Pavel A. Aronov

Urine Metabolomics Analysis for Kidney Cancer Detection and Biomarker Discovery

Renal cell carcinoma (RCC) accounts for 11,000 deaths per year in the United States. When detected early, generally serendipitously by imaging conducted for other reasons, long term survival is generally excellent. When detected with symptoms, prognosis is poor. Under these circumstances, a screening biomarker has the potential for substantial public health benefit. The purpose of this study was to evaluate the utility of urine metabolomics analysis for metabolomic profiling, identification of biomarkers, and ultimately for devising a urine screening test for RCC. Fifty urine samples were obtained from RCC and control patients from two institutions, and in a separate study, urine samples were taken from 13 normal individuals. Hydrophilic interaction chromatography-mass spectrometry was performed to identify small molecule metabolites present in each sample. Cluster analysis, principal components analysis, linear discriminant analysis, differential analysis, and variance component analysis were used to analyze the data. Previous work is extended to confirm the effectiveness of urine metabolomics analysis using a larger and more diverse patient cohort. It is now shown that the utility of this technique is dependent on the site of urine collection and that there exist substantial sources of variation of the urinary metabolomic profile, although group variation is sufficient to yield viable biomarkers. Surprisingly there is a small degree of variation in the urinary metabolomic profile in normal patients due to time since the last meal, and there is little difference in the urinary metabolomic profile in a cohort of pre- and postnephrectomy (partial or radical) renal cell carcinoma patients, suggesting that metabolic changes associated with RCC persist after removal of the primary tumor. After further investigations relating to the discovery and identity of individual biomarkers and attenuation of residual sources of variation, our work shows that urine metabolomics analysis has potential to lead to a diagnostic assay for RCC.

Regulation of Ceramide Biosynthesis by TOR Complex 2

Ceramides and sphingoid long-chain bases (LCBs) are precursors to more complex sphingolipids and play distinct signaling roles crucial for cell growth and survival. Conserved reactions within the sphingolipid biosynthetic pathway are responsible for the formation of these intermediates. Components of target of rapamycin complex 2 (TORC2) have been implicated in the biosynthesis of sphingolipids in S. cerevisiae; however, the precise step regulated by this complex remains unknown. Here we demonstrate that yeast cells deficient in TORC2 activity are impaired for de novo ceramide biosynthesis both in vivo and in vitro. We find that TORC2 regulates this step in part by activating the AGC kinase Ypk2 and that this step is antagonized by the Ca2+/calmodulin-dependent phosphatase calcineurin. Because Ypk2 is activated independently by LCBs, the direct precursors to ceramides, our data suggest a model wherein TORC2 signaling is coupled with LCB levels to control Ypk2 activity and, ultimately, regulate ceramide formation.

Vitamin D Intake Needed to Maintain Target Serum 25-Hydroxyvitamin D Concentrations in Participants with Low Sun Exposure and Dark Skin Pigmentation Is Substantially Higher Than Current Recommendations

Cutaneous cholecalciferol synthesis has not been considered in making recommendations for vitamin D intake. Our objective was to model the effects of sun exposure, vitamin D intake, and skin reflectance (pigmentation) on serum 25-hydroxyvitamin D (25[OH]D) in young adults with a wide range of skin reflectance and sun exposure. Four cohorts of participants (n = 72 total) were studied for 7-8 wk in the fall, winter, spring, and summer in Davis, CA [38.5 degrees N, 121.7 degrees W, Elev. 49 ft (15 m)]. Skin reflectance was measured using a spectrophotometer, vitamin D intake using food records, and sun exposure using polysulfone dosimeter badges. A multiple regression model (R(2) = 0.55; P < 0.0001) was developed and used to predict the serum 25(OH)D concentration for participants with low [median for African ancestry (AA)] and high [median for European ancestry (EA)] skin reflectance and with low [20th percentile, approximately 20 min/d, approximately 18% body surface area (BSA) exposed] and high (80th percentile, approximately 90 min/d, approximately 35% BSA exposed) sun exposure, assuming an intake of 200 iu/d (5 ug/d). Predicted serum 25(OH)D concentrations for AA individuals with low and high sun exposure in the winter were 24 and 42 nmol/L and in the summer were 40 and 60 nmol/L. Corresponding values for EA individuals were 35 and 60 nmol/L in the winter and in the summer were 58 and 85 nmol/L. To achieve 25(OH)D > or =75 nmol/L, we estimate that EA individuals with high sun exposure need 1300 iu/d vitamin D intake in the winter and AA individuals with low sun exposure need 2100-3100 iu/d year-round.

Role of soluble epoxide hydrolase phosphatase activity in the metabolism of lysophosphatidic acids

The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), which has two distinct enzyme activities: epoxide hydrolase (Cterm-EH) and phosphatase (Nterm-phos). The Cterm-EH is involved in the metabolism of epoxides from arachidonic acid and other unsaturated fatty acids, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, inflammation and pain. While recent findings suggested complementary biological roles for Nterm-phos, its mode of action is not well understood. Herein, we demonstrate that lysophosphatidic acids are excellent substrates for Nterm-phos. We also showed that sEH phosphatase activity represents a significant (20-60%) part of LPA cellular hydrolysis, especially in the cytosol. This possible role of sEH on LPA hydrolysis could explain some of the biology previously associated with the Nterm-phos. These findings also underline possible cellular mechanisms by which both activities of sEH (EH and phosphatase) may have complementary or opposite roles.

Identification of two epoxide hydrolases in Caenorhabditis elegans that metabolize mammalian lipid signaling molecules

We have identified two genes in the genomic database for Caenorhabditis elegans that code for proteins with significant sequence similarity to the mammalian soluble epoxide hydrolase (sEH). The respective transcripts were cloned from a mixed stage cDNA library from C. elegans. The corresponding proteins obtained after recombinant expression in insect cells hydrolyzed standard epoxide hydrolase substrates, including epoxyeicosatrienoic acids (EETs) and leukotoxins (EpOMEs). The enzyme activity was inhibited by urea-based compounds originally designed to inhibit the mammalian sEH. In vivo inhibition of the enzymes using the most potent of these compounds resulted in elevated levels of the EpOMEs in the nematode. These results suggest that the hydrolases are involved in the metabolism of possible lipid signaling molecules in C. elegans.

Soluble Epoxide Hydrolase Homologs in Strongylocentrotus purpuratus Suggest a Gene Duplication Event and Subsequent Divergence

The mammalian soluble epoxide hydrolase (sEH) is a multidomain enzyme composed of C- and N-terminal regions that contain active sites for epoxide hydrolase (EH) and phosphatase activities, respectively. We report the cloning of two 60 kDa multidomain enzymes from the purple sea urchin Strongylocentrotus purpuratus displaying significant sequence similarity to both the N- and C-terminal domains of the mammalian sEH. While one urchin enzyme did not exhibit EH activity, the second enzyme hydrolyzed several lipid messenger molecules metabolized by the mammalian sEH, including the epoxyeicosatrienoic acids. Neither of the urchin enzymes displayed phosphatase activity. The urchin EH was inhibited by small molecule inhibitors of the mammalian sEH and is the likely ancestor of the enzyme. Sequence comparisons suggest that the urchin sEH homologs are the result of a gene fusion event between a gene encoding for an EH and a gene for an enzyme of undetermined function. This fusion event was followed by a duplication event to produce the urchin enzymes.