Original article
Ferritin subunits in CSF are decreased in restless legs syndrome

https://doi.org/10.1016/j.lab.2005.06.011Get rights and content

Restless legs syndrome (RLS) is a neurological disorder that may be related to iron misregulation at the level of the central nervous system. Evidence that iron is involved in RLS comes from magnetic resonance imaging data, autopsy studies, analyses of cerebrospinal fluid (CSF), and correlations of symptoms with serum ferritin. To further examine the possibility that brain iron status is insufficient in RLS, we determined ferritin levels in the CSF. Specifically, we differentiated between the H- and L-subunits of ferritin, because these peptides are expressed from different chromosomes and have different functions. We measured H- and L-ferritin subunit levels in control and RLS human CSF using immunoblot analysis and found that both H- and L-ferritin are significantly decreased in early but not late-onset RLS. Additionally, we quantified total protein in each CSF sample to establish that the decrease in ferritin subunits in RLS did not reflect a decrease in total protein in CSF. Furthermore, we used equal amounts of total CSF protein in the immunoblot analyses, in contrast to previously published studies that provided only volumetric data, to determine which approach was more accurate for quantifying the amount of ferritin relative to other proteins in CSF. Our results establish a protein standard in RLS, provide a comparative analysis of protein-controlled versus volumetric immunoblot techniques, and argue for a profound loss of iron storage capacity in the brain in RLS, specifically in the early onset RLS phenotype. These data suggest that CSF ferritin levels may provide a biomarker for assisting in the diagnosis of RLS.

Section snippets

Patient characteristics

This is an Institutional Review Board-approved study. The research was carried out according to the principles of the Declaration of Helsinki. Informed consent was obtained from all participants. We used CSF collected from a total of 39 persons at Johns Hopkins University, 25 who were found to have RLS (12 early onset RLS and 13 late-onset RLS) and 14 control subjects who did not have RLS. The LPs were all performed at 10 pm in the evening at a time when symptoms are maximal. The patients fit

Results

The specificity of the H- and L-monoclonal antibodies is shown in Fig 1. Human spleen ferritin was used as a control for the primary antibodies on both blots, because it contains both H- and L-ferritins, in a ratio of approximately 10% to 20% H-ferritin and 80% to 90% L-ferritin.20, 28, 29, 30, 31 The H-ferritin antibody is less sensitive than the L-ferritin antibody in detecting human spleen ferritin, consistent with the ratio of H-ferritin to L-ferritin and with previous characterizations of

Discussion

We have established that both H- and L-ferritin subunits are decreased in early onset RLS. These data are consistent with previous reports4 and extend the findings to the individual ferritin subunits. The data provide further support for the notion that RLS is a disorder related to insufficient iron at the level of the CNS.4, 32 We also established that total protein amounts in RLS CSF are normal—an important piece of information in elucidating the pathology of RLS. Changes in total CSF protein

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  • Cited by (0)

    Supported by NIH/NCRR Grant M01-RR-02719 (Johns Hopkins GCRC) and by NIH Grant RO1-NS038704, Dopaminergic Function in Restless Legs Syndrome (C. Earley, PI).

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