Sphingosine-1-phosphate in the lymphatic fluid determined by novel methods

BACKGROUND: Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that regulates many physiological and pathological processes. It has been suggested that S1P gradient with high concentrations in the blood and lymphatic fluid and low concentrations in the peripheral tissue plays important roles in immune cell trafficking and potentially cancer progression. However, only a few reports have assessed S1P levels in the lymphatic fluid due to lack of an established easy-to-use method. Here, we report a simple technique for collection of lymphatic fluid to determine S1P. MATERIALS AND METHODS: Lymphatic fluid was collected directly with a catheter needle (classical method) or was absorbed onto filter paper after incision of cisterna chyli (new method) in murine models. Blood, lymphatic fluid and mesenteric lymph nodes were corrected from wild type and sphingosine kinase 2 (SphK2) knockout mice to determine S1P levels by mass spectrometry. RESULTS: The volume of lymphatic fluid collected by the new method was at least three times greater than those collected by the classical method. S1P concentrations in lymphatic fluid are lower than in blood and higher than in lymph nodes. Interestingly, S1P levels in lymphatic fluid from SphK2 knockout mice were significantly higher than those in wild type, suggesting an important role of SphK2 and/or SphK1 to regulate S1P levels in lymphatic fluid. CONCLUSIONS: In agreement with the previous theory, our results confirm "S1P gradient" among blood, lymphatic fluid and peripheral lymphatic tissues. Convenient methods for collection and measurement of sphingolipids in lymphatic fluid are expected to provide new insights on functions of sphingolipids.