105 NEAR-INFRARED SPECTROSCOPY AND AQUAPHOTOMICS ANALYSIS OF SERUM FROM MARES EXPOSED TO THE FUNGAL MYCOTOXIN ZEARALENONE

Abstract

Aquaphotomics is a branch of near-infrared (NIR) spectroscopy in which bond vibrations from organic molecules and water create unique spectral absorbance patterns to profile complex aqueous mixtures. Aquaphotomics has been shown to detect virus infected soybean plants from extracts, classify probiotic bacteria, and detect contamination of aquatic ecosystems. We have used aquaphotomics to characterise serum profiles from horses in various phases of the reproductive cycle such as oestrus and diestrus. Because serum is a complex solution of biomolecules, various modes of serum processing (e.g. large protein removal for proteomics or mass spectrometry) may provide different NIR spectral profiles for quantitative analysis of specific compounds or their effects. Zearalenone is a fungal mycotoxin that may have estrogenic potential in mares and is found in feedstuffs. The objectives of this study were to (1) establish NIR spectral profiles of serum and protein-precipitated serum (PPS) collected at peak oestrus from mares; (2) determine if NIR profiles correlate and quantify E₂ concentrations in serum or PPS; and (3) determine if NIR can detect differences in serum chemistry of zearalenone-treated mares. Mares were fed zearalenone daily at low (2 mg, 2 mares, 5 cycles) and high (8 mg, 1 mare, 3 cycles) concentrations, plus control (0 mg, 1 mare, 3 cycles). Oestrus cycles were monitored by ultrasound and serum hormone analysis. Serum collected at peak oestrus had E₂ values determined by radioimmunoassay (range 0.02–16.87 pg mL⁻¹). Protein precipitated serum had high and medium MW proteins removed with acetonitrile. NIR spectra, collected in triplicate with a 1 mm quartz cuvette and ASD FieldSpec^®3 (Boulder, CO, USA), were pre-treated with a Savitsky-Golay 1^st derivative for inspection of spectral features, principal component analysis, and partial least-squares regression (PLS) to investigate spectral correlations to E₂ concentrations and zearalenone treatment effects. The NIR profiles contrasting serum and PPS at oestrus had distinct spectral features differing significantly at 1320, 1491, 1536, and 1566 nm in the NIR water spectrum, and principal component-1 accounted for 97% of principal component analysis variance in spectra from serum compared to PPS. In the PLS cross-validation linear fit regression model, NIR predicted E₂ concentrations (validated by RIA) from serum (slope = 0.89, SECV = 1.92, R² = 0.81, 3 factors), and from PPS (slope = 0.61; SECV = 1.84, R² = 0.76, 4 factors). Spectral predictions were poorest at the low E₂ threshold, E₂ = 0.02 pg mL⁻¹. The PLS model validation metrics of zearalenone dose-dependent effects were also evident in serum (slope = 0.88, SECV = 1.26, R² = 0.86) and in PPS (slope = 0.67, SECV = 1.96, R² = 0.66). Correlations of quantitative values of E₂ and zearalenone were both better for spectra taken of serum compared to PPS. In summary, NIR spectral profiles of serum chemistry may be able to map E₂ hormone levels during reproductive cycling, and these spectra may also have correlations that reflect exposure of mares to estrogenic toxins such as zearalenone.

Research was supported by USDA-ARS Biophotonics grant #58-6402-3-018.