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Htec 500 detector

Manufactured by Eicom
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The HTEC 500 detector is a laboratory instrument designed to detect and measure the presence of specific analytes in a sample. It functions by employing advanced sensor technology to identify and quantify the target substances. The HTEC 500 provides accurate and reliable data to support various analytical applications.

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Lab products found in correlation

Hr 80, by Thermo Fisher Scientific (1 mentions) Bds hypersil, by Thermo Fisher Scientific (1 mentions) Adsorbosil, by Grace Bio-Labs (1 mentions)

9 protocols using htec 500 detector

1

Quantification of 3-Hydroxykunurenine by HPLC

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For 3-HK determination, 20 μL of the supernatant were applied to a 3 μm C18 reverse phase HPLC column (HR-80; 80 mm × 4.6 mm; Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase consisted of 1.5% acetonitrile, 0.9% trimethylamine, 0.59% phosphoric acid, 0.27 mM EDTA and 8.9 mM sodium heptane sulfonic acid. 3-HK was eluted at a flow rate of 0.5 ml/min and detected electrochemically using an HTEC 500 detector (Eicom, San Diego, CA, USA; oxidation potential: + 0.5 V). The retention time of 3-HK was approximately 11 min.
Notarangelo F.M., Beggiato S, & Schwarcz R. (2019). Assessment of Prenatal Kynurenine Metabolism using Tissue Slices: Focus on the Neosynthesis of Kynurenic Acid in Mice. Developmental neuroscience, 41(1-2), 102-111.
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2

Measurement of Kynurenine Pathway Enzyme Activity

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Cell pellets were thawed and homogenized by sonication in 400 µL of ultrapure water (Branson Ultrasonics, Brookfield, CT, USA). The homogenate was further diluted (1:2, v/v) in 100 mM Tris-HCl (pH 8.1) containing 10 mM KCl and 1 mM EDTA. Then, 80 µL of the preparation was incubated for 40 min at 37 °C in a solution containing 1 mM NADPH, 3 mM glucose-6-phosphate, 1 U/mL glucose-6 phosphate dehydrogenase, 100 µM L-kynurenine, 10 mM KCl and 1 mM EDTA, in a total volume of 200 µL. Blanks were obtained by including the specific enzyme inhibitor Ro 61-8048 (100 μM) in the incubation solution. The reaction was stopped by the addition of 50 μL of 6% perchloric acid. The samples were centrifuged at 16,000× g, 15 min, and the resulting supernatants were diluted as needed. Then, 20 μL of the solution was applied to a 3 μm HPLC column (HR-80; 80 mm × 4.6 mm, ESA), using a mobile phase consisting of 1.5% acetonitrile, 0.9% triethylamine, 0.59% phosphoric acid, 0.27 mM EDTA and 8.9 mM sodium heptane sulfonic acid. In the eluate, the reaction product, 3-HK, was detected electrochemically using an Eicom HTEC 500 detector (oxidation potential: +0.5 V; flow rate: 0.5 mL/min). The retention time of 3-HK was ~11 min. Protein was determined according to Lowry et al. [36 (link)], using bovine serum albumin as a standard.
Swaih A.M., Breda C., Sathyasaikumar K.V., Allcock N., Collier M.E., Mason R.P., Feasby A., Herrera F., Outeiro T.F., Schwarcz R., Repici M, & Giorgini F. (2022). Kynurenine 3-Monooxygenase Interacts with Huntingtin at the Outer Mitochondrial Membrane. Biomedicines, 10(9), 2294.
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3

Measuring Kynurenine Metabolism in Brain Tissues

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Rat and human brain tissues were weighed while frozen and then homogenized (rat: 1:15, w/v; human: 1:25, w/v) by sonication (Branson Ultrasonics) in 100 mM Tris–HCl buffer (pH 8.1) containing 10 mM KCl and 1 mM EDTA. Eighty μl of the preparation were incubated for 40 min at 37°C in a solution containing 1 mM NADPH, 3 mM glucose-6-phosphate, 1 U/ml glucose-6 phosphate dehydrogenase, 100 μM kynurenine, 10 mM KCl and 1 mM EDTA, in a total volume of 200 μl. To examine the effect of NAC, 20 μl of the drug solution (pH 7.8) were added to the incubation mixture. Blanks were obtained by adding the KMO inhibitor Ro 61-8048 (final concentration: 100 μM). The reaction was stopped by the addition of 50 μl of 6% perchloric acid. After centrifugation (16,000 x g, 15 min), 20 μl of the supernatant were applied to a 3 μm HPLC column (HR-80; 80 mm x 4.6 mm; ESA, Chelmsford, MA, USA), using a mobile phase consisting of 1.5% acetonitrile, 0.9% triethylamine, 0.59% phosphoric acid, 0.27 mM EDTA and 8.9 mM sodium heptane sulfonic acid, and a flow rate of 0.5 ml/min. In the eluate, the reaction product, 3-HK, was detected electrochemically using a HTEC 500 detector (Eicom Corp., San Diego, CA, USA; oxidation potential: +0.5 V) (Heyes & Quearry 1988 (link)). The retention time of 3-HK was ~10 min.
Ayala T.B., Sathyasaikumar K.V., Uys J.D., Peréz-de-la-Cruz V., Pidugu L.S, & Schwarcz R. (2020). N-Acetylcysteine Inhibits Kynurenine Aminotransferase II. Neuroscience, 444, 160-169.
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4

Quantification of 5-HIAA in Plasma

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A volume of 50 μL of plasma was mixed with 150 μL of ultrapure water and deproteinized by the addition of 50 μL 6% perchloric acid. Following centrifugation (16,000× g, 15 min) and recentrifugation of the resulting supernatant (again at 16,000× g, 5 min), the final supernatant was diluted (1:5, v/v) with ultrapure water, and 20 μL was injected onto a 3 μm HPLC column (BDS HyperSil; 150 mm x 3 mm; Thermo Fisher Scientific, Pittsburgh, PA, USA). 5-HIAA was eluted using a mobile phase containing 75 mM sodium monophosphate, 0.01% triethylamine, 25 μM EDTA, 1.7 mM octane sulfonic acid and 5% acetonitrile (pH 3.0) at a flow rate of 0.4 mL/min. In the eluate, 5-HIAA was measured electrochemically using an HTEC 500 detector (Eicom Corp., San Diego, CA, USA; oxidation potential: 450 mV) [90 (link)]. The retention time of 5-HIAA was ~12.5 min.
Sathyasaikumar K.V., Notarangelo F.M., Kelly D.L., Rowland L.M., Hare S.M., Chen S., Mo C., Buchanan R.W, & Schwarcz R. (2022). Tryptophan Challenge in Healthy Controls and People with Schizophrenia: Acute Effects on Plasma Levels of Kynurenine, Kynurenic Acid and 5-Hydroxyindoleacetic Acid. Pharmaceuticals, 15(8), 1003.
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5

Quantification of 3-Hydroxykyanurenine in Brain and Liver

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Brain (1:5, w/v) and liver (1:5, w/v) were homogenized, and plasma was diluted (1:2, v/v), in ultrapure water. Twenty-five μL of perchloric acid (6%) were then added to 100 μL of the preparation, and precipitated proteins were removed by centrifugation (16,000 × g, 15 min). Twenty μL of the resulting supernatant were injected onto a 3 μM HPLC column (80 mm × 4.6 mm; ESA), using a mobile phase consisting of 1.5% acetonitrile, 0.9% triethylamine, 0.59% phosphoric acid, 0.27 mM EDTA and 8.9 mM sodium heptanesulfonic acid, and a flow rate of 0.5 mL/min. In the eluate, 3-HK was detected electrochemically using an HTEC 500 detector (Eicom, San Diego, CA, USA; oxidation potential: + 0.5 V). The retention time of 3-HK was approximately 11 min.
Notarangelo F.M., Wang X.D., Horning K.J, & Schwarcz R. (2016). Role of D-amino acid oxidase in the production of kynurenine pathway metabolites from D-tryptophan in mice. Journal of neurochemistry, 136(4), 804-814.
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6

HPLC Detection of Xanthurenic Acid

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Twenty μL of the same supernatant used for KYNA analysis were applied to a 5 μm C18 reverse-phase HPLC column (Adsorbosil; 150 mm × 4.6 mm; Grace, Deerfield, IL, USA), and XA was isocratically eluted using a mobile phase containing citric acid buffer (pH 3.0), 7–8% acetonitrile at a flow rate of 600 μL/min (Okech et al., 2006 (link)). In the eluate, XA was detected electrochemically using a HTEC 500 detector (Eicom Corp., San Diego, CA, USA; oxidation potential: +0.85 V). The retention time of XA was ~9 min.
In pilot experiments, we also used UV detection to measure XA. To this end, 20 μl of the supernatant were applied to a 5 pm C18 reverse-phase HPLC column (150 mm × 4.6 mm; Adsorbosil; Grace, Deerfield, IL, USA), and XA was isocratically eluted using a mobile phase containing 0.1 mM citric acid buffer (pH 3.0) and 8% acetonitrile at a flow rate of 800 μL/min. In the eluate, XA was detected by UV absorbance at 340 nm (SPD-10A VP series; Shimadzu Corp., Columbia, MD, USA). The retention time of XA was ~8 min.
Sathyasaikumar K.V., Tararina M., Wu H.Q., Neale S.A., Weisz F., Salt T.E, & Schwarcz R. (2017). Xanthurenic Acid Formation from 3-Hydroxykynurenine in the Mammalian Brain: Neurochemical Characterization and Physiological Effects. Neuroscience, 367, 85-97.
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7

HPLC Analysis of 3-Hydroxykinurenine

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Cortical samples were weighed, sonicated (1:5, w/v) in ultrapure water and aliquoted. Twenty-five μl of 6% perchloric acid were used to acidify 100 μl of diluted sample. After centrifugation (12,000 × g, 10 min), 20 μl of the supernatant was subjected to HPLC. 3-HK was eluted from a 3-μm HPLC column (HR-80; 80 × 4.6 mm; ESA) using a mobile phase of 1.5% acetonitrile, 0.9% triethylamine, and 0.59% phosphoric acid at a flow rate of 0.5 mL/min and detected electrochemically using an HTEC 500 detector (Eicom, San Diego, CA, USA; oxidation potential: + 0.5 V).
Baratta A.M., Buck S.A., Buchla A.D., Fabian C.B., Chen S., Mong J.A, & Pocivavsek A. (2018). Sex Differences in Hippocampal Memory and Kynurenic Acid Formation Following Acute Sleep Deprivation in Rats. Scientific Reports, 8, 6963.
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8

HPLC-EC Detection of 3-Hydroxykinurenine

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Fetal brain (1:10, w/v) and placenta (1:20, w/v) were homogenized, and maternal (1:2, v/v) and fetal plasma (1:4, v/v) were diluted, in ultrapure water. Twenty-five μL of 25% perchloric acid were then added to 100 μL of each preparation, and precipitated proteins were removed by centrifugation (16,000 × g, 15 min). Twenty μL of the resulting supernatant were then injected onto a 3 μM HPLC column (80 mm × 4.6 mm; Thermo Fisher Scientific, Waltham, MA, USA), using a mobile phase consisting of 1.5% acetonitrile, 0.9% triethylamine, 0.59% phosphoric acid, 0.27 mM EDTA and 8.9 mM sodium heptane sulfonic acid, and a flow rate of 0.5 mL/min. In the eluate, 3-HK was detected electrochemically using an HTEC 500 detector (Eicom, San Diego, CA, USA; oxidation potential: + 0.5 V).
Goeden N., Notarangelo F.M., Pocivavsek A., Beggiato S., Bonnin A, & Schwarcz R. (2017). Prenatal dynamics of kynurenine pathway metabolism in mice: focus on kynurenic acid. Developmental neuroscience, 39(6), 519-528.
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9

Quantification of 3-Hydroxykhynurenine in Forebrain

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Forebrain was homogenized (1:5, w/v), and plasma was diluted (1:2, v/v), in ultrapure water. Twenty-five μL of 6% perchloric acid were added to 100 μL of the samples. After thorough mixing, the precipitated proteins were removed by centrifugation (16,000 × g, 15 min). Twenty μL of the resulting supernatant were applied to a 3 μm HPLC column (HR-80; 80 mm × 4.6 mm; ESA, Chelmsford, MA, USA), using a mobile phase consisting of 1.5 % acetonitrile, 0.9 % triethylamine, 0.59 % phosphoric acid, 0.27 mM EDTA and 8.9 mM sodium heptane sulfonic acid, and a flow rate of 0.5 mL/min. In the eluate, 3-HK was detected electrochemically using a HTEC 500 detector (Eicom Corp., San Diego, CA, USA; oxidation potential: +0.5 V). The retention time of 3-HK was ~11 min.
Larkin P.B., Sathyasaikumar K.V., Notarangelo F.M., Funakoshi H., Nakamura T., Schwarcz R, & Muchowski P.J. (2016). Tryptophan 2,3-Dioxygenfase and Indoleamine 2,3-Dioxygenase 1 Make Separate, Tissue-Specific Contributions to Basal and Inflammation-Induced Kynurenine Pathway Metabolism in Mice. Biochimica et biophysica acta, 1860(11 Pt A), 2345-2354.
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