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

Эта работа финансируется Национальным институтом здоровья R01 NR011209-03

1. Взятия пробы и перерабатывающей <ol><li> Сбор крови в 6 мл K3E ЭДТА K3 трубку, которая хранится на льду. </li><li> В течение 2 мин сбора, центрифуги образцов при температуре 4 ° С в 1500 г за 10 мин. </li><li> Передача супернатант (плазмы) в 1,5 мл трубки микроцентрифужных. </li><li> Центрифуга при 4 ° С при 18 000 г н?...

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Методы, описанные здесь разрешение оценки неонатального ишемии гипоксии. Этот протокол комбинирует измерения маркеров энергии (АТФ), лишения, окислительный стресс, окислительные повреждения, и активность ферментов, чтобы получить общую биохимическую картину наличия или даже степень ...

<table border="1"><tbody><tr><th> Название реагента </th><th> Компания </th><th> Номер в каталоге </th><th> Комментарии (необязательно) </th></tr><tr><td> 6 мл K3E ЭДТА K3 трубки </td><td> Fisher Scientific </td><td> 2204061 </td><td></td></tr><tr><td> 5702R центрифуги </td><td> Fisher Scientific </td><td> 05413319 </td><td> С 13 и 16 мм адаптер </td></tr><tr><td> 1,5 мл трубки микроцентрифужных </td><td> США Научная </td><td> 1615-5599 </td><td></td></tr><tr><td> 2-аминопурин </td><td> Sigma-Aldrich </td><td> A3509 </td><td></td></tr><tr><td> Varian Cary 100 спектрофотометр </td><td> Agilant Технологии </td><td> 0010071500 </td><td></td></tr><tr><td> Savant SpeedVac </td><td> Thermo Scientific </td><td> SC210A-115 </td><td></td></tr><tr><td> Micron центробежный фильтр устройства </td><td> Fisher Scientific </td><td> UFC501596 </td><td></td></tr><tr><td> Supelcosil LC-18-S колонка </td><td> Sigma-Aldrich </td><td> 58931 </td><td></td></tr><tr><td> Supelcosil LC-18-S Supelguard картридж и держатель </td><td> Sigma-Aldrich </td><td> 59629 </td><td></td></tr><tr><td> ВЭЖХ </td><td> Воды </td><td></td><td></td></tr><tr><td> GCMS флакон </td><td> Fisher Scientific </td><td> 03376607 </td><td></td></tr><tr><td> DL-Аллантоин-5 -13 С; 1 -15 N </td><td> CDN Изотопы </td><td> M-2307 </td><td> Лот № L340P9 </td></tr><tr><td> МТБСТФА </td><td> Thermo Scientific </td><td> 48920 </td><td></td></tr><tr><td> Пиридин </td><td> Sigma-Aldrich </td><td> 270970 </td><td></td></tr><tr><td> 5973E GC / MSD </td><td> Компания Agilent Technologies </td><td> G7021A </td><td> Часть # для 5975E ГХ / МС </td></tr><tr><td> 3-Ethoxymethacrolein </td><td> Sigma-Aldrich </td><td> 232548 </td><td></td></tr><tr><td> Едкий натр </td><td> Sigma-Aldrich </td><td> S5881 </td><td></td></tr><tr><td> Дихлорметан </td><td> Sigma-Aldrich </td><td> 270997 </td><td></td></tr><tr><td> Бензол </td><td> Sigma-Aldrich </td><td> 401765 </td><td></td></tr><tr><td> Диизопропил эфир </td><td> Sigma-Aldrich </td><td> 38270 </td><td></td></tr><tr><td> BHT </td><td> Sigma-Aldrich </td><td> B1378 </td><td></td></tr><tr><td> Этанол </td><td> Sigma-Aldrich </td><td> 459844 </td><td></td></tr><tr><td> Фенилгидразином </td><td> Sigma-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

Описан метод для измерения биохимических маркеров неонатальной гипоксии-ишемии. Подход использует высокого давления жидкостной хроматографии (ВЭЖХ) и газовой хроматографии-масс-спектрометрии (GC / MS).

Биохимические измерения новорожденных Гипоксия

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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