Patent Reports

We synthesized a hydroquinone glucoside (HG) as a potential skin-whitening agent using Leuconostoc mesenteroides (B-1299CB BF563) dextransucrase with hydroquinone (HQ) as an acceptor and sucrose as a donor. The product was purified using butanol partitioning and silica gel column chromatography. The structure of the purified HG was determined by nuclear magnetic resonance and the ionic product was observed at m/z 295 (C12, H16, O7 Na)⁺. HG was identified as 4-hydroxyphenyl-α-d-glucopyranoside. The optimum condition of HG synthesis, determined using a response surface methodology, was 450 mM HQ, 215 mM sucrose, and 0.55 U/mL dextransucrase; the final HG produced was 544 mg/L. The IC₅₀ of diphenylpicryl-hydrazyl scavenging activity was 3.85 mM indicating a higher antioxidant activity compared to β-arbutin (IC₅₀ = 6.04 mM). HG-mediated inhibition of lipid peroxidation was 3.51% that of HQ (100%) and much higher than that of β-arbutin (0.81% of HQ). In addition the IC₅₀ value of nitrite-scavenging activity was 14.76 mM showing a superior scavenging activity to that of β-arbutin (IC₅₀ = 27.09 mM).     

Pilot-scale production of 1,3-propanediol using Klebsiella pneumoniae

The conversion of glycerol to 1,3-propanediol (PDO) using Klebsiella pneumoniae M5al under anaerobic condition was scaled up from scale 5 to 5000 l in series. A simple strategy for scale-up was to transfer the optimized conditions of a lab scale bioreactor to pilot-scale fermentation. Multistage inocula were developed and their fermentation abilities were assessed in a small-scale fermenter. The experimental results showed that inoculum development in the early steps of a scale-up process could influence the outcomes of a large scale fermentation. Through three-stage liquid inoculum development and a pulse addition of (NH₄)₂SO₄ and yeast extract at 30 h of fermentation, the best results in a 5000 l fermentation were achieved leading to 58.8 g l⁻¹ 1,3-propanediol with a yield of 0.53 mol mol⁻¹ glycerol and productivity of 0.92 g l⁻¹ h⁻¹. This is the first report on pilot-scale 1,3-propanediol production using K. pneumoniae.     

Direct and residual effects of nitrogen fertilization, foliar application of potassium and plant growth retardant on Egyptian cotton growth, seed yield, seed viability and seedling vigor

In order to obtain high productivity for a cotton crop, one of the major requirements is to establish an adequate plant population. The use of good-quality seed may ultimately be the best approach to attain this goal problem. The objective of this research was to study the effect of N-fertilization (at rates of 95.2 and 142.8 kg of N ha^?1), foliar application of K (at rates of 0, 0.38, 0.77, 1.15 kg of K₂O ha^?1, applied twice during square initiation and boll development stages) and the plant growth retardant (PGR), mepiquat chloride (applied twice, 75 days after planting at 0.0 [control] and 0.048 kg a.i. ha^?1, and 90 days after planting at 0.0 [control] and 0.024 kg a.i. ha^?1), on seed yield, viability, and seedling vigor of Egyptian cotton (Gossypium barbadense cv. Giza 86). A field experiment was conducted at the Agricultural Research Center, Giza, Egypt in two growing seasons. Growth, mineral uptake, seed yield per plant and per ha, seed weight, seed viability, seedling vigor and cool germination test performance were all found to increase significantly due to the addition of the high N-rate, the foliar application of three potassium concentrations, and the PGR mepiquat chloride. The N and K rates as well as application of mepiquat chloride had no significant effect on the germination rate index in both seasons. Under the conditions of this study, applying N at a rate of 142.8 kg ha^?1 combined with spraying cotton plants with K₂O at 1.15 kg ha^?1 and with mepiquat chloride at 0.048 + 0.024 kg ha^?1 were found to improve seed yield as well as seed viability and seedling vigor in the next season.     

Partial purification and characterization of a copper-induced anionic peroxidase of sunflower roots

Treatment of 14-day-old sunflower seedlings with a toxic amount of copper (50 μM of CuSO₄) during 5 days caused significant increase in peroxidase activity in roots. Qualitative analysis of soluble proteins using native anionic PAGE followed by detection of peroxidase activity with guaïacol as electron donor in the presence of H₂O₂ revealed five stimulated peroxidases, named A1, A2, A3, A4, and A5. These peroxidases had differential behavior during the period of treatment. A1, A2, A3 and A4 were stimulated in the first period of stress, but rapidly suppressed at 72 h. A5 showed a progressive stimulation which was even increased at 120 h. A1 was partially purified, identified using liquid chromatography coupled to mass spectrometry (LC-MS/MS), and characterized. Effects of pH and temperature on its activity were determined with guaïacol as electron donor. Optima were obtained at pH 8 and at 40 °C. Analysis of substrate specificity showed that A1 was active on coniferyl alcohol but not on IAA. Enzymatic activity was inhibited by a high concentration of H₂O₂.     

Effects of dwarf bamboo (Fargesia denudata) density on biomass, carbon and nutrient distribution pattern

Dense dwarf bamboo population is a structurally and functionally important component in many subalpine forest systems. To characterize the effects of stem density on biomass, carbon and majority nutrients (N, P, K, Ca and Mg) distribution pattern, three dwarf bamboo (Fargesia denudata) populations with different stem densities (Dh with 220 ± 11 stems m^?2, Dm with 140 ± 7 stems m^?2, and Dl with 80 ± 4 stems m^?2, respectively) were selected beneath a bamboo-fir (Picea purpurea) forest in Wanglang National Nature Reserve, Sichuan, China. Leaf, branch, rhizome, root and total biomass of dwarf bamboo increased with the increase of stem density, while carbon and nutrient concentrations in bamboo components decreased. Percentages of below-ground biomass and element stocks to total biomass and stocks decreased with the increase of stem density, whereas above-ground biomass and element stocks exhibited the opposite tendency. Moreover, more above-ground biomass and elements were allocated to higher part in the higher density population. In addition, percentages of culm biomass, above-ground biomass and element stocks below 100 cm culm height (H₁₀₀) increased with the increase of stem density, while percentages of branch and leaf biomass below H₁₀₀ decreased. Pearson’s correlation analyses revealed that root biomass, above-ground biomass, below-ground biomass and total biomass significantly correlated to leaf biomass in H_100?200 and total leaf biomass within high density population, while they significantly correlated to leaf biomass in H_50?150 within low density population. The results suggested that dwarf bamboo performed an efficient adaptive strategy to favor limited resources by altering biomass, carbon and nutrients distribution pattern in the dense population.     

Advantage of menadione-catalyzed chemiluminescent assay for the determination of viable mammalian cell number

A chemiluminescent assay composed of TCPO [bis(2,4,6-trichlorophenyl)oxalate] and harmless rhodamine B is proposed to be superior in the determination of menadione-catalyzed hydrogen peroxide (H₂O₂) production by viable mammalian cells to that composed of TCPO and harmful pyrene [Anal. Biochem. 207 (1992) 255–260]. In tests, the proposed assay showed that the measurable concentration of H₂O₂ and the viable cell number ranged from 10^?9 to 10^?3 M and from 2 × 10² to 2 × 10⁶ cells/100 μl/well in the presence of 10% bovine serum, respectively. The measuring time was approximately 10 min. On the other hand, the measurable cell numbers by the colorimetric WST-1 and MTT assays requiring several hours ranged only from 10³ to 10⁴ cells/100 μl/well and from 10⁴ to 10⁵ cells/100 μl/well, respectively. The cytotoxicity of sodium dodecyl sulfate was also observed at intervals of 1 min by the proposed assay, but not by the above colorimetric assays.     

Scaling-up of a B-phycoerythrin production and purification bioprocess involving aqueous two-phase systems: Practical experiences

One of the most attractive segments in food and cosmetic industries is that of natural pigments. Since some synthetic pigments have been reported to be hazardous for humans, natural pigments obtained through biotechnological processes represent an attractive alternative. Our research group has previously worked on the development of an aqueous two-phase system (ATPS)-based prototype process for the recovery of B-phycoerythrin (BPE), a natural high-value pigment obtained from Porphyridium cruentum. Detailed studies describing the scaling up of ATPS processes from bench scale to pilot plant facilities are not common. In this paper experiences derived from the scale-up of a previously developed process for production and recovery of highly purified (purity defined as the absorbance ratio A₅₄₅/A₂₈₀ > 4) BPE are described, where a scale-up factor of 850× was implemented. Characterization of cell disruption with a pilot-scale bead mill allowed efficient BPE release at 2900 rpm, 10% (w/v) sample load, 60% (v/v) bead load and 0.5 mm glass beads and 22 min of residence time with a yield of 1.35 mg BPE/g of wet biomass. BPE was recovered and purified using a strategy comprising isoelectric precipitation, aqueous two-phase fractionation and ultrafiltration. A 54% global BPE recovery yield, with final purity of 4.1, was achieved under optimal process conditions. Considering total costs for raw materials and energy expenditures for one batch, it was determined that the production cost of BPE was of $1.17 USD/mg, which is underneath the commercial price of a BPE standard (>$30 USD/mg).     

Novel 1H-pyrrolo[2,3-c]pyridines as acid pump antagonists (APAs)

A series of 1H-pyrrolo[2,3-c]pyridines as acid pump antagonists (APAs) was synthesized and the inhibitory activities against H⁺/K⁺ ATPase isolated from hog gastric mucosa were determined. After elaborating on substituents at N1, C5, and C7 position of 1H-pyrrolo[2,3-c]pyridine scaffold, we have observed that compounds 14f and 14g are potent APAs with H⁺/K⁺ ATPase IC₅₀ = 28 and 29 nM, respectively.     

Magnetic nanocomposite of anti-human IgG/COOH-multiwalled carbon nanotubes/Fe₃O₄ as a platform for electrochemical immunoassay

An electrochemical immunosensing method was developed based on a magnetic nanocomposite. The multiwalled carbon nanotubes (MWCNTs) were treated with nitric acid to produce carboxyl groups at the open ends. Then, Fe₃O₄ nanoparticles were deposited on COOH–MWCNTs by chemical coprecipitation of Fe²⁺ and Fe³⁺ salts in an alkaline solution. Goat anti-human IgG (anti-hIgG) was covalently attached to magnetic nanocomposite through amide bond formation between the carboxylic groups of MWCNTs and the amine groups of anti-hIgG. The prepared bio-nanocomposite was used for electrochemical sensing of human tetanus IgG (hIgG) as a model antigen. The anti-hIgG magnetic nanocomposite was fixed on the surface of a gold plate electrode using a permanent magnet. The hIgG was detected using horseradish peroxidase (HRP)-conjugated anti-hIgG in a sandwich model. Electrochemical detection of hIgG was carried out in the presence of H₂O₂ and KI as substrates of HRP. Using this method, hIgG was detected in a concentration range from 30 to 1000 ng ml^?1 with a correlation coefficient of 0.998 and a detection limit of 25 ng ml^?1 (signal/noise = 3). The designed immunosensor was stable for 1 month.     

Analytical measurement of discrete hydrogen sulfide pools in biological specimens

Hydrogen sulfide (H₂S) is a ubiquitous gaseous signaling molecule that plays a vital role in numerous cellular functions and has become the focus of many research endeavors, including pharmacotherapeutic manipulation. Among the challenges facing the field is the accurate measurement of biologically active H₂S. We have recently reported that the typically used methylene blue method and its associated results are invalid and do not measure bona fide H₂S. The complexity of analytical H₂S measurement reflects the fact that hydrogen sulfide is a volatile gas and exists in the body in various forms, including a free form, an acid-labile pool, and bound as sulfane sulfur. Here we describe a new protocol to discretely measure specific H₂S pools using the monobromobimane method coupled with RP-HPLC. This new protocol involves selective liberation, trapping, and derivatization of H₂S. Acid-labile H₂S is released by incubating the sample in an acidic solution (pH 2.6) of 100 mM phosphate buffer with 0.1 mM diethylenetriaminepentaacetic acid (DTPA), in an enclosed system to contain volatilized H₂S. Volatilized H₂S is then trapped in 100 mM Tris–HCl (pH 9.5, 0.1 mM DTPA) and then reacted with excess monobromobimane. In a separate aliquot, the contribution of the bound sulfane sulfur pool was measured by incubating the sample with 1 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride), a reducing agent, to reduce disulfide bonds, in 100 mM phosphate buffer (pH 2.6, 0.1 mM DTPA), and H₂S measurement was performed in a manner analogous to the one described above. The acid-labile pool was determined by subtracting the free hydrogen sulfide value from the value obtained by the acid-liberation protocol. The bound sulfane sulfur pool was determined by subtracting the H₂S measurement from the acid-liberation protocol alone compared to that of TCEP plus acidic conditions. In summary, our new method allows very sensitive and accurate measurement of the three primary biological pools of H₂S, including free, acid-labile, and bound sulfane sulfur, in various biological specimens.     

Energy metabolism and thermoregulation in pygmy lorises (Nycticebus pygmaeus) from Yunnan Daweishan Nature Reserve

The pygmy loris (Nycticebus pygmaeus) is a small prosimian living in Vietnam, Laos, eastern Cambodia and the south part of China. In China it is only found in Pingbian, Hekou, Jinping, Luchun of Yunnan. As N. pygmaeus is seriously threatened by hunting, trade and habitat destruction, it is listed in Appendix II of CITES, and in 2006 the IUCN classified it as “vulnerable”. In order to understand the characteristics of energy metabolism and thermoregulation of N. pygmaeus, the resting metabolic rate (RMR) and body temperature (T_b) at different ambient temperature (T_a) of pygmy lorises, as well as body mass, energy intake, digestable energy intake, digestability and the thermal conductance were measured in captivity. The results obtained mainly are as follows: (1) Pygmy loris feed dry food averaged 12.90 ± 1.02 g/d. They could gain 214.87 ± 16.65 kJ/d from food intake, and earned 200.15 ± 16.36 kJ digestable energy intake per day with 90.13 ± 1.34% of the digestability. (2) The T_b at room temperatures was a little low (35.23 ± 0.16 °C) and varied with T_a from 25 °C to 35 °C. There was a positive relationship between T_b and T_a, which was described as: T_b = 27.22 + 0.34T_a (r = 0.880). (3) The resting metabolic rate (RMR) of the pygmy loris was 0.3844 ± 0.0162 mlO₂/g/h, which was 51.91 ± 1.90% of the previous predicted rate by Kleiber (1961) [21]. (4) The average thermal conductance of the pygmy loris (N. pygmaeus) was 0.0449 ± 0.0031 mlO₂/g/h/°C. These characteristics of energy metabolism and thermoregulation of N. pygmaeus in Yunnan Daweishan Nature Reserve might be considered as the adaptive characteristics to their environment in tropical semi-evergreen forests and secondary forests.     

Synthesis and CYP24A1 inhibitory activity of N-(2-(1H-imidazol-1-yl)-2-phenylethyl)arylamides

A series of N-(2-(1H-imidazol-1-yl)-2-phenylethyl)arylamides were prepared, using an efficient three- to five-step synthesis, and evaluated for their inhibitory activity against human cytochrome P450C24A1 (CYP24A1) hydroxylase. Inhibition ranged from IC₅₀ 0.3–72 μM compared with the standard ketoconazole IC₅₀ 0.52 μM, with the styryl derivative (11c) displaying enhanced activity (IC₅₀ = 0.3 μM) compared with the standard, providing a useful preliminary lead for drug development.     

Synthesis,biological evaluation,and docking studies of novel heterocyclic diaryl compounds as selective COX-2 inhibitors

Three novel series of diaryl heterocyclic derivatives bearing the 2-oxo-5H-furan, 2-oxo-3H-1,3-oxazole, and 1H-pyrazole moieties as the central heterocyclic ring were synthesized and their in vitro inhibitory activities on COX-1 and COX-2 isoforms were evaluated using a purified enzyme assay. The 2-oxo-5H-furan derivative 6b was identified as potent COX inhibitor with selectivity toward COX-1 (COX-1 IC₅₀ = 0.061 μM and COX-2 IC₅₀ = 0.325 μM; selectivity index (SI) = 0.19). Among the 1H-pyrazole derivatives, 11b was found to be a potent COX-2 inhibitor, about 38 times more potent than Rofecoxib (COX-2 IC₅₀ = 0.011 μM and 0.398 μM, respectively), but showed no selectivity for COX-2 isoform. Compound 11c demonstrated strong and selective COX-2 inhibitory activity (COX-1 IC₅₀ = 1 μM, COX-2 IC₅₀ = 0.011 μM; SI = ～92). Molecular docking studies of compounds 6b and 11b–d into the binding sites of COX-1 and COX-2 allowed to shed light on the binding mode of these novel COX inhibitors.     

Purification and crystallization of the ABC-type transport substrate-binding protein OppA from Thermoanaerobacter tengcongensis

Di- and oligopeptide- binding protein OppAs play important roles in solute and nutrient uptake, sporulation, biofilm formation, cell wall muropeptides recycling, peptide-dependent quorum-sensing responses, adherence to host cells, and a variety of other biological processes. Soluble OppA from Thermoanaerobacter tengcongensis was expressed in Escherichia coli. The protein was found to be >95% pure with SDS–PAGE after a series of purification steps and the purity was further verified by mass spectrometry. The protein was crystallized using the sitting-drop vapour-diffusion method with PEG 400 as the precipitant. Crystal diffraction extended to 2.25 Å. The crystal belonged to space group C222₁, with unit-cell parameters of a = 69.395, b = 199.572, c = 131.673 Å, and α = β = γ = 90°.     

Effects of shell morphological traits on the weight traits of Manila clam (Ruditapes philippinarum)

The shell length, height, and width, live body weight, and edible tissue weight of Manila clam of 1, 2, and 3 years of age were measured, and their correlation coefficients were calculated. The shell morphological traits were used as independent variables, and live body weight or edible tissue weigh used as a dependent variable for calculating the path coefficients, correlation index and determination coefficients. The results showed that the correlation coefficients between each shell morphological trait and the live body weight or edible tissue weight were all highly significant (P < 0. 01). The shell height at 1-year old clams was highly correlated with the live body weight and edible tissue weight. The shell width of 2- to 3-year-old clams was strongly associated with the live body weight, while the shell length was closely linked to the edible tissue weight. The results of coefficients of determination for the morphological traits against weight traits agreed well with the results of path analysis. The correlation indices for all morphological traits against weight traits were approximately the same as determination coefficients regardless of clam age. The correlation indices (R²) of morphological traits against the live body weight of clams of all ages and edible tissue weight of 1-year-old clams were larger than 0.85, but R² of morphological traits against the edible tissue weight of 2- and 3-year-old clams was smaller than 0.85, indicating that some other factors might be associated with the edible tissue weight of 2- and 3-year-old clams. Multiple regression equations were obtained to estimate shell length X₁ (cm), shell height X₂ (cm), shell width X₃ (cm) against live body weight Y (g), edible tissue weight Z (g): for 1-year-old clams: Y = ?4.317 + 0.18X₁ + 0.147X₂, (X₁ < 0.01, X₂ < 0.01), Z = ?1.011 + 0.095X₂, (X₂ < 0.01); for 2-year-old clams: Y = ?15.119 + 0.249X₁ + 0.176X₂ + 0.688X₃, (X₁ < 0.01, X₃ < 0.01), Z = ?4.248 + 0.198X₁, (X₁ < 0.05, X₃ < 0.01); and for 3-year-old clams: Y = ?25.013 + 0.415X₁ + 1.184X₃, (X₁ < 0.01, X₃ < 0.01), Z = ?7.082 + 0.119X₁ + 0.332X₃, (X₁ < 0.05, X₃ < 0.01).     

Synthesis and evaluation of tricyclic derivatives containing a non-aromatic amide as inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1)

Highly potent poly(ADP-ribose)polymerase-1 (PARP-1) inhibitors, including 9-hydroxy-1,2-dihydro-4H-thiopyrano[3,4-c]quinolin-5(6H)-one derivatives with a non-aromatic A-ring, were synthesized. Among the derivatives, 12a showed low nanomolar enzyme and cellular activity (IC₅₀ = 42 nM, ED₅₀ = 220 nM) with good water solubility. Further, 12a exhibited microsomal stability in vitro and brain permeability in vivo.     

Viral safety of C1-inhibitor NF

We studied the efficacy of virus reduction by three process steps (polyethylene glycol 4000 (PEG) precipitation, pasteurization, and 15 nm virus filtration) in the manufacturing of C1-inhibitor NF. The potential prion removing capacity in this process was estimated based on data from the literature. Virus studies were performed using hepatitis A virus (HAV) and human immunodeficiency virus (HIV) as relevant viruses and bovine viral diarrhea virus (BVDV), canine parvovirus (CPV) and pseudorabies virus (PRV) as model viruses, respectively. In the PEG precipitation step, an average reduction in infectious titer of 4.5 log₁₀ was obtained for all five viruses tested. Pasteurization resulted in reduction of infectious virus of >6 log₁₀ for BVDV, HIV, and PRV; for HAV the reduction factor was limited to 2.8 log₁₀ and for CPV it was zero. Virus filtration (15 nm) reduced the infectious titer of all viruses by more than 4.5 log₁₀. The overall virus reducing capacity was >16 log₁₀ for the LE viruses. For the NLE viruses CPV and HAV, the overall virus reducing capacities were >8.7 and >10.5 log₁₀, respectively. Based on literature and theoretical assumptions, the prion reducing capacity of the C1-inhibitor NF process was estimated to be >9 log₁₀.     

The kinetics and regulation of phosphofructokinase from Teladorsagia circumcincta

Phosphofructokinase (PFK-1) activity was examined in L₃ and adult Teladorsagia circumcincta, both of which exhibit oxygen consumption. Although activities were higher in the adult stage, the kinetic properties of the enzyme were similar in both life cycle stages. T. circumcincta PFK-1 was subject to allosteric inhibition by high ATP concentration, which increased both the Hill coefficient (from 1.4 ± 0.2 to 1.7 ± 0.2 in L₃s and 2.0 ± 0.3 to 2.4 ± 0.4 in adults) and the K_½ for fructose 6 phosphate (from 0.35 ± 0.02 to 0.75 ± 0.05 mM in L₃s and 0.40 ± 0.03 to 0.65 ± 0.05 mM in adults). The inhibitory effects of high ATP concentration could be reversed by fructose 2,6 bisphosphate and AMP, but glucose 1,6 bisphosphate had no effect on activity. Similarly, phosphoenolpyruvate had no effect on activity, while citrate, isocitrate and malate exerted mild inhibitory effects, but only at concentrations exceeding 2 mM. The observed kinetic properties for T. circumcincta PFK-1 were very similar to those reported for purified Ascaris suum PFK-1, though slight differences in sensitivity to ATP concentration suggests there may be subtle variations at the active site. These results are consistent with the conservation of properties of PFK-1 amongst nematode species, despite between species variation in the ability to utilise oxygen.     

Pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate by human serum albumin: pH-dependence of rates of individual steps

Human serum albumin (HSA) displays esterase activity reflecting multiple irreversible chemical modifications rather than turnover. Here, kinetics of the pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate (NphOAc) are reported. Under conditions where [HSA] ? 5×[NphOAc] and [NphOAc] ? 5×[HSA], the HSA-catalyzed hydrolysis of NphOAc is a first-order process for more than 95% of its course. From the dependence of the apparent rate constants k_app and k_obs on [HSA] and [NphOAc], respectively, values of K_s, k₊₂, and k₊₂/K_s were determined. Values of K_s, k₊₂, and k₊₂/K_s obtained at [HSA] ? 5×[NphOAc] and [NphOAc] ? 5×[HSA] are in good agreement, the deacylation step being rate limiting in catalysis. The pH-dependence of k₊₂/K_s, k₊₂, and K_s reflects the acidic pK_a shift of the Tyr411 catalytic residue from 9.0 ± 0.1 in the substrate-free HSA to 8.1 ± 0.1 in the HSA:NphOAc complex. Accordingly, diazepam inhibits competitively the HSA-catalyzed hydrolysis of NphOAc by binding to Tyr411.     

High-performance liquid chromatography-tandem mass spectrometry assay of fatty acid amide hydrolase (FAAH) in blood: FAAH inhibition as clinical biomarker

Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for the degradation of the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA). FAAH inhibitors may be useful in treating many disorders involving inflammation and pain. Although brain FAAH may be the relevant target for inhibition, rat studies show a correlation between blood and brain FAAH inhibition, allowing blood FAAH activity to be used as a target biomarker. Building on experience with a rat leukocyte FAAH activity assay using [³H]AEA, we have developed a human leukocyte assay using stably labeled [²H₄]AEA as substrate. The deuterium-labeled ethanolamine reaction product ([²H₄]EA) was analyzed by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) in the positive electrospray ionization (ESI) mode. The response for [²H₄]EA was linear from 10 nM to 10 μM, and the analysis time was less than 6 min/sample. Results using the [²H₄]AEA and HPLC–MS/MS method agreed well with those obtained using the [³H]AEA radiometric assay. In addition to using a nonradioactive substrate, the HPLC–MS/MS method had increased sensitivity with lower background. Importantly, the assay preserved partial FAAH inhibition resulting from ex vivo treatment with a time-dependent irreversible inhibitor, suggesting its utility with clinical samples. The assay has been used to profile the successful inhibition of FAAH in recent clinical trials.