RESUMEN
BACKGROUND: Clinical practice guidelines recommend estimation of glomerular filtration rate (eGFR) using validated equations based on serum creatinine (eGFRcr), cystatin C (eGFRcys), or both (eGFRcr-cys). However, when compared with the measured GFR (mGFR), only eGFRcr-cys meets recommended performance standards. Our goal was to develop a more accurate eGFR method using a panel of metabolites without creatinine, cystatin C, or demographic variables. METHODS: An ultra-performance liquid chromatography-tandem mass spectrometry assay for acetylthreonine, phenylacetylglutamine, pseudouridine, and tryptophan was developed, and a 20-day, multiinstrument analytical validation was conducted. The assay was tested in 2424 participants with mGFR data from 4 independent research studies. A new GFR equation (eGFRmet) was developed in a random subset (n = 1615) and evaluated in the remaining participants (n = 809). Performance was assessed as the frequency of large errors [estimates that differed from mGFR by at least 30% (1 - P30); goal <10%]. RESULTS: The assay had a mean imprecision (≤10% intraassay, ≤6.9% interassay), linearity over the quantitative range (r 2 > 0.98), and analyte recovery (98.5%-113%). There was no carryover, no interferences observed, and analyte stability was established. In addition, 1 - P30 in the validation set for eGFRmet (10.0%) was more accurate than eGFRcr (13.1%) and eGFRcys (12.0%) but not eGFRcr-cys (8.7%). Combining metabolites, creatinine, cystatin C, and demographics led to the most accurate equation (7.0%). Neither equation had substantial variation among population subgroups. CONCLUSIONS: The new eGFRmet equation could serve as a confirmatory test for GFR estimation.
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Cromatografía Liquida/métodos , Tasa de Filtración Glomerular , Espectrometría de Masas en Tándem/métodos , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Glutamina/análogos & derivados , Glutamina/sangre , Humanos , Masculino , Persona de Mediana Edad , Seudouridina/sangre , Reproducibilidad de los Resultados , Treonina/análogos & derivados , Treonina/sangre , Triptófano/sangreRESUMEN
Precision medicine, taking account of human individuality in genes, environment, and lifestyle for early disease diagnosis and individualized therapy, has shown great promise to transform medical care. Nontargeted metabolomics, with the ability to detect broad classes of biochemicals, can provide a comprehensive functional phenotype integrating clinical phenotypes with genetic and nongenetic factors. To test the application of metabolomics in individual diagnosis, we conducted a metabolomics analysis on plasma samples collected from 80 volunteers of normal health with complete medical records and three-generation pedigrees. Using a broad-spectrum metabolomics platform consisting of liquid chromatography and GC coupled with MS, we profiled nearly 600 metabolites covering 72 biochemical pathways in all major branches of biosynthesis, catabolism, gut microbiome activities, and xenobiotics. Statistical analysis revealed a considerable range of variation and potential metabolic abnormalities across the individuals in this cohort. Examination of the convergence of metabolomics profiles with whole-exon sequences (WESs) provided an effective approach to assess and interpret clinical significance of genetic mutations, as shown in a number of cases, including fructose intolerance, xanthinuria, and carnitine deficiency. Metabolic abnormalities consistent with early indications of diabetes, liver dysfunction, and disruption of gut microbiome homeostasis were identified in several volunteers. Additionally, diverse metabolic responses to medications among the volunteers may assist to identify therapeutic effects and sensitivity to toxicity. The results of this study demonstrate that metabolomics could be an effective approach to complement next generation sequencing (NGS) for disease risk analysis, disease monitoring, and drug management in our goal toward precision care.
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Voluntarios Sanos , Metaboloma , Plasma , Medicina de Precisión , Cromatografía Liquida , Estudios de Cohortes , Cromatografía de Gases y Espectrometría de Masas , HumanosRESUMEN
OBJECTIVE: To interrogate the metabolic profile of five subjects from three families with rare, nonsense and missense mutations in SLC13A5 and Early Infantile Epileptic Encephalopathies (EIEE) characterized by severe, neonatal onset seizures, psychomotor retardation and global developmental delay. METHODS: Mass spectrometry of plasma, CSF and urine was used to identify consistently dysregulated analytes in our subjects. RESULTS: Distinctive elevations of citrate and dysregulation of citric acid cycle intermediates, supporting the hypothesis that loss of SLC13A5 function alters tricarboxylic acid cycle (TCA) metabolism and may disrupt metabolic compartmentation in the brain. SIGNIFICANCE: Our results indicate that analysis of plasma citrate and other TCA analytes in SLC13A5 deficient patients define a diagnostic metabolic signature that can aid in diagnosing children with this disease.
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Ciclo del Ácido Cítrico , Espasmos Infantiles/metabolismo , Simportadores/deficiencia , Simportadores/genética , Niño , Ácido Cítrico/sangre , Femenino , Humanos , Recién Nacido , Masculino , Espectrometría de Masas , Metaboloma , Metabolómica/métodos , Mutación , Mutación Missense , Convulsiones/metabolismo , Espasmos Infantiles/diagnóstico , Secuenciación del ExomaRESUMEN
We sought to determine the molecular composition of human cerebrospinal fluid (CSF) and identify the biochemical pathways represented in CSF to understand the potential for untargeted screening of inborn errors of metabolism (IEMs). Biochemical profiles for each sample were obtained using an integrated metabolomics workflow comprised of four chromatographic techniques followed by mass spectrometry. Secondarily, we wanted to compare the biochemical profile of CSF with those of plasma and urine within the integrated mass spectrometric-based metabolomic workflow. Three sample types, CSF (N=30), urine (N=40) and EDTA plasma (N=31), were analyzed from retrospectively collected pediatric cohorts of equivalent age and gender characteristics. We identified 435 biochemicals in CSF representing numerous biological and chemical/structural families. Sixty-three percent (273 of 435) of the biochemicals detected in CSF also were detected in urine and plasma, another 32% (140 of 435) were detected in either plasma or urine, and 5% (22 of 435) were detected only in CSF. Analyses of several metabolites showed agreement between clinically useful assays and the metabolomics approach. An additional set of CSF and plasma samples collected from the same patient revealed correlation between several biochemicals detected in paired samples. Finally, analysis of CSF from a pediatric case with dihydropteridine reductase (DHPR) deficiency demonstrated the utility of untargeted global metabolic phenotyping as a broad assessment to screen samples from patients with undifferentiated phenotypes. The results indicate a single CSF sample processed with an integrated metabolomics workflow can be used to identify a large breadth of biochemicals that could be useful for identifying disrupted metabolic patterns associated with IEMs.
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Proteínas del Líquido Cefalorraquídeo/genética , Proteínas del Líquido Cefalorraquídeo/metabolismo , Líquido Cefalorraquídeo/química , Líquido Cefalorraquídeo/metabolismo , Metaboloma , Metabolómica/métodos , Adolescente , Biomarcadores/sangre , Biomarcadores/orina , Proteínas del Líquido Cefalorraquídeo/análisis , Proteínas del Líquido Cefalorraquídeo/química , Niño , Preescolar , Dihidropteridina Reductasa/sangre , Dihidropteridina Reductasa/genética , Dihidropteridina Reductasa/metabolismo , Dihidropteridina Reductasa/orina , Femenino , Humanos , Lactante , Masculino , Espectrometría de Masas/métodos , Errores Innatos del Metabolismo/diagnóstico , Fenotipo , Estudios Retrospectivos , Adulto JovenRESUMEN
Global metabolic profiling currently achievable by untargeted mass spectrometry-based metabolomic platforms has great potential to advance our understanding of human disease states, including potential utility in the detection of novel and known inborn errors of metabolism (IEMs). There are few studies of the technical reproducibility, data analysis methods, and overall diagnostic capabilities when this technology is applied to clinical specimens for the diagnosis of IEMs. We explored the clinical utility of a metabolomic workflow capable of routinely generating semi-quantitative z-score values for ~900 unique compounds, including ~500 named human analytes, in a single analysis of human plasma. We tested the technical reproducibility of this platform and applied it to the retrospective diagnosis of 190 individual plasma samples, 120 of which were collected from patients with a confirmed IEM. Our results demonstrate high intra-assay precision and linear detection for the majority compounds tested. Individual metabolomic profiles provided excellent sensitivity and specificity for the detection of a wide range of metabolic disorders and identified novel biomarkers for some diseases. With this platform, it is possible to use one test to screen for dozens of IEMs that might otherwise require ordering multiple unique biochemical tests. However, this test may yield false negative results for certain disorders that would be detected by a more well-established quantitative test and in its current state should be considered a supplementary test. Our findings describe a novel approach to metabolomic analysis of clinical specimens and demonstrate the clinical utility of this technology for prospective screening of IEMs.
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Biomarcadores/análisis , Errores Innatos del Metabolismo/diagnóstico , Metabolómica/métodos , Tamizaje Neonatal/métodos , Humanos , Recién Nacido , Reproducibilidad de los Resultados , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
Drug-induced liver injury (DILI) is a significant consideration for drug development. Current preclinical DILI assessment relying on histopathology and clinical chemistry has limitations in sensitivity and discordance with human. To gain insights on DILI pathogenesis and identify potential biomarkers for improved DILI detection, we performed untargeted metabolomic analyses on rats treated with thirteen known hepatotoxins causing various types of DILI: necrosis (acetaminophen, bendazac, cyclosporine A, carbon tetrachloride, ethionine), cholestasis (methapyrilene and naphthylisothiocyanate), steatosis (tetracycline and ticlopidine), and idiosyncratic (carbamazepine, chlorzoxasone, flutamide, and nimesulide) at two doses and two time points. Statistical analysis and pathway mapping of the nearly 1900 metabolites profiled in the plasma, urine, and liver revealed diverse time and dose dependent metabolic cascades leading to DILI by the hepatotoxins. The most consistent change induced by the hepatotoxins, detectable even at the early time point/low dose, was the significant elevations of a panel of bile acids in the plasma and urine, suggesting that DILI impaired hepatic bile acid uptake from the circulation. Furthermore, bile acid amidation in the hepatocytes was altered depending on the severity of the hepatotoxin-induced oxidative stress. The alteration of the bile acids was most evident by the necrosis and cholestasis hepatotoxins, with more subtle effects by the steatosis and idiosyncratic hepatotoxins. Taking together, our data suggest that the perturbation of bile acid homeostasis is an early event of DILI. Upon further validation, selected bile acids in the circulation could be potentially used as sensitive and early DILI preclinical biomarkers.
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Ácidos y Sales Biliares/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Estrés Oxidativo/fisiología , Toxinas Biológicas/toxicidad , Animales , Ácidos y Sales Biliares/sangre , Ácidos y Sales Biliares/orina , Biomarcadores/sangre , Biomarcadores/metabolismo , Biomarcadores/orina , Cromatografía Líquida de Alta Presión , Cromatografía de Gases y Espectrometría de Masas , Hepatocitos/metabolismo , Masculino , Metabolómica/métodos , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem , Toxinas Biológicas/administración & dosificaciónRESUMEN
Our objective was to identify metabolic pathways affected by ALS using non-targeted metabolomics in plasma, comparing samples from healthy volunteers to those from ALS patients. This discovery could become the basis for the identification of therapeutic targets and diagnostic biomarkers of ALS. Two distinct cross-sectional studies were conducted. Plasma was collected from 62 (Study 1) and 99 (Study 2) participants meeting El Escorial criteria for possible, probable, or definite ALS; 69 (Study 1) and 48 (Study 2) healthy controls samples were collected. Global metabolic profiling was used to detect and evaluate biochemical signatures of ALS. Twenty-three metabolites were significantly altered in plasma from ALS patients in both studies. These metabolites include biochemicals in pathways associated with neuronal change, hypermetabolism, oxidative damage, and mitochondrial dysfunction, all of which are proposed disease mechanisms in ALS. The data also suggest possible hepatic dysfunction associated with ALS. In conclusion, the data presented here provide insight into the pathophysiology of ALS while suggesting promising areas of focus for future studies. The metabolomics approach can generate novel hypotheses regarding ALS disease mechanisms with the potential to identify therapeutic targets and novel diagnostic biomarkers.
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Esclerosis Amiotrófica Lateral/sangre , Esclerosis Amiotrófica Lateral/fisiopatología , Biomarcadores/sangre , Adulto , Anciano , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Estudios Transversales , Suplementos Dietéticos , Femenino , Humanos , Masculino , Metabolómica/métodos , Persona de Mediana Edad , Fármacos Neuroprotectores/uso terapéutico , Riluzol/uso terapéuticoRESUMEN
BACKGROUND: The application of whole-exome sequencing for the diagnosis of genetic disease has paved the way for systems-based approaches in the clinical laboratory. Here, we describe a clinical metabolomics method for the screening of metabolic diseases through the analysis of a multi-pronged mass spectrometry platform. By simultaneously measuring hundreds of metabolites in a single sample, clinical metabolomics offers a comprehensive approach to identify metabolic perturbations across multiple biochemical pathways. METHODS: We conducted a single- and multi-day precision study on hundreds of metabolites in human plasma on 4, multi-arm, high-throughput metabolomics platforms. RESULTS: The average laboratory coefficient of variation (CV) on the 4 platforms was between 9.3 and 11.5% (median, 6.5-8.4%), average inter-assay CV on the 4 platforms ranged from 9.9 to 12.6% (median, 7.0-8.3%) and average intra-assay CV on the 4 platforms ranged from 5.7 to 6.9% (median, 3.5-4.4%). In relation to patient sample testing, the precision of multiple biomarkers associated with IEM disorders showed CVs that ranged from 0.2 to 11.0% across 4 analytical batches. CONCLUSIONS: This evaluation describes single and multi-day precision across 4 identical metabolomics platforms, comprised each of 4 independent method arms, and reproducibility of the method for the measurement of key IEM metabolites in patient samples across multiple analytical batches, providing evidence that the method is robust and reproducible for the screening of patients with inborn errors of metabolism.
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Errores Innatos del Metabolismo/sangre , Errores Innatos del Metabolismo/diagnóstico , Metaboloma , Metabolómica/métodos , Metabolómica/normas , Adolescente , Biomarcadores , Niño , Preescolar , Cromatografía Liquida , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Redes y Vías Metabólicas , Errores Innatos del Metabolismo/etiología , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem , Adulto JovenRESUMEN
AIMS: We wished to determine the efficacy of using urine as an analyte to screen for a broad range of metabolic products associated with multiple different types of inborn errors of metabolism (IEMs), using an automated mass spectrometry-based assay. Urine was compared with plasma samples from a similar cohort analyzed using the same assay. Specimens were analyzed using two different commonly utilized urine normalization methods based on creatinine and osmolality, respectively. METHODS: Biochemical profiles for each sample (from both affected and unaffected subjects) were obtained using a mass spectrometry-based platform and population-based statistical analyses. RESULTS: We identified over 1200 biochemicals from among 100 clinical urine samples and identified clear biochemical signatures for 16 of 18 IEM diseases tested. The two diseases that did not result in clear signatures, X-linked creatine transporter deficiency and ornithine transcarbamylase deficiency, were from individuals under treatment, which masked biomarker signatures. Overall the process variability and coefficient of variation for isolating and identifying biochemicals by running technical replicates of each urine sample was 10%. CONCLUSIONS: A single urine sample analyzed with our integrated metabolomic platform can identify signatures of IEMs that are traditionally identified using many different assays and multiple sample types. Creatinine and osmolality-normalized data were robust to the detection of the disorders and samples tested here.
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Biomarcadores/orina , Errores Innatos del Metabolismo/orina , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Espectrometría de Masas/métodos , Errores Innatos del Metabolismo/diagnóstico , Metabolómica/métodos , Análisis por Matrices de Proteínas/métodosRESUMEN
Bladder cancer (BCa) is a common malignancy worldwide and has a high probability of recurrence after initial diagnosis and treatment. As a result, recurrent surveillance, primarily involving repeated cystoscopies, is a critical component of post diagnosis patient management. Since cystoscopy is invasive, expensive and a possible deterrent to patient compliance with regular follow-up screening, new non-invasive technologies to aid in the detection of recurrent and/or primary bladder cancer are strongly needed. In this study, mass spectrometry based metabolomics was employed to identify biochemical signatures in human urine that differentiate bladder cancer from non-cancer controls. Over 1000 distinct compounds were measured including 587 named compounds of known chemical identity. Initial biomarker identification was conducted using a 332 subject sample set of retrospective urine samples (cohort 1), which included 66 BCa positive samples. A set of 25 candidate biomarkers was selected based on statistical significance, fold difference and metabolic pathway coverage. The 25 candidate biomarkers were tested against an independent urine sample set (cohort 2) using random forest analysis, with palmitoyl sphingomyelin, lactate, adenosine and succinate providing the strongest predictive power for differentiating cohort 2 cancer from non-cancer urines. Cohort 2 metabolite profiling revealed additional metabolites, including arachidonate, that were higher in cohort 2 cancer vs. non-cancer controls, but were below quantitation limits in the cohort 1 profiling. Metabolites related to lipid metabolism may be especially interesting biomarkers. The results suggest that urine metabolites may provide a much needed non-invasive adjunct diagnostic to cystoscopy for detection of bladder cancer and recurrent disease management.
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Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/orina , Metabolómica , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/orina , Anciano , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Neoplasias de la Vejiga Urinaria/diagnósticoRESUMEN
Our objective was to identify plasma biomarkers of ALS that can aid in distinguishing patients with ALS from those with disease mimics. In this multi-center study, plasma samples were collected from 172 patients recently diagnosed with ALS, 50 healthy controls, and 73 neurological disease mimics. Samples were analyzed using metabolomics. Using all identified biochemicals detected in > 50% of all samples in the metabolomics analysis, samples were classified as ALS or mimic with 65% sensitivity and 81% specificity by LASSO analysis (AUC of 0.76). A subset panel of 32 candidate biomarkers classified these diagnosis groups with a specificity of 90%/sensitivity 58% (AUC of 0.81). Creatinine was lower in subjects with lower revised ALS Functional Rating Scale (ALSFRS-R) scores. In conclusion, ALS can be distinguished from neurological disease mimics by global biochemical profiling of plasma samples. Our analysis identified ALS versus mimics with relatively high sensitivity. We identified a subset of 32 metabolites that identify patients with ALS with a high specificity. Interestingly, lower creatinine correlates significantly with a lower ALSFRS-R score. Finally, molecules previously reported to be important in disease pathophysiology, such as urate, are included in our metabolite panel.
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Esclerosis Amiotrófica Lateral/sangre , Esclerosis Amiotrófica Lateral/diagnóstico , Biomarcadores/sangre , Adulto , Anciano , Área Bajo la Curva , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Masculino , Persona de Mediana Edad , Examen Neurológico , Sensibilidad y Especificidad , Máquina de Vectores de SoporteRESUMEN
Genetically modified (GM) crops currently constitute a significant and growing part of agriculture.An important aspect of GM crop adoption is to demonstrate safety; identifying differences in end points with respect to conventional crops is a part of the safety assessment process [corrected]. Untargeted metabolomics has the ability to profile diverse classes of metabolites and thus could be an adjunct for identification of differences between the GM crop and its conventional counterpart [corrected].To account for environmental effects and introgression of GM traits into diverse genetic backgrounds, we propose that the assessment for GM crop metabolic composition should be understood within the context of the natural variation for the crop. Using a non-targeted metabolomics platform, we profiled 169 metabolites and established their dynamic ranges from the seeds of 49 conventional soybean lines representing the current commercial genetic diversity. We further demonstrated that the metabolome of a GM line had no significant deviation from natural variation within the soybean metabolome, with the exception of changes in the targeted engineered pathway.
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Glycine max/genética , Glycine max/metabolismo , Metaboloma , Metabolómica , Semillas/genética , Semillas/metabolismo , Análisis por Conglomerados , Biología Computacional , Plantas Modificadas GenéticamenteRESUMEN
Pentamethyl-6-chromanol (PMCol), a chromanol-type compound related to vitamin E, was proposed as an anticancer agent with activity against androgen-dependent cancers. In repeat dose-toxicity studies in rats and dogs, PMCol caused hepatotoxicity, nephrotoxicity, and hematological effects. The objectives of this study were to determine the mechanisms of the observed toxicity and identify sensitive early markers of target organ injury by integrating classical toxicology, toxicogenomics, and metabolomic approaches. PMCol was administered orally to male Sprague-Dawley rats at 200 and 2000 mg/kg daily for 7 or 28 days. Changes in clinical chemistry included elevated alanine aminotransferase, total bilirubin, cholesterol and triglycerides-indicative of liver toxicity that was confirmed by microscopic findings (periportal hepatocellular hydropic degeneration and cytomegaly) in treated rats. Metabolomic evaluations of liver revealed time- and dose-dependent changes, including depletion of total glutathione and glutathione conjugates, decreased methionine, and increased S-adenosylhomocysteine, cysteine, and cystine. PMCol treatment also decreased cofactor levels, namely, FAD and increased NAD(P)+. Microarray analysis of liver found that differentially expressed genes were enriched in the glutathione and cytochrome P450 pathways by PMCol treatment. Reverse transcription-polymerase chain reaction of six upregulated genes and one downregulated gene confirmed the microarray results. In conclusion, the use of metabolomics and toxicogenomics demonstrates that chronic exposure to high doses of PMCol induces liver damage and dysfunction, probably due to both direct inhibition of glutathione synthesis and modification of drug metabolism pathways. Depletion of glutathione due to PMCol exposure ultimately results in a maladaptive response, increasing the consumption of hepatic dietary antioxidants and resulting in elevated reactive oxygen species levels associated with hepatocellular damage and deficits in liver function.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Cromanos/toxicidad , Hígado/efectos de los fármacos , Animales , Biomarcadores/sangre , Biomarcadores/orina , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Cromanos/sangre , Cromanos/orina , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Hígado/metabolismo , Hígado/patología , Masculino , Metabolómica , Estructura Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , ToxicogenéticaRESUMEN
BACKGROUND: Periodontal disease has been studied primarily from clinical outcomes in lengthy human studies. Comprehensive biochemical profiling (metabolomics) has become a powerful tool for disease characterization and biomarker discovery. In a previous study, we performed a metabolomic analysis of gingival crevicular fluid collected from healthy, gingivitis, and periodontitis sites. Many metabolites associated with inflammation, oxidative stress, tissue degradation, and bacterial metabolism were found to be significantly induced by the diseases. METHODS: A panel of 10 markers was selected from the previous metabolomic study based on their statistical significance. Thirty-nine chronic periodontitis subjects were randomly assigned to a toothpaste regimen: control dentifrice (n = 21) or triclosan-containing dentifrice ([CT] n = 18). Subjects were instructed to use their assigned dentifrice twice daily for 6 weeks. Gingival crevicular fluid samples from six healthy, six gingivitis, and three periodontitis sites were collected from each subject at baseline, 1 week, and 6 weeks. The relative levels of the markers in the samples were determined by mass spectrometry. One-sided matched-paired t tests were performed to compare data from healthy, gingivitis, and periodontitis sites. RESULTS: Statistical analysis indicates that CT significantly decreased the levels of inosine, lysine, putrescine, and xanthine at the gingivitis sites as early as week 1. In contrast, control dentifrice had little effect. CONCLUSIONS: This result provides biochemical confirmation for the therapeutic effects of CT on gingivitis. Biomarkers were significantly altered by CT before clinical changes were observed, suggesting that the markers have predicative value for disease state assessment.