A Blood Spot Method for Detecting Fumonisin-Induced Changes in Putative Sphingolipid Biomarkers in LM/Bc Mice and Humans

Authors: Riley, Ronald; Showker, Adele; LEE, CHRISTINE - University Of Georgia; ZIPPERER, CODY - University Of Georgia; Mitchell, Trevor; Voss, Kenneth; ZITOMER, NICHOLAS - Former ARS Employee; TORRES, OLGA - National Institute Of Public Health (INSP); MATUTE, JORGE - National Institute Of Public Health (INSP); GREGORY, SIMON - Duke University Medical Center; ASHLEY-KOCH, ALLISON - Duke University Medical Center; MADDOX, JOYCE - Creighton University; GARDNER, NICOLE - Creighton University; GELINEAU-VAN WAES, JANEE - Creighton University

Submitted to: Journal of Food Additives & Contaminants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/2/2015
Publication Date: 4/10/2015
Citation: Riley, R.T., Showker, A.J., Lee, C.M., Zipperer, C.E., Mitchell, T.R., Voss, K.A., Zitomer, N.C., Torres, O., Matute, J., Gregory, S.G., Ashley-Koch, A.E., Maddox, J.R., Gardner, N., Gelineau-Van Waes, J. 2015. A Blood Spot Method for Detecting Fumonisin-Induced Changes in Putative Sphingolipid Biomarkers in LM/Bc Mice and Humans. Journal of Food Additives & Contaminants. 32(6):934-949.

Interpretive Summary: Fumonisins (FB) are mycotoxins found in maize. They have been hypothesized to be potential environmental risk factors for neural tube defects (NTDs) in humans living in areas of the world where maize is a dietary staple and infection with Fusarium verticillioides is likely. In LM/Bc mice, FB1-treatment of pregnant dames 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 potential biomarker for FB1 inhibition of ceramide synthase in humans. Collection of blood spots on absorbent paper from finger sticks is a relatively non-invasive and acceptable way to obtain blood samples for biomarkers analysis. One short coming of blood spots is that the volume of blood collected can be variable. The objective of the present study was to develop a method to estimate the actual volume of blood collected as blood spots on absorbent paper so as to allow quantification of the mass amount of sphingoid base 1-phosphates normalized to blood volume. To accomplish this objective, known amounts of blood were collected from male LM/Bc mice and human volunteers and applied to two types of absorbent paper. The dried blood spots were extracted with acetonitrile:water 5% formic acid and the sphingoid base 1-phosphates, absorbance maximum at 270 nm (A270), and total protein content of the extracts (Bradford) were determined. The results show that the A270, total protein, and blood volume are closely correlated and that the volume of blood spotted can be calculated accurately and directly (without additional reagents) using only the A270 of the extracts.

Technical Abstract: Fumonisin B1 (FB1) is a toxic chemical produced by molds. The molds that produce fumonisin are common in corn. Consumption of contaminated corn by farm animals has been shown to be the cause of disease. Fumonisin has been hypothesized to be an environmental risk factor for diseases in humans in countries where corn is a dietary staple and infection with the mold is likely. In order to determine if fumonisin contributes to disease in humans, we have developed methods to measure changes in the urine and blood levels of chemicals that are indicators of changes indicative of pre-disease states. For measuring these changes we use very small volumes of blood collected on absorbent paper. Collection of blood spots from finger sticks is a relatively non-invasive and acceptable way to obtain blood samples. One short coming of blood spots is that the volume of blood collected can be variable. The objective of the present study was to develop a method to estimate the actual volume of blood collected on the absorbent paper so as to allow quantification of the amounts and concentrations of the chemical indicators (biomarkers) in the blood. To accomplish this objective, known amounts of blood were collected from male mice and human volunteers and applied to two types of absorbent paper. The biomarkers were extracted from the blood spots and it was found that the total blood volume in each blood spot could be accurately estimated based on a simple process that estimated the total protein in the blood spot which was proportional to the blood volume applied. This procedure will be used to assess the concentration of fumonisin biomarkers in humans and to relate the levels of the biomarkers to specific disease states.