Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 2014; Early Online: 1–6
ORIGINAL ARTICLE
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Serum ferritin is elevated in amyotrophic lateral sclerosis patients
XIAOWEI W. SU1, STACEY L. CLARDY2, HELEN E. STEPHENS2, ZACHARY SIMMONS2 & JAMES R. CONNOR1 Departments of 1Neurosurgery and 2Neurology, Penn State College of Medicine, Hershey, Pennsylvania, USA
Abstract Our objective was to measure serum ferritin levels, which reflect iron metabolism, in ALS patients versus healthy and disease controls, and determine whether serum ferritin levels correlate with survival. We retrospectively analyzed data from 138 ALS patients, 152 healthy controls, and 82 disease controls. Gender, age, site of onset, and dates of symptom onset and death were recorded. Survival was defined as the time from symptom onset to death. Serum ferritin levels were measured using immunoassay. ANOVA and Pearson’s correlation were used to compare ferritin levels between groups and test the association between ferritin levels and age and survival. Ferritin levels were categorized into high and low groups, and Kaplan-Meier analysis performed. Results showed that gender proportions differed between ALS patients versus healthy and disease controls, and gender affected serum ferritin levels. Ferritin comparisons were stratified for gender. In both males and females, ferritin levels were higher in ALS patients versus healthy and disease controls. However, ferritin levels were unrelated to survival in either gender, by tests of association or survival analysis. In conclusion, ALS patients have altered iron metabolism that is not simply due to the presence of neurological disease. Serum ferritin levels alone are not sufficient to predict survival. Key words: Amyotrophic lateral sclerosis, serum ferritin, survival, neurological disease control, iron metabolism
Introduction Abnormalities in a number of pathways are implicated in amyotrophic lateral sclerosis (ALS) pathophysiology. These include RNA metabolism, protein clearance, excitatory neurotransmission, cellular trafficking, mitochondrial function, and iron dyshomeostasis (1). However, the mechanisms underlying most cases remain unknown. Insight into processes specific to ALS may advance biomarker research for diagnosis or prognosis and suggest targets for novel therapies. One potential pathogenic pathway that has received attention is dysregulated iron metabolism (2). HFE iron regulatory gene variants are present in up to 30% of patients with ALS (3–9). Iron dyshomeostasis affects pathways implicated in ALS, including oxidative stress (10,11). Increased serum levels of the iron storage protein ferritin are associated with ALS diagnosis (12,13), accelerated disease progression (14), and decreased survival (15). Ferritin also may be a potential ALS biomarker, as levels of L-ferritin, which primarily stores iron and
lacks ferroxidase activity, have been used successfully in both diagnostic (16) and prognostic (17) models. Expression of mutant superoxide dismutase (SOD1), implicated in familial ALS, as well as overexpression of wild-type SOD1, are known to alter iron metabolism and increase serum ferritin levels (18). Despite evidence correlating ferritin with ALS diagnosis, progression and survival, it remains uncertain whether elevated ferritin is specific to ALS, or indicative of neurological disease in general. Previous studies linking serum ferritin levels to ALS compared patients with ALS to healthy controls. We extended these studies by also analyzing serum ferritin in patients with non-ALS neurological disease. We also determined whether ferritin correlates with survival in patients with ALS.
Patients and methods We retrospectively analyzed data from patients with possible, probable, laboratory-supported probable, or definite ALS (19); healthy controls; and patients
Correspondence: J. R. Connor, Department of Neurosurgery, Mailcode H110, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033-0850, USA. Fax: 717 531 0091. E-mail: [email protected] (Received 20 June 2014 ; accepted 2 November 2014 ) ISSN 2167-8421 print/ISSN 2167-9223 online © 2014 Informa Healthcare DOI: 10.3109/21678421.2014.984723
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X. W. Su et al. Table I. Clinical characteristics of all study subjects. ALS (n ⫽ 138)
Clinical variable
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Gender, number of males (% males) Ethnicity, number non-white (% non-white) Age at sample (yrs), med. (range) H63D HFE carriers, number (%)
87 1 62.1 28
with other neurological diseases (disease controls) seen at a university-based multidisciplinary ALS clinic or part of a cohort that provided samples to the Northeast ALS Consortium (NEALS)/Neurological Clinical Research Institute (NCRI) ALS Biofluid Repository (http://www.alsconsortium.org/ neals_samples.php). Other neurological diseases were varied, and included multiple sclerosis, chronic
Healthy controls Disease controls (n ⫽ 152) (n ⫽ 82)
(63.0) 49 (32.2) 36 (43.9) (0.7) 5 (3.2) 2 (2.4) (30.8 – 82.7) 39.9 (20.0 – 81.0) 54.5 (23.1– 81.5) (27.5) 34 (32.7) N/A
migraine, peripheral neuropathy, meningitis, dementia, Parkinson’s disease and Alzheimer’s disease. Gender, ethnicity and age were recorded for all subjects. For those with ALS, we also noted site of onset, pattern of ALS inheritance, time of patientidentified symptom onset and time of death. Total disease duration (survival) was defined as the time from patient-identified symptom onset to death.
Table II. Disease control diagnoses. No. of males
Disease Alzheimer ’s disease Aseptic meningitis Ataxic disorder Ataxic dysarthria and biparietal lobe dysfunction Bell’s palsy Cognitive deficits and vertigo Dementia Disc herniation Distal neuropathy Downbeat nystagmus Gait disorder Gait dystaxia, multifocal lacunar disease, and dementia Headache Headache and sensory loss Herpes simplex virus meningitis Hydrocephalus Intermittent paroxysmal headache Lumbar pain Lyme disease Memory loss Migraine headaches Multiple sclerosis Muscle weakness Myopathy Pain unspecified Parkinson’s disease Peripheral neuropathy Periventricular lesions Spinal injury and paraplegia Trigeminal neuralgia Undetermined
0 1 0 0 0 0 1 0 0 1 1 0 0 0 1 1 0 1 1 0 1 4 1 0 1 1 2 1 0 0 17
No. of females 1 0 1 1 1 1 0 1 2 0 0 1 1 1 0 0 1 0 1 1 2 14 0 1 0 0 0 0 1 1 13
Total number 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 2 1 3 18 1 1 1 1 2 1 1 1 30
Table III. ALS patient characteristics. Clinical variable Age at onset (yrs), med. (range) Onset to sample time (mo), med. (range) ALS type, number familial, (% familial) Site of onset, number bulbar, (% bulbar) Total disease duration (mo), med. (range) H63D HFE carriers, number (%)
Males (n ⫽ 87) 58.2 23.6 1 26 45.3 19
(30.2 – 81.3) (3.3 –246.7) (1.1) (29.9) (7.3 –193.7) (31.1)
Females (n ⫽ 51) 60.4 (23.3 –79.6) 20.5 (9.0 –166.3) 3 (5.9) 23 (45.1) 35.2 (18.6 –206.3) 9 (22)
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Elevated serum ferritin in ALS patients
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Familial cases were determined by family history. The study was approved by our Institutional Review Board. All patients provided informed consent. Because the H63D HFE polymorphism has been associated with ALS, H63D HFE genotyping was performed as previously described (4) on samples as available. In brief, genomic DNA was purified from leukocytes using QIAamp DNA Mini Kit (Qiagen, Valencia, CA). Polymerase chain reaction followed by restriction fragment length analysis and confirmation DNA sequencing was used to analyze H63D HFE status. Analysis of serum ferritin levels was performed in the clinical laboratory at Penn State Hershey Medical Center. In brief, ferritin was bound to biotinylated antibody, captured by streptavidin and detected using horseradish peroxidase-labeled monoclonal antibody using the Vitros 5600 automated immunoassay system (Ortho Clinical Diagnostics, Rochester, NY). Ferritin levels between groups were compared using one-way ANOVA. Pearson’s correlation was used to analyze the associations between ferritin versus age and ferritin versus survival. Ferritin levels were categorized into low (below the median) and high (equal to or above the median) groups and Kaplan-Meier survival analysis with log rank analysis was conducted. All tests were two-sided with significance set at the p ⬍ 0.05 level. Results Clinical variables from 138 patients with ALS, 152 healthy controls and 82 disease controls were obtained. The majority of subjects were Caucasian. Gender proportions were different between groups, and thus subsequent results were stratified by gender. Age at serum sample collection was different between groups. However, age and ferritin values were not correlated (Pearson’s r ⫽ 0.10, p-value ⬎ 0.05), and thus results were not stratified by age. H63D HFE genotyping was available for 102 patients with ALS and 104 healthy controls; genotyping was not available for disease controls. HFE genotyping indicated 27.5% of patients with ALS harbored H63D HFE polymorphism, consistent with previous reports (3–9) (Table I), whereas 32.7% of healthy controls harbored H63D HFE. The majority of H63D HFE carriers were heterozygous; 6/28 patients with ALS harboring H63D HFE and 2/32 healthy controls harboring H63D HFE were homozygous (Table I). Disease controls had a variety of diagnoses with multiple sclerosis the most common in both males and females (Table II). Clinical characteristics of patients with ALS were representative of the general ALS patient population, with the vast majority of patients classified as having sporadic ALS. H63D HFE genotyping was available for 61 of 87 males and 41 of 51 females with ALS. Of those harboring H63 HFE, two males and four females
Figure 1. Serum ferritin is elevated in patients with ALS. (A) Males with ALS have higher levels of serum ferritin (mean 286.6 ng/ml) versus healthy controls (mean 160.8 ng/ml, p ⬍ 0.001) or disease controls (mean 164.5 ng/ml, p ⫽ 0.003). (B) Females with ALS have higher levels of serum ferritin (mean 142.6 ng/ml) versus healthy controls (mean 69.3 ng/ml, p ⬍ 0.001) or disease controls (mean 77.5 ng/ml, p ⬍ 0.001). Box plot with interquartile range (left), dot plot (middle) and bar graph with means showing standard error of the mean (right) depicted for each group.
were homozygous, with the remainder heterozygous for the allele (Table III). Serum ferritin levels were elevated in males and females with ALS versus healthy controls or disease controls (Figure 1). Kaplan-Meier survival analysis, stratified by gender, was performed. Serum ferritin category did not affect survival in either gender (Figure 2). Pearson’s correlation using raw values also did not indicate an association between ferritin levels and survival in either gender (Figure 3).
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Discussion This study found that serum ferritin levels in patients with ALS were elevated versus healthy controls, consistent with previous research (12,13,15). Serum ferritin levels in ALS patients were also elevated compared to patients with other neurological diseases, suggesting the effect is not simply due to neurological disease. In the circulation, ferritin is an acute phase reactant that reflects systemic inflammation (20), as well as a well-studied indicator of stored iron (21). Previous research demonstrated levels of C-reactive protein and other acute phase reactants were not correlated with serum ferritin in patients with ALS (13), suggesting that ferritin was specific to ALS rather than being indicative of an inflammatory reaction associated with neurological
disease in general. This is further supported by our data for patients with multiple sclerosis. This autoimmune disorder was the most common diagnosis in the disease control group, suggesting that serum ferritin is elevated in ALS not solely because it is a marker of inflammation. The relationship of ferritin to iron status is well established. Iron dyshomeostasis induces oxidative stress by generating reactive oxygen species, and oxidative damage is a hallmark of ALS (22). Ferritin binds to and sequesters iron and other trivalent metals, preventing oxidative damage (23). In transgenic SOD1-G93A mice, expression of both H- and L-ferritin decreased iron catalyzed free radical formation, limiting oxidative damage to lipids, proteins, and nucleic acids possibly caused by mutant
Figure 2. Serum ferritin categories do not predict survival in patients with ALS. (A) Males with serum ferritin levels below the median value (202.0 ng/ml) have similar survival to those with levels equal to or above the median value (median 45.3 versus 44.0 months, p ⫽ 0.778). (B) Females with serum ferritin levels below the median value (123.0 ng/ml) have similar survival to those with levels equal to or above the median value (median 30.4 versus 36.0 months, p ⫽ 0.360).
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Elevated serum ferritin in ALS patients SOD1-induced mitochondrial dysfunction (24). These results suggest elevated serum ferritin may be an adaptive response to increased levels of oxidative stress in ALS. Our finding that serum ferritin levels were not associated with disease duration in patients with ALS contrasts with previous reports suggesting that higher ferritin levels correlate with accelerated disease progression (14) and decreased survival (15). One possible reason for this may be that serum ferritin, or any single biomarker, is insufficient to reliably and consistently reflect the complex neurodegenerative processes involved in ALS. Multivariable approaches involving panels of biomarkers spanning different biologic pathways and tissue compartments may better predict prognosis. In a multivariable analysis, elevated serum ferritin was part of a panel of proteins that predicted longer disease duration in patients with ALS (17). This is consistent with the discussion in the preceding paragraph suggesting that, in animal models, an enhanced capacity to detoxify iron and prevent further oxidative damage could promote neuronal survival in response to oxidative stress. It is possible that other factors confound the use of ferritin as a predictor of disease. For example, even though most patients with ALS receive riluzole treatment as the standard of care, other pharmacotherapies likely differ between patient populations. Differences in pharmacotherapy may influence serum ferritin levels independent of effects on survival, or alternatively, uncouple the association between ferritin levels and survival via unrecognized biochemical mechanisms. In our own study, 17 out of 138 patients with ALS provided samples through the NEALS/NCRI ALS Biofluid Repository, and were part of two separate clinical trials of novel agents. Perhaps effects on serum ferritin and other biomarkers may be useful in assessing the efficacy of therapeutic strategies given the heterogeneous nature of disease progression in ALS. Although the percentage of patients receiving enteral nutrition and the iron content of enteral formulas were not reported in previous studies or available in our study, iron overload exacerbated by enteral nutrition may play a role in the divergent disease course results. Body iron homeostasis is affected by environmental factors, including diet. Dysphagia affects up to 80% of patients with ALS, many of whom receive enteral nutrition (25), which may exacerbate iron overload. The recommended daily iron intake for healthy individuals is 10 mg/day; however, the iron content of typical enteral formulas ranges from 13 to 24 mg/l, and most patients with ALS receiving enteral nutrition consume at least one liter of formula per day (26). Enteral nutrition may worsen iron overload in select individuals with ALS; for example, in the approximately 30% of patients with ALS that carry the HFE gene variant associated with elevated cellular iron uptake (3–9,27). Our results indicate 27.5% of patients with ALS harbor
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Figure 3. Serum ferritin levels are not associated with survival in patients with ALS. Pearson’s correlation indicates no association between serum ferritin levels and survival in males (A) Pearson’s r ⫽ 0.011, p ⫽ 0.933) or females (B) Pearson’s r ⫽ 0.197, p ⫽ 0.237).
H63D HFE, further arguing for the importance of this polymorphism in the disease, and the need to investigate the molecular and physiological consequences of HFE polymorphism in ALS. There are a number of limitations to our study. Serum ferritin was the only marker of iron metabolism analyzed in this study. Other studies have demonstrated significant differences in transferrin and the transferrin saturation coefficient in patients with ALS versus healthy controls (15). However, elevated serum ferritin is the most robust finding across a number of studies (12,13,15), with conflicting results reported for other markers of iron metabolism, including transferrin and transferrin saturation (13). Thus, we chose to study serum ferritin. Our study was cross-sectional, and did not follow serum ferritin
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levels over time. This has been a limitation of all studies investigating ferritin levels in ALS, and ALS biomarkers research in general. Longitudinal analysis may uncover the temporal dynamics of ferritin in ALS, allowing more precise delineation of its role in pathophysiology. In summary, our finding of increased serum ferritin levels in patients with ALS may reflect increasing cellular iron load, or alternatively, up-regulation of adaptive mechanisms for buffering iron and other trivalent trace metals (23). The lack of correlation between ferritin levels and survival in our study may reflect the limitation of any single biomarker to adequately capture disease prognosis that could stem from differences in pharmacotherapy (including enteral feeding), genotype that would influence environmental interaction, and body iron status. It also could be an important indicator of a loss of ferritindependent iron buffering capacity in patients with increasingly severe disease. Acknowledgements The authors would like to thank Robert Lawson and the Northeast Amyotrophic Lateral Sclerosis Consortium for providing serum samples for this study. The study was supported in part by the Paul and Harriett Campbell Fund for ALS Research; the Zimmerman Family Love Fund; and the ALS Association, Greater Philadelphia Chapter. Declaration of interest: The authors report no confl icts of interest. The authors alone are responsible for the content and writing of the paper. References 1. Robberecht W, Philips T. The changing scene of amyotrophic lateral sclerosis. Nat Rev Neurosci. 2013;14: 248–64. 2. Nandar W, Connor JR. HFE gene variants affect iron in the brain. J Nutr. 2011;141:S729–39. 3. Wang XS, Lee S, Simmons Z, Boyer P, Scott K, Liu W, et al. Increased incidence of the Hfe mutation in amyotrophic lateral sclerosis and related cellular consequences. J Neurol Sci. 2004;227:27–33. 4. Goodall EF, Greenway MJ, van Marion I, Carroll CB, Hardiman O, Morrison KE. Association of the H63D polymorphism in the hemochromatosis gene with sporadic ALS. Neurology. 2005;65:934–7. 5. Restagno G, Lombardo F, Ghiglione P, Calvo A, Cocco E, Sbaiz L, et al. HFE H63D polymorphism is increased in patients with amyotrophic lateral sclerosis of Italian origin. J Neurol Neurosurg Psychiatry. 2007;78:327. 6. Sutedja NA, Sinke RJ, van Vught PW, van der Linden MW, Wokke JH, van Duijn CM, et al. The association between H63D mutations in HFE and amyotrophic lateral sclerosis in a Dutch population. Arch Neurol. 2007;64:63–7. 7. He X, Lu X, Hu J, Xi J, Zhou D, Shang H, et al. H63D polymorphism in the hemochromatosis gene is associated with sporadic amyotrophic lateral sclerosis in China. Eur J Neurol. 2011;18:359–61.
8. Praline J, Blasco H, Vourc’h P, Rat V, Gendrot C, Camu W, et al. Study of the HFE gene common polymorphisms in French patients with sporadic amyotrophic lateral sclerosis. J Neurol Sci. 2012;317:58–61. 9. van Rheenen W, Diekstra FP, van Doormaal PT, Seelen M, Kenna K, McLaughlin R, et al. H63D polymorphism in HFE is not associated with amyotrophic lateral sclerosis. Neurobiol Aging. 2013;34:1517, e5–7. 10. Liu Y, Lee SY, Neely E, Nandar W, Moyo M, Simmons Z, et al. Mutant HFE H63D protein is associated with prolonged endoplasmic reticulum stress and increased neuronal vulnerability. J Biol Chem. 2011;286:13161–70. 11. Nandar W, Neely EB, Unger E, Connor JR. A mutation in the HFE gene is associated with altered brain iron profiles and increased oxidative stress in mice. Biochim Biophys Acta. 2013;1832:729–41. 12. Qureshi M, Brown RH Jr, Rogers JT, Cudkowicz ME. Serum ferritin and metal levels as risk factors for amyotrophic lateral sclerosis. Open Neurol J. 2008;2:51–4. 13. Goodall EF, Haque MS, Morrison KE. Increased serum ferritin levels in amyotrophic lateral sclerosis patients. J Neurol. 2008;255:1652–6. 14. Ikeda K, Hirayama T, Takazawa T, Kawabe K, Iwasaki Y. Relationships between disease progression and serum levels of lipid, urate, creatinine and ferritin in Japanese patients with amyotrophic lateral sclerosis: a cross-sectional study. Intern Med. 2012;51:1501–8. 15. Nadjar Y, Gordon P, Corcia P, Bensimon G, Pieroni L, Meininger V, et al. Elevated serum ferritin is associated with reduced survival in amyotrophic lateral sclerosis. PLoS One. 2012;7:e45034. 16. Mitchell RM, Simmons Z, Beard JL, Stephens HE, Connor JR. Plasma biomarkers associated with ALS and their relationship to iron homeostasis. Muscle Nerve. 2010; 42:95–103. 17. Su XW, Simmons Z, Mitchell RM, Kong L, Stephens HE, Connor JR. Biomarker-based predictive models for prognosis in amyotrophic lateral sclerosis. JAMA Neurol. 2013; 70:1505–11. 18. Danzeisen R, Achsel T, Bederke U, Cozzolino M, Crosio C, Ferri A, et al. Superoxide dismutase-1 modulates expression of transferrin receptor. J Biol Inorg Chem. 2006;11: 489–98. 19. Brooks BR, Miller RG, Swash M, Munsat TL. El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord. 2000;1:293–9. 20. Wang W, Knovich MA, Coffman LG, Torti FM, Torti SV. Serum ferritin: past, present and future. Biochim Biophys Acta. 2010;1800:760–9. 21. Torti FM, Torti SV. Regulation of ferritin genes and protein. Blood. 2002;99:3505–16. 22. Barber SC, Shaw PJ. Oxidative stress in ALS: key role in motor neuron injury and therapeutic target. Free Radic Biol Med. 2010;48:629–41. 23. Joshi JG, Sczekan SR, Fleming JT. Ferritin: a general metal detoxicant. Biol Trace Elem Res. 1989;21:105–10. 24. Olsen MK, Roberds SL, Ellerbrock BR, Fleck TJ, McKinley DK, Gurney ME. Disease mechanisms revealed by transcription profiling in SOD1-G93A transgenic mouse spinal cord. Ann Neurol. 2001;50:730–40. 25. Greenwood DI. Nutrition management of amyotrophic lateral sclerosis. Nutr Clin Pract. 2013;28:392–9. 26. Molfino A, Kushta I, Tommasi V, Rossi Fanelli F, Muscaritoli M. Amyotrophic lateral sclerosis, enteral nutrition and the risk of iron overload. J Neurol. 2009;256:1015–6. 27. Lee SY, Patton SM, Henderson RJ, Connor JR. Consequences of expressing mutants of the hemochromatosis gene (HFE) into a human neuronal cell line lacking endogenous HFE. FASEB J. 2007;21:564–76.
ORIGINAL ARTICLE
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Serum ferritin is elevated in amyotrophic lateral sclerosis patients
XIAOWEI W. SU1, STACEY L. CLARDY2, HELEN E. STEPHENS2, ZACHARY SIMMONS2 & JAMES R. CONNOR1 Departments of 1Neurosurgery and 2Neurology, Penn State College of Medicine, Hershey, Pennsylvania, USA
Abstract Our objective was to measure serum ferritin levels, which reflect iron metabolism, in ALS patients versus healthy and disease controls, and determine whether serum ferritin levels correlate with survival. We retrospectively analyzed data from 138 ALS patients, 152 healthy controls, and 82 disease controls. Gender, age, site of onset, and dates of symptom onset and death were recorded. Survival was defined as the time from symptom onset to death. Serum ferritin levels were measured using immunoassay. ANOVA and Pearson’s correlation were used to compare ferritin levels between groups and test the association between ferritin levels and age and survival. Ferritin levels were categorized into high and low groups, and Kaplan-Meier analysis performed. Results showed that gender proportions differed between ALS patients versus healthy and disease controls, and gender affected serum ferritin levels. Ferritin comparisons were stratified for gender. In both males and females, ferritin levels were higher in ALS patients versus healthy and disease controls. However, ferritin levels were unrelated to survival in either gender, by tests of association or survival analysis. In conclusion, ALS patients have altered iron metabolism that is not simply due to the presence of neurological disease. Serum ferritin levels alone are not sufficient to predict survival. Key words: Amyotrophic lateral sclerosis, serum ferritin, survival, neurological disease control, iron metabolism
Introduction Abnormalities in a number of pathways are implicated in amyotrophic lateral sclerosis (ALS) pathophysiology. These include RNA metabolism, protein clearance, excitatory neurotransmission, cellular trafficking, mitochondrial function, and iron dyshomeostasis (1). However, the mechanisms underlying most cases remain unknown. Insight into processes specific to ALS may advance biomarker research for diagnosis or prognosis and suggest targets for novel therapies. One potential pathogenic pathway that has received attention is dysregulated iron metabolism (2). HFE iron regulatory gene variants are present in up to 30% of patients with ALS (3–9). Iron dyshomeostasis affects pathways implicated in ALS, including oxidative stress (10,11). Increased serum levels of the iron storage protein ferritin are associated with ALS diagnosis (12,13), accelerated disease progression (14), and decreased survival (15). Ferritin also may be a potential ALS biomarker, as levels of L-ferritin, which primarily stores iron and
lacks ferroxidase activity, have been used successfully in both diagnostic (16) and prognostic (17) models. Expression of mutant superoxide dismutase (SOD1), implicated in familial ALS, as well as overexpression of wild-type SOD1, are known to alter iron metabolism and increase serum ferritin levels (18). Despite evidence correlating ferritin with ALS diagnosis, progression and survival, it remains uncertain whether elevated ferritin is specific to ALS, or indicative of neurological disease in general. Previous studies linking serum ferritin levels to ALS compared patients with ALS to healthy controls. We extended these studies by also analyzing serum ferritin in patients with non-ALS neurological disease. We also determined whether ferritin correlates with survival in patients with ALS.
Patients and methods We retrospectively analyzed data from patients with possible, probable, laboratory-supported probable, or definite ALS (19); healthy controls; and patients
Correspondence: J. R. Connor, Department of Neurosurgery, Mailcode H110, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033-0850, USA. Fax: 717 531 0091. E-mail: [email protected] (Received 20 June 2014 ; accepted 2 November 2014 ) ISSN 2167-8421 print/ISSN 2167-9223 online © 2014 Informa Healthcare DOI: 10.3109/21678421.2014.984723
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X. W. Su et al. Table I. Clinical characteristics of all study subjects. ALS (n ⫽ 138)
Clinical variable
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Gender, number of males (% males) Ethnicity, number non-white (% non-white) Age at sample (yrs), med. (range) H63D HFE carriers, number (%)
87 1 62.1 28
with other neurological diseases (disease controls) seen at a university-based multidisciplinary ALS clinic or part of a cohort that provided samples to the Northeast ALS Consortium (NEALS)/Neurological Clinical Research Institute (NCRI) ALS Biofluid Repository (http://www.alsconsortium.org/ neals_samples.php). Other neurological diseases were varied, and included multiple sclerosis, chronic
Healthy controls Disease controls (n ⫽ 152) (n ⫽ 82)
(63.0) 49 (32.2) 36 (43.9) (0.7) 5 (3.2) 2 (2.4) (30.8 – 82.7) 39.9 (20.0 – 81.0) 54.5 (23.1– 81.5) (27.5) 34 (32.7) N/A
migraine, peripheral neuropathy, meningitis, dementia, Parkinson’s disease and Alzheimer’s disease. Gender, ethnicity and age were recorded for all subjects. For those with ALS, we also noted site of onset, pattern of ALS inheritance, time of patientidentified symptom onset and time of death. Total disease duration (survival) was defined as the time from patient-identified symptom onset to death.
Table II. Disease control diagnoses. No. of males
Disease Alzheimer ’s disease Aseptic meningitis Ataxic disorder Ataxic dysarthria and biparietal lobe dysfunction Bell’s palsy Cognitive deficits and vertigo Dementia Disc herniation Distal neuropathy Downbeat nystagmus Gait disorder Gait dystaxia, multifocal lacunar disease, and dementia Headache Headache and sensory loss Herpes simplex virus meningitis Hydrocephalus Intermittent paroxysmal headache Lumbar pain Lyme disease Memory loss Migraine headaches Multiple sclerosis Muscle weakness Myopathy Pain unspecified Parkinson’s disease Peripheral neuropathy Periventricular lesions Spinal injury and paraplegia Trigeminal neuralgia Undetermined
0 1 0 0 0 0 1 0 0 1 1 0 0 0 1 1 0 1 1 0 1 4 1 0 1 1 2 1 0 0 17
No. of females 1 0 1 1 1 1 0 1 2 0 0 1 1 1 0 0 1 0 1 1 2 14 0 1 0 0 0 0 1 1 13
Total number 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 2 1 3 18 1 1 1 1 2 1 1 1 30
Table III. ALS patient characteristics. Clinical variable Age at onset (yrs), med. (range) Onset to sample time (mo), med. (range) ALS type, number familial, (% familial) Site of onset, number bulbar, (% bulbar) Total disease duration (mo), med. (range) H63D HFE carriers, number (%)
Males (n ⫽ 87) 58.2 23.6 1 26 45.3 19
(30.2 – 81.3) (3.3 –246.7) (1.1) (29.9) (7.3 –193.7) (31.1)
Females (n ⫽ 51) 60.4 (23.3 –79.6) 20.5 (9.0 –166.3) 3 (5.9) 23 (45.1) 35.2 (18.6 –206.3) 9 (22)
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Elevated serum ferritin in ALS patients
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Familial cases were determined by family history. The study was approved by our Institutional Review Board. All patients provided informed consent. Because the H63D HFE polymorphism has been associated with ALS, H63D HFE genotyping was performed as previously described (4) on samples as available. In brief, genomic DNA was purified from leukocytes using QIAamp DNA Mini Kit (Qiagen, Valencia, CA). Polymerase chain reaction followed by restriction fragment length analysis and confirmation DNA sequencing was used to analyze H63D HFE status. Analysis of serum ferritin levels was performed in the clinical laboratory at Penn State Hershey Medical Center. In brief, ferritin was bound to biotinylated antibody, captured by streptavidin and detected using horseradish peroxidase-labeled monoclonal antibody using the Vitros 5600 automated immunoassay system (Ortho Clinical Diagnostics, Rochester, NY). Ferritin levels between groups were compared using one-way ANOVA. Pearson’s correlation was used to analyze the associations between ferritin versus age and ferritin versus survival. Ferritin levels were categorized into low (below the median) and high (equal to or above the median) groups and Kaplan-Meier survival analysis with log rank analysis was conducted. All tests were two-sided with significance set at the p ⬍ 0.05 level. Results Clinical variables from 138 patients with ALS, 152 healthy controls and 82 disease controls were obtained. The majority of subjects were Caucasian. Gender proportions were different between groups, and thus subsequent results were stratified by gender. Age at serum sample collection was different between groups. However, age and ferritin values were not correlated (Pearson’s r ⫽ 0.10, p-value ⬎ 0.05), and thus results were not stratified by age. H63D HFE genotyping was available for 102 patients with ALS and 104 healthy controls; genotyping was not available for disease controls. HFE genotyping indicated 27.5% of patients with ALS harbored H63D HFE polymorphism, consistent with previous reports (3–9) (Table I), whereas 32.7% of healthy controls harbored H63D HFE. The majority of H63D HFE carriers were heterozygous; 6/28 patients with ALS harboring H63D HFE and 2/32 healthy controls harboring H63D HFE were homozygous (Table I). Disease controls had a variety of diagnoses with multiple sclerosis the most common in both males and females (Table II). Clinical characteristics of patients with ALS were representative of the general ALS patient population, with the vast majority of patients classified as having sporadic ALS. H63D HFE genotyping was available for 61 of 87 males and 41 of 51 females with ALS. Of those harboring H63 HFE, two males and four females
Figure 1. Serum ferritin is elevated in patients with ALS. (A) Males with ALS have higher levels of serum ferritin (mean 286.6 ng/ml) versus healthy controls (mean 160.8 ng/ml, p ⬍ 0.001) or disease controls (mean 164.5 ng/ml, p ⫽ 0.003). (B) Females with ALS have higher levels of serum ferritin (mean 142.6 ng/ml) versus healthy controls (mean 69.3 ng/ml, p ⬍ 0.001) or disease controls (mean 77.5 ng/ml, p ⬍ 0.001). Box plot with interquartile range (left), dot plot (middle) and bar graph with means showing standard error of the mean (right) depicted for each group.
were homozygous, with the remainder heterozygous for the allele (Table III). Serum ferritin levels were elevated in males and females with ALS versus healthy controls or disease controls (Figure 1). Kaplan-Meier survival analysis, stratified by gender, was performed. Serum ferritin category did not affect survival in either gender (Figure 2). Pearson’s correlation using raw values also did not indicate an association between ferritin levels and survival in either gender (Figure 3).
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Discussion This study found that serum ferritin levels in patients with ALS were elevated versus healthy controls, consistent with previous research (12,13,15). Serum ferritin levels in ALS patients were also elevated compared to patients with other neurological diseases, suggesting the effect is not simply due to neurological disease. In the circulation, ferritin is an acute phase reactant that reflects systemic inflammation (20), as well as a well-studied indicator of stored iron (21). Previous research demonstrated levels of C-reactive protein and other acute phase reactants were not correlated with serum ferritin in patients with ALS (13), suggesting that ferritin was specific to ALS rather than being indicative of an inflammatory reaction associated with neurological
disease in general. This is further supported by our data for patients with multiple sclerosis. This autoimmune disorder was the most common diagnosis in the disease control group, suggesting that serum ferritin is elevated in ALS not solely because it is a marker of inflammation. The relationship of ferritin to iron status is well established. Iron dyshomeostasis induces oxidative stress by generating reactive oxygen species, and oxidative damage is a hallmark of ALS (22). Ferritin binds to and sequesters iron and other trivalent metals, preventing oxidative damage (23). In transgenic SOD1-G93A mice, expression of both H- and L-ferritin decreased iron catalyzed free radical formation, limiting oxidative damage to lipids, proteins, and nucleic acids possibly caused by mutant
Figure 2. Serum ferritin categories do not predict survival in patients with ALS. (A) Males with serum ferritin levels below the median value (202.0 ng/ml) have similar survival to those with levels equal to or above the median value (median 45.3 versus 44.0 months, p ⫽ 0.778). (B) Females with serum ferritin levels below the median value (123.0 ng/ml) have similar survival to those with levels equal to or above the median value (median 30.4 versus 36.0 months, p ⫽ 0.360).
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Elevated serum ferritin in ALS patients SOD1-induced mitochondrial dysfunction (24). These results suggest elevated serum ferritin may be an adaptive response to increased levels of oxidative stress in ALS. Our finding that serum ferritin levels were not associated with disease duration in patients with ALS contrasts with previous reports suggesting that higher ferritin levels correlate with accelerated disease progression (14) and decreased survival (15). One possible reason for this may be that serum ferritin, or any single biomarker, is insufficient to reliably and consistently reflect the complex neurodegenerative processes involved in ALS. Multivariable approaches involving panels of biomarkers spanning different biologic pathways and tissue compartments may better predict prognosis. In a multivariable analysis, elevated serum ferritin was part of a panel of proteins that predicted longer disease duration in patients with ALS (17). This is consistent with the discussion in the preceding paragraph suggesting that, in animal models, an enhanced capacity to detoxify iron and prevent further oxidative damage could promote neuronal survival in response to oxidative stress. It is possible that other factors confound the use of ferritin as a predictor of disease. For example, even though most patients with ALS receive riluzole treatment as the standard of care, other pharmacotherapies likely differ between patient populations. Differences in pharmacotherapy may influence serum ferritin levels independent of effects on survival, or alternatively, uncouple the association between ferritin levels and survival via unrecognized biochemical mechanisms. In our own study, 17 out of 138 patients with ALS provided samples through the NEALS/NCRI ALS Biofluid Repository, and were part of two separate clinical trials of novel agents. Perhaps effects on serum ferritin and other biomarkers may be useful in assessing the efficacy of therapeutic strategies given the heterogeneous nature of disease progression in ALS. Although the percentage of patients receiving enteral nutrition and the iron content of enteral formulas were not reported in previous studies or available in our study, iron overload exacerbated by enteral nutrition may play a role in the divergent disease course results. Body iron homeostasis is affected by environmental factors, including diet. Dysphagia affects up to 80% of patients with ALS, many of whom receive enteral nutrition (25), which may exacerbate iron overload. The recommended daily iron intake for healthy individuals is 10 mg/day; however, the iron content of typical enteral formulas ranges from 13 to 24 mg/l, and most patients with ALS receiving enteral nutrition consume at least one liter of formula per day (26). Enteral nutrition may worsen iron overload in select individuals with ALS; for example, in the approximately 30% of patients with ALS that carry the HFE gene variant associated with elevated cellular iron uptake (3–9,27). Our results indicate 27.5% of patients with ALS harbor
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Figure 3. Serum ferritin levels are not associated with survival in patients with ALS. Pearson’s correlation indicates no association between serum ferritin levels and survival in males (A) Pearson’s r ⫽ 0.011, p ⫽ 0.933) or females (B) Pearson’s r ⫽ 0.197, p ⫽ 0.237).
H63D HFE, further arguing for the importance of this polymorphism in the disease, and the need to investigate the molecular and physiological consequences of HFE polymorphism in ALS. There are a number of limitations to our study. Serum ferritin was the only marker of iron metabolism analyzed in this study. Other studies have demonstrated significant differences in transferrin and the transferrin saturation coefficient in patients with ALS versus healthy controls (15). However, elevated serum ferritin is the most robust finding across a number of studies (12,13,15), with conflicting results reported for other markers of iron metabolism, including transferrin and transferrin saturation (13). Thus, we chose to study serum ferritin. Our study was cross-sectional, and did not follow serum ferritin
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levels over time. This has been a limitation of all studies investigating ferritin levels in ALS, and ALS biomarkers research in general. Longitudinal analysis may uncover the temporal dynamics of ferritin in ALS, allowing more precise delineation of its role in pathophysiology. In summary, our finding of increased serum ferritin levels in patients with ALS may reflect increasing cellular iron load, or alternatively, up-regulation of adaptive mechanisms for buffering iron and other trivalent trace metals (23). The lack of correlation between ferritin levels and survival in our study may reflect the limitation of any single biomarker to adequately capture disease prognosis that could stem from differences in pharmacotherapy (including enteral feeding), genotype that would influence environmental interaction, and body iron status. It also could be an important indicator of a loss of ferritindependent iron buffering capacity in patients with increasingly severe disease. Acknowledgements The authors would like to thank Robert Lawson and the Northeast Amyotrophic Lateral Sclerosis Consortium for providing serum samples for this study. The study was supported in part by the Paul and Harriett Campbell Fund for ALS Research; the Zimmerman Family Love Fund; and the ALS Association, Greater Philadelphia Chapter. Declaration of interest: The authors report no confl icts of interest. The authors alone are responsible for the content and writing of the paper. References 1. Robberecht W, Philips T. The changing scene of amyotrophic lateral sclerosis. Nat Rev Neurosci. 2013;14: 248–64. 2. Nandar W, Connor JR. HFE gene variants affect iron in the brain. J Nutr. 2011;141:S729–39. 3. Wang XS, Lee S, Simmons Z, Boyer P, Scott K, Liu W, et al. Increased incidence of the Hfe mutation in amyotrophic lateral sclerosis and related cellular consequences. J Neurol Sci. 2004;227:27–33. 4. Goodall EF, Greenway MJ, van Marion I, Carroll CB, Hardiman O, Morrison KE. Association of the H63D polymorphism in the hemochromatosis gene with sporadic ALS. Neurology. 2005;65:934–7. 5. Restagno G, Lombardo F, Ghiglione P, Calvo A, Cocco E, Sbaiz L, et al. HFE H63D polymorphism is increased in patients with amyotrophic lateral sclerosis of Italian origin. J Neurol Neurosurg Psychiatry. 2007;78:327. 6. Sutedja NA, Sinke RJ, van Vught PW, van der Linden MW, Wokke JH, van Duijn CM, et al. The association between H63D mutations in HFE and amyotrophic lateral sclerosis in a Dutch population. Arch Neurol. 2007;64:63–7. 7. He X, Lu X, Hu J, Xi J, Zhou D, Shang H, et al. H63D polymorphism in the hemochromatosis gene is associated with sporadic amyotrophic lateral sclerosis in China. Eur J Neurol. 2011;18:359–61.
8. Praline J, Blasco H, Vourc’h P, Rat V, Gendrot C, Camu W, et al. Study of the HFE gene common polymorphisms in French patients with sporadic amyotrophic lateral sclerosis. J Neurol Sci. 2012;317:58–61. 9. van Rheenen W, Diekstra FP, van Doormaal PT, Seelen M, Kenna K, McLaughlin R, et al. H63D polymorphism in HFE is not associated with amyotrophic lateral sclerosis. Neurobiol Aging. 2013;34:1517, e5–7. 10. Liu Y, Lee SY, Neely E, Nandar W, Moyo M, Simmons Z, et al. Mutant HFE H63D protein is associated with prolonged endoplasmic reticulum stress and increased neuronal vulnerability. J Biol Chem. 2011;286:13161–70. 11. Nandar W, Neely EB, Unger E, Connor JR. A mutation in the HFE gene is associated with altered brain iron profiles and increased oxidative stress in mice. Biochim Biophys Acta. 2013;1832:729–41. 12. Qureshi M, Brown RH Jr, Rogers JT, Cudkowicz ME. Serum ferritin and metal levels as risk factors for amyotrophic lateral sclerosis. Open Neurol J. 2008;2:51–4. 13. Goodall EF, Haque MS, Morrison KE. Increased serum ferritin levels in amyotrophic lateral sclerosis patients. J Neurol. 2008;255:1652–6. 14. Ikeda K, Hirayama T, Takazawa T, Kawabe K, Iwasaki Y. Relationships between disease progression and serum levels of lipid, urate, creatinine and ferritin in Japanese patients with amyotrophic lateral sclerosis: a cross-sectional study. Intern Med. 2012;51:1501–8. 15. Nadjar Y, Gordon P, Corcia P, Bensimon G, Pieroni L, Meininger V, et al. Elevated serum ferritin is associated with reduced survival in amyotrophic lateral sclerosis. PLoS One. 2012;7:e45034. 16. Mitchell RM, Simmons Z, Beard JL, Stephens HE, Connor JR. Plasma biomarkers associated with ALS and their relationship to iron homeostasis. Muscle Nerve. 2010; 42:95–103. 17. Su XW, Simmons Z, Mitchell RM, Kong L, Stephens HE, Connor JR. Biomarker-based predictive models for prognosis in amyotrophic lateral sclerosis. JAMA Neurol. 2013; 70:1505–11. 18. Danzeisen R, Achsel T, Bederke U, Cozzolino M, Crosio C, Ferri A, et al. Superoxide dismutase-1 modulates expression of transferrin receptor. J Biol Inorg Chem. 2006;11: 489–98. 19. Brooks BR, Miller RG, Swash M, Munsat TL. El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord. 2000;1:293–9. 20. Wang W, Knovich MA, Coffman LG, Torti FM, Torti SV. Serum ferritin: past, present and future. Biochim Biophys Acta. 2010;1800:760–9. 21. Torti FM, Torti SV. Regulation of ferritin genes and protein. Blood. 2002;99:3505–16. 22. Barber SC, Shaw PJ. Oxidative stress in ALS: key role in motor neuron injury and therapeutic target. Free Radic Biol Med. 2010;48:629–41. 23. Joshi JG, Sczekan SR, Fleming JT. Ferritin: a general metal detoxicant. Biol Trace Elem Res. 1989;21:105–10. 24. Olsen MK, Roberds SL, Ellerbrock BR, Fleck TJ, McKinley DK, Gurney ME. Disease mechanisms revealed by transcription profiling in SOD1-G93A transgenic mouse spinal cord. Ann Neurol. 2001;50:730–40. 25. Greenwood DI. Nutrition management of amyotrophic lateral sclerosis. Nutr Clin Pract. 2013;28:392–9. 26. Molfino A, Kushta I, Tommasi V, Rossi Fanelli F, Muscaritoli M. Amyotrophic lateral sclerosis, enteral nutrition and the risk of iron overload. J Neurol. 2009;256:1015–6. 27. Lee SY, Patton SM, Henderson RJ, Connor JR. Consequences of expressing mutants of the hemochromatosis gene (HFE) into a human neuronal cell line lacking endogenous HFE. FASEB J. 2007;21:564–76.
