Phospholipase A2 products predict the hematopoietic support capacity of horse serum.

Horse serum is commonly used as an additive to support the maintenance of hematopoietic progenitor cells in culture. However, the wide variability in the performance of different lots calls for parallel testing of multiple batches over extended periods of culture. Identification of the serum components that determine hematopoietic support would therefore save considerable time and effort and would help to standardize culture procedures. We report here that the ability of horse serum to support the self-renewal of multipotent murine hematopoietic progenitor FDCP-Mix cells is correlated to the concentration of specific fatty acid products of phospholipase A2 and more closely to the spectrum of eicosanoids generated by their further processing through cyclooxygenase and lipoxygenase pathways. Supportive sera have low levels of lysophosphatidylcholine and inflammatory eicosanoids. This links known markers of inflammation, infection and platelet activation to the ability of serum to maintain progenitor cells in an undifferentiated state, providing a means for prospective identification of suitable sera as well as quality control of the production process.

Determination of the Phosphatidylcholine/Lysophosphatidylcholine Ratio in Intact Serum by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry with Prior Enzymatic Albumin Digestion.

Lysophosphatidylcholine (lysoPtdCho) is a well-known biomarker in body fluids for inflammation and oxidative stress and provides a possible clinical screening marker for certain diseases where inflammation is involved. It was shown in our previous article that the measurement of intact serum using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) provides the phosphatidylcholine (PtdCho)/lysoPtdCho ratios faster than the measurements after organic extraction, while the standard deviations of those "intact" measurements are even smaller. Surprisingly, the PtdCho/lysoPtdCho ratio is about two times higher in the intact serum MALDI-TOF MS measurement than in the MALDI-TOF MS analysis of the organic extracts. Albumin binding of lysoPtdCho seems to be a very likely reason for increased PtdCho/lysoPtdCho ratios in the intact serum measurements. In this article, this hypothesis is tested on horse serum as a biological sample. Albumin (equine and bovine) addition to serum shows an increase in the PtdCho/lysoPtdCho ratio detected by MALDI-TOF MS. Further experiments with a comparable lipid model suspension verify that pepsin and trypsin are able to liberate the bound lipids. Under different conditions, the effects of both enzymes on the lipid model suspension are compared. Finally, an improved MALDI-TOF MS measurement of the PtdCho/lysoPtdCho ratio in intact serum after a prior pepsin digestion step was established. As is known that lysoPtdCho is cytotoxic and albumin is capable of decreasing this cytotoxicity by binding lysoPtdCho, this study proposes to consider both PtdCho/lysoPtdCho ratios-with and without albumin-bound lysoPtdCho-that could be superior diagnostic markers for inflammation and oxidative stress.