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Drug Monitoring and Toxicology |
1
Laboratory of Biochemistry (University Hospital) and
2
Department of Nephrology, University of Ioannina Medical School, GR 455 00 Ioannina, Greece.
a Address correspondence to this author at: Laboratory of Biochemistry, Regional University General Hospital of Ioannina, GR-455 00 Ioannina, Greece. Fax 30-651-99418; e-mail ebairakt{at}cc.uoi.gr.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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PQ-related primary lesions, from both acute and chronic exposure, occur in the lung. The ability of the lung to accumulate this herbicide probably causes the selectivity of the PQ-related toxicity. A mouthful of the herbicidal compound usually results in death from caustic burns, renal tubular necrosis, and circulatory failure due to pulmonary fibrosis. Intoxication with PQ still has a low prognosis because of no efficient treatment.
High-resolution proton nuclear magnetic resonance (H NMR) spectroscopy has been extensively applied for the analysis of the composition of biological fluids in endogenous and drug metabolites (4)(5)(6)(7). NMR provides quantitative information of the low molecular weight metabolites present in the specimen studied and allows the detection of unexpected constituents related to disease or tissue damage.
Urinalysis by NMR spectroscopy has led to the detailed investigation of the excretion pattern in various physiological and pathological situations (8)(9)(10)(11)(12). Renal damage as a consequence of acute toxic exposure in drugs or other xenobiotics has been studied extensively in experimental animals by Nicholson and co-workers (13)(14)(15)(16)(17). These studies showed that the NMR pattern appears to be dependent stringently on the type of toxin to which an animal has been exposed. Each toxic compound or class of compounds produces characteristic changes in the concentrations and patterns of endogenous metabolites in biofluids that provide information on the sites and basic mechanisms of the toxic process. Although there are many and detailed NMR studies in experimental animals concerning renal damage, only a few studies have been reported in humans (18)(19)(20).
In the present study, we used H NMR spectroscopy and conventional clinical chemistry methods to explore the changes in low molecular weight metabolites in urine and localize the renal injury in two patients suffering from paraquat intoxication after overdose with suicidal intent.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The first patient had taken ~150 mL of "Gramoxone" (200 g/L paraquat), whereas for the second patient, it was reported that he had taken a large amount of the same herbicidal compound.
To eliminate the poison, the patients were immediately submitted to sequential sessions of hemoperfusion, because, in both of them, an adequate gastric lavage had been performed at a local hospital. The first patient received a total of 11 sessions of hemoperfusion, and the second patient received one. In addition, supportive measures, including maintenance of water, electrolytes, and acid-base balance, were taken. In addition to this treatment, 1 g of methylprednisolone was administered intravenously to the second patient. Urine and serum samples were collected until death (80 h after poisoning for the first patient due to gastrointestinal hemorrhage and 12 h for the second, who succumbed to heart failure).
Samples were centrifuged and stored at -35 °C until NMR analysis. Standard clinical chemistry routine analysis of serum and urine was made the same day of the collection. For urine protein analysis, the samples were stored at 4 °C, and the measurements were performed within 3 days.
nmr analysis
In a 0.5-mL volume of crude urine, 50 µL of
H2O was added containing
sodium-3-trimethylsilyl-[2,2,3,3-H4]-1-propionate
(TSP) as a chemical shift reference (
= 0.0). The final
concentration of TSP in the working solution was 0.193 mmol/L.
H NMR measurements were made on a Bruker AMX400
spectrometer (Bruker Analytische Messtechnik), operating at a field
strength of 9.4 Tesla (400 MHz H frequency), at 22 °C
(NMR Center, University of Ioannina). A continuous secondary
irradiation field at the resonance frequency of water was applied to
suppress the intense H2O signal. Typically for each sample,
64 free induction decays were collected into 16 384 computer points.
The assignments of resonances were confirmed by consideration of
chemical shifts and the addition of standards. NMR analysis of the
urine of healthy individuals was used for comparison. Quantitation of
the metabolites was made against internal TSP. For method comparison,
we related the creatinine results obtained by NMR spectroscopy with
those of conventional clinical chemistry assay. The intramethod
correlation ranged between 0.89 and 1.08 (Table 1
).
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conventional clinical chemistry analysis
A standard profile of serum composition was performed, including
glucose, urea, creatinine, total proteins, albumin, bilirubin,
aspartate aminotransferase, alanine aminotransferase, 
-glutamyl
transferase, alkaline phosphatase, lactate dehydrogenase, creatine
kinase, sodium, potassium, calcium, magnesium, and phosphorus on a
Olympus AU560 analyzer (Olympus Diagnostica GmbH) by standard
procedures. Urine was also tested for the following analytes: glucose,
urea, creatinine, sodium, potassium, calcium, phosphorus, and
magnesium. Urinary total protein was measured by two methods: a manual
photometric method using sulfosalicylic acid and an automated
nephelometric method on a Behring BN100 nephelometer (Behring
Diagnostics GmbH) using trichloroacetic acid. Analysis of the urine
protein composition was performed by measuring albumin, IgG, and
1-microglobulin, specific markers for selective
glomerular, nonselective glomerular, and tubular proteinuria,
respectively. All three proteins were measured on a Behring BN100
nephelometer using specific antibodies.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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(aliphatic region, : 0.5–4.5 ppm). The main constituents of
the spectrum in the aliphatic region are creatinine, which gives rise
to two intense peaks at 3.05 and 4.12 ppm, hippurate, glycine,
N-methylated metabolites, citrate, small amounts of alanine, lactate,
and other metabolites in lower concentrations
(9)(18).
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In the NMR urinalysis performed by Nicholson and co-workers after renal damage in experimental animals (13)(14)(15)(16)(17), the excretion of altered metabolite concentrations has been described in detail. Proximal tubular injury is associated with glucosuria, aminoaciduria, lactic aciduria, and 3-D-hydroxybutyric aciduria, along with reduced excretion of citric acid cycle intermediates, such as citrate and succinate (indicative of impaired oxidative metabolism), whereas medullary damage leads to the early appearance of trimethylamine-N-oxide and dimethylamine, followed by increased excretion of acetate and succinate.
The urine spectrum of the two patients, after paraquat intoxication,
was markedly altered compared with the unaffected urine (Fig. 1
). Renal
damage was manifested in the first urine collection after poisoning
(12–18 h for the first and 8–12 h for the second patient,
respectively). The excretions of glucose, lactate, alanine, valine, and
glutamine were markedly increased. The excretion of creatinine was
reduced, whereas the resonances of hippurate were completely suppressed
and those of citrate were detected only in traces (Table 1
).
This NMR pattern is similar to that observed after exposure of the experimental animals to proximal tubule toxins and closer to that observed after mercury chloride intoxication, which is well known to result in necrosis of the S3 region of renal tubules (pars recta) (13)(14).
Increased excretions of the lipid peroxidation products,
formaldehyde, acetaldehyde, malondialdehyde, and mainly acetone
detected by gas chromatography–mass spectroscopy and HPLC in rats, has
been associated with paraquat intoxication (21). In the NMR
spectrum of the first patient, an intense signal from acetone was
detected in the urine collection 12–18 h after intoxication (Fig. 1
).
In addition, in this urine collection, an intense signal at 3.23 ppm
was noticed, derived from the N-trimethyl group of the
molecule of carnitine, which is a carrier molecule in the transport of
fatty acids from the cytoplasm into the mitochondria across the
membranes for ß-oxidation. This signal at 3.23 ppm is hidden by the
double-double resonance of the ß-C2 proton of glucose at 3.2–3.3
ppm. We confirmed the presence of carnitine in the specimen by two
ways: (a) by adding an amount of carnitine and re-recording
the spectrum; and (b) by using temperature variation to
separate the resonances of glucose and carnitine (unpublished data).
The increased concentrations of carnitine in urine after paraquat
intoxication have never been reported previously.
Conventional clinical chemistry analysis has further supported the NMR
findings (Table 2
). Reduced creatinine clearance and glucose reabsorption are
indicative of progressive renal failure. Mild proteinuria was measured
(~1.5 g/L) with a IgG/albumin ratio of 0.11, suggesting a selective
proteinuria due to glomerular damage. Furthermore, the excretion of
1-microglobulin, a specific marker for tubular
damage, was progressively increased.
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In addition, in the urine of the second patient, a high concentration
of unmodified toxic compound (1,1'-dimethyl-4,4'-bipyridylium
dichloride) was detected. Fig. 2
shows the NMR spectrum of the aromatic region in the urine of
healthy subject and the two patients. The resonances of hippurate
present in the healthy urine spectrum were suppressed in the two
patients, as mentioned above. Two well-resolved doublets due to the
pyridylium ring of paraquat were detected in the urine of the second
patient, who was admitted in the hospital soon after the episode. This
spectrum was identical to the spectrum of the commercial product
Gramoxone (data not shown).
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The concentrations of lactate, glucose, and amino acids were maintained
high in all urine collections during the 3 days of the first patient's
life, whereas the creatinine concentration was further reduced, also
confirmed by conventional clinical chemistry methods (Fig. 3
). The NMR pattern of the urine sample at 44–50 h of the first
patient was similar to that of the second patient (Fig. 1
), whose
survival after intoxication was shorter. This is probably the pattern
of the end stage of paraquat injury.
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Based on the above findings, we can conclude that the rapid screening of urine by NMR spectroscopy provides information about both the identity of the poison and the resulting abnormal pattern of endogenous metabolites. This pattern is related to the site and severity of toxicity within the kidney, reveals alterations in unusual metabolites that are not commonly measured, and can be used as a noninvasive identification procedure for paraquat poisoning.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  W. G. E. J. Schoonen, C. P. A. M. Kloks, J.-P. H. T. M. Ploemen, G. J. Horbach, M. J. Smit, P. Zandberg, J.-R. Mellema, C. T.-v. Zuylen, A. C. Tas, J. H. J. van Nesselrooij, et al. Sensitivity of 1H NMR Analysis of Rat Urine in Relation to Toxicometabonomics. Part I: Dose-Dependent Toxic Effects of Bromobenzene and Paracetamol Toxicol. Sci., July 1, 2007; 98(1): 271 - 285. [Abstract] [Full Text] [PDF]
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 E. Bairaktari, K. Seferiadis, G. Liamis, N. Psihogios, O. Tsolas, and M. Elisaf Rhabdomyolysis-related Renal Tubular Damage Studied by Proton Nuclear Magnetic Resonance Spectroscopy of Urine Clin. Chem., July 1, 2002; 48(7): 1106 - 1109. [Full Text] [PDF]
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