Integrative Analysis of the Inflammatory Bowel Disease Serum Metabolome Improves Our Understanding of Genetic Etiology and Points to Novel Putative Therapeutic Targets.

BACKGROUND & AIMS: Polygenic and environmental factors are underlying causes of inflammatory bowel disease (IBD). We hypothesized that integration of the genetic loci controlling a metabolite's abundance, with known IBD genetic susceptibility loci, may help resolve metabolic drivers of IBD. METHODS: We measured the levels of 1300 metabolites in the serum of 484 patients with ulcerative colitis (UC) and 464 patients with Crohn's disease (CD) and 365 controls. Differential metabolite abundance was determined for disease status, subtype, clinical and endoscopic disease activity, as well as IBD phenotype including disease behavior, location, and extent. To inform on the genetic basis underlying metabolic diversity, we integrated metabolite and genomic data. Genetic colocalization and Mendelian randomization analyses were performed using known IBD risk loci to explore whether any metabolite was causally associated with IBD. RESULTS: We found 173 genetically controlled metabolites (metabolite quantitative trait loci, 9 novel) within 63 non-overlapping loci (7 novel). Furthermore, several metabolites significantly associated with IBD disease status and activity as defined using clinical and endoscopic indexes. This constitutes a resource for biomarker discovery and IBD biology insights. Using this resource, we show that a novel metabolite quantitative trait locus for serum butyrate levels containing ACADS was not supported as causal for IBD; replicate the association of serum omega-6 containing lipids with the fatty acid desaturase 1/2 locus and identify these metabolites as causal for CD through Mendelian randomization; and validate a novel association of serum plasmalogen and TMEM229B, which was predicted as causal for CD. CONCLUSIONS: An exploratory analysis combining genetics and unbiased serum metabolome surveys can reveal novel biomarkers of disease activity and potential mediators of pathology in IBD.

Gut microbial metabolites facilitate anticancer therapy efficacy by modulating cytotoxic CD8+ T cell immunity.

Recent studies in both mice and humans have suggested that gut microbiota could modulate tumor responsiveness to chemo- or immunotherapies. However, the underlying mechanism is not clear yet. Here, we found that gut microbial metabolites, especially butyrate, could promote the efficacy of oxaliplatin by modulating CD8+ T cell function in the tumor microenvironment. Butyrate treatment directly boosted the antitumor cytotoxic CD8+ T cell responses both in vitro and in vivo in an ID2-dependent manner by promoting the IL-12 signaling pathway. In humans, the oxaliplatin responder cancer patients exhibited a higher amount of serum butyrate than did non-responders, which could also increase ID2 expression and function of human CD8+ T cells. Together, our findings suggest that the gut microbial metabolite butyrate could promote antitumor therapeutic efficacy through the ID2-dependent regulation of CD8+ T cell immunity, indicating that gut microbial metabolites could be effective as a part of cancer therapy.

Increased plasma concentrations of an antidyslipidemic drug pemafibrate co-administered with rifampicin or cyclosporine A in cynomolgus monkeys genotyped for the organic anion transporting polypeptide 1B1.

In vitro permeability and in vivo pharmacokinetics of pemafibrate were investigated in human intestinal and animal models untreated or pretreated with cyclosporine A or rifampicin to evaluate any drug interactions. Ratios of basal to apical apparent permeability (Papp) over apical to basal Papp in the presence of pH gradients decreased from 0.37 to 0.080 on rifampicin co-incubation, suggesting active transport of pemafibrate from basal to apical sides in intestinal models. Plasma concentrations of intravenously administered pemafibrate were enhanced moderately in control mice but only marginally in humanized-liver mice by oral pretreatment with rifampicin [an organic anion transporting polypeptide (OATP) 1B1 inhibitor] 1 h before the administration of pemafibrate. In three cynomolgus monkeys genotyped as wild-type OATP1B1 (2 homozygous and 1 heterozygous), oral dosing of cyclosporine A 4 h or rifampicin 1 h before pemafibrate administration significantly increased the areas under the plasma concentration-time curves (AUC) of intravenously administered pemafibrate by 4.9- and 7.4-fold, respectively. Plasma AUC values of three pemafibrate metabolites in cynomolgus monkeys were also increased by cyclosporine A or rifampicin. These results suggested that pemafibrate was actively uptaken in livers and rapidly cleared from plasma in cynomolgus monkeys; this rapid clearance was suppressible by OATP1B1 inhibitors.

Systemic short chain fatty acids limit antitumor effect of CTLA-4 blockade in hosts with cancer.

Gut microbiota composition influences the clinical benefit of immune checkpoints in patients with advanced cancer but mechanisms underlying this relationship remain unclear. Molecular mechanism whereby gut microbiota influences immune responses is mainly assigned to gut microbial metabolites. Short-chain fatty acids (SCFA) are produced in large amounts in the colon through bacterial fermentation of dietary fiber. We evaluate in mice and in patients treated with anti-CTLA-4 blocking mAbs whether SCFA levels is related to clinical outcome. High blood butyrate and propionate levels are associated with resistance to CTLA-4 blockade and higher proportion of Treg cells. In mice, butyrate restrains anti-CTLA-4-induced up-regulation of CD80/CD86 on dendritic cells and ICOS on T cells, accumulation of tumor-specific T cells and memory T cells. In patients, high blood butyrate levels moderate ipilimumab-induced accumulation of memory and ICOS + CD4 + T cells and IL-2 impregnation. Altogether, these results suggest that SCFA limits anti-CTLA-4 activity.

The microbe-derived short-chain fatty acids butyrate and propionate are associated with protection from chronic GVHD.

Studies of the relationship between the gastrointestinal microbiota and outcomes in allogeneic hematopoietic stem cell transplantation (allo-HCT) have thus far largely focused on early complications, predominantly infection and acute graft-versus-host disease (GVHD). We examined the potential relationship of the microbiome with chronic GVHD (cGVHD) by analyzing stool and plasma samples collected late after allo-HCT using a case-control study design. We found lower circulating concentrations of the microbe-derived short-chain fatty acids (SCFAs) propionate and butyrate in day 100 plasma samples from patients who developed cGVHD, compared with those who remained free of this complication, in the initial case-control cohort of transplant patients and in a further cross-sectional cohort from an independent transplant center. An additional cross-sectional patient cohort from a third transplant center was analyzed; however, serum (rather than plasma) was available, and the differences in SCFAs observed in the plasma samples were not recapitulated. In sum, our findings from the primary case-control cohort and 1 of 2 cross-sectional cohorts explored suggest that the gastrointestinal microbiome may exert immunomodulatory effects in allo-HCT patients at least in part due to control of systemic concentrations of microbe-derived SCFAs.

Integrative Analysis of Circulating Metabolite Profiles and Magnetic Resonance Imaging Metrics in Patients with Traumatic Brain Injury.

Recent evidence suggests that patients with traumatic brain injuries (TBIs) have a distinct circulating metabolic profile. However, it is unclear if this metabolomic profile corresponds to changes in brain morphology as observed by magnetic resonance imaging (MRI). The aim of this study was to explore how circulating serum metabolites, following TBI, relate to structural MRI (sMRI) findings. Serum samples were collected upon admission to the emergency department from patients suffering from acute TBI and metabolites were measured using mass spectrometry-based metabolomics. Most of these patients sustained a mild TBI. In the same patients, sMRIs were taken and volumetric data were extracted (138 metrics). From a pool of 203 eligible screened patients, 96 met the inclusion criteria for this study. Metabolites were summarized as eight clusters and sMRI data were reduced to 15 independent components (ICs). Partial correlation analysis showed that four metabolite clusters had significant associations with specific ICs, reflecting both the grey and white matter brain injury. Multiple machine learning approaches were then applied in order to investigate if circulating metabolites could distinguish between positive and negative sMRI findings. A logistic regression model was developed, comprised of two metabolic predictors (erythronic acid and myo-inositol), which, together with neurofilament light polypeptide (NF-L), discriminated positive and negative sMRI findings with an area under the curve of the receiver-operating characteristic of 0.85 (specificity = 0.89, sensitivity = 0.65). The results of this study show that metabolomic analysis of blood samples upon admission, either alone or in combination with protein biomarkers, can provide valuable information about the impact of TBI on brain structural changes.

Changes in Plasma Short-Chain Fatty Acid Levels after Dietary Weight Loss Among Overweight and Obese Adults over 50 Weeks.

Gut microbial-derived short-chain fatty acids (SCFAs) may regulate energy homeostasis and exert anti-carcinogenic, immunomodulatory and anti-inflammatory effects. Smaller trials indicate that dietary weight loss may lead to decreased SCFA production, but findings have been inconclusive. SCFA concentrations were measured by HPLC-MS/MS in plasma samples of 150 overweight or obese adults in a trial initially designed to evaluate the metabolic effects of intermittent (ICR) versus continuous (CCR) calorie restriction (NCT02449148). For the present post hoc analyses, participants were classified by quartiles of weight loss, irrespective of the dietary intervention. Linear mixed models were used to analyze weight-loss-induced changes in SCFA concentrations after 12, 24 and 50 weeks. There were no differential changes in SCFA levels across the initial study arms (ICR versus CCR versus control) after 12 weeks, but acetate concentrations significantly decreased with overall weight loss (mean log-relative change of -0.7 ± 1.8 in the lowest quartile versus. -7.6 ± 2 in the highest, p = 0.026). Concentrations of propionate, butyrate and other SCFAs did not change throughout the study. Our results show that weight-loss, achieved through calorie restriction, may lead to smaller initial decreases in plasma acetate, while plasma SCFAs generally remain remarkably stable over time.

Plasma concentrations of pemafibrate with co-administered drugs predicted by physiologically based pharmacokinetic modeling in virtual populations with renal/hepatic impairment.

Pharmacokinetic profiles of pemafibrate with virtual drug and/or disease interactions were assessed by creating a detailed physiologically based pharmacokinetic (PBPK) model.Passive diffusion clearance in liver was experimentally determined as 0.013 mL/min/106 human hepatocytes. In vitro intrinsic clearance values for pemafibrate by cytochromes P450 2C8, 2C9, and 3A4 were 54, 26, and 16 µL/min/mg protein, respectively. Values for the effective permeability and the intrinsic clearance of hepatic uptake by organic anion transporting polypeptide (OATP) 1B1 were optimized in a simulator platform.This PBPK model was subsequently validated using reported maximum pemafibrate plasma concentration and area under the curve values in reported interaction studies in healthy subjects co-administered with rifampicin.For subjects with Child-Pugh A and B liver cirrhosis, the intrinsic clearance of hepatic uptake of pemafibrate by OATP1B1 were modeled using 53% and 31% of that of healthy subjects, respectively. Virtual co-administrations of rifampicin and sacubitril (OATP1B inhibitors) in subjects with renal impairment and liver cirrhosis resulted in 11- to 13-folds (rifampicin) and 1.1- to 1.3-folds (sacubitril) increased plasma exposures of pemafibrate.The current PBPK model and simulations revealed different pharmacokinetic profiles for pemafibrate following co-administration of rifampicin or sacubitril in virtual subjects with or without renal/hepatic impairment.

Predictive Modeling of Type 1 Diabetes Stages Using Disparate Data Sources.

This study aims to model genetic, immunologic, metabolomics, and proteomic biomarkers for development of islet autoimmunity (IA) and progression to type 1 diabetes in a prospective high-risk cohort. We studied 67 children: 42 who developed IA (20 of 42 progressed to diabetes) and 25 control subjects matched for sex and age. Biomarkers were assessed at four time points: earliest available sample, just prior to IA, just after IA, and just prior to diabetes onset. Predictors of IA and progression to diabetes were identified across disparate sources using an integrative machine learning algorithm and optimization-based feature selection. Our integrative approach was predictive of IA (area under the receiver operating characteristic curve [AUC] 0.91) and progression to diabetes (AUC 0.92) based on standard cross-validation (CV). Among the strongest predictors of IA were change in serum ascorbate, 3-methyl-oxobutyrate, and the PTPN22 (rs2476601) polymorphism. Serum glucose, ADP fibrinogen, and mannose were among the strongest predictors of progression to diabetes. This proof-of-principle analysis is the first study to integrate large, diverse biomarker data sets into a limited number of features, highlighting differences in pathways leading to IA from those predicting progression to diabetes. Integrated models, if validated in independent populations, could provide novel clues concerning the pathways leading to IA and type 1 diabetes.

Maternal Soluble Fiber Diet during Pregnancy Changes the Intestinal Microbiota, Improves Growth Performance, and Reduces Intestinal Permeability in Piglets.

Increasing evidence suggests that maternal diet during pregnancy modifies an offspring's microbiota composition and intestinal development in a long-term manner. However, the effects of maternal soluble fiber diet during pregnancy on growth traits and the developing intestine are still underexplored. Sows were allocated to either a control or 2.0% pregelatinized waxy maize starch plus guar gum (SF) dietary treatment during gestation. Growth performance, diarrhea incidence, gut microbiota composition and metabolism, and gut permeability and inflammation status of 14-day-old suckling piglets were analyzed. The maternal SF diet improved the growth rate and decreased the incidence of diarrhea in the piglets. Next-generation sequencing analysis revealed that the intestinal microbiota composition was altered by a maternal SF diet. The fecal and plasma levels of acetate and butyrate were also increased. Furthermore, a maternal SF diet reduced the levels of plasma zonulin and fecal lipocalin-2 but increased the plasma concentrations of interleukin 10 (IL-10) and transforming growth factor ß (TGF-ß). Additionally, the increased relative abundances of Lactobacillus spp. in SF piglets were positively correlated with growth rate, while the decreased abundances of Bilophila spp. were positively correlated with fecal lipocalin-2 levels. Our data reveal that a maternal SF diet during pregnancy has remarkable effects on an offspring's growth traits and intestinal permeability and inflammation, perhaps by modulating the composition and metabolism of gut microbiota.IMPORTANCE Although the direct effects of dietary soluble fiber on gut microbiota have been extensively studied, the more indirect effects of maternal nutrition solely during pregnancy on the development of the offspring's intestine are until now largely unexplored. Our data show that a maternal soluble fiber diet during pregnancy is independently associated with changes in the intestinal microbiota composition and metabolism of suckling piglets. These findings have direct implications for refining dietary recommendations in pregnancy. Moreover, a maternal soluble fiber diet reduces intestinal permeability and prevents intestinal inflammation and an excessive systemic immune response of suckling piglets. Therefore, the suckling piglets' resistance to disease was enhanced, diarrhea was reduced, and weight gain was raised. Additionally, the changes in gut microbiota in response to a maternal soluble fiber diet may also be directly correlated with the offspring's growth and gut development.

Imbalance of gut microbiome and intestinal epithelial barrier dysfunction in patients with high blood pressure.

Recent evidence indicates a link between gut pathology and microbiome with hypertension (HTN) in animal models. However, whether this association exists in humans is unknown. Thus, our objectives in the present study were to test the hypotheses that high blood pressure (BP) patients have distinct gut microbiomes and that gut-epithelial barrier function markers and microbiome composition could predict systolic BP (SBP). Fecal samples, analyzed by shotgun metagenomics, displayed taxonomic and functional changes, including altered butyrate production between patients with high BP and reference subjects. Significant increases in plasma of intestinal fatty acid binding protein (I-FABP), lipopolysaccharide (LPS), and augmented gut-targetting proinflammatory T helper 17 (Th17) cells in high BP patients demonstrated increased intestinal inflammation and permeability. Zonulin, a gut epithelial tight junction protein regulator, was markedly elevated, further supporting gut barrier dysfunction in high BP. Zonulin strongly correlated with SBP (R2 = 0.5301, P<0.0001). Two models predicting SBP were built using stepwise linear regression analysis of microbiome data and circulating markers of gut health, and validated in a separate cohort by prediction of SBP from zonulin in plasma (R2 = 0.4608, P<0.0001). The mouse model of HTN, chronic angiotensin II (Ang II) infusion, was used to confirm the effects of butyrate and gut barrier function on the cardiovascular system and BP. These results support our conclusion that intestinal barrier dysfunction and microbiome function are linked to HTN in humans. They suggest that manipulation of gut microbiome and its barrier functions could be the new therapeutic and diagnostic avenues for HTN.

Effect of chickpea husk dietary supplementation on blood and cecal parameters in rats.

Chickpea husk was functionally evaluated for antioxidant status, blood parameters, cecal fermentation and microbial profiles in rats. Fifteen male rats (5 weeks of age) were divided into three groups; they were individually housed and fed one of the following diets for 3 weeks: purified diet containing 5% cellulose (Cellulose), an identical diet in which cellulose was replaced by corn starch (Starch) or by chickpea husk (Chick). Rats were sacrificed to obtain blood and cecal digesta samples. Chickpea husk contained high polyphenolic content and significant superoxide dismutase and 2,2-diphenyl-picrylhydrazyl scavenging activities. In a feeding experiment, Chick showed lowered cholesterol levels and improved antioxidant activity represented by reduced thiobarbituric acid reactive substances in blood. Chick showed increased cecal levels of total short chain fatty acids and butyrate, leading to a lower pH. Chick presented with lowered cecal indole and skatole concentrations, as did Cellulose. Cecal bacterial changes were notable in Chick, evidenced by differences in denaturing gradient gel electrophoresis banding patterns. However, representative bacteria quantified by real-time PCR assay did not support this bacterial change. These results indicate that chickpea husk feeding can improve the antioxidative status of rats through its polyphenolic components and modulate the hindgut environment by its fibrous components.

Effects of whole-grain cereal foods on plasma short chain fatty acid concentrations in individuals with the metabolic syndrome.

OBJECTIVE: Short chain fatty acids (SCFAs) derived from dietary fiber fermentation by gut microbiota have been identified as one of the mechanisms behind the association between habitual whole-grain intake and a lower risk of cardiometabolic diseases. The aims of the present work are: (1) to evaluate whether a whole-grain wheat-based diet may increase SCFAs concentration, and (2) to identify possible associations between SCFAs and metabolic changes observed after the nutritional intervention. METHODS: Fifty-four subjects participated in the trial. They underwent a 12-wk dietary intervention based on whole-grain or refined cereal products. At baseline and after the intervention, glucose, insulin, triacylglycerol, inflammatory markers (hs-CRP, IL-1 ra, IL-6, and TNF-α), and SCFAs plasma concentrations were evaluated. RESULTS: After the intervention, in the whole-grain group fasting plasma propionate concentrations were higher than at baseline, whereas a reduction was detected in the control group. The absolute changes (end of trial minus baseline) in fasting plasma propionate concentrations were significantly different between the two groups (P = 0.048). The absolute changes of fasting propionate correlated with cereal fiber intake (r = 0.358, P = 0.023), but no significant correlations with clinical outcomes were found. However, postprandial insulin was significantly decreased in the group having the absolute changes of fasting propionate concentration above the median value (P = 0.022 versus subjects with fasting propionate changes below the median value). CONCLUSIONS: A 12-wk whole-grain wheat-based diet increases fasting plasma propionate. This increase correlates with the cereal fiber intake and is associated with lower postprandial insulin concentrations.

Hepatic Uptake of Rectally Administered Butyrate Prevents an Increase in Systemic Butyrate Concentrations in Humans.

BACKGROUND: Short-chain fatty acids (SCFAs), fermentation products of undigested fibers, are considered beneficial for colonic health. High plasma concentrations are potentially harmful; therefore, information about systemic SCFA clearance is needed before therapeutic use of prebiotics or colonic SCFA administration. OBJECTIVE: The aim of this study was to investigate the effect of rectal butyrate administration on SCFA interorgan exchange. METHODS: Twelve patients (7 men; age: 66.4 ± 2.0 y; BMI 24.5 ± 1.4 kg/m(2)) undergoing upper abdominal surgery participated in this randomized placebo-controlled trial. During surgery, 1 group received a butyrate enema (100 mmol sodium butyrate/L; 60 mL; n = 7), and the other group a placebo (140 mmol 0.9% NaCl/L; 60 mL; n = 5). Before and 5, 15, and 30 min after administration, blood samples were taken from the radial artery, hepatic vein, and portal vein. Plasma SCFA concentrations were analyzed, and fluxes from portal-drained viscera, liver, and splanchnic area were calculated and used for the calculation of the incremental area under the curve (iAUC) over a 30-min period. RESULTS: Rectal butyrate administration led to higher portal butyrate concentrations at 5 min compared with placebo (92.2 ± 27.0 µmol/L vs. 14.3 ± 3.4 µmol/L, respectively; P < 0.01). In the butyrate-treated group, iAUCs of gut release (282.8 ± 133.8 µmol/kg BW · 0.5 h) and liver uptake (-293.7 ± 136.0 µmol/kg BW · 0.5 h) of butyrate were greater than in the placebo group [-16.6 ± 13.4 µmol/kg BW · 0.5 h (gut release) and 16.0 ± 13.8 µmol/kg BW · 0.5 h (liver uptake); P = 0.01 and P < 0.05, respectively]. As a result, splanchnic butyrate release did not differ between groups. CONCLUSION: After colonic butyrate administration, splanchnic butyrate release was prevented in patients undergoing upper abdominal surgery. These observations imply that therapeutic colonic SCFA administration at this dose is safe. The trial was registered at clinicaltrials.gov as NCT02271802.

Quantitative determination of decursin, decursinol angelate, and decursinol in mouse plasma and tumor tissue using liquid-liquid extraction and HPLC.

The pyranocoumarin compound decursin and its isomer decursinol angelate (DA) are the major hydrophobic phytochemicals in the root of Angelica gigas Nakai (AGN, Korean Angelica), a major traditional medicinal herb. The ethanol extract of AGN and especially the purified decursin and DA have been shown to exhibit antitumor activities by our collaborative team and others. Although decursinol has been identified as a major hydrolysis metabolite of decursin and DA in vivo in previous pharmacokinetic studies with mouse and rat, other recently published results sharply disputed this conclusion. In this study, we set up a practical method for the concurrent analysis of decursin, DA, and decursinol in mouse plasma and tumor tissues by liquid-liquid extraction and HPLC-UV and applied the method to several animal experiments. Plasma or tumor homogenate was extracted directly with ethyl acetate. The extraction efficiency for decursin/DA (quantitated together) and decursinol was between 82-95 % in both mouse plasma and tumor homogenate. The lower limit of quantitation (LLOQ) was approximately 0.25 µg/mL for decursin/DA and 0.2 µg/mL for decursinol in mouse plasma. In a pilot pharmacokinetic study, male C57BL/6 mice were given a single dose of 4.8 mg decursin/DA mixture (~240 mg/kg) per mouse either by oral gavage or intraperitoneal injection. Maximum plasma concentrations for decursin/DA and decursinol were 11.2 and 79.7 µg/mL, respectively, when decursin/DA was administered via intraperitoneal injection, and 0.54 and 14.9 µg/mL via oral gavage. Decursin/DA and decursinol contents in the tumor tissues from nude mouse xenografts correlated very well with those in plasma. Overall, our results confirm the conclusion that the majority of decursin/DA hydrolyze to decursinol in rodent models with a tiny fraction remaining as the intact compounds administered.

Olive oil-based fat emulsion versus soy oil-based fat emulsion in abdominal oncologic surgery.

In parenteral nutrition (PN), essential fatty acids are provided by soy oil-based fat emulsions, which may exert adverse effects on the immune system and lipid peroxidation. Olive oil -based fat emulsions have been said to prevent these undesired effects. This study compares effects of olive oil - and soy oil -based fat emulsions in 22 patients who underwent abdominal surgery for cancer. The first group (n = 10) received soy oil -based fat emulsion; the second group (n = 10) received olive oil -based fat emulsion. Body temperature, body mass index, (BMI) and biochemical variables were measured on days 0 and 7. There were no differences between the groups with regard to BMI or temperature. On day 7, the first group (compared with day 0) had significant increases in plasma alkaline phosphatase (81.70 ± 16.03 vs 117.60 ± 11.1), γ-glutamyl transferase (39.90 ± 15.40 vs 137.70 ± 24.09), and mean body temperature (36.72°C ± 0.14°C vs 37.20°C ± 0.17°C) (P < .01). Second group had increases in alkaline phosphatase (85.80 ± 13.46 vs 147.20 ± 34.17), γ-glutamyl transferase (48.40 ± 12.86 vs 129.40 ± 42.03), total protein (5.14 ± 0.19 vs 6.06 ± 0.49), and albumin (2.62 ± 0.14 vs 3.00 ± 0.18) (P < .05). Changes in thiobutyric acid levels were not statistically significant in either group. In postoperative cancer patients, olive oil-based fat emulsion had similar effects on BMI, body temperature, biochemical values, and thiobutyric acid levels as soy oil-based fat emulsions.

Determination of tributyrin and its metabolite butyrate in Wistar rat plasma samples by gas chromatography/mass spectrometry.

A gas chromatographic (GC) method with mass spectrometric (MS) detection was developed for the determination of tributyrin and its metabolite butyrate in rat plasma. Following precipitation of plasma protein with acetonitrile, the analytes in the samples were separated on a DB-5ms capillary column with helium as carrier gas. Phenylmethylsulfonyl fluoride (PMSF), an inhibitor for serine proteases, papain and acetylcholinesterase, was found to be essential to inhibit the activity of enzyme(s) responsible for the hydrolysis of tributyrin in both rat and human blood samples. The enzyme inhibitor in 5 mM (final concentration) was added immediately into the blood samples after collection to prevent the hydrolysis. The linear concentration ranges for tributyrin and butyrate were 0.1-2.0 and 1-20 microM, respectively. The coefficients of variation for intra-day and inter-day assays for tributyrin were all <10%, and those for butyrate were also <10%, except for the lowest concentration (1 microM), which was less than 20%. The accuracy of all concentration determinations ranged from 96.0-110.0%. The limit of quantification (LOQ) was 0.1 microM for tributyrin and 1.0 microM for butyrate. This method could detect tributyrin and butyrate simultaneously, and represents an improvement in sensitivity for the detection of tributyrin compared with the previous gas chromatography-flame ionization detection (GC-FID) method.

New insight into butyrate metabolism.

Butyrate is a C4 acid produced by microbial fermentation of carbohydrates and protein in the large intestine of all animal species. The factor of prime importance for the production rate of butyrate in the lower gut is type and levels of non-digestible carbohydrates entering the large intestine. It was previously believed that 85-90 % of the butyrate produced in the gut was cleared when passing the gut epithelium, but recent studies with catheterised pigs have shown that the concentration of butyrate in the portal vein is strongly influenced by the production rate in the large intestine. Increased gut production of butyrate further raises the circulating level of butyrate. For good reason it is not possible with current technologies to perform direct measurements of the variation in the butyrate concentration in the portal vein of human subjects, but short-chain fatty acid levels in portal blood from sudden-death victims, subjects undergoing emergency surgery or planned surgery have indicated a higher gut production and absolute and relative concentration of butyrate in non-fasted as compared with fasted human subjects. However, despite an expected higher gut production of butyrate when feeding a high-fibre rye-bread-based diet as compared with a low-fibre wheat-bread-based diet, there was no difference in absolute or relative levels of butyrate in the peripheral blood of human subjects.

Acetate, propionate and butyrate in plasma: determination of the concentration and isotopic enrichment by gas chromatography/mass spectrometry with positive chemical ionization.

This study describes a rapid and simple method to determine short-chain fatty acid (SCFA) concentrations and their isotopic enrichments (M(0) + 1 and M(0) + 2) in human plasma. Sample preparation involves SCFA extraction and derivatization with 1-(tert-butyldimethylsilyl)imidazole. Gas chromatography/mass spectrometry was performed using chemical ionization with ammonia as the reagent gas. Outstanding resolution, excellent linearity and good detection limits were obtained. Inter-assay and intra-assay repeatability was below 10% and 3% respectively for SCFA concentration. Inter-assay repeatability was below 5%, 4%, 6%, and 14% for isotopic enrichment determination of [1-(13)C]acetate and [1,2-(13)C(2)]acetate, [1-(13)C]propionate and [1-(13)C]butyrate respectively, with intra-assay being below 6%. Such SCFA concentrations and isotopic enrichments were determined in the plasma of rats infused with a (13)C-labeled SCFA. The turnovers of acetate, propionate and butyrate in rats were 19 micromol kg(-1) min(-1), 2.6 micromol kg(-1) min(-1), 0.3 micromol kg(-1) min(-1) respectively.

Potato and high-amylose maize starches are not equivalent producers of butyrate for the colonic mucosa.

Portal appearance of short-chain fatty acids (SCFA) produced from fermentation of three different resistant starch (RS) sources (raw potato starch, high-amylose maize starch and retrograded high-amylose maize starch) was investigated in pigs. The catheterization technique coupled with determination of portal blood flow was used to estimate SCFA uptake by the colonic mucosa. Our hypothesis was that these three RS were not equivalent butyrate providers for the colonic mucosa and that butyrate uptake would therefore be different after in vivo fermentation of each starch. The starches induced different patterns of appearance of SCFA in the portal blood; raw potato starch was the only RS source to show a significant appearance of butyrate in the portal blood. Thus, uptake of butyrate by the colonic mucosa apparently differed between starches. This finding suggests that butyrate uptake does not only depend on the flow of butyrate appearing in the lumen. Indeed, for unexplained reasons, utilization of butyrate by the colonic mucosa appeared to be less efficient when the butyrate was produced from fermentation of potato starch than when it was produced from fermentation of the other RS sources.