Metabolic flux analysis of branched-chain amino and keto acids (BCAA, BCKA) and ß-hydroxy ß-methylbutyric acid across multiple organs in the pig.

Branched-chain amino acids (BCAA) and their metabolites the branched-chain keto acids (BCKA) and ß-hydroxy ß-methylbutyric acid (HMB) are involved in the regulation of key signaling pathways in the anabolic response to a meal. However, their (inter)organ kinetics remain unclear. Therefore, branched-chain amino acids (BCAA) [leucine (Leu), valine (Val), isoleucine (Ile)], BCKA [α-ketoisocaproic acid (KIC), 3-methyl-2-oxovaleric acid (KMV), 2-oxoisovalerate (KIV)], and HMB across organ net fluxes were measured. In multi-catheterized pigs (n = 12, ±25 kg), net fluxes across liver, portal drained viscera (PDV), kidney, and hindquarter (HQ, muscle compartment) were measured before and 4 h after bolus feeding of a complete meal (30% daily intake) in conscious state. Arterial and venous plasma were collected and concentrations were measured by LC- or GC-MS/MS. Data are expressed as mean [95% CI] and significance (P < 0.05) from zero by the Wilcoxon Signed Rank Test. In the postabsorptive state (in nmol/kg body wt/min), the kidney takes up HMB (3.2[1.3,5.0]) . BCKA is taken up by PDV (144[13,216]) but no release by other organs. In the postprandial state, the total net fluxes over 4 h (in µmol/kg body wt/4 h) showed a release of all BCKA by HQ (46.2[34.2,58.2]), KIC by the PDV (12.3[7.0,17.6]), and KIV by the kidney (10.0[2.3,178]). HMB was released by the liver (0.76[0.49,1.0]). All BCKA were taken up by the liver (200[133,268]). Substantial differences are present in (inter)organ metabolism and transport among the BCAA and its metabolites BCKA and HMB. The presented data in a translation animal model are relevant for the future development of optimized clinical nutrition.NEW & NOTEWORTHY Branched-chain amino acids (BCAA) and their metabolites the branched-chain keto acids (BCKA) and ß-hydroxy ß-methylbutyric acid (HMB) are involved in the regulation of key signaling pathways in the anabolic response to a meal. Substantial differences are present in (inter)organ metabolism and transport among the BCAA and its metabolites BCKA and HMB. The presented data in a translation animal model are relevant for the future development of optimized clinical nutrition.

Development of an inhalation unit risk factor for isoprene.

A unit risk factor (URF) was developed for isoprene based on evaluation of three animal studies with adequate data to perform dose-response modeling (NTP, 1994, 1999; Placke et al., 1996). Ultimately, the URF of 6.2E-08 per ppb (2.2E-08 per µg/m(3)) was based on the 95% lower confidence limit on the effective concentration corresponding to 10% extra risk for liver carcinoma in male B6C3F1 mice after incorporating appropriate adjustment factors for species differences in target tissue metabolite concentrations and inhalation dosimetry. The corresponding lifetime air concentration at the 1 in 100,000 no significant excess risk level is 160 ppb (450 µg/m(3)). This concentration is almost 4400 times lower than the lowest exposure level associated with statistically increased liver carcinoma in B6C3F1 mice in the key study (700 ppm in Placke et al., 1996) and is above typical isoprene breath concentrations reported in the scientific literature. Continuous lifetime environmental exposure to the 1 in 100,000 excess risk level of 160 ppb would be expected to raise the human blood isoprene area under the curve (AUC) less than one-third of the standard deviation of the endogenous mean blood AUC. The mean for ambient air monitoring sites in Texas (2005-2014) is approximately 0.13 ppb.

A quantitative study of the influence of inhaled compounds on their concentrations in exhaled breath.

Throughout the development of breath analysis research, there has been interest in how the concentrations of trace compounds in exhaled breath are related to their concentrations in the ambient inhaled air. In considering this, Phillips introduced the concept of 'alveolar gradient' and judged that the measured exhaled concentrations of volatile organic compounds should be diminished by an amount equal to their concentrations in the inhaled ambient air. The objective of the work described in this paper was to investigate this relationship quantitatively. Thus, experiments have been carried out in which inhaled air was polluted by seven compounds of interest in breath research, as given below, and exhaled breath has been analysed by SIFT-MS as the concentrations of these compounds in the inhaled air were reduced. The interesting result obtained is that all the exogenous compounds are partially retained in the exhaled breath and there are close linear relationships between the exhaled and inhaled air concentrations for all seven compounds. Thus, retention coefficients, a, have been derived for the following compounds: pentane, 0.76 ± 0.09; isoprene, 0.66 ± 0.04; acetone, 0.17 ± 0.03; ammonia, 0.70 ± 0.13, methanol, 0.29 ± 0.02; formaldehyde, 0.06 ± 0.03; deuterated water (HDO), 0.09 ± 0.02. From these data, correction to breath analyses for inhaled concentration can be described by coefficients specific to each compound, which can be close to 1 for hydrocarbons, as applied by Phillips, or around 0.1, meaning that inhaled concentrations of such compounds can essentially be neglected. A further deduction from the experimental data is that under conditions of the inhalation of clean air, the measured exhaled breath concentrations of those compounds should be increased by a factor of 1/(1 - a) to correspond to gaseous equilibrium with the compounds dissolved in the mixed venous blood entering the alveoli. Thus, for isoprene, this is a factor of 3, which we have confirmed experimentally by re-breathing experiments.

A drug-in-adhesive matrix based on thermoplastic elastomer: evaluation of percutaneous absorption, adhesion, and skin irritation.

A novel drug-in-adhesive matrix was designed and prepared. A thermoplastic elastomer, styrene-isoprene-styrene (SIS) block copolymer, in combination with tackifying resin and plasticizer, was employed to compose the matrix. Capsaicin was selected as the model drug. The drug percutaneous absorption, adhesion properties, and skin irritation were investigated. The results suggested that the diffusion through SIS matrix was the rate-limiting step of capsaicin percutaneous absorption. [SI] content in SIS and SIS proportions put important effects on drug penetration and adhesion properties. The chemical enhancers had strong interactions with the matrix and gave small effect on enhancement of drug skin permeation. The in vivo absorption of samples showed low drug plasma peaks and a steady and constant plasma level for a long period. These results suggested that the possible side effects of drug were attenuated, and the pharmacological effects were enhanced with an extended therapeutic period after application of SIS matrix. The significant differences in pharmacokinetic parameters produced by different formulations demonstrated the influences of SIS copolymer on drug penetrability. Furthermore, the result of skin toxicity test showed that no skin irritation occurred in guinea pig skin after transdermal administration of formulations.

Natural prenylated resveratrol analogs arachidin-1 and -3 demonstrate improved glucuronidation profiles and have affinity for cannabinoid receptors.

RATIONALE: The therapeutic promise of trans-resveratrol (tRes) is limited by poor bioavailability following rapid metabolism. We hypothesise that trans-arachidin-1 (tA1) and trans-arachidin-3 (tA3), peanut hairy root-derived isoprenylated analogs of tRes, will exhibit slower metabolism/enhanced bioavailability and retain biological activity via cannabinoid receptor (CBR) binding relative to their non-prenylated parent compounds trans-piceatannol (tPice) and tRes, respectively. RESULTS: The activities of eight human UDP-glucuronosyltransferases (UGTs) toward these compounds were evaluated. The greatest activity was observed for extrahepatic UGTs 1A10 and 1A7, followed by hepatic UGTs 1A1 and 1A9. Importantly, an additional isoprenyl and/or hydroxyl group in tA1 and tA3 slowed overall glucuronidation. CBR binding studies demonstrated that all analogs bound to CB1Rs with similar affinities (5-18 µM); however, only tA1 and tA3 bound appreciably to CB2Rs. Molecular modelling studies confirmed that the isoprenyl moiety of tA1 and tA3 improved binding affinity to CB2Rs. Finally, although tA3 acted as a competitive CB1R antagonist, tA1 antagonised CB1R agonists by both competitive and non-competitive mechanisms. CONCLUSIONS: Prenylated stilbenoids may be preferable alternatives to tRes due to increased bioavailability via slowed metabolism. Similar structural analogs might be developed as novel CB therapeutics for obesity and/or drug dependency.

The role of mathematical modeling in VOC analysis using isoprene as a prototypic example.

Isoprene is one of the most abundant endogenous volatile organic compounds (VOCs) contained in human breath and is considered to be a potentially useful biomarker for diagnostic and monitoring purposes. However, neither the exact biochemical origin of isoprene nor its physiological role is understood in sufficient depth, thus hindering the validation of breath isoprene tests in clinical routine. Exhaled isoprene concentrations are reported to change under different clinical and physiological conditions, especially in response to enhanced cardiovascular and respiratory activity. Investigating isoprene exhalation kinetics under dynamical exercise helps to gather the relevant experimental information for understanding the gas exchange phenomena associated with this important VOC. The first model for isoprene in exhaled breath has been developed by our research group. In this paper, we aim at giving a concise overview of this model and describe its role in providing supportive evidence for a peripheral (extrahepatic) source of isoprene. In this sense, the results presented here may enable a new perspective on the biochemical processes governing isoprene formation in the human body.

Kinetic and spectroscopic characterization of type II isopentenyl diphosphate isomerase from Thermus thermophilus: evidence for formation of substrate-induced flavin species.

Type II isopentenyl diphosphate (IPP) isomerase catalyzes the interconversion of IPP and dimethylallyl diphosphate (DMAPP). Although the reactions catalyzed by the type II enzyme and the well-studied type I IPP isomerase are identical, the type II protein requires reduced flavin for activity. The chemical mechanism, including the role of flavin, has not been established for type II IPP isomerase. Recombinant type II IPP isomerase from Thermus thermophilus HB27 was purified by Ni2+ affinity chromatography. The aerobically purified enzyme was inactive until the flavin cofactor was reduced by NADPH or dithionite or photochemically. The inactive oxidized flavin-enzyme complex bound IPP in a Mg2+-dependent manner for which KD approximately KmIPP, suggesting that the substrate binds to the inactive oxidized and active reduced forms of the protein with similar affinities. N,N-Dimethyl-2-amino-1-ethyl diphosphate (NIPP), a transition state analogue for the type I isomerase, competitively inhibits the type II enzyme, but with a much lower affinity. pH-dependent spectral changes indicate that the binding of IPP, DMAPP, and a saturated analogue isopentyl diphosphate promotes protonation of anionic reduced flavin. Electron paramagnetic resonance (EPR) and UV-visible spectroscopy show a substrate-dependent accumulation of the neutral flavin semiquinone during both the flavoenzyme reduction and reoxidation processes in the presence of IPP and related analogues. Redox potentials of IPP-bound enzyme indicate that the neutral semiquinone state of the flavin is stabilized thermodynamically relative to free FMN in solution.

A new method for estimating the retention of selected smoke constituents in the respiratory tract of smokers during cigarette smoking.

This report describes a new method for estimating the retention of selected mainstream smoke constituents in the respiratory tract of adult smokers during cigarette smoking. Both particulate-phase (PP) constituents including nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N'-nitrosonornicotine (NNN), two tobacco-specific nitrosamines (TSNA), and gas-vapor-phase (GVP) constituents including carbon monoxide (CO), isoprene (IP), acetaldehyde (AA), and ethylene, were studied. To estimate the amounts of smoke constituents delivered during smoking, we used predetermined linear relationships between the measured cigarette filter solanesol content and machine-generated mainstream deliveries of these selected compounds. To determine the amounts of smoke constituents exhaled, the expired breath was directed through a Cambridge filter pad (CFP) attached to an infrared spectrometer. PP compounds were trapped on the CFP for later analysis and GVP compounds were analyzed in near real time. The smokers' respiratory parameters during smoking, such as inhalation/exhalation volume and time, were monitored using LifeShirt(R), a respiratory inductive plethysmography (RIP) device. The retention of each smoke constituent, expressed as a percentage, was then calculated as the difference between the amount delivered (estimated) and the amount exhaled relative to the amount delivered. We studied 16 adult male smokers who smoked cigarettes according to 3 predefined smoking patterns: no inhalation (pattern A), normal inhalation (pattern B), and deep inhalation (pattern C). For the three PP constituents, the mean retentions for pattern A ranged between 10 and 20%; and while the mean retentions of the two TSNAs were significantly higher for pattern C (84% for NNK and 97% for NNN) than those for pattern B (63% for NNK and 84% for NNN), the mean retentions of nicotine were basically the same between patterns B and C, which were both greater than 98%. For the GVP constituents, the retentions were similar between pattern B and pattern C, although different constituents were retained to different degrees (average values of 33%, 52%, 79%, and 99% for ethylene, IP, CO, and AA, respectively). The differences in the retention between different constituents could be interpreted in terms of each constituent's physical properties such as volatility and solubility. In conclusion, the method described is suitable for studying the retention of selected mainstream smoke constituents in the respiratory tract of smokers.