Studies have identified that several amino acids, in particular, branched-chain amino acids (BCAAs), have increased significantly in obese individuals when compared to lean individuals. Additionally, these metabolites were strongly associated with future diabetes, which rendered them prognostic markers suitable for obese populations. Here we report a metabonomic study that reveals new findings on the role of these amino acid markers, particularly BCAAs, in a Chinese cohort including 106 healthy obese and 105 healthy lean participants. We found that the BCAAs were correlated with insulin resistance and differentially expressed in obese men, but not in obese women. The results were verified with two independent groups of participants (Chinese, n = 105 and American, n = 72) and demonstrate that the serum metabolite profiles of the obese population are gender-dependent. The study supports the previous findings of a panel of several key metabolites as prognostic markers of the obese population and highlights the need to take into account gender differences when using these markers for risk assessment.Keywords: branched-chain amino acid; GC−MS; gender; insulin resistance; metabonomics; obesity; UPLC-MS;

The hypolipidemic effect of simvastatin varies greatly among patients. In the current study, we investigated the gut microbial-involved mechanisms underlying the different responses to simvastatin. Male C57BL/6J mice were divided into control (Con), high-fat/cholesterol diet (HFD), antibiotic (AB), simvastatin (SV) and antibiotic_simvastatin (AB_SV) groups, respectively. At the end of the experiment, serum samples were collected for lipids and metabolomic analysis, and liver tissues for histology, gene and protein expression analysis. The results showed that antibiotic treatment not only altered the composition of gut microbiota, but attenuated the hypolipidemic effect of SV. A total of 16 differential metabolites between SV and HFD groups were identified with metabolomics, while most of them showed no statistical differences between AB_SV and HFD groups, and similar changes were also observed in bile acids profile. The expressions of several genes and proteins involved in regulating bile acids synthesis were significantly reversed by SV, but not AB_SV in HFD fed mice. In summary, our current study indicated that the hypolipidemic effect of SV was correlated with the composition of the gut microbiota, and the attenuated hypolipidemic effect of SV by gut microbiota modulation was associated with a suppression of bile acids synthesis from cholesterol.Keywords: antibiotic; gut microbiota; hypolipidemic effect; metabolomics; simvastatin;

Recent studies suggest that biofluid-based metabonomics may identify metabolite markers promising for colorectal cancer (CRC) diagnosis. We report here a follow-up replication study, after a previous CRC metabonomics study, aiming to identify a distinct serum metabolic signature of CRC with diagnostic potential. Serum metabolites from newly diagnosed CRC patients (N = 101) and healthy subjects (N = 102) were profiled using gas chromatography time-of-flight mass spectrometry (GC–TOFMS) and ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC–QTOFMS). Differential metabolites were identified with statistical tests of orthogonal partial least-squares-discriminant analysis (VIP > 1) and the Mann–Whitney U test (p < 0.05). With a total of 249 annotated serum metabolites, we were able to differentiate CRC patients from the healthy controls using an orthogonal partial least-squares-discriminant analysis (OPLS-DA) in a learning sample set of 62 CRC patients and 62 matched healthy controls. This established model was able to correctly assign the rest of the samples to the CRC or control groups in a validation set of 39 CRC patients and 40 healthy controls. Consistent with our findings from the previous study, we observed a distinct metabolic signature in CRC patients including tricarboxylic acid (TCA) cycle, urea cycle, glutamine, fatty acids, and gut flora metabolism. Our results demonstrated that a panel of serum metabolite markers is of great potential as a noninvasive diagnostic method for the detection of CRC.Keywords: colorectal cancer; diagnostic markers; metabolomics/metabonomics; serum metabolites;

Chronic ethanol consumption is associated with not only the alteration of metabolic profiles in biofluids but also the composition of the gut microbiome. Our understanding of the importance of the intestinal microbiota as well as the disturbances elicited by ethanol intervention is limited by the fact that previous analyses have primarily focused on biofluids and liver tissue metabolome; the metabolic profiles of the gastrointestinal (GI) contents are rarely investigated. In this study, we applied a metabonomics approach using a high performance liquid chromatography–time-of-flight mass spectrometry (HPLC–TOF MS) and gas chromatography–mass spectrometry (GC–MS) to characterize the metabolic alterations of the contents within the GI tract (stomach, duodenum, jejunum, ileum, cecum, colon, and rectum) in male Sprague–Dawley rats following 8 weeks of ethanol exposure. We obtained a snapshot of the distinct changes of the intestinal content metabolite composition in rats with ethanol exposure, which indicated a profound impact of ethanol consumption on the intestinal metabolome. Many metabolic pathways that are critical for host physiology were affected, including markedly altered bile acids, increased fatty acids and steroids, decreased carnitines and metabolites involved in lipid metabolism, a significant decrease of all amino acids and branched chain amino acids, and significantly decreased short chain fatty acids except for acetic acid, which rapidly elevated as a product of ethanol metabolism. These results provide an improved understanding of the systemic alteration of intestinal content metabolites in mammals and the interplay between the host and its complex resident microbiota and may aid in the design of new therapeutic strategies that target these interactions.Keywords: branched chain amino acid; chronic ethanol consumption; gas chromatography mass spectrometry; gastrointestinal tract; gut microbiota; high performance liquid chromatography mass spectrometry; metabonomics; short chain fatty acid;

Bile acids (BAs) are a group of important physiological agents for cholesterol metabolism, intestinal nutrient absorption, and biliary secretion of lipids, toxic metabolites, and xenobiotics. Extensive research in the last two decades has unveiled new functions of BAs as signaling molecules and metabolic regulators that modulate hepatic lipid, glucose, and energy homeostasis through the activation of nuclear receptors and G-protein-coupled receptor signaling in gut-liver metabolic axis involving host-gut microbial co-metabolism. Therefore, investigation of serum BA profiles, in healthy human male and female subjects with a wide range of age and body mass index (BMI), will provide important baseline information on the BA physiology as well as metabolic homeostasis among human subjects that are regulated by two sets of genome, host genome, and symbiotic microbiome. Previous reports on age- or gender-related changes on BA profiles in animals and human showed inconsistent results, and the information acquired from various studies was highly fragmentary. Here we profiled the serum BAs in a large population of healthy participants (n = 502) and examined the impact of age, gender, and BMI on serum BA concentrations and compositions using a targeted metabonomics approach with ultraperformance liquid chromatography triple–quadrupole mass spectrometry. We found that the BA profiles were dependent on gender, age, and BMI among study subjects. The total BAs were significantly higher in males than in females (p < 0.05) and higher in obese females than in lean females (p < 0.05). The difference in BA profiles between male and female subjects was decreased at age of 50–70 years, while the difference in BA profiles between lean and obese increased for subjects aged 50–70 years. The study provides a comprehensive understanding of the BA profiles in healthy subjects and highlights the need to take into account age, gender, and BMI differences when investigating pathophysiological changes of BAs resulting from gastrointestinal diseases.Keywords: age; bile acids; BMI; gender; LC−TQMS; metabonomics;

Similar symptoms of the different types of arthritis have continued to confound the clinical diagnosis and represent a clinical dilemma making treatment choices with a more personalized or generalized approach. Here we report a mass spectrometry-based metabolic phenotyping study to identify the global metabolic defects associated with arthritis as well as metabolic signatures of four major types of arthritis—rheumatoid arthritis (n = 27), osteoarthritis (n = 27), ankylosing spondylitis (n = 27), and gout (n = 33)—compared with healthy control subjects (n = 60). A total of 196 metabolites were identified from serum samples using a combined gas chromatography coupled with time-of-flight mass spectrometry (GC–TOF MS) and ultraperformance liquid chromatography quadrupole-time-of-flight mass spectrometry (UPLC–QTOF MS). A global metabolic profile is identified from all arthritic patients, suggesting that there are common metabolic defects resulting from joint inflammation and lesion. Meanwhile, differentially expressed serum metabolites are identified constituting an unique metabolic signature of each type of arthritis that can be used as biomarkers for diagnosis and patient stratification. The results highlight the applicability of metabonomic phenotyping as a novel diagnostic tool for arthritis complementary to existing clinical modalities.Keywords: ankylosing spondylitis; gouty arthritis; metabonomics; osteoarthritis; rheumatoid arthritis;

Host-gut microbial interactions contribute to human health and disease states and an important manifestation resulting from this cometabolism is a vast diversity of bile acids (BAs). There is increasing interest in using BAs as biomarkers to assess the health status of individuals and, therefore, an increased need for their accurate separation and identification. In this study, the negative ion fragmentation behaviors of C24 BAs were investigated by UPLC-ESI-QTOF-MS. The step-by-step fragmentation analysis revealed a distinct fragmentation mechanism for the unconjugated BAs containing a 12-hydroxyl group. The unconjugated BAs lacking 12-hydroxylation fragmented via dehydration and dehydrogenation. In contrast, the 12-hydroxylated ones, such as deoxycholic acid (DCA) and cholic acid (CA), employed dissociation routes including dehydration, loss of carbon monoxide or carbon dioxide, and dehydrogenation. All fragmentations of the 12-hydroxylated unconjugated BAs, characterized by means of stable isotope labeled standards, were associated with the rotation of the carboxylate side chain and the subsequent rearrangements accompanied by proton transfer between 12-hydroxyl and 24-carboxyl groups. Compared to DCA, CA underwent further cleavages of the steroid skeleton. Accordingly, the effects of stereochemistry on the fragmentation pattern of CA were investigated using its stereoisomers. Based on the knowledge gained from the fragmentation analysis, a novel BA, 3β,7β,12α-trihydroxy-5β-cholanic acid, was identified in the postprandial urine samples of patients with nonalcoholic steatohepatitis. The analyses used in this study may contribute to a better understanding of the chemical diversity of BAs and the molecular basis of human liver diseases that involve BA synthesis, transport, and metabolism.

Recent metabonomic studies have identified an important role of bile acids in patients with liver cirrhosis. Serum bile acids, such as glycocholate (GCA), glycochenodeoxycholate (GCDCA), taurocholate (TCA), and taurochenodeoxycholate (TCDCA), increased significantly in liver cirrhosis patients. Our recently published urinary metabonomic study showed that glycocholate 3-glucuronide, taurohyocholate, TCA, glycolithocholate 3-sulfate, and glycoursodeoxycholate (GUDCA) were markedly increased in hepatitis B-induced cirrhotic patients (n = 63) compared with healthy controls (n = 31). The urinary levels of GUDCA were able to differentiate among three stages of cirrhotic patients with Child-Pugh (CP) score A, B, and C. In this study, we recruited two new cohorts of patients with hepatitis-B-induced cirrhosis and healthy control subjects and quantitatively profiled their serum bile acids using ultra-performance liquid chromatography triple quadrupole mass spectrometry. Serum bile acid profile and corresponding differential bile acids were characterized, in addition to the blood routine, liver, and renal function tests. The alterations of bile acids contributing to the intergroup variation between healthy controls and cirrhotic patients and among pathological stages of CP grade A, B and C were also investigated. Five bile acids, GCA, GCDCA, TCA, TCDCA, and GUDCA, were significantly altered among different stages of liver cirrhosis (n = 85), which was validated with an independent cohort of cirrhotic patients (n = 53). Our results show that dynamic alteration of serum bile acids is indicative of an exacerbated liver function, highlighting their potential as biomarkers for staging the liver cirrhosis and monitoring its progression.

Glucocorticoids are commonly used in anti-inflammatory and immunomodulatory therapies, but glucocorticoid withdrawal can result in life-threatening risk of adrenal insufficiency. Chinese patented pharmaceutical product Jinkui Shenqi pill (JKSQ) has potent efficacy on clinical adrenal insufficiency resulting from glucocorticoid withdrawal. However, the underlying molecular mechanism remains unclear. We used an animal model to study JKSQ-induced metabolic changes under adrenal insufficiency and healthy conditions. Sprague–Dawley rats were treated with hydrocortisone for 7 days with or without 15 days of JKSQ pretreatment. Sera were collected after 72 h hydrocortisone withdrawal and used for global and free fatty acids (FFAs)-targeted metabolomics analyses using gas chromatography/time-of-flight mass spectrometry and ultraperformance liquid chromatography/quadruple time-of-flight mass spectrometry. Rats without hydrocortisone treatment were used as controls. JKSQ pretreatment normalized the significant changes of 13 serum metabolites in hydrocortisone-withdrawal rats, involving carbohydrates, lipids, and amino acids. The most prominent effect of JKSQ was on the changes of FFAs and some [product FFA]/[precursor FFA] ratios, which represent estimated desaturase and elongase activities. The opposite metabolic responses of JKSQ in adrenal insufficiency rats and normal rats highlighted the “Bian Zheng Lun Zhi” (treatment based on ZHENG differentiation) guideline of TCM and suggested that altered fatty acid metabolism was associated with adrenal insufficiency after glucocorticoid withdrawal and the protective effects of JKSQ.

Biliary atresia (BA) is a rare neonatal cholestatic disorder caused by obstruction of extra- and intra-hepatic bile ducts. If untreated, progressive liver cirrhosis will lead to death within 2 years. Early diagnosis and operation improve the outcome significantly. Infants with neonatal hepatitis syndrome (NHS) present similar symptoms, confounding the early diagnosis of BA. The lack of noninvasive diagnostic methods to differentiate BA from NHS greatly delays the surgery of BA infants, thus deteriorating the outcome. Here we performed a metabolomics study in plasma of BA, NHS, and healthy infants using gas chromatography–time-of-flight mass spectrometry. Scores plots of orthogonal partial least-squares discriminant analysis clearly separated BA from NHS and healthy infants. Eighteen metabolites were found to be differentially expressed between BA and NHS, among which seven (l-glutamic acid, l-ornithine, l-isoleucine, l-lysine, l-valine, l-tryptophan, and l-serine) were amino acids. The altered amino acids were quantitatively verified using ultraperformance liquid chromatography–tandem mass spectrometry. Ingenuity pathway analysis revealed the network of “Cellular Function and Maintenance, Hepatic System Development and Function, Neurological Disease” was altered most significantly. This study suggests that plasma metabolic profiling has great potential in differentiating BA from NHS, and amino acid metabolism is significantly different between the two diseases.

Biliary atresia (BA) is a severe chronic cholestasis disorder of infants that leads to death if not treated on time. Neonatal hepatitis syndrome (NHS) is another leading cause of neonatal cholestasis confounding the diagnosis of BA. Recent studies indicate that altered bile acid metabolism is closely associated with liver injury and cholestasis. In this study, we systematically measured the bile acid metabolome in plasma of BA, NHS, and healthy controls. Liver bile acids were also measured using biopsy samples from 48 BA and 16 NHS infants undergoing operative cholangiography as well as 5 normal adjacent nontumor liver tissues taken from hepatoblastoma patients as controls. Both BA and NHS samples had significantly elevated bile acid levels in plasma compared to normal controls. BA patients showed a distinct bile acid profile characterized by the higher taurochenodeoxycholic acid (TCDCA) level and lower chenodeoxycholic acid (CDCA) level than those in NHS patients. The ratio of TCDCA to CDCA in plasma was significantly higher in BA compared to healthy infants (p < 0.001) or NHS (p < 0.001). The area under receiver operating characteristic curve for TCDCA/CDCA to differentiate BA from NHS was 0.923 (95% CI: 0.862–0.984). These findings were supported by significantly altered expression levels of bile acid transporters and nuclear receptors in liver including farnesoid X receptor (FXR), small heterodimer partner (SHP), bile salt export pump (BSEP), and multidrug resistant protein 3 (MDR3) in BA compared to NHS. Taken together, the plasma bile acid profiles are distinct in BA, NHS, and normal infants, as characterized by the ratio of TCDCA/CDCA differentially distributed among the three groups of infants.

A number of metabolic conditions, including hypoglycemia, high blood pressure (HBP), dyslipidemia, nerve damage and amputation, and vision problems, occur as a result of uncontrolled blood glucose levels over a prolonged period of time. The different components of diabetic complications are not independent but rather interdependent of each other, rendering the disease difficult to diagnose and control. The underlying pathogenesis of those components cannot be easily elucidated because of the heterogeneous, polygenic, and multifactorial nature of the disease. Metabonomics offers a snapshot of distinct biochemical variations that may reflect the unique metabolic phenotype under pathophysiological conditions. Here we report a mass-spectrometry-based metabonomic study designed to identify the distinct metabolic changes associated with several complications of type 2 diabetes mellitus (T2DM). The 292 patients recruited in the study were divided into five groups, including T2DM with HBP, T2DM with nonalcoholic fatty liver disease (NAFLD), T2DM with HBP and NAFLD, T2DM with HBP and coronary heart disease (CHD), and T2DM with HBP, NAFLD, and CHD. Serum differential metabolites were identified in each group of T2DM complication, mainly involving bile acid, fatty acid, amino acid, lipid, carbohydrate, steroids metabolism, and tricarboxylic acids cycle. These broad-spectrum metabolic changes emphasize the complex abnormalities present among these complications with elevated blood glucose levels, providing a novel strategy for stratifying patients with T2DM complications using blood-based metabolite markers.

American ginseng (Panax quinquefolius L.) is one of the most commonly used herbal medicines in the West. It has been reported to possess significant antitumor effects that inhibit the process of carcinogenesis. However, the mechanisms underlying its anticancer effects remain largely unresolved. In this study, we investigated the cancer chemopreventive effects of American ginseng on the progression of high fat (HF) diet-enhanced colorectal carcinogenesis with a genetically engineered ApcMin/+ mouse model. The metabolic alterations in sera of experimental mice perturbed by HF diet intervention as well as the American ginseng treatment were measured by gas chromatography time-of-flight mass spectrometry (GC-TOFMS) and liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) analysis. American ginseng treatment significantly extended the life span of the ApcMin/+ mouse. Significant alterations of metabolites involving amino acids, organic acids, fatty acids, and carbohydrates were observed in ApcMin/+ mouse in sera, which were attenuated by American ginseng treatment and concurrent with the histopathological improvement with significantly reduced tumor initiation, progression and gut inflammation. These metabolic changes suggest that the preventive effect of American ginseng is associated with attenuation of impaired amino acid, carbohydrates, and lipid metabolism. It also appears that American ginseng induced significant metabolic alterations independent of the ApcMin/+ induced metabolic changes. The significantly altered metabolites induced by American ginseng intervention include arachidonic acid, linolelaidic acid, glutamate, docosahexaenoate, tryptophan, and fructose, all of which are associated with inflammation and oxidation. This suggests that American ginseng exerts the chemopreventive effects by anti-inflammatory and antioxidant mechanisms.

Patients with pancreatic cancer (PC) are usually diagnosed at late stages, when the disease is nearly incurable. Sensitive and specific markers are critical for supporting diagnostic and therapeutic strategies. The aim of this study was to use a metabonomics approach to identify potential plasma biomarkers that can be further developed for early detection of PC. In this study, plasma metabolites of newly diagnosed PC patients (n = 100) and age- and gender-matched controls (n = 100) from Connecticut (CT), USA, and the same number of cases and controls from Shanghai (SH), China, were profiled using combined gas and liquid chromatography mass spectrometry. The metabolites consistently expressed in both CT and SH samples were used to identify potential markers, and the diagnostic performance of the candidate markers was tested in two sample sets. A diagnostic model was constructed using a panel of five metabolites including glutamate, choline, 1,5-anhydro-d-glucitol, betaine, and methylguanidine, which robustly distinguished PC patients in CT from controls with high sensitivity (97.7%) and specificity (83.1%) (area under the receiver operating characteristic curve [AUC] = 0.943, 95% confidence interval [CI] = 0.908–0.977). This panel of metabolites was then tested with the SH data set, yielding satisfactory accuracy (AUC = 0.835; 95% CI = 0.777–0.893), with a sensitivity of 77.4% and specificity of 75.8%. This model achieved a sensitivity of 84.8% in the PC patients at stages 0, 1, and 2 in CT and 77.4% in the PC patients at stages 1 and 2 in SH. Plasma metabolic signatures show promise as biomarkers for early detection of PC.

Colorectal cancer (CRC) is one of the most common cancers in the world, having both high prevalence and mortality. It is usually diagnosed at advanced stages due to the limitations of current screening methods used in the clinic. There is an urgent need to develop new biomarkers and modalities to detect, diagnose, and monitor the disease. Metabonomics, an approach that involves the comprehensive profiling of the full complement of endogenous metabolites in a biological system, has demonstrated its great potential for use in the early diagnosis and personalized treatment of various cancers including CRC. By applying advanced analytical techniques and bioinformatics tools, the metabolome is mined for biomarkers that are associated with carcinogenesis and prognosis. This review provides an overview of the metabonomics workflow and studies, with a focus on recent advances and findings in biomarker discovery for the early diagnosis and prognosis of CRC.

Nutrition research is increasingly concerned with the complex interactions between multicomponent dietary ingredients and the human metabolic regulatory system. The substantiation of nutritional health benefits is challenged by the intrinsic complexity of macro- and micronutrients and individualized human metabolic responses. Metabonomics, uniquely suited to assess metabolic responses to deficiencies or excesses of nutrients, is used to characterize the metabolic phenotype of individuals integrating genetic polymorphisms, metabolic interactions with commensal and symbiotic partners such as gut microbiota, as well as environmental and behavioral factors including dietary preferences. The two profiling strategies, metabolic phenotyping (metabotyping) and phytochemical profiling (phytoprofiling), greatly facilitate the measurement of these important health determinants and the discovery of new biomarkers associated with nutritional requirements and specific phytochemical interventions. This paper presents an overview of the applications of these two profiling approaches for personalized nutrition research, with a focus on recent advances in the study of the role of phytochemicals in regulating the human or animal metabolic regulatory system.

Asymmetric dimethylarginine (ADMA) is synthesized by protein arginine methyltransferases during methylation of protein arginine residues and released into blood upon proteolysis. Higher concentrations of ADMA in blood have been observed in patients with metabolic diseases and certain cancers. However, the role of ADMA in colon cancer has not been well investigated. ADMA serum levels in human patients diagnosed with colon cancer were found to be higher than those present in healthy subjects. ADMA treatment of LoVo cells, a human colon adenocarcinoma cell line, attenuated serum starvation-induced apoptosis and suppressed the activation of the Fas (APO-1/CD95)/JNK (SAPK) (c-Jun N terminal protein kinase/stress-activated protein kinase)pathway. ADMA also suppressed the activation of JNK triggered by death receptor ligand anti-Fas mAb and exogenous C2-ceramide. Moreover, we demonstrated that ADMA pretreatment protected LoVo cells from doxorubicin hydrochloride-induced cell death and activation of the Fas/JNK pathway. In summary, our results suggest that the elevated ADMA in colon cancer patients may contribute to the blocking of apoptosis of cancer cells in response to stress and chemotherapy.

Background and aimsBiliary atresia (BA) is a severe neonatal cholestasis disease that is caused by obstruction of extra bile ducts. Liver fibrosis progresses dramatically in BA, and the underlying molecular mechanism is largely unknown.MethodsAmino acids and biogenic amines were quantified by targeted metabolomic methods in livers of 52 infants with BA and 16 infants with neonatal hepatitis syndrome (NHS). Normal adjacent nontumor liver tissues from 5 hepatoblastoma infants were used as controls. Orthogonal partial least-squares discriminant analysis was used to identify the differences between BA, NHS, and control tissues. Histamine metabolism enzymes and receptors were analyzed by immunohistochemistry and Western blot.ResultsThe orthogonal partial least-squares discriminant analysis clearly separated BA from NHS and the controls using amino acid and biogenic amine profiles. Histamine was significantly increased in the livers of BA infants and was positively correlated with the severity of fibrosis. This finding was supported by the elevated l-histidine decarboxylase and reduced monoamine oxidase type B expressions in the BA infants with severe fibrosis. Furthermore, histamine receptor H1 was observed in the cholangiocytes of BA livers.ConclusionsHistamine was positively correlated with fibrosis and may be a potential target to prevent liver fibrosis in BA.