We determined the concentration of urinary CAs (noradrenaline and adrenaline) and their metabolites by treatment with sulfatase from Helix pomatia Type H-2 (Sigma Aldrich, St. Louis, MO) according to a previous report.(20 (link)) Briefly, the urine was heated for 10 min following incubation with 500 U/ml of the enzyme at 37°C for 1 h. After the addition of isoprenaline (Sigma Aldrich, Milwaukee, WI) as the internal standard, CAs were purified using Monospin PBA® (GL Sciences, Tokyo Japan). The HPLC system (Prominance HPLC System Shimazu Corporation, Kyoto Japan) consisted of a quaternary pump with a vacuum degasser, thermostatted column compartment, and an autosampler equipped with an electrochemical detector (ECD 700 S; Eicom Corporation, Kyoto, Japan). A reverse-phase column (Inertsil ODS-4, 250 × 3.0 mm ID, 5 μm; GL Sciences) was used and the column temperature was maintained at 35°C. The HPLC mobile phase was 24 mM acetate-citrate buffer (pH 3.5, -CH3CN, 100/14.1 v/v). The mobile phase flow rate was 0.3 ml/min and the injection volume 20 μl. The eluents were detected and analyzed at 500 mV. Excretion of CA was expressed as a ratio with the urinary creatinine concentration measured using Laboassay creatinine (FUJIFILM Wako Pure Chemical Corporation).
Monospin pba
Manufactured by GL Sciences
Sourced in Japan
The Monospin PBA is a laboratory centrifuge designed for the separation of suspended particles and liquids. It utilizes a compact and efficient motor to generate a controlled rotational force, enabling the effective segregation of components within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Inertsil ods 4, by GL Sciences (2 mentions)
Isoprenaline, by Merck Group (2 mentions)
Sulfatase from helix pomatia type h 2, by Merck Group (2 mentions)
Ecd 700 s, by Eicom (2 mentions)
Laboassay creatinine, by Fujifilm (1 mentions)
Fisherbrand microhematocrit capillary tubes, by Thermo Fisher Scientific (1 mentions)
Htec 510, by Eicom (1 mentions)
Labassay creatinine, by Fujifilm (1 mentions)
Labassay creatinine kit, by Fujifilm (1 mentions)
Inertsil ods 4 column, by GL Sciences (1 mentions)
Ultrasonic homogenizer, by Yamato Scientific (1 mentions)
5 protocols using monospin pba
1
Measurement of Urinary Catecholamines and Metabolites
2
Measuring Catecholamine Levels in Plasma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood was collected from the ophthalmic artery using a glass tube (Fisherbrand Microhematocrit Capillary Tubes, Fisher Scientific) under isoflurane inhalation. Isoflurane was given via the swivel-connected tube during 2g load. Plasma noradrenaline and adrenaline were extracted using manufactural kits (MonoSpin® PBA, GLSciences) and were measured using high-performance liquid chromatography (HTEC-510, Eicom).
3
Quantification of Urinary Catecholamines and Metabolites
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We determined the concentration of urinary CAs, such as noradrenaline (NA), adrenaline (AD) and their metabolites, by treatment with the enzyme (sulfatase from Helix pomatia Type H-2, Sigma–Aldrich, St. Louis, MO, USA) according to the previous report [43 (link)]. Briefly, the urine was heated for 10 min following incubation with 500 U/mL of enzyme at 37 °C for one hour. After addition of isoprenaline (Sigma-Aldrich, St. Louis, MO, USA) as the internal standard, CAs were purified using a Monospin PBA (GL sciences, Tokyo Japan). The HPLC system (Prominance HPLC System Shimazu Corporation, Kyoto, Japan) consisted of a quaternary pump with a vacuum degasser, thermostatted column compartment, autosampler, and equipped with an electrochemical detector (ECD 700 S, Eicom Corporation, Kyoto, Japan). A reverse-phase column (Inertsil ODS-4; 250 × 3.0 mm ID, 5 µm, GL Sciences, Tokyo, Japan) was used, and the column temperature was maintained at 35 °C. The HPLC mobile phase was 24 mM acetate–citrate buffer (pH 3.5)-CH3CN (100/14.1, v/v). The mobile phase flow rate was 0.3 mL/min, and the injection volume was 20 μL. The eluents were detected and analyzed at 500 mV. Excretion of CAs were expressed as a ratio, with urinary creatinine concentration measured using a LabAssay Creatinine (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan).
4
Quantifying Catecholamine Levels in Mouse Urine
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Twenty-four-hour urine (for catecholamine measurements) was collected into collection vials from 11- to 12-week-old mice individually housed in metabolic cages. To avoid spontaneous oxidation of catecholamine, 60 μL of hydrochloric acid (6 mol/L) was added to the collection vials (Moreira-Rodrigues et al., 2012 (link)). The collected urine was purified using MonoSpin PBA (GL Sciences, Tokyo, Japan) and urine catecholamines were measured by HPLC as previously described (Tsunoda et al., 2011 ). In brief, Chromatography was performed using an Inertsil ODS-4 column (5 μm, 250 × 20 mm i.d., GL Science) with acetate-citrate buffer at a flow rate of 0.5 mL/min and detected by electrochemical detection. The concentration of urine creatinine was measured using a LabAssay™ Creatinine kit (Wako Pure Chemical Industries, Ltd.).
5
Quantification of l-DOPA and Dopamine in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
UPLC was used to quantitate the amount of l -DOPA and DA. The right brain of each mouse was divided into the brain stem, cerebellum and cerebrum (including striatum and olfactory bulb) and each region was homogenized using a ultrasonic homogenizer (Yamato Scientific co., Tokyo Japan). The obtained suspension was centrifuged to remove insoluble materials.
The supernatant of the rough extraction was purified with a spin column (MonoSpin PBA, GL Sciences, Torrance, CA, USA). The supernatant of the rough extraction was applied to the spin column and centrifuged at 10,000 × g for 2 min at room temperature (RT). A 0.5 mL volume of 0.1 M HEPES buffer (pH 8.5) was applied to the spin column and centrifuged at 5,000 × g for 1 min to remove any residues. Finally, 0.1 mL of 1% acetic acid was added to the spin column and centrifuged at 10,000 × g for 1 min at RT to elute the targetl -DOPA and dopamine.
The obtained extract was analyzed using a UPLC system (ACQUITY, Waters, USA) consisting of a binary pump, degasser, autosampler, thermostated column oven and fluorescence detector (ex/em 230/300 nm). An ODS column (ACQUITY BEH Shield RP18, 1.7 μm, 2.1 × 50 mm, Waters) was used. Ammonium acetate-methanol-trifluoroacetic acid (20 mM, 80:0.9:1, v/v/v) was employed as the mobile phase at a flow rate of 0.5 mL/min and run time of 5 min.
The supernatant of the rough extraction was purified with a spin column (MonoSpin PBA, GL Sciences, Torrance, CA, USA). The supernatant of the rough extraction was applied to the spin column and centrifuged at 10,000 × g for 2 min at room temperature (RT). A 0.5 mL volume of 0.1 M HEPES buffer (pH 8.5) was applied to the spin column and centrifuged at 5,000 × g for 1 min to remove any residues. Finally, 0.1 mL of 1% acetic acid was added to the spin column and centrifuged at 10,000 × g for 1 min at RT to elute the target
The obtained extract was analyzed using a UPLC system (ACQUITY, Waters, USA) consisting of a binary pump, degasser, autosampler, thermostated column oven and fluorescence detector (ex/em 230/300 nm). An ODS column (ACQUITY BEH Shield RP18, 1.7 μm, 2.1 × 50 mm, Waters) was used. Ammonium acetate-methanol-trifluoroacetic acid (20 mM, 80:0.9:1, v/v/v) was employed as the mobile phase at a flow rate of 0.5 mL/min and run time of 5 min.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!