Super Oxide Scavenging Assays With Microhydrin

The superoxide mediated reduction of cytochrome c showed that the reduced silicate mineral inhibited the superoxide-mediated reduction of cytochrome c, indicating that the reduced silicate is reducing, or scavenging, the superoxide radical also:

Mic (H:-) + O.2- + H+ ½ Mic + H2O2

In this assay Microhydrin was found to inhibit and prevent the reduction of cytochrome c by superoxide:

O.2- + Cyt c(Fe+3) ½ O2 + Cyt c(Fe+2)

An alternative assay to further elucidate the activity of the reduced silicate mineral, observed the oxidation of epinephrine to adrenochrome by superoxide (unpublished data).

Microhydrin
O.2- + epinephrine ½ H2O2 + adrenochrome

Data is shown in Table 1 for superoxide dismutase-like (SOD) activity based on the oxidation of epinephrine by superoxide.

In this assay the reduced hydrogen mineral silicate (Microhydrin) scavenged the superoxide ion again. Quantitatively, half-maximal inhibition was achieved at a Microhydrin concentration of 90 µg/ml (Table 1). When concentrations of Microhydrin were at 60 µg/ml or When concentrations of Microhydrin were at 60 µg/ml or greater, epinephrine oxidation was inhibited showing that the silicate mineral reduced the superoxide free radical. The assay conditions utilized superoxide dismutase at a concentration of 50µg/ml with a conversion rate of 0.15 µg/ml (unpublished data) (Table 1, Figure 2).

NAD Reduction With Microhydrin

The ability of Microhydrin to reduce NAD⁺ to NADH was also examined. The redox potential for this reaction is –0.32 V (– 320 mV), requiring a fairly strong reducing agent. Microhydrin reduced NAD⁺ to NADH. The reduction of NAD⁺ was verified by scanning the absorption spectrum from 200 nm to 500 nm. In all subsequent studies NAD⁺ reduction was monitored as the difference between two wavelengths (340 nm to 500 nm).

The 500 nm wavelength served as a reference (independent of NAD+ reduction, but controlling for turbidity changes due to the light scattering effect of the silicate) (Figure 3). NAD⁺ could be titrated by sequential addition of aliquots of Microhydrin in a linear fashion.

Microhydrin was suspended for 30 min. at 10mg/ml. Microhydrin was added incrementally to 60µM NAD⁺(Figure 3).