Efficient production and characterization of homopolymeric poly(3-hydroxyvalerate) produced by Bacillus strain PJC48.

Aliphatic polyester, poly(3-hydroxyvalerate) (PHV), is commonly produced as a granular component in bacterial cells of various species. Based on 16S rDNA gene sequence analysis, strain PJC48 was identified as a Bacillus species. The current study is aimed to screen for a high-yield strain that can produce PHV efficiently and to increase PHV product yield by optimizing the fermentative process. We identified a high-producer strain based on Nile red staining. Characterization of the PHV produced by PJC48 by nuclear magnetic resonance spectroscopy revealed that it consisted of (R)-3-hydroxyvalerate monomers. The suggested model was validated by response surface methodology. Optimization of the PHV yield resulted in an increase of 32.75% compared to control, with a maximum production of 1.64 g/L after 48 H.

Metabolites of milk intake: a metabolomic approach in UK twins with findings replicated in two European cohorts.

PURPOSE: Milk provides a significant source of calcium, protein, vitamins and other minerals to Western populations throughout life. Due to its widespread use, the metabolic and health impact of milk consumption warrants further investigation and biomarkers would aid epidemiological studies. METHODS: Milk intake assessed by a validated food frequency questionnaire was analyzed against fasting blood metabolomic profiles from two metabolomic platforms in females from the TwinsUK cohort (n = 3559). The top metabolites were then replicated in two independent populations (EGCUT, n = 1109 and KORA, n = 1593), and the results from all cohorts were meta-analyzed. RESULTS: Four metabolites were significantly associated with milk intake in the TwinsUK cohort after adjustment for multiple testing (P < 8.08 × 10-5) and covariates (BMI, age, batch effects, family relatedness and dietary covariates) and replicated in the independent cohorts. Among the metabolites identified, the carnitine metabolite trimethyl-N-aminovalerate (ß = 0.012, SE = 0.002, P = 2.98 × 10-12) and the nucleotide uridine (ß = 0.004, SE = 0.001, P = 9.86 × 10-6) were the strongest novel predictive biomarkers from the non-targeted platform. Notably, the association between trimethyl-N-aminovalerate and milk intake was significant in a group of MZ twins discordant for milk intake (ß = 0.050, SE = 0.015, P = 7.53 × 10-4) and validated in the urine of 236 UK twins (ß = 0.091, SE = 0.032, P = 0.004). Two metabolites from the targeted platform, hydroxysphingomyelin C14:1 (ß = 0.034, SE = 0.005, P = 9.75 × 10-14) and diacylphosphatidylcholine C28:1 (ß = 0.034, SE = 0.004, P = 4.53 × 10-16), were also replicated. CONCLUSIONS: We identified and replicated in independent populations four novel biomarkers of milk intake: trimethyl-N-aminovalerate, uridine, hydroxysphingomyelin C14:1 and diacylphosphatidylcholine C28:1. Together, these metabolites have potential to objectively examine and refine milk-disease associations.

Faecal metabolite profiling identifies medium-chain fatty acids as discriminating compounds in IBD.

BACKGROUND: Bacteria play a role in the onset and perpetuation of intestinal inflammation in IBD. Compositional alterations may also change the metabolic capacities of the gut bacteria. OBJECTIVE: To examine the metabolic activity of the microbiota of patients with Crohn's disease (CD), UC or pouchitis compared with healthy controls (HC) and determine whether eventual differences might be related to the pathogenesis of the disease. METHODS: Faecal samples were obtained from 40 HC, 83 patients with CD, 68 with UC and 13 with pouchitis. Disease activity was assessed in CD using the Harvey-Bradshaw Index, in UC using the UC Disease Activity Index and in pouchitis using the Pouchitis Disease Activity Index. Metabolite profiles were analysed using gas chromatography-mass spectrometry. RESULTS: The number of metabolites identified in HC (54) was significantly higher than in patients with CD (44, p<0.001), UC (47, p=0.042) and pouchitis (43, p=0.036). Multivariate discriminant analysis predicted HC, CD, UC and pouchitis group membership with high sensitivity and specificity. The levels of medium-chain fatty acids (MCFAs: pentanoate, hexanoate, heptanoate, octanoate and nonanoate), and of some protein fermentation metabolites, were significantly decreased in patients with CD, UC and pouchitis. Hexanoate levels were inversely correlated to disease activity in CD (correlation coefficient=-0.157, p=0.046), whereas a significant positive correlation was found between styrene levels and disease activity in UC (correlation coefficient=0.338, p=0.001). CONCLUSIONS: Faecal metabolic profiling in patients with IBD relative to healthy controls identified MCFAs as important metabolic biomarkers of disease-related changes. TRIAL REGISTRATION NO: NCT 01666717.

Quantitative determination of ß-hydroxymethylbutyrate and leucine in culture media and microdialysates from rat brain by UHPLC-tandem mass spectrometry.

The main objective of the present work was to develop a method to determine ß-hydroxymethylbutyrate (HMB) and leucine (Leu) in culture media and brain microdialysates. An accurate, selective, and cost-effective method, based on the use of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), was developed for the identification and quantification of both compounds. The method consisted of sample dilution, direct injection onto the chromatographic equipment, and quantification with a triple quadrupole mass spectrometer using an electrospray ionization interface in positive mode. The procedure and the UHPLC-MS/MS parameters were accurately optimized to achieve the highest recoveries and to enhance the analytical characteristics of the method. For chromatographic separation, an Acquity UPLC BEH Hilic column using acetonitrile-water gradient with formic acid as additive was employed. The total run time was 4 min. The limits of detection (LODs) obtained ranged from 0.01 to 0.04 µg mL(-1), and the limits of quantification (LOQs) ranged from 0.04 to 0.12 µg mL(-1). Precision (expressed as relative standard deviation) was lower than 15 %, and the determination coefficient (R (2)) was higher than 99.0 % with a residual deviation for each calibration point lower than ±25 %. Mean recoveries were between 85 and 115 %. The method was successfully applied to the analysis of both compounds, HMB and Leu, in samples obtained from an experiment of blood-brain barrier (BBB) passage in vitro and to an experiment of brain microdialysis in rats in vivo after an oral challenge with HMB to detect its appearance in the brain.

Determination of calcium ß­hydroxy-ß-methylbutyrate content in special medical formula food and sports nutrition food by solid-phase extraction and high performance liquid chromatography.

Calcium ß­hydroxy-ß-methylbutyrate (CaHMB) can promote muscle growth, prevent muscle atrophy, and enhance immunity, therefore, it is widely used as a nutritional supplement in special medical formula food and sports nutrition food. Many methods for the detection of CaHMB have been reported, but the pretreatment method for these reported literatures directly involves extraction using hydrochloric acid solution, without any purification steps. A method for accurately determining CaHMB in special medical formula food and sports nutrition food was established for the first time using solid-phase extraction (SPE) purification and high-performance liquid chromatography method (HPLC). The samples were extracted and precipitated protein using methanol-water solution, purified using SPE method and analyzed by HPLC on diode array detector (DAD) mode under external standard method. The method obtained excellent calibration linearity (r2>0.9993) and a satisfactory analysis of the targeted compound, which were evaluated with calibration standards over the range of 0.020-2.00 mg/mL. The limit of quantifications (LOQs), which defined as the lowest spiking level, were set at 0.4 g/100 g (special medical formula food) and 1.0 g/100 g (sports nutrition food). The average recoveries were within 92.9-104% for the analytes, and the relative standard deviations (RSDs) were below 3.93%, measured at low, medium, and high concentrations. Moreover, the positive sample analysis results indicated that CaHMB was detected on 10 real special medical formula food and sports nutrition food products, the contents of which were generally consistent with their labeled values, with measured values ranging from 97.1 % to 119 % of the labeled values. These results suggested that the developed highly sensitive and specific method is highly feasible for monitoring of the target analyte in special medical formula food and sports nutrition food.

Mode of delivery and early nutrition modulate microbial colonization and fermentation products in neonatal piglets.

Colonization of the intestinal microbiota after birth plays an important role in development of the neonatal gastrointestinal and immune systems. Two key environmental factors that influence the colonization pattern are delivery mode and nutrition. In this study, the impact of delivery mode and nutrition on microbial colonization and metabolic activity was investigated in the pig model. Vaginally (VD) or caesarean- (CD) delivered piglets were sow-reared (SR) or fed formula alone (FF) or with 4 g/L prebiotics [1:1 ratio of short-chain fructo-oligosaccharides (scFOS) and polydextrose (PDX); FP]. Intestinal contents were collected on d 7 and 14. SR piglets harbored different microbial populations from FF and FP piglets in ileum and ascending colon (AC). On d 7, FF piglets had a greater abundance of Clostridium XIVa in AC, but lower total bacteria, Clostridium XIVa, and Lactobacillus spp. in ileum and Fecalibacterium prausnitzii in AC compared with FP piglets. On d 14, total bacteria were more abundant in FP than FF piglets. Butyrate, isobutyrate, valerate, and isovalerate concentrations in AC were greater in SR piglets compared with FF or FP piglets. At both sampling days, acetate concentrations in AC were similar between the SR and FF groups, whereas propionate was higher in the SR compared with FF group. Delivery mode also significantly affected microbial populations. Bacterial densities differed in AC for Bacteroides-Prevotella at d 7 and Clostridium XIVa at d 14, being higher in VD piglets. Correspondingly, VD piglets had higher propionate in ileum and propionate and butyrate in AC compared with CD piglets. Our results indicate that both delivery mode and nutrition affect microbial composition and metabolic activity. Supplementation of scFOS/PDX to formula modulates microbial colonization and produces a SCFA pattern closer to that of SR piglets.

Biosynthesis and characterization of a new copolymer, poly(3-hydroxyvalerate-co-5-hydroxydecenoate), from Pseudomonas aeruginosa.

Using a new isolate of Pseudomonas aeruginosa, we obtained 7 g cell dry wt (CDW/l) using 5 % (w/v) glucose. Crude polyhydroxyalkanoates were obtained at 14.6 % of CDW. FTIR and NMR analysis confirmed that this was a new co-polymer: 3-hydroxyvalerate-co-5-hydroxydecenoate. Differential scanning calorimetry analysis showed two different melting temperatures of the copolymer and also indicated the glass transition temperature to be 4 °C. The polydispersity index of the polymer was 1.059.

Multiomic features associated with mucosal healing and inflammation in paediatric Crohn's disease.

BACKGROUND: The gastrointestinal microbiota has an important role in mucosal immune homoeostasis and may contribute to maintaining mucosal healing in Crohn's disease (CD). AIM: To identify changes in the microbiota, metabolome and protease activity associated with mucosal healing in established paediatric CD METHODS: Twenty-five participants aged 3-18 years with CD, disease duration of over 6 months, and maintenance treatment with biological therapy were recruited. They were divided into a low calprotectin group (faecal calprotectin <100 µg/g, "mucosal healing," n = 11), and a high calprotectin group (faecal calprotectin >100 µg/g, "mucosal inflammation," n = 11). 16S gene-based metataxonomics, 1 H-NMR spectroscopy-based metabolic profiling and protease activity assays were performed on stool samples. RESULTS: Relative abundance of Dialister species was six-times greater in the low calprotectin group (q = 0.00999). Alpha and beta diversity, total protease activity and inferred metagenomic profiles did not differ between groups. Pentanoate (valerate) and lysine were principal discriminators in a machine-learning model which differentiated high and low calprotectin samples using NMR spectra (R2 0.87, Q2 0.41). Mean relative concentration of pentanoate was 1.35-times greater in the low calprotectin group (95% CI 1.03-1.68, P = 0.036) and was positively correlated with Dialister. Mean relative concentration of lysine was 1.54-times greater in the high calprotectin group (95% CI 1.05-2.03, P = 0.028). CONCLUSIONS: This multiomic study identified an increase in Dialister species and pentanoate, and a decrease in lysine, in patients with "mucosal healing." It supports further investigation of these as potential novel therapeutic targets in CD.

Sex attractant of sugar beet wireworm: identification and biological activity.

The sex attractant produced by adult females of the sugar beet wire-worm, Limonius californicus (Mannerheim) has been isolated and identified as valeric acid. In the laboratory, male wireworm beetles are repelled by the pure attractant but are drawn with intense sexual excitement to its dilute solutions; in the field, male beetles are lured from a distance of 12 meters. The pheromone occurs in unusually large amounts in the female's body.

Ruminant-like digestion of the langur monkey.

The adaptation of langur monkeys to a laboratory environment has made possible a detailed investigation of their digestive physiology. The diverticular form of the langur stomach permits a bacterial fermentation of the leafy diet, which results in important contributions to the nutrition of these primates. The demonstration of a ruminant-like digestion in langurs extends the known taxonomic distribution of this digestive adaptation.

Triacylglycerols characteristic of porpoise acoustic tissues: molecular structures of diisovaleroylglycerides.

More than two-thirds of the triacylglycerols from the acoustic tissues of the porpoise (Tursiops gilli) consist of 2 moles of isovaleric acid for every 1 mole of long-chain acids. Cranial blubber, which has no distinct acoustic function, does not contain these unusual glycerides. The presence of large amounts of diisovaleroylisopentadecanoylglycerol suggests that this structure may be particularly important in sound transmission through lipid-protein matrices.

High-titer production of monomeric hydroxyvalerates from levulinic acid in Pseudomonas putida.

Hydroxyacids represent an important class of compounds that see application in the production of polyesters, biodegradable plastics and antibiotics, and that serve as useful chiral synthetic building blocks for other fine chemicals and pharmaceuticals. An economical, high-titer method for the production of 4-hydroxyvalerate (4HV) and 3-hydroxyvalerate (3HV) from the inexpensive and renewable carbon source levulinic acid was developed. These hydroxyvalerates were produced by periodically feeding levulinate to Pseudomonas putida KT2440 expressing a recombinant thioesterase II (tesB) gene from Escherichia coli K12. The titer of 4HV in shake flask culture reached 13.9+/-1.2 g L(-1) from P. putida tesB(+) cultured at 32 degrees C in LB medium periodically supplemented with glucose and levulinate. The highest 3HV titer obtained was 5.3+/-0.1 g L(-1) in M9 minimal medium supplemented with glucose and levulinate.

The effect of propionic to acetic acid ratio on anaerobic-aerobic (low dissolved oxygen) biological phosphorus and nitrogen removal.

In this paper, three lab-scale sequencing batch reactors (SBR-A, B, and C) operated with anaerobic/aerobic (low dissolved oxygen, 0.15-0.45 mg L(-1)) configuration were long-term cultured, respectively with single acetic acid and propionic/acetic acid of 1/1 and 2/1 (carbon molar ratio), and the comparisons of anaerobic and aerobic transformations of phosphorus and nitrogen among them were made. With the increase of propionic/acetic acid, lower anaerobic phosphorus release and higher phosphorus release to short-chain fatty acids uptake ratio were observed, and less anaerobic and aerobic transformations of glycogen and poly-3-hydroxybutyrate as well as total polyhydroxyalkanoates occurred, but the transformations of poly-3-hydroxyvalerate and poly-3-hydroxy-2-methyvalerate increased. The phosphorus removal efficiency was respectively 81, 94 and 97% in SBR-A, B and C. Almost all ammonium was removed and no significant nitrite was accumulated at different propionic/acetic acid ratios. However, the nitrate accumulation and total nitrogen removal were observed to be affected by propionic/acetic acid ratio. The total nitrogen removal efficiency was 61, 68 and 82%, and the aerobic end nitrate concentration was 8.05, 6.40 and 3.54 mg L(-1) in three SBRs, respectively. All the above studies indicated that the sole acetic acid caused more nitrate accumulation than propionic and acetic acids mixture, and a pertinent increase of wastewater propionic/acetic acid ratio was of benefit to both nitrogen and phosphorus removal in an anaerobic/aerobic (low dissolved oxygen) biological wastewater treatment process.

Proton HR-MAS NMR spectroscopic characterization of metabolites in various human organ tissues: pancreas, brain and liver from trauma cases.

High-Resolution Magic Angle Spinning (HR-MAS) NMR has been employed to characterize various metabolites of human pancreas, liver and brain tissues from trauma cases. The potential usefulness of NMR in identifying the metabolites in human tissues has been explored using a combination of one- and two-dimensional experiments. The complete resonance assignments of pancreas tissue have been carried out for the first time. Two new metabolites, alpha-hydroxyisovalarate and alpha-hydroxybutarate were identified in all the tissue specimens. The metabolites information of these human tissues can further be utilized in correlating several diseases associated with pathological manifestations as well in distinguishing traumatic tissues along with control tissues of pancreas, liver and brain.

Solid phase extraction, multidimensional gas chromatography mass spectrometry determination of four novel aroma powerful ethyl esters. Assessment of their occurrence and importance in wine and other alcoholic beverages.

A method for the quantitative determination of four powerful aromatic ethyl esters recently identified in some wines has been developed, validated and applied to the determination of these compounds in different samples of wine, whisky and brandy. Ethyl 2-, 3-, and 4-methylpentanoate and ethyl cyclohexanoate are extracted from 100ml of sample by solid phase extraction (SPE) on a 200mg LiChrolut EN bed. Major compounds are eliminated by rinsing with a water-methanol (50:50) solution containing 1% sodium bicarbonate, and analytes are eluted with 1.5ml of dichloromethane. Fifty microlitres of this extract are then injected in a multidimensional gas chromatography-mass spectromety (GC-GC-MS) system. Recoveries in the SPE are quantitative. Method repeatability is satisfactory (5-12% for a 5-10ngl(-1) level, and less than 7% for 25-50ngl(-1) level), the method linearity holds along the whole range of occurrence of analytes (2-2700ngl(-1)), and the signal is independent on the matrix. Method detection limits are below 1ngl(-1) in all cases. Results suggest that these compounds are formed by the slow esterification with ethanol of the corresponding acids formed by different microorganisms. The levels of these compounds are above the corresponding thresholds in most samples of aged wines or distillates, but are particularly high in some sweet wines, whiskeys and brandies where they may constitute the most important contributors to the sweet-fruity notes reaching concentrations up to 85-350 times higher than the corresponding odor thresholds.

RIFM fragrance ingredient safety assessment, isobornyl isovalerate, CAS registry number 7779-73-9.

This material was evaluated for genotoxicity, repeated dose toxicity, developmental toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization potential, as well as, environmental safety. Data from the suitable read across analog isobornyl acetate (CAS # 125-12-2) show that this material is not genotoxic, provided a MOE > 100 for the repeated dose, developmental and reproductive endpoints, and does not have skin sensitization potential. The local respiratory toxicity endpoint was completed using the TTC (threshold of Toxicological Concern) for a Cramer Class II material (0.47 mg/day). The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra. The environmental endpoint was completed as described in the RIFM Framework.

Identification of three novel compounds in wine by means of a laboratory-constructed multidimensional gas chromatographic system.

A laboratory-made off-line multidimensional GC-GC system has been optimized and applied to the isolation and identification of three novel aroma compounds in wine. The system comprises two independent chromatographs equipped with four-port manually actuated valves and fast capillary connectors that make it possible to quickly transfer a capillary trapping loop from the first to the second system. The first system includes a split/splitless injector, a thick phase semi-capillary column, the valve and both a flame ionization detection (FID) system and a sniffing port. The trapping loop lies outside the chromatograph and it is immersed in a Dewar with liquid nitrogen. The second system includes a programmable temperature vaporization (PTV) injector, the valve, the analytical column and two parallel detectors (a MS system and a sniffing port). The injection of large sample volumes (90 microl) in the first GC system and the general operation of the system have been optimized. The extract was obtained by purging the wine at 37 degrees C with a stream of nitrogen and trapping the volatiles in a bed containing 1g LiChrolut EN. Fatty acids and alcohols were removed by washing the resins with a water/methanol solution containing 1% NaHCO3, and volatiles were recovered with dichloromethane. The GC-MS analysis of the cuts obtained in the first GC system made it possible to identify by first time in wine the strong smelling compounds ethyl 2-, 3- and 4-methylpentanoate.

Applications of ion mobility spectrometry (IMS) to the analysis of gamma-hydroxybutyrate and gamma-hydroxyvalerate in toxicological matrices.

The predator drug gamma-hydroxybutyrate (GHB) and its lactone form gamma-butyrolactone (GBL) continue to present significant analytical challenges to forensic toxicologists and chemists. The five-carbon analogue (gamma hydroxyvalerate GHV) and the corresponding lactone GVL) are emerging as substitutes for GHB, adding further complications. Ion mobility spectrometry (IMS) was investigated as a method of screening urine and breath for the presence of these drugs and their degradation products. Sample was introduced into the instrument via a programmable split/splitless injection port with thermal desorption. The injection method in effect replaces problematic solvent extraction methods with a physical extraction, an efficient method in the present case considering the hydrophilic nature of GHB. No chromatography was employed and results were obtained within a few seconds. The negative ion mode showed the greatest sensitivity with detection limits in the low parts-per-million range for GHB and GHV. Because GHB is often delivered in alcoholic beverages, ethanol and acetaldehyde, along with potential interfering compounds methanol, isopropanol, and acetone, were also analyzed. None were found to interfere. The thermally induced ring opening prevented differentiation of GHB and GBL using direct injection/thermal desorption protocol, but IMS does show promise as a rapid, simple, and affordable screening technique for GHB and related compounds.

The effect of glyoxalase I on the metabolism of 4,5-dioxovaleric acid.

Biosynthesis of 5-aminolevulinic acid in mammalian cells is catalyzed by aminolevulinic acid synthase in a condensation reaction utilizing glycine and succinyl X coenzyme A. An alternate pathway in mammalian cells may involve the biosynthesis of aminolevulinic acid via a transamination reaction in which L-alanine is the amino donor and 4,5-dioxovaleric acid is the acceptor. This transamination reaction, or one very similar, is employed by plants for the biosynthesis of aminolevulinic acid which is ultimately converted to chlorophyll. The effect of glyoxalase I on the diversion of dioxovaleric acid to other products was tested using both purified glyoxalase I and crude tissue homogenates. Glyoxalase I is a metalloenzyme and glutathione is a co-substrate. Purified glyoxalase I reduced the amount of aminolevulinic acid formed in the presence of dioxovaleric acid, L-alanine, glutathione, and purified L-alanine: 4,5-dioxovaleric acid aminotransferase (dioxovalerate transaminase). The conversion of dioxovaleric acid to aminolevulinic acid was inhibited by the addition of glutathione when a dialyzed bovine liver homogenate served as the source of both glyoxalase I and dioxovalerate transaminase. Removal of metals from bovine liver homogenates produced an 85% decrease in glyoxalase I activity. These 'metal-free' homogenates still affected the conversion of dioxovaleric acid to aminolevulinic acid after preincubation with MgSO4. The effect of glyoxalase I on the metabolism of dioxovaleric acid was also studied using a fluorometric enzyme assay for the quantification of dioxovaleric acid via a coupled enzyme reaction converting it to uroporphyrin. Homogenates of both liver and barley diminished the amount of dioxovaleric acid detected by the coupled assay, but this effect could be prevented by dialysis of the homogenates. Addition of glutathione to dialyzed homogenates markedly reduced the amount of uroporphyrin generated from dioxovaleric acid. Metal-free homogenates supplemented with glutathione reduced the conversion of dioxovaleric acid to uroporphyrin in the coupled assay, but preincubation with MgSO4 greatly augmented this effect. These studies point out the difficulty in evaluating dioxovaleric acid as a heme precursor using whole cell homogenates.

Analysis of GHB and 4-methyl-GHB in postmortem matrices after long-term storage.

Postmortem heart blood, peripheral blood, vitreous humor, urine, and bile specimens from 26 autopsy cases were analyzed for the presence of gamma-hydroxybutyric acid (GHB) and gamma-methyl gamma-hydroxybutyric acid (4-Me-GHB) after long-term freezer storage. Cases were selected for which exogenous GHB, gamma-butyrolactone (GBL), gamma valerolactone (GVL), or 1,4-butanediol use was not suspected. One documented positive GHB case subjected to the same storage conditions was also evaluated for comparison. Specimens did not contain any preservatives or additives except heart blood, which contained sodium fluoride (2% w/v). The results of the analysis for GHB in vitreous humor (n = 26) demonstrated, with one exception, concentrations below the limit of detection for the method (5 mg/L). In the exception case, the value was determined to be 7 mg/L. Documented cases of GHB positive fatalities showed vitreous humor concentrations (n = 6) that exceeded this range by a factor of 12 or more. There was no apparent relationship between storage times and GHB concentrations. The data developed in this study demonstrate a postmortem endogenous range for GHB in vitreous humor that is less than or equal to 7 mg/L. Studies of the stored GHB-positive case demonstrated no significant change in concentration over the time period studied. None of the specimens analyzed in this study contained detectable amounts of 4-Me-GHB. This would support the contention that when 4-Me-GHB is detected, it is most likely due to the exogenous consumption of GVL.