A blood spot method for detecting fumonisin-induced changes in putative sphingolipid biomarkers in LM/Bc mice and humans

Abstract

Fumonisins (FB) are mycotoxins found in maize. They are hypothesised risk factors for neural tube defects (NTDs) in humans living where maize is a dietary staple. In LM/Bc mice, FB₁-treatment of pregnant dams induces NTDs and results in increased levels of sphingoid base 1-phosphates in blood and tissues. The increased level of sphingoid base 1-phosphates in blood is a putative biomarker for FB₁ inhibition of ceramide synthase in humans. Collection of blood spots on paper from finger sticks is a relatively non-invasive way to obtain blood for biomarker analysis. The objective of this study was to develop and validate in an animal model, and ultimately in humans, a method to estimate the volume of blood collected as blood spots on absorbent paper so as to allow quantification of the molar concentration of sphingoid base 1-phosphates in blood. To accomplish this objective, blood was collected from unexposed male LM/Bc and FB₁-exposed pregnant LM/Bc mice and humans and applied to two types of absorbent paper. The sphingoid base 1-phosphates, absorbance at 270 nm (A₂₇₀), and total protein content (Bradford) were determined in the acetonitrile:water 5% formic acid extracts from the dried blood spots. The results show that in both mouse and human the A₂₇₀, total protein, and blood volume were closely correlated and the volume of blood spotted was accurately estimated using only the A₂₇₀ of the extracts. In mouse blood spots, as in tissues and embryos, the FB₁-induced changes in sphingolipids were correlated with urinary FB₁. The half-life of FB₁ in the urine was short (<24 h) and the elevation in sphingoid base 1-phosphates in blood was also short, although more persistent than the urinary FB₁.

Keywords:

• sphingolipid
• fumonisin
• biomarkers
• LC-MS
• blood spots
• maize
• corn
• sphinganine 1-phosphate

Acknowledgements

The authors thank Ms. Melinda Vongkunthong for her assistance in mouse model development. We also thank all the women and men who participated in the human studies, the Ministry of Health of Guatemala, the leaders in the communities of Chimaltenango and Escuintla and the leadership of the USDA-ARS South Atlantic Area and Russell Research Center without whose cooperation these studies could not have been performed. The authors also thank Adela Ruiz, Rosa Chovix and Waldemar González for the field work and sample collection in Guatemala, Marta María Méndez, Cecilia de Mayorga, Luis Rodríguez and Flor Díaz for the processing of samples in Guatemala. For all animal studies Institutional Animal Care and Use Committee approved protocols were followed and for human studies Institutional Review Board-approved protocols were followed.

Additional information

Funding

This work was supported by USDA-ARS NP108 in house project 6612-42000-012-00D and Award Number RC4HD067971-01 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Eunice Kennedy Shriver National Institute of Child Health & Human Development or the National Institutes of Health.