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1Division of Biochemistry, Department of Basic Sciences, Loma Linda University, 2Division of Physiology, Department of Basic Sciences, Loma Linda University
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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).
Plank, M. S., Calderon, T. C., Asmerom, Y., Boskovic, D. S., Angeles, D. M. Biochemical Measurement of Neonatal Hypoxia. J. Vis. Exp. (54), e2948, doi:10.3791/2948 (2011).
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.
1. Sample Collection and Processing
2. Preparing Internal Standard, 2-Aminopurine (2-AP), for Purine and XO Analysis
3. HPLC Measurement of Purines
Table 1. Solvent changes for HPLC measurement of purine compounds.
|Retention Time*||Observed λmax*||Reported λmax19||Reported εmax19|
|Uric Acid||~3.5 min||288||283 ||11,500 |
|Hypoxanthine||~7.0 min||248||248||10,800 |
|Xanthine||~9.5 min||267||267 ||10,200 |
|2-Aminopurine||~12.5 min||305||314 ||4,000 |
Table 2. Typical retention times and λmax for purines and internal standard. *Determined on HPLC using isocratic 50mM ammonium formate buffer (pH 5.5) with a flow rate of 1mL/min. pH is in [ ].
4. GC/MS Measurement of Allantoin
5. Preparing Internal Standard, Methyl Malondialdehyde (MMDA), for MDA Analysis
6. GC/MS Measurement of MDA
7. HPLC Measurement of Xanthine Oxidase
8. Representative Results:
An example of the HPLC quantification of purine compounds is shown in Figure 1A. The specific retention times and emission wavelengths of hypoxanthine, xanthine, and uric acid permit the simultaneous quantification of purine compounds (Table 2). When the assay is run correctly, the compounds will have adequate separation and the peak shape will be sharp and unimodal. These peaks are then converted into concentrations, ranges shown in Table 3, through the use of standard curves. Because sample processing for this assay is minimal, the only sample based problems which may arise would be the lysing of the red blood cells. If the red blood cells lyse before the samples are centrifuged, the plasma will take on an orange/red color and cannot be used for evaluating hypoxic ischemia. The other issues which may arise when measuring purines involves the HPLC system and the column (Figure 1B). If there are air bubbles in the HPLC system the retention times will shift and the HPLC pressure will fluctuate dramatically. If the guard cartridge needs to be changed, the pressure will increase and the peaks will widen and become bi or tri-modal.
|Hypoxanthine (μM)||Xanthine (μM)||Uric Acid (μM)||Xanthine Oxidase ((nmol product)/min)||Malondialdehyde (μM)||Allantoin (μM)|
|Term Normoxic||1.13-19.3 (64)||0.02-3.69 (61)||107.20-726.12 (63)||2.47x10-6 - 3.92x10-3 (20)||0.44-3.76 (53)||NA|
|Term Respiratory Distress||1.78-12.59 (27)||0.07-11.8 (24)||225.40-653.32 (27)||0 - 1.03x10-5 (8)||0.82-2.73 (24)||NA|
|Term Hypoxic||0.38-31.80 (13)||0.11-2.88 (13)||235.65-1348.13 (13)||1.20x10-5 -236.44 (9)||0.95-2.15 (7)||NA|
|Preterm Normoxic||1.54-4.39 (9)||0.03-1.77 (9)||178.92-593.49 (9)||2.46x10-5 - 5.44x10-4 (2)||0.95-2.74 (8)||2.30-5.26 (67)|
|Preterm Respiratory Distress||3.04-8.04 (2)||NA||327.56-365.11 (2)||NA||2.40-3.46 (3)||NA|
Table 3. Representative ranges for purines, xanthine oxidase, malondialdehyde, and allantoin.
An example of the GC/MS quantification of Allantoin is shown in Figure 2. Because the mass of derivitized allantoin and derivitized heavy allantoin is known, select ion mode can be used to identify these compounds on the mass spec. If the assay is done correctly, two peaks will be observed at the same retention time. One corresponding to allantoin (398.00 m/z) and the other to heavy allantoin (400.00 m/z). These peaks are then converted into concentrations, ranges shown in Table 3, through the use of a standard curve If the assay is run incorrectly, and samples were not derivitized properly, the peaks may not be present or may not be quantitatively representative. Once again, if the red blood cells lysed the plasma cannot be used to evaluate oxidative stress in neonatal hypoxic ischemia.
The results for the quantification of MDA are similar to those for allantoin with the exception that the two peaks are observed at different retention times. At ~ 3.5 minutes retention time, a 144.00 m/z peak for MDA and at ~4 minutes retention time, a 158.00 m/z peak for MMDA is observed (Figure 3). These peaks are then converted into concentrations, ranges shown in Table 3, through the use of a standard curve. If the assay is run incorrectly, or samples are not derivitized properly, no peaks may be observed when selecting for 144.00m/z and 158.00m/z. It should be noted that if there is an excess of lipid in the plasma from bolus oral feedings or intravenous lipid administration, the plasma will take on a milky appearance and cannot be used to evaluate oxidative stress in neonatal hypoxic ischemia.
An example of the HPLC-based quantification of xanthine oxidase function is shown in Figure 4. If the assay is run correctly, three peaks should be observed with the fluorescent detector, one for pterin, one for isoxanthopterin, and one for 2-AP. These peaks are then converted into concentrations, ranges shown in Table 3, through the use of a standard curve. There should also be a peak corresponding to isoxanthopterin and 2-AP on the spectrum generated from the PDA detector. If the enzyme activity is absent, the peak corresponding to isoxanthopterin will not be seen. Because this assay measures enzyme function, repeated freezing and thawing of the sample may alter this.
Figure 1. HPLC spectrum for the identification of purine compounds. A). Representative results if the assay was run correctly. B). representative results if there is a problem with the HPLC, column, or guard cartridge.
Figure 2. GC/MS spectrum for the quantification of Allantoin. The peak at ion 398.00 m/z corresponds to allantoin. The peak at ion 400.00 m/z corresponds to heavy allantoin.
Figure 3. GC/MS spectrum for the quantification of MDA. The peak at ion 144.00 m/z corresponds to MDA. The peak at ion 158.00 m/z corresponds to MMDA.
Figure 4. HPLC spectrum for the measurement of XO activity. A) Representative results for the 0 min incubation time and B) representative results for the 4 hour incubation time. Note the higher isoxanthopterine peak (at ~17 min) for the 4 hour incubation time. In the fluorescence spectrum, 2-AP elutes first at ~ 5min and pterine elutes next at ~10min.
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 children, but it becomes a concern when working with neonates, especially those who are premature. We work around this issue by prioritizing assays and diluting some of the samples if necessary. Secondly, it can be difficult to synthesize MMDA; however, deuterated MDA can be used as a substitute for MMDA as an internal standard.
Despite these limitations, the methods described provide a useful tool in evaluating neonatal hypoxia ischemia as well as other disorders associated with unbalanced redox homeostasis. Moreover, all of these markers, with the exception of XO, can be measured in urine, providing a non-invasive means of monitoring neonates.
No conflicts of interest declared.
This work is funded by National Institutes of Health R01 NR011209-03
|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|
|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|
|GCMS Vial||Fisher Scientific||03376607|
|DL-Allantoin-5-13C;1-15N||CDN Isotopes||M-2307||Lot #L340P9|
|MTBSTFA||Thermo Fisher Scientific, Inc.||48920|
|5973E GC/MSD||Agilent Technologies||G7021A||Part # for 5975E GC/MS|
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