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Institute of Animal Nutrition, Physiology, and Metabolism, Christian-Albrechts-University Kiel, 24098 Kiel, Germany
2To whom correspondence should be addressed. E-mail: blank{at}aninut.uni-kiel.de.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • bioavailability • excretion • mycotoxins • ochratoxin A • ochratoxin B
The worldwide occurrence of ochratoxin A (OA)3 contamination of raw agricultural products has been amply documented; it occurs in a variety of plant products such as cereals, coffee beans, and dried fruits (1–3). The average contamination level of feedstuffs is in the range of 0.1–0.5 mg/kg but can be >5 mg/kg under unfavorable storage conditions (1). Furthermore, contamination of animal products such as pigs’ kidney, blood sausages, meat, and milk occurs as a result of feeding animals OA-contaminated feed (3–6). With respect to the carryover of OA to humans, animal products derived from monogastric species such as pigs are of greater relevance than those derived from ruminants (7).
Due to striking similarities between the porcine nephropathy caused by OA and the Balkan Endemic Nephropathy including the development of urinary tract tumors in humans (8), OA is assumed to be a causative agent in the development of the human disease as well, although other risk factors may be involved (9). The International Agency for Research on Cancer (IARC) classified OA as a putative human carcinogen (10). Because of the ubiquitous occurrence of OA in improperly stored food and feedstuffs, there is a high risk of exposure for humans and animals. Effective methods are required in animal production either to detoxify OA-containing feedstuffs or to reduce its absorption and/or to enhance its elimination from the body.
According to previous studies (11), OA is subjected to extensive reabsorption along the nephron, resulting in an accumulation of OA in the kidney. This is of toxicologic importance because renal reabsorption contributes to the long half-life of the mycotoxin in the body. From microinfusion and microperfusion studies in rats (12), it was concluded that reabsorption of OA along the nephron results mainly from nonionic diffusion and in a minor way from other carrier-mediated mechanisms (H+-dipeptide cotransporter, organic anion transporter). These findings indicate that urine alkalinization may accelerate renal OA excretion and thus reduce its toxicity. Thus, the present study investigated the effect of urine alkalinization by means of dietary sodium bicarbonate (NaHCO3) supplementation on the systemic availability and excretion of OA in pigs and its dechloro-analog ochratoxin B (OB), which often occurs with OA in contaminated feedstuffs.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, and Oß were produced from inoculated wheat as previously described (13,14). All other chemicals were derived from the following sources: chloroform, 2-propanol (Mallinckrodt Baker), magnesium chloride (Sigma-Aldrich), hydrochloric acid (Merck), methanol, and ortho-phosophoric acid (Roth). Animals. Castrated male pigs (n = 7; cross-breed, German Hybrid Pig Breeding Programme) surgically equipped with a permanent jugular vein catheter (Cook Deutschland) and a mean body weight of 36.7 ± 2.0 kg were used. Twice each day, the pigs were restrictively fed (1400 g/d) a commercially available pig diet4 (Fa. Plambeck) based on barley, wheat, soybean meal, and corn. During the study, pigs were kept in metabolism crates with free access to tap water.
Treatment groups and diets. Control pigs (n = 3) were fed the commercially available standard diet. The treated group (NaHCO3, n = 4) received the same diet except that it contained 2% NaHCO3. The dietary electrolyte balance (dEB, Na + K –Cl) of the control and NaHCO3 diets were 170 and 400 mEq/kg diet.
Throughout d 3 and 4 of the feeding period, urinary pH was monitored several times. On d 5, the pigs received a single portion of 150 g of wheat containing 6.0 µmol OA, 1.0 µmol OB, 0.12 µmol ochratoxin (O), and 0.45 µmol ochratoxin ß (Oß). After exposure to OA, jugular blood samples were collected for 96 h. Urine and feces were collected each day for 4 d and stored frozen until analysis. The total daily urine volume was recorded before OA exposure to differentiate between effects of NaHCO3 and OA on daily urine volume because both NaHCO3 and OA may induce polyuria (15,16). Animal care and experimental procedures were conducted according to the German Guidelines and Regulations on Animal Care (Deutsches Tierschutzgesetz 1986, Durchführung von Tierversuchen).
Analytical procedures. Serum samples prepared from blood were stored at –20°C until analysis. Feces were freeze-dried and ground through a 0.2-mm screen before analysis. Urine was thawed and an aliquot of 10 mL was taken for analysis. Extraction of ochratoxins from blood serum, feces, and urine and analysis by HPLC were performed as described previously (17).
Statistical analysis. Data are presented as means ± SD. Bartlett’s test for homogeneity of variances revealed that variances for all data among groups were equal. Serum concentrations, water intake, urine volume, as well as daily fecal and urinary excretion of the ochratoxins, were subjected to repeated-measures analysis using the MIXED procedure of SAS vers. 6.12 (SAS Institute). A spatial covariance structure was used, which assumes a reduction in correlation in relation to the power of the distance between time points. The model included treatment, time, and their interaction. The LSMEANS follow-up test was used for comparison of means. Urinary pH, growth performance, total fecal and urinary excretion of the ochratoxins and the area under the curve (AUC) of the concentration-time profiles of OA and OB in serum, calculated according to the trapezoidal rule, were analyzed using the General Linear Model procedure. The Tukey-Kramer test was used for comparison of means. Differences were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Due to clogging of the jugular vein catheter in 2 pigs after administration of OA, the serum concentrations of control and the NaHCO3 group are derived only from 2 and 3 pigs, respectively. The serum appearance/disappearance curves of OA and OB (Fig. 1) revealed that both mycotoxins were rapidly absorbed within 5–8 h after ingestion. The maximum serum concentrations for OA and OB were obtained between 7 and 8 h and 5 and 7 h, respectively, after ingestion of contaminated wheat (Table 1). After 12 h, serum concentrations of OA and OB were significantly lower in the NaHCO3-treated group (Fig. 1). The relative bioavailability of the mycotoxins, calculated as the AUC (control = 100%), was reduced (P < 0.05) by 25% for OA and 33% for OB due to NaHCO3 treatment.
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Ochratoxin and Oß are formed by microbial hydrolytic cleavage of the phenylalanine group of OA and OB, respectively. Urinary excretion of O in the NaHCO3 group was greater than in controls on d 1 after exposure, leading to an increased total excretion (0.34 ± 0.06 vs. 0.23 ± 0.02 µmol). Urinary excretion of Oß was not affected by NaHCO3. Total excretion of OA, O, and Oß in feces was not affected by treatment but decreased with time, whereas fecal excretion of OB was significantly reduced by the NaHCO3 treatment (data not shown). This might be explained by a more pronounced renal elimination, which in turn might result in lower biliary secretion of OB.
Intact OA was excreted mainly in the urine and was 2 times higher in the NaHCO3 group than in the control group (Table 2). The low proportional urinary excretion of O, which is derived mainly from OA, indicates that neither postabsorptive generation nor intestinal absorption of O from the large intestine is of major importance. In addition, the high fecal excretion of O indicates that microbial hydrolysis of nonabsorbed OA in the large intestine was nearly complete. Total fecal recovery of OA and O in feces was in the range of 40–45% of the ingested dose, indicating that the intestinal absorption of OA is 50–60% of an oral dose. For OB, total urinary and fecal excretion (OB + Oß) was 42% higher than intake (Table 2), because contaminated wheat also contained additional 0.12 µmol O (2.1% of the total OA dose) and 0.45 µmol Oß (45% of the OB dose). Consequently, the total intake of OB and Oß was excreted within 4 d in the NaHCO3 group, whereas in the control group, excretion of OB and Oß was 
69% of the total ingested OB and Oß. Because fecal excretion of Oß was similar in both groups, the missing fraction in the control group may have been due to conversion of OB or Oß to other metabolites during liver passage (conjugation, methylation, and hydroxylation), which were not analyzed.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Because the groups did not differ in the serum concentrations of OA and OB during the first 12 h after feeding, absorption of ochratoxins from the gastrointestinal tract appears not to be affected by NaHCO3 treatment. This is consistent with the finding that the supplementation of 2.6% NaHCO3 to the diet of pigs did not affect the pH of gastrointestinal contents (16).
In the present study, fecal excretion of OA and O was not affected by NaHCO3 supplementation. In both groups, mainly O and only very small amounts of OA were excreted with the feces. Hydrolysis of OA seems to occur mainly in the large intestine and cecum, indicating that the microbial population in the lower portion of the gastrointestinal tract is responsible for the hydrolysis (23). In the present study, fecal excretion of OA and O was 45% of the dose fed, indicating an apparent absorption of OA of 55%, which is in good agreement with previous results obtained in rats, mice, pigs, and monkeys (24).
Renal elimination of OA contributes up to 50% to its total clearance (25). Carrier-mediated transport of OA by organic anion exchangers located in the basolateral membrane of epithelial cells lining the proximal tubulus is the major pathway for renal secretion of OA (26). According to microperfusion studies in rats (12), 40% of the reabsorption of OA along the nephron results from nonionic diffusion, 25% is mediated by pH-dependent H+-dipeptide cotransporter, and a further 25% is mediated by the pH-independent apical organic anion transporter OAT-K1. In the present study, renal elimination of OA increased 2-fold after NaHCO3 treatment. Because OA is a weak acid with a pKa value of 7.1, >90% of the OA exists in its ionized form at a urinary pH of 8 (obtained by NaHCO3 treatment in the present study) and thus cannot be reabsorbed passively. The increased sodium intake from NaHCO3 increased the urine volume. A sodium-induced diuresis would be another possible explanation for the enhanced renal elimination of OA. Previous studies in mice, comparing the effect of NaHCO3 vs. ammonium chloride on OA toxicity, showed that NaHCO3 reduced mortality and the number of renal lesions by 20%, whereas ammonium chloride, which leads to urinary acidification and diuresis, did not have any beneficial effect (27). This implies that alkalinization of urine is the main mechanism for the increased elimination of OA. This conclusion is further substantiated by the fact that renal elimination of OA is due mainly to volume-insensitive transcellular secretion along the nephron rather than to filtration in the glomeruli (11,12). The bioavailability and excretion pattern of OB followed the same pattern as OA, indicating that excretion of OB is affected in the same way as OA by NaHCO3 supplementation. Changes in urinary pH can be calculated from dEB (28), which might be useful as a feedstuff-related tool to estimate urinary OA excretion under practical farm situations.
In conclusion, urinary alkalinization by means of 2% NaHCO3 supplementation to pig diets accelerates the urinary excretion of OA by inhibiting the pH-dependent tubular reabsorption, thereby reducing its systemic toxicity. In addition, local toxicity of OA may be influenced by tubular pH as indicated by a reduced activation of caspase-3 and DNA ladder formation in Madin Darby canine kidney cells (MDCK-C7) at a slightly basic extracellular pH compared with slightly acidic conditions (29).
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FOOTNOTES |
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3 Abbreviations used; AUC, area under the curve; dEB, dietary electrolyte balance; OA, ochratoxin A; O, ochratoxion ; OB, ochratoxin B; Oß, ochratoxin ß.
4 Proximate composition of the standard diet (per kg diet): 174 g crude protein, 35 g crude fat, 40 g crude fiber, 13.5 MJ metabolizable energy, 880 g dry matter. Macro mineral and vitamin composition (per kg diet): calcium, 7.8 g; phosphorus, 5.6 g; sodium, 1.9 g; potassium, 8.2 g; chloride, 4.3 g; Mg 1.5 g; retinyl acetate, 58 mg; cholecalciferol, 80 mg; -tocopheryl acetate, 80 mg.
Manuscript received 31 March 2004. Initial review completed 29 April 2004. Revision accepted 11 June 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Kuiper-Goodman, T. & Scott, P. M. (1989) Risk assessment of the mycotoxin ochratoxin A. Biomed. Environ. Sci. 2:179-248.[Medline]
2. Studer-Rohr, I., Dietrich, D. R., Schlatter, J. & Schlatter, C. (1995) The occurrence of ochratoxin A in coffee. Food Chem. Toxicol. 33:341-355.[Medline]
3. MacDonald, S., Wilson, P., Barnes, K., Damant, A., Massey, R., Mortby, E. & Shepherd, M. J. (1999) Ochratoxin A in dried vine fruit: method development and survey. Food Addit. Contam. 16:253-260.[Medline]
4. Gareis, M. & Scheuer, R. (2000) Ochratoxin A in meat and meat products. Arch. Lebensmittelhyg. 51:102-104.
5. Breitholtz-Emanuelsson, A., Olsen, M., Oskarsson, A., Palminger, I. & Hult, K. (1993) Ochratoxin A in cow’s milk and in human milk with corresponding human blood samples. J. Assoc. Off. Anal. Chem. Int. 76:842-846.
6. Skaug, M. A. (1999) Analysis of Norwegian milk and infant formulas for ochratoxin A. Food Addit. Contam. 26:75-78.
7. Blank, R. (2002) Contribution of animal products to human dietary mycotoxin intake. Z. Umweltchem. Ökotox. 14:104-109.
8. Krogh, P. (1992) Role of ochratoxin A in disease causation. Food. Chem. Toxicol. 30:213-224.[Medline]
9. Tatu, C. A., Orem, W. H., Finkelman, R. B. & Feder, G. L. (1998) The etiology of Balkan Endemic Nephropathy: still more questions than answers. Environ. Health Perspect. 106:689-700.
10. IARC (1993) IARC Monographs on evaluation of carcinogenic risks to humans. Some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins 56:489-521.
11. Zingerle, M., Silbernagl, S. & Gekle, M. (1997) Reabsorption of the nephrotoxin ochratoxin A along the rat nephron in vivo. J. Pharmacol. Exp. Ther. 280:220-224.
12. Dahlmann, A., Dantzler, W. H., Silbernagl, S. & Gekle, M. (1998) Detailed mapping of ochratoxin A reabsorption along the rat nephron in vivo: the nephrotoxin can be reabsorbed in all nephron segments by different mechanisms. J. Pharmacol. Exp. Ther. 286:157-162.
13. Xiao, H., Marquardt, R. R., Frohlich, A. A. & Ling, Y. Z. (1995) Synthesis and structural elucidation of analogs of ochratoxin A. J. Agric. Food Chem. 43:524-530.
14. Xiao, H., Marquardt, R. R., Abramson, D. & Frohlich, A. A. (1996) Metabolites of ochratoxins in rat urine and in a culture of Aspergillus ochraceus. Appl. Environm. Microbiol. 62:648-655.[Abstract]
15. Tapia, M. O. & Seawright, A. A. (1984) Experimental ochratoxicosis A in pigs. Austr. Vet. J. 61:219-222.
16. Patience, J. F., Austic, R. E. & Boyd, R. D. (1986) The effect of sodium bicarbonate on acid-base status and protein and energy digestibility in swine. Nutr. Res. 6:263-273.
17. Blank, R., Rolfs, J.-P., Südekum, K.-H., Frohlich, A. A., Marquardt, R. R. & Wolffram, S. (2003) Effects of chronic ingestion of ochratoxin A on blood levels and excretion of the mycotoxin in sheep. J. Agric. Food Chem. 51:6899-6905.[Medline]
18. Kumagai, S. (1988) Effects of plasma ochratoxin A and luminal pH on the jejunal absorption of ochratoxin A in rats. Food Chem. Toxicol. 26:753-758.[Medline]
19. Kumagai, S. & Aibara, K. (1982) Intestinal absorption and secretion of ochratoxin A in the rat. Toxicol. Appl. Pharmacol. 64:94-102.[Medline]
20. Hagelberg, S., Hult, K. & Fuchs, R. (1989) Toxicokinetics of ochratoxin A in several species and its plasma-binding properties. J. Appl. Toxicol. 9:91-96.[Medline]
21. Roth, A., Chakor, K., Creppy, E. E., Kane, A., Röschenthaler, R. & Dirheimer, G. (1988) Evidence for an enterohepatic circulation of ochratoxin A in mice. Toxicology 48:293-308.[Medline]
22. Rang, H. P., Dale, M. M., Ritter, J. M. & Moore, P. K. (2003) Pharmacology 5th ed. 2003 Churchill Livingstone Edinburgh, UK.
23. Madhyastha, M. S., Marquardt, R. R. & Frohlich, A. A. (1992) Hydrolysis of ochratoxin A by the microbial activity of digesta in the gastrointestinal tract of rats. Arch. Environ. Contam. Toxicol. 23:468-472.[Medline]
24. Galtier, P. (1991) Pharmacokinetics of ochratoxin A in animals. Castegnaro, M. Ple
tina, R. Dirheimer, G. Chernozemsky, I. N. Bartsch, H. eds. Mycotoxins, Endemic Nephropathy and Urinary Tract Tumors 1991:187-200 International Agency for Research on Cancer Lyon, France. .
25. Li, S., Marquardt, R. R., Frohlich, A. A., Vitti, T. G. & Crow, G. (1997) Pharmacokinetics of ochratoxin A and its metabolites in rats. Toxicol. Appl. Pharmacol. 145:82-90.[Medline]
26. Gekle, M. & Silbernagl, S. (1994) The role of the proximal tubule in ochratoxin A nephrotoxicity in vivo: toxodynamic and toxokinetic aspects. Renal Physiol. Biochem. 17:40-49.[Medline]
27. Yong, S., Albassam, M. & Prior, M. (1987) Protective effects of sodium bicarbonate on murine ochratoxicosis. J. Environ. Sci. Health B 22:455-470.[Medline]
28. Patience, J. F. (1990) A review of the role of acid-base balance in amino acid nutrition. J. Anim. Sci. 68:398-408.[Abstract]
29. Schwerdt, G., Freudinger, R., Schuster, C., Silbernagl, S. & Gekle, M. (2003) Inhibition of mitochondria and extracellular acidification enhance ochratoxin A-induced apoptosis in renal collecting duct-derived MDCK-C7 cells. Cell. Physiol. Biochem. 14:47-56.
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Different from NaHCO3, P < 0.05