Malondialdhyde
Hemoglobin Back Extraction

Revision: 08/29/06


Figure 1.

Introduction
The presence of hemoglobin (Hb)in the sample interferes with the analysis of malondialdehyde (MDA). Figure 1 shows the MDA results of a set of plasma samples, measured using the NWLSS™ NWK-MDA01 assay, with varying levels on hemolysis. There is a high correlation between Hb concentration and the reported MDA concentration, r2=0.975, indicating the high probability that Hb is causing significant overestimation of the MDA concentration.

Badcock (Clin Chem 42:1655-1657, 1997) described reduction of interferences in the MDA-TBA reaction using butanol (BuOH) extraction of the TBA Rxm followed by back-extraction with sodium hydroxide (NaOH). The protocol to apply the Badcock extraction to the NWLSS™ NWK-MDA01 assay is shown below. The protocol is followed by a summary of the experimental data used to validate the method.

Protocol
  1. Add 250 µL sample or calibrator to tube
  2. Add 10 µL BHT to each tube
  3. Add 250 µL Acid Reagent to each tube
  4. Add 250 µL TBA Reagent to each tube
  5. Stopper
  6. Vortex
  7. Incubate at 60°C for 60 minutes
  8. Cool to room temperature
  9. Add 500µL BuOH to each tube
  10. Stopper
  11. Vortex
  12. Centrifuge (10,000 xg, 2-3 minutes)
  13. Transfer 400 µL of the BuOH fraction (upper, e.g. pink 4µM calibrator) to a second tube
  14. Add 500µL 1N NaOH to each tube
  15. Stopper
  16. Vortex
  17. Collect 400 µL of the NaOH fraction (lower) a third tube
  18. Add 100 µL 3.7 M phosphoric acid (25%) to all tubes
  19. Obtain absorption spectrum from 400-700 nm for each sample and calibrator (recommended)
  20. Analyze the data using Derivative Analysis or the Two Point method.

Experimental Results
The thiobarbituric (TBA) reaction mixture (Rxm), in the presence of Hb, produces a complex absorption spectra with a significant non-linear baseline. Of particular concern is the presence of the 540 nm absorption peak for methemoglobin which is in close proximity to the 532 nm peak for the MDA-TBA2 chromophore.

The hemoglobin interference issue was investigated by measuring the MDA in samples containing 0, 1.7 and 5.3 mg/mL hemoglobin (lysate) that were spiked with 2 µM of the MDA precursor, tetramethoxypropane (TMOP).


Figure 2.
Figure 3.
Table 1. Butanol Extraction
Method MDA Hemoglobin
1.7 mg/mL 5.2 mg/mL
Single Point
(532 nm)		 µM Added 0 2 0 2
µM Measured 6.00 7.68 16.09 19.32
%Recovery 84 162
Two Point
(532-573 nm)		 µM Added 0 2 0 2
µM Measured 2.03 4.40 6.37 8.81
%Recovery 118 122
Derivative µM Added 0 2 0 2
µM Measured 0.96 3.00 2.93 4.55
%Recovery 102 81

Following butanol (BuOH) extraction, the absorption spectra was measured. Figure 2 shows the zero order spectra for the reaction mixtures. The MDA concentration was estimated using the 532 nm absorption values (TBARS, Single Point) and the Two Point method (Jentzsch, FRBM 20:251-256; 1996). The Single Point results significantly over-estimate the MDA concentration and shows poor recovery. The Two Point calculation achieves a reasonable recovery but the samples without TMOP over-estimate MDA by 5-7 fold (see NaOH Extraction below). Figure 3 is the 3rd-derivative spectra for the 5.2 mg/mL Hb sample. The derivative results are an improvement but over-estimate the MDA by a factor of 3.

Figure 4 shows that much of the absorbance contributed by Hb has been removed using the Badcock back-extraction. The recoveries using both the Single point and Two Point methods are significantly better; the Two Point MDA estimate in the TMOP free sample is within 0.1 micro;M of the Derivative value.


Figure 4.
Figure 5.
Table 1. Sodium Hydroxide Back-Extraction
Method MDA Hemoglobin
1.7 mg/mL 5.2 mg/mL
Single Point µM Added 0 2 0 2
µM Measured 1.19 2.98 3.64 5.35
%Recovery 90 86
Two Point µM Added 0 2 0 2
µM Measured 0.40 2.15 1.15 3.08
%Recovery 103 97
Derivative µM Added 0 2 0 2
µM Measured 0.29 2.25 0.88 2.77
%Recovery 98 95

The 3rd-derivative (Figure 5) is considered the best estimate of the true MDA concentration in the lysate; however, there's probably little difference with the Two Point calculation. The derivative provides evidence that the MDA measured in the non-spiked lysate samples is authentic MDA because the derivative peaks (568 nm) are the same for TMOP, the spiked and non-spiked samples. This is not the case in Figure 3. Note that the derivative peaks are shifted to the right because of the alkaline conditions. The NaOH extract is not stable and needs to be acidified. Following acidification, the derivative peaks are all at 541 nm (data not shown).