The overall goal of the following experiment is to measure biochemical markers of neonatal hypoxic ischemia. This is accomplished by first collecting blood samples from infants to isolate the plasma. Next, some of the plasma is filtered, combined with an internal standard and analyzed for hypoxanthine, santine, and uric acid concentrations on the HPLC two.
More plasma aliquots are then differentially processed and analyzed separately. For allantoin concentration and MDA levels on the GCMS, the final step of the procedure is to combine a portion of plasma with a substrate to initiate the enzymatic reaction after quenching the reaction. The activity of xanthe oxidase can be determined on an HPLC equipped with a fluorescent detector.
Ultimately, results are obtained that show chromatograms of peak areas, which can be converted into molar concentrations of the desired biochemical markers or enzyme activity via standard curves. The implications of this technique extend towards the therapy and diagnosis of neonatal hypoxia ischemia because this method combines measurements of markers of energy deprivation, oxidative stress, oxidative damage, and enzyme activity to generate an overall biochemical picture of the presence and even the degree of hypoxic ischemia Prior to the start of this procedure. A human blood sample is collected from a newborn immediately after birth in a six milliliter EDTA tube within less than five minutes.
After collection, centrifuge the sample at four degrees Celsius and 1, 500 times G for 10 minutes. Transfer the plasma, which is present in the supinate into a 1.5 milliliter micro centrifuge tube and centrifuge for 30 minutes. Remove the supinate taking care not to contaminate the samples with red blood cells.
Eloqua the samples into separate micro centri tubes for purine lanin, MDA and xanthe oxidase analysis for each PLC measurement of purines. Transfer 200 microliters of the plasma to a centrifugal filter device. Centrifuge of the plasma at four degrees Celsius and 14, 000 times G for 1.5 hours.
Remove the filtrate and transfer to a micro centrifuge tube containing two aino purine. Be sure to record the volume of filtrate added to the micro centrifuge tube containing two AP.To accurately determine the concentration. Vortex the tube for 10 to 20 seconds.
Analyze the samples with an HPLC as described in the written protocol using three 50 microliter injections for each sample from the resulting chromatograms. Determine the concentration of purines in the sample. Quantify hypoxanthine, xanthine and uric acid by obtaining peak areas at their corresponding retention times and wavelengths.
Next, quantify the peak area of two ap. From this information, the area ratios of hyper xanthine and uric acid to two AP can be calculated. Then convert the ratios to micromolar concentrations using standard curves To perform measurement of lanin, add 50 microliters of the internal standard and 100 microliters of aceto nitrile to 50 microliters of plasma.
After vortexing the mixture for 10 to 20 seconds. Centrifuge for 10 minutes following centrifugation. Remove the supinate and place it into a GCM S vial.
Dry the liquid under nitrogen gas. Once the liquid is dry, add 25 microliters of izing agents M-T-B-S-T-F-A and 25 microliters of pyridine. Cap the vial and incubate it at 50 degrees Celsius for two hours.
Then transfer the sample into another GCMS vial with a 300 microliter insert. Analyze the samples on the GCMS using an auto sampler. Perform compound separation using helium as the carrier gas as a flow rate of 1.5 milliliters per minute.
To begin the GCMS analysis, input the parameters for the run. Set the initial column temperature to 100 degrees Celsius and hold that temperature for two minutes before increasing it to 180 degrees Celsius at a rate of 10 degrees Celsius per minute. Hold the temperature for four minutes, then increase it to 260 degrees Celsius at a rate of 20 degrees Celsius per minute.
Maintain this temperature until the end of the run. Use the autos sampler to inject one microliter of the DERIVATIZED product in split mode and perform the run between each sample run. Have the auto sampler clean the column with two injections of one to one M-T-B-S-T-F-A pyridine.
To quantify a LAN toin, you select ion monitoring mode and monitor the 3 98 master charge ratio ion for atlanto in and the 400 master charge ratio for the heavy atlanto in. Then convert the ion abundance ratios of atlanto in to heavy atlanto in to micromolar concentrations of atlanto in using a prepared standard curve prior to the measurement of MDA, prepare butylated hydroxyl toluene, and 50 millimolar phenyl hydrazine solutions as described in the written protocol. ADD M-M-D-A-B-H-T and sodium citrate to the 100 microliter plasma sample.
Dilute the final mixture to 480 microliters by adding distilled deionized water. Derivatize the solution by adding 20 microliters of 50 millimolar phenol hydrazine. After capping the vial, incubate the solution on an orbital shaker.
Following incubation, add one milliliter of hexane vortex the mixture for one minute and centrifuge at 3000 rotations per minute for 10 minutes. After centrifugation, transfer the organic layer to a micro centrifuge tube. Then concentrate the solution to 100 microliters by evaporating under a stream of nitrogen gas and transfer it to A-G-C-M-S file with a 300 microliter insert.
Next, analyze the samples on A-G-C-M-S using the autos sampler performing compound separation and quantification of MDA as described in the written procedure. Begin detection of santhe oxidase activity by preparing two vials for plasma samples. One to incubate with the substrate for zero minutes as the control, and one to incubate for four hours.
As the experiment, add buffer and substrate to each tube. Pre incubate the samples at 37 degrees Celsius for five minutes. Initiate the enzyme reaction by adding 60 microliters of plasma to each tube.
Immediately add 300 microliters of 4%per chloric acid to the zero incubation tube. Conversely, allow the other mixture to incubate for four hours at 37 degrees Celsius before quenching the reaction after the addition of chloric acid. Vortex the zero incubation control tube and follow with centrifugation.
Remove 500 microliters of the supinate and add it to a tube containing 20 microliters of five molar potassium carbonate to neutralize the reaction vortex the neutralized sample and centrifuge for a second time. Then add 350 microliters of the neutralized solution to a micro centrifuge tube containing two ap. After incubating the four hour sample, quench the reaction by adding 300 microliters of 4%per chloric acid.
Continue processing of the sample as performed for the control sample. Analyze the samples on an HPLC equipped with a scanning fluorescence detector. Enter parameters using is Socratic conditions at a flow rate of one milliliter per minute.
To obtain adequate peak separation and identification, set the fluorescence detector excitation wavelength at 340 nanometers and the emission wavelength of 410 nanometers three 50. Microliter injections are run on the HPLC for each sample following the HPLC run. Determine the ratio of ISO terin to two AP by obtaining peak areas from the fluorescence detector spectrum.
The first peak in the spectrum at approximately five minutes corresponds to two ap. The second and largest peak will be terran. The ISO antho terin peak elutes last, obtain the difference in the ISO ANTOIN two AP peak area ratios between the zero and the four hour incubation time points.
Use this value to calculate the xanthine oxidase activity from the standard curves. An example of the HPLC quantification of puring compounds is shown the specific retention times and emission wavelengths of uric acid, hypoxanthine, and xanthine permit the simultaneous quantification of purine compounds. When the assay is run correctly, the compounds will have adequate separation and the peak shape will be sharp and unimodal.
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 by or trimodal as shown in this example here. An example of the GCMS quantification of lanin is shown because the massive derivatized, lanin and derivatized, heavy lanin is known.
Select ion mode can be used to identify these compounds on the mass spectrometer. If the assay is done correctly, two peaks will be observed at the same retention time. One peak corresponds to Alan Toin and the other to heavy toin.
The results for the quantification of MDA are similar to those for Alan Toin, with the exception that the two peaks are observed at different retention times. Here, a 144 master charge ratio peak for MDA and a 158 master charge ratio peak for MMDA are observed. An example of the HPLC based quantification of xanthe oxidase function is demonstrated at the zero minute incubation time and the four hour incubation time.
If the assay is run correctly, three peaks should be observed with the fluorescent detector, one for two ap, one for Terran, and one for ISO terin. Note, the higher ISO terin peak for the four hour incubation time due to increased santhe oxidase enzyme activity. Because this assay measures enzyme function repeated freezing and thawing of the sample may alter this.
After watching this video, you should have a good understanding of how to accurately measure several biochemical markers of neonatal hypoxia ischemia.
