Some minerals and vitamins have been reported as micronutrients whose deficiencies might cause DNA damage and accelerate mitochondrial decay associated to degenerative diseases of aging  and cancer . In humans, the mineral absorption could be improved by complexation with organic compounds such as gluconate, as reported for Zinc . However, controversial results accounted for the bioavailability of these salts, indicating a different behavior, which depends on target cells or tissue and on pathological conditions. In anemic cancer patients the intravenous supplementation with Fe-gluconate increased the levels of blood hemoglobin more than oral Iron therapy . However, in healthy female volunteers, orally administrated Zn-bis-glycinate exhibited an higher Zinc bioavailability than Zn-gluconate . Moreover, in the rat liver Zn-enriched and Cu-enriched yeasts were more bioavailable (i.e. absorbed and found detected in a greater concentration) than Zn-gluconate and Cu-gluconate . Therefore, among ten different organic and inorganic Magnesium salts orally administered in Mg-depleted rats, Mg-gluconate exhibited the highest Magnesium bioavailability at intestinal level , slightly superior to Mg-pidolate and Mg-citrate.
In the present work we study Mn, another essential mineral that is a required component of a number of mitochondrial enzymes including MnSOD, and whose inadequate levels increase mitochondrial oxidants and subsequent mitochondrial decay .
The following points resume the results obtained by treating polarized Caco-2 cells with two different organic Mn salts. 1) MnGluc and MnOxP does not affect cell viability, as demonstrated by unaltered morphology and by the transient raise of pERK expression. 2) In brief experiments, in the presence of MnOxP and MnGluc, the mitochondrial activity is significantly lower with respect to untreated cells at 5 min only. In long experiments (24 h) both metal supplements moderately improve the mitochondrial oxidative activity with respect to untreated Caco-2 cells. 3) In comparison to Caco-2 cells treated with MnOxP and untreated cells, MnGluc-treated cells apically release a higher amount of ATP at 5 min, and display a lower ATP intracellular content at 24 h. 4) In treated cells MnSOD expression is variable but sustained at all time points. 5) Both βNADPH and NADH diaphorase activities are maintained by Caco-2 cells upon metal supplementation and seem more sustained than in untreated cells at few time points.
Taken together, in this in vitro model of intestinal epithelium, our findings demonstrate that MnGluc and MnOxP at the concentration of 12.5 μM are able to elicit a rapid metabolic response, which relates to mitochondrial activation. In metal supplemented respect to untreated Caco-2 cells, the mitochondrial oxidative activity is not significantly impaired, but slightly increases after 24 h of treatment, as indicated by MT fluorescence intensity. The limited changes in mitochondrial oxidation, i.e. tendency of MT fluorescence to decrease during brief treatment, appear counter-balanced by MnSOD upregulation in both MnOxP- and MnGluc-treated cells. MnSOD, an antioxidant defense mitochondrial metalloenzyme, is considered a good parameter to monitorate Mn exposure and protection in humans . The increase in MnSOD could scavenge superoxide anion, protecting the cells from oxidative damage and apoptosis, and ultimately self-regulating the oxidative stress . Of note, the mitochondrial activity appears higher in the presence of both MnOxP and MnGluc than in untreated cells, as shown by MT and by MnSOD.
In so far the MnSOD localization has been associated to NADH-diaphorase reactivity on gut tissue . The untreated polarized Caco-2 confluent monolayers do not display elevate histochemical signal for NADH- nor βNADPH-diaphorase, as previously reported for DT-diaphorase . Indeed, in MnOxP- and MnGluc-treated cells NADH-diaphorase is highest and increases with time, whereas βNADPH-diaphorase weakly and transiently increases, suggesting a modest activation of nitric oxide synthase . Although we cannot exclude a contribute of other enzymes of the diaphorase family to enzymatic activity observed in Caco-2 cells, the enhancement following Mn supplementation supports the activation of a reductase-based metabolism protective against oxygen radical production.
The adverse effects of Mn supplementation depend on the cell type (picomoles are toxic for astrocytes , whereas micromoles are safe for enterocytes ) and on the administration way . In our model, a sustained pERK activation, which is associated to abnormal mitochondria  and to superoxide anion production , is absent. This is demonstrated by the few more pERK granules transiently appearing inside Caco-2 cytoplasm during the first 15 min of incubation either with MnOxP or MnGluc, which revert within 60 min to the level of untreated monolayers. A transient polarization of mitochondria in MnOxP-treated monolayers accompanied the increase in pERK supportive of a mitochondria response. Indeed, long experiments demonstrated a general preservation of mitochondrial distribution in Mn-supplemented Caco-2 cells with respect to untreated cells. This observation excludes a commitment to cell death via mitochondrial dysfunction, is in agreement with morphological data, and supports the hypothesis that metal supplements may not affect Caco-2 cells viability, i.e. in these conditions MnGluc and MnOxP are not toxic for enterocytes. However, our findings are to be considered preliminary respect to the effects of MnOxP and MnGluc on gut cells in vivo. Although the general principles of nutrient risk assessment have been established, the value of controlled human studies on nutrient metabolism needs to be improved . In the nutritional field, despite the common use of supplements, the dietary quantity is crucial; a safe and adequate daily dietary intake estimated 1.8-2.3 mg Mn/day for adults . High levels of Mn are associated with neurodegenerative phenomena , whereas low doses were claimed as beneficial in prevention and treatment of osteoporosis in women .
Interestingly MnOxP does not deplete ATP content in Caco-2 cell, whereas MnGluc decreases moderately ATP levels in all the considered compartments. Together with the moderate reduction of mitochondria oxidative activity detected by MT, these findings suggest a decreased production of ATP by still viable Caco-2 in the presence of MnGluc, but an increased production in the presence of MnOxP, less affecting cell healthiness. These results support the hypothesis of a possible better bioavailability in vivo for MnOxP than for MnGluc.