Plasma metabolites associated with arterial stiffness in patients with type 2 diabetes.

BACKGROUND: Although an increased arterial stiffness has been associated with traditional coronary risk factors, the risk factors and pathology of arterial stiffness remain unclear. In this study, we aimed to identify the plasma metabolites associated with arterial stiffness in patients with type 2 diabetes mellitus. METHODS: We used the metabolomic data of 209 patients with type 2 diabetes as the first dataset for screening. To form the second dataset for validation, we enlisted an additional 31 individuals with type 2 diabetes. The non-targeted metabolome analysis of fasting plasma samples using gas chromatography coupled with mass spectrometry and the measurement of brachial-ankle pulse wave velocity (baPWV) were performed. RESULTS: A total of 65 annotated metabolites were detected. In the screening dataset, there were statistically significant associations between the baPWV and plasma levels of indoxyl sulfate (r = 0.226, p = 0.001), mannitol (r = 0.178, p = 0.010), mesoerythritol (r = 0.234, p = 0.001), and pyroglutamic acid (r = 0.182, p = 0.008). Multivariate regression analyses revealed that the plasma levels of mesoerythritol were significantly (ß = 0.163, p = 0.025) and that of indoxyl sulfate were marginally (ß = 0.124, p = 0.076) associated with baPWV, even after adjusting for traditional coronary risk factors. In the independent validation dataset, there was a statistically significant association between the baPWV and plasma levels of indoxyl sulfate (r = 0.430, p = 0.016). However, significant associations between the baPWV and plasma levels of the other three metabolites were not confirmed. CONCLUSIONS/INTERPRETATION: The plasma levels of indoxyl sulfate were associated with arterial stiffness in Japanese patients with type 2 diabetes. Although the plasma levels of mannitol, mesoerythritol, and pyroglutamic acid were also associated with arterial stiffness, further investigation is needed to verify the results.

Mammalian metabolism of erythritol: a predictive biomarker of metabolic dysfunction.

PURPOSE OF REVIEW: To summarize recent advances in our understanding of mammalian erythritol metabolism and its use as a predictive biomarker of cardiometabolic disease risk. RECENT FINDINGS: Elevated serum erythritol predicts future central adiposity gain and type 2 diabetes mellitus in healthy adults. Erythritol is a newly recognized human metabolic product of glucose, synthesized through the pentose phosphate pathway. The final conversion of this metabolic pathway is catalyzed by the enzymes sorbitol dehydrogenase and alcohol dehydrogenase 1. Erythritol is also a well characterized nonnutritive sweetener. Recent studies show that dietary erythritol can be metabolized to erythrose or erythronate in humans before excretion. SUMMARY: Elevated serum erythritol predicts risk for cardiometabolic disease, but more research is required to maximize its utility as a biomarker, including characterizing the determinants of endogenous erythritol synthesis from glucose. New insights into dietary erythritol metabolism also highlight the need to evaluate the effects of long-term erythritol consumption.

Erythritol is a pentose-phosphate pathway metabolite and associated with adiposity gain in young adults.

Metabolomic markers associated with incident central adiposity gain were investigated in young adults. In a 9-mo prospective study of university freshmen (n = 264). Blood samples and anthropometry measurements were collected in the first 3 d on campus and at the end of the year. Plasma from individuals was pooled by phenotype [incident central adiposity, stable adiposity, baseline hemoglobin A1c (HbA1c) > 5.05%, HbA1c < 4.92%] and assayed using GC-MS, chromatograms were analyzed using MetaboliteDetector software, and normalized metabolite levels were compared using Welch's t test. Assays were repeated using freshly prepared pools, and statistically significant metabolites were quantified in a targeted GC-MS approach. Isotope tracer studies were performed to determine if the potential marker was an endogenous human metabolite in men and in whole blood. Participants with incident central adiposity gain had statistically significantly higher blood erythritol [P < 0.001, false discovery rate (FDR) = 0.0435], and the targeted assay revealed 15-fold [95% confidence interval (CI): 13.27, 16.25] higher blood erythritol compared with participants with stable adiposity. Participants with baseline HbA1c > 5.05% had 21-fold (95% CI: 19.84, 21.41) higher blood erythritol compared with participants with lower HbA1c (P < 0.001, FDR = 0.00016). Erythritol was shown to be synthesized endogenously from glucose via the pentose-phosphate pathway (PPP) in stable isotope-assisted ex vivo blood incubation experiments and through in vivo conversion of erythritol to erythronate in stable isotope-assisted dried blood spot experiments. Therefore, endogenous production of erythritol from glucose may contribute to the association between erythritol and obesity observed in young adults.

De novo transcriptome sequencing in Bixa orellana to identify genes involved in methylerythritol phosphate, carotenoid and bixin biosynthesis.

BACKGROUND: Bixin or annatto is a commercially important natural orange-red pigment derived from lycopene that is produced and stored in seeds of Bixa orellana L. An enzymatic pathway for bixin biosynthesis was inferred from homology of putative proteins encoded by differentially expressed seed cDNAs. Some activities were later validated in a heterologous system. Nevertheless, much of the pathway remains to be clarified. For example, it is essential to identify the methylerythritol phosphate (MEP) and carotenoid pathways genes. RESULTS: In order to investigate the MEP, carotenoid, and bixin pathways genes, total RNA from young leaves and two different developmental stages of seeds from B. orellana were used for the construction of indexed mRNA libraries, sequenced on the Illumina HiSeq 2500 platform and assembled de novo using Velvet, CLC Genomics Workbench and CAP3 software. A total of 52,549 contigs were obtained with average length of 1,924 bp. Two phylogenetic analyses of inferred proteins, in one case encoded by thirteen general, single-copy cDNAs, in the other from carotenoid and MEP cDNAs, indicated that B. orellana is closely related to sister Malvales species cacao and cotton. Using homology, we identified 7 and 14 core gene products from the MEP and carotenoid pathways, respectively. Surprisingly, previously defined bixin pathway cDNAs were not present in our transcriptome. Here we propose a new set of gene products involved in bixin pathway. CONCLUSION: The identification and qRT-PCR quantification of cDNAs involved in annatto production suggest a hypothetical model for bixin biosynthesis that involve coordinated activation of some MEP, carotenoid and bixin pathway genes. These findings provide a better understanding of the mechanisms regulating these pathways and will facilitate the genetic improvement of B. orellana.

Is serum or plasma more appropriate for intersubject comparisons in metabolomic studies? An assessment in patients with small-cell lung cancer.

In clinical analyses, the most appropriate biofluid should be analyzed for optimal assay performance. For biological fluids, the most readily accessible is blood, and metabolomic analyses can be performed either on plasma or serum. To determine the optimal agent for analysis, metabolic profiles of matched human serum and plasma were assessed by gas chromatography/time-of-flight mass spectrometry and ultrahigh-performance liquid chromatography mass spectrometry (in positive and negative electrospray ionization modes). Comparison of the two metabolomes, in terms of reproducibility, discriminative ability and coverage, indicated that they offered similar analytical opportunities. An analysis of the variation between 29 small-cell lung cancer (SCLC) patients revealed that the differences between individuals are markedly similar for the two biofluids. However, significant differences between the levels of some specific metabolites were identified, as were differences in the intersubject variability of some metabolite levels. Glycerophosphocholines, erythritol, creatinine, hexadecanoic acid, and glutamine in plasma, but not in serum, were shown to correlate with life expectancy for SCLC patients, indicating the utility of metabolomic analyses in clinical prognosis and the particular utility of plasma in relation to the clinical management of SCLC.

Erythritol is a sweet antioxidant.

OBJECTIVE: Hyperglycemia, oxidative stress, and the onset and progression of diabetic complications are strongly linked. Reduction of oxidative stress could be of utmost importance in the long-term treatment of diabetic patients. The chronic nature of the disease calls for a mode of antioxidant intake that can be sustained easily, e.g., by the diet. Erythritol, a simple polyol, could be such a compound. It is orally available, well tolerated, and its chemical structure resembles that of mannitol, a well-known hydroxyl radical (HO*) scavenger. METHODS: We studied the antioxidant properties of erythritol in vitro and subsequently determined its antioxidant activity and its vasoprotective effect in the streptozotocin diabetic rat. RESULTS: Erythritol was shown to be an excellent HO* radical scavenger and an inhibitor of 2,2'-azobis-2-amidinopropane dihydrochloride-induced hemolysis but inert toward superoxide radicals. High-performance liquid chromatographic and electron spin resonance spectroscopy studies showed that the reaction of erythritol with hydroxyl radicals resulted in the formation of erythrose and erythrulose by abstraction of a carbon-bound hydrogen atom. In the streptozotocin diabetic rat, erythritol displayed an endothelium-protective effect and, in accordance with the in vitro experiments, erythrose was found in the urine of erythritol-consuming rats. CONCLUSION: Erythritol acts as an antioxidant in vivo and may help protect against hyperglycemia-induced vascular damage.

[Quantitative gas chromatography-mass spectrometry determination of pentaerythrityl tetranitrate metabolites pentaerythrityl trinitrate, pentaerythrityl dinitrate and pentaerythrityl in human plasma]. / Quantitative gaschromatographische/massenspektrometrische Bestimmung der Pentaerithrityltetranitrat-Metaboliten Pentaerithrityltrinitrat, Pentaerithrityldinitrat und Pentaerithritylmononitrat in Humanplasma.

Assay methods based on gas chromatography/mass spectroscopy (GC-MS) may be used to measure PE1N (pentaerithrityl mononitrate, CAS 1607-00-7), PE2N (pentaerithrityl dinitrate, CAS 1607-01-8) and intermediate pentaerithrityltrinitrate (PE3N, CAS 1607-17-6) in human plasma. Based on this method a simplified method to quantify the metabolites of PETN (pentaerithrityl tetranitrate, CAS 78-11-5, Pentalong) is described. In the present study a consistent method to extract the metabolites of human plasma and following derivatisation is described. Since PE1N can be quantified up to 150 ng/ml, PE2N and PE3N up to 30 ng/ml in human plasma, a dilution of the plasma samples can be avoided. The mean recovery rate is not so high as in other described methods, and inaccuracy is about 10%. Therefore a calibration range between 0.2-30 ng/ml of PE2N and 1-150 ng/ml of PE1N has to be considered. The described method offers an alternative and applicable option to quantify the PETN-metabolites and elucidate their part as NO-donors.

Fate of erythritol after single oral administration to rats and dogs.

The absorption and distribution of radiolabeled erythritol were studied in Wistar rats and beagle dogs after a single oral administration in doses ranging from 0.125 to 2.0 g erythritol/kg body wt. Erythritol concentrations in blood and plasma of rats reached their maxima 1 hr after administration and then declined biexponentially. In the blood and plasma of dogs, the highest concentrations occurred after 1/2 hr followed by a similar decline. Blood plasma distribution ratios and plasma protein binding ratios increased as blood plasma levels declined. At 120 hr after administration, 95.68 +/- 2.25% of the radioactivity had been excreted in the urine of dogs and 92.70 +/- 0.44% in the urine of rats; 0.33 +/- 0.05% had been excreted in the feces of dogs and 1.19 +/- 0.09% in the feces of rats; 1.17 +/- 0.04% had been excreted in expired air from dogs and 4.80 +/- 0.32% in expired air from rats. The results demonstrate that erythritol is rapidly absorbed and excreted, principally through the urinary pathway.

Plasma and urine kinetics of erythritol after oral ingestion by healthy humans.

The plasma and urine kinetics of erythritol and the effect of erythritol on plasma glucose and insulin levels were studied in human volunteers administered a single oral dose of 1 g erythritol/kg body wt. The plasma level of erythritol increased during the first 30 to 40 min, reaching a maximum value of approximately 2.2 mg/ml after 90 min. Plasma levels of erythritol then declined gradually to approximately 1.5 to 1.7 mg/ml at the end of the 3-hr sampling period. An average of 30% of the ingested amount of erythritol was excreted unchanged in the urine during the first 3 hr. Total urinary excretion increased to 78% after 24 hr. Renal clearance of erythritol was approximately half that of creatinine, indicating tubular reabsorption of erythritol by the kidney. Mean plasma glucose and insulin levels, measured for up to 3 hr after ingestion, were unaffected by erythritol. The results of this study indicate that erythritol was readily absorbed following oral administration and was excreted unchanged in the urine. Less than 20% of erythritol remained unabsorbed and was available for colonic fermentation and potential production of short-chain fatty acids. Its caloric value was estimated to be < or = 0.4 kcal/g.

Gastrointestinal response and plasma and urine determinations in human subjects given erythritol.

This study was undertaken to examine the influence of erythritol on certain plasma and urinary parameters and to assess the gastrointestinal response of humans given erythritol at single oral doses of 0.4 or 0.8 g/kg body wt/day. Three groups of six healthy volunteers each received a midmorning snack containing the equivalent of 0.4 or 0.8 g erythritol/kg body wt or 0.8 g sucrose/kg body wt. A fourth group received no snack and served as a negative control group. Consumption of erythritol did not affect plasma osmolarity, water consumption, or diuresis, and no significant variations in plasma or urine electrolyte balance were observed. Plasma glucose and insulin concentrations also were not affected by erythritol. Gastrointestinal responses to erythritol were comparable to those of sucrose. Plasma and urine erythritol concentrations increased within 2 hr of ingestion in proportion to the amount ingested. Approximately 60% of the erythritol dose was eliminated in the urine within 22 hr. The results of this study demonstrate that ingestion of erythritol at doses of up to 0.8 g/kg body wt does not alter plasma or urine osmolarity or electrolyte balance and is well tolerated by the digestive tract.

Serum glucose and insulin levels and erythritol balance after oral administration of erythritol in healthy subjects.

OBJECTIVES: To investigate the effect of an oral administration of erythritol on serum glucose and insulin levels in healthy subjects and estimate available energy of erythritol in human. DESIGN: Ingestion of erythritol (0.3 g/kg body weight) or the same dose of glucose as a control. SETTING: Omiya Research Lab., Nikken Chemicals Co., Japan. SUBJECTS: 5 healthy male volunteers aged 45-58 years. MAIN OUTCOME MEASURES: Serum glucose, insulin and erythritol levels after erythritol ingestion. Urinary erythritol excretion. RESULTS: Erythritol did not increase serum levels of glucose or insulin, while the same dose of glucose increased rapidly glucose and insulin levels within 30 min. Erythritol did not induce any significant effects on serum levels of total cholesterol, triacylglycerol, free fatty acids, Na, K and Cl. Also, urinary Na, K and Cl were not affected by erythritol ingestion. Serum levels of erythritol reached the maximum concentration of 426.5 +/- 113.4 micrograms/ml at 30 min and declined to 13.5 +/- 3.2 micrograms/ml at 24 h. Total urinary excretion of erythritol was 85.8 +/- 4.6% for 24 h and 90.3 +/- 4.5% for 48 h, respectively. CONCLUSIONS: Erythritol did not affect serum levels of glucose, insulin or other serum constituents. More than 90% of ingested erythritol was readily absorbed and excreted in urine without degradation. This fact suggests that available energy of erythritol in human is less than 1.7 kJ/g (0.4 kcal/g). DESCRIPTORS: erythritol, glucose, insulin, low energy sweetener.

Determination of polyols in serum by selected ion monitoring.

We used gas chromatography-chemical ionization mass spectrometry with selected ion monitoring of the (M-59)+ ions to determine polyols (as their peracetyl derivatives) in serum. The internal standard was 2-deoxygalactitol. Mean (and SD) polyol concentrations (mg/L) in 33 normal sera were: erythritol 0.45 (0.14), threitol 0.20 (0.06), adonitol 0.06 (0.02), arabinitol 0.37 (0.12), xylitol 0.05 (0.02), mannitol 0.41 (0.45), galactitol 0.15 (0.11), sorbitol 0.16 (0.11). Arabinitol determined in the same 33 normal sera by trimethylsilylation was 0.39 (0.13) mg/L. In 32 samples from cancer patients with normal creatinine, all polyol concentrations were in the normal range except for seven increased erythritol and one increased threitol concentrations. In all six patients with renal dysfunction we found increased erythritol and arabinitol, and increased threitol and mannitol in four of the six. Initial results showed that only arabinitol concentrations increased in invasive candidiasis.

A sensitive assay for the pharmacokinetic investigation of a rapidly hydrolyzing alkylating agent Lycurim (ritrosulfan; R-74; NSC-122 402).

Lycurim [1,4-di(2'-methanesulfonyloxyethylamino)-1,4-dideoxymesoerythri tol dimethane sulfonate] is rapidly hydrolyzed in aqueous media to inactive products according to a second-order reaction. The respective rate constants are: k1 = 9.82 X 10(-2) and k2 = 1.76 X 10(-2) mumol/ml/min. The concentrations of the parent compound and alkylating intermediate(s) were measured by chemical trapping with N,N-diethyldithiocarbamic acid (DDTC). The rate of this reaction is substantially higher: k1 = 2.61 X 10(-1) and K2 = 4.76 X 10(-2) mumol/ml/min. By using 35S-labeled DDTC, alkylating compounds in concentrations as low as 0.04 microgram/ml could be detected in spiked plasma samples. After intracavitary application of 60 mg Lycurim no alkylating activity could be demonstrated in the plasma of patients at any time point.

Pharmacological and physiological doses of insulin and determinants of bile flow in dogs.

The role of insulin in control of bile secretion is uncertain. To study the mechanism of choleresis produced by large doses of insulin, bile was collected through modified Thomas cannulas from dogs anesthetized with pentobarbital. Animals received pipenzolate methylbromide, sodium taurocholate, and [14C]erythritol. After bile flow had stabilized three animals received infusions of insulin at 2, 4, 13, 26, 35, and 70 mU . kg-1 . min-1 for 40 min each. Bile and [14C]erythritol clearance increased (P less than 0.005), but bile salt output remained constant, suggesting that the choleresis was mainly due to enhanced bile salt-independent canalicular flow. Plasma insulin and glucagon levels also rose when insulin was infused. To exclude the possible effects of glucagon three additional animals received somatostatin (800 ng . kg-1 . min-1) along with infusions of insulin. Bile flow and [14C]erythritol clearance again increased significantly, but glucagon levels remained low, suggesting that the effects on bile flow were due to insulin alone. To determine whether physiological doses of insulin altered bile flow dogs were anesthetized with pentobarbital and received pipenzolate methylbromide, taurocholate, [14C]erythritol, and somatostatin (800 ng . kg-1 . min-1). Insulin (0.2 and 0.8 mU . kg-1 . min-1) was infused through the portal vein for 1 h each. Bile flow and [14C]erythritol clearance increased with insulin (0.8 mU . kg-1 . min-1; P less than 0.02), suggesting that the choleresis may have been due to bile salt-independent canalicular flow. Plasma insulin rose to physiological postprandial levels. These studies demonstrate that pharmacological and physiological levels of insulin administered to dogs produce a significant choleresis. Thus insulin may play an important role in the regulation of bile secretion.

Choleresis and bile pigment excretion in the nutria.

Mean excretory rates of endogenous biliverdin (BV) and bilirubin (BR) in nutria were determined to be 6.0 +/- 1.0 and 0.67 +/- 0.10 microgram/min/kg, respectively. Endogenous bile flow was 33 +/- 7.0 microliter/min/kg. Infusion of sodium taurocholate at rates above 0.1 mumol/min/kg resulted in cholestasis. Hepatic transport maxima for BR conjugates following the infusion of unconjugated BR were 120 and 130 micrograms/min/kg. Although nutria excrete predominantly BV like avian and reptilian species, they differ by readily excreting an exogenous load of unconjugated BR.

General and transport properties of hypotonic and isotonic preparations of resealed erythrocyte ghosts.

Resealed human erythrocyte ghosts are regarded as valuable tools for the study of membrane properties. In order to investigate to what extent preparation procedures affect the yield of ghosts, their general properties, and their permeability, ghosts prepared by lysis at low (hypotonic media) and high (isotonic media) ionic strength were compared with each other and with native erythrocytes. For isotonic lysis, cells were either subjected to dielectric breakdown or suspended in isotonic NH4Cl solutions. In spite of very different characteristics of the lysis and the resealing process in the three types of preparations, the resulting ghosts do not differ in a number of features except for somewhat varying yields and for properties resulting from the mode of lysis. Specific transport properties, as characterized by the mediated fluxes of m-erythritol, L-arabinose, L-lactate, and sulfate, proved to be unaltered with a few unsystematic exceptions. The simple nonmediated fluxes of all these permeants, as measured in the presence of inhibitors, however, were enhanced between 1.5- and 4-fold, indicating a somewhat increased ground permeability (of the lipid domain) in all ghost membranes.

Dual effect of membrane cholesterol on simple and mediated transport processes in human erythrocytes.

The influence of cholesterol on simple and facilitated transport processes across the membrane of intact human erythrocytes was studied after graded depletion or enrichment of membrane cholesterol by incubation of the cells in phospholipid or phospholipid/cholesterol suspensions. 1. The carrier-mediated transfer of L-lactate and of L-arabinose proved to be enhanced in this effect. In contrast, the self-exchange of SO4(2-), mediated by the inorganic anion-exchange system, and the simple diffusion of erythritol via the lipid phase of the membrane are inhibited by cholesterol. 2. Reversibility of these two opposite effects of cholesterol was demonstrated by measurements on cells depleted again after cholesterol enrichment and enriched again after previous depletion. 3. Certain phospholipids used for preparing the lipid dispersions that are required for cholesterol variation have effects on permeability of their own, due, for example, to traces of contaminants. A discrimination of such artifacts due, for example, to traces of contaminants. A discrimination of such artifacts from the effects of cholesterol is only possible by demonstrating reversibility. 4. The opposite effects of cholesterol on various facilitated transfer processes, which have a correlation in the opposite effects of other modifications of the membrane lipid phase (Deuticke, B., Grunze, M. and Haest, C.W.M. (1979) Alfred Benzon Symposium 14, Munksgaard, Copenhagen, in the press), are indicative of different types of lipid-protein interaction in the erythrocyte membrane.

Effects of anesthetic alcohols on membrane transport processes in human erythrocytes.

1. Anesthetic alcohols (pentanol, hexanol and heptanol) were found to increase the fluidity of red cell membrane lipids as monitored by the fluorescence depolarization of diphenylhexatriene. The relative potency of the alcohols was found to be parallel to their relative membrane/water partition coefficients. 2. Hexanol had biphasic effect on erythritol uptake by simple diffusion by red cells. At concentrations less than 9 mM, there was an approximately linear increase in erythritol permeability with increasing alcohol concentration. 3. The facilitated transport of uridine was markedly inhibited by hexanol. Hexanol at 6 mM produced a 65% inhibition of uridine (4 mM) uptake. Hexanol decreased both the apparent Km and V values for the equilibrium exchange of uridine. 4. The facilitated transport of galactose was only slightly inhibited by hexanol. 5. Hexanol was without effect on the passive and active fluxes of Na+ and K+ in red cells with altered cation contents. Cells that were slightly depleted of K+ and cells that were highly K+ -depleted were both insensitive to hexanol.

Gas chromatographic separation and mass spectrometric identification of polyols in human cerebrospinal fluid and plasma.

Seven polyols, erythritol, arabinitol, anhydroflucitol, mannitol, sorbitol, myoinositol and possibly ribitol were identified in human cerebrospinal fluid by means of gas-liquid chromatography and mass spectrometry. Quantitative data were obtained for five polyols, arabinitol, anhydroglucitol, mannitol, sorbitol and myoinositol, by screening of 205 CSF samples. These five polyols represented 90-95 per cent of the polyol-concentration which was 340 +/- 105 mumol/1 in the total series. The concentration of polyols in the CSF was two times higher than that in the plasma (148 +/- 30 mumol/1), where only anhydroglucitol and myoinositol could be quantitated. The variations noted were not associated with age, sex or the plasma concentrations of polyols. The polyols of the CSF most likely originate from brain tissue and/or spinal cord since penetration from the plasma against a gradient seems unlikely.

Effect of atropine on bile flow and biliary excretion of 3H-ouabain in the isolated perfused rat liver.

Effects of choleresis produced by atropine (4-10(-4)M) on the biliary elimination of 3H-ouabain and on the biliary clearance rate of 14C-erythritol were studied in the isolated perfused rat liver. The increased bile flow produced by atropine was associated with decreased level of 3H-ouabain in the bile. The cumulative biliary elimination of ouabain was not affected. Atropine choleresis caused an increase in the erythritol clearance rate and a reduction in the bile to plasma ratios of erythritol as compared to the controls. The results suggest that the choleresis induced by atropine is not entirely of canalicular origin but possibly reabsorption mechanisms in the bile ducts or ductules are also involved.