Development of a shotgun lipidomics approach for analysis of lipids in perennial ryegrass : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Chemistry at Massey University, Manawatu, New Zealand

Mass Spectrometry is a powerful analytical tool which is used for identification and quantitation of compounds within samples for a variety of sample matrices. One example of this is to look into the lipid profile (lipidome) of perennial ryegrass (lolium perenne). The lipid profile tells us many things about the inner workings of rye grass which can lead to better understanding of mechanisms behind desirable traits (such as lipid quantity and composition). Traditional high performance liquid chromatography (HPLC) is the most widely used chromatographic technique when researching into the lipidome of different plants (Burgos et al., 2011; Chen, Markham, & Cahoon, 2012; Degenkolbe et al., 2012). Shotgun lipidomics applies major principles of the traditional methods but differs in the delivery of the sample to the Mass Spectrometer and data analysis; providing considerable advantages, disadvantages and challenges. A shotgun lipidomics method for analysing the lipids in perennial ryegrass has been developed. This involved first determining the most efficient extraction protocol and then establishing a methodology (based on one found in the literature for animal samples) for shotgun lipidomic analysis of perennial ryegrass. The shotgun data was problematic to analyse using traditional methods so LCMS data was investigated and the results were transferred to the shotgun data. Investigation was conducted to find the limiting factor for the analysis of the shotgun data. This limiting factor was found to be pheophytin a and other chlorophyll derivatives. The high abundance and ion suppression effects attributed to pheophytin a and other chlorophyll derivatives contributed to unfavourable conditions for analysing the lipidome of perennial ryegrass. The major outcomes of this study are the annotation of 118 lipids in perennial ryegrass using LCMS, with 27 of those being found in the shotgun data also and also the understanding of the limitations of using shotgun techniques for perennial ryegrass. With this understanding further research can be conducted to enhance the methodologies detailed herein

Rolling cans: the answer

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Short-term hematological effects upon completion of a four-week simulated altitude camp

Hemoglobin mass (tHb) is considered to be a main factor for sea-level performance after “live high–train low” (LHTL) altitude training, but little research has focused on the persistence of tHb following cessation of altitude exposure. The aim of the case study was to investigate short-term effects of various hematological measures including tHb upon completion of a simulated altitude camp. Five female cyclists spent 26 nights at simulated altitude (LHTL, 16.6 ± 0.4 h/d, 3000 m in an altitude house) where tHb was measured at baseline, at cessation of the camp, and 9 d thereafter. Venous blood measures (hemoglobin concentration, hematocrit, %reticulocytes, serum erythropoietin, ferritin, lactate dehydrogenase, and haptoglobin) were determined at baseline; on day 21 during LHTL; and at days 2, 5, and 9 after LHTL. Hemoglobin mass increased by 5.5% (90% confidence limits [CL] 2.5 to 8.5%, very likely) after the LHTL training camp. At day 9 after simulated LHTL, tHb decreased by 3.0% (90%CL −5.1 to −1.0%, likely). There was a substantial decrease in serum EPO (−34%, 90%CL −50 to −12%) at 2 d after return to sea level and a rise in ferritin (23%, 90%CL 3 to 46%) coupled with a decrease in %reticulocytes (−23%, 90%CL −34 to −9%) between day 5 and 9 after LHTL. Our findings show that following a hypoxic intervention with a beneficial tHb outcome, there may be a high probability of a rapid tHb decrease upon return to normoxic conditions. This highlights a rapid component in red-cell control and may have implications for the appropriate timing of altitude training in relation to competition