-
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230
5-ALA-PROMOTED
METABOLIC
size that ALA may be maintained somehow in the keto, nonautoxidizable form, for example by association with plasma proteins. That the position of a keto-enol equilibrium is influenced by the solvent polarity and the two form s under equilibrium are partitioned between the aqueous and lipid phases of interfaces was demonstrated recently in studies by Ueno et al. (27) with use of a-benzoylacetanilide-liposomes. Therefore, one could predict that, in the lipid phase of membranes, which is less polar than water and lo-fold richer in 0, than the aqueous phase, the oxidation of ALA should be more important because it could initiate lipoperoxidation. Perhaps this could explain the demyelinization (6) and inhibition of K+-stimulated release of y-aminobutyric acid from preloaded synaptosomes (5) promoted by ALA and then could be related to the typical neurological manifestation of AIP and plumbism. We thank Prof. G. Cilento (Instituto de Quimica, University of Sao Paulo) for reading the manuscript. The study was supported by the FundaeBo de Amparo & Pesquisa do Estado de 5350 Paulo, the Conselho National de Desenvolvimento Cientifico e Tecnologico, and the Financiadora de Estudos e Projetos. Address for reprint requests: E. J. H. Bechara, Dept. of Biochemistry, Instituto de Q&mica, Universidade de Sao Paulo, CP20780,01498 Sao Paulo, SP, Brazil.
CHANGES
cal effort 2. Metabolic changes induced by acute exercise. Physiol. Behav.
11. DAVIES, 12.
13. 14. 15.
16. 17. 18. 19.
Received 26 December 1990; accepted in final form 4 September 1991.
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l
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