Name: biochemical measurement of neonatal hypoxia
Uploaded: 2011-08-24T13:00:00
Description: Watch this Scientific Journal Video about Biochemical Measurement of Neonatal Hypoxia at JoVE.com

इस काम - R01 NR011209 03 स्वास्थ्य के राष्ट्रीय संस्थान द्वारा वित्त पोषित है

1. नमूना संग्रह और प्रसंस्करण <ol><li> 6ml K3E EDTA K3 ट्यूब जो बर्फ पर रखा जाता है में खून का नमूना ले लीजिए. </li><li> संग्रह के 2 मिनट के भीतर, 4 में नमूना अपकेंद्रित्र ° C 10 मिनट के लिए 1500 ग्राम में. </li><li> एक 1.5ml microcentrifuge ट्यूब स्थाना?...

<ol>
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I., Sustrova, M., Garaiova, I., Muchova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uric+acid+and+allantoin+levels+in+Down+syndrome:+antioxidant+and+oxidative+stress+mechanisms?.">Uric acid and allantoin levels in Down syndrome: antioxidant and oxidative stress mechanisms?.</a> Clin Chim Acta. 341, 139-146 (2004).</li><li>Siciarz, A., Weinberger, B., Witz, G., Hiatt, M., Hegyi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+thiobarbituric+acid-reacting+substances+as+potential+biomarkers+of+intrauterine+hypoxia.">Urinary thiobarbituric acid-reacting substances as potential biomarkers of intrauterine hypoxia.</a> Arch Pediatr Adolesc Med. 155, 718-722 (2001).</li><li>Buonocore, G., Perrone, S., Longini, M., Terzuoli, L., Bracci, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+hydroperoxide+and+advanced+oxidation+protein+products+in+preterm+hypoxic+babies.">Total hydroperoxide and advanced oxidation protein products in preterm hypoxic babies.</a> Pediatr Res. 47, 221-224 (2000).</li><li>Berne, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+nucleotides+in+hypoxia:+possible+role+in+regulation+of+coronary+blood+flow.">Cardiac nucleotides in hypoxia: possible role in regulation of coronary blood flow.</a> Am J Physiol. 204, 317-322 (1963).</li><li>Harkness, R. A., Lund, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+concentrations+of+hypoxanthine,+xanthine,+uridine+and+inosine:+high+concentrations+of+the+ATP+metabolite,+hypoxanthine,+after+hypoxia.">Cerebrospinal fluid concentrations of hypoxanthine, xanthine, uridine and inosine: high concentrations of the ATP metabolite, hypoxanthine, after hypoxia.</a> J Clin Pathol. 36, 1-8 (1983).</li><li>Plank, M. S., Boskovic, D. S., Sowers, L. C., Angeles, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+markers+of+neonatal+hypoxia.">Biochemical markers of neonatal hypoxia.</a> Pediatric Health. 2, 485-501 (2008).</li><li>Cighetti, G., Allevi, P., Anastasia, L., Bortone, L., Paroni, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+methyl+malondialdehyde+as+an+internal+standard+for+malondialdehyde+detection:+validation+by+isotope-dilution+gas+chromatography-mass+spectrometry.">Use of methyl malondialdehyde as an internal standard for malondialdehyde detection: validation by isotope-dilution gas chromatography-mass spectrometry.</a> Clin Chem. 48, 2266-2269 (2002).</li><li>Paroni, R., Fermo, I., Cighetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+methyl+malondialdehyde+as+internal+standard+for+malondialdehyde+detection+by+capillary+electrophoresis.">Validation of methyl malondialdehyde as internal standard for malondialdehyde detection by capillary electrophoresis.</a> Anal Biochem. 307, 92-98 (2002).</li><li>Cighetti, G., Debiasi, S., Ciuffreda, P., Allevi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-ethoxyacrolein+contamination+increases+malondialdehyde+inhibition+of+milk+xanthine+oxidase+activity.">Beta-ethoxyacrolein contamination increases malondialdehyde inhibition of milk xanthine oxidase activity.</a> Free Radic Biol Med. 25, 818-825 (1998).</li><li>Cighetti, G., Debiasi, S., Paroni, R., Allevi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+and+total+malondialdehyde+assessment+in+biological+matrices+by+gas+chromatography-mass+spectrometry:+what+is+needed+for+an+accurate+detection.">Free and total malondialdehyde assessment in biological matrices by gas chromatography-mass spectrometry: what is needed for an accurate detection.</a> Anal Biochem. 266, 222-229 (1999).</li><li>Yamamoto, T., Moriwaki, Y., Takahashi, S., Tsutsumi, Z., Yamakita, J., Nasako, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+human+plasma+xanthine+oxidase+activity+by+high-performance+liquid+chromatography.">Determination of human plasma xanthine oxidase activity by high-performance liquid chromatography.</a> J Chromatogr B Biomed Appl. 681, 395-400 (1996).</li><li>Fasman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Biochemistry and Molecular Biology. , (1988).</li><li>Chen, X. B., Calder, A. G., Prasitkusol, P., Kyle, D. J., Jayasuriya, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+15N+isotopic+enrichment+and+concentrations+of+allantoin+and+uric+acid+in+urine+by+gas+chromatography/mass+spectrometry.">Determination of 15N isotopic enrichment and concentrations of allantoin and uric acid in urine by gas chromatography/mass spectrometry.</a> J Mass Spectrom. 33, 130-137 (1998).</li></ol>

यहाँ वर्णित तरीकों नवजात hypoxia ischemia के मूल्यांकन की अनुमति. इस प्रोटोकॉल की उपस्थिति एक समग्र जैव रासायनिक चित्र या यहाँ तक कि hypoxic ischemia की डिग्री हासिल करने के लिए ऊर्जा के अभाव (एटीपी), oxidative तनाव, oxidative नुकसान, और...

<table border="1"><tbody><tr><th> अभिकर्मक के नाम </th><th> कंपनी </th><th> सूचीपत्र संख्या </th><th> टिप्पणियां (वैकल्पिक) </th></tr><tr><td> 6ml K3E EDTA K3 ट्यूब </td><td> फिशर साइंटिफिक </td><td> 2204061 </td><td></td></tr><tr><td> 5702R अपकेंद्रित्र </td><td> फिशर साइंटिफिक </td><td> 05413319 </td><td> के साथ 13 और 16mm एडाप्टर </td></tr><tr><td> 1.5ml microcentrifuge ट्यूब </td><td> संयुक्त राज्य अमेरिका वैज्ञानिक </td><td> 1615-5599 </td><td></td></tr><tr><td> 2-Aminopurine </td><td> सिग्मा Aldrich </td><td> A3509 </td><td></td></tr><tr><td> Varian 100 स्पेक्ट्रोफोटोमीटर Cary </td><td> Agilant टेक्नोलॉजीज </td><td> 0010071500 </td><td></td></tr><tr><td> पंडित SpeedVac </td><td> थर्मो वैज्ञानिक </td><td> SC210A-115 </td><td></td></tr><tr><td> माइक्रोन केन्द्रापसारक फिल्टर डिवाइस </td><td> फिशर साइंटिफिक </td><td> UFC501596 </td><td></td></tr><tr><td> Supelcosil LC-18-एस स्तंभ </td><td> सिग्मा Aldrich </td><td> 58931 </td><td></td></tr><tr><td> Supelcosil LC-18-एस Supelguard कारतूस और धारक </td><td> सिग्मा Aldrich </td><td> 59629 </td><td></td></tr><tr><td> HPLC </td><td> वाटर्स </td><td></td><td></td></tr><tr><td> GCMS शीशी </td><td> फिशर साइंटिफिक </td><td> 03376607 </td><td></td></tr><tr><td> डीएल-allantoin 5 -13 सी, 1 -15 एन </td><td> CDN आइसोटोप </td><td> M-+२३०७ </td><td> L340P9 # लूत </td></tr><tr><td> MTBSTFA </td><td> थर्मो वैज्ञानिक </td><td> 48920 </td><td></td></tr><tr><td> Pyridine </td><td> सिग्मा Aldrich </td><td> 270970 </td><td></td></tr><tr><td> 5973E जीसी / एमएसडी </td><td> Agilent टेक्नोलॉजीज </td><td> G7021A </td><td> # पार्ट 5975E जीसी / एमएस के लिए </td></tr><tr><td> 3 - Ethoxymethacrolein </td><td> सिग्मा Aldrich </td><td> 232548 </td><td></td></tr><tr><td> सोडियम हीड्राकसीड </td><td> सिग्मा Aldrich </td><td> S5881 </td><td></td></tr><tr><td> Dichloromethane </td><td> सिग्मा Aldrich </td><td> 270997 </td><td></td></tr><tr><td> बेंजीन </td><td> सिग्मा Aldrich </td><td> 401765 </td><td></td></tr><tr><td> Diisopropyl ईथर </td><td> सिग्मा Aldrich </td><td> 38270 </td><td></td></tr><tr><td> BHT </td><td> सिग्मा Aldrich </td><td> B1378 </td><td></td></tr><tr><td> इथेनॉल </td><td> सिग्मा Aldrich </td><td> 459844 </td><td></td></tr><tr><td> Phenylhydrazine </td><td> सिग्मा Aldrich </td><td> P26252 </td><td></td></tr></tbody></table>

biochemical measurement of neonatal hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

Watch this Scientific Journal Video about Biochemical Measurement of Neonatal Hypoxia at JoVE.com

Biochemical Measurement of Neonatal Hypoxia

एक विधि नवजात hypoxia-ischemia के जैव रासायनिक मार्करों के उपाय वर्णित है. दृष्टिकोण उच्च दबाव तरल क्रोमैटोग्राफी (HPLC) और गैस क्रोमैटोग्राफी मास स्पेक्ट्रोमेट्री (जीसी / एमएस) का इस्तेमाल करता.

नवजात Hypoxia की बायोकेमिकल मापन

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

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