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

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.

Application of a Novel Metallomics Tool to Probe the Fate of Metal-Based Anticancer Drugs in Blood Plasma: Potential, Challenges and Prospects.

Although metallodrugs are used to treat a variety of human disorders and exhibit a remarkable diversity of therapeutic properties, they constitute only a tiny minority of all medicinal drugs that are currently on the market. This undesirable situation must be partially attributed to our general lack of understanding the fate of metallodrugs in the extremely ligand-rich environment of the bloodstream. The challenge of gaining insight into these bioinorganic processes can be overcome by the application of 'metallomics tools', which involve the analysis of biological fluids (e.g., blood plasma) with a separation method in conjunction with multi-element specific detectors. To this end, we have developed a metallomics tool that is based on size-exclusion chromatography (SEC) hyphenated to an inductively coupled plasma atomic emission spectrometer (ICP-AES). After the successful application of SEC-ICPAES to analyze plasma for endogenous copper, iron and zinc-metalloproteins, it was subsequently applied to probe the metabolism of a variety of metal-based anticancer drugs in plasma. The versatility of this metallomics tool is exemplified by the fact that it has provided insight into the metabolism of individual Pt-based drugs, the modulation of the metabolism of cisplatin by sulfur-containing compounds, the metabolism of two metal-based drugs that contain different metals as well as a bimetallic anticancer drug, which contained two different metals. After adding pharmacologically relevant doses of metallodrugs to plasma, the temporal analysis of aliquots by SEC-ICP-AES allows to observe metal-protein adducts, metallodrug-derived degradation products and the parent metallodrug(s). This unique capability allows to obtain comprehensive insight into the fate of metal-based drugs in plasma and can be extended to in vivo studies. Thus, the application of this metallomics tool to probe the fate of novel metalcomplexes that exert the desired biological activity in plasma has the potential to advance more of these to animal/preclinical studies to fully explore the potential that metallodrugs inherently offer.

Sample preparation of blood plasma enables baseline separation of iron metalloproteins by SEC-GFAAS.

The analysis of human plasma for biomarkers holds promise to revolutionize disease diagnosis, but is hampered by the inherent complexity of the plasma proteome. One way to overcome this problem is to analyze plasma for a sub-proteome, such as the metalloproteome. Previous studies employing size-exclusion chromatography (SEC) coupled on-line to an inductively coupled plasma-atomic emission spectrometer (ICP-AES) have revealed that plasma contains ~12 copper, iron and zinc metalloproteins. This included the iron metalloproteins transferrin (Tf) and a recently identified haptoglobin-hemoglobin (Hp-Hb) complex, which is formed in plasma when red blood cells rupture. Since this SEC-ICP-AES method required a sample volume of 500 µL to generate diagnostically useful results, we sought to develop an alternative SEC-based hyphenated approach using a smaller SEC column (150 × 5 mm I.D.) and a graphite furnace atomic absorption spectrometer (GFAAS) as the iron-specific detector. A designed interface enabled the integration of the SEC system with the GFAAS. Baseline separation between the Hp-Hb complex and Tf was achieved by developing a sample preparation procedure which involved the chelating agent-based mobilization of Fe from Tf to a small molecular weight Fe complex. Spiking of human plasma (1.0 mL) with red blood cell lysate (1-2 µL) increased only the intensity of the Fe peak corresponding to the Hp-Hb complex, but not that of Tf. Since the developed SEC-GFAAS method requires only 50 µL of plasma for analysis, it can now be employed for the cost-effective quantification of the clinically relevant Hb-Hp complex in human plasma in <50 min.

SEC hyphenated to a multielement-specific detector unravels the degradation pathway of a bimetallic anticancer complex in human plasma.

The bimetallic metal complex Titanocref exhibits relevant anticancer activity, but it is unknown if it is stable to reach target tissues intact. To gain insight, a pharmacologically relevant dose was added to human blood plasma and the mixture was incubated at 37 °C. The obtained mixture was analyzed 5 and 60 min later by size-exclusion chromatography hyphenated to an inductively coupled plasma atomic emission spectrometer (SEC-ICP-AES). We simultaneously detected several titanium (Ti), gold (Au) and sulfur (S)-peaks, which corresponded to a Ti degradation product that eluted partially, and a Au degradation product that eluted entirely bound to plasma proteins (both time points). Although ~70% of the intact Titanocref was retained on the column after 60 min, our results allowed us to establish - for the first time - its likely degradation pathway in human plasma at near physiological conditions. These results suggest that ~70% of Titanocref remain in plasma after 60 min, which supports results from a recent in vivo study in which mice were treated with Titanocref and revealed Ti:Au molar ratios in tumors and organs close to 1:1. Thus, our stability studies suggest that the intact drug is able to reach target tissue. Overall, our results exemplify that SEC-ICP-AES enables the execution of intermediate in vitro studies with human plasma in the context of advancing bimetallic metal-based drugs to more costly clinical studies.

Identification of a haptoglobin-hemoglobin complex in human blood plasma.

Blood plasma metalloproteins that contain copper (Cu), iron (Fe), zinc (Zn) and/or other metals/metalloids are potential disease biomarkers because the bloodstream is in permanent contact with organs. Their quantification and/or the presence of additional metal-entities or the absence of certain metalloproteins in blood plasma (e.g. in Wilson's disease) may provide insight into the dyshomeostasis of the corresponding metal (s) to gain insight into disease processes. The first step in investigating if the determination of plasma metalloproteins is useful for the diagnosis of diseases is their definitive qualitative identification. To this end, we have added individual highly pure Cu, Fe or Zn-containing metalloproteins to plasma (healthy volunteer) and analyzed this mixture by size-exclusion chromatography (SEC) coupled to an inductively coupled plasma atomic spectrometer (ICP-AES), simultaneously monitoring the emission lines of Cu, Fe and Zn. The results clearly identified ceruloplasmin (Cp), holo-transferrin (hTf), and α2-macroglobulin (α2M), which verifies our previous assignments. Interestingly, another major Fe-peak in plasma was identified as a haptoglobin (Hp)-hemoglobin (Hb) complex. This Hp-Hb complex is formed after Hb, which is released during the hemolysis of erythrocytes, binds to the plasma protein Hp. The Hp-Hb complex formation is known to be one of the strongest interactions in biochemistry (Kd≈1pmol/L) and is critical because it prevents kidney toxicity of free Hb. Hence, the simultaneous determination of Cp, hTf, α2M and the Hp-Hb complex in plasma in <25min has the potential to provide new insight into disease processes associated with the bioinorganic chemistry of Cu, Fe and Zn.

Remarkable differences in the biochemical fate of Cd2+, Hg2+, CH3Hg+ and thimerosal in red blood cell lysate.

Humans are environmentally exposed to potentially toxic Cd and Hg species and to the Hg compound thimerosal (THI), an antibactericidal vaccine additive. Previous studies have revealed that Cd2+, Hg2+ and CH3Hg+ are taken up by red blood cells (RBCs) and bind to cytosolic glutathione (GSH) and/or hemoglobin (Hb). Since interactions in the cytosol of RBCs may be linked to their hemolysis, a more comprehensive characterization of these interactions was sought. After the addition of each Cd and Hg species to RBC lysate, the mixtures were analyzed after 5 min, 2 h and 6 h by size-exclusion chromatography (SEC) coupled on-line to an inductively coupled plasma atomic emission spectrometer (ICP-AES). In contrast to previous studies, however, reducing conditions were maintained by employing a 100 mM Tris buffer mobile phase (pH 7.4), which contained ∼2.5 mM of glutathione (GSH). At ≥2 h, ∼85% of Cd2+ weakly interacted with hemoglobin (Hb), while ∼13% eluted as (GS)xCd and ∼2% bound to a ≥70 kDa Cd-binding protein. In contrast, ∼6% of Hg2+ co-eluted with Hb at all time points, while ∼94% eluted as (GS)xHg. The results for CH3Hg+ showed that ∼5% of Hg co-eluted with Hb, while for THI this percentage gradually increased to 12% (6 h). The remaining Hg eluted as GS-HgCH3 and GS-HgCH2CH3. Our results revealed remarkable differences in the interaction of the investigated Cd and Hg species with cytosolic RBC constituents. The formation of (Hb)xHg species, regardless of which Hg compound was added, suggests their mammalian toxicology to be intertwined with the metabolism of Fe.

Environmentally relevant concentrations of aminopolycarboxylate chelating agents mobilize Cd from humic acid.

Although Cd is a pollutant of public health relevance, many dietary sources from which it can be absorbed into human tissues remain unknown. While it is well established that the biogeochemical cycle of Cd involves its complexation with environment-derived ligands (e.g., humic acids, HAs) and anthropogenic ones (e.g., chelating agents, CAs), the interaction of Cd with both of these ligands is less well understood. To gain insight, a HA-Cd complex was injected on a size-exclusion chromatography (SEC) column coupled on-line with a flame atomic absorption spectrometer (FAAS) using 10mmol/L Tris buffer (pH8.0) as the mobile phase. This approach allowed us to observe the intact HA-Cd complex and the retention behavior of Cd as a function of 2-20µmol/L concentrations of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) or methylglycinediacetic acid (MGDA) that were added to the mobile phase. An increase of the retention time of Cd was indicative of a partial or complete abstraction of Cd from HA. Our results revealed that all CAs abstracted Cd from the HA-Cd complex at concentrations of 5µmol/L, while MGDA and DTPA were effective at 2µmol/L. The bioavailability of some of the on-column formed CA-Cd complexes explains the previously reported increased accumulation of Cd in periphyton in the ecosystem downstream of wastewater treatment plants. In addition, our results imply that the use of effluents which contain CAs and Cd for the irrigation of food crops can introduce Cd into the food supply and compromise food safety.