While the bulk of the findings from this study described reports of iron levels in AD, several important findings relevant to zinc and copper were noteworthy. No significant change in bulk neocortical zinc levels was found, although a modest elevation in the parietal lobe was noted. The data on zinc levels in the neocortex was heterogeneous and no clear explanation for the heterogeneity could be deduced from the meta-data. However, some of the included studies indicated that their brain samples contained equal portions of white and grey matter, while others were more ambiguous; the study reporting the largest increase in zinc levels sampled from temporal lobe cortex (Religa et al 2006
). Consistent with this pattern, we recently reported that zinc levels were unchanged in AD in temporal lobe white matter, but were significantly increased in the overlying cortical ribbon (Schrag et al 2010b
). It is therefore possible that the heterogeneity of these results is due to differences in tissue sampling.
The available evidence suggests that copper is generally depleted in AD (although copper was noted to increase in the putamen by one study). A single discordant study originating from the U of K (excluded from this analysis) indicated a more than four-fold increased copper levels in AD (potentially a fixation artifact) -- this study is the most cited paper on the subject of copper in AD and appears to be the source for numerous articles reporting that copper levels are (several fold) increased in AD (Bush 2000
, Cuajungco et al 2000
, Filiz et al 2008
, Lovell et al 1998
, Rottkamp et al 2001
). It is important to emphasize that the overwhelming number of studies report that copper is not increased in AD brain. Of note, a clinical trial of D-penicillamine, a copper chelator, was unable to produce any clinical improvement in the treated cohort of AD patients (in fact patients trended toward worse outcomes), although subjects experienced numerous toxicities resulting in one subject death and the early suspension of the trial (Squitti et al 2002
Regarding iron, we conclude that Alzheimer’s disease does not appear to alter neocortical iron levels. Iron was modestly elevated in the AD putamen over controls, but no other brain region appeared to be affected. The increases in tissue iron in deep grey matter may be a significant component of Alzheimer’s disease pathology, but certainly do not account for the neocortical dysfunction observed in this disease. Moreover, AD is commonly comorbid with some degree of Lewy body disease which is strongly associated with increases in basal ganglia iron -- the findings in the putamen may be more reflective of this disease process (Chavhan et al 2009
, Dexter et al 1991
). Gradient echo T2* (GRE-T2*) and susceptibility weighted imaging (SWI) are iron-sensitive sequences that have been used to follow brain iron levels in AD patients. Several large studies of this technology have been conducted to evaluate the usefulness of following brain iron levels as a biomarker of AD. In these studies the putamen was the only region consistently found to contain increased levels of iron in AD (Ding et al 2009
, Kirsch et al 2009
, Zhu et al 2009
). This is remarkably consistent with the results of this meta-analysis and validates the utility of this technology for noninvasively estimating tissue iron levels. However, increases in putamen iron are not specific to AD and therefore may not be particularly helpful in establishing a diagnosis. Few studies described the levels of iron in males versus females with AD, but the limited data available does not show any significant difference between sexes (Magaki et al 2007
). Finally, none of the included studies specifically analyzed iron levels in brains from patients with early-onset and familial forms of AD.
Focal alterations in metals distribution have been suggested by several studies to be associated with the primary pathologies of AD. All three metals evaluated here are reported to accumulate within senile plaques, although other studies call into question the consistency of this observation. One study found 30% of plaques had no detectable iron in them while a few of the largest plaques had high concentrations of redox-active magnetite. Because of the inhomogeneous distribution of metals in the brain, it is important to cautiously interpret the finding that bulk levels of iron are unchanged – an underlying alteration in iron metabolism may still be present and if this were to result in an increased fraction of poorly liganded iron it is reasonable that it could account for the oxidative injuries observed in Alzheimer’s disease. Never-the-less, based upon these cumulative findings and because of the disproportionate impact of outlier data on the literature, we feel it will be important to re-evaluate brain metals-overload hypotheses particularly when considering additional clinical trials of metal chelating/modulating therapies. It is fundamentally important that the application of metal-targeted pharmacology restores not only normal metal levels, but also normal transition metal physiology.
Importantly, the data from this meta-analysis indicates that there is a wide-spread misconception in the scientific literature regarding the levels of several transition metals, most prominently iron, in AD brain. Less than 1% of the review articles analyzed in this study reported that iron levels were not increased. This family of studies spanning fifty years serves as a case-study in the development of a dogma and we tried to understand what factors contributed to the development of the distortion and what strategies might be reasonable to modify this risk. The misconception appears to have arisen as a result of significant citation bias (p<0.0001) in the absence of publication bias which amplified the contributions of one laboratory which were significantly different from all other published reports.
Systematic evidence describing citation bias is limited. The bulk of available evidence describing the phenomenon is focused on clinical studies where it is obviously of great importance – selective citation may misguide clinical and health-policy decision making which can immediately endanger patients. Citation bias has been reported in a wide range of topics, including literature describing smoking rate among schizophrenics (Chapman et al 2009
), in clinical trials for various hepato-biliary diseases (Kjaergard et al 2002
) and the effect of anti-inflammatory drugs on rheumatoid arthritis (Gotsche et al 1989
) among others topics. Evidence for citation bias in basic science literature is more limited, although an elegant study recently demonstrated extensive bias in studies reporting the presence or absence of amyloid in muscle tissue in inclusion body myositis (Greenberg 2010
). Citation bias is generally thought to favor studies reporting significant findings (what has sometimes been termed optimism bias), although there is evidence for mixed biases, with certain fields favoring over-citation of negative associations (Hutchison et al 1995
, Nieminem et al 2007
). Understanding how citation bias develops is important in developing strategies to control it. One reasonable hypothesis that has been presented is that studies reporting significant findings are likely to be published in higher impact factor journals and are therefore more visible which could account for higher citation rates. However, one of the earliest analyses of citation bias reported no significant correlation between citation rates of individual articles and journal impact factor (Seglen 1989
). These findings are consistent with the results of our study – negative results were just as likely to be published in high impact factor journals. Another hypothesis suggests that a regional citation bias favors citation of articles originating in the United States or in English-speaking countries. Paris et al argued that Italian scientific contributions to the field of environmental science were systematically under-cited despite being published in journals with strong impact factors relative to the field (1998
). We could not exclude some component of regional citation bias in our results. While the studies that received the greatest number of citations in our analysis originated in North America, those reporting —negative findings were cited at comparable rates regardless of the geographical origin of the publication – of course, this study is probably far too small to effectively identify such a bias. Finally, some evidence has previously suggested that narrative review articles were particularly prone to citation bias (Schmidt et al 2006
). This pattern was strikingly evident in our analysis as well. Among other purposes, the narrative review is a venue to distill current research to frame scientific hypotheses. Unfortunately, the tacit assumption of unbiased literature sampling in the formulation of review literature has proved to be unreliable. This is probably not so conspiratorial as it is a human and technological reality which underscores the need for more rigorous systematic analysis of basic science data to provide the foundation for clinical trials in a given field.
Using meta-analysis and systemic review methodologies we have identified the wide-spread misconception in AD literature that iron, and to a lesser degree zinc and copper, levels are increased in AD brain. In addition to misleading research studies, this may also prove to be dangerous to AD patients. Therapies based on this largely unsupported dogma could result in unexpected toxicities and failure to be of therapeutic value. In light of our findings it will be important to re-evaluate the brain metal-overload hypothesis in AD and critically review related research and review articles.