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Archived Comments for: Fluorescent nanodiamonds as a relevant tag for the assessment of alum adjuvant particle biodisposition

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  1. Lumogallion as the only known fluorescent probe for the identification of aluminium in living cells

    Matthew Mold, Keele University

    3 August 2015

    Research performed in our group has recently highlighted the use of lumogallion as an excellent fluorescent molecular probe for aluminium that has provided for the first time unequivocal evidence of particulate aluminium in any human cell line (Mold et al., 2014, Sci. Rep. 4, 6287). Further published studies have used lumogallion for the successful identification of intracellular aluminium in human spermatozoa (Klein et al., 2014, Reprod. Toxicol. 50, 43-48) and aluminium adjuvants at a muscular level, in vaccinated rat models (Kashiwagi et al., 2014, Vaccine, 32, 3393-3401). The published report by Eidi and co-workers has highlighted what they describe as “limitations” in the use of lumogallion for this purpose, of which inaccuracies raised in their recent publication must be addressed. The authors state that lumogallion may bind to the metal gallium to give fluorescence, similar to that of the Al-lumogallion complex. Gallium is a non-essential metal and is not considered to be essential to life nor is it known to have any natural metabolic role. Furthermore, the presence of gallium in human tissues has been reported to be present at nanomolar concentrations at levels lower than 10-4 to 10-3 ppm (André and Mäcke, 2003, J. Inorg. Biochem. 97, 315-323). It is further unlikely at physiological pH that trace if any amount of the metal ion would be available to bind to lumogallion as the necessary hydrated trivalent metal cation.


    Eidi et al., also draw the conclusion that lumogallion “stains” for iron thereby suggesting that iron can act as a false positive when using lumogallion to stain for aluminium in human tissues. Whilst it is known that iron binds to lumogallion, it is of critical importance that the resultant complex formed is non-fluorescent which therefore occludes iron from contributing to the fluorescence signal produced in the presence of aluminium (Hydes and Liss, 1976, Analyst. 101, 922-931). In solution, the binding of lumogallion to metal ions is mainly driven by kinetic processes of which the binding of iron to the fluorophore has been shown to interfere with the quantitative measurement of Al, when added in excess (Hydes and Liss, 1976). Importantly however, in the staining procedures utilised by our group and others, lumogallion is always present in excess and as such remains available to bind to aluminium.


    The published report by Eidi and co-workers highlights the use of modified fluorescently functionalised nanodiamonds (mfNDs) that alternatively are chemically attached to aluminium adjuvant preparations. As the fluorescence signal from the lumogallion-aluminium complex overlays the spectral region of mfNDs, this prevented the use of the only known fluorophore demonstrated to provide unequivocal evidence of intracellular aluminium. The only evidence provided of aluminium remaining associated or bound with mfNDs in vivo, in the current study is provided using the fluorescent stain, morin. As highlighted by the authors, morin is known to bind to calcium and magnesium that are commonly found in physiological media, to produce false positives (Browne, McColl and Driscoll, 1990, J. Environ. Qual. 19, 65-72). In order to overcome this a hydrochloric acid pre-rinse step is often used prior to staining. As this was not performed in this study the contributive effects of both divalent metal ions cannot be excluded from the tissue staining performed. Furthermore, the binding of morin to aluminium is relatively weak which is especially true at circum-neutral pH (Exley et al., 1994, J. Inorg. Biochem. 54, 297-304) further hindering its use in identifying aluminium deposited in human or animal tissues.


    Whilst it remains a fundamental necessity to establish the fluorescence signal from fluorophore-stained tissues prior to the administration of aluminium in either particulate or solubilised form, results for which are near never-provided in published studies. The mere presentation of autofluorescence (i.e. the fluorescence signal produced in the absence of any added fluorophore or stain) is not sufficient nor adequate to unequivocally demonstrate that the signal observed is solely due to the fluorophore binding to aluminium. Unless controls of this nature are provided, then the sole use of morin for the detection of aluminium adjuvants in either animal or human tissues remains equivocal and a regression in current methodological advances in this area.    

    Competing interests

    None declared