Role of Copper in the Onset of Alzheimer’s Disease Compared to Other Metals

1Alzheimer’s disease (AD) is a progressive neurodegenerative disorder described in 1907 by Alois Alzheimer. He observed amyloid plaques and neurofibrillary tangles (NFTs) in the brain of patients showing signs of dementia. Today, AD is the most prevalent neurodegenerative disease affecting 10% of people aged 65+ and 50% of people aged 80+. In 2016, it was estimated that there were about 47 million AD patients in the world and this number is expected to increase to more than 130 million by 2050. The World Alzheimer Report evaluated the annual social and economic cost of dementia to be US$ 818 billion worldwide in 2015 and this amount is expected to increase to one trillion by 2018.

2Alzheimer’s disease is ultimately lethal, characterized by the developing damage of neuronal tissues in the brain. Signs include memory loss, paranoia, loss of reasoning powers and confusion. Unfortunately, AD is recognized only after the manifestation of cognitive signs, which may be too late for effective treatment. Moreover, approved drugs have inconsiderable effects on patients’ well-being, which may be because many factors are responsible for AD. Indeed, there are several theories to demonstrate the cause of AD supported by empirical data, which have been reviewed by.

3In brain regions affected in AD, such as the cortex and hippocampus, extracellular senile plaques, and intracellular NFTs accumulate. Senile plaques or amyloid plaques, as the name implies, are mainly composed of small peptides called β-amyloid. The latter is produced from β-amyloid precursor protein (APP) through successive cleavages first by β-secretase at residue 671 and then by γ-secretase at residues 711 or 713 (residue numbering according to the APP770 isoform) in amyloidogenic pathway. Alternatively, APP molecules can be cleaved by α-secretase within the β-amyloid domain at residue 687 and prevent β-amyloid production in non-amyloidogenic pathway. There are three main isoforms of APP including APP695, APP751, and APP770 with APP695 being expressed at high levels in brain compared to other isoforms. Furthermore, NFTs, mainly composed of tau proteins, is the other important factor that accumulate in AD brain. Tau proteins mostly expressed in neurons have the ability to induce microtubule assembly in vitro. In fact, microtubules, one of the main components of the cytoskeletal system, are involved in the maintenance of neuronal morphology and the formation of axonal and dendritic processes. One of the first and most severely injured brain areas in AD is the hippocampus, which is associated with neurogenesis and long-term memory storage. It is also thought to be more susceptible to metal disturbance than other brain areas. Another brain region that suffers from damage in AD due to plaque pathology is the cortex, associated with functions such as argumentation, feeling, and language.

4β-amyloid aggregations into senile plaques are one of the main characteristics of AD. A considerable co-localization of adenosine receptors and β-amyloid has been reported in senile plaques. Adenosine, a purine ribonucleoside that has neuromodulatory and neuroprotective properties, affects various important brain functions such as sleep, cognition, memory, and neurodegeneration. Adenosine is involved in numerous neurological disorders including AD. It exerts its various effects via its receptors, and thus managing its receptor agonists and antagonists significantly influences learning and memory. On one hand, deamination of adenosine to inosine by adenosine deaminase (ADA) is one of the metabolic pathways for the catabolism of adenosine in the brain. On the other hand, ADA acts as an allosteric modulator of adenosine receptors. Because of ADA’s involvement in different health disorders, the development of ADA inhibitors as feasible therapeutic agents has been considered in many studies. Recently, ADA inhibitors have been proposed in perinatal hypoxia–ischemia brain injury treatment.

5Polyvalent metal cations such as copper, zinc, and iron are found in high concentrations in senile plaques in AD patients’ brain. Furthermore, some studies in mouse models of AD revealed that in spite of accumulation of copper in senile plaques in the mouse models with neurodegeneration including 5 × FAD and CVN, no copper accumulation is observed in PSAPP mouse model with slight neurodegeneration. Considerable data point to dyshomeostasis of zinc and copper ions as the main factor of AD pathogenesis and indicate that copper metabolism proteins are associated with AD.

6Different authors have put forward various models of the toxicity of copper involvement in AD. The most accredited one proposes the gain-of-function of β-amyloid after binding Cu2+. Alternative and more recent hypotheses propose a protective role of β-amyloid against an excess of toxic metals within the brain, designating β-amyloid loss-of-function as a pathogenic process in the disease. APP is thought to possess a normal function in metal export from neurons, and a putative loss of the soluble, functional β-amyloid monomer could cause copper build-up in the cell.

7Scientific evidence has shown that metal ion binding to β-amyloid accelerates amyloid aggregation, which could finally damage the neurons in AD. The involvement of copper in AD is controversial, as some studies show copper deficiency in AD, and consequently a need to enhance copper levels, while other data point to copper overload and therefore a need to reduce copper levels. An aberrant copper homeostasis with an increase in the labile pool of copper and a decrease in the copper bound to protein is the main up-dated interpretation. In this paper, the role of metal ions, particularly copper, in AD is reviewed and discussed.

8The key event in AD is the formation of fibrils and plaques in AD patients’ brain. Plaques are mainly made of β-amyloid peptide, the natural peptide that is produced in the brain and exists at nanomolar concentration levels in cerebrospinal fluid (CSF) and serum. On the other hand, a high concentration of trace metals, including copper, is observed in amyloid plaques. Interestingly, some data show that copper distribution in the brain does not correspond to β-amyloid plaques distribution in TASTPM mice model with plaque pathology but not appreciable neuronal loss. Copper is a necessary trace metal in nervous system development since disruption of its homeostasis leads to neurodegenerative disorders like Menkes and Wilson’s diseases. Cu2+ ions bind to β-amyloid peptides with high affinity and increase the proportions of β-sheet and α-helix structures in amyloid peptides, which can be responsible for β-amyloid aggregation. Various concentrations of Cu2+ ions enhance fibril formation while binding of copper ions to β-amyloid noticeably increases its toxicity for cells. In addition, substoichiometric concentrations of Cu2+ are more toxic to cells.

9Fibril formation is highly pH-dependent and Cu2+ ions cause it to occur at physiological pH. However, the formation of amorphous aggregates dominates in acidic conditions. In a proton-rich environment, β-amyloid (Aβ40) possesses two copper binding sites, and its second bound Cu2+ion causes the formation of amorphous aggregates by preventing the conformational transition of β-amyloid into amyloid fibrils.

10The production of Reactive Oxygen Species (ROS) is a key factor in β-amyloid toxicity toward neurons, which is dependent on metal ion redox properties. Copper ions in complex with β-amyloid fibrils produce hydrogen peroxide, in the presence of biological reducing agents. When the ratio of copper to peptide increases, hydrogen peroxide levels and the production of hydroxyl radicals increase, and the morphology of aggregates changes from fibrillar to amorphous. Although previous studies have presented ROS as fatal molecules provoking neurodegeneration, the accumulated evidence shows that some ROS act as essential molecules in processes underlying cognition and memory formation. On the other hand, some results imply that the copper-amyloid complex produces fewer ROS than free copper ions. According to in vitro data, oligomeric and fibrillar forms of β-amyloid inhibit H2O2 generation at higher concentrations of Cu2+. In addition, the fibrillar form generates less H2O2 than the oligomeric form.

11Copper toxicity in AD brains is attributed to the oxidized form of copper ions, i.e., Cu2+,. In contrast, other data show that copper ions are only transported in their reduced form, i.e., Cu1+,. Some studies suggest that Cu2+ bypasses the liver. Otherwise, some data show that the removal of Cu1+ from β-amyloid, hinders the formation of oligomers and prevents ROS production. A study of the affinity of the soluble copper-binding domain of the β-amyloid peptide for Cu1+shows that it binds to β-amyloid stronger than Cu2+ suggesting Cu1+ is the relevant in vivo oxidation state. Both Cu1+ and Cu2+ inhibit β-amyloid degradation by insulin-degrading enzyme, but Cu1+ cations act as irreversible inhibitors. Copper ion by reduction from Cu2+ to Cu1+ protects proteins against free radicals.

12A meta-analysis and subsequent studies demonstrated that the concentration of total copper is decreased in the brain of AD patients, while the concentration of labile copper is increased in the most affected regions of the AD brain. In addition, AD cortical tissues and the cortex of mice with Traumatic Brain Injury show an elevated binding capacity for Cu2+. Another study shows that in APPsw/0 mouse model of AD, which shows parenchymal plaques but no neuronal loss, unlike in the control mouse in which the metal accumulates in the capillaries, copper ions accumulate in brain parenchyma. These ions could bind to β-amyloid and stimulate β-sheet conformation, aggregation, and toxicity.

13In the “amyloid cascade hypothesis,” plaque formation is a main event in AD pathology but it is sometimes preceded by neurodegeneration, and plaque clearance by immunization of AD patients does not prevent disease progression. Moreover, some studies show that senile plaques exist in cognitively normal people and, despite an equivalent plaque presence, the concentration of brain amyloid oligomers is higher in AD patients than in normal cases. The “Toxic oligomers hypothesis” explains these events by suggesting that small, diffusible oligomers are responsible for toxicity, and not the amyloid plaques. The oligomers derived from cell culture have unusually high chemical stability and resist degradation into monomers by various degrading agents, supporting the existence of covalent cross-links between the oligomers. Based on in vitro experiments, Cu2+ binding to β-amyloid can lead to the formation of dityrosine-linked dimers of β-amyloids found in AD. In the presence of Cu2+, the dimer conformation changes from parallel to anti-parallel and is stabilized by the occupied copper binding sites. However, the same authors showed later that Cu2+ at nanomolar concentrations has no effect on peptide–peptide affinity in the amyloid dimer. Other authors have demonstrated that binding of Cu2+ ions induces structural changes in the amyloid dimer resulting in N-termini interactions within it. The mutant dimer that is unable to produce cross-links provides supporting evidence for the toxicity of Cu2+cross-linked dimers because the mutant dimer’s properties are the same as those of the wild type dimer except that it has no neurotoxicity (; Figure 1). In addition, other studies suggest that the toxicity of the cross-linked dimer is due to enhanced membrane binding.

14Elimination of Cu2+ from β-amyloid prevents amyloid aggregation in vitro, and promotes β-amyloid degradation, and prevents H2O2 formation. Hence, it also decreases cell mortality. Because of these positive effects of copper elimination, some studies have targeted copper chelators as suitable drugs. However, the most recently published review on copper chelation therapy states that the results in human clinical trials are discouraging, even though some authors have refuted this interpretation. Furthermore, studies in Tg2576 mouse model of AD, which shows parenchymal plaques but no neuronal loss, show that although the use of chelator helps to prevent AD, it is inefficient in AD treatment suggesting that systemic copper removal is useful only in the early stages of the disease. Interestingly, there is some evidence that making changes in brain copper uptake in the primary stages can have a considerable effect on amyloid pathology.

15Until 2012, a number of ambiguous results published previously fueled a debate about copper levels in AD patients. Overall, six meta-analyses have been carried out in the last 6 years to evaluate copper concentrations in AD in different biological matrices (serum, plasma, and cerebrospinal fluid). These meta-analyses, combining data collected from studies published between 1984 and 2017, provide unequivocal results: total copper and “free” copper are higher in the serum–plasma of AD patients in comparison with healthy controls. More specifically, the large stand most recent meta-analysis (total pool of subjects analyzed: 2128 AD vs. 2889 healthy controls) includes a total of 35 studies: 18 report an increase, 14 no difference, and one a decrease in values of copper in the serum–plasma in AD compared to healthy controls. Three additional studies appeared after the publication of this consensus result, reporting increased concentrations of Cu2+ in AD patients vs. controls.

16Recent studies have contributed to unraveling further the initial controversy, demonstrating that the increased concentration of serum copper in AD can be explained by the increased concentrations of the plasma fraction of the “free” copper pool in the blood, which is detected in only 50–60% of AD patients. An older study also indicated that serum copper concentration rises in a special type of AD. Some studies have proposed a genetic basis for this AD subtype as an explanation of this observation.

17A significant reduction in copper ion levels is observed in the hippocampus and amygdale areas of AD patients compared to age-matched control subjects. In addition, a reduction in net copper is found in the brain of TgCRND8 AD mice model, and this model exhibits parenchymal amyloid deposition but no neuronal loss. As mentioned above, a meta-analysis indicates that copper is significantly decreased in the brain of AD patients. An analysis of the human brains of deceased patients with dementia concludes that defective regions have a very low copper content. The copper content of aged human brains has a significant negative correlation with the degree of severity of amyloid plaques. Based on the results showing a significant reduction in copper ion in AD patients compared to controls, it has been hypothesized that AD is a result of copper deficiency. An alternative, and more comprehensive, interpretation, which can explain the copper quantification results of meta-analyses in serum–plasma and the meta-analysis in the brain, is that the copper decrease in the brain is a sign of an aberrant copper homeostasis, which resembles Wilson’s disease. Interestingly, some data indicate that copper deficiency in AD patients is independent of their diet.

18In the presence and absence of copper, APP molecules are cleaved in non-amyloidogenic and amyloidogenic pathways, respectively. The latter pathway results in amyloid production. Some data show that copper ions inhibit amyloid production by interacting with a γ-secretase complex or by affecting APP dimerization. Otherwise, copper ions enhance APP exposure on the cell surface by both increasing its exocytosis and decreasing its endocytosis. Cu deficiency, as observed in AD patients, enhances β-amyloid production and accumulation by inducing the amyloidogenic processing of APP. However, its molecular mechanism is still unclear.

19The mutations in genes encoding proteins required for copper ion uptake in mammalian systems lead to early-onset familial AD. On the other hand, enhancement of intracellular copper levels through addition of dietary copper in APP/PS1 transgenic AD mice and APP23 transgenic mice with parenchymal plaques but no neuronal loss, causes a reduction in AD pathology. Based on the report of low brain copper in several neurodegenerative disorders, impairment in copper protection against free radicals has been proposed as the main cause of these disorders.

20A significant elevation in Zn2+ is found in the AD hippocampus and amygdale area, and in the AD neuropil compared to controls, although some data indicate an elevation in Zn2+ content in the AD brain cortex. Accordingly, a meta-analysis revealed no significant changes in zinc content in the AD neocortex. Nevertheless, three meta-analyses were recently carried out on plasma and serum zinc levels in AD patients and healthy controls. One study (the pooled sample size included 777 AD vs. 1728 healthy controls) analyzed a total of 16 studies and concludes that AD patients show a decrease in serum zinc levels compared to healthy controls. Ventriglia et al.’ (2015) study (the pooled sample size included 287 AD vs. 166 healthy controls) analyzed a total of five studies on plasma zinc: two indicate a decrease and three no significant differences in plasma zinc in AD patients compared to controls. The other study (the pool of subjects included 862 AD patients vs. 1705 controls) analyzed a total of 17 studies: 10 indicate a decrease, and seven no significant differences in serum zinc in AD patients compared to controls. The most recent meta-analysis by analyzed 22 studies with a total pool of 1027 patients with AD and 1949 healthy controls: three studies indicate an increase, 18 studies a decrease, and one study no differences in serum zinc between AD and healthy controls. Interestingly, an older study found that a rise in serum Zn2+ occurs in a special type of AD. As the absence of fibrinogen in serum is the main difference between serum and plasma compositions and fibrinogen-β-amyloid interactions are involved in AD progression, it is likely that the reduction in serum Zn2+ in AD patients is due to its interactions with fibrinogen. The rise in zinc levels is accompanied by a rise in tissue amyloid levels. AD brain tissue contains hot spots of metal ions especially enriched by copper and zinc ions. In 2006, it was reported for the first time that β-amyloid plaques and hot spots of accumulated metal ions co-localize.

21It has been suggested that zinc ions induce β-amyloid aggregation in vitro whereas Cu2+ ions inhibit it through competing with zinc for histidine residues. The strongest inhibitory effect occurs at a copper: β-amyloid molar ratio of about four. Above this value, copper ions themselves induce aggregation. Interestingly, previous studies have shown that β-amyloid binds to three or four Cu2+ ions at pH 7.0. Studies on synthetic Aβ(1–40) and Aβ(1–42) peptides show that at physiological pH, β-amyloid binds the same ratio of copper and zinc ions, whereas in acidic conditions copper ions replace zinc ions (Figure 2;; ). Cu2+ and Zn2+ ions inhibit β-amyloid fibrillization, promoting instead the formation of non-fibrillar aggregates in vitro. In addition, zinc ions have a threefold stronger inhibitory effect than copper ions. Both Cu2+ and Zn2 ions prevent the formation of soluble fibrils if incubated with Aβ(1–42) in vitro. Some authors have also reported that small, soluble oligomers play the main role in β-amyloid neurotoxicity. Cu2+ ions form stable and soluble 1:1 complexes with β-amyloid (Aβ40) while Zn2+ ions cause their partial aggregation. Cu2+-induced aggregates are toxic to neurons only in the presence of ascorbate, while monomers and zinc-induced aggregates are not toxic.

22The role of zinc supplementation in AD remains controversial according to a systematic review in 2012 reporting that Zn2+ in the diet has no effect on cognitive decline even though the Zenith and the Zincage studies suggested some effects. However, 3xTg-AD mice model with background neuropathology similar to AD patients displayed a delay in memory impairment. Furthermore, Tg2576, TgCRND8 and CRND8/E4 models exhibited a potentiationin memory impairment. Moreover, Tg2576 mice and Sprague Dawley rats showed a lower brain copper and amyloid burden, and a higher cognitive performance, respectively.

23Selective release of Cu2+ and Zn2+ ions inside the cells by complexes can decrease the level of β-amyloids. B-amyloid peptides in the presence of physiological concentrations of copper and zinc ions are degraded but at higher concentrations of these cations they aggregate. In agreement with this finding, the use of a copper–zinc chelator rapidly causes a considerable decrease in the deposition of brain amyloid in APP2576 transgenic mice. Other authors suggest that the addition of chelators to β-amyloid aggregates induces their rapid fibrillization in vitro. In addition, a long incubation of non-fibrillar aggregates transforms them into fibrillar forms. These findings confirm that the fibrillar form of β-amyloid is its most stable state. Zn2+ and Cu2+ ions induce refolding of β-sheets into α-helix structures leading to oligomerization and membrane penetration of amyloid peptides. Copper and zinc ions both induce a β-amyloid conformational change but zinc has a greater effect.

24Iron is a redox active metal and its brain level varies according to specific regions of the brain. Commonly, the brain areas responsible for motor functions are found to have higher iron concentrations than non-motor related areas. Iron also has a high concentration at the periphery of amyloid plaques. Nevertheless, there is controversy about the level of iron outside plaques in the AD brain. Contrary to copper and zinc, very few structural studies have been reported on iron coordination to β-amyloid. While copper and zinc co-purify with β-amyloid extracted from plaques, iron does not co-localize within β-amyloid deposits and in vitro studies have shown that β-amyloid binds to iron with low affinity. After Louis Goodman’s case studies performed in the 1950s, a relationship between regions of AD pathology and iron accumulation was proposed. Some data show that β-amyloid reduces Fe3+–Fe2+ in solution in vitro. While some authors argue that the direct coordination of Fe3+ to β-amyloid is impossible at natural pH and that Fe2+ is air-sensitive and oxidizes to Fe3+ during measurements, others report a potential pro-aggregating function for Fe2+ and Fe3+ and some data show a higher affinity for β-amyloid to Fe2+ relative to transferrin. Three meta-analyses have been carried out in the last 4 years to assess iron concentrations in AD in serum, cerebrospinal fluid, and brain. meta-analysis (total pool of subjects analyzed: 1813 AD vs. 2401 healthy controls) includes a total of 43 studies; 21 consider serum iron; seven consider CSF iron and 19 investigate iron in various brain areas in AD. Their results shows that serum iron significantly decreases in AD compared to controls. CSF iron shows no difference while some specific brain areas show an increase in iron concentration. meta-analysis (total pool of subjects analyzed: 1084 AD vs. 1319 healthy controls) includes a total of 18 studies showing no difference in serum iron but, after exclusion of the study producing high heterogeneity, their results conclude that serum iron levels are significantly lower in AD subjects. meta-analysis (total pool of subjects analyzed: 1379 AD vs. 1664 healthy controls) includes 25 studies that show overall no significant difference in serum iron between AD and controls but, after excluding two studies with high heterogeneity, serum iron is significantly lower in AD cases.

25There is considerable evidence that the amyloidogenic pathway takes place in lipid rafts, which are specific membrane domains enriched in cholesterol. The enzymes that cleave APP to β-amyloid peptides are found in lipid rafts. Cellular copper deficiency results in an accumulation of copper ions in cholesterol-rich lipid rafts. In fact, the copper level in lipid rafts is inversely related to cellular copper, which results in enhanced copper-amyloid complex formation under copper deficiency conditions in AD.

26Clearly, diet is a critical factor in the progression of AD. Several studies indicate the key role of fat in AD. The effective role of Cu2+ and cholesterol overload in neurodegeneration has been reported. In rats treated with copper and cholesterol, a significant change in visuo-spatial memory is detected while the administration of dietary cholesterol plus copper-supplemented drinking water (in the form of copper sulfate) induces an accumulation of β-amyloid in rabbit brain. The binding of Cu2+ ions to β-amyloid causes oxidation of cholesterol, and the generation of H2O2 and other lipid peroxidation products accumulating in AD patients’ brain and in Tg2576 transgenic mouse model. In fact, previous unsuccessful therapeutic attempts and recent findings regarding β-amyloid accumulation at lipid rafts have led to a new hypothesis that neurotoxicity in AD is the result of the association of small soluble amyloid oligomers with the plasma membrane. B-amyloid membrane binding is mediated by its interactions with phosphatidylserine.

27A highly toxic isoform of β-amyloid that accumulates in AD patients’ brain has a great capacity to induce lipid peroxidation. It also alters the calcium influx by binding to cell membranes. This isoform does not lead to an increase in ROS but it causes instead a reduction in plasma membrane integrity and an increase in dityrosine-β-amyloid oligomers. Other studies have suggested that the toxicity of the cross-linked dimer is due to enhanced membrane binding.

28Simulation experiments show that increased cell membrane cholesterol results in some changes in the membrane; namely, an enhancement of surface hydrophobicity and a reduction in bilayer mobility. These membrane changes induce amyloid binding to the cell membrane and cause β-amyloid to refold into a helical or unstructured form. B-amyloid is stabilized on the membrane surface or inserted into the bilayer with the help of calcium ions. In fact, β-amyloid peptides compose the oligomeric pores in the membrane via the cholesterol-binding domain. Channel formation is cholesterol-dependent and occurs in the presence of at least 30% cholesterol in lipid bilayer membranes. These pores are ion channels that disrupt calcium homeostasis in neural cells and have led to the return of the “calcium hypothesis” of AD (,; Figure 3). In the absence of copper, β-amyloids are cleared to the blood even if there are increased cholesterol levels, while in the presence of copper β-amyloid accumulate in the brain. Otherwise, copper ions in the absence of β-amyloid are not toxic to cells.

29Alzheimer’s disease is a progressive degenerative disease characterized by the presence of senile plaques in the AD patient’s brain. It is a multifactorial disease with a number of genetic and environmental factors likely involved. Previous studies have identified these plaques as toxic elements in AD. However, other studies have demonstrated that 20–40% of healthy people possess senile plaques in their brains. Moreover, cell death sometimes precedes plaque formation. A number of studies have focused on ROS generation by a copper-amyloid complex and propose ROS as toxic elements in AD. Still other authors have shown that ROS are the basis of memory formation. In addition, fibrils and oligomers at higher copper ion concentrations inhibit the production of ROS. As an alternative, the toxic oligomers hypothesis seems strong, showing that soluble oligomers interact with cell membranes, inducing in them the formation of calcium channels. Collectively, it seems that a disruption of copper control mechanisms occurs in AD, affecting the compartmentalization of the metal in different tissues and organs. Some authors have observed that the increased levels of the labile pool of copper in the brain and in the periphery bring about a deficiency of copper in the brain and an increase in the labile pool of copper in the brain and in “free copper” in the blood. This is the picture implied by the conditions of Wilson’s disease. A possible interpretation is that a copper deficiency in brain cells is harmful because of amyloid production, and then copper deficiency leads to a rise in copper levels on lipid rafts. In conditions of copper deficiency, the amyloid-copper complex increases in lipid rafts because of higher levels of both copper and amyloid as well as the high affinity of amyloid peptides for copper. Copper ion binding and proximity to the cell membrane induce the refolding of amyloid peptides, and finally their oligomerization and interactions with cell membranes. The root of copper deficiency in the brain cells seems to be an important factor in AD. Since it is accompanied by copper enrichment in lipid rafts, one can argue that an elevation in lipid raft domains could lead to copper deficiency in the brain, thus targeting lipid rafts could be an effective therapeutic approach. Indeed, some data show that disrupting lipid rafts (by omega-3 fatty acids) delays the incidence of the disease.

30The review was written by SB with assistance and feedback from AS, RS, TH, and MS. All authors approved the final version of the manuscript.

31The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.