Correlation between plasma 5-aminolevulinic acid concentrations and indicators of oxidative stress in lead-exposed workers.
Evidence for the involvement of free radicals in the pathophysiology of saturnism is growing . Bechara et al.  elsewhere demonstrated that ALA produces reactive oxygen species during metal-catalyzed aerobic oxidation, and is able to (a) induce lipid peroxidation and the release of encapsulated carboxyfluorescein from cardio-lipin-rich liposomes , (b) liberate iron from ferritin , (c) induce single-strand breaks in plasmid pBR222 DNA  and guanosine oxidation in calf thymus DNA , (d) cause iron-catalyzed calcium-dependent oxidative damage to the inner membrane of rat liver mitochondria [18, 19], (e) increase the glycolytic metabolism of rats during chronic treatment , and (f) associate with increased activities of erythrocytic superoxide dismutase (SOD) and glutathione peroxidase (GSPx) found in saturnism [21, 22]. Hiraku and Kawanishi  reported recently that free radicals generated by copper-catalyzed oxidation of ALA can cause oxidative damage to DNA fragments obtained from c-Ha-ras protooncogene.
Quinlan et al. [241 also have reported that lead (and aluminum) stimulates iron-dependent lipoperoxidation of membranes, thus also implicating deleterious reactive oxygen species in the physiopathology of plumbism. That lead ions can directly accelerate oxidation of oxyhemoglobin (oxyHb) to methemoglobin (metHb) and inactivate several thiol enzymes has also been established . Furthermore, several electron donors (e.g., phenols, arylamines, dithionite, nitrite)  or nucleophiles (e.g., [N.sup.-.sub.3], [SCN.sup.-], [F.sup.-], [Cl.sup.-])  can promote oxidation of oxyHb to metHb plus either [H.sub.2][O.sub.2] or superoxide species, respectively. ALA, an easily oxidized [alpha]-aminoketone, may be viewed as belonging to the former class of hemoglobin reactants . Moreover, a high frequency of methemoglobinemia and increased erythrocytic SOD and GSPx concentrations were found in residents of Cubatao, a highly polluted town in the State of Sao Paulo, Brazil . The increase in blood antioxidant enzymes was interpreted as a protective response against oxidative injury promoted by superoxide and [H.sub.2][O.sub.2] generated by pollutant-induced oxidation of oxyHb.
SOD is an enzyme used extensively as a biochemical indicator of pathological states associated with oxidative stress  because of the protective role it plays against deleterious effects triggered by superoxide radical anion, iron ions , and peroxynitrite . Mercury poisoning , lead poisoning , smoking-related erythrocyte peroxidation , multiple myeloma , hyperthyroidism , schizophrenia and manic depression , air pollution-related methemoglobinemia , and aging  are among the conditions with altered SOD activities. Monteiro et al.  demonstrated that the concentrations of SOD activity are higher in individuals exposed to lead, suggesting a correlation between accumulation of heme precursors and adaptation to reactive oxygen species.
Alternatively, evaluation of oxidative stress by low-activity chemiluminescence (CL) measurements has proven useful in several disorders for assessing excited species formed during protein and lipid oxidation in biological systems [39, 40], including fluids like urine . In this case, CL probably arises from spontaneous decomposition of peroxide derivatives .
This study characterizes the involvement of ALA as a prooxidant compound in lead poisoning and evaluates the possibility of using plasma ALA as an index of lead exposure. Our approach was to measure plasma ALA in lead-exposed subjects and correlate these data with indicators of oxidative stress (erythrocytic SOD and metHb and urine CL) and lead exposure (blood lead and protoporphyrin IX (PP-IX)).
Materials and Methods
ALA-HCI, EDTA, perchloric acid, Triton X-100, bovine serum albumin, Folin & Ciocalteu s phenol reagent, purine, cytochrome c, xanthine oxidase, and heparin were purchased from Sigma Chemical Co., St. Louis, MO. All other reagents and solvents used were analytical grade from Merck, Darmstadt, Germany. Acetonitrile for the mobile phase was chromatographic grade. Water was doubly distilled and subsequently deionized in a MilliQ system (Waters Associates, Milford, MA).
HPLC. Isocratic liquid determinations of the ALA-OPA derivative were performed on a HPLC system that consisted of a LC10AD pump coupled to a LECD 6A electrochemical detector from Shimadzu Corp., Kyoto, Japan. The detector working electrode was maintained at +0.6 V vs Ag/AgCl, and its signal was delivered to a 386 ASA computer with data collection and handling provided by Scientific Software, San Ramon, CA. All analyses were performed with 15.0 cm X 3.9 mm (i.d.) Water Associates 4.0-[micro]m [C.sub.18] columns. The mobile phase was phosphate buffer (pH 7.0)/acetonitrile (90:10 by vol) containing EDTA, 2.4 mmol/L. The samples were introduced with a 20-[micro]L external loop from Rheodyne, Cotati, CA, and eluted with the mobile phase circulating at 1.0 mL/min.
Spectrometry. We used either a Hitachi Koki U2000 or a Beckman Instruments (Brea, CA) DU 70 spectrophotometer. For atomic absorption spectroscopy we used an AA 5000 Perkin-Elmer (Norwalk, CT) spectrometer equipped with a graphite furnace (pyrolytic tube and atomization from L'vov platform) and automated sampler (Model AS 40).
CL. A LKB Wallac (Helsinki, Finland) Model 1211 Rackbeta scintillation counter was used for urine CL measurements.
Population and samples. The blood and urine samples of the subjects involved in this study were collected from healthy white and mulatto men, ages 18-53, taking no medication. Control group samples were collected from police preparatory school students, and the exposed group samples were from workers at a pottery manufacturing plant, which uses lead-containing paints. Blood samples from healthy subjects and lead-exposed workers were collected in plastic tubes containing heparin, under informed consent and in accordance with the ethical standards of the revised Helsinki Declaration of 1983. Aliquots of whole-blood samples were separated for assaying PP-IX, lead, and metHb. The remaining blood was centrifuged immediately, and the plasma and erythrocytes were separated for later analysis. All aliquots were kept at -20 [degrees]C until assayed. The urine samples collected from all the subjects involved in this study were the first of the morning, as standardized by Lissi et al. .
Chromatographic assays. The determination of ALA in calibrators and plasma was performed by HPLC after prederivatization of the sample with o-phthalaldehyde (OPA). OPA reagent was made up in the proportions described by Lindroth and Mopper , and derivatization of the samples was done by mixing 10 [micro]L of calibrator solution or deproteinized plasma, 5 [micro]L of OPA reagent (36 mmol/L), and 35 [micro]L of water. The sample was then incubated for 1 min at room temperature, after which an aliquot of 20 [micro]L was injected into the HPLC. The plasma was deproteinized with 0.8 mol/L perchloric acid, and the supernatant obtained by centrifugation (8008) was neutralized by addition of NaHC[O.sub.3] crystals (~pH 7.4).
Spectrometric assays. MetHb was measured according to Hegesh et al. , SOD by the method of Oberley and Oberley , PP-IX according to Heller et al. , lead by the method described by Subramanian , creatinine according to Heinegard and Tinderstrom , and protein by the method of Lowry et al. .
CL assays. Urine CL was measured by the method of Lissi et al. .
Table 1 shows the mean [+ or -] SD of variables related to either oxidative stress or exposure to lead, measured in samples from the control group and exposed workers. All of the indicators analyzed were significantly higher in the subjects exposed to lead (P <0.0002). On the basis of these data, we investigated the relationship of plasma ALA with the variables for lead exposure (blood lead and PP-IX) and oxidative stress (SOD, metHb, CL). To facilitate correlation analysis, all data concerning plasma ALA and blood lead were grouped in concentration ranges and subsequently submitted to Dixon's test to eliminate outliers. The distribution profile of the number of samples used to make the correlation plots varied in each range of concentration selected, but was not fewer than 6.
Fig. 1, top, represents the correlation between ALA and metHb, expressed in terms of the blood content of metHb vs plasma ALA concentrations. By scatter analysis the data shown in Fig. 1, top, are best represented by the equation y = 0.984 + 0.053x (r = 0.984). Fig. 1, middle and bottom panels, depict the relation between plasma ALA and either PP-IX or lead, expressed in terms of variation of erythrocyte PP-IX or lead with plasma ALA concentration. The scatter analysis of these data showed that ALA vs PP-IX and ALA vs lead correlate linearly and may be represented by the equations y = 137.2 + 28.9x (r = 0.891) and y = 38.93 + 3.9x (r = 0.992), respectively. The relationship between lead and PP-IX, SOD, or metHb is shown in Fig. 2. The scatter analysis of these data showed linear correlations, with equations y = 246.14 + 10.14x (r = 0.993) for lead vs PP-IX, y = 224.35 + 40.39x (r = 0.948) for lead vs SOD, and y = 0.178 + 0.004x (r = 0.993) for lead vs metHb. Fig. 3 represents the relationship between plasma ALA and urine CL; its scatter analysis showed a linear correlation whose equation is best represented by y = 1550 +1790x (r = 0.987).
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
This work indicates that in the lead-exposed workers there is, on average, a 6-fold increase in the circulating concentrations of ALA. The importance of increased circulating ALA concentrations produced by lead lies in its pharmacological and neurological effects [2, 12]. The heterogeneity of the control group studied does not allow a definition concerning the limits within which ALA concentrations may be affected by age, race, or medication. The mean control group concentration of plasma ALA found here is in agreement with measurements carried out by gas-liquid chromatographic (GLC) analysis  and 10 times less than the value described in the literature for measurements by HPLC with fluorescence detection . Previous analysis of the samples by the fluorescence method did not show any substantial difference in plasma ALA concentrations between lead-exposed and control groups , in contrast to our data that reveal a 6-fold difference between the two groups. This disagreement may be related to differences in detection limits of the fluorescence method regarding the plasma ALA concentrations in individuals not exposed to lead, since its values differed from those observed by both GLC  and HPLC coupled to electrochemical detection (this work). The discrepancy with the fluorescence data may also be related to the low concentrations of lead exposure of the subjects in that study . The analysis of plasma ALA concentrations by our method (Table 1) agreed with the data obtained by the GLC method for the control group and revealed a relationship between plasma ALA concentrations and biochemical indicators of oxidative stress (SOD, metHb, urine CL) or biological indexes of exposure to lead (lead, PP-IX) (Fig. 1, top and bottom, and Fig. 3).
[FIGURE 3 OMITTED]
Chisolm  stated that concentrations of lead in blood are reliable indicators for the so-called internal lead dose. This author suggests, however, that blood lead does not represent the best chemical indicator for judging the exposure to lead of subjects with high risk of plumbism, but instead recommends the determination of heme precursors that accumulate in erythroblasts because they reflect better the effect of lead on bone marrow. Chisolm  did not find a linear correlation between urinary ALA excretion and blood lead. In the present study, we verified a correlation between plasma ALA and either blood lead or PP-IX, pointing to a possible use of plasma ALA concentrations as a toxicological indicator of exposure to lead. Such measurements would be useful especially if future clinical studies demonstrate a relationship between plasma ALA and the symptoms of plumbism. Some authors associate the increase of PP-IX concentrations with some of the main manifestations observed in plumbism, e.g., renal failure, hematopoietic damage, and neurological disturbances . However, a positive correlation with PP-IX was observed only for the hematopoietic damage.
Overload of ALA seems to be involved in the neurological disturbances observed in plumbism. Besides leading to the inhibition of GABA release from synaptosomes of rat brain and blocking GABA receptors , ALA also can cause oxidative damage to the brain . The involvement of ALA as a prooxidant in the cellular damage observed in plumbism has been studied extensively by Bechara . Monteiro et al.  demonstrated that individuals exposed to lead have increased antioxidant defenses (SOD and GSPx) and found a nonlinear relationship between blood lead and SOD. Here, we obtained a linear correlation (r = 0.948) for these variables, probably because of the larger sample size. The biochemical basis for explaining the correlation between lead and SOD is not evident. However, the increase of the antioxidant defenses in response to lead exposure may reflect a protective response to the deleterious effects of oxyradicals generated by tissue ALA oxidation . The involvement of reactive oxygen species in lead poisoning has also been addressed recently by Ercal et al. , who demonstrated a decrease in the concentrations of reduced glutathione (GSH) and an increase in the concentrations of oxidized glutathione (GSSG) and malondialdehyde in lead acetate-treated mice. In addition, they observed that these effects are reduced by treatment of these animals with N-acetylcysteine, a precursor of GSH, which opens the possibility of antioxidant therapy for individuals exposed to lead. Glutathione is considered an important component of the antioxidant defense system in mammalian cells, and GSH/GSSG is rated a sensitive indicator of oxidative stress .
Hemoglobin, a Fenton-type reagent under special conditions , is considered a main biological source of oxyradicals in erythrocytes . The mechanism of Hb oxidation to metHb and superoxide, followed by SOD-catalyzed dismutation of superoxide to [H.sub.2][O.sub.2], has long been known . It is tempting to propose that the high metHb values found in the lead-exposed workers reflect both lead-induced direct oxyHb oxidation and co-oxidation of oxyHb with ALA . This hypothesis is supported by the linear correlation found for lead vs metHb (r = 0.993) and ALA vs metHb (r = 0.984). The positive correlation for ALA vs lead (r = 0.992) and ALA vs PP-IX (r = 0.891) argues in favor of plasma ALA as an useful index of lead exposure.
According to Lissi et al.  the measurement of weak CL in urine has become a very useful method of evaluating noninvasively the degree of oxidative stress. Indeed, this approach has revealed abnormally high concentrations of urine CL increases in several pathological states associated with oxidative stress, e.g., hyperthyroidism , Duchenne muscular dystrophy , and cigarette smoking . Similarly, the increase of urine CL associated with lead exposure may thus be seen as reflecting increased oxidative stress in plumbism.
Together with previous results showing oxidative stress associated with accumulation of ALA, the current data provide strong evidence for the contribution of ALA to the prooxidant effects and toxicity of lead in humans and other animals. In addition, we find that plasma concentrations of ALA provide a useful index of the physiological response to lead exposure.
Received November 5, 1996; revised March 20, 1997; accepted March 27, 1997.
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CRISTINE A. COSTA, (1) GILMAR C. TRIVELATO, (2) ADRIANA M. P. PINTO, (3) and ETELVINO J. H. BECHARA (3)*
(1) Departamento de Toxicologia, Faculdade de Cieancias Farmaceauticas da Universidade de Sao Paulo, (2) Fundacao Jorge Duprat de Figueiredo e Medicina do Trabalho, Fundacentro, and (3) Departamento de Bioquimica, Instituto de Quimica, Universidade de Sao Paulo, CP 26077, 05599-970 Sao Paulo, SP, Brazil.
* Author for correspondence. Fax 5511-8155579; e-mail firstname.lastname@example.org.
(4) Nonstandard abbreviations: ALA, 5-aminolevulinic acid; OPA, o-phthalaldehyde; GLC, gas liquid chromatography; CL, chemiluminescence; PP-IX, protoporphyrin IX; metHb, methemoglobin; SOD, superoxide dismutase; GSPx, glutathione peroxidase; oxyHb, oxyhemoglobin; GABA, [gamma]-aminobutyric acid; GSH, reduced glutathione; GSSG, oxidized glutathione.
Table 1. Mean [+ or -] SD of either oxidative stress or lead exposure indicators. Control (a) Exposed (b) MetHb (% total Hb) 0.56 [+ or -] 0.18 0.86 [+ or -] 0.31 (c) PP-IX ([micro] g/L) 1762.0 [+ or -] 132 2770 [+ or -] 147.8 (c) SOD (U/mg) 88.3 [+ or -] 29.1 375 [+ or -] 169.3 (c) ALA ([micro] mol/L) 0.26 [+ or -] 0.08 1.9 [+ or -] 1.0 (c) Lead ([micro] g/L) 63.0 [+ or -] 2.3 534.0 [+ or -] 12.1 (c) Urine CL ([10.sup.3] cpm) 9.2 [+ or -] 1.6 15.4 [+ or -] 3.6 (c) (a) n = 30. (b) n = 60. (c) P = 0.0002, compared with the control group by Student's Rest.
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|Title Annotation:||Drug Monitoring and Toxicology|
|Author:||Costa, Cristine A.; Trivelato, Gilmar C.; Pinto, Adriana M.P.; Bechara, Etelvino J.H.|
|Date:||Jul 1, 1997|
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