Evidence of ochratoxin A conjugates in urine samples from infants and adults

Abstract

Ochratoxin A (OTA), a mycotoxin with nephrotoxic and carcinogenic properties, is an important contaminant of food and feed. Analysis of OTA in human biological fluids (blood, urine, or breast milk) has documented frequent exposure to this mycotoxin, yet at quite variable levels in different population groups across the world. Urine is the preferred matrix in biomonitoring since sample collection is non-invasive and better accepted by study participants. As only a small fraction of the ingested OTA is excreted in urine, determination of urinary OTA requires sensitive analytical techniques, and phase-II-metabolites should be also considered as biomarkers of exposure. Yet, data published so far on the presence of OTA-glucuronide/sulfate in human urine have been contradictory. In this study, urines (n = 38) from two groups of breastfed infants (German and Turkish) and from German adults were now analysed for the presence of OTA glucuronides or sulfates by an indirect method, i.e. by comparing the levels of OTA (aglycone) in urines without and after enzymatic hydrolysis with ß-glucuronidase/arylsulfatase. Additionally, ochratoxin A-8-β-glucuronide and open lactone ochratoxin A-8-β-glucuronide were synthesized to serve as reference materials for metabolite analysis. Attempts for definitive confirmation of glucuronides of OTA via direct identification in LC-MS/MS analysis were hampered by the lower ionizability of the conjugates compared to the parent compound. Considerable increases in OTA levels were found after enzymatic hydrolysis in several (not all) urine samples and provide clear evidence for the excretion of OTA-conjugates. The latter observation is of importance, since OTA phase-II-metabolites may escape detection when direct methods are applied for urinary biomarker analysis. In conclusion, enzymatic hydrolysis of urine samples is highly advisable in order to avoid an underestimation of the OTA-exposure.

Studies on the Presence of Mycotoxins in Biological Samples: An Overview

“…High method sensitivity is of most importance since the concentration of these analytes in urine samples is often present in a very low concentration range. From the relatively few studies performed on one single mycotoxin in urine, OTA was the most common one, extracted from human urine by IAC [ 60 , 61 , 62 ], an automated multi-fiber SPME system [ 63 ], or the classical LLE with CHCl 3 -isopropanol [ 64 ].…”

Aflatoxin, fumonisin, ochratoxin, zearalenone and deoxynivalenol biomarkers in human biological fluids: A systematic literature review, 2001–2018

“…Nonetheless OTA can be deactivated through sulphate and glutathione conjugation, which will lead to their secretion (Ringot et al 2006;Wu et al 2011). Recent studies demonstrated that ß-glucuronidase/arylsulfatase enzymatic hydrolysis of phase II metabolites returned significantly higher values for OTα in urine samples indicating that glucuronidation, and possibly sulfation, is involved in the detoxification process of OTα (Duarte et al 2011;Klapec et al 2012;Munoz et al 2017).…”

Mycotoxin Biomarkers of Exposure: A Comprehensive Review

“…However, phase II metabolites of OTA were also observed in biological fluids such as urine, serum, and milk. Glucuronidated conjugates of OTA were more likely to be present in animal and human urine, as after enzymatic hydrolysis of the urine samples the levels of OTA and OTα were considerably (up to 6‐fold) higher (Muñoz, Cramer, Dopstadt, Humpf, & Degen, ; Pena, Seifrtova, Lino, Silveira, & Solich, ; Solfrizzo, Gambacorta, Lattanzio, Powers, & Visconti, ). Although there was clear evidence of OTA‐glucuronides, no glucuronidated forms have been identified yet.…”

“…Ochratoxin A‐8‐β‐glucuronide (OTA‐8‐glucuronide) and open lactone‐ochratoxin A‐8‐β‐glucuronide (open lactone OTA‐8‐glucuronide) have been synthesized as reference standards, but they have not been observed in human urine. Probably this phenomenon was attributed to a low sensitivity and ionization efficiency revealed in spiked analysis by LC‐MS/MS compared to OTA (Muñoz et al., ).…”