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Eno 20

Manufactured by Eicom
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Sourced in Japan

The ENO-20 is a laboratory equipment designed for the measurement and analysis of diverse parameters. It features a compact and sturdy construction, ensuring reliable performance in various laboratory settings.

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47 protocols using eno 20

1

Sensitive HPLC Quantification of Nitrite and Nitrate

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A HPLC system dedicated to assessment of nitrite and nitrate (ENO-20; EiCom, Japan) and attached to an auto-sampler (840, EiCom, Kyoto, Japan) was used. The method is sensitive and specific to nitrite and nitrate, and is based on the separation of nitrate by reverse-phase/ion exchange chromatography, followed by inline reduction of nitrate to nitrite with cadmium and reduced copper. Derivatization of reduced nitrate was performed with Griess reagent, and the level of diazo compounds was measured at 540 nm. The reactor solution was freshly prepared before analysis. A standard curve was prepared from sodium nitrite and sodium nitrate, diluted with carrier solution in the range of 0.1–20 µM. Aliquots of standards were stored at −20 °C. The slope was examined and used to calculate the concentration in the samples.
Immediately before running the samples in the HPLC, 100 µL of the samples were transferred to a 96 well plate with conical wells (Costar, nitrate- and nitrite-free). To avoid evaporation and also contamination from the auto sampler that contains a lot of electrical components releasing NO to the surrounding air, we sealed the plate with a film that was sterile and easy for the auto sampler needle to penetrate. The samples were kept at 4 °C by a cooling device in the auto sampler. The data collected and analyzed using the PowerChrom software (V 2.7.9, eDAQ).
Montenegro M.F., Sundqvist M.L., Nihlén C., Hezel M., Carlström M., Weitzberg E, & Lundberg J.O. (2016). Profound differences between humans and rodents in the ability to concentrate salivary nitrate: Implications for translational research. Redox Biology, 10, 206-210.
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2

Quantifying Nitrate and Nitrite Levels

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Plasma samples were analyzed for their levels of nitrate and nitrite, using a dedicated high performance liquid chromatography (HPLC) system (ENO-20; Eicom) [27] (link).
Paulo L.L., Cruz J.C., Zhuge Z., Carvalho-Galvão A., Brandão M.C., Diniz T.F., Haworth S.M., Athayde-Filho P.F., Lemos V.S., Lundberg J.O., Montenegro M.F., Braga V.A, & Carlström M. (2017). The novel organic mononitrate NDHP attenuates hypertension and endothelial dysfunction in hypertensive rats. Redox Biology, 15, 182-191.
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3

Quantitative Biomarker Analysis Protocol

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Blood glucose was measured using Accu-Chek Aviva (Roche Diagnostics, Tokyo, Japan) immediately after blood samples were collected. Serum triglyceride (TG) and non-esterified fatty acid (NEFA) levels were determined using specific assay kits (Wako Pure Chemical). After serum and urine samples were filtered through a 10-kDa molecular weight cutoff membrane filtration unit (Amicon Ultra; Merck Millipore, Darmstadt, Germany), serum and urinary H2O2 concentrations were determined using a quantitative H2O2 assay kit (Oxis International, Portland, OR, USA). The concentration of plasma NO metabolites (NOx: NO2− and NO3−) was determined using the method described by Matsumoto et al. [34 (link)]. Briefly, 0.03 ml of plasma sample was mixed with 0.03 ml of 100% methanol and centrifuged at 3500×g for 10 min at 4 °C. Plasma NOx was then determined using an automated NO detector/high-performance liquid chromatography system (ENO20; Eicom, Kyoto, Japan).
Ishida K., Misawa K., Yamamoto M, & Shimotoyodome A. (2018). Hydroxyhydroquinone impairs fat utilization in mice by reducing nitric oxide availability. The Journal of Physiological Sciences, 68(6), 855-864.
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4

Aortic NOx Detection via Chromatography

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The total NOx (nitrate + nitrite) level was detected in the aorta using an automated NO detector/high-performance liquid chromatography system (ENO20; Eicom, Kyoto, Japan) according to the manufacturer's protocol (Ishida et al. 2016 (link), Taguchi et al. 2016 , 2017a ,b, 2018) . Each aorta was cut into transverse rings of 4 mm in length. These were placed in KHS at 37°C and treated with vehicle, Control MPs or DM MPs (10 µL; as above) for 30 min, and then stimulated with ACh (10 -6 mol/L) for 20 min. The amount of NOx was expressed as follows: non-stimulated NOx or ACh-stimulated NOx (10 -5 mol/20 (min) g (weight of the aorta)).
Taguchi K., Narimatsu H., Matsumoto T, & Kobayashi T. (2019). ERK-containing microparticles from a diabetic mouse induce endothelial dysfunction. The Journal of endocrinology, 241(3).
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5

Nitrate and Nitrite Quantification in Frozen Tissue

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Blood samples collected for dissection were immediately immersed in liquid nitrogen and stored at –80 °C until use. The samples were treated with plasma: methanol (1:1, volume/volume) to remove proteins and were then centrifuged (10,000× g, 5 min, room temperature). The nitrate and nitrite ion concentrations were measured using an HPLC system (ENO-20, Eicom, Kyoto, Japan) for NOx measurement based on the Griess method [55 (link),56 (link)].
Sonoda K., Kono Y., Kitamori K., Ohtake K., Shiba S., Kasono K, & Kobayashi J. (2022). Beneficial Effects of Dietary Nitrite on a Model of Nonalcoholic Steatohepatitis Induced by High-Fat/High-Cholesterol Diets in SHRSP5/Dmcr Rats: A Preliminary Study. International Journal of Molecular Sciences, 23(6), 2931.
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6

Quantification of Plasma Nitrate and Nitrite

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Plasma levels of nitrate and nitrite were assessed using a dedicated high-performance liquid chromatography system (ENO-20; Eicom) as previously described. 39 (link) The method is based on the separation of nitrate by reverse-phase or ion-exchange chromatography, followed by online reduction of nitrate to nitrite with cadmium and reduced copper. Derivatization of reduced nitrite was performed with Griess reagent, and the level of diazo compounds was measured at 540 nm.
Montenegro M.F., Sundqvist M.L., Larsen F.J., Zhuge Z., Carlström M., Weitzberg E, & Lundberg J.O. (2017). Blood Pressure-Lowering Effect of Orally Ingested Nitrite Is Abolished by a Proton Pump Inhibitor. Hypertension (Dallas, Tex. : 1979), 69(1).
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7

Nitrite Quantification in Pulmonary Arteries

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pulmonary arteries were bathed isolated, blotted dry and weighed and then placed in 200μl saline containing Ca2+/Mg2+ at 37°C. After 15mins equilibration, A23187 (5μM) was added for a further 15min. 100μl buffer was carefully aspirated and nitrite levels measured by the Griess assay coupled with HPLC detection using the ENO-20 (EiCOM) as previously described (Samal et al. 2012 (link)).
Honavar J., Bradley E., Bradley K., Oh J.Y., Vallejo M.O., Kelley E.E., Cantu-Medellin N., Doran S., Dell’italia L.J., Matalon S, & Patel R.P. (2014). Chlorine gas exposure disrupts nitric oxide homeostasis in the pulmonary vasculature. Toxicology, 321, 96-102.
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8

Quantification of Nitric Oxide Metabolites

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For the analysis of nitric oxide (NO) metabolites, blood and tissue were collected as described previously [8 (link)]. At the beginning of the experiment, mice were anaesthetised with isoflurane (2.0%) and blood was taken by heart punction and collected in a heparinized syringe. Afterwards the blood was transferred directly into tubes containing N-ethylmaleimide (NEM)/EDTA (10:1 v/v), dissolved in phosphate-buffered solution (PBS) at pH 7.4 (final concentrations: 10 mM NEM, 2 mM EDTA), and centrifuged immediately for 3 min at 3000g. Tissue was removed after 1 min of perfusion with ice cold 10 mM NEM/2 mM EDTA in PBS pH 7.4, blotted dry on filter paper, weighed, snap frozen in liquid nitrogen, and kept at − 80 °C until later analysis. Nitrosated (S-nitroso and N-nitroso) products (RXNO) and NO haem were quantified by gas phase chemiluminescence as described [19 (link)]. The analysis of nitrite/nitrate was done in deproteinized NEM-treated samples with ice-cold methanol (1:1 v/v), cleared by centrifugation and subjected to analysis a gas phase chemiluminescence-based technique as well as a high pressure liquid chromatography (HPLC) using a dedicated nitrite/nitrate analyser (ENO20, Eicom) as described [8 (link), 33 (link)]. Data are given for the respective volume of plasma and RBC in 1 mL blood volume normalised to haematocrit in mice with comparable body weight.
Wischmann P., Kuhn V., Suvorava T., Muessig J.M., Fischer J.W., Isakson B.E., Haberkorn S.M., Flögel U., Schrader J., Jung C., Cortese-Krott M.M., Heusch G, & Kelm M. (2020). Anaemia is associated with severe RBC dysfunction and a reduced circulating NO pool: vascular and cardiac eNOS are crucial for the adaptation to anaemia. Basic Research in Cardiology, 115(4), 43.
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9

Quantifying Plasma and Urinary Nitric Oxide

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Insulin ELISAs were purchased from Mercodia (Uppsala, Sweden). Multiplex proinflammatory 7 cytokine kit and custom made metabolic kit (Glucagon, active GLP-1, Insulin, and Leptin) were purchased from Mesoscale Discoveries (Rockville, MD, USA). T3 and T4 ELISA kits were purchased from Calbiotech (Spring Valley, CA, USA). cGMP EIA was purchased from GE Healthcare. All kits were run according to manufacturers' instructions. Plasma nitrite and nitrate and urinary nitrate were analyzed by HPLC (ENO-20) and autosampler (840, EiCom, Kyoto, Japan). Plasma was extracted using methanol (1:2) then centrifuged for 10 min 4 °C 10g. Urine samples were initially diluted (1:50) using carrier solution containing 10% methanol. Nitrate and nitrite were separated by reverse phase/ion exchange chromatography followed by nitrate reduction to nitrite by cadmium and reduced copper. The nitrite was then derivatized using Griess reagent to form diazo compounds and analyzed by detection at 540 nm.
Hezel M.P., Liu M., Schiffer T.A., Larsen F.J., Checa A., Wheelock C.E., Carlström M., Lundberg J.O, & Weitzberg E. (2015). Effects of long-term dietary nitrate supplementation in mice. Redox Biology, 5, 234-242.
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10

Plasma Nitrate and Nitrite Analysis

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Mice were anaesthetized using 5% isoflurane and 4 L/min oxygen, and blood was collected via intracardiac puncture into EDTA-containing anticoagulant tubes containing 100 µL 10 mM N-ethylmaleimide. Plasma was generated by centrifugation at 2000 × g for 10 min at room temperature, then snap-frozen using liquid nitrogen and stored at −80°C until analysis. Plasma concentrations of nitrate and nitrite were determined by a dedicated HPLC system (ENO-20; Eicom), as previously reported (28 (link)).
Ntessalen M., Procter N.E., Schwarz K., Loudon B.L., Minnion M., Fernandez B.O., Vassiliou V.S., Vauzour D., Madhani M., Constantin‐Teodosiu D., Horowitz J.D., Feelisch M., Dawson D., Crichton P.G, & Frenneaux M.P. (2019). Inorganic nitrate and nitrite supplementation fails to improve skeletal muscle mitochondrial efficiency in mice and humans. The American Journal of Clinical Nutrition, 111(1), 79-89.
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