ABTS assay of phenol oxidase activity in soil

Phenol oxidases (PO) are involved in degradation of many recalcitrant aromatic compounds and may be sensitive to some pollutants. Hence, their activities may be a useful indicator for evaluating soil quality and health. To this end, the aim of this study was to develop a simple method to assay PO activity directly in bulk samples by spectrophotometric test using 2,2′-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) as the substrate. Three Mediterranean soils were used as models. For each soil, we studied the kinetic parameters and the effects of certain factors (i.e. amount of soil, pH, temperature, incubation time and substrate concentration) in order to determine the optimum conditions for the ABTS assay. Results showed that PO attain their optimum activities when incubating 0.1 g of soil at 30 °C for 5 min with 10 ml of a Modified Universal Buffer (MUB) at pH 2 and 200 μl of a 0.1 M ABTS solution.     

Identification and characterization of a hemocyanin-derived phenoloxidase from the crab Charybdis japonica

To determine effective activators of crab hemocyanin (Hc) and the properties of Hc-derived phenoloxidase (HdPO), Hc, for the first time, was purified from hemolymph of Charybdis japonica, and the properties of activated HdPO were studied by using L-DOPA as a substrate. Three distinct subunits were isolated, and each had a molecular mass of about 80, 75 and 70 kDa, respectively. SDS and HLS were much effective in conversion of Hc into HdPO whose PO activity was optimal at pH 7.0 and temperature of 40 °C. The Km value of the HdPO was 2.90 mM for L-DOPA and 7.33 mM for tyrosine. The PO activity of HdPO was most sensitive to 1-phenyl-2-thiourea, cysteine and ascorbic acid, and much sensitive to thio urea and sodium sulfite. Based on its inhibition characteristics and the substrate specificity, this HdPO could be classified as a kind of tyrosinase-type phenoloxidase. The PO activity of HdPO was also strongly inhibited by Cu²⁺, Zn²⁺, ethylenediaminetetraacetic acid (EDTA) and diethyldithiocarbamate (DETC). The results with EDTA, DETC, and some metal ions, combined with the perfect recovery effect of Cu²⁺ on DETC-inhibited PO activity, indicate that the HdPO is a kind of copper-containing metalloenzyme. All these imply that the Hc, as an oxygen carrier, can be activated to have PO activities by SDS or HLS, and the activated HdPO has the properties of a tyrosinase-type copper-containing phenoloxidase. This study makes us to understand more easily the multifunctions of crustacean Hc in oxygen carrier and melaninization at certain stresses in host defence as well.     

Screening of tree leaves as annual renewable green biomass for phenol oxidase production and biochemical characterization of mulberry (Morus alba) leaf phenol oxidases

Fruit tree leaf tissues were screened in a search for determination of an alternative source(s) for commercial phenol oxidase (PO) production considering the importance of utilization of green biomass for production of value-added products. Mulberry, pear, sour cherry and apricot leaves were identified as promising PO production sources, due to their comparable enzyme activities with respect to mushroom (Agaricus bisporus), a well-known PO source. Within the scope of this research, further biochemical characterization was only performed for mulberry (Morus alba) leaf tissue due to its high PO activity (ca. 19 EU g⁻¹ tissue) and also its known non-toxic and edible nature which are important properties of an enzyme source to be used without detailed purification. In mulberry leaves, presence of three different PO activities, laccase, peroxidase and catechol oxidase of 62–64 kDa molecular weights, were identified. Since simple extraction/concentration steps without fractionation/purification was aimed as PO production process, operational parameters such as optimal temperature, pH and kinetic studies of overall PO activity were investigated using concentrated crude extract. The highest PO activity against 4-methyl catechol was observed at 45°C and pH 7. Michaelis–Menten kinetic parameters, K _m and V _max, of PO activity were determined as 6 mM 4-methyl catechol and 2.2 μmol quinone produced min⁻¹ ml⁻¹, respectively. PO activity of mulberry leaves increased up to late November. Consequently, mulberry leaves seem as a suitable PO source for industrial applications in which a wide range of substrate utilization is necessary.     

Phenoloxidase activity and thermostability of Cancer pagurus and Limulus polyphemus hemocyanin

The intrinsic and inducible o-diphenoloxidase (o-diPO) activity of Cancer pagurus hemocyanin (CpH) and Limulus polyphemus hemocyanin (LpH) were studied using catechol, l-Dopa and dopamine as substrates. The kinetic analysis shows that dopamine is a more specific substrate for CpH than catechol and l-Dopa (K_m value of 0.01 mM for dopamine versus 0.67 mM for catechol, and 2.14 mM for l-Dopa), while k_cat is highest for catechol (2.44 min^? 1 versus 0.67 min^? 1 for l-Dopa and 0.71 min^? 1 for dopamine). On treatment with 4 mM sodium dodecyl sulfate (SDS) or by proteolysis the o-diPO activity of CpH increases about twofold. In contrast, native LpH shows no o-diPO activity, and exhibits only a slight activity after incubation with SDS. Neither CpH nor LpH show intrinsic mono-PO activity with l-tyrosine and tyramine as substrates. To explore the possible correlation between the degree of PO activity and protein stability of arthropod hemocyanins, the thermal stability of CpH and LpH was investigated by differential scanning calorimetry and Fourier transform infrared spectroscopy. CpH is found to be less thermostable (T_m ~ 80 °C), suggesting that the dicopper active sites are more accessible, thereby allowing the hemocyanin to show PO activity in the native state. The LpH, on the other hand, is more thermostable (T_m ~ 92 °C), suggesting the existence of a correlation between the thermal stability and the intrinsic PO activity of arthropod hemocyanins.     

Knee flexor strength and bicep femoris electromyographical activity is lower in previously strained hamstrings

The aim of this study was to determine if athletes with a history of hamstring strain injury display lower levels of surface EMG (sEMG) activity and median power frequency in the previously injured hamstring muscle during maximal voluntary contractions. Recreational athletes were recruited, 13 with a history of unilateral hamstring strain injury and 15 without prior injury. All athletes undertook isokinetic dynamometry testing of the knee flexors and sEMG assessment of the biceps femoris long head (BF) and medial hamstrings (MHs) during concentric and eccentric contractions at ±180 and ±60° s^?1. The knee flexors on the previously injured limb were weaker at all contraction speeds compared to the uninjured limb (+180° s^?1 p = 0.0036; +60° s^?1 p = 0.0013; ?60° s^?1 p = 0.0007; ?180° s^?1 p = 0.0007) whilst sEMG activity was only lower in the BF during eccentric contractions (?60° s^?1 p = 0.0025; ?180° s^?1 p = 0.0003). There were no between limb differences in MH sEMG activity or median power frequency from either BF or MH in the injured group. The uninjured group showed no between limb differences in any of the tested variables. Secondary analysis comparing the between limb difference in the injured and the uninjured groups, confirmed that previously injured hamstrings were mostly weaker (+180° s^?1 p = 0.2208; +60° s^?1 p = 0.0379; ?60° ^?1 p = 0.0312; ?180° s^?1 p = 0.0110) and that deficits in sEMG were confined to the BF during eccentric contractions (?60° s^?1 p = 0.0542; ?180° s^?1 p = 0.0473). Previously injured hamstrings were weaker and BF sEMG activity was lower than the contralateral uninjured hamstring. This has implications for hamstring strain injury prevention and rehabilitation which should consider altered neural function following hamstring strain injury.     

Phenoloxidase and its zymogen are required for the larval–pupal transition in Bactrocera dorsalis (Diptera: Tephritidae)

Phenoloxidases (POs) play a key role in melanin production, are involved in invertebrate immune mechanisms, and are considered important enzymes in the insect development process. In the present study, we report the developmental stage and tissue-specific expression patterns of BdPPO1 and PO activity from Bactrocera dorsalis. The results showed that the activity of PO and its zymogen expression were closely related to the development of B. dorsalis during the larval–pupal transition, particularly in the integument. Additionally, biochemical characterization showed that PO from different developmental stages and tissues all had maximum activity at pH 7.5 and 37 °C. After feeding a metal ion-containing artificial diet, the activity of PO and expression of BdPPO1 were significantly increased, indicating that PO was a metalloprotein and it could be activated by Zn²⁺, Mg²⁺, Ca²⁺, and Cu²⁺. The functional analysis showed that the expression of BdPPO1 could be regulated by 20-hydroxyecdysone (20E) after injection. Furthermore, injection of the double-stranded RNA of BdPPO1 into the 3rd instar larvae significantly reduced mRNA levels after 24 h and 48 h, and resulted in a lower pupation rate and abnormal phenotype. These results expand the understanding of the important role of PO and its zymogen in the growth of B. dorsalis.     

Purification and characterization of a small cationic protein from the tobacco hornworm Manduca sexta

The prophenoloxidase (proPO) activation system is an important defense mechanism in arthropods, and activation of proPO to active phenoloxidase (PO) involves a serine proteinase cascade. Here, we report the purification and characterization of a small cationic protein CP8 from the tobacco hornworm, Manduca sexta, which can stimulate proPO activation. BLAST search showed that Manduca CP8 is similar to a fungal proteinase inhibitor-1 (AmFPI-1), an inducible serine proteinase inhibitor-1 (ISPI-1), and other small cationic proteins with unknown functions. However, we showed that Manduca CP8 did not inhibit proteinase activity, but stimulated proPO activation in plasma. When small amount (0.1 μg) of purified native CP8 or BSA was added to cell-free plasma samples and incubated for 20 min, low PO activity was observed in both groups. But significantly higher PO activity was observed in the CP8-group than in the BSA-group when more proteins (0.5 μg) were added and incubated for 20 min. However, when the plasma samples were incubated with proteins for 30 min, high PO activity was observed in both the CP8 and BSA groups regardless of the amount of proteins added. Moreover, when PO in the plasma was pre-activated with Micrococcus luteus, addition of CP8 did not have an effect on PO activity, and CP8/bacteria mixture did not stimulate PO activity to a higher level than did BSA/bacteria. These results suggest that CP8 helps activate proPO more rapidly at the initial stage. CP8 mRNA was specifically expressed in fat body and its mRNA level decreased when larvae were injected with saline or bacteria. However, CP8 protein concentration in hemolymph did not change significantly in larvae injected with saline or microorganisms.     

Effects of the dietary administration of sodium alginate on the immune responses and disease resistance of Taiwan abalone,Haliotis diversicolor supertexta

Sodium alginate extracted from brown algae was reported to enhance the immune response and resistance of fish and shrimp. In this study, survival rates of the abalone, Haliotis diversicolor supertexta, against Vibrio parahaemolyticus, and its non-specific immune parameters such as the total haemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts, superoxide dismutase (SOD) activity, phagocytic activity, and clearance efficiency to V. parahaemolyticus by H. diversicolor supertexta were determined when abalone (4.5 ± 0.4 g) were fed diets containing sodium alginate at 0, 1.0, 2.0, and 3.0 g kg^?1. Abalone fed a diet containing sodium alginate at 2.0 and 3.0 g kg^?1 for 14 days and at 1.0 g kg^?1 for 21 days had significantly higher survival rates than those fed the control diet after challenge with V. parahaemolyticus. The relative survival percentages of abalone fed the 1.0, 2.0, and 3.0 g kg^?1 sodium alginate-containing diets for 14 and 21 days were 16.1%, 40.0%, and 48.0%, and 63.6%, 27.3% and 22.6%, respectively. The PO activity, respiratory bursts, SOD activity, and phagocytic activity and clearance efficiency of V. parahaemolyticus of abalone fed the sodium alginate-containing diets at 1.0, 2.0, and 3.0 g kg^?1 were significantly higher than those of abalone fed the control diet for 14 days. After 21 days, the PO activity, respiratory bursts, SOD activity, and phagocytic activity and clearance efficiency of V. parahaemolyticus by abalone fed the sodium alginate-containing diet at 1.0 g kg^?1 were significantly higher than those of abalone fed the other diets. It was concluded that sodium alginate can be used as an immunostimulant for abalone through dietary administration to enhance immune responses of abalone and resistance against V. parahaemolyticus, which were related to the dose and timing of administration.     

Design,synthesis, and preliminary pharmacological evaluation of new imidazolinones as l-DOPA prodrugs

l-DOPA, the immediate biological precursor of dopamine, is still considered the drug of choice in the treatment of Parkinson’s disease. However, therapy with l-DOPA is associated with a number of acute problems. With the aim to increase the bioavailability after oral administration, we designed a multi-protected l-DOPA prodrugs able to release the drug by both spontaneous chemical or enzyme catalyzed hydrolysis. The new compounds have been synthesized and preliminarily evaluated for their water solubility, log P, chemical stability, and enzymatic stability. The results indicate that the incorporation of the amino acidic moiety of l-DOPA into an imidazoline-4-one ring provides prodrugs sufficiently stable to potentially cross unchanged the acidic environment of the stomach, and to be absorbed from the intestine. They also might be able to release l-DOPA in human plasma after enzymatic hydrolysis. The ability of prodrugs 6a–b to increase basal levels of striatal DA, and influence brain neurochemistry associated with dopaminergic activity following oral administration, as well as the radical-scavenging activity against DPPH for compounds 6a–b and 15a are also reported.     

Determination of GDC-0449, a small-molecule inhibitor of the Hedgehog signaling pathway,in human plasma by solid phase extraction-liquid chromatographic-tandem mass spectrometry

To support clinical development, a solid phase extraction (SPE) liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method for the determination of GDC-0449 concentrations in human plasma has been developed and validated. Samples (200 μl) were extracted using an Oasis MCX 10 mg 96-well SPE plate and the resulting extracts were analyzed using reverse-phase chromatography coupled with a turbo-ionspray interface. The method was validated over calibration curve range 5–5000 ng/mL. Quadratic regression and 1/x² weighing were used. Within-run relative standard deviation (%RSD) was within 10.1% and accuracy ranged from 88.6% to 109.0% of nominal. Between-run %RSD was within 8.6% and accuracy ranged from 92.4% to 105.3% of nominal. Extraction recovery of GDC-0449 was between 88.3% and 91.2% as assessed using quality control sample concentrations. GDC-0449 was stable in plasma for 315 days when stored at ?70 °C and stable in reconstituted sample extracts for 117 h when stored at room temperature. Quantitative matrix effect/ion suppression experiment was performed and no significant matrix ion suppression was observed. This assay allows for the determination of GDC-0449 plasma concentrations over a sufficient time period to determine pharmacokinetic parameters at relevant clinical doses.     

Changes in membrane-associated H+-ATPase activities and amounts in young grape plants during the cross adaptation to temperature stresses

Proton pumps make a critical contribution to the physiology of plants, although it remains unclear whether or not membrane-associated H⁺-ATPase is involved in the cross adaptation to different temperature stresses. This experiment investigated the changes in membrane-associated H⁺-ATPase activities that associated with chilling-treated plants after heat acclimation (HA, 38 °C/10 h) and with heat-treated plants after cold acclimation (CA, 8 °C/2.5 days) in annual young grape plants (Vitis vinifera L. cv. Jingxiu) using biochemical and electron microscopic cytochemical assay methods in which cerium trichloride (CeCl₃) precipitation was adopted. The results indicated that plasma membrane H⁺-ATPase activity increased as a result of both pretreatments, while V-type and F-type H⁺-ATPase activity hardly changed. Under subsequent cross temperature stresses, however, the three H⁺-ATPase types did maintain higher activity levels than that of the control. This finding suggests that either a HA or CA pretreatment may promote stability in membrane-associated H⁺-ATPase. A western-blotting assay of the plasma membrane H⁺-ATPase (P-H⁺-ATPase) indicated that the immuno-signal intensity of a 100 kDa peptide was visibly stronger in the HA and CA pretreated plants than in the control both before and after stress. This suggests that the HA- or CA-induced P-type H⁺-ATPase activation can be partly attributed to a new synthesis of the enzyme protein. Further, the results also suggested that membrane-associated H⁺-ATPase was involved in the HA-induced chilling resistance and the CA-induced thermo-tolerance in grape plants and that they had a similar regulating mechanism.     

Quantitation of bergenin in human plasma by liquid chromatography/tandem mass spectrometry

This paper reports the development and validation of an assay for quantitation of bergenin in human plasma using liquid chromatography/tandem mass spectrometry (LC–MS/MS). Bergenin and the internal standard (I.S.), 5-bromo-2,4(1H,3H)-pyrimidinedione (5-BrU), were separated by reversed phase HPLC and quantitated by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the negative ion mode. The most intense [M?H]^? MRM transition of bergenin at m/z 326.9 → 312.3 was used for quantitation and the transition at m/z 188.9 → 42.2 was used to monitor 5-BrU. Stability issues with bergenin required the addition of ascorbic acid to plasma samples prior to storage and analysis within 10 days storage at ?80 °C. The method was linear in the range 3–1000 ng/mL with intra- and inter-day precision of 3.94–5.96 and 1.62–8.31%, respectively, and accuracy <2.33%. The assay was successfully applied to a pharmacokinetic study in healthy volunteers after administration of a single 250 mg oral dose.     

Developing and validating a modified enzyme linked immunosorbent assay method for detecting HEV IgG antibody from dried blood spot (DBS) samples in endemic settings

Serological analysis is an integral part of laboratory practice nowadays. The present study was aimed to develop and validate a modified Enzyme linked Immunosorbent Assay (ELISA) for determination of IgG antibody against Hepatitis E Virus (HEV) using dried blood spots (DBS) and corresponding plasma samples. A total of 65 samples (45 HEV patients, 20 healthy controls) were analyzed. DBS and plasma samples demonstrated equivalent optical densities for detecting anti-HEV IgG. A highly significant correlation was observed between plasma and DBS sample absorbances (R² = 0.98; p < 0.001) at dilution 1:200, indicating true agreement between the two procedures. The assay exhibited decent linearity and showed no effect of physiological hematocrit on assay performance. Data suggested recommendable promise in using DBS as a suitable alternative to plasma samples to determine HEV IgG antibody evidenced by significant correlation with plasma results. Therefore, identical method for processing DBS specimens including it's proper storage is recommended for implementation of a modified ELISA in different settings.     

White shrimp Litopenaeus vannamei that received the hot-water extract of Gracilaria tenuistipitata showed earlier recovery in immunity after a Vibrio alginolyticus injection

White shrimp Litopenaeus vannamei which had been immersed in seawater containing the hot-water extract of Gracilaria tenuistipitata at 0 (control), 200, 400, and 600 mg L^?1 for 3 h, were challenged with Vibrio alginolyticus at 4.6 × 10⁶ colony-forming units (CFU) shrimp^?1 and then placed in normal seawater (34‰). The survival rates of shrimp immersed in 200, 400, and 600 mg L^?1 of the hot-water extract were significantly higher than those of control shrimp over 48–120 h. In another experiment, L. vannamei which had been immersed in the hot-water extract at 0, 200, 400, and 600 mg L^?1 for 3 h, were challenged with V. alginolyticus at 4.0 × 10⁶ CFU shrimp^?1, and the immune parameters examined included the haemocyte count, phenoloxidase (PO) activity, respiratory burst (RB), and superoxide dismutase (SOD) activity at 12–120 h post-challenge after shrimp had been released into normal seawater. Shrimp not exposed to the hot-water extract or V. alginolyticus served as the background control. Results indicated that the haemocyte count, PO activity, RB, and SOD activity of shrimp immersed in 600 mg L^?1 were significantly higher than those of control shrimp at 12–72 h post-challenge. Results also indicated that total haemocyte count (THC), PO activity, RB and SOD activity of shrimp immersed in 400 and 600 mg L^?1 of the hot-water extract returned to the background values at 96, 48, 48, and 72 h, whereas these parameters of control shrimp returned to the original values at >120, >120, 96, and 96 h post-challenge, respectively. It was therefore concluded that L. vannamei that had been immersed in seawater containing the hot-water extract of G. tenuistipitata exhibited protection against V. alginolyticus as evidenced by the earlier recovery of immune parameters.     

Innate immunity of the white shrimp Litopenaeus vannamei weakened by the combination of a Vibrio alginolyticus injection and low-salinity stress

White shrimp Litopenaeus vannamei were reared at a salinity of 35‰ without a Vibrio alginolyticus injection (unchallenged group), and other shrimp were reared at 35‰, injected with tryptic-soy broth (TSB)-grown V. alginolyticus at 1.8 × 10⁵ colony-forming units (cfu) shrimp^?1 (challenged group), and then examined for the hyaline cell (HC) count, granular cell (GC, including semi-granular cell) count, total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB) and superoxide dismutase (SOD) activity after transfer to 35‰ (control), 25‰, 20‰, and 15‰ for 1, 6, 12, 24, 72, and 120 h. Results indicated that the haemocyte count, PO activity, RB, and SOD activity of unchallenged shrimp and challenged shrimp that were transferred to low-salinity levels all began to significantly decrease at 6, 6, 6, and 1 h, respectively, and reached the lowest levels at 12 h. HC, GC, the THC, PO activity, RB, and SOD activity of unchallenged shrimp that were transferred to 15‰ decreased by 53%, 41%, 49%, 68%, 39%, and 62%, whereas those parameters of challenged shrimp that were transferred to 15‰ decreased by 79%, 78%, 79%, 82%, 54%, and 72%, respectively after 12 h compared to control shrimp. These immune parameters began to recover after 24–72 h for both unchallenged shrimp and challenged shrimp. We concluded that the innate immunity was weakened in white shrimp L. vannamei that received combined stresses of a V. alginolyticus injection, and low-salinity transfer. It was also concluded that shrimp with respectively 21%, 18%, 46%, and 28% lower THC, PO activity, RB, and SOD activity of the original values would be killed due to decreases in their immunity, and resistance to V. alginolyticus infection. Shrimp farming should be maintained at a constant high salinity level to prevent exacerbated decreases in innate immune parameters of shrimp when infected by a pathogen coupled with low-salinity stress leading to mortality.     

Microbial surface displaying formate dehydrogenase and its application in optical detection of formate

In the present work, NAD⁺-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD⁺, and the production of NADH can be detected spectrometrically at 340 nm. After induction for 24 h in Luria-Bertani medium containing isopropyl-β-d-thiogalactopyranoside, over 80% of MBP-INP-FDH fusion protein present on the surface of E. coli cells. The cell-surface-displayed FDH showed optimal temperature of 50 °C and optimal pH of 9.0. Additionally, the cell-surface-displayed FDH retained its original enzymatic activity after incubation at 4 °C for one month with the half-life of 17 days at 40 °C and 38 h at 50 °C. The FDH activity could be inhibited to different extents by some transition metal ions and anions. Moreover, the E. coli cells expressing FDH showed different tolerance to solvents. The recombinant whole cell exhibited high formate specificity. Finally, the E. coli cell expressing FDH was used to assay formate with a wide linear range of 5–700 μM and a low limit of detection of 2 μM. It is anticipated that the genetically engineered cells may have a broad application in biosensors, biofuels and cofactor regeneration system.     

Analytical variables affecting exchangeable copper determination in blood plasma

To resolve discrepancies observed in the determination of plasma exchangeable Cu (also called direct reacting Cu or loosely bound Cu) by several methods, plasma storage techniques and various aspects of a stable isotope dilution procedure for exchangeable Cu were evaluated. Results indicated that the exchangeable Cu fraction of plasma increased with storage at room temperature, at 5°C and when subjected to repeated freeze/thaw cycles. Samples could be safely stored at −65°C. Exchange between added ⁶⁵Cu²⁺ and endogenous plasma Cu rapidly went to completion in the isotope dilution procedure. Analytical results were unaffected by shaking method, sample size or the presence of heparin. A small difference was observed between serum and plasma. The determination of exchangeable Cu did not vary over a period of 4 h when plasma was exposed to 1.6 × 10⁻⁴-M sodium diethyldithiocarbamate (used in the isotope dilution method) but steadily increased when exposed to 1.1 × 10⁻²-M sodium diethyldithiocarbamate, which suggested that tightly bound Cu (probably in ceruloplasmin) was exchanging with isotopic tracer at the higher concentration. Determination of exchangeable Cu was constant from pH 7.2–8.5 but increased substantially at higher pH. Complete recovery of natural Cu added to plasma was obtained. Studies in solution indicated that ⁶⁵Cu²⁺ exchanged readily with albumin- and amino acid-bound Cu. Ultrafiltration of plasma yielded a Cu fraction about half that of the exchangeable Cu fraction. We conclude that the stable isotope dilution procedure for plasma exchangeable Cu yields reliable, physiologically meaningful results.     

A novel validated procedure for the determination of nicotine,eight nicotine metabolites and two minor tobacco alkaloids in human plasma or urine by solid-phase extraction coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry

A novel validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) procedure was developed and fully validated for the simultaneous determination of nicotine-N-β-d-glucuronide, cotinine-N-oxide, trans-3-hydroxycotinine, norcotinine, trans-nicotine-1′-oxide, cotinine, nornicotine, nicotine, anatabine, anabasine and cotinine-N-β-d-glucuronide in human plasma or urine. Target analytes and corresponding deuterated internal standards were extracted by solid-phase extraction and analyzed by LC–MS/MS with electrospray ionization (ESI) using multiple reaction monitoring (MRM) data acquisition. Calibration curves were linear over the selected concentration ranges for each analyte, with calculated coefficients of determination (R²) of greater than 0.99. The total extraction recovery (%) was concentration dependent and ranged between 52–88% in plasma and 51–118% in urine. The limits of quantification for all analytes in plasma and urine were 1.0 ng/mL and 2.5 ng/mL, respectively, with the exception of cotinine-N-β-d-glucuronide, which was 50 ng/mL. Intra-day and inter-day imprecision were ≤14% and ≤17%, respectively. Matrix effect (%) was sufficiently minimized to ≤19% for both matrices using the described sample preparation and extraction methods. The target analytes were stable in both matrices for at least 3 freeze–thaw cycles, 24 h at room temperature, 24 h in the refrigerator (4 °C) and 1 week in the freezer (?20 °C). Reconstituted plasma and urine extracts were stable for at least 72 h storage in the liquid chromatography autosampler at 4 °C. The plasma procedure has been successfully applied in the quantitative determination of selected analytes in samples collected from nicotine-abstinent human participants as part of a pharmacokinetic study investigating biomarkers of nicotine use in plasma following controlled low dose (7 mg) transdermal nicotine delivery. Nicotine, cotinine, trans-3-hydroxycotinine and trans-nicotine-1′-oxide were detected in the particular sample presented herein. The urine procedure has been used to facilitate the monitoring of unauthorized tobacco use by clinical study participants at the time of physical examination (before enrollment) and on the pharmacokinetic study day.     

Cryopreservation of sperm from seven-band grouper,Epinephelus septemfasciatus

In the present study, we examined methods for the cryopreservation of Epinephelus septemfasciatus spermatozoa. The percent motility, average path velocity, and linearity of movement (LIN) of fresh and corresponding post-thaw sperm were evaluated. Sperm motility was investigated using computer-assisted sperm analysis. Five percent dimethyl sulphoxide (Me₂SO) with 95% fetal bovine serum (FBS) was the most successful cryoprotectant diluent with a comparative post-thaw motility of 77.6 ± 8.5%; 5% dimethyl formamide was also effective. Fetal bovine serum was significantly better as an extender when compared with artificial seminal plasma, glucose, and trehalose solution. Sperm tolerated a wide range of cooling rates (from 27.1 to 94.3 °C min^?1); however, the post-thaw motility of sperm cooled to ?30 °C was significantly lower than that of other cooled temperatures (?40 to ?70 °C). The velocity of post-thaw sperm was significantly lower than that of fresh sperm, although LIN remained the same. For effective cryopreservation of seven-band grouper sperm, samples should be diluted in 5% Me₂SO with 95% FBS and cooled to at least ?40 °C before immersion in liquid nitrogen.     

Development and validation of an LC–MS/MS method for quantification of cyclic guanosine 3′,5′-monophosphate (cGMP) in clinical applications: A comparison with a EIA method

An LC–MS/MS method was developed and validated to quantify endogenous cyclic guanosine 3′,5′-monophosphate (cGMP) in human plasma. The LC–MS/MS and competitive enzyme immunoassay (EIA) assays were compared. cGMP concentrations of 20 human plasma samples were measured by both methods. For the MS-based assay, plasma samples were subjected to a simple protein precipitation procedure by acetonitrile prior to analysis by electrospray ionization LC–MS/MS. De-protonated analytes generated in negative ionization mode were monitored through multiple reaction monitoring (MRM). A stable isotope-labeled internal standard, ¹³C₁₀,¹⁵N₅-cGMP, which was biosynthesized in-house, was used in the LC–MS/MS method. The competitive EIA was validated using a commercially available cGMP fluorescence assay kit. The intra-assay accuracy and precision for MS-based assay for cGMP were 6–10.1% CV and ?3.6% to 7.3% relative error (RE), respectively, while inter-assay precision and accuracy were 5.6–8.1% CV and ?2.1% to 6.3% RE, respectively. The intra-assay accuracy and precision for EIA were 17.9–27.1% CV and ?4.9% to 24.5% RE, respectively, while inter-assay precision and accuracy were 15.1–39.5% CV and ?30.8% to 4.37% RE, respectively. Near the lower limits of detection, there was little correlation between the cGMP concentration values in human plasma generated by these two methods (R² = 0.197, P = 0.05). Overall, the MS-based assay offered better selectivity, recovery, precision and accuracy over a linear range of 0.5–20 ng/mL. The LC–MS/MS method provides an effective tool for the quantitation of cGMP to support clinical mechanistic studies of curative pharmaceuticals.