Plasma metabolomic profiles reveal sex- and maturation-dependent metabolic strategies in sea lamprey (Petromyzon marinus)

Plasma metabolomic profiles reveal sex- and maturation-dependent metabolic strategies in sea lamprey (Petromyzon marinus) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Plasma metabolomic profiles reveal sex- and maturation-dependent metabolic strategies in sea lamprey (Petromyzon marinus) Sonam Tamrakar, Belinda Huerta, Yu-Wen Chung-Davidson, Weiming Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1606386/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Introduction Adult sea lamprey ( Petromyzon marinus ) cease feeding and migrate to spawning streams where males build nests, undergo final sexual maturation, and subsequently produce and release large quantities of bile acid pheromones that attract mature females. These animals are predicted to rearrange their metabolic pathways drastically to support their reproductive strategies, presenting advantageous opportunities to examine how sex and the maturation processes affect metabolism. Objectives The objective is to investigate the metabolic differences between sexes and maturation states in sea lamprey that support changes in physiological functions. Methods We compared plasma metabolomes of spawning and prespawning sea lamprey in both sexes using both non-targeted and targeted metabolomics approaches using UPLC/MS-MS with electrospray ionization in both positive and negative modes. The data were processed using Progenesis QI, Compound Discoverer and XCMS softwares for alignment, peak picking, and deconvolution of the peaks. Principle component analyses (PCA) and partial least squares discriminant analyses (PLS-DA) were performed using SIMCA and Metaboanalyst softwares to identify discriminating features, followed by fragmentation matching with extensive database search and pathway mapping. Results The pheromonal bile acid biosynthesis was upregulated significantly in males compared to females. Spermiating males further upregulated bile acid biosynthesis by altering amino acid metabolisms, upregulating cofactors and nucleotide metabolisms, but downregulating carbohydrate and energy metabolisms. Conclusion Plasma metabolomes are sex- and maturation-dependent and reflect the special metabolic demands at each life stage and reproductive strategy. bile acid biosynthesis targeted analyses untargeted analyses metabolic pathways sexual selection agnathan Figures Figure 1 Figure 2 1 Introduction Adult sea lamprey ( Petromyzon marinus ) must rearrange their metabolomes drastically to support the demand for reproduction after they cease feeding. One of the pronounced physiological changes during their spawning migration is that they stop feeding and the lipid reserves function as the primary energy source during this period (Sheridan 1988 ; Bird et al. 1993 ). It provides a unique opportunity to study how animals alter metabolic processes to mobilize energy reserves to fulfill biological functions. In early spring adult sea lamprey cease feeding and migrate to spawning grounds (Applegate, 1951 ) where sexually mature spermiating males (SM) release large quantities of bile acid pheromones that attract ovulatory females (OF) to the nest for spawning (Buchinger et al., 2015 ; Chung-Davidson et al., 2010 ; Fissette et al., 2021 ; Johnson et al., 2006 , 2012; Li et al., 2002 ; Scott et al., 2019 ). One SM can release up to 0.5 mg/h of a major component of the male sex pheromone, 3-keto petromyzonol sulfate (3kPZS; Li et al., 2002 ). On the contrary, females and PSM only release trace or non-detectable amounts of bile acids (Chung-Davidson et al., 2021 ). Since bile acid pheromones are synthesized in the liver, transported via blood circulation to the gills, and released into the water (Brant et al., 2013 ; Fissette et al., 2021 ; Li et al., 2002 ; Siefkes et al., 2003 ), comparing sea lamprey plasma metabolomes between sexes and maturation states may help to infer metabolic strategies that sustain high pheromone production and release in SM. The intricate networks of metabolic pathways are coordinated to meet the needs of the whole organism, i.e. , to generate ATP for energy consumption and the building blocks for biosynthesis. ATP is generated by oxidation of fuel molecules such as glucose, fatty acids and amino acids, whereas the building blocks for biosynthesis are usually intermediates from various metabolic pathways (Stryer, 1995 ). We expect that SM employ effective metabolic strategies to generate ATP and building blocks for massive bile acid biosynthesis. Since blood plasma mediate biological functions between organs, plasma metabolomes provide a proxy for the metabolic status of an organism. We hypothesize that adult sea lamprey alter metabolic pathways to facilitate the different biological functions necessitated by sex and maturation status, resulting in dramatic differences in plasma metabolomes in preovulatory females (POF), PSM, OF and SM. Using both non-targeted and targeted approaches, we found that sea lamprey exhibited distinct plasma metabolomes in different sexes and maturation states, and SM maximized pheromone production by downregulating energy metabolisms but upregulating bile acid biosynthesis. There is an apparent trade-off between sexual signals (bile acid pheromones) and energy consumption in sea lamprey. 2 Methods 2.1 Animals Migratory adult sea lampreys were collected from Ocqueoc River (Presque Isle, Michigan, USA) in the summer of 2019 by agents of the US Geological Survey at Hammond Bay Biological Station, Great Lakes Science Center, Millersburg, Michigan, USA. Pre-spawning lamprey were held in the lower Ocqueoc River (Presque Isle County, Michigan, USA) to induce sexual maturation, which was assessed daily by visual inspection of secondary sex characteristics and gentle expression of gametes (Brant et al., 2013 ). All animals were then transferred to Michigan State University (East Lansing, Michigan, USA) where samples were collected immediately upon arrival. Standard operating procedures for transporting, maintaining, handling, anesthetizing, and euthanizing sea lampreys were approved by the Institutional Committee on Animal Use and Care of Michigan State University (AUF # Li-02-17-030-99) and in compliance with standards defined by the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, 2011 ). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. 2.2 Chemicals HPLC grade methanol, acetonitrile, chloroform, and ammonium acetate, cholic acid (CA), deoxycholic acid (DCA), taurochenodeoxycholic acid (TCDCA), and MS222 were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Petromyzonol sulfate (PZS), 3-keto petromyzonol sulfate (3kPZS), petromyzonamine disulfate (PADS), petromyzonamine-24-monosulfate (PAMS-24), allocholic acid (ACA or 5α-cholic acid), 3-keto allocholic acid (3kACA) and deuterated 3-keto petromyzonol sulfate ([ 2 H 5 ]-3kPZS) were custom-synthesized from Bridge Organic, Inc. (Vicksburg, Michigan, USA). 2.3 Sample analyses 2.3.1 Sample preparation Blood samples (n = 10 each for POF, PSM, OF, and SM) were collected by cardiac puncture with 10 mL heparinized vacutainers after animals were anesthetized with 0.02% MS222. Plasma samples (supernatant) were obtained by centrifugation at 1000 x g, 4°C for 20 min. Sample proteins were precipitated by adding 600 µl of ice-cold methanol to 300 µl of plasma (2:1, v/v), incubated at -20°C overnight and centrifuged at 15800 x g at 4°C for 15 min. The resulting supernatant was mixed with 600 µl of chloroform at 4°C (on ice) at 100 rpm for 20 min, incubated at -20°C for 30 min, and then centrifuged at 10,000 x g at 4°C for 30 min. The aqueous (top) layer was freeze dried and then reconstituted in 100 µl of 50% methanol in water. Quality control (QC) samples for male and female groups were prepared separately by pooling equal volumes of PSM and SM samples for male QC, and POF and OF for female QC. 2.3.2 Untargeted analyses Samples were analyzed on a UPLC Q-Exactive Orbitrap system equipped with a heated electrospray ionization source (HESI). Chromatographic separations were optimized (detail see Supplementary Information) using an Acquity UPLC BEH C18 column (100 x 2.1 mm; 1.7 µm particle) with 10 mM ammonium acetate in water as solvent A and acetonitrile as solvent B. The injection volume was 10 µl. The mobile phase gradient was maintained as follows, 0 min: 5% B; 5 min: 38% B; 7 min: 55% B; 10 min: 70% B; 16 min: 95% B; 21 min: 100% B; 21.5 ~ 25 min: 5% B. The flow rate was maintained at 0.3 ml/min and the column temperature at 55°C throughout the analyses. Samples were analyzed in both positive and negative ionization modes using the full scan MS with data-dependent (dd)-MS2 acquisition mode. The Orbitrap parameters for the full scan acquisition were set as follows, resolution: 17,500; AGC target: 3e 6 ; maximum injection time (IT): 50 ms; scan range: 80 to 1200 m/z. The parameters for dd-MS2 were set as follows, resolution: 17,500; AGC target: 1e 5 ; IT: 50 ms; scan range: 200 to 2000 m/z; normalized collision energy: 10, 30, and 60. Source parameters were as follows, sheath gas flow rate: 48 AU; spray voltage: 3.5 kV; capillary temperature: 256°C; auxiliary gas flow rate: 11; sweep gas flow rate: 2; auxiliary gas heater temperature: 413°C. 2.3.3 Targeted analyses Nine bile acids (including 6 sea lamprey-specific bile acids) were quantified in all samples. A Waters Acquity H-Class UPLC system connected to a Xevo TQ-S Triple Quadrupole mass spectrometer was used for the analyses. Separation was achieved using a Waters BEH C18 column (2.1 x 100 mm; 1.7 µm particle size) coupled to an Acquity UPLC BEH C18 VanGuard Pre-column (2.1 x 5 mm; 1.7 µm particle size). For sea lamprey-specific bile acids (3kPZS, PZS, PADS, PAMS-24, ACA, and 3kACA), 10 mM triethylamine (TEA) in water was used as mobile phase A and methanol was used as mobile phase B. The gradient was maintained as follows, 0 min: 40% B; 7 ~ 9 min: 99% B; 10 ~ 12 min: 40% B. The flow rate was maintained at 0.25 µL/min and the column temperature at 35°C (Wang et al., 2015 ). For DCA, CA, and TCDCA, water containing 7.5 mM ammonium acetate and 0.1% formic acid was used as solvent A, and acetonitrile/methanol (1:9; v/v) containing 7.5 mM ammonium acetate and 0.1% formic acid was used as solvent B. The gradient was maintained as follows, 0 min: 40% B; 7 min: 74% B; 7.01 ~ 9 min: 100% B; 9.01 ~ 12 min: 40% B. The flow rate was maintained at 0.25 µL/min and the column temperature at 30°C. The injection volume was 10 µL in both cases (Li et al., 2015 ). All bile acids were analyzed by electrospray ionization in the negative mode. The ESI-MS/MS parameters were set as follows, capillary voltage: 2.60 kV; extractor voltage: 5 V; source temperature: 150°C; desolvation temperature: 500°C; desolvation gas flow: 800 L/h (N 2 , 99.9% purity). Argon (99.9% purity) was introduced as the collision gas into the collision cell at a flow rate of 0.15 mL/min. The multiple reaction monitoring (MRM) and other parameters are provided in Supplementary Information (Table S1). MassLynx 4.2 software was used for data acquisition and data were processed using TargetLynx XS (Waters Corp., Milford, MA, USA). 2.4 Data processing 2.4.1 Untargeted analyses The workflow applied for data processing is illustrated in Fig. S1. Progenesis QI and Compound Discoverer 3.1 softwares were used to maximize the number of metabolite identification. Briefly, the spectra were selected from the raw data and then aligned with a retention time (RT) tolerance of 0.2 min and mass error of 5 ppm. The discriminating features were selected based on the ANOVA p-value ( 1.2). Compound annotations of significant metabolites were based on the fragment matching with available databases, especially Human Metabolome Database (HMDB) ( https://hmdb.ca/ ) and KEGG ( https://www.genome.jp/kegg ). Principal component analyses (PCA) and partial least squares discriminant analyses (PLS-DA) were performed by exporting the data to SIMCA 17 software (Umetrics, Sweden). Raw files were converted to .mzXLM files using ProteoWizard (MS Converter; Chambers et al., 2012 ). Converted files were uploaded into an online platform XCMS (Smith et al., 2006 ) to produce a data matrix following the same discrimination criteria as described above (p-value 1.2, and abundance > 10,000). 2.4.2 Targeted analyses Data were processed using MassLynx 4.2 software (Waters Corp., Milford, MA, USA). The software produced a table with m/z, RT, and intensity (peak area) values for each variable in each sample. Linear relationship calculations between peak areas and concentrations were adopted by weighted least squares regression. For targeted analyses, FC between groups was calculated as the ratio of the mean concentrations, e.g., dividing the mean concentration of SM by the mean concentration of PSM. 2.4.3 Statistical analyses Multivariate data analyses were performed to find the similarities and differences between sample groups. PCA, an unsupervised dimensionality-reduction tool, was used for feature selection and classification, i.e., to identify patterns in the data and to check the trends and outliers, using SIMCA 17 software (Umetrics, Sweden) (Smilowitz et al., 2013 ). PLS-DA, a supervised machine learning dimensionality-reduction tool, was also performed as it is suited for metabolomics data with large number of features, noise and missing data, and fewer samples than features (Ruiz-Perez et al., 2020 ). The PCA and PLS-DA models were evaluated in terms of their goodness of fit (R2Xcum, R2Ycum) and goodness of prediction (Q2cum). R2Xcum is the cumulative modeled variation in X, R2Ycum is the cumulative variation in X correlated to Y, and Q2cum estimates the cumulative predictive ability of the model. A Q2cum value > 0.5 is considered a good model for metabolomics analyses. 2.4.4 Pathway analyses Potential impacted pathways were assessed by pathway analyses using MetaboAnalyst 4.0 ( http://www.metaboanalyst.ca/ ) and the Human Metabolome Database (HMDB, https://hmdb.ca/ ) (Chong et al., 2019 ). The platform iPath v.3 was used to map the impacted pathways whenever the annotated metabolites were listed in its database (Darzi et al., 2018 ). 3. Results 3.1 Metabolomic profiles showed sex differences and dramatic changes during sexual maturation LC-HRMS analyses revealed 6554 significant features of metabolic fingerprints in ESI- mode and 7105 features in ESI + mode. PCA analyses in both negative and positive modes showed significant differences between PSM vs. SM, POF vs. OF, OF vs. SM, and POF vs. SM (Figs. 1 and S2; Table S2). The goodness of fit for these models resulted in R2X = 0.69 for male sexual maturation (PSM vs. SM), R2X = 0.715 for female sexual maturation (POF vs. OF), and sex differences in mature adults (OF vs. SM: R2X = 0.751) and immature adults (POF vs. PSM: R2X = 0.917). In each comparison, the goodness of prediction (Q2) was greater than 0.5, indicating that the model was acceptable for metabolomic analysis (Table S2). PLS-DA plots also confirmed significant differences between PSM vs. SM, POF vs. OF, OF vs. SM, and POF vs. SM (Figs. S3 and S4). The goodness of fit for each comparison (PSM vs. SM; POF vs. OF; POF vs. PSM; OF vs. SM) resulted in R2X > 0.5, R2Y > 0.8, and Q2 > 0.5 (Table S2). Discriminant analyses revealed dramatic upregulations in sea lamprey-specific bile acids in the comparisons between sexes and male sexual maturation (Table 1 ). For example, PSM contained 244-fold (↑) PZS compared to POF, SM contained 155-fold (↑) PZ and 53-fold (↑) ACA compared to OF, and SM contained 314-fold (↑) ACA and 201-fold (↑) PZ compared to PSM. Other bile acids, including cholic acid, lithocholic acid and chenodeoxycholic acid-3-sulfate were also upregulated hundreds of folds in SM compared to PSM or OF. Changes in nucleotide and amino acid metabolisms were apparent in all comparisons. SM showed increases in adenine (↑ 77-fold) but decreases in creatine (↓ 81-fold) compared to OF. On the other hand, OF showed downregulations of fatty acid metabolism compared to POF (11-dehydrothromboxane: ↓ 4-fold; palmitic acid: ↓ 2-fold). Table 1 List of metabolic pathways and the most discriminant metabolites in the comparison of different sea lamprey groups between sexes and maturation states Group Metabolism pathway Metabolite Regulation Fold Change p-value q-value PSM vs SM Secondary bile acid biosynthesis Lithocholic acid ↑ 152.9 9.66E-11 2.55E-08 - Petromyzonol sulfate ↑ 212.3 1.38E-10 3.37E-08 - Chenodeoxycholic acid 3-sulfate ↑ 264.2 1.83E-09 3.68E-07 - Petromyzonol ↑ 201.0 4.45E-09 6.49E-07 Purine metabolism Adenine ↑ 46.8 2.21E-07 4.95E-05 Amino acid metabolism 3-Hydroxypropenoate ↓ 8.4 1.63E-06 2.11E-04 Primary and Secondary bile acid biosynthesis Cholic acid ↑ 134.0 2.44E-04 2.72E-03 Secondary bile acid biosynthesis 5α-Cholic acid ↑ 313.9 1.07E-04 4.26E-03 - D-Xylulosonic acid ↑ 11.2 1.64E-04 6.07E-03 Tyrosine metabolism Tyrosine ↑ 1.9 2.61E-02 7.76E-02 Amino acid metabolism Arginine ↓ 2.2 5.64E-02 1.08E-01 Lysine degradation Lysopine ↓ 1.5 6.98E-02 1.18E-01 Amino acid metabolism - Glutathione metabolism Ornithine ↑ 1.5 1.04E-01 1.44E-01 Arginine and proline metabolism Octopine ↓ 1.2 4.52E-01 3.02E-01 Heme catabolism (bile pigment) Biliverdin ↑ 1.4 5.14E-01 3.25E-01 Purine metabolism Hypoxanthine ↑ 1.2 7.15E-01 3.97E-01 Histidine metabolism Formimino-L-glutamic acid ↑ 1.2 7.29E-01 4.01E-01 Pyrimidine metabolism Cytosine ↑ 37.2 4.70E-05 1.10E-03 Amino acid metabolism Glutamic acid ↑ 1.7 4.50E-03 2.60E-02 Histidine metabolism Methylhistidine ↑ 2.8 1.00E-02 5.10E-02 Amino acid metabolism Glutamine ↑ 4.4 7.40E-05 3.80E-03 Amino acid metabolism Glutamic acid ↑ 1.5 1.60E-03 3.30E-02 - N3,N4-Dimethyl-L-arginine ↓ 7.1 2.70E-03 1.90E-02 TCA cycle Citric acid ↓ 2.0 6.20E-04 7.10E-03 Biosynthesis of ansamycins AminoDHQ ↓ 2.1 3.03E-03 2.00E-02 Amino acid metabolism 2-Aminoadipic acid ↑ 2.1 2.10E-03 1.70E-02 Phenylalanine metabolism 2-Oxo-4-pentenoic acid ↓ 2.9 2.30E-03 4.30E-02 POF vs OF Lysine biosynthesis N-acetyl-LL-2,6-diaminopimelic acid ↑ 12.2 7.22E-07 9.39E-06 - N,N-dimethylarginine ↓ 6.4 7.83E-07 1.01E-05 - 7a,12a-Dihydroxy-3-oxo-4-cholenoic acid ↓ 11.0 4.13E-05 1.64E-04 Cysteine and methionine metabolism Methionine ↑ 4.0 1.26E-03 2.34E-03 - Tryptophan ↑ 3.0 2.21E-03 3.76E-03 - D-Xylulosonic acid ↑ 6.6 3.22E-04 6.17E-03 - N-(3-Carboxypropyl)-L-glutamine ↓ 19.5 1.96E-03 1.46E-02 - Prostaglandin C1 ↓ 2.8 2.60E-03 1.79E-02 Arachidonic acid metabolism 11-Dehydrothromboxane B2 ↓ 4.4 5.21E-03 2.06E-02 Valine, leucine, and isoleucine biosynthesis Acetyl lactic acid ↑ 4.3 5.40E-03 2.10E-02 Amino acid metabolism Creatine ↓ 4.9 2.60E-02 1.2E-01 Arginine and proline metabolism Creatinine ↓ 15.2 2.20E-02 1.1E-02 Alanine, aspartate, and glutamate metabolism Aspartic acid ↓ 9.5 1.40E-02 8.20E-02 - Glutamyl-glutamic acid ↓ 2.2 7.90E-02 2.20E-01 Pyrimidine metabolism 3-Ureidopropionic acid ↓ 2.2 1.10E-01 2.70E-01 Phenylalanine, tyrosine, and tryptophan biosynthesis Pretyrosine ↓ 13.0 1.40E-03 2.50E-02 Fatty acid metabolism Palmitic acid ↓ 1.7 3.60E-02 1.90E-01 Pyrimidine metabolism Methylmalonic acid ↓ 2.1 2.30E-01 4.40E-01 Pentose phosphate pathway (2R)-2,3-Dihydroxypropanoic acid ↓ 1.8 2.54E-02 1.60E-01 Fatty acid degradation Glutaric acid ↓ 1.8 2.40E-02 1.60E-01 - Petromyzonol ↑ 155.1 3.54E-09 3.16E-07 Purine metabolism Adenine ↑ 76.7 2.04E-07 2.08E-05 Riboflavin metabolism Riboflavin ↑ 4.9 8.20E-06 1.63E-04 TCA cycle Isocitric acid ↓ 2.1 6.50E-04 4.89E-03 OF vs SM Bile secretion Carnitine ↓ 20.9 2.12E-03 6.67E-03 Primary and Secondary bile acid biosynthesis Cholic acid ↑ 93.7 4.27E-03 1.09E-02 Pyrimidine metabolism Uridine ↓ 4.6 5.96E-03 1.57E-02 Glycine, serine, and threonine metabolism Creatine ↓ 81.0 9.32E-03 1.82E-02 Secondary bile acid biosynthesis 5a-Cholic acid ↑ 53.4 9.86E-03 2.25E-02 Biosynthesis of unsaturated fatty acids Behenic acid ↓ 1.4 6.50E-02 6.94E-02 - N-glycoloyl-beta-D-glucosamine ↑ 81.0 7.23E-02 7.54E-02 Biosynthesis of amino acids N-acetyl-LL-2,6-diaminopimelic acid ↓ 117.2 3.7E-15 1.4E-12 Norleucine ↑ 1.7 1.8E-03 1.5E-02 Biosynthesis of amino acids Tryptophan ↑ 2.8 6.5E-04 7.5E-03 POF vs PSM Tyrosine metabolism Tyramine ↑ 1.7 3.2E-02 1.0E-01 Petromyzonol sulfate ↑ 244.4 1.1E-02 5.1E-02 Riboflavin metabolism Riboflavin ↑ 4.9 9.2E-07 5.2E-05 Purine metabolism Adenine ↑ 10.2 8.7E-02 2.6E-01 3,6-Dideoxy-L-galactose ↑ 2.5 8.8E-03 6.2E-02 Down regulation (↓): SM < PSM; OF < POF; SM < OF; PSM PSM; OF > POF; SM > OF; PSM > POF. –: Not identified in KEGG. OF: ovulatory females; POF: preovulatory females; PSM: prespermiating males; SM: spermiating males. 3.2 Males reduced sugar and energy metabolisms and altered amino acid metabolisms in favor of bile acid biosynthesis during sexual maturation Pathway analyses revealed that the most prominent changes between SM and PSM were the metabolites in primary and secondary bile acid biosynthesis (Table 1 ); e.g., lithocholic acid (↑ 153-fold) and cholic acid (↑ 134-fold). Interestingly, lamprey-specific bile acids were upregulated at least 2 times more than those common bile acids (Table 1 ); e.g., ACA (5α-cholic acid ↑ 314-fold), PZ (↑ 201-fold), and PZS (↑ 212-fold). Several amino acid metabolic pathways were affected during male sexual maturation (impact > 0.15, Table S5); e.g., tyrosine (↑ 2-fold), arginine (↓ 2-fold), glutamate (↑ 2-fold), and glutamine (↑ 4-fold). Significantly changed metabolites are listed in Table 1 , and their associated primary metabolic pathways are shown in Fig. S5. In general, metabolic pathway maps indicated that SM reduced TCA cycle-related activities and carbohydrate and energy metabolisms, and at the same time increased biosynthesis of bile acids, cofactors, and vitamins (Fig. S5). On the contrary, most metabolic pathways were downregulated in OF compared to POF, including lipid metabolism (e.g., palmitate ↓ 2-fold; Tables 1 and S5, and Fig. S6). Notably, two eicosanoid metabolites were downregulated (Table 1 ); i.e., prostaglandin C1 (↓ 3-fold) and 11-dehydrothromboxane B2 (↓ 4-fold). Other notable changes were amino acid metabolisms (Table 1 ); e.g., methionine (↑ 4-fold), tryptophan (↑ 3-fold), creatine (↑ 5-fold), and pretyrosine (↓ 13-fold). Interestingly, D-aspartate but not L-aspartate were downregulated (↓ 10-fold, Table 1 ). Sex differences are most prominent in the pathways involved in biosynthesis of bile acids, cofactors, and vitamins (Tables 1 and S5, and Fig. S7: OF vs. SM and Fig. S8: POF vs. PSM), as males contained more bile acids than females (Tables S3 and S4); e.g., PZS: ↑ 244-fold in PSM vs. POF; PZ: ↑ 155-fold in SM vs. OF (Tables 1 and S5). Since bile acid biosynthetic pathways showed the most dramatic changes between sexes and during male sexual maturation, we used targeted analyses to confirm the results from untargeted analyses. Indeed, SM plasma contained the highest levels of most bile acids, except TCDCA (Tables S3 and S4). PZS was the most abundant bile acid in SM and PSM, with concentrations of 19 µg/ml and 2 µg/ml, respectively (Table S3). Upregulations of bile acids were seen during sexual maturation (PSM vs. SM and POF vs. OF, Tables S3 and S4), and the most dramatic changes were observed between sexes (males > females; OF vs. SM and POF vs. PSM; Tables S3 and S4), consistent with the untargeted analysis results. A putative sea lamprey bile acid biosynthetic pathway is shown in Fig. 2 . Higher levels of bile acids in SM vs. OF (Fig. 2 and Tables S3 and S4) were exemplified by PZS (↑ 388-fold), ACA (↑ 138-fold), and 3kACA (↑ 83-fold). For pre-spawning adults, higher levels of bile acids in PSM vs POF (Tables S3 and S4) were found in PZS (↑ 388-fold), 3kPZS (↑ 255-fold), and PAMS-24 (↑ 69-fold). ACA was probably important for sea lamprey sexual maturation since it was the most upregulated metabolite in PSM vs. SM (↑ 87-fold; Table S4) and POF vs. OF (↑ 15-fold; Table S4). 4. Discussion One striking difference was found in the comparisons of sea lamprey plasma metabolomes between sexes and maturation states, i.e., males contain hundreds-folds more bile acids than females, and SM contains the highest amounts of bile acids in all groups. Bile acids/salts are the major end metabolites of cholesterol and are important in lipid, protein, and sugar metabolisms (Hagey et al., 2010 ). The details of bile acid biosynthetic pathways are only known in mammals such as humans and rodents and have not been resolved in other species, including sea lamprey (Hagey et al., 2010 ). Based on our metabolomics results and what was available in the literature (Chiang, 2004 ; Chung-Davidson et al., 2021 ; Heubi et al., 2007 ; Pandak and Kakiyama, 2019 ; Setchell et al., 1988 ), we posited a putative sea lamprey bile acid synthetic pathway. It appears that SM has evolved a strategy that downregulates the biosynthesis of other lipid species (i.e., fatty acids and leukotrienes) and upregulates cholesterol-derived bile acid biosynthesis. At the same time, SM lowers carnitine that transports long-chain activated fatty acids into the mitochondrial matrix for β-oxidation (Stryer, 1995 ). Interestingly, citrate carries acetyl groups from mitochondria to the cytosol for fatty acid biosynthesis (Stryer, 1995 ). SM has higher bile acid levels compared to both PSM and OF, but lower citrate and 12-epi leukotriene B4 compared to PSM, and lower isocitrate compared to OF. Taken together, SM seems to lower fatty acid β-oxidation, limit other lipid biosynthesis, but maximize pheromonal bile acid biosynthesis. On the other hand, PSM swims long distance during migration and searching for suitable spawning ground, which demands ATP consumptions. There is an apparent trade-off between pheromone synthesis and other metabolic pathways, and consistent with many results in life history and chemical ecology of sea lamprey that only SM release sex pheromones that attract OF (Brant et al., 2013 ; Chung-Davidson et al., 2021 ; Li et al., 2002 ). We found that OF had lower fatty acid metabolism and lower prostaglandin C1 and 11-dehydro thromboxane B2 compared to POF. Since eicosanoid hormones such as leukotrienes, prostaglandins, and thromboxanes are derived from polyunsaturated fatty acids, it seems that OF downregulated the pathways involved in biosynthesis of the precursors (fatty acids) and the end products (prostaglandins and thromboxanes). Interestingly, arachidonic acid, a precursor for eicosanoid production that modulate steroid synthesis and spermiation during sexual maturation (Wade et al. 1994 ; Norambuena et al. 2013 ), is also critical to producing vitellogenin, a high-density lipoprotein which is synthesized in the liver and transported to the eggs (Wade et al. 1994 ; Norambuena et al. 2013 ; Tocher 2003 ). The high investment of resources for reproduction is to mobilize lipid reserves from liver and muscles to gonads during sexual maturation and spawning migration (Zaboukas et al. 2006 ; Sutharshiny and Sivashanthini 2011 ; Singh et al. 2012 ). In the sea lamprey, gonad development occurs when they stop feeding and initiate the atrophy of the digestive tract (Beamish et al., 1979). Consequently, gonadal growth and the energy expenditure for migration depend on mobilization of lipid and protein reserves accumulated during the parasitic phase (Hardisty 2006 ) and are not restored during the process of reproductive migration after feeding has ceased (Martins et al., 2019 ). A drawback of this study is that we only measured more polar molecules due to the extraction and optimized analytical methods. Further lipidomics analyses will provide more information in lipid metabolisms during sexual maturation in sea lamprey. Pheromone communication consists of two components: (1) the biosynthesis, storage, and release of the pheromones; (2) the reception and translation of the chemical message into a behavioral pattern (Kittredge and Takahashi, 1972 ). In a sexual selection context for sea lamprey, the sender is SM, broadcasting information about aspects of its quality towards rival SM and/or OF. Apparently, SM evolved metabolic strategies to produce and release massive amounts of bile acid pheromones to signal spawning. Intersexual selection through female choice is generally regarded the most important driver of signal evolution (Andersson and Iwasa, 1996 ; Ord et al., 2001 ). Indeed, OF prefer higher pheromone levels on spawning grounds (Johnson et al., 2009 ; Fissette et al., 2020 ). In addition, SM increases 3kPZS release virtually instantaneously upon detection of 3kPZS, indicating presence of competitor males (Fissette et al., 2020 ). Male signals are typically costly to produce, and thus display frequency may provide a measure of male attractiveness (Davie et al., 2010 ). The sex- and maturation-dependent metabolomic profiles of sea lamprey support these notions. High inter-male variation in 3kPZS release and female preference for pheromone plumes with higher 3kPZS concentrations indicate 3kPZS likely guides female mate choice (Buchinger et al., 2017 ; Fissette et al., 2020 ; Johnson et al., 2009 ). Indeed, biosynthesis of 3kPZS and possible precursors appears to be under sexual selection (Buchinger et al., 2019 ). Interestingly, 3kPZS signaling may also mediate interactions among males, as intrasexual competition increases male 3kPZS release which may have consequences on female mate choice (Fissette et al., 2020 )). Therefore, changes in metabolomic profiles and chemical signaling strategies resulting from male competition may influence mate choice (Fissette et al., 2020 ). Our metabolomics results indicate that SM maximizes pheromonal bile acid production, likely benefits reproduction and increase fitness. In conclusion, adult sea lamprey drastically alter their plasma metabolomic profile and energy expenditure to support migration and reproduction. Spermiating male sea lamprey upregulate bile acid biosynthesis, producing a pheromone known to increase mate attraction and reproductive success. Declarations Author Conflict of Interest Statement Funding: This study was funded by Great Lakes Fishery Commission. Conflict of Interest: All authors declare that they have no conflict of interest. Author Contribution Statement YWCD and WML conceived and designed research. YWCD and ST conducted experiments. BH and ST contributed analytical tools and analyzed data. BH, ST and YWCD wrote the manuscript. WL edited the manuscript. Compliance with Ethical Standards : All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Data Availability Statement: The metabolomics and metadata reported in this paper are available via GNPS at doi:10.25345/C5V29C under the MSV000088417. Acknowledgments: We thank the staff in the Mass Spectrometry and Metabolomics Core Facility of Michigan State University for their technical support. We thank Skye D. Fissette and his summer technicians, and Dr. Nicholas S. Johnson and the staff of the U.S. Geological Survey, Great Lakes Science Center, Hammond Bay Biological Station for lamprey procurement. We thank Dr. Anne M. Scott for help with sea lamprey plasma collections. References Andersson, M., & Iwasa, Y. (1996). Sexual selection. Trends in Ecology & Evolution , 11, 53–58. https://doi.org/10.1016/0169-5347(96)81042-1 Applegate, V. C. (1951). The Sea Lamprey in the Great Lakes. The Scientific Monthly , 72, 275–281 Beamish, F. W. H. (1979). Migration and spawning energetics of the anadromous sea lamprey, Petromyzon marinus . Environmental Biology of Fishes , 4, 3–7 Bird, D. 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J., Bussy, U., Fissette, S. D., Johnson, N. S., & Li, W. (2015). Quantification of 15 bile acids in lake charr feces by ultra-high performance liquid chromatography–tandem mass spectrometry. Journal of Chromatography B , 1001, 27–34. https://doi.org/10.1016/j.jchromb.2015.07.028 Li, W., Scott, A. P., Siefkes, M. J., Yan, H., Liu, Q., Yun, S. S., & Gage, D. A. (2002). Bile Acid Secreted by Male Sea Lamprey That Acts as a Sex Pheromone. Science , 296, 138–141. https://doi.org/10.1126/science.1067797 Martins, E., Almeida, P. R., Quintella, B. R., da Silva, M. G., & Lança, M. J. (2019). Muscle fatty acid profiles of sea lamprey ( Petromyzon marinus L.) indicate the use of fast metabolized energy during ontogenesis. Fish Physiology and Biochemistry , 45, 849–862 Norambuena, F., Morais, S., Estévez, A., Bell, J. G., Tocher, D. R., Navarro, J. C. … Duncan, N. (2013).Dietary modulation of arachidonic acid metabolism in Senegalese sole ( Solea senegalensis ) broodstock reared in captivity. Aquaculture 372, 80–88 Norlin, M., & Wikvall, K. (2007). Enzymes in the conversion of cholesterol into bile acids. CMM , 7, 199–218. https://doi.org/10.2174/156652407780059168 Ord, T. J., Blumstein, D. T., & Evans, C. S. (2001). Intrasexual selection predicts the evolution of signal complexity in lizards. Proc. R. Soc. Lond. B 268, 737–744. https://doi.org/10.1098/rspb.2000.1417 Pandak, W. M., & Kakiyama, G. (2019). The acidic pathway of bile acid synthesis: Not just an alternative pathway. Liver Research , 3, 88–98 Ruiz-Perez, D., Guan, H., Madhivanan, P., Mathee, K., & Narasimhan, G. (2020). So you think you can PLS-DA? BMC Bioinformatics , 21(Suppl 1), 2 https://doi.org/10.1186/s12859-019-3310-7 Scott, A. M., Zhang, Z., Jia, L., Li, K., Zhang, Q., Dexheimer, T. … Li, W. (2019). Spermine in semen of male sea lamprey acts as a sex pheromone. PLoS Biol , 17, e3000332. https://doi.org/10.1371/journal.pbio.3000332 Sheridan, M. A. (1988). Lipid dynamics in fish: aspects of absorption, transportation, deposition and mobilization. Comparative Biochemistry and Physiology , 90B, 679–690 Singh, R., Singh, A. K., & Tripathi, M. (2012). Melatonin induced changes in specific growth rate, gonadal maturity and lipid and protein production in Nile tilapia Oreochromis niloticus (Linnaeus, 1758). Asian-Australian Journal of Animal Sciences , 25, 37–43 Setchell, K. D., Suchy, F. J., Welsh, M. B., Zimmer-Nechemias, L., Heubi, J., & Balistreri, W. F. (1988). Delta 4-3-oxosteroid 5 beta-reductase deficiency described in identical twins with neonatal hepatitis. A new inborn error in bile acid synthesis. Journal of Clinical Investigation , 82(6), 2148–2157 Siefkes, M. J., Scott, A. P., Zielinski, B., Yun, S. S., & Li, W. (2003). Male Sea Lampreys, Petromyzon marinus L., Excrete a Sex Pheromone from Gill Epithelia1. Biology of Reproduction , 69, 125–132. https://doi.org/10.1095/biolreprod.102.014472 Smilowitz, J. T., Zivkovic, A. M., Wan, Y. J. Y., Watkins, S. M., Nording, M. L., Hammock, B. D., & German, J. B. (2013). Nutritional lipidomics: Molecular metabolism, analytics, and diagnostics. Mol. Nutr. Food Res , 57, 1319–1335. https://doi.org/10.1002/mnfr.201200808 Smith, C. A., Want, E. J., O’Maille, G., Abagyan, R., & Siuzdak, G. (2006). Processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal. Chem , 78, 779–787. https://doi.org/10.1021/ac051437y Stryer (1995). Integration of metabolism, Biochemistry Sutharshiny, S., & Sivashanthini, K. (2011). Lipid reserves of Scomberoides lysan (Pisces: Carangidae) from the Sri Lankan waters. International Journal of Biological Chemistry , 5, 170–183 Tocher, D. R. (2003). Metabolism and functions of lipids and fatty acids in teleost fish. Reviews in Fisheries Science , 11, 107–184 Wade, M. G., Van der Kraak, G., Gerrits, M. F., & Ballantyne, J. S. (1994). 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Supplementary Files MetabolomicsSupplementaryInformation.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 27 Sep, 2022 Reviews received at journal 24 Jun, 2022 Reviewers agreed at journal 09 Jun, 2022 Reviewers invited by journal 06 May, 2022 Editor assigned by journal 28 Apr, 2022 Submission checks completed at journal 28 Apr, 2022 First submitted to journal 28 Apr, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1606386","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":102289074,"identity":"7b638f14-9b69-4861-a813-a2829cc72a38","order_by":0,"name":"Sonam Tamrakar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYNCCAwx2/BAWM2HFPFAtyZINpGph3HCAWC327KfTPvw4Y8dsfO34MwmGCuvEBoK28ORuntlzI5nP7HaOmQTDmXQitDDkbmbg+cDMDNTCJsHYdpgILfxvNzP++VDPuHl2+jMJxn/EaJHI3czMc+Mw4wbpBDMJxgZitNx4u5lZ5szxZInbOcYWCcfSjQlqYe/P3cz45li1Hf/s9Ic3PtRYyxLUggoSSFM+CkbBKBgFowAXAADJ2T2hrsh1xQAAAABJRU5ErkJggg==","orcid":"","institution":"Michigan State University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Sonam","middleName":"","lastName":"Tamrakar","suffix":""},{"id":102289075,"identity":"9db06c2d-3568-408b-8c11-20c47d00917e","order_by":1,"name":"Belinda Huerta","email":"","orcid":"","institution":"Dublin City University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Belinda","middleName":"","lastName":"Huerta","suffix":""},{"id":102289078,"identity":"007e62d4-6076-46e7-8b47-ffc5430f3a06","order_by":2,"name":"Yu-Wen Chung-Davidson","email":"","orcid":"","institution":"Michigan State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu-Wen","middleName":"","lastName":"Chung-Davidson","suffix":""},{"id":102289079,"identity":"918e08e0-ce62-4911-b002-91e9aae4bc2b","order_by":3,"name":"Weiming Li","email":"","orcid":"","institution":"Michigan State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weiming","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2022-04-29 00:29:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1606386/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1606386/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":21118599,"identity":"3efba49f-4172-4350-aa60-16571527891b","added_by":"auto","created_at":"2022-05-05 16:05:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":312281,"visible":true,"origin":"","legend":"\u003cp\u003ePCA score plot of metabolic fingerprints in the negative mode: (a) prespermiating males (PSM) vs. spermiating males (SM); (b) preovulatory females (POF) vs. ovulatory females (OF); (c) OF vs. SM; (d) POF vs. PSM. Blue circles: PSM; purple circles: SM; yellow circles: QC males; pink circles: POF; red circles: OF; green circles: QC females. QC: quality control samples were pooled from each sample in the group (detailed see Methods 2.3.1).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1606386/v1/4a6cd3a20b03da934217d9c5.png"},{"id":21118598,"identity":"e685b383-d0bb-4e76-9505-50834142003b","added_by":"auto","created_at":"2022-05-05 16:05:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":126866,"visible":true,"origin":"","legend":"\u003cp\u003ePutative bile acid synthetic pathway map comparing plasma metabolites in mature male and female sea lamprey. The metabolites in bold letters represent the bile acids quantified by targeted analysis in sea lamprey plasma. The number below each box denotes the fold change for the respective metabolite and the red color indicates upregulation (SM \u0026gt; OF). OF: ovulatory females; SM: spermiating males.\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1606386/v1/6bcb311cb132ee92516ba15b.png"},{"id":21118601,"identity":"b11ab746-4f38-4c5a-803c-1c7ead33e083","added_by":"auto","created_at":"2022-05-05 16:05:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":472718,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1606386/v1/1bd61934-6968-4fc7-bd1d-8bc8cd48a10c.pdf"},{"id":21118600,"identity":"9aff6512-67eb-4eb2-8f7b-cda8eb36ece6","added_by":"auto","created_at":"2022-05-05 16:05:36","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":4772576,"visible":true,"origin":"","legend":"","description":"","filename":"MetabolomicsSupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-1606386/v1/357ff3e9bc4f66673eedaacf.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Plasma metabolomic profiles reveal sex- and maturation-dependent metabolic strategies in sea lamprey (Petromyzon marinus)","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eAdult sea lamprey (\u003cem\u003ePetromyzon marinus\u003c/em\u003e) must rearrange their metabolomes drastically to support the demand for reproduction after they cease feeding. One of the pronounced physiological changes during their spawning migration is that they stop feeding and the lipid reserves function as the primary energy source during this period (Sheridan \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Bird et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). It provides a unique opportunity to study how animals alter metabolic processes to mobilize energy reserves to fulfill biological functions.\u003c/p\u003e \u003cp\u003eIn early spring adult sea lamprey cease feeding and migrate to spawning grounds (Applegate, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1951\u003c/span\u003e) where sexually mature spermiating males (SM) release large quantities of bile acid pheromones that attract ovulatory females (OF) to the nest for spawning (Buchinger et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Chung-Davidson et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Fissette et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Johnson et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, 2012; Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Scott et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). One SM can release up to 0.5 mg/h of a major component of the male sex pheromone, 3-keto petromyzonol sulfate (3kPZS; Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). On the contrary, females and PSM only release trace or non-detectable amounts of bile acids (Chung-Davidson et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Since bile acid pheromones are synthesized in the liver, transported via blood circulation to the gills, and released into the water (Brant et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Fissette et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Siefkes et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), comparing sea lamprey plasma metabolomes between sexes and maturation states may help to infer metabolic strategies that sustain high pheromone production and release in SM.\u003c/p\u003e \u003cp\u003eThe intricate networks of metabolic pathways are coordinated to meet the needs of the whole organism, \u003cem\u003ei.e.\u003c/em\u003e, to generate ATP for energy consumption and the building blocks for biosynthesis. ATP is generated by oxidation of fuel molecules such as glucose, fatty acids and amino acids, whereas the building blocks for biosynthesis are usually intermediates from various metabolic pathways (Stryer, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). We expect that SM employ effective metabolic strategies to generate ATP and building blocks for massive bile acid biosynthesis. Since blood plasma mediate biological functions between organs, plasma metabolomes provide a proxy for the metabolic status of an organism.\u003c/p\u003e \u003cp\u003eWe hypothesize that adult sea lamprey alter metabolic pathways to facilitate the different biological functions necessitated by sex and maturation status, resulting in dramatic differences in plasma metabolomes in preovulatory females (POF), PSM, OF and SM. Using both non-targeted and targeted approaches, we found that sea lamprey exhibited distinct plasma metabolomes in different sexes and maturation states, and SM maximized pheromone production by downregulating energy metabolisms but upregulating bile acid biosynthesis. There is an apparent trade-off between sexual signals (bile acid pheromones) and energy consumption in sea lamprey.\u003c/p\u003e"},{"header":"2 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals\u003c/h2\u003e \u003cp\u003eMigratory adult sea lampreys were collected from Ocqueoc River (Presque Isle, Michigan, USA) in the summer of 2019 by agents of the US Geological Survey at Hammond Bay Biological Station, Great Lakes Science Center, Millersburg, Michigan, USA. Pre-spawning lamprey were held in the lower Ocqueoc River (Presque Isle County, Michigan, USA) to induce sexual maturation, which was assessed daily by visual inspection of secondary sex characteristics and gentle expression of gametes (Brant et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). All animals were then transferred to Michigan State University (East Lansing, Michigan, USA) where samples were collected immediately upon arrival. Standard operating procedures for transporting, maintaining, handling, anesthetizing, and euthanizing sea lampreys were approved by the Institutional Committee on Animal Use and Care of Michigan State University (AUF # Li-02-17-030-99) and in compliance with standards defined by the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Chemicals\u003c/h2\u003e \u003cp\u003eHPLC grade methanol, acetonitrile, chloroform, and ammonium acetate, cholic acid (CA), deoxycholic acid (DCA), taurochenodeoxycholic acid (TCDCA), and MS222 were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Petromyzonol sulfate (PZS), 3-keto petromyzonol sulfate (3kPZS), petromyzonamine disulfate (PADS), petromyzonamine-24-monosulfate (PAMS-24), allocholic acid (ACA or 5α-cholic acid), 3-keto allocholic acid (3kACA) and deuterated 3-keto petromyzonol sulfate ([\u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e5\u003c/sub\u003e]-3kPZS) were custom-synthesized from Bridge Organic, Inc. (Vicksburg, Michigan, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Sample analyses\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Sample preparation\u003c/h2\u003e \u003cp\u003eBlood samples (n\u0026thinsp;=\u0026thinsp;10 each for POF, PSM, OF, and SM) were collected by cardiac puncture with 10 mL heparinized vacutainers after animals were anesthetized with 0.02% MS222. Plasma samples (supernatant) were obtained by centrifugation at 1000 x g, 4\u0026deg;C for 20 min. Sample proteins were precipitated by adding 600 \u0026micro;l of ice-cold methanol to 300 \u0026micro;l of plasma (2:1, v/v), incubated at -20\u0026deg;C overnight and centrifuged at 15800 x g at 4\u0026deg;C for 15 min. The resulting supernatant was mixed with 600 \u0026micro;l of chloroform at 4\u0026deg;C (on ice) at 100 rpm for 20 min, incubated at -20\u0026deg;C for 30 min, and then centrifuged at 10,000 x g at 4\u0026deg;C for 30 min. The aqueous (top) layer was freeze dried and then reconstituted in 100 \u0026micro;l of 50% methanol in water. Quality control (QC) samples for male and female groups were prepared separately by pooling equal volumes of PSM and SM samples for male QC, and POF and OF for female QC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Untargeted analyses\u003c/h2\u003e \u003cp\u003eSamples were analyzed on a UPLC Q-Exactive Orbitrap system equipped with a heated electrospray ionization source (HESI). Chromatographic separations were optimized (detail see Supplementary Information) using an Acquity UPLC BEH C18 column (100 x 2.1 mm; 1.7 \u0026micro;m particle) with 10 mM ammonium acetate in water as solvent A and acetonitrile as solvent B. The injection volume was 10 \u0026micro;l. The mobile phase gradient was maintained as follows, 0 min: 5% B; 5 min: 38% B; 7 min: 55% B; 10 min: 70% B; 16 min: 95% B; 21 min: 100% B; 21.5\u0026thinsp;~\u0026thinsp;25 min: 5% B. The flow rate was maintained at 0.3 ml/min and the column temperature at 55\u0026deg;C throughout the analyses. Samples were analyzed in both positive and negative ionization modes using the full scan MS with data-dependent (dd)-MS2 acquisition mode. The Orbitrap parameters for the full scan acquisition were set as follows, resolution: 17,500; AGC target: 3e\u003csup\u003e6\u003c/sup\u003e; maximum injection time (IT): 50 ms; scan range: 80 to 1200 m/z. The parameters for dd-MS2 were set as follows, resolution: 17,500; AGC target: 1e\u003csup\u003e5\u003c/sup\u003e; IT: 50 ms; scan range: 200 to 2000 m/z; normalized collision energy: 10, 30, and 60. Source parameters were as follows, sheath gas flow rate: 48 AU; spray voltage: 3.5 kV; capillary temperature: 256\u0026deg;C; auxiliary gas flow rate: 11; sweep gas flow rate: 2; auxiliary gas heater temperature: 413\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 Targeted analyses\u003c/h2\u003e \u003cp\u003eNine bile acids (including 6 sea lamprey-specific bile acids) were quantified in all samples. A Waters Acquity H-Class UPLC system connected to a Xevo TQ-S Triple Quadrupole mass spectrometer was used for the analyses. Separation was achieved using a Waters BEH C18 column (2.1 x 100 mm; 1.7 \u0026micro;m particle size) coupled to an Acquity UPLC BEH C18 VanGuard Pre-column (2.1 x 5 mm; 1.7 \u0026micro;m particle size). For sea lamprey-specific bile acids (3kPZS, PZS, PADS, PAMS-24, ACA, and 3kACA), 10 mM triethylamine (TEA) in water was used as mobile phase A and methanol was used as mobile phase B. The gradient was maintained as follows, 0 min: 40% B; 7\u0026thinsp;~\u0026thinsp;9 min: 99% B; 10\u0026thinsp;~\u0026thinsp;12 min: 40% B. The flow rate was maintained at 0.25 \u0026micro;L/min and the column temperature at 35\u0026deg;C (Wang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). For DCA, CA, and TCDCA, water containing 7.5 mM ammonium acetate and 0.1% formic acid was used as solvent A, and acetonitrile/methanol (1:9; v/v) containing 7.5 mM ammonium acetate and 0.1% formic acid was used as solvent B. The gradient was maintained as follows, 0 min: 40% B; 7 min: 74% B; 7.01\u0026thinsp;~\u0026thinsp;9 min: 100% B; 9.01\u0026thinsp;~\u0026thinsp;12 min: 40% B. The flow rate was maintained at 0.25 \u0026micro;L/min and the column temperature at 30\u0026deg;C. The injection volume was 10 \u0026micro;L in both cases (Li et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll bile acids were analyzed by electrospray ionization in the negative mode. The ESI-MS/MS parameters were set as follows, capillary voltage: 2.60 kV; extractor voltage: 5 V; source temperature: 150\u0026deg;C; desolvation temperature: 500\u0026deg;C; desolvation gas flow: 800 L/h (N\u003csub\u003e2\u003c/sub\u003e, 99.9% purity). Argon (99.9% purity) was introduced as the collision gas into the collision cell at a flow rate of 0.15 mL/min. The multiple reaction monitoring (MRM) and other parameters are provided in Supplementary Information (Table S1). MassLynx 4.2 software was used for data acquisition and data were processed using TargetLynx XS (Waters Corp., Milford, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Data processing\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Untargeted analyses\u003c/h2\u003e \u003cp\u003eThe workflow applied for data processing is illustrated in Fig. S1. Progenesis QI and Compound Discoverer 3.1 softwares were used to maximize the number of metabolite identification. Briefly, the spectra were selected from the raw data and then aligned with a retention time (RT) tolerance of 0.2 min and mass error of 5 ppm. The discriminating features were selected based on the ANOVA p-value (\u0026lt;\u0026thinsp;0.01) and fold change (FC\u0026thinsp;\u0026gt;\u0026thinsp;1.2). Compound annotations of significant metabolites were based on the fragment matching with available databases, especially Human Metabolome Database (HMDB) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hmdb.ca/\u003c/span\u003e\u003cspan address=\"https://hmdb.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e and KEGG (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp/kegg\u003c/span\u003e\u003cspan address=\"https://www.genome.jp/kegg\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Principal component analyses (PCA) and partial least squares discriminant analyses (PLS-DA) were performed by exporting the data to SIMCA 17 software (Umetrics, Sweden). Raw files were converted to .mzXLM files using ProteoWizard (MS Converter; Chambers et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Converted files were uploaded into an online platform XCMS (Smith et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) to produce a data matrix following the same discrimination criteria as described above (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01, FC\u0026thinsp;\u0026gt;\u0026thinsp;1.2, and abundance\u0026thinsp;\u0026gt;\u0026thinsp;10,000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Targeted analyses\u003c/h2\u003e \u003cp\u003eData were processed using MassLynx 4.2 software (Waters Corp., Milford, MA, USA). The software produced a table with m/z, RT, and intensity (peak area) values for each variable in each sample. Linear relationship calculations between peak areas and concentrations were adopted by weighted least squares regression. For targeted analyses, FC between groups was calculated as the ratio of the mean concentrations, e.g., dividing the mean concentration of SM by the mean concentration of PSM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Statistical analyses\u003c/h2\u003e \u003cp\u003eMultivariate data analyses were performed to find the similarities and differences between sample groups. PCA, an unsupervised dimensionality-reduction tool, was used for feature selection and classification, i.e., to identify patterns in the data and to check the trends and outliers, using SIMCA 17 software (Umetrics, Sweden) (Smilowitz et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). PLS-DA, a supervised machine learning dimensionality-reduction tool, was also performed as it is suited for metabolomics data with large number of features, noise and missing data, and fewer samples than features (Ruiz-Perez et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The PCA and PLS-DA models were evaluated in terms of their goodness of fit (R2Xcum, R2Ycum) and goodness of prediction (Q2cum). R2Xcum is the cumulative modeled variation in X, R2Ycum is the cumulative variation in X correlated to Y, and Q2cum estimates the cumulative predictive ability of the model. A Q2cum value\u0026thinsp;\u0026gt;\u0026thinsp;0.5 is considered a good model for metabolomics analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.4 Pathway analyses\u003c/h2\u003e \u003cp\u003ePotential impacted pathways were assessed by pathway analyses using MetaboAnalyst 4.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.metaboanalyst.ca/\u003c/span\u003e\u003cspan address=\"http://www.metaboanalyst.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e and the Human Metabolome Database (HMDB, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hmdb.ca/\u003c/span\u003e\u003cspan address=\"https://hmdb.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Chong et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The platform iPath v.3 was used to map the impacted pathways whenever the annotated metabolites were listed in its database (Darzi et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Metabolomic profiles showed sex differences and dramatic changes during sexual maturation\u003c/h2\u003e \u003cp\u003eLC-HRMS analyses revealed 6554 significant features of metabolic fingerprints in ESI- mode and 7105 features in ESI\u0026thinsp;+\u0026thinsp;mode. PCA analyses in both negative and positive modes showed significant differences between PSM vs. SM, POF vs. OF, OF vs. SM, and POF vs. SM (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S2; Table S2). The goodness of fit for these models resulted in R2X\u0026thinsp;=\u0026thinsp;0.69 for male sexual maturation (PSM vs. SM), R2X\u0026thinsp;=\u0026thinsp;0.715 for female sexual maturation (POF vs. OF), and sex differences in mature adults (OF vs. SM: R2X\u0026thinsp;=\u0026thinsp;0.751) and immature adults (POF vs. PSM: R2X\u0026thinsp;=\u0026thinsp;0.917). In each comparison, the goodness of prediction (Q2) was greater than 0.5, indicating that the model was acceptable for metabolomic analysis (Table S2). PLS-DA plots also confirmed significant differences between PSM vs. SM, POF vs. OF, OF vs. SM, and POF vs. SM (Figs. S3 and S4). The goodness of fit for each comparison (PSM vs. SM; POF vs. OF; POF vs. PSM; OF vs. SM) resulted in R2X\u0026thinsp;\u0026gt;\u0026thinsp;0.5, R2Y\u0026thinsp;\u0026gt;\u0026thinsp;0.8, and Q2\u0026thinsp;\u0026gt;\u0026thinsp;0.5 (Table S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDiscriminant analyses revealed dramatic upregulations in sea lamprey-specific bile acids in the comparisons between sexes and male sexual maturation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For example, PSM contained 244-fold (\u0026uarr;) PZS compared to POF, SM contained 155-fold (\u0026uarr;) PZ and 53-fold (\u0026uarr;) ACA compared to OF, and SM contained 314-fold (\u0026uarr;) ACA and 201-fold (\u0026uarr;) PZ compared to PSM. Other bile acids, including cholic acid, lithocholic acid and chenodeoxycholic acid-3-sulfate were also upregulated hundreds of folds in SM compared to PSM or OF. Changes in nucleotide and amino acid metabolisms were apparent in all comparisons. SM showed increases in adenine (\u0026uarr; 77-fold) but decreases in creatine (\u0026darr; 81-fold) compared to OF. On the other hand, OF showed downregulations of fatty acid metabolism compared to POF (11-dehydrothromboxane: \u0026darr; 4-fold; palmitic acid: \u0026darr; 2-fold).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of metabolic pathways and the most discriminant metabolites in the comparison of different sea lamprey groups between sexes and maturation states\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetabolism pathway\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eMetabolite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRegulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFold Change\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eq-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"26\" rowspan=\"27\"\u003e \u003cp\u003ePSM vs SM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eSecondary bile acid biosynthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLithocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e152.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.66E-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.55E-08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePetromyzonol sulfate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e212.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.38E-10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.37E-08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChenodeoxycholic acid 3-sulfate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e264.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.83E-09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.68E-07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePetromyzonol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e201.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.45E-09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.49E-07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePurine metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAdenine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.21E-07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.95E-05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAmino acid metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3-Hydroxypropenoate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.63E-06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.11E-04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePrimary and Secondary bile acid biosynthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e134.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.44E-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.72E-03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eSecondary bile acid biosynthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5α-Cholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e313.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.07E-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.26E-03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eD-Xylulosonic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.64E-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.07E-03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eTyrosine metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTyrosine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.61E-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7.76E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAmino acid metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eArginine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.64E-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.08E-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eLysine degradation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLysopine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.98E-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.18E-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAmino acid metabolism - Glutathione metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOrnithine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.04E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.44E-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eArginine and proline metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOctopine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.52E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.02E-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eHeme catabolism (bile pigment)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBiliverdin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.14E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.25E-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePurine metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypoxanthine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.15E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.97E-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eHistidine metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFormimino-L-glutamic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.29E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.01E-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePyrimidine metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCytosine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.70E-05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.10E-03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAmino acid metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlutamic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.50E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.60E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eHistidine metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMethylhistidine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.00E-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5.10E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAmino acid metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlutamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.40E-05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.80E-03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAmino acid metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlutamic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.60E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.30E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN3,N4-Dimethyl-L-arginine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.70E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.90E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eTCA cycle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCitric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.20E-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7.10E-03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eBiosynthesis of ansamycins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAminoDHQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.03E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.00E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAmino acid metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2-Aminoadipic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.10E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.70E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePhenylalanine metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2-Oxo-4-pentenoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.30E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.30E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"11\" rowspan=\"12\"\u003e \u003cp\u003ePOF vs OF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eLysine biosynthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-acetyl-LL-2,6-diaminopimelic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.22E-07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9.39E-06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN,N-dimethylarginine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.83E-07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.01E-05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7a,12a-Dihydroxy-3-oxo-4-cholenoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.13E-05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.64E-04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eCysteine and methionine metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMethionine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.26E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.34E-03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTryptophan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.21E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.76E-03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eD-Xylulosonic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.22E-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.17E-03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-(3-Carboxypropyl)-L-glutamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e19.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.96E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.46E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProstaglandin C1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.60E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.79E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eArachidonic acid metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11-Dehydrothromboxane B2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.21E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.06E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eValine, leucine, and isoleucine biosynthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAcetyl lactic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.40E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.10E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAmino acid metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCreatine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.60E-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.2E-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eArginine and proline metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCreatinine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.20E-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.1E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAlanine, aspartate, and glutamate metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAspartic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.40E-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8.20E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlutamyl-glutamic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.90E-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.20E-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePyrimidine metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3-Ureidopropionic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.10E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.70E-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePhenylalanine, tyrosine, and tryptophan biosynthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePretyrosine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.40E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.50E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eFatty acid metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePalmitic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.60E-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.90E-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePyrimidine metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMethylmalonic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.30E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.40E-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePentose phosphate pathway\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(2R)-2,3-Dihydroxypropanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.54E-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.60E-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eFatty acid degradation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGlutaric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.40E-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.60E-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePetromyzonol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e155.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.54E-09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.16E-07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePurine metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAdenine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e76.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.04E-07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.08E-05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eRiboflavin metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRiboflavin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.20E-06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.63E-04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eTCA cycle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIsocitric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.50E-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.89E-03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOF vs SM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eBile secretion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.12E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.67E-03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePrimary and Secondary bile acid biosynthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e93.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.27E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.09E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePyrimidine metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUridine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.96E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.57E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGlycine, serine, and threonine metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCreatine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e81.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.32E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.82E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eSecondary bile acid biosynthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5a-Cholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e53.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.86E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.25E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eBiosynthesis of unsaturated fatty acids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBehenic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.50E-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.94E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-glycoloyl-beta-D-glucosamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e81.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.23E-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7.54E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eBiosynthesis of amino acids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN-acetyl-LL-2,6-diaminopimelic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e117.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.7E-15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.4E-12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNorleucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.8E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.5E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eBiosynthesis of amino acids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTryptophan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.5E-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7.5E-03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePOF vs PSM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eTyrosine metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTyramine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.2E-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.0E-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePetromyzonol sulfate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e244.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.1E-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5.1E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eRiboflavin metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRiboflavin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.2E-07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5.2E-05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePurine metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAdenine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.7E-02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.6E-01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3,6-Dideoxy-L-galactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.8E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6.2E-02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eDown regulation (\u0026darr;): SM\u0026thinsp;\u0026lt;\u0026thinsp;PSM; OF\u0026thinsp;\u0026lt;\u0026thinsp;POF; SM\u0026thinsp;\u0026lt;\u0026thinsp;OF; PSM\u0026thinsp;\u0026lt;\u0026thinsp;POF. Up regulation (\u0026uarr;): SM\u0026thinsp;\u0026gt;\u0026thinsp;PSM; OF\u0026thinsp;\u0026gt;\u0026thinsp;POF; SM\u0026thinsp;\u0026gt;\u0026thinsp;OF; PSM\u0026thinsp;\u0026gt;\u0026thinsp;POF. \u0026ndash;: Not identified in KEGG. OF: ovulatory females; POF: preovulatory females; PSM: prespermiating males; SM: spermiating males.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 Males reduced sugar and energy metabolisms and altered amino acid metabolisms in favor of bile acid biosynthesis during sexual maturation\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePathway analyses revealed that the most prominent changes between SM and PSM were the metabolites in primary and secondary bile acid biosynthesis (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e); e.g., lithocholic acid (\u0026uarr; 153-fold) and cholic acid (\u0026uarr; 134-fold). Interestingly, lamprey-specific bile acids were upregulated at least 2 times more than those common bile acids (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e); e.g., ACA (5α-cholic acid \u0026uarr; 314-fold), PZ (\u0026uarr; 201-fold), and PZS (\u0026uarr; 212-fold). Several amino acid metabolic pathways were affected during male sexual maturation (impact\u0026thinsp;\u0026gt;\u0026thinsp;0.15, Table S5); e.g., tyrosine (\u0026uarr; 2-fold), arginine (\u0026darr; 2-fold), glutamate (\u0026uarr; 2-fold), and glutamine (\u0026uarr; 4-fold). Significantly changed metabolites are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and their associated primary metabolic pathways are shown in Fig. S5. In general, metabolic pathway maps indicated that SM reduced TCA cycle-related activities and carbohydrate and energy metabolisms, and at the same time increased biosynthesis of bile acids, cofactors, and vitamins (Fig. S5).\u003c/p\u003e \u003cp\u003eOn the contrary, most metabolic pathways were downregulated in OF compared to POF, including lipid metabolism (e.g., palmitate \u0026darr; 2-fold; Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S5, and Fig. S6). Notably, two eicosanoid metabolites were downregulated (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e); i.e., prostaglandin C1 (\u0026darr; 3-fold) and 11-dehydrothromboxane B2 (\u0026darr; 4-fold). Other notable changes were amino acid metabolisms (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e); e.g., methionine (\u0026uarr; 4-fold), tryptophan (\u0026uarr; 3-fold), creatine (\u0026uarr; 5-fold), and pretyrosine (\u0026darr; 13-fold). Interestingly, D-aspartate but not L-aspartate were downregulated (\u0026darr; 10-fold, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSex differences are most prominent in the pathways involved in biosynthesis of bile acids, cofactors, and vitamins (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S5, and Fig. S7: OF vs. SM and Fig. S8: POF vs. PSM), as males contained more bile acids than females (Tables S3 and S4); e.g., PZS: \u0026uarr; 244-fold in PSM vs. POF; PZ: \u0026uarr; 155-fold in SM vs. OF (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S5).\u003c/p\u003e \u003cp\u003eSince bile acid biosynthetic pathways showed the most dramatic changes between sexes and during male sexual maturation, we used targeted analyses to confirm the results from untargeted analyses. Indeed, SM plasma contained the highest levels of most bile acids, except TCDCA (Tables S3 and S4). PZS was the most abundant bile acid in SM and PSM, with concentrations of 19 \u0026micro;g/ml and 2 \u0026micro;g/ml, respectively (Table S3). Upregulations of bile acids were seen during sexual maturation (PSM vs. SM and POF vs. OF, Tables S3 and S4), and the most dramatic changes were observed between sexes (males\u0026thinsp;\u0026gt;\u0026thinsp;females; OF vs. SM and POF vs. PSM; Tables S3 and S4), consistent with the untargeted analysis results. A putative sea lamprey bile acid biosynthetic pathway is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Higher levels of bile acids in SM vs. OF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Tables S3 and S4) were exemplified by PZS (\u0026uarr; 388-fold), ACA (\u0026uarr; 138-fold), and 3kACA (\u0026uarr; 83-fold). For pre-spawning adults, higher levels of bile acids in PSM vs POF (Tables S3 and S4) were found in PZS (\u0026uarr; 388-fold), 3kPZS (\u0026uarr; 255-fold), and PAMS-24 (\u0026uarr; 69-fold). ACA was probably important for sea lamprey sexual maturation since it was the most upregulated metabolite in PSM vs. SM (\u0026uarr; 87-fold; Table S4) and POF vs. OF (\u0026uarr; 15-fold; Table S4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOne striking difference was found in the comparisons of sea lamprey plasma metabolomes between sexes and maturation states, i.e., males contain hundreds-folds more bile acids than females, and SM contains the highest amounts of bile acids in all groups. Bile acids/salts are the major end metabolites of cholesterol and are important in lipid, protein, and sugar metabolisms (Hagey et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The details of bile acid biosynthetic pathways are only known in mammals such as humans and rodents and have not been resolved in other species, including sea lamprey (Hagey et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Based on our metabolomics results and what was available in the literature (Chiang, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Chung-Davidson et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Heubi et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Pandak and Kakiyama, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Setchell et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1988\u003c/span\u003e), we posited a putative sea lamprey bile acid synthetic pathway. It appears that SM has evolved a strategy that downregulates the biosynthesis of other lipid species (i.e., fatty acids and leukotrienes) and upregulates cholesterol-derived bile acid biosynthesis. At the same time, SM lowers carnitine that transports long-chain activated fatty acids into the mitochondrial matrix for β-oxidation (Stryer, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Interestingly, citrate carries acetyl groups from mitochondria to the cytosol for fatty acid biosynthesis (Stryer, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). SM has higher bile acid levels compared to both PSM and OF, but lower citrate and 12-epi leukotriene B4 compared to PSM, and lower isocitrate compared to OF. Taken together, SM seems to lower fatty acid β-oxidation, limit other lipid biosynthesis, but maximize pheromonal bile acid biosynthesis. On the other hand, PSM swims long distance during migration and searching for suitable spawning ground, which demands ATP consumptions. There is an apparent trade-off between pheromone synthesis and other metabolic pathways, and consistent with many results in life history and chemical ecology of sea lamprey that only SM release sex pheromones that attract OF (Brant et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Chung-Davidson et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe found that OF had lower fatty acid metabolism and lower prostaglandin C1 and 11-dehydro thromboxane B2 compared to POF. Since eicosanoid hormones such as leukotrienes, prostaglandins, and thromboxanes are derived from polyunsaturated fatty acids, it seems that OF downregulated the pathways involved in biosynthesis of the precursors (fatty acids) and the end products (prostaglandins and thromboxanes). Interestingly, arachidonic acid, a precursor for eicosanoid production that modulate steroid synthesis and spermiation during sexual maturation (Wade et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Norambuena et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), is also critical to producing vitellogenin, a high-density lipoprotein which is synthesized in the liver and transported to the eggs (Wade et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Norambuena et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tocher \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The high investment of resources for reproduction is to mobilize lipid reserves from liver and muscles to gonads during sexual maturation and spawning migration (Zaboukas et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Sutharshiny and Sivashanthini \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In the sea lamprey, gonad development occurs when they stop feeding and initiate the atrophy of the digestive tract (Beamish et al., 1979). Consequently, gonadal growth and the energy expenditure for migration depend on mobilization of lipid and protein reserves accumulated during the parasitic phase (Hardisty \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and are not restored during the process of reproductive migration after feeding has ceased (Martins et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A drawback of this study is that we only measured more polar molecules due to the extraction and optimized analytical methods. Further lipidomics analyses will provide more information in lipid metabolisms during sexual maturation in sea lamprey.\u003c/p\u003e \u003cp\u003ePheromone communication consists of two components: (1) the biosynthesis, storage, and release of the pheromones; (2) the reception and translation of the chemical message into a behavioral pattern (Kittredge and Takahashi, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1972\u003c/span\u003e). In a sexual selection context for sea lamprey, the sender is SM, broadcasting information about aspects of its quality towards rival SM and/or OF. Apparently, SM evolved metabolic strategies to produce and release massive amounts of bile acid pheromones to signal spawning. Intersexual selection through female choice is generally regarded the most important driver of signal evolution (Andersson and Iwasa, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Ord et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Indeed, OF prefer higher pheromone levels on spawning grounds (Johnson et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Fissette et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, SM increases 3kPZS release virtually instantaneously upon detection of 3kPZS, indicating presence of competitor males (Fissette et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Male signals are typically costly to produce, and thus display frequency may provide a measure of male attractiveness (Davie et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The sex- and maturation-dependent metabolomic profiles of sea lamprey support these notions.\u003c/p\u003e \u003cp\u003eHigh inter-male variation in 3kPZS release and female preference for pheromone plumes with higher 3kPZS concentrations indicate 3kPZS likely guides female mate choice (Buchinger et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Fissette et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Johnson et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Indeed, biosynthesis of 3kPZS and possible precursors appears to be under sexual selection (Buchinger et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Interestingly, 3kPZS signaling may also mediate interactions among males, as intrasexual competition increases male 3kPZS release which may have consequences on female mate choice (Fissette et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)). Therefore, changes in metabolomic profiles and chemical signaling strategies resulting from male competition may influence mate choice (Fissette et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Our metabolomics results indicate that SM maximizes pheromonal bile acid production, likely benefits reproduction and increase fitness.\u003c/p\u003e \u003cp\u003eIn conclusion, adult sea lamprey drastically alter their plasma metabolomic profile and energy expenditure to support migration and reproduction. Spermiating male sea lamprey upregulate bile acid biosynthesis, producing a pheromone known to increase mate attraction and reproductive success.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Conflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding: This study was funded by Great Lakes Fishery Commission.\u003c/p\u003e\n\u003cp\u003eConflict of Interest: All authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYWCD and WML conceived and designed research. YWCD and ST conducted experiments. BH and ST contributed analytical tools and analyzed data. BH, ST and YWCD wrote the manuscript. WL edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eAll applicable international, national, and/or institutional guidelines for the care and use of animals were followed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The metabolomics and metadata reported in this paper are available via GNPS at doi:10.25345/C5V29C under the MSV000088417.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eWe thank the staff in the Mass Spectrometry and Metabolomics Core Facility of Michigan State University for their technical support. We thank Skye D. Fissette and his summer technicians, and Dr. Nicholas S. Johnson and the staff of the U.S. Geological Survey, Great Lakes Science Center, Hammond Bay Biological Station for lamprey procurement. We thank Dr. Anne M. Scott for help with sea lamprey plasma collections.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAndersson, M., \u0026amp; Iwasa, Y. (1996). Sexual selection. \u003cem\u003eTrends in Ecology \u0026amp; Evolution\u003c/em\u003e, 11, 53\u0026ndash;58. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0169-5347(96)81042-1\u003c/span\u003e\u003cspan address=\"10.1016/0169-5347(96)81042-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eApplegate, V. C. (1951). The Sea Lamprey in the Great Lakes. \u003cem\u003eThe Scientific Monthly\u003c/em\u003e, 72, 275\u0026ndash;281\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeamish, F. W. H. (1979). 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Y., Li, K., Chung-Davidson, Y. W., \u0026amp; Li, W. (2015). An UPLC\u0026ndash;MS/MS method for quantitative profiling of bile acids in sea lamprey plasma and tissues. \u003cem\u003eJournal of Chromatography B\u003c/em\u003e, 980, 72\u0026ndash;78. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jchromb.2014.12.018\u003c/span\u003e\u003cspan address=\"10.1016/j.jchromb.2014.12.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZaboukas, N., Miliou, H., Megalofonou, P., \u0026amp; Moraitou-Apostolopoulou, M. (2006). Biochemical composition of the Atlantic bonito \u003cem\u003eSarda sarda\u003c/em\u003e from the Aegean Sea (eastern Mediterranean Sea) in different stages of sexual maturity. \u003cem\u003eJournal of Fish Biology\u003c/em\u003e, 69, 347\u0026ndash;362\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"metabolomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mebo","sideBox":"Learn more about [Metabolomics](http://link.springer.com/journal/11306)","snPcode":"11306","submissionUrl":"https://submission.nature.com/new-submission/11306/3","title":"Metabolomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"bile acid biosynthesis, targeted analyses, untargeted analyses, metabolic pathways, sexual selection, agnathan","lastPublishedDoi":"10.21203/rs.3.rs-1606386/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1606386/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction\u0026nbsp;\u0026nbsp;\u003c/strong\u003eAdult sea lamprey (\u003cem\u003ePetromyzon marinus\u003c/em\u003e) cease feeding and migrate to spawning streams where males build nests, undergo final sexual maturation, and subsequently produce and release large quantities of bile acid pheromones that attract mature females. These animals are predicted to rearrange their metabolic pathways drastically to support their reproductive strategies, presenting advantageous opportunities to examine how sex and the maturation processes affect metabolism.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eObjectives \u003c/strong\u003eThe objective is to investigate the metabolic differences between sexes and maturation states in sea lamprey that support changes in physiological functions.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eWe compared plasma metabolomes of spawning and prespawning sea lamprey in both sexes using both non-targeted and targeted metabolomics approaches using UPLC/MS-MS with electrospray ionization in both positive and negative modes. The data were processed using Progenesis QI, Compound Discoverer and XCMS softwares for alignment, peak picking, and deconvolution of the peaks. Principle component analyses (PCA) and partial least squares discriminant analyses (PLS-DA) were performed using SIMCA and Metaboanalyst softwares to identify discriminating features, followed by fragmentation matching with extensive database search and pathway mapping. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eThe pheromonal bile acid biosynthesis was upregulated significantly in males compared to females. Spermiating males further upregulated bile acid biosynthesis by altering amino acid metabolisms, upregulating cofactors and nucleotide metabolisms, but downregulating carbohydrate and energy metabolisms. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion \u003c/strong\u003ePlasma metabolomes are sex- and maturation-dependent and reflect the special metabolic demands at each life stage and reproductive strategy.\u003c/p\u003e","manuscriptTitle":"Plasma metabolomic profiles reveal sex- and maturation-dependent metabolic strategies in sea lamprey (Petromyzon marinus)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-05-05 16:05:34","doi":"10.21203/rs.3.rs-1606386/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-09-27T15:01:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-06-25T00:34:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"fb10c569-66af-4e35-b9ad-0a74e5253d27","date":"2022-06-09T14:15:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-05-06T07:53:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-04-29T03:09:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-04-29T03:09:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Metabolomics","date":"2022-04-29T00:25:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"metabolomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mebo","sideBox":"Learn more about [Metabolomics](http://link.springer.com/journal/11306)","snPcode":"11306","submissionUrl":"https://submission.nature.com/new-submission/11306/3","title":"Metabolomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3f4e29ae-a1f4-442b-8b18-4a255dd2cf11","owner":[],"postedDate":"May 5th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-10-29T15:59:18+00:00","versionOfRecord":[],"versionCreatedAt":"2022-05-05 16:05:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1606386","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1606386","identity":"rs-1606386","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}