Microdialysis-integrated HPLC system with dual-electrode detection using track-etched membrane electrodes for in vivo monitoring of dopamine dynamics.

A capillary high-performance liquid chromatography (HPLC) system equipped with a dual-electrode detector utilizing track-etched membrane electrodes (TEMEs) was combined with a microdialysis sampling setup. The electrochemical detector benefits from the high electrolysis efficiency of TEMEs, allowing for calibration-free coulometric detection and simplifying data analysis to determine the dopamine recovery through a dialysis probe. Additionally, this system was used for in vivo monitoring of dopamine in the right striatum of a mouse brain. Temporal changes in dopamine levels, including an exponential decay immediately after the dialysis probe insertion and an excess release of dopamine induced by a high concentration of potassium ions, confirmed the system's proper operation. Furthermore, subsequent measurements following the intraperitoneal injection of mirtazapine showed no increase in dopamine levels in the right dorsal striatum. The dual-electrode system displayed characteristic dopamine detection behavior, with anodic and cathodic peak pairs indicative of reversible electrochemical reactions. This capability facilitated the identification of the dopamine peak within the complex chromatogram of the mouse brain dialysate. The consistency between dopamine collection efficiency from standard solutions and dialysate indicated the absence of interfering electroactive substances overlapping with the dopamine peak in the chromatogram. This integrated analysis system successfully tracked temporal fluctuations in dopamine concentration within the mouse brain.

Effects of pellet starch levels in automatic milking systems on rumen fermentation, plasma metabolites, and milk production in mid-lactation cows.

The aim of this study was to evaluate whether the starch levels in pellets fed to cows in automatic milking systems (AMS) affect subacute ruminal acidosis (SARA) occurrence and metabolite parameters. Twenty-four lactating cows (124.4 ± 49.9 days in milk) were studied in a crossover design with two periods of 21 days each and two treatment groups-a control group fed AMS pellets containing 30.0% of starch dry matter (DM) and an experimental group fed AMS pellets containing 23.5% of starch DM. All cows received the same partial mixed ration (PMR). The 1-hr mean ruminal pH in both groups decreased over 4 hr after feeding on PMR but recovered by the next morning. The ruminal pH was unaffected by either treatment, and both groups developed SARA. The groups had no significant differences in the concentrations of ruminal volatile fatty acids, lipopolysaccharides, plasma acute-phase proteins, other metabolites, and hormones. The milk yield and composition were not different in both groups. Feeding low-starch pellets in the AMS did not contribute to the risk of SARA occurrence in cows and had no additive effects on rumen fermentation, plasma metabolites, or milk production.

Conjugation monitoring of gold nanoparticles with alkanedithiols by capillary zone electrophoresis.

Alkanedithiols were used for the conjugation of gold nanoparticles (AuNP) prepared by a solution plasma process. Capillary zone electrophoresis was utilized for the monitoring of the conjugated AuNP. When 1,6-hexanedithiol (HDT) was used as a linker, a resolved peak from the AuNP was detected in the electropherogram; the resolved peak was attributed to the conjugated AuNP. The resolved peak was developed with increasing concentrations of HDT, while the peak of the AuNP decreased complementary. The resolved peak also tended to develop along with the standing time at least up to 7 weeks. The electrophoretic mobility of the conjugated AuNP was almost identical over the HDT concentrations examined, suggesting that the conjugation of the AuNP did not proceed further, such as aggregate/agglomerate formation. The conjugation monitoring was also examined with some dithiols and monothiols. Resolved peak of the conjugated AuNP was also detected with 1,2-ethanedithiol and 2-aminoethanethiol.

Effects of feeding wood kraft pulp silage containing sweet-potato shochu distillery by-product on feed intake, feed digestibility, rumen fermentation, blood components, and growth performance in Japanese Black fattening steers during early period.

The effects of long-term feeding of wood kraft pulp (KP) silage containing sweet-potato shochu distillery by-product (SDP) on feed intake, feed digestibility, rumen fermentation, and growth performance of Japanese Black steers were investigated during the early fattening period. Ten Japanese Black steers (9.8 ± 0.6 months of age) were used in this study. Five steers (KP group) were fed KP silage as a replacement for 10% timothy hay (dry matter bases), in contrast to the other five (control group). KP silage consisted of 92.9% KP and 7.1% SDP (dry matter bases). The experiment was conducted for 18 weeks. No significant differences were observed in terms of feed intake, feed digestibility, or daily body weight gain between the groups. In addition, diurnal changes in the rumen pH and ruminal lipopolysaccharide activity did not differ between the groups. However, the plasma concentration of aspartate transaminase in the KP group was slightly lower (P = 0.078) than that in the control group. Thus, our study suggested that feeding KP silage does not reduce feed intake or affect the rumen fermentation or growth performance of Japanese Black fattening steer.

Kinetic analyses of two-steps oxidation from l-tyrosine to l-dopaquinone with tyrosinase by capillary electrophoresis/dynamic frontal analysis.

Tyrosinase catalyzes the oxidation of l-tyrosine in two stages to produce l-dopa and l-dopaquinone stepwise, and l-dopaquinone is subsequently converted to dopachrome. Most of the conventional analyses subjected only one-step reaction from l-tyrosine to l-dopa or from l-dopa to l-dopaquinone. In this study, kinetic analyses of two-steps oxidation of l-tyrosine with tyrosinase were made by capillary electrophoresis/dynamic frontal analysis (CE/DFA). When l-dopa was introduced into a capillary as a sample plug in a CE/DFA format, the enzymatic oxidation continuously occurred during the electrophoresis, and the product l-dopaquinone was subsequently converted to dopachrome which was detected as a plateau signal. A Michaelis-Menten constant of the second-step kinetic reaction, Km,Do, was determined as 0.45 ± 0.03 mmol L-1. In the analysis of the first-step kinetic reaction from l-tyrosine to l-dopa, l-dopa was not resolved by CE/DFA because both l-tyrosine and l-dopa are electrically neutral. The l-dopa formed and co-migrated at the l-tyrosine zone was calibrated beforehand with the final product of dopachrome detected as a plateau signal. Constantly formed l-dopa was successfully detected as a plateau signal of dopachrome, and a Michaelis-Menten constant of Km,Ty was also determined as 0.061 ± 0.009 mmol L-1 by the CE/DFA. CE/DFA is applicable to two-steps enzymatic reactions.

Highly stable gold nanoparticles in an aqueous solution without any stabilizer prepared by a solution plasma process evaluated through capillary zone electrophoresis.

Gold nanoparticles (AuNP) were prepared by a solution plasma process in the presence of H2O2, and they were dispersed in an aqueous solution without any stabilizer generally used. The dispersion stability of the AuNP in an aqueous solution was evaluated by capillary zone electrophoresis (CZE). An anionic broad peak was detected with the AuNP by CZE based on its wide variations in size and net charge. The broad peak also suggests that the AuNP were well dispersed in an aqueous solution. The dispersion stability of AuNP was evaluated from the viewpoints of long-term dispersion, salt concentration, and organic co-solvent. The anionic broad peak attributed to the dispersed AuNP was successfully detected for at least 55 weeks from the preparation with less shot signals of the aggregates. The AuNP was also well dispersed in aqueous NaCl solutions with its concentrations up to 30 mmol L-1, as well as with ethanol co-solvent up to 40%(v/v). The AuNP prepared by the solution plasma process was proved to be highly stable in an aqueous solution.

Effects of wood kraft pulp as a partial replacement for roughage on rumen fermentation and productivity in dairy cows.

In this study, we evaluated the partial replacement of roughage with wood kraft pulp (KP) on rumen fermentation and productivity of dairy cows. Eighteen cows were divided into control and KP groups. The KP group started adaptation to KP 3 weeks before calving; after calving, they were fed a total mixed ration for 12 weeks, wherein 18% Timothy hay was replaced with KP. The dry matter intake, body weight, and milk yield and composition were similar in the control and KP groups. The average daily rumen pH was higher with KP feeding, and the average daily ruminal temperature remained lower at 16 days after calving (P < 0.05). The concentration of volatile fatty acids remained unaltered, the molar proportion of acetic acid decreased, and the molar proportion of propionic acid increased, indicating a low acetic acid:propionic acid ratio (P < 0.05). Lipopolysaccharide activity in the rumen fluid was higher in the KP group (P < 0.05); however, the rumen microbiota were unaffected. The digestibility of dry matter and neutral detergent fiber increased 12 weeks after calving, whereas excretion of urinary nitrogen decreased (P < 0.05). Partial replacement of roughage with KP did not suppress rumen fermentation and maintained postpartum productivity.

Effects of wood kraft pulp feeding on feed digestibility and rumen fermentation of Japanese Black steer in the middle fattening stage.

We investigated the effects of wood kraft pulp (KP) feeding on feed digestibility and rumen fermentation of Japanese Black (JB) steer in the middle fattening stage. The feeding experiment was carried out a replicated 3 × 3 Latin square design using six JB steers (16.2 ± 0.8 months of age). Steers were fed rice straw and three concentrated feeds: commercial formula feed (control), formula feed containing 7.5% KP on a dry matter (DM) basis (7.5% KP), and formula feed containing 15% KP on a DM basis (15% KP). Feed digestibility and rumen fermentation parameters were recorded for 3 days during the measurement period, after an adaptation phase of 11 days. There was no significant difference in DM intake per day among diets. A slightly higher digestibility of neutral detergent fiber was observed in steers fed 15% KP, compared with those fed other diets (P = 0.059). The butyric acid ratio in rumen fluid was higher in steers fed KP diets than in steers fed control (P = 0.083). The average ruminal pH was unaffected by KP diets. This study suggests that replacing 15% of concentrated feed with KP has little effect on feed digestibility and rumen fermentation in JB fattening steer.

Anti-lipopolysaccharide antibody mitigates ruminal lipopolysaccharide release without acute-phase inflammation or liver transcriptomic responses in Holstein bulls.

Anti-lipopolysaccharide (LPS) antibody administration has the potential benefits of neutralizing and consequently controlling rumen-derived LPS during subacute ruminal acidosis. Four Holstein bulls were used in this crossover study with a 2-week wash-out period. Anti-LPS antibody (0 or 4 g) was administered once daily for 14 days. Significantly lower ruminal LPS and higher 1-h mean ruminal pH were identified in the 4 g group. However, blood metabolites, acute-phase proteins, cytokines, and hepatic transcriptomes were not different between the two groups. Therefore, anti-LPS antibody administration mitigated ruminal LPS release and pH depression without accompanying responses in acute-phase inflammation or hepatic transcriptomic expression.

Anti-lipopolysaccharide antibody administration mitigates ruminal lipopolysaccharide release and depression of ruminal pH during subacute ruminal acidosis challenge in Holstein bull cattle.

The effects of anti-lipopolysaccharide (LPS) antibody on rumen fermentation and LPS activity were investigated during subacute ruminal acidosis (SARA) challenge. Eleven Holstein cattle (164 ± 14 kg) were used in a 3 × 3 Latin square design. Cattle were fed a roughage diet on days -11 to -1 (pre-challenge) and day 2 (post-challenge), and a high-grain diet on days 0 and 1 (SARA challenge). For 14 days, 0-, 2-, or 4-g of anti-LPS antibody was administered once daily through a rumen fistula. Ruminal pH was measured continuously, and rumen fluid and blood samples were collected on days -1, 0, 1, and 2. Significantly lower ruminal LPS activity on day 1 was observed in the 2- and 4-g groups than those in the 0-g group. In addition, significantly higher 1-hr mean ruminal pH on SARA challenge period (days 0 and 1) was identified in the 4-g group than in the 0-g group. However, rumen fermentation measurements (total volatile fatty acid [VFA], VFA components, NH3-N and lactic acid) and peripheral blood metabolites (glucose, free fatty acid, beta-hydroxybutyrate, total cholesterol, blood urea nitrogen, aspartate aminotransferase and gamma-glutamyl transferase) were not different among the groups during the experimental periods. Therefore, anti-LPS antibody administration mitigates LPS release and pH depression without the depression of rumen fermentation and peripheral blood metabolites during SARA challenge in Holstein cattle.

Kinetic analysis of the transphosphorylation with creatine kinase by pressure-assisted capillary electrophoresis/dynamic frontal analysis.

Kinetic reactions of the transphosphorylation with creatine kinase (CK) were individually investigated between creatine (Cr) and creatine phosphate (CrP) by pressure-assisted capillary electrophoresis/dynamic frontal analysis (pCE/DFA). The transphosphorylations are reversible between Cr and CrP, and reverse reactions inevitably accompany in general batch analyses. In pCE/DFA, the kinetic reaction proceeds in a separation capillary and the product is continuously resolved from the substrate zone. Therefore, the formation rate is kept constant at the substrate zone without the reverse reaction, and the product is detected as a plateau signal. This study demonstrates the direct and individual analyses of both the forward and the backward kinetic reactions with CK by pCE/DFA. A plateau signal was detected in the pCE/DFA with ADP or ATP as one of the products on either the forward or the backward reactions. The Michaelis-Menten constants of Km,ATP (from Cr to CrP) and Km,ADP (from CrP to Cr) were successfully determined through the plateau signal. Determined values of Km,ATP and Km,ADP by pCE/DFA were smaller than the ones obtained by the pre-capillary batch analyses. The results agree with the fact that the reverse reaction is excluded in the analysis of the kinetic reactions. The proposed pCE/DFA is useful on individual analyses of both forward and backward kinetic reactions without any interference from the reverse reaction.

An Improved Reflection Colorimeter Integrated with a Coaxial Optical-fiber Cable for Highly Sensitive Solid-phase Colorimetry Using a Membrane Filter.

Highly sensitive solid-phase colorimetry for nickel ion was demonstrated using an improved reflection colorimeter equipped with a coaxial optical-fiber cable. The nickel complex with α-furil dioxime was collected on a small-size membrane filter embedded in a disposable syringe filter unit. The leading edge of the optical-fiber cable was connected to the syringe filter unit via a Luer-lock fitting, and the color intensity of the sample on the filter was evaluated accurately. The detection limit was 0.8 ng in 2.5 mL of the complex solution (0.3 µg L-1). This improved configuration is applicable to highly sensitive on-site analysis without expensive instruments nor high laboratory skills.

Effects of grazing adaptation on intake, ruminal fermentation, blood metabolites, and body weight change in dairy cows after turning out to pasture in early spring.

This study investigated the effect of adaptation to grazing in early spring on herbage intake, ruminal fermentation parameters, blood metabolite concentrations, and body weight change in dairy cows. The experiment was conducted on eight rumen-cannulated non-lactating cows in the early spring period. Four cows were adapted to grazing by stocking for 4 hr for 1 week (ADP group). The other cows were kept in a barn during the period (CON group). Then, both groups of cows were stocked together throughout a day on a 1 ha pasture for 3 weeks (experimental period). In the first week of the experimental period, compared to the CON group, the ADP group had a higher herbage intake, ruminal NH3 -N and total VFA concentration, and blood urea concentration, but the NEFA concentration was lower in the ADP group (p < .01). During the subsequent weeks, there were little differences in ruminal fermentation parameters and blood metabolites. Cows in the ADP group maintained their body weight, but cows in the CON group lost 60 kg of body weight in the first week of the experimental period.

Kinetic analysis of an enzymatic hydrolysis of p-nitrophenyl acetate with carboxylesterase by pressure-assisted capillary electrophoresis/dynamic frontal analysis.

An enzymatic hydrolysis of p-nitrophenyl acetate with carboxylesterase was analyzed by capillary electrophoresis/dynamic frontal analysis (CE/DFA). A plateau signal was expected with the anionic product of p-nitrophenol by the CE/DFA applying in-capillary reaction and the continuous CE resolution of the product from the substrate zone. However, the plateau height was not sufficient, and/or the plateau signal fluctuated and drifted. Therefore, a pressure assist was utilized in the CE/DFA to detect the product zone fast and to average the fluctuated plateau signal by mixing in a laminar flow. The plateau signal became relatively flat and its height was developed by the pressure-assisted capillary electrophoresis/dynamic frontal analysis (pCE/DFA). The plateau height was used for the Michaelis-Menten analysis, and a Michaelis-Menten constant was determined as KM = 0.83 mmol L-1. An enzyme inhibition was also examined with bis(p-nitrophenyl) phosphate by adding it in the separation buffer. The height of the plateau signal decreased by the inhibition, and a 50% inhibitory concentration was determined as IC50 = 0.79 µmol L-1. The values of KM and IC50 obtained in this study agreed well with the reported values. Since the proposed pCE/DFA includes electrophoretic migration of the substrate zone in a capillary, it is also noticed that the deactivation of the enzyme by ethanol on the preparation of the substrate solution can be avoided, as well as the exclusion of the inhibition by the product.

Effects of short-term fasting on ruminal pH and volatile fatty acids in cattle fed high-roughage versus high-concentrate diets.

We evaluated whether the dietary roughage-to-concentrate ratio affects ruminal pH and volatile fatty acids (VFAs) in response to a one-time morning fast. Four healthy rumen-cannulated Holstein steers 4-5 months old were used. Cattle were subjected to 2 weeks of adaptation (high-roughage or high-concentrate diet), and morning feed restriction was performed on the day after the adaptation period ended (Day 0). Thereafter, each diet was reintroduced on the evening of Day 0. Our results showed that the 1-hr mean ruminal pH from 0800 to 1900 on Day 0 was higher, and that from 1700 to 1900 on Day 1 was lower (P<0.05) than pH on 1 day before fasting (Day -1) in cattle fed both diets. On Day 0, total VFA levels decreased after morning fasting and were lower (P<0.05) than those on Day -1 irrespective of evening refeeding. Furthermore, blood non-esterified fatty acid and beta-hydroxybutyric acid levels on Day 0 increased and decreased, respectively, compared to Day -1 in cattle fed both diets. These results indicate that even a one-time feed restriction can disrupt ruminal fermentation, and the changes can persist to the next day after fasting.

Kinetic analysis of substrate competition in enzymatic reactions with ß-D-galactosidase by capillary electrophoresis / dynamic frontal analysis.

Competitive inhibition between two substrates with an enzyme is investigated by capillary electrophoresis/dynamic frontal analysis (CE/DFA). Enzymatic hydrolyses of o-nitrophenyl ß-D-galactopyranoside and p-nitrophenyl ß-D-galactopyranoside with ß-D-galactosidase were examined as a model competitive reaction. A sample solution containing the two substrates was injected into a capillary filled with a separation buffer containing an enzyme. Enzymatic hydrolysis occurred during the electrophoresis, and the products of o-nitrophenol and p-nitrophenol were continuously formed and resolved from the sample zone. Two-steps plateau signal was detected with the two-substrate solutions based on the difference in the effective electrophoretic mobility of o-nitrophenol and p-nitrophenol. Michaelis-Menten constants and inhibition constants were determined with the plateau heights. Usefulness of CE/DFA on competitive inhibition analysis is demonstrated in this study.

Determination of acid dissociation constants of flavin analogues by capillary zone electrophoresis.

Acid dissociation constants (pKa ) of nine kinds of flavin analogues as molecular catalyst candidates were determined by CZE. Although some of the analogues are instable and degradable under the light exposure or in alkaline aqueous solutions, the effective electrophoretic mobility of the flavin analogue of interest has been measured with the residual substance. The pKa values of the flavin analogues were analyzed through the changes in the effective electrophoretic mobility with varying pH of the separation buffer. One or two steps pKa values were determined by the analysis. One of the degraded species from the flavin analogues, lumichrome, was also detected in the CZE analysis, and its pKa values were also determined. While coexisting impurities generated over the storage conditions were found in some analogues, the pKa values of the target analogues were successfully determined with the help of the CZE separations. A pressure-assisted CZE was utilized for the determination or the estimation of the pKa values of such analogues as possessing carboxylic acid moiety.

Track-etched membrane-based dual-electrode coulometric detector for microbore/capillary high-performance liquid chromatography.

The electrochemical flow cell containing track-etched microporous membrane electrodes was applied to a dual-electrode coulometric detector for microbore/capillary HPLC with a small injection volume and low eluent flow rate. The proposed flow cell with a 0.1-mm diameter inlet channel gave a detection volume of 0.08 nL per electrode, which was determined by the eluent flow through the electrode. For the dual-electrode detector, the calculated volume was 0.24 nL. The efficiency of electrooxidation of l-ascorbic acid increased as the flow rate decreased and was close to 100% when the flow rate was below 50 µL min-1, which is a common flow rate in microbore or capillary liquid chromatography. Catecholamines, such as noradrenaline, adrenaline, and dopamine, were detected by total conversion with two-electron oxidation in the potential range from 0.8 to 1.0 V vs. Ag/AgCl after separation with a microbore column. These peaks were accompanied by corresponding cathodic peaks derived from quasi-stable electrooxidation products of the catecholamines. The detection limits of noradrenaline, adrenaline, and dopamine were 0.1, 0.1, and 0.2 µM, respectively. The RSD values for five replicate measurements of 5.0 µM of these compounds were 0.9%, 0.7%, and 1.5%, respectively. Coulometric detection was also demonstrated by determination of catecholamines in pharmaceuticals.

Capillary Electrophoretic Characterization of Water-soluble Carbon Nanodots Formed from Glutamic Acid and Boric Acid under Microwave Irradiation.

Water-soluble carbon nanodots (CND) were synthesized under microwave irradiation from glutamic acid or glutamic acid-boric acid mixture. The CNDs were collected in an aqueous solution through size fractionation by centrifugal filtration. The CNDs thus prepared were subjected to characterization by capillary electrophoresis (CE). A peak signal of anionic substance was detected in the electropherogram, and it was found to be a major component of the CNDs. The effective electrophoretic mobility of the major component was almost identical over the pH range between 6.7 and 11.6, suggesting that the functional group of amine or boric acid moiety was not included in the CNDs. The effective electrophoretic mobility decreased at an acidic pH of less than 5, and it was suggested that carboxylate moiety was included in the CNDs. A signal of less-charged CNDs was also detected in the electropherogram, and the CNDs were characterized by a CE format of micellar electrokinetic chromatography. Two or four peaks were detected just after the electroosmotic flow; the less-charged CNDs were thus hydrophilic. The affinity interaction was also examined between the major anionic CNDs and a hydrophobic pairing cation. The peak signal of the major anionic CNDs broadened, and its theoretical number of plates decreased in the presence of tetrabutylammonium ion in the separation buffer. A small portion of the anionic CNDs were a little hydrophobic at different degrees, and their effective electrophoretic mobility decreased by the hydrophobic interaction, resulting in peak broadening of the anionic CNDs.

Capillary Electrophoresis/Dynamic Frontal Analysis for the Enzyme Assay of 4-Nitrophenyl Phosphate with Alkaline Phosphatase.

A substrate of 4-nitrophenyl phosphate was enzymatically hydrolyzed by alkaline phosphatase (ALP) in a capillary tube, while an injected zone of the substrate was electrophoretically migrating in the separation buffer containing the enzyme by capillary electrophoresis (CE). During CE migration of the substrate from the start time of the electrophoresis to the detection time of the substrate, the substrate was continuously hydrolyzed by ALP to form a product of 4-nitrophenolate, and a plateau signal of 4-nitrophenolate was detected as a result of the zero-order kinetic reaction. The height of the plateau signal was directly related to the reaction rate, and it was used for the determination of a Michaelis-Menten constant through Lineweaver-Burk plots. Since the plateau signal is attributed to the dynamic formation of the product by the enzymatic reaction in CE, this analysis method is named as capillary electrophoresis/dynamic frontal analysis (CE/DFA). In CE/DFA, the CE separation is included on detecting the plateau signal, and the hydrolysis product before the sample injection is resolved from the dynamically and continuously formed product. The inhibition of the enzyme with the product is also eliminated in CE/DFA by the CE separation.