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.
Highlights
Disease specific molecular biomarkers can be quantified in a variety of human bodily fluids, including urine, saliva, cerebrospinal fluid and blood [1]
The iron % recovery values for the individual plasma samples (Table 1) displayed an average of 70 ± 23% for multiple sclerosis (MS) participants (n = 18), 68±11% for AIS participants (n = 7) and 86±21 for healthy controls (n = 12)
We have previously reported that the addition of different Hp isoforms (Hp 1–1, Hp 1–2, Hp 2–2) to red blood cell lysate followed by size exclusion chromatography (SEC)-inductively coupled plasma atomic emission spectrometer (ICP-AES) analysis resulted in an Fe-peak that corresponded to a Hp bound to Hb (Hp-Hb) complex which had a ~100 s larger retention time for Hp 1–1 compared to Hp 1–2 and Hp 2–2 [19]
Summary
Disease specific molecular biomarkers can be quantified in a variety of human bodily fluids, including urine, saliva, cerebrospinal fluid and blood [1]. Among these biofluids, plasma is one of the most information-rich [2] and its analysis is relevant from an analytical point of view as it receives biomolecules that are released from specific organs and/or red blood cells as a result of disease processes [3,4,5]. Since plasma is accessible and may harbor diagnostically relevant biomarkers [6,7,8,9], the development of advanced methods for specific molecular biomarkers is a vibrant area of research [10]. Hp is found in the plasma of all mammals, only humans display a Hp polymorphism which is associated with the presence of Hp isoforms 1–1 (a dimer) or the larger Hp 1–2 and Hp 2–2 isoforms [12]
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