(A) Scatter plot for total CoQ10 level in serum in the MSA group (n = 18), PD group (n = 20), and control group (n = 18). Mean levels of total CoQ10 were lowest in the MSA group (593.2 nmol/l) and highest in the Control group (985.3 nmol/l), although no significant difference was seen between groups. (B) Scatter plot for total CoQ10/T-Cho ratio in serum in the three groups. A significant difference was seen between groups. Post-hoc analysis showed that mean level was significantly lower in the MSA group (3.04) than in the Control group (5.92). (C) The scatter plot for total CoQ10/T-Cho ratio in serum in the three groups after excluding an outlier from the Control group.

<p>Similar statistical results to those shown in Graph B were also found. Bars indicate mean values. One participant in the Control group showed an extremely high total CoQ10/T-Cho ratio (Graph B). Graph C therefore shows participants after excluding this outlier. P values over the columns were obtained using the Kruskal-Wallis test. When the Kruskal-Wallis test yielded significant findings, post-hoc analysis was conducted; the results of which are shown on the right side of the graph.</p

Decrease in Plasma Levels of α-Synuclein Is Evident in Patients with Parkinson’s Disease after Elimination of Heterophilic Antibody Interference

<div><p>There is substantial biochemical, pathological, and genetic evidence that α-synuclein (A-syn) is a principal molecule in the pathogenesis of Parkinson disease (PD). We previously reported that total A-syn levels in cerebrospinal fluid (CSF), measured with the specific enzyme-linked immunosorbent assay (ELISA) developed by ourselves, were decreased in patients with PD, and suggested the usefulness of A-syn in CSF and plasma as a biomarker for the diagnosis of PD. After our report, a considerable number of studies have investigated the levels A-syn in CSF and in blood, but have reported inconclusive results. Such discrepancies have often been attributed not only to the use of different antibodies in the ELISAs but also to interference from hemolysis. In this study we measured the levels of A-syn in CSF and plasma by using our own sandwich ELISA with or without heterophilic antibody (HA) inhibitor in 30 patients with PD and 58 age-matched controls. We thereby revealed that HA interfered with ELISA measurements of A-syn and are accordingly considered to be an important confounder in A-syn ELISAs. HA produced falsely exaggerated signals in A-syn ELISAs more prominently in plasma samples than in CSF samples. After elimination of HA interference, it was found that hemolysis did not have a significant effect on the signals obtained using our A-syn ELISA. Furthermore, plasma levels of A-syn were significantly lower in the PD group compared with the control group following elimination of HA interference with an HA inhibitor. Our results demonstrate that HA was a major confounder that should be controlled in A-syn ELISAs, and that plasma A-syn could be a useful biomarker for the diagnosis of PD if adequately quantified following elimination of HA interference.</p></div

Biomarker repurposing: Therapeutic drug monitoring of serum theophylline offers a potential diagnostic biomarker of Parkinson’s disease

<div><p>Caffeine has been considered a neuroprotective agent against Parkinson’s disease (PD). Recent metabolomic analysis showed that levels of caffeine and its metabolites were decreased in sera from patients with PD compared with those from healthy controls. We focused on theophylline, which is one of the primary caffeine metabolites, as a candidate biomarker of PD because: (1) its serum level can be measured in hospital laboratories by standardized immunoassay kits for therapeutic drug monitoring and (2) because it is less markedly affected by caffeine intake. This was a pilot study to measure the levels of theophylline in sera of 31 patients with PD and 33 age-matched disease controls using an immunoassay kit. We confirmed the previous finding of significantly lower levels of serum theophylline in the PD group compared with control group (PD: 0.07±0.09 μg/mL, control: 0.18±0.24 μg/mL, p<0.05). Using such an approach of applying known medical biomarkers for neurodegenerative diseases may allow us to skip the process from the discovery phase to clinical application, and subsequently shorten the period of time necessary for biomarker development.</p></div

Concept of biomarker repurposing.

<p>The process of biomarker development is shown. Biomarker repurposing, the novel application of existing biomarkers for other purposes, enables the skipping of the development of quantification and standardization processes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201260#pone.0201260.ref027" target="_blank">27</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201260#pone.0201260.ref028" target="_blank">28</a>].</p

Correlations between A-syn levels in CSF and plasma samples obtained at the same time.

<p>Scatter plots of A-syn levels in CSF versus plasma in the PD group (A), control group (B), and the combination of PD patients and the controls (C). Closed and open circles indicate samples from the PD and the control groups, respectively. The solid lines indicate regression analyses. There was a significant positive correlation in the PD group (A) (p = 0.005). In the control group (B), no significant correlation was observed (p = 0.50). Dashed lines in (C) indicate the mean values of CSF (29.07 ng/ml) and plasma (31.72 ng/ml) A-syn in the controls, and are plotted to clarify the discrepancy between the PD and control groups with respect to the relationship between CSF and plasma A-syn levels. There was a tendency for patients whose CSF A-syn levels were lower than 29.07 ng/ml to exhibit plasma A-syn levels less than 31.72 ng/ml in the PD group (closed circles, Fig 5C); however, no such a tendency was observed in the control group (open circles, Fig 5C).</p

Characteristics of patients with PD and disease controls.

<p>Characteristics of patients with PD and disease controls.</p

Effects of HA on measurements of CSF and plasma A-syn levels using the 211-FL140 ELISA.

<p>Levels of CSF-A-syn (A) and plasma-A-syn (B) were measured using the 211-FL140 ELISA in the absence and presence of 5% HAI. HAI pretreatment slightly increased A-syn signals in the majority of CSF samples (A), while the A-syn signals were remarkably decreased in most of the plasma samples (B). (C) In order to detect HA in plasma samples, the primary antibodies (C211 and FL-140) were separated by blue native-PAGE and immunoblotted with two plasma samples. The plasma sample on the left exhibited high HA activity, while the sample on the right had low HA activity in previous experiments that had determined the effects of HAI on the 211-FL140 ELISA. The plasma with high HA activity clearly reacted with both the 211 and FL-140 antibodies, while the plasma with low HA activity did not react with either.</p

Determination of the optimal HAI concentration for the A-syn 211-FL140 ELISA.

<p>Chemiluminescence signals obtained from serially diluted recombinant A-syn in the presence of various HAI concentrations (0, 5, 16.7 and 50%) with the 211-FL140 ELISA. ELISA performance was not affected by 5% HAI, and was reduced in the presence of 16.7% and 50% HAI.</p

Correlations between Hb and A-syn levels in plasma and CSF.

<p>(A, B) Scatter plots of the levels of Hb versus those of A-syn in plasma measured without (A) or with (B) HAI pretreatment. (C, D) Scatter plots of Hb versus A-syn in CSF measured without (C) or with (D) HAI pretreatment. Closed rectangles and triangles indicate samples from the PD group, open rectangles and triangles are those from the control group. The horizontal axis is shown on a logarithmic scale. There were no significant correlations between the levels of Hb and plasma A-syn without (A; p = 0.86) or with (B; p = 0.39) HAI pretreatment. Significant correlations were not also observed between Hb and CSF A-syn without (C; p = 0.93) or with (D; p = 0.65) HAI pretreatment.</p

Theophylline levels in sera of the PD and control groups.

<p>Fig 1(A) Scatter plot showing the levels of serum theophylline in the control (n = 33) and PD (n = 31) groups. Bars indicate median values. The levels of theophylline in the PD group were significantly lower than those in the control group (P = 0.0383). Fig 1(B) ROC curve showing serum levels of theophylline to discriminate PD patients from controls. The AUC value was 0.65.</p