The effects of thawing on the plasma metabolome: evaluating differences between thawed plasma and multi-organ samples

Introduction

Post-collection handling, storage and transportation can affect the quality of blood samples. Pre-analytical biases can easily be introduced and can jeopardize accurate profiling of the plasma metabolome. Consequently, a mouse study must be carefully planned in order to avoid any kind of bias that can be introduced, in order not to compromise the outcome of the study. The storage and shipment of the samples should be made in such a way that the freeze–thaw cycles are kept to a minimum. In order to keep the latent effects on the stability of the blood metabolome to a minimum it is essential to study the effect that the post-collection and pre-analytical error have on the metabolome.

Objectives

The aim of this study was to investigate the effects of thawing on the metabolic profiles of different sample types.

Methods

In the present study, a metabolomics approach was utilized to obtain a thawing profile of plasma samples obtained on three different days of experiment. The plasma samples were collected from the tail on day 1 and 3, while retro-orbital sampling was used on day 5. The samples were analysed using gas chromatography time-of-flight mass spectrometry (GC TOF-MS).

Results

The thawed plasma samples were found to be characterized by higher levels of amino acids, fatty acids, glycerol metabolites and purine and pyrimidine metabolites as a result of protein degradation, cell degradation and increased phospholipase activity. The consensus profile was thereafter compared to the previously published study comparing thawing profiles of tissue samples from gut, kidney, liver, muscle and pancreas.

Conclusions

The comparison between thawed organ samples and thawed plasma samples indicate that the organ samples are more sensitive to thawing, however thawing still affected all investigated sample types.

     

Metabolomics characterization of energy metabolism reveals glycogen accumulation in gut-microbiota-lacking mice

Microbiota in the gut are considered an important environmental factor associated with host metabolism and physiology. Although gut microbiota are known to contribute to hepatic lipogenesis and fat storage, little is known about how the condition influences the deposition of glycogen in the liver. To better understand and characterize the host energy metabolism in guts lacking microbiota, we compared the liver metabolome of specific pathogen-free and germ-free mice by gas chromatography-mass spectrometry combined with partial least-squares discriminant analysis. We identified 30 of 52 highly reproducible peaks in chromatograms of liver tissue extracts from the two groups of mice. The two groups showed significant differences in metabolic profile. Changes in liver metabolism involved metabolites such as amino acids, fatty acids, organic acids and carbohydrates. The metabolic profile of germ-free mice suggests that they synthesize glycogen and accumulate it in the liver through gluconeogenesis and glycogenesis. Our findings shed light on a new perspective of the role of gut microbiota in energy metabolism and will be useful to help study probiotics, obesity and metabolic diseases.     

Mesenteric lymph system constitutes the second route in gut–liver axis and transports metabolism-modulating gut microbial metabolites

The gut–liver axis denotes the intricate connection and interaction between gut microbiome and liver, in which compositional and functional shifts in gut microbiome affect host metabolism. Hepatic portal vein of the blood circulation system has been thought to be the major route for metabolite transportation in the gut–liver axis, but the existence and importance of other routes remain elusive. Here, we perform metabolome comparison in blood circulation and mesenteric lymph systems and identify significantly shifted metabolites in serum and mesentery. Using cellular assays, we find that the majority of decreased metabolites in lymph system under high-fat diet are effective in alleviating metabolic disorders, indicating a high potential of lymph system in regulating liver metabolism. Among those, a representative metabolite, L-carnitine, reduces diet-induced obesity in mice. Metabolic tracing analysis identifies that L-carnitine is independently transported by the mesenteric lymph system, serving as an example that lymph circulation comprises a second route in the gut–liver axis to modulate liver metabolism. Our study provides new insights into metabolite transportation via mesenteric lymph system in the gut–liver axis, offers an extended scope for the investigations in host-gut microbiota metabolic interactions and potentially new targets in the treatment of metabolic disorders.     

Integrative Metabolic Signatures for Hepatic Radiation Injury

Background

Radiation-induced liver disease (RILD) is a dose-limiting factor in curative radiation therapy (RT) for liver cancers, making early detection of radiation-associated liver injury absolutely essential for medical intervention. A metabolomic approach was used to determine metabolic signatures that could serve as biomarkers for early detection of RILD in mice.

Methods

Anesthetized C57BL/6 mice received 0, 10 or 50 Gy Whole Liver Irradiation (WLI) and were contrasted to mice, which received 10 Gy whole body irradiation (WBI). Liver and plasma samples were collected at 24 hours after irradiation. The samples were processed using Gas Chromatography/Mass Spectrometry and Liquid Chromatography/Mass Spectrometry.

Results

Twenty four hours after WLI, 407 metabolites were detected in liver samples while 347 metabolites were detected in plasma. Plasma metabolites associated with 50 Gy WLI included several amino acids, purine and pyrimidine metabolites, microbial metabolites, and most prominently bradykinin and 3-indoxyl-sulfate. Liver metabolites associated with 50 Gy WLI included pentose phosphate, purine, and pyrimidine metabolites in liver. Plasma biomarkers in common between WLI and WBI were enriched in microbial metabolites such as 3 indoxyl sulfate, indole-3-lactic acid, phenyllactic acid, pipecolic acid, hippuric acid, and markers of DNA damage such as 2-deoxyuridine. Metabolites associated with tryptophan and indoles may reflect radiation-induced gut microbiome effects. Predominant liver biomarkers in common between WBI and WLI were amino acids, sugars, TCA metabolites (fumarate), fatty acids (lineolate, n-hexadecanoic acid) and DNA damage markers (uridine).

Conclusions

We identified a set of metabolomic markers that may prove useful as plasma biomarkers of RILD and WBI. Pathway analysis also suggested that the unique metabolic changes observed after liver irradiation was an integrative response of the intestine, liver and kidney.     

Metabonomic and microbiological analysis of the dynamic effect of vancomycin-induced gut microbiota modification in the mouse

The effects of the antibiotic vancomycin (2 x 100 mg/kg/day) on the gut microbiota of female mice (outbred NMRI strain) were studied, in order to assess the relative contribution of the gut microbiome to host metabolism. The host's metabolic phenotype was characterized using (1)H NMR spectroscopy of urine and fecal extract samples. Time-course changes in the gut microbiotal community after administration of vancomycin were monitored using 16S rRNA gene PCR and denaturing gradient gel electrophoresis (PCR-DGGE) analysis and showed a strong effect on several species, mostly within the Firmicutes. Vancomycin treatment was associated with fecal excretion of uracil, amino acids and short chain fatty acids (SCFAs), highlighting the contribution of the gut microbiota to the production and metabolism of these dietary compounds. Clear differences in gut microbial communities between control and antibiotic-treated mice were observed in the current study. Reduced urinary excretion of gut microbial co-metabolites phenylacetylglycine and hippurate was also observed. Regression of urinary hippurate and phenylacetylglycine concentrations against the fecal metabolite profile showed a strong association between these urinary metabolites and a wide range of fecal metabolites, including amino acids and SCFAs. Fecal choline was inversely correlated with urinary hippurate. Metabolic profiling, coupled with the metagenomic study of this antibiotic model, illustrates the close inter-relationship between the host and microbial "metabotypes", and will provide a basis for further experiments probing the understanding of the microbial-mammalian metabolic axis.     

Tissue Distribution of Berberine and Its Metabolites after Oral Administration in Rats

Berberine (BBR) has been confirmed to have multiple bioactivities in clinic, such as cholesterol-lowering, anti-diabetes, cardiovascular protection and anti- inflammation. However, BBR’s plasma level is very low; it cannot explain its pharmacological effects in patients. We consider that the in vivo distribution of BBR as well as of its bioactive metabolites might provide part of the explanation for this question. In this study, liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LC/MSⁿ-IT-TOF) as well as liquid chromatography that coupled with tandem mass spectrometry (LC-MS/MS) was used for the study of tissue distribution and pharmacokinetics of BBR in rats after oral administration (200 mg/kg). The results indicated that BBR was quickly distributed in the liver, kidneys, muscle, lungs, brain, heart, pancreas and fat in a descending order of its amount. The pharmacokinetic profile indicated that BBR’s level in most of studied tissues was higher (or much higher) than that in plasma 4 h after administration. BBR remained relatively stable in the tissues like liver, heart, brain, muscle, pancreas etc. Organ distribution of BBR’s metabolites was also investigated paralleled with that of BBR. Thalifendine (M1), berberrubine (M2) and jatrorrhizine (M4), which the metabolites with moderate bioactivity, were easily detected in organs like the liver and kidney. For instance, M1, M2 and M4 were the major metabolites in the liver, among which the percentage of M2 was up to 65.1%; the level of AUC _(0-t) (area under the concentration-time curve) for BBR or the metabolites in the liver was 10-fold or 30-fold higher than that in plasma, respectively. In summary, the organ concentration of BBR (as well as its bioactive metabolites) was higher than its concentration in the blood after oral administration. It might explain BBR’s pharmacological effects on human diseases in clinic.     

Metabolic profiling of a Schistosoma mansoni infection in mouse tissues using magic angle spinning-nuclear magnetic resonance spectroscopy

In order to enhance our understanding of physiological and pathological consequences of a patent Schistosoma mansoni infection in the mouse, we examined the metabolic responses of different tissue samples recovered from the host animal using a metabolic profiling strategy. Ten female NMRI mice were infected with ∼80 S. mansoni cercariae each, and 10 uninfected age- and sex-matched animals served as controls. At day 74 post infection (p.i.), mice were killed and jejunum, ileum, colon, liver, spleen and kidney samples were removed. We employed ¹H magic angle spinning-nuclear magnetic resonance spectroscopy to generate tissue-specific metabolic profiles. The spectral data were analyzed using multivariate modelling methods including an orthogonal signal corrected-projection to latent structure analysis and hierarchical principal component analysis to assess the differences and/or similarities in metabolic responses between infected and non-infected control mice. Most tissues obtained from S. mansoni-infected mice were characterized by high levels of amino acids, such as leucine, isoleucine, lysine, glutamine and asparagine. High levels of membrane phospholipid metabolites, including glycerophosphoryl choline and phosphoryl choline were found in the ileum, colon, liver and spleen of infected mice. Additionally, low levels of energy-related metabolites, including lipids, glucose and glycogen were observed in ileum, spleen and liver samples of infected mice. Energy-related metabolites in the jejunum, liver and renal medulla were found to be positively correlated with S. mansoni worm burden upon dissection. These findings show that a patent S. mansoni infection causes clear disruption of metabolism in a range of tissues at a molecular level, which can be interpreted in relation to the previously reported signature in a biofluid (i.e. urine), giving further evidence of the global effect of the infection.     

Effects of potassium deficiency on potassium,polyamines and amino acids in mouse tissues

Sexual dimorphism in potassium content was found in plasma, kidney, heart and skeletal muscle of CD1 mice. We observed that feeding mice with a K(+)-deficient diet had an uneven and gender-dependent effect on organ weight and tissue potassium concentrations. Treatment produced a marked decrease in plasma, pancreas and skeletal muscle K(+) levels in both sexes, and a reduction in kidney, liver and heart potassium concentrations in females. Moreover, K(+) deficiency produced a 2-3-fold increase in the concentrations of cationic amino acids, such as arginine and lysine in both heart and skeletal muscle of the two sexes, a slight increase ( approximately 37%) in renal arginine in the male mice. The concentrations of these amino acids in plasma and other tissues in both sexes remained unaltered. Polyamine levels in heart, liver, skeletal muscle and pancreas from male and female mice were not affected by K(+) deficiency. However, in the male kidney potassium deficiency was accompanied by an increase of putrescine and spermidine concentration, and a reduction of putrescine excretion into the urine, even though renal K(+) concentration was not significantly affected and ornithine decarboxylase activity was dramatically decreased. The general lack of correlation between tissue potassium decrease and the increase in organic cations suggests that it is unlikely that the changes observed could be related with an attempt of the tissues to compensate for the reduction in cellular positive charge produced by the fall in K(+) content. The mechanisms by which these changes are produced are discussed, but their physiological implications remain to be determined.     

Gut microbiota–bile acid–skeletal muscle axis

The gut microbiota represents a ‘metabolic organ’ that can regulate human metabolism. Intact gut microbiota contributes to host homeostasis, whereas compositional perturbations, termed dysbiosis, are associated with a wide range of diseases. Recent evidence demonstrates that dysbiosis, and the accompanying loss of microbiota-derived metabolites, results in a substantial alteration of skeletal muscle metabolism. As an example, bile acids, produced in the liver and further metabolized by intestinal microbiota, are of considerable interest since they regulate several host metabolic pathways by activating nuclear receptors, including the farnesoid X receptor (FXR). Indeed, alteration of gut microbiota may lead to skeletal muscle atrophy via a bile acid–FXR pathway. This Review aims to suggest a new pathway that connects different mechanisms, involving the gut–muscle axis, that are often seen as unrelated, and, starting from preclinical studies, we hypothesize new strategies aimed at optimizing skeletal muscle functionality.     

Metabolomic analysis of a human oral glucose tolerance test reveals fatty acids as reliable indicators of regulated metabolism

Gas chromatography/mass spectrometry-based metabolomics was applied to investigate dynamic changes in the plasma metabolome upon an oral glucose tolerance test (OGTT). The OGTT is a frequently used diagnostic test of glucose homeostasis and diabetes. Diabetes is diagnosed either when glucose levels ≥7.0 mM in the fasting state or ≥11.0 mM at 2 h after oral glucose intake. The accuracy of the OGTT would, however, most likely improve if additional variables could be identified. In the present study, plasma samples were drawn every 15 min for 2 h after an oral glucose load of 75 g preceded by an overnight fast in healthy individuals. Blood plasma levels of more than 200 putative metabolites were measured. Multivariate modelling was used to distinguish metabolic regulation due to the glucose challenge from that of other variability. Two data scaling methods were applied, yielding similar results when evaluated by appropriate diagnostic tools. Fatty acid levels were found to be strongly decreased during the OGTT. Also, the levels of amino acids were shown to decrease. However, technical and uninduced biological variations were found to affect the amino acid levels to a greater extent than the fatty acid levels, making the fatty acids more reliable as indicators of metabolic regulation. Levels of several metabolites correlated with the quadratic glucose profile and two were found having an inverse correlation. Raw data plots of all identified significantly altered metabolites confirmed the excellent performance of the multivariate models. Using this approach, a better understanding of the metabolic response to an OGTT can be achieved, paving the way for inclusion of other variables describing appropriate metabolic control.     

Gut microflora is a key player in host energy homeostasis

Gut microflora is now considered as a key organ involved in host energy homeostasis. Recent data suggest that the alterations of the gut bacteria ecosystem could contribute to the development of metabolic disorders such as type 2 diabetes and obesity. First, gut microflora may increase energy efficiency of non digested food via the fermentation, thus providing more energy to the host. Secondly, fatty acids flux and storage in the adipose tissue is under the control of the fasting-induced adipocyte factor FIAF, which expression depends on gut microflora. Third, high-fat diet feeding changes gut bacteria profile, leading to a drop in bifidobacteria content, which correlates with a higher LPS plasma levels, thereby participating to the onset of inflammation, insulin resistance and type 2 diabetes associated with obesity. Changing gut microflora composition could be a useful tool to prevent or to treat high-fat/low fibres diet-induced metabolic syndrome. double dagger.     

Systemic multicompartmental effects of the gut microbiome on mouse metabolic phenotypes

To characterize the impact of gut microbiota on host metabolism, we investigated the multicompartmental metabolic profiles of a conventional mouse strain (C3H/HeJ) (n=5) and its germ‐free (GF) equivalent (n=5). We confirm that the microbiome strongly impacts on the metabolism of bile acids through the enterohepatic cycle and gut metabolism (higher levels of phosphocholine and glycine in GF liver and marked higher levels of bile acids in three gut compartments). Furthermore we demonstrate that (1) well‐defined metabolic differences exist in all examined compartments between the metabotypes of GF and conventional mice: bacterial co‐metabolic products such as hippurate (urine) and 5‐aminovalerate (colon epithelium) were found at reduced concentrations, whereas raffinose was only detected in GF colonic profiles. (2) The microbiome also influences kidney homeostasis with elevated levels of key cell volume regulators (betaine, choline, myo‐inositol and so on) observed in GF kidneys. (3) Gut microbiota modulate metabotype expression at both local (gut) and global (biofluids, kidney, liver) system levels and hence influence the responses to a variety of dietary modulation and drug exposures relevant to personalized health‐care investigations.     

Systems responses of rats to aflatoxin B1 exposure revealed with metabonomic changes in multiple biological matrices

Exposure to aflatoxins causes liver fibrosis and hepatocellular carcinoma posing a significant health risk for human populations and livestock. To understand the mammalian systems responses to aflatoxin-B1 (AFB1) exposure, we analyzed the AFB1-induced metabonomic changes in multiple biological matrices (plasma, urine, and liver) of rats using (1)H NMR spectroscopy together with clinical biochemistry and histopathologic assessments. We found that AFB1 exposure caused significant elevation of glucose, amino acids, and choline metabolites (choline, phosphocholine, and glycerophosphocholine) in plasma but reduction of plasma lipids. AFB1 also induced elevation of liver lipids, amino acids (tyrosine, histidine, phenylalanine, leucine, isoleucine, and valine), choline, and nucleic acid metabolites (inosine, adenosine, and uridine) together with reduction of hepatic glycogen and glucose. AFB1 further caused decreases in urinary TCA cycle intermediates (2-oxoglutarate and citrate) and elevation of gut microbiota cometabolites (phenylacetylglycine and hippurate). These indicated that AFB1 exposure caused hepatic steatosis accompanied with widespread metabolic changes including lipid and cell membrane metabolisms, protein biosynthesis, glycolysis, TCA cycle, and gut microbiota functions. This implied that AFB1 exposure probably caused oxidative-stress-mediated impairments of mitochondria functions. These findings provide an overview of biochemical consequences of AFB1 exposure and comprehensive insights into the metabolic aspects of AFB1-induced hepatotoxicity in rats.     

High sucrose diet-induced dysbiosis of gut microbiota promotes fatty liver and hyperlipidemia in rats

Excess sucrose intake has been found to be a major factor in the development of metabolic syndrome, especially in promoting nonalcoholic fatty liver disease. The excess fructose is believed to targets the liver to promote de novo lipogenesis, as described in major biochemistry textbooks. On the contrary, in this study, we explored the possible involvement of gut microbiota in excess sucrose-induced lipid metabolic disorders, to validate a novel mechanism by which excess sucrose causes hepatic lipid metabolic disorders via alterations to the gut microbial community structure. Wistar male rats were fed either a control starch diet or a high-sucrose diet for 4 weeks. Half of the rats in each group were treated with an antibiotic cocktail delivered via drinking water for the entire experimental period. After 4 weeks, rats fed with the high-sucrose diet showed symptoms of fatty liver and hyperlipidemia. The architecture of cecal microbiota was altered in rats fed with high-sucrose diet as compared to the control group, with traits including increased ratios of the phyla Bacteroidetes/Firmicutes, reduced α-diversity, and diurnal oscillations changes. Antibiotic administration rescued high-sucrose diet-induced lipid accumulation in the both blood and liver. Levels of two microbial metabolites, formate and butyrate, were reduced in rats fed with the high-sucrose diet. These volatile short-chain fatty acids might be responsible for the sucrose-induced fatty liver and hyperlipidemia. Our results indicate that changes in the gut microbiota induced by a high-sucrose diet would promote the development of nonalcoholic fatty liver disease via its metabolites, such as short-chain fatty acids.     

Responses of digestive enzymes of tambaqui (Colossoma macropomum) to dietary cornstarch changes and metabolic inferences

Digestive enzyme responses plus metabolic implications were studied in tambaqui (Colossoma macropomum) fed isoproteic diets containing 28% crude protein, 3300 kcal of gross energy/kg and different amounts of cornstarch (30, 40 and 50%). Amylase, maltase, acid protease, trypsin and chymotrypsin from the alimentary tract were assayed. Plasma, liver and white muscle metabolites were gauged to profile metabolism of the fish. The alimentary tract of tambaqui is compartmentalized morphologically and enzymatically. Amylase was present through the gut; acid protease was detected in stomach; maltase, trypsin and chymotrypsin were found in pyloric caeca and proximal and distal intestine sections. Increase of cornstarch levels from 40 to 50% in the diet resulted in an increase in amylase and maltase. Trypsin and chymotrypsin were unresponsive to starch levels. Acid protease follows the protein/carbohydrate ratio decrease. The increase of dietary cornstarch resulted in liver glycogenesis and the increase in plasma triglycerides is suggestive of lipogenesis. Digestive biochemical responses of tambaqui correlated with changes of feeding plus the analyses of metabolic profile are assumed as a tool for optimizing diet formulation and are a clue to other feeding optimizations for freshwater tropical species.     

The interplay between host cellular and gut microbial metabolism in NAFLD development and prevention

Metabolism regulation centred on insulin resistance is increasingly important in nonalcoholic fatty liver disease (NAFLD). This review focuses on the interactions between the host cellular and gut microbial metabolism during the development of NAFLD. The cellular metabolism of essential nutrients, such as glucose, lipids and amino acids, is reconstructed with inflammation, immune mechanisms and oxidative stress, and these alterations modify the intestinal, hepatic and systemic environments, and regulate the composition and activity of gut microbes. Microbial metabolites, such as short-chain fatty acids, secondary bile acids, protein fermentation products, choline and ethanol and bacterial toxicants, such as lipopolysaccharides, peptidoglycans and bacterial DNA, play vital roles in NAFLD. The microbe–metabolite relationship is crucial for the modulation of intestinal microbial composition and metabolic activity. The intestinal microbiota and their metabolites participate in epithelial cell metabolism via a series of cell receptors and signalling pathways and remodel the metabolism of various cells in the liver via the gut–liver axis. Microbial metabolic manipulation is a promising strategy for NAFLD prevention, but larger-sampled clinical trials are required for future application.     

Effects of myocardial ischemia/reperfusion injury on plasma metabolomic profile during aging

BackgroundHeart disease is a frequent cause of hospitalization and mortality for elderly patients. A common feature of both heart disease and aging itself is the involvement of metabolic organ alterations ultimately leading to changes in circulating metabolite levels. However, the specific contribution of aging and ischemic injury to the metabolic dysregulation occurring in older adults with ischemic heart disease is still unknown.AimTo evaluate the effects of aging and ischemia/reperfusion (I/R) injury on plasma metabolomic profiling in mice.MethodsYoung and aged mice were subjected to a minimally invasive model of I/R injury or sham operation. Complete evaluation of cardiac function and untargeted plasma metabolomics analysis were performed.ResultsWe confirmed that aged mice from the sham group had impaired cardiac function and augmented left ventricular (LV) dimensions compared to young sham‐operated mice. Further, we found that ischemic injury did not drastically reduce LV systolic/diastolic function and dyssynchrony in aged compared to young mice. Using an untargeted metabolomics approach focused on aqueous metabolites, we found that ischemic injury does not affect the plasma metabolomic profile either in young or old mice. Our data also demonstrate that age significantly affects circulating metabolite levels (predominantly amino acids, phospholipids and organic acids) and perturbs several pathways involved in amino acid, glucid and nucleic acid metabolism as well as pyridoxal‐5′‐phosphate salvage pathway in both sham and ischemic mice.ConclusionsOur approach increases our understanding of age‐associated plasma metabolomic signatures in mice with and without heart disease excluding confounding factors related to metabolic comorbidities.     

Morphological and physiological responses to altitude in deer mice Peromyscus maniculatus.

Individuals within a species, living across a wide range of habitats, often display a great deal of phenotypic plasticity for organ mass and function. We investigated the extent to which changes in organ mass are variable, corresponding to environmental demand, across an altitudinal gradient. Are there changes in the mass of oxygen delivery organs (heart and lungs) and other central processing organs (gut, liver, kidney) associated with an increased sustainable metabolic rate that results from decreased ambient temperatures and decreased oxygen availability along an altitudinal gradient? We measured food intake, resting metabolic rate (RMR), and organ mass in captive deer mice (Peromyscus maniculatus bairdii) at three sites from 1,200 to 3,800 m above sea level to determine whether energy demand was correlated with organ mass. We found that food intake, gut mass, and cardiopulmonary organ mass increased in mice living at high altitudes. RMR was not correlated with organ mass differences along the altitudinal gradient. While the conditions in this study were by no means extreme, these results show that mice living at high altitudes have higher levels of energy demand and possess larger cardiopulmonary and digestive organs than mice living at lower altitudes.     

Absorption and metabolism of butyric acid in rabbit hind gut

Butyrate absorption in the large intestine of the rabbit was evaluated by the variation of concentrations in the bowel, the arterio-venous plasma and the intestinal loops. The metabolic transformations were studied with (3-4 C14) butyrate. The caeco-colonic epithelium oxidized negligible quantities of butyrate to ketone bodies and other metabolic pathways were found. These pathways were of different intensity according to the region of the gut and both phases of the excretory cycle. A portion, which may be large, was metabolized in the caeco-colonic wall and in the liver where radioactivity was found in free amino acids, carboxylic acids and sugars. The oxidation to CO2 in TCA cycle yields energy for metabolic activities. This study of metabolism takes account of the endoflora participation.     

Transport of bacterial end products from the colon of Periplaneta americana

The possible contribution of fermentation products produced by the anaerobic bacterial hindgut flora of Periplaneta americana was examined with respect to the physiology of the host. The microbial flora of the ileum and colon produced readily detectable quantities of short chain acids in vivo. Experiments where [¹⁴C] acids were injected into the hindgut indicated these products were transported out through the gut wall in vitro and in vivo. Other data underscore the importance of considering the metabolic capabilities of the gut microflora when using dyes as controls in studies on insect gut function.