This work is funded by National Institutes of Health R01 NR011209-03

1. Sample Collection and Processing
<ol>
<li>Collect blood sample in a 6ml K3E EDTA K3 tube which is kept on ice.</li>
<li>Within 2 min of collection, centrifuge the sample at 4&deg;C at 1500 g for 10 min.</li>
<li>Transfer the supernatant (plasma) to a 1.5ml microcentrifuge tube.</li>
<li>Centrifuge at 4&deg;C at 18000 g for 30 min.</li>
<li>Remove the supernatant aliquots and transfer them into separate microcentrifuge tubes for purine (200&mu;l), allantoin (50&mu;l), MDA (100&mu;l), and XO (120&mu;l) analysis. Be c...

<ol>
	<li>Harkness, R. A., Whitelaw, A. G., Simmonds, 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=Intrapartum+hypoxia:+the+association+between+neurological+assessment+of+damage+and+abnormal+excretion+of+ATP+metabolites.">Intrapartum hypoxia: the association between neurological assessment of damage and abnormal excretion of ATP metabolites.</a> J Clin Pathol. 35, 999-1007 (1982).</li><li>Shalak, L., Perlman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxic-ischemic+brain+injury+in+the+term+infant-current+concepts.">Hypoxic-ischemic brain injury in the term infant-current concepts.</a> Early Hum Dev. 80, 125-141 (2004).</li><li>Webster, W. S., Abela, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+hypoxia+in+development.">The effect of hypoxia in development.</a> Birth Defects Res C Embryo Today. 81, 215-228 (2007).</li><li>Engerson, T. D., McKelvey, T. G., Rhyne, D. B., Boggio, E. B., Snyder, S. J., Jones, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+xanthine+dehydrogenase+to+oxidase+in+ischemic+rat+tissues.">Conversion of xanthine dehydrogenase to oxidase in ischemic rat tissues.</a> J Clin Invest. 79, 1564-1570 (1987).</li><li>Choi, E. Y., Stockert, A. L., Leimkuhler, S., Hille, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+mechanism+of+action+of+xanthine+oxidase.">Studies on the mechanism of action of xanthine oxidase.</a> J Inorg Biochem. 98, 841-848 (2004).</li><li>Godber, B. L., Schwarz, G., Mendel, R. R., Lowe, D. J., Bray, R. C., Eisenthal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+human+xanthine+oxidoreductase:+the+enzyme+is+grossly+deficient+in+molybdenum+and+substantially+deficient+in+iron-sulphur+centres.">Molecular characterization of human xanthine oxidoreductase: the enzyme is grossly deficient in molybdenum and substantially deficient in iron-sulphur centres.</a> Biochem J. 388, 501-508 (2005).</li><li>Gruber, J., Tang, S. Y., Jenner, A. M., Mudway, I., Blomberg, A., Behndig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allantoin+in+human+plasma,+serum,+and+nasal-lining+fluids+as+a+biomarker+of+oxidative+stress:+avoiding+artifacts+and+establishing+real+in+vivo+concentrations.">Allantoin in human plasma, serum, and nasal-lining fluids as a biomarker of oxidative stress: avoiding artifacts and establishing real in vivo concentrations.</a> Antioxid Redox Signal. 11, 1767-1776 (2009).</li><li>Zitnanova, I., Korytar, P., Aruoma, O. 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>

The methods described here permit the evaluation of neonatal hypoxia ischemia. This protocol combines the measurements of markers of energy (ATP) deprivation, oxidative stress, oxidative damage, and enzyme activity to gain an overall biochemical picture of the presence or even the degree of hypoxic ischemia. Despite the usefulness of this method, there are potential limitations. Firstly, it takes roughly 1-2 ml of blood to collect enough plasma to run all of the assays. This will not be a problem in adults or childre...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>6ml K3E EDTA K3 tube</td>
			<td>Fisher Scientific</td>
			<td>2204061</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5702R centrifuge</td>
			<td>Fisher Scientific</td>
			<td>05413319</td>
			<td>With 13&amp;16MM adaptor</td>
		</tr>
		<tr>
			<td>1.5ml microcentrifuge tube</td>
			<td>USA Scientific</td>
			<td>1615-5599</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2-Aminopurine</td>
			<td>Sigma-Aldrich</td>
			<td>A3509</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Varian Cary 100 spectrophotometer</td>
			<td>Agilant Technologies</td>
			<td>0010071500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Savant SpeedVac</td>
			<td>Thermo Scientific</td>
			<td>SC210A-115</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micron centrifugal filter device</td>
			<td>Fisher Scientific</td>
			<td>UFC501596</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Supelcosil LC-18-S Column</td>
			<td>Sigma-Aldrich</td>
			<td>58931</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Supelcosil LC-18-S Supelguard cartridge and holder</td>
			<td>Sigma-Aldrich</td>
			<td>59629</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HPLC</td>
			<td>Waters</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GCMS Vial</td>
			<td>Fisher Scientific</td>
			<td>03376607</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DL-Allantoin-5-13C;1-15N</td>
			<td>CDN Isotopes</td>
			<td>M-2307</td>
			<td>Lot #L340P9</td>
		</tr>
		<tr>
			<td>MTBSTFA</td>
			<td>Thermo Scientific</td>
			<td>48920</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pyridine </td>
			<td>Sigma-Aldrich</td>
			<td>270970</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5973E GC/MSD</td>
			<td>Agilent Technologies</td>
			<td>G7021A</td>
			<td>Part # for 5975E GC/MS</td>
		</tr>
		<tr>
			<td>3-Ethoxymethacrolein</td>
			<td>Sigma-Aldrich</td>
			<td>232548</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S5881</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Benzene</td>
			<td>Sigma-Aldrich</td>
			<td>401765</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Diisopropyl ether</td>
			<td>Sigma-Aldrich</td>
			<td>38270</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BHT</td>
			<td>Sigma-Aldrich</td>
			<td>B1378</td>
			<td>&nbsp;</td>
		</tr>
		
		<tr>
			<td>Ethanol </td>
			<td>Sigma-Aldrich</td>
			<td>459844</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phenylhydrazine</td>
			<td>Sigma-Aldrich</td>
			<td>P26252</td>
			<td>&nbsp;</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

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

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

biochemical-measurement-of-neonatal-hypoxia

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Medicine

14.0K vues.  Loma Linda University. Une méthode est décrite pour mesurer les marqueurs biochimiques du hypoxie-ischémie néonatale. L'approche utilise la chromatographie liquide à haute pression (HPLC) et chromatographie en phase gazeuse spectrométrie de masse (GC / MS).