Microhydrin Increases Mitochondrial NADH Production and Enhances Mitochondrial Membrane Potential in Intact Liver Cells

Microhydrin (200 ug/ml) was introduced to cultured 90% viable rat hepatocytes (500,000cells/ 4ml medium). Blue autofluorescence of mitochondrial NADH was visualized by a Zeiss LSM 410 inverted laser scanning confocal microscope using a 40X water immersion lens and 356/365 nm excitation light from a UV argon laser. Under the conditions used, autofluorescence arises primarily from mitochondrial NADH. Oxidation of NADH to NAD causes loss of fluorescence since only NADH is fluorescent.

The line graph summarizes data from 3 Microhydrin and 3 vehicle (control) experiments. In the Microhydrin group NADH increased 20% over 20 minutes while the vehicle group showed a decrease in NADH fluorescence by about 30%. These preliminary experiments suggest that Microhydrin promotes electron transfer to NAD in intact living hepatocytes. Moreover, Microhydrin prevented the spontaneous oxidation (or bleaching) of NADH that generally occurs during an incubation of this type (see vehicle plot) thereby indicating a continuous recharging of the pyridine nucleotide (NADH).

Mitochondrial membrane potential was monitored using overnight cultured hepatocytes similar to the NADH experiment and which were loaded for 20 min. with the fluorescent probe tetramethylrhodamine methylester (TMRM). The medium was adjusted to pH 7.4 to assure that the previously noticed increase in membrane potential was not due to a pH effect. The TMRM-loaded cells were imaged with a Zeiss 410 inverted laser scanning confocal microscope through a 63X objective lens. In these experiments, an increase of the mitochondrial fluorescence of TMRM represents an increase of mitochondrial depolarization (more negative membrane potential). The line graph summarizes data from 3 Microhydrin and 4 vehicle experiments. In the vehicle group, TMRM fluorescence decreased by about 6% over 20 minutes. In the Microhydrin group, TMRM increased about 25%. These preliminary experiments suggest that Microhydrin enhances mitochondrial membrane potential in intact living hepatocytes. The combination of increased mitochondrial membrane potential and increased NADH suggests an enhancement of bioenergetic capacity of the mitochondria when Microhydrin is present in the cell suspension (Unpublished data 1999). Microhydrin appears to be providing electrons or H- available to the cofactors that are able to utilize these for cellular energy production. NADH provides electrons to the mitochondria electron transport chain directly producing H₂0 and ATP, the primary cellular energy source for numerous biochemical reactions throughout the cell.

Evaluation of Microhydrin as a Scavenger of Free Radicals, Report I

Tests on Microhydrin were conducted using electron spin resonance techniques (ESR) by a scientist who specializes in the evaluation of antioxidants at a major university. The following is quoted from the test report:

"We have made a thorough investigation of the antioxidant activity of Microhydrin by several experimental methods. Hydroxyl radical scavenging activity was found in two different Microhydrin preparations provided."

"Our conclusions are that Microhydrin has antioxidant activity towards hydroxyl radicals. Hydroxyl radicals are among the most dangerous of oxygen free radicals that occur in biological systems. They are the same types of radicals that can be produced by exposure to ionizing radiation. Therefore, it can be stated that Microhydrin has antioxidant activity in this regard." (Personal Communication: Lester Packer, Ph.D., University of California at Berkley, 1999)

Evaluation of Microhydrin as a Scavenger of Free Radicals, Report II

Tests on Microhydrin were conducted by a scientist who specializes in the evaluation of antioxidants at a separate major university. The following is quoted from the test report: "When assayed in the ‘standard’ assay for super oxide dismutase activity based upon the reduction of cytochrome c by xanthine (see J. Biol. Chem. 244: 6049-6055, 1969), Microhydrin showed two characteristics:

1. Microhydrin can directly reduce cytochrome c, showing that it is a reducing agent (or an antioxidant).
2. Microhydrin can inhibit the superoxide-mediated reduction of cytochrome c, indicating that it can scavenge the superoxide free radical.”

"When assayed with an alternative method based on the ability of superoxide to oxidize epinephrine to adrenochrome, Microhydrin also showed its ability to scavenge the superoxide radical and inhibit the process." (Personal Communication: Joe McCord, Ph.D., University of Colorado Health Sciences Center, 1998). An abstract of this in vitro antioxidant data was presented and published in the Proceedings of the National Hydrogen Association 10th Annual Meeting. pg. 595-610; 1999)

Physician Using Microhydrin Climbs Mount Everest Without Oxygen

In 1991, at age 39, Denis Brown M.D. of British Columbia attempted to climb Mt. Everest without supplemental oxygen. He reached 26,000 feet. Again in 1994 he made the climb and achieved 28,000 feet before he surrendered to the extreme conditions and turned back. In 1999, at age 47, he added Microhydrin to his supplement regime. Microhydrin helps to reduce the build-up of free radicals and lactic acid, and supports production of ATP. He succeeded in reaching the peak of the South Summit at 28,750 ft. without supplemental oxygen and reported that this time he felt stronger and had more energy than on previous climbs.

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