Quantification of human plasma metalloproteins in multiple sclerosis, ischemic stroke and healthy controls reveals an association of haptoglobin-hemoglobin complexes with age

Abstract

Advanced analytical methods play an important role in quantifying serum disease biomarkers. The problem of separating thousands of proteins can be reduced by analyzing for a 'sub-proteome', such as the 'metalloproteome', defined as all proteins that contain bound metals. We employed size exclusion chromatography (SEC) coupled to an inductively coupled plasma atomic emission spectrometer (ICP-AES) to analyze plasma from multiple sclerosis (MS) participants (n = 21), acute ischemic stroke (AIS) participants (n = 17) and healthy controls (n = 21) for Fe, Cu and Zn-metalloproteins. Using ANOVA analysis to compare the mean peak areas among the groups revealed no statistically significant differences for ceruloplasmin (p = 0.31), α2macroglobulin (p = 0.51) and transferrin (p = 0.31). However, a statistically significant difference was observed for the haptoglobin-hemoglobin (Hp-Hb) complex (p = 0.04), being driven by the difference between the control group and AIS (p = 0.012), but not with the MS group (p = 0.13), based on Dunnes test. A linear regression model for Hp-Hb complex with the groups now adjusted for age found no statistically significant differences between the groups (p = 0.95), but was suggestive for age (p = 0.057). To measure the strength of association between the Hp-Hb complex and age without possible modifications due to disease, we calculated the Spearman rank correlation in the healthy controls. The latter revealed a positive association (r = 0.39, 95% Confidence Interval = (-0.05, 0.83), which suggests that either the removal of Hp-Hb complexes from the blood circulation slows with age or that the release of Hb from red blood cells increases with age. We also observed that the Fe-peak corresponding to the Hp-Hb complex eluted ~100 s later in ~14% of all study samples, which was not correlated with age or disease diagnosis, but is consistent with the presence of the smaller Hp (1-1) isoform in 15% of the population.

Fig 1. Fe-specific chromatograms obtained after the analysis of plasma from MS participants, AIS participants and healthy controls.

Column: Superdex 200 Increase 10/300 GL (30 x 1.0 cm I.D., 8 μm particle size), Temperature: 22°C, Mobile phase: 150 mM PBS buffer (pH 7.4), Flow rate: 0.75 mL/min, Injection volume: 500 μL. ICP-AES detection at 259.940 nm (Fe). Retention times of molecular weight markers are depicted on top. 750 μL of plasma was diluted with 250 μL of PBS buffer and 500 μL of the obtained solution were injected on the SEC column.

Fig 2. Plot of the age of the participants (AIS, MS and control: x-axis) against the square root of the obtained Fe-peak areas corresponding to the Hp-Hb complex (y-axis).

The regression line is for all data (adjusted R² of 0.11, F(1,57) = 8.58, p = 0.005) and ignores which group people are in, but clearly depicts a positive association between the age of the individual and the Hb-Hp peak areas.

Fig 3. Plot of the age of the control participants (x-axis) against the square root of the obtained Fe-peak areas corresponding to the Hp-Hb complex (y-axis).

The regression line is for all controls (adjusted R² of 0.14, F(1,19) = 4.12, p = 0.057) and depicts a positive association between the age of the individual and the Hp-Hb peak area.

Fig 4. Cu-specific chromatograms obtained after the analysis of plasma from MS participants, AIS participants and healthy controls.

Column: Superdex 200 Increase 10/300 GL (30 x 1.0 cm I.D., 8 μm particle size), Temperature: 22°C, Mobile phase: 150 mM PBS buffer (pH 7.4), Flow rate: 0.75 mL/min, Injection volume: 500 μL. ICP-AES detection at 324.75 nm (Cu). Retention times of molecular weight markers are depicted on top. 750 μL of plasma was diluted with 250 μL of PBS buffer and 500 μL of the obtained solution were injected on the SEC column.

Fig 5. Zn-specific chromatograms obtained after the analysis of plasma from MS participants, AIS participants and healthy controls.

Column: Superdex 200 Increase 10/300 GL (30 x 1.0 cm I.D., 8 μm particle size), Temperature: 22°C, Mobile phase: 150 mM PBS buffer (pH 7.4), Flow rate: 0.75 mL/min, Injection volume: 500 μL. ICP-AES detection at 213.86 nm (Zn). Retention times of molecular weight markers are depicted on top. 750 μL of plasma was diluted with 250 μL of PBS buffer and 500 μL of the obtained solution were injected on the SEC column.