Figures
Figure 1. Graphical abstract. Plasma lipid and hormone profiling in a polycystic ovarian syndrome (PCOS) rat model. PCOS symptoms include excessive body or facial hair, irregular periods, reduced fertility, and reoccurring pregnancy loss. To validate the use of rat models for PCOS, we have optimized a mass spectrometry-based targeted lipidomics workflow. Our work provides new insights into altered lipid and hormone profiles (red boxes - increased, blue boxes - decreased abundance) associated with human disease, recapitulated partially in PCOS rat models.
Figure 2. Optimized lipid chromatography and experimental workflow for the analysis of steroids, steroid hormones and bioactive lipids in rat plasma. (a) The dynamic multiple reaction monitoring (dMRM) chromatograms for both quantitation and qualification ions for all the lipids analyzed with this methodology. The lipids are analyzed at the same on-column concentration of 300 fmol/L, the difference in counts for each lipid highlights the differences in ionization efficiencies of the lipids (molecular structures of analyzed lipids are in Supplementary Fig. 1, jem.elmerpub.com). (b) Analyses were conducted using an ultra-high-performance liquid chromatography-dMRM-mass spectrometry (UPLC-dMRM-MS) analysis. Rats were treated with either letrozole (LET) or dihydrotestosterone (DHT) to induce polycystic ovarian syndrome (PCOS)-like physiology. At age 49 days, blood was taken from rats, and plasma was obtained by lithium-heparin separation. Lipid extraction from plasma was performed with methanol/methyl-tert-butyl-ether (50:50, v:v). Lipid extracts were subsequently dried and reconstituted into methanol prior to LC-MS analysis.
Figure 3. Rat plasma profiles modulated by letrozole (LET) and dihydrotestosterone (DHT). (a) Analysis of lipids in rat plasma after LET treatment. LET induced polycystic ovarian syndrome (PCOS) in rats modulates steroid hormone biosynthesis, steroid hormone degradation, eicosanoid and sphingolipid pathways. The letrozole control (LET-C) is on the left and the LET treated is on the right. Black dots are each data point (technical triplicate of biological triplicates, n = 9). The statistical analysis is the two-sample (or unpaired) t-test. A P-value less than 0.05 (*) suggests that the results have highly significant differences. If there is no significant difference, it is not labelled. Values for mean, standard deviation (SD) and population number (n) are described in the Supplementary Materials section. (b) Analysis of lipids in rat plasma after DHT treatment. DHT induced PCOS in rats modulates steroid hormone biosynthesis, steroid hormone degradation, eicosanoid and sphingolipid pathways. Graphs represent the mean (line) with the standard error of the mean (error bars). The DHT control is on the left and the DHT treated is on the right. Black dots are each data point (technical triplicate of biological triplicates, n = 9). The statistical analysis is the two-sample (or unpaired) t-test. A P-value less than 0.05 (*) suggests that the results have highly significant differences. If there is no significance difference, it is not labelled. Values for mean, standard deviation (SD) and population number (n) are described in the Supplementary Materials section. (c) Analysis of lipids in rat plasma after LET or DHT treatment. Mean percentage change of lipids in rat plasma, not-treated control (control) and treated with LET or DHT. Heat maps display normalized lipid abundances relative to the mean concentration of the respective non-treated control samples (control), which is represented by the white boxes. For LET or DHT treatment, blue results are for a decrease in concentration, in percentage, white is for no change and red is for an increase in concentration in percentage.
Figure 4. Letrozole (LET) affects metabolic networks. LET treatment pathway analysis. Lipid profile after treatment with LET. Converting enzymes are in boxes and the lipids in this study are circled. Lipids circled in grey have either no change compared to non-treated control rat plasma or were not detected with this methodology; lipids circled in blue have a decreased concentration compared to non-treated control rats; and lipids circled in red have an increased concentration compared to non-treated control rats. Lipids with a significant difference between non-treated control and LET treated (a P-value of less than 0.05) are indicated by a black star and inhibition of CYP19A activity by LET with a black cross.
Figure 5. Dihydrotestosterone (DHT) affects metabolic networks. DHT treatment pathway analysis. Lipid profile after treatment with DHT. Converting enzymes are in boxes and the lipids in this study are circled. Lipids circled in grey have either no change compared to non-treated control rat plasma or was not detected with this methodology; lipids circled in blue have a decreased concentration compared to non-treated control rats; and lipids circled in red have an increased concentration compared to non-treated control rats. Lipids with a significant difference between non-treated control and DHT treated (a P-value of less than 0.05) are indicated by a black star.