Dimethylguanidino valeric acid is a marker of liver fat and predicts diabetes.

Unbiased, "nontargeted" metabolite profiling techniques hold considerable promise for biomarker and pathway discovery, in spite of the lack of successful applications to human disease. By integrating nontargeted metabolomics, genetics, and detailed human phenotyping, we identified dimethylguanidino valeric acid (DMGV) as an independent biomarker of CT-defined nonalcoholic fatty liver disease (NAFLD) in the offspring cohort of the Framingham Heart Study (FHS) participants. We verified the relationship between DMGV and early hepatic pathology. Specifically, plasma DMGV levels were correlated with biopsy-proven nonalcoholic steatohepatitis (NASH) in a hospital cohort of individuals undergoing gastric bypass surgery, and DMGV levels fell in parallel with improvements in post-procedure cardiometabolic parameters. Further, baseline DMGV levels independently predicted future diabetes up to 12 years before disease onset in 3 distinct human cohorts. Finally, we provide all metabolite peak data consisting of known and unidentified peaks, genetics, and key metabolic parameters as a publicly available resource for investigations in cardiometabolic diseases.

Metabolic syndrome increases dietary α-tocopherol requirements as assessed using urinary and plasma vitamin E catabolites: a double-blind, crossover clinical trial.

Background: Vitamin E supplementation improves liver histology in patients with nonalcoholic steatohepatitis, which is a manifestation of the metabolic syndrome (MetS). We reported previously that α-tocopherol bioavailability in healthy adults is higher than in those with MetS, thereby suggesting that the latter group has increased requirements.Objective: We hypothesized that α-tocopherol catabolites α-carboxyethyl hydroxychromanol (α-CEHC) and α-carboxymethylbutyl hydroxychromanol (α-CMBHC) are useful biomarkers of α-tocopherol status.Design: Adults (healthy or with MetS; n = 10/group) completed a double-blind, crossover clinical trial with four 72-h interventions during which they co-ingested 15 mg hexadeuterium-labeled RRR-α-tocopherol (d6-α-T) with nonfat, reduced-fat, whole, or soy milk. During each intervention, we measured α-CEHC and α-CMBHC excretions in three 8-h urine collections (0-24 h) and plasma α-tocopherol, α-CEHC, and α-CMBHC concentrations at various times ≤72 h.Results: During the first 24 h, participants with MetS compared with healthy adults excreted 41% less α-CEHC (all values are least-squares means ± SEMs: 0.6 ± 0.1 compared with 1.0 ± 0.1 µmol/g creatinine, respectively; P = 0.002), 63% less hexadeuterium-labeled (d6)-α-CEHC (0.04 ± 0.02 compared with 0.13 ± 0.02 µmol/g creatinine, respectively; P = 0.002), and 58% less d6-α-CMBHC (0.017 ± 0.004 compared with 0.041 ± 0.004 µmol/g creatinine, respectively; P = 0.0009) and had 52% lower plasma d6-α-CEHC areas under the concentration curves [area under the curve from 0 to 24 h (AUC0-24h): 27.7 ± 7.9 compared with 58.4 ± 7.9 nmol/L × h, respectively; P = 0.01]. d6-α-CEHC peaked before d6-α-T in 77 of 80 paired plasma concentration curves. Urinary d6-α-CEHC 24-h concentrations were associated with the plasma AUC0-24 h of d6-α-T (r = 0.53, P = 0.02) and d6-α-CEHC (r = 0.72, P = 0.0003), and with urinary d6-α-CMBHC (r = 0.88, P < 0.0001), and inversely with the plasma inflammation biomarkers C-reactive protein (r = -0.70, P = 0.0006), interleukin-10 (r = -0.59, P = 0.007), and interleukin-6 (r = -0.54, P = 0.01).Conclusion: Urinary α-CEHC and α-CMBHC are useful biomarkers to noninvasively assess α-tocopherol adequacy, especially in populations with MetS-associated hepatic dysfunction that likely impairs α-tocopherol trafficking. This trial was registered at clinicaltrials.gov as NCT01787591.

Carcinogenicity assessment of the pan-caspase inhibitor, emricasan, in Tg.rasH2 mice.

Emricasan, formerly IDN-6556, is a small molecule currently being evaluated in clinical trials to reduce hepatic injury and liver fibrosis. Since emricasan is an irreversible pan-caspase inhibitor that potently inhibits caspase-mediated apoptosis and inflammation, its carcinogenic potential was evaluated in a humanized mouse model. Tg.rasH2 mice received LabDiet formulated with 0, 10, 25, and 75mg/kg/day of emricasan, for 26weeks. At terminal sacrifice, blood was collected for clinical pathology analysis and tissues were collected, processed, and evaluated microscopically. There were no treatment related deaths or overt signs of toxicity for the duration of the study. There was no evidence of a carcinogenic effect in the peripheral blood leukocyte counts. Liver microgranulomas, which are background lesions, were slightly increased, especially in males. Increases in the incidence of the activated germinal centers were seen in the spleens and mesenteric lymph nodes of males and females, and in the mandibular lymph nodes of male mice. Atrophy of ovaries and testicular degeneration were also seen in emricasan treated animals. Although several non-neoplastic lesions were observed, there was no evidence of emricasan-related tumor formation in any tissue. In addition, the non-neoplastic lesions were not considered pre-neoplastic. Thus, emricasan is not considered carcinogenic.

PET imaging-based evaluation of hepatobiliary transport in humans with (15R)-11C-TIC-Me.

UNLABELLED: It is well accepted that drug transporters play a pivotal role in hepatobiliary excretion of anionic drugs, in which drug-drug interactions and genetic polymorphisms are known to cause variations. However, PET probes for in vivo functional characterization of these transporters have not been established yet. We used PET to investigate hepatic uptake and subsequent canalicular efflux of (11)C-labeled (15R)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin methyl ester [(15R)-(11)C-TIC-Me)] in healthy subjects. METHODS: Serial PET scans of the abdominal region in healthy male subjects were obtained with or without the organic anion-transporting polypeptide (OATP) inhibitor rifampicin after intravenous injection of (15R)-(11)C-TIC-Me as a radiotracer. Venous blood samples and PET images were obtained at frequent intervals up to 30 min after administration of the PET tracer. Dynamic imaging data were evaluated by integration plots of data collected for 2-10 min and for 10-30 min after tracer administration for the determination of tissue uptake clearance and biliary efflux clearance, respectively. RESULTS: After rapid hydrolysis in blood, the acid form-(11)C-labeled (15R)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin [(15R)-(11)C-TIC]-accumulated in the liver (37% of the dose by 17 min), and the radioactivity was then excreted into the bile (6.2% by 30 min). Rifampicin (600 mg by mouth), a potent OATP inhibitor, significantly reduced the radioactivity excreted into the bile (by 44%) by inhibiting both uptake (by 45%) and subsequent canalicular efflux (by 62%). (15R)-(11)C-TIC is an in vitro substrate of OATP1B1 and OATP1B3, and clinically relevant concentrations of rifampicin inhibited uptake by OATP1B1 and OATP1B3. These results demonstrated that in humans, (15R)-(11)C-TIC-associated radioactivity is excreted into the bile by organic anion transport systems. CONCLUSION: We demonstrated that PET image analysis with (15R)-(11)C-TIC-Me is useful for investigating variations in OATP function in the human hepatobiliary transport system.

Postoperative infusion of amino acids induces a positive protein balance independently of the type of analgesia used.

BACKGROUND: Net loss of body protein is a prominent feature of the catabolic response to surgical tissue trauma. Epidural analgesia with hypocaloric dextrose has been demonstrated to attenuate leucine oxidation but was unable to make protein balance positive. The current study was set to determine whether an infusion of amino acids on the second day after colon surgery would revert the catabolic state and promote protein synthesis while maintaining glucose homeostasis in patients receiving epidural analgesia as compared with patient-controlled analgesia with morphine (PCA). METHODS: Sixteen patients undergoing colorectal surgery were randomly assigned to receive epidural blockade or PCA as analgesic techniques and underwent a 6-h stable isotope infusion study (3 h fasted, 3 h fed) on the second postoperative day. Whole body glucose kinetics and protein turnover were measured using [6,6-2H2]glucose and l-[1-13C]leucine as tracer. RESULTS: The infusion of amino acids caused a decrease in endogenous glucose rate of appearance in both groups (P < 0.05), with greater changes in the PCA group (P < 0.05). Administration of amino acids suppressed the appearance of leucine from protein breakdown in both groups (P < 0.05), although the decrease was greater in the PCA group (P < 0.05). Leucine oxidation increased in both groups (P < 0.05), with greater change in the epidural group (P < 0.05). Protein synthesis increased to the same extent in both groups (P < 0.05). Protein balance became positive after the infusion of amino acids, and the effect was greater in the PCA group (P < 0.05). CONCLUSIONS: Infusion of amino acids decreased the endogenous glucose production and induced a positive protein balance independent of the type of anesthesia provided, although such effects were greater in the PCA group.

Improvement of glucose tolerance in Zucker diabetic fatty rats by long-term treatment with the dipeptidyl peptidase inhibitor P32/98: comparison with and combination with rosiglitazone.

AIM: The aim of this study was to investigate the effect of long-term treatment with the dipeptidyl peptidase inhibitor P32/98 and its combination with rosiglitazone on blood glucose control and islet of Langerhans histology in male Zucker diabetic fatty (ZDF) rats, when treatment begins before or after the development of overt diabetes. METHODS: ZDF rats were treated with P32/98 from the age of 9, 12 or 15 weeks. Rosiglitazone maleate was given to a separate group from the age of 13 weeks. P32/98 was given to all of these rosiglitazone-treated rats from 16 weeks of age. Rosiglitazone maleate was also given from 16 weeks of age to half the rats that were given P32/98 from 9 weeks of age. The compounds were given by oral gavage until the rats were 14 weeks old and then in the diet. The experiment was terminated at the age of 20-21 weeks. Blood glucose, plasma insulin and oral glucose tolerance were measured at intervals; islet histology was assessed terminally. RESULTS: P32/98 improved glucose tolerance after both single and multiple doses when treatment started at 9 weeks of age, also after the third week of treatment when treatment began at 12 or 15 weeks of age. P32/98 reduced daytime blood glucose when treatment began at 12 weeks. Treatment with rosiglitazone increased food intake and body weight, and after 2 weeks, reduced daytime blood glucose, water intake and the area under the glucose tolerance curve. A single dose of P32/98 markedly improved glucose tolerance in rosiglitazone-treated rats. When treatment had begun at 9 weeks of age, P32/98 stimulated insulin secretion in some glucose tolerance tests. Neither P32/98 nor rosiglitazone affected pancreatic insulin content, nor did they have clear effects on islet histology. CONCLUSION: P32/98 elicited a sustained improvement in glucose tolerance in both prediabetic and diabetic ZDF rats. The effects of P32/98 on glucose and insulin were similar to those of rosiglitazone, and in contrast to rosiglitazone, P32/98 did not increase food intake or body weight. However, neither compound was especially effective at improving diabetes in ZDF rats when treatment began at 9, 12 or 15 (P32/98) or 13 (rosiglitazone) weeks of age.

Isovaleric acidemia with promyelocytic myeloproliferative syndrome.

Isovaleric acidemia, an autosomal recessive disorder, is due to isovaleryl-coenzyme A dehydrogenase deficiency and is one of the branched-chain aminoacidopathies. Isovaleric acidemia may present in the neonatal period with an acute episode of severe metabolic acidosis, ketosis, and vomiting and may lead to coma and death in the first 2 months of life. This report concerns an infant who presented at 10 days of age because of lethargy, poor feeding, hypothermia, cholestasis, and thrombocytopenia, leukopenia, and profound pancytopenia. Death occurred at 19 days of age. Autopsy showed mild fatty change in the liver and extramedullary hematopoiesis, generalized Escherichia coli sepsis, and myelodysplasia of the bone marrow with arrest of the myeloid series at the promyelocytic stage. The appearance resembled promyelocytic leukemia, but the diagnostic 15:17 translocation was not present. The maturation arrest in granulopoiesis in isovaleric acidemia appears to be most likely due to a direct metabolic effect on granulocyte precursor cells.

Deconvolution gas chromatography/mass spectrometry of urinary organic acids--potential for pattern recognition and automated identification of metabolic disorders.

The National Institute of Standards and Technology (NIST) Automated Mass Spectral Deconvolution and Identification System (AMDIS) is applied to a selection of data files obtained from the gas chromatography/mass spectrometry (GC/MS) analysis of urinary organic acids. Mass spectra obtained after deconvolution are compared with a special user library containing both the mass spectra and retention indices of ethoxime-trimethylsilyl (EO-TMS) derivatives of a set of organic acids. Efficient identification of components is achieved and the potential of the procedure for automated diagnosis of inborn errors of metabolism and for related research is demonstrated.

Assay of the enantiomers of 1,2-propanediol, 1,3-butanediol, 1,3-pentanediol, and the corresponding hydroxyacids by gas chromatography-mass spectrometry.

We developed gas chromatographic-mass spectrometric assays for the enantiomers of 1,2-propanediol, 1,3-butanediol, 1,3-pentanediol, and their corresponding hydroxyacids, lactate, beta-hydroxybutyrate, and beta-hydroxypentanoate (3-hydroxyvalerate) in biological fluids. The corresponding ketoacids, acetoacetate and beta-ketopentanoate, can be assayed simultaneously by pretreating the samples with NaB2H4. The assays involve spiking the samples with deuterated internal standards, deproteinization, ether extraction, and derivatization of the carboxyl groups with (R,S)-2-butanol/HCl and of the hydroxyl groups with chiral (S)-(+)-2-phenylbutyryl chloride. Mass spectrometric analysis is conducted under ammonia positive chemical ionization. We used these assays to follow the metabolism of diol enantiomers in dogs. For (R,S)-1,3-butanediol and (R,S)-1,3-pentanediol, the uptakes from dog plasma of the R and S enantiomer of each diol were identical. In contrast, the metabolism of (S)-1,2-propanediol was faster than that of (R)-1,2-propanediol. (R)-1,2-Propanediol is formed during acetone metabolism, while (R,S)-1,3-butanediol and (R,S)-1,3-pentanediol are potential nutrients. The assays developed will allow further investigations of the metabolisms of acetone, (R)-lactate, and artificial nutrients derived from the 1,3-butanediol and 1,3-pentanediol enantiomers.

Thromboxane A2 receptor antagonism and synthase inhibition in essential hypertension.

Short-term effects of ridogrel, a combined thromboxane synthase inhibitor and receptor antagonist, were investigated in 16 patients with uncomplicated essential hypertension. After a 2-week placebo period without antihypertensive medication, patients were admitted to the hospital overnight on two occasions 3 weeks apart. On each occasion, they received two doses of either placebo or ridogrel (300 mg) 12 hours apart according to a double-blind crossover protocol. Renal and systemic thromboxane A2 and prostacyclin biosynthesis were investigated by measuring urinary excretion of thromboxane B2, 6-oxo-prostaglandin F1 alpha, and their respective 2,3-dinor metabolites using gas chromatography/mass spectrometry. Responses of platelets to a thromboxane A2 mimetic and to adenosine diphosphate were studied turbidometrically. Blood pressure was measured automatically at 20-minute intervals. Ridogrel reduced excretion of 2,3-dinor-thromboxane B2 and thromboxane B2 compared with placebo (21 +/- 6 versus 279 +/- 28 and 14 +/- 4 versus 39 +/- 9 ng/g creatinine, respectively; P < .0001 and P < .05). Excretion of 2,3-dinor-6-oxoprostaglandin F1 alpha and 6-oxoprostaglandin F1 alpha was increased by ridogrel compared with placebo (184 +/- 20 versus 146 +/- 11 and 86 +/- 9 versus 58 +/- 6 ng/g creatinine, respectively; P < .05). Ridogrel selectively antagonized platelet aggregation to the thromboxane mimetic (P < .0001). Blood pressure did not differ significantly between ridogrel and placebo treatment periods. Thus, in patients with essential hypertension, acute administration of ridogrel reduces renal and extrarenal thromboxane A2 biosynthesis, increases renal and extrarenal prostacyclin biosynthesis, inhibits thromboxane receptor-activated platelet aggregation, but has no effect on systemic arterial pressure.

Ridogrel in the setting of percutaneous transluminal coronary angioplasty.

The safety of the combination of heparin and ridogrel therapy and its antiplatelet efficacy was examined in the setting of percutaneous transluminal coronary angioplasty (PTCA). In 32 patients without known aspirin intake for 10 days before PTCA, therapy with ridogrel (300-mg intravenous bolus) was begun just before PTCA and continued orally at a dose of 300 mg twice daily until discharge. Heparin was administered as a 10,000 IU bolus dose before PTCA and followed by an intravenous infusion at a rate of 1,000 IU/hour for 24 hours. Bleeding problems at the arterial entry site occurred in 13 patients, which required a blood transfusion in only 2 patients. One patient underwent emergency bypass surgery without specific problems of hemostasis. Ridogrel virtually eliminated thromboxane B2 from the serum (29,990 +/- 6,555 pg/0.1 ml before vs 63 +/- 7 pg/0.1 ml at 2 hours after ridogrel), with a concomitant increase in serum 6-keto-prostaglandin F1 alpha (511 +/- 34 pg/0.1 ml before vs 1,190 +/- 146 pg/0.1 ml at 24 hours after ridogrel). There were no acute reocclusions in the ridogrel-treated patients, whereas acute reocclusions occurred in 5.6% of the patients taking the standard aspirin + heparin regimen during the same period. Furthermore, at 6-month clinical follow-up patients treated with ridogrel compared favorably with those receiving standard treatment.

Fanconi syndrome in a patient with a variant of isovaleric acidemia.

Fanconi syndrome with proximal renal tubular acidosis is caused by a variety of anatomic, functional and metabolic disorders. We report a patient with a variant of isovaleric acidosis who developed proximal tubular acidosis. This patient was able to acidify the urine during metabolic acidosis, developed a hyperchloremic metabolic acidosis, and needed 24 mEq/kg/day of bicarbonate to maintain normal serum bicarbonate. She had a FE Bicarbonate of 12 +/- 4% during bicarbonate infusion. Isovaleric acidosis may be another toxic cause of proximal RTA.

Thymidine 5'-O-pivaloate: evidence for prodrug action in the rat and rhesus monkey.

Thymidine (TdR) 5'-O-pivaloate was administered to rats and a rhesus monkey to assess its prodrug characteristics. Within 15 minutes after a 2000-mg/kg oral or sc bolus in rats, free TdR was detected in the serum of liquid chromatography. At 1 hour the TdR level reached a plateau of approximately 0.6 mM with oral administration and 0.1 mM with sc treatment. In the monkey experiment, radioactivity was detected in the serum 15 minutes after oral administration of 200 mg/kg of [3H]TdR 5'-O-pivaloate. Thin-layer chromatographic analysis showed that this consisted of unchanged ester as well as free TdR and thymine. The concentration of intact ester reached a peak value of 5.4 microM after 40 minutes, before declining to a steady level of 0.9--1.0 microM over the next 6 hours. Serum [3H]TdR and [3H]thymine followed a similar course, except that both remained in the range of 1.0--1.2 microM for as long as 24 hours. Total recovery of radioactivity in the urine over the first 6 hours was 2.5% of the administered dose, will all compounds present in equal proportions. The results of these studies are consistent with absorption of intact pivaloate ester from the stomach, followed by gradual cleavage to TdR and catabolism of TdR to thymine and subsequent products. With a single oral dose of ester, it was possible to achieve, and maintain for up to 24 hours, a level (1 microM) of free TdR in the serum which has previously been found to protect normal tissues during high-dose methotrexate therapy.