RESUMO
Recent advances in methodology have made phosphopeptide analysis a tractable problem for many proteomics researchers. There are now a wide variety of robust and accessible enrichment strategies to generate phosphoproteomes while free or inexpensive software tools for quantitation and site localization have simplified phosphoproteome analysis workflow tremendously. As a research group under the Association for Biomolecular Resource Facilities umbrella, the Proteomics Standards Research Group has worked to develop a multipathway phosphopeptide standard based on a mixture of heavy-labeled phosphopeptides designed to enable researchers to rapidly develop assays. This mixture contains 131 mass spectrometry vetted phosphopeptides specifically chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling networks: AMPK signaling, death and apoptosis signaling, ErbB signaling, insulin/insulin-like growth factor-1 signaling, mTOR signaling, PI3K/AKT signaling, and stress (p38/SAPK/JNK) signaling. Here, we describe a characterization of this mixture spiked into a HeLa tryptic digest stimulated with both epidermal growth factor and insulin-like growth factor-1 to activate the MAPK and PI3K/AKT/mTOR pathways. We further demonstrate a comparison of phosphoproteomic profiling of HeLa performed independently in five labs using this phosphopeptide mixture with data-independent acquisition. Despite different experimental and instrumentation processes, we found that labs could produce reproducible, harmonized datasets by reporting measurements as ratios to the standard, while intensity measurements showed lower consistency between labs even after normalization. Our results suggest that widely available, biologically relevant phosphopeptide standards can act as a quantitative "yardstick" across laboratories and sample preparations enabling experimental designs larger than a single laboratory can perform. Raw data files are publicly available in the MassIVE dataset MSV000090564.
Assuntos
Fosfopeptídeos , Proteínas Proto-Oncogênicas c-akt , Fosforilação , Fosfopeptídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fosfoproteínas/metabolismoRESUMO
Synthesis of multiple deuterium-labeled CCR2 antagonist JNJ-26131300, that is, [4-(1H-indol-3-yl)-piperidin-1-yl]-{1-[3-(3,4,5-trifluoro-phenyl)-acryloyl]-piperidin-4-yl}-acetic acid, is described. First, condensation of indole-D7 with 4-piperidone produced 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole-D5 , which subsequently underwent catalytic hydrogenation to give 3-piperidin-4-yl-1H-indole-D5 . Next, bromo-{1-[3-(3,4,5-trifluoro-phenyl)-acryloyl]-piperidin-4-yl}-acetic acid was prepared through multiple steps from 3-(3,4,5-trifluoro-phenyl)-acrylic acid and bromo-piperidin-4-yl-acetic acid ethyl ester. Nucleophilic coupling of 3-piperidin-4-yl-1H-indole-D5 with bromo-{1-[3-(3,4,5-trifluoro-phenyl)-acryloyl]-piperidin-4-yl}-acetic acid afforded the desired compound [4-(1H-indol-3-yl)-piperidin-1-yl]-{1-[3-(3,4,5-trifluoro-phenyl)-acryloyl]-piperidin-4-yl}-acetic acid-D5 .
Assuntos
Ácido Acético , Indóis , Deutério , Indóis/farmacologiaRESUMO
During drug development, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry is used for visually elucidating the distribution of substances such as biomarkers, candidate compounds, and metabolites in the tissues. However, it is difficult to make relative comparisons between tissue sections and there are still many challenges. Here, we report a new method of "triple spray" for the comparison of analyte distribution in multiple tissue slices. This method targets amino acids and amines, and it incorporates the application of the internal standard in the on-tissue derivatization step. With further development, it has the potential to alleviate problems caused by the matrix effect. Initially, we measured three serial sections of rat brain to verify the efficacy of this method. In the hypothalamus, where gamma-aminobutyric acid (GABA) is known to be present in high concentration, the GABA levels of the three serial section showed little variation (CV = 1.62%). Subsequently, we compared the GABA level in the brain between stroke-prone spontaneous hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats with three individuals each. It showed significant differences between these models at the pre-selected region of interest (p < 0.05). Our results show that the triple spray allows for relative comparison among multiple tissue slices with high reproducibility. Graphical abstract.
Assuntos
Aminoácidos/análise , Química Encefálica , Neurotransmissores/análise , Animais , Indicadores e Reagentes , Marcação por Isótopo/métodos , Masculino , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Ácido gama-Aminobutírico/análiseRESUMO
Parkinson disease (PD) is a neurodegenerative disorder characterized by the accumulation of α-synuclein into Lewy bodies. 3-Benzylidine-indolin-2-one represents a class of compounds, which are known to inhibit the accumulation of α-synuclein. In this paper, we report the synthesis of [13 C] and [15 N] labelled 1-benzyl-(Z)-3-(benzylidene)indolin-2-one from commercially available [13 C2 ]-chloroacetic acid and [15 N]-aniline in five steps. The product will be used to study its metabolites in human liver microsomes by liquid chromatography-tandem mass spectrometry.
Assuntos
Isótopos de Carbono/química , Indóis/química , Indóis/síntese química , Isótopos de Nitrogênio/química , Técnicas de Química Sintética , Humanos , Indóis/metabolismo , Microssomos Hepáticos/metabolismo , RadioquímicaRESUMO
Cell secretome, the complex set of proteins that are secreted by the cells, is a fundamental mechanism of cell-cell communication both in vitro and in vivo. In vivo, the analysis of proteins secreted into body fluids can bring to the identification of biomarkers for important physiopathological conditions. However, due to the complexity of the protein content of body fluids, a better understanding of the secreted proteins by different cell types is highly desirable and can be performed in vitro for dissection. To this aim, microfluidic culture systems could be particularly relevant because of the accumulation of extrinsic endogenous signals at microliter scale, which better preserves the self-regulation occurring in the small interstitial spaces in vivo. In this work, we perform a quantitative study to compare the secretome in microfluidics and in a standard well plate. Human foreskin fibroblasts are used as a case study. This work also represents an important technological advance in terms of feasibility of high-throughput quantitative protein analyses in microfluidics.
Assuntos
Técnicas de Cultura de Células/instrumentação , Fibroblastos/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Proteoma/metabolismo , Proteômica/instrumentação , Transdução de Sinais , Linhagem Celular , Meios de Cultivo Condicionados/metabolismo , Desenho de Equipamento , Humanos , Proteoma/análise , Espectrometria de Massas em TandemRESUMO
For rapid and effective clinical translation, polymer-based anticancer therapeutics need long circulating conjugates that produce a sustained concentration gradient between the vasculature and solid tumor. To this end, we designed second-generation backbone-degradable diblock N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer carriers and evaluated sequential combination therapy of HPMA copolymer-paclitaxel and HPMA copolymer-gemcitabine conjugates against A2780 human ovarian carcinoma xenografts. First, extensive in vitro assessment of administration sequence impact on cell cycle, viability, apoptosis, migration, and invasion revealed that treatment with paclitaxel conjugate followed by gemcitabine conjugate was the most effective scheduling strategy. Second, in an in vivo comparison with first-generation (nondegradable, molecular weight below the renal threshold) conjugates and free drugs, the second-generation degradable high-molecular weight conjugates showed distinct advantages, such as favorable pharmacokinetics (three- to five-times half-life compared with the first generation), dramatically enhanced inhibition of tumor growth (complete tumor regression) by paclitaxel and gemcitabine conjugate combination, and absence of adverse effects. In addition, multimodality imaging studies of dual-labeled model conjugates confirmed the efficacy of second-generation conjugates by visualizing more than five-times enhanced tumor accumulation, rapid conjugate internalization, and effective intracellular release of payload. Taken together, the results indicate that the second-generation degradable HPMA copolymer carrier can provide an ideal platform for the delivery of a range of antitumor compounds, which makes it one of the most attractive candidates for potential clinical application.
Assuntos
Acrilamidas/química , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Ciclo Celular , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Ovarianas/patologia , Paclitaxel/administração & dosagem , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios X , GencitabinaRESUMO
Canagliflozin (Invokana, JNJ-28431754) is an orally bioavailable and selective SGLT2 (subtype 2 sodium-glucose transport protein) inhibitor approved for the treatment of type 2 diabetes. Herein, we report the synthesis of 13 C and 14 C-labeled canagliflozin. Stable isotope-labeled [13 C6 ]canagliflozin was synthesized in 4 steps starting from [13 C6 ]-labeled glucose. The [14 C]-Labeled canagliflozin was synthesized by incorporation of [14 C] into the benzylic position between the thiophene and benzene rings of the compound. Detailed synthesis of the isotope-labeled compounds is reported.
Assuntos
Canagliflozina/química , Canagliflozina/síntese química , Inibidores do Transportador 2 de Sódio-Glicose , Canagliflozina/farmacologia , Isótopos de Carbono/química , Radioisótopos de Carbono/química , Técnicas de Química Sintética , Marcação por IsótopoRESUMO
Two regioisomeric glucuronide metabolites of dapagliflozin (BMS-512148) were synthesized and used to elucidate the structures of dapagliflozin metabolites observed in human urine samples. The structures of the synthetic metabolites were assigned by heteronuclear multiple-bond correlation, ROESY, and total correlation spectroscopy experiments. Analogues of these metabolites containing carbon-13 as a stable label were also prepared for use as internal standards for the analysis of urine samples obtained from patients participating in clinical studies.
Assuntos
Compostos Benzidrílicos/química , Compostos Benzidrílicos/síntese química , Glucosídeos/química , Glucosídeos/síntese química , Glucuronídeos/metabolismo , Compostos Benzidrílicos/metabolismo , Técnicas de Química Sintética , Glucosídeos/metabolismo , Marcação por Isótopo , EstereoisomerismoRESUMO
Several biologically active peptides contain a d- amino acid in a well-defined position, which is position 2 in all peptide epimers isolated to date from vertebrates and also some from invertebrates. The detection of such D- residues by standard analytical techniques is challenging. In tandem mass spectrometric (MS) analysis, although fragment masses are the same for all stereoisomers, peak intensities are known to depend on chirality. Here, we observe that the effect of a d- amino acid in the second N-terminal position on the fragmentation pattern in matrix assisted laser desorption time-of-flight spectrometry (MALDI-TOF/TOF MS) strongly depends on the peptide sequence. Stereosensitive fragmentation (SF) is correlated to a neighborhood effect, but the d- residue also exerts an overall effect influencing distant bonds. In a fingerprint analysis, multiple peaks can thus serve to identify the chirality of a sample in short time and potentially high throughput. Problematic variations between individual spots could be successfully suppressed by cospotting deuterated analogues of the epimers. By identifying the [d-Leu2] isomer of the predicted peptide GH-2 (gene derived bombininH) in skin secretions of the toad Bombina orientalis, we demonstrated the analytical power of SF-MALDI-TOF/TOF measurements. In conclusion, SF-MALDI-TOF/TOF MS combines high sensitivity, versatility, and the ability to complement other methods.
Assuntos
Aminoácidos/análise , Peptídeos/química , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Estereoisomerismo , Animais , Peptídeos Catiônicos Antimicrobianos/análise , Peptídeos Catiônicos Antimicrobianos/química , Anuros , Deutério , Pele/metabolismoRESUMO
SCH 900567 is a specific inhibitor of tumor necrosis factor-alpha converting enzyme and is a potential candidate for the treatment of rheumatoid arthritis. [(3) H]SCH 900567 was synthesized to support the initial drug metabolism and pharmacokinetics studies. Stable isotope-labeled [(13) C3 , (15) N]SCH 900567 was requested by the bioanalytical group as an internal standard for Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method development as well as by the drug metabolism and pharmacokinetics group for a potential microdose study. [(13) C3 , (15) N]SCH 900567 is synthesized via a linear sequence of seven steps from commercially available materials in 2.6% overall yield. [(14) C]SCH 900567 was needed for a quantitative whole body autoradiography studies and was prepared from unlabeled Active Pharmaceutical Ingredient (API) via hydrolysis of the hydantoin moiety followed by rebuilding the hydantoin ring using potassium [(14) C]cyanate to give the desired product in 42.8% overall yield. Activation of the hydantoin moiety of SCH 900567 to achieve hydrolysis followed by derivatization of the resulting amino acid to avoid decarboxylation during cyclization is also discussed.
Assuntos
Proteínas ADAM/antagonistas & inibidores , Hidantoínas/síntese química , Isoindóis/síntese química , Compostos Radiofarmacêuticos/síntese química , Proteína ADAM17 , Radioisótopos de Carbono/química , Hidantoínas/farmacocinética , Isoindóis/farmacocinética , Radioisótopos de Nitrogênio/química , Trítio/químicaRESUMO
Syntheses of stable and radioactive isotope-labeled anticonvulsant agent, JNJ-26990990, that is, N-(benzo[b]thien-3-ylmethyl)-sulfamide and its metabolites are described. [(13)C(15)N]Benzo[b]thiophene-3-carbonitrile was first prepared by coupling of 3-bromo-benzo[b]thiophene with [(13)C(15)N]-copper cyanide. The resultant [(13)C(15)N]benzo[b]thiophene-3-carbonitrile was reduced with lithium aluminum deuteride to give [(13)CD2(15)N]benzo[b]thiophen-3-yl-methylamine; which was then coupled with sulfamide to afford [(13)CD2(15)N]-N-(benzo[b]thien-3-ylmethyl)-sulfamide, the stable isotope-labeled compound with four stable isotope atoms. Direct oxidation of [(13)CD2(15)N]-N-(benzo[b]thien-3-ylmethyl)-sulfamide with hydrogen peroxide and peracetic acid gave the stable isotope-labeled sulfoxide and sulfone metabolites. On the other hand, radioactive (14)C-labeled N-(benzo[b]thien-3-ylmethyl)-sulfamide was prepared conveniently by sequential coupling of 3-bromo-benzo[b]thiophene with [(14)C]-copper cyanide, reduction of the carbonitrile to carboxaldehyde, and reductive amination with sulfamide.
Assuntos
Anticonvulsivantes/síntese química , Deutério/química , Compostos Radiofarmacêuticos/síntese química , Sulfonamidas/síntese química , Tiofenos/síntese química , Isótopos de Carbono/química , Radioisótopos de Carbono/química , Técnicas de Química Sintética/métodos , Marcação por Isótopo/métodos , Isótopos de Nitrogênio/químicaRESUMO
Diverse metabolic disorders can disrupt brain growth, and analyzing metabolism in animal models of microcephaly may reveal new mechanisms of pathogenesis. The metabolism of functioning cells in a living organism is constantly changing in response to a changing environment, circadian rhythms, consumed food, drugs, progressing sicknesses, aging, and many other factors. Metabolic profiling can give important insights into the working machinery of the cell. However, a frozen snapshot of the interconnected, complex network of reactions gives very limited information about this system. Flux analysis using stable isotope labels enables more robust metabolic studies that consider interrogate metabolite processing and changes in molecular concentrations over time.
Assuntos
Doenças Metabólicas , Microcefalia , Animais , Camundongos , Ritmo Circadiano/fisiologia , Isótopos , Doenças Metabólicas/diagnóstico , Metabolômica , Microcefalia/complicaçõesRESUMO
The fragmentation mechanism of D-glucose was investigated in detail by two different fragmentation techniques, namely, collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) using all six 13 C-labeled isotopomers and 2 H-labeled isotopomers. For both CID and IRMPD energy-resolved measurements were carried out. Individual fragmentation pathways were studied at MS2 and MS3 levels. Additionally, we have developed an HPLC-tandem MS method to separate the anomers of D-glucose using a HILIC column and investigated their fragmentation patterns individually. We propose a complete fragmentation landscape of D-glucose, demonstrating that a rather simple multifunctional molecule displays extreme complexity in gas phase dissociation, following multiple parallel fragmentation routes yielding a total of 23 distinct fragment ions. The results allowed a detailed formulation of the complex fragmentation mechanism of D-glucose. The results have immediate consequences for the full structure analysis of complex carbohydrates.
Assuntos
Carboidratos , Glucose , Espectrometria de Massas/métodos , Íons/química , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
A UPLC-MS/MS method was developed for the detection of tropomyosin (TM) in shrimp and crab. After simple extraction, the samples were purified by immunoaffinity column and then digested by trypsin. The obtained sample was separated by Easy-nLC 1000-Q Exactive. The obtained spectrums were analyzed by Thermo Proteome Discoverer 1.4 software and then ANIQLVEK with high sensitivity was selected as the quantitative signature peptide. Isotope-labeled internal standard was used in the quantitative analysis. The method showed good linearity in the range of 5-5,000 µg/L with a limit of quantification (LOQ) of 0.1 mg/kg. The average recoveries were 77.22-95.66% with RSDs ≤ 9.97%, and the matrix effects were between 88.53 and 112.60%. This method could be used for rapid screening and quantitative analysis of TM in shrimp and crab. Thus, it could provide technical support for self-testing of TM by food manufacturers and promote further improvement of allergen labeling in China.
RESUMO
The introduction of stable isotopes in vivo via metabolic labeling approaches (SILAC or 15N-labeling) allows, after combination of differentially treated labeled and unlabeled cells or protein extracts, for correction of protein quantification errors implemented during elaborated sample preparation workflows. The SILAC-based approach uses heavy arginine and lysine to incorporate the label into bacterial strains and cell lines, whereas 15N-metabolic labeling is achieved by cultivation in 15N-salt containing media. In case of Clostridioides difficile, the lack in arginine and lysine auxotrophy as well as the Stickland dominated metabolism makes metabolic labeling challenging. Here, a step-by-step guideline for the metabolic labeling of C. difficile is described, which combines cultivation in liquid 15N-substituted medium followed by cultivation steps on solid 15N-substituted medium. The described procedure results in a label incorporation rate higher than 97%. Cells prepared by the following method can be used as standard for relative quantification approaches of, e.g., the membrane or surface proteome of C. difficile.
Assuntos
Proteínas de Bactérias/análise , Clostridioides difficile/metabolismo , Marcação por Isótopo , Proteômica , Clostridioides difficile/crescimento & desenvolvimento , Projetos de PesquisaRESUMO
The herbicide metamitron is frequently detected in the environment, and its degradation in soil differs from that in aquatic sediments. In this study, we applied 13C6-metamitron to investigate the differences in microbial activity, metamitron mineralization and metamitron degrading microbial communities between soil and water-sediment systems. Metamitron increased soil respiration, whereas it suppressed respiration in the water-sediment system as compared to controls. Metamitron was mineralized two-fold faster in soil than in the water-sediment. Incorporation of 13C from 13C6-metamitron into Phospholipid fatty acids (PLFAs) was higher in soil than in sediment, suggesting higher activity of metamitron-degrading microorganisms in soil. During the accelerated mineralization of metamitron, biomarkers for Gram-negative, Gram-positive bacteria and actinobacteria dominated within the 13C-PLFAs in soil. Gram-negative bacteria dominated among the metamitron degraders in sediment throughout the incubation period. Actinobacteria, and actinobacteria and fungi were the main consumers of necromass of primary degraders in soil and water-sediment, respectively. This study clearly showed that microbial groups involved in metamitron degradation depend on the system (soil vs. water-sediment) and on time. It also indicated that the turnover of organic chemicals in complex environments is driven by different groups of synthropic degraders (primary degraders and necromass degraders) rather than by a single degrader.
Assuntos
Microbiota , Solo , Microbiologia do Solo , Triazinas , ÁguaRESUMO
The simultaneous effects of biochar on soil organic matter (SOM, C4) and sweet potato (SP) residue (Ipomoea batatas, C3) mineralization were studied over 180 days via 13C and 14C isotopic label partitioning. Upon concomitant SP residue addition, biochar mineralization decreased by 11% of the total added biochar-C. Compared to positive priming effects induced by biochar amendment alone on SOM (0.46 mg C g-1 soil) at 180 days, amendment solely with SP residues induced significantly larger effects (1.5 mg C g-1 soil). Combination biochar and SP residue addition reduced SOM mineralization by 20.5% and increased SP residue mineralization by 10.1%. Biochar addition caused preferential uptake of SP residues over SOM by microbes. Thus, the lower priming effects on SOM and CO2 emission induced by biochar amendment with or without SP residues compared to that from SP residue addition alone may result in crop residues being partly pyrolyzed to biochar in the cropland.
Assuntos
Isótopos de Carbono/química , Radioisótopos de Carbono/química , Carvão Vegetal/química , Ipomoea batatas/química , Cinética , Solo/químicaRESUMO
Bioavailability/bioequivalence studies supporting clinical drug development or commercial supply of drug formulations are often time, cost, and resource intensive. The drug's pharmacokinetic (PK) variability, systemic half-life, and safety issues may pose additional challenges. The stable isotope label (SIL) approach provides a useful tool to significantly reduce the study size in clinical PK studies. Tafenoquine (TQ) is an 8-aminoquinoline under development for preventing Plasmodium vivax malaria relapse. This SIL study assessed the impact of differences in the in vitro dissolution profiles on in vivo exposure of TQ tablets. Fourteen healthy volunteers received a single dose of 300 mg TQ Intermediate Aged or 300 mg TQ Control formulations in this single-center, two-arm, randomized, open-label, parallel-group study. Endpoints included the geometric means ratio of the area under the concentration-time curve (AUC(0-t) and AUC(0-∞); primary endpoint) and maximum plasma concentration (Cmax) for Intermediate Aged versus Control TQ; correlation of PK parameters for venous versus peripheral (via microsample) blood samples; and safety and tolerability endpoints. Geometric mean ratios for PK parameters (AUC and Cmax) and their 90% confidence intervals fell well within standard bioequivalence limits (0.80-1.25). Only one mild adverse event (skin abrasion) was reported. In summary, this SIL methodology-based study demonstrates that the observed differences in the in vitro dissolution profiles between the Control and Intermediate Aged TQ tablets have no clinically relevant effect on systemic TQ exposure. The SIL approach was successfully implemented to enable the setting of a clinically relevant dissolution specification. CLINICAL TRIAL: This study (GSK study number 201780) is registered at clinicaltrials.gov with identifier NCT02751294.
Assuntos
Aminoquinolinas/farmacocinética , Antimaláricos/farmacocinética , Liberação Controlada de Fármacos , Administração Oral , Adulto , Aminoquinolinas/administração & dosagem , Aminoquinolinas/química , Antimaláricos/administração & dosagem , Área Sob a Curva , Disponibilidade Biológica , Isótopos de Carbono , Feminino , Meia-Vida , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Isótopos de Nitrogênio , Solubilidade , Comprimidos , Equivalência TerapêuticaRESUMO
AIM: A traditional oral fatty acid challenge assesses absorption of triacylglycerol (TG) into the periphery through the intestines, but cannot distinguish the composition or source of fatty acid in the TG. Stable isotope-labeled tracers combined with LC-MRM can be used to identify and distinguish TG synthesized with dietary and stored fatty acids. RESULTS: Concentrations of three abundant TGs (52:2, 54:3 and 54:4) were monitored for incorporation of one or two (2)H11-oleate molecules per TG. This method was subjected to routine assay validation and meets typical requirements for an assay to be used to support clinical studies. CONCLUSION: Calculations for the fractional appearance rate of TG in plasma are presented along with the intracellular enterocyte precursor pool for 12 study participants.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Mucosa Intestinal/metabolismo , Triglicerídeos/análise , Adolescente , Adulto , Deutério/análise , Dieta , Humanos , Marcação por Isótopo/métodos , Masculino , Ácido Oleico/análise , Ácido Oleico/sangue , Ácido Oleico/metabolismo , Triglicerídeos/sangue , Triglicerídeos/metabolismo , Adulto JovemRESUMO
We contrasted the seasonal use of simulated large rain events (24 mm) by three native species of the arid Colorado Plateau: the perennial grass Hilaria jamesii and two shrubs Artemesia filifolia and Coleogyne ramosissima. Deuterium-enriched water was used to distinguish shallow "pulse" water from water in deeper soil layers that were unaffected by the water input. We also measured the leaf gas exchange rates of watered and unwatered control plants for 5 days after the rain event. H. jamesii had twice the pulse water proportion in its xylem than the two shrubs in spring (approx. 70% vs 35%). In summer, the pulse water proportions of all species were around 70%. The increase in the relative pulse water uptake of the two shrubs was caused primarily by a reduction in the rate of water uptake from deeper sources, consistent with the decrease in the availability of stored winter water. Rain increased the rates of gas exchange in C. ramosissima in both seasons, in H. jamesii only in summer and had no significant effect on A. filifolia. In H. jamesii, summer rain also increased water use efficiency. This suggests three principle mechanisms for rainwater use: (1) immediate increase in gas exchange via stomatal opening (C. ramisissima), (2) immediate increase in water use efficiency through restoration of the photosynthetic apparatus (H. jamesii) and (3) conservation of deeper soil water, potentially extending photosynthetic activity into later drought periods (A. filifolia). On a ground-area basis, A. filifolia was by far the largest consumer of spring and summer rain, due to its greater ground cover, while rain use by H. jamesii was negligible. We hypothesize that a population's fraction of the total community Leaf Area Index, more than species identity, determines which species takes up most of the spring and summer precipitation and we discuss this idea in the context of Walter and Stadelmann's (1974, In: Brown JW Jr (ed) Desert biology. Academic Press, New York, pp 213-310) water partitioning hypothesis.