Biochemical Measurement of Neonatal Hypoxia

Summary

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).

Abstract

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.

Protocol

1. Sample Collection and Processing

1. Collect blood sample in a 6ml K3E EDTA K3 tube which is kept on ice.
2. Within 2 min of collection, centrifuge the sample at 4°C at 1500 g for 10 min.
3. Transfer the supernatant (plasma) to a 1.5ml microcentrifuge tube.
4. Centrifuge at 4°C at 18000 g for 30 min.
5. Remove the supernatant aliquots and transfer them into separate microcentrifuge tubes for purine (200μl), allantoin (50μl), MDA (100μl), and XO (120μl) analysis. Be careful not to contaminate the samples with red blood cells. You may need to adjust the volumes of plasma for MDA, XO, and purines based upon the total volume of plasma a....

Discussion

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.......

Materials

List of materials used in this article

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