N-Tert-Butyl Hydroxlamine (NtBHA)

A Mitochondrial-targeted Antioxidant

According to anti-aging specialist, Dr. Ward Dean, "The preliminary studies with NtBHA are intriguing. NtBHA may prove to be a potent anti-aging compound capable of reversing age-related changes in mitochondrial enzymes, proteins and DNA".

• NtBHA is an experimental chemical with great potential for maintaining mitochondrial health by reversing mitochondrial damage and controlling aging at the cellular level.
• NtBHA and related hydroxylamines are being extensively researched as anti-aging compounds.
• NtBHA is an antioxidant and possible metabolite of the nitrone spin-trap PBN.
• NtBHA is recycled by the mitochondrial electron transport chain and prevents free radical-induced toxicity to mitochondria.

Age-related changes in mitochondria

Aging is known to cause a decline in several basic functions of the mitochondria, including the Respiratory Control Ratio (RCR, a measure of how tightly coupled oxygen uptake is to the phosphorylation of ADP to ATP), and the activities of complexes I, III, and IV of the electron transport chain, as a result of a lifetime of oxidative damage to key enzymes.

Protects mitochondrial enzymes and proteozomes against oxidative damage

NtBHA prevented the loss of the mitochondrial enzyme glutamate dehydrogenase (GDH), decreased glutathione-mixed disulfides (markers for oxidative stress which increase with age), increased the free glutathione/ glutathione-mixed disulfide ratio in liver proteins, and improved the RCR. Aconitase, a key Kreb's cycle enzyme is known to decline in an age-dependent manner due to its susceptibility to attack by superoxide and peroxynitrite radicals. This decline is prevented by administering NtBHA.

These results indicate that NtBHA is a powerful antioxidant. It protects mitochondria and proteins from oxidative damage, while simultaneously reversing existing damage and returning enzyme markers to youthful levels.

Proteasomal (organelles within cells that degrade and process proteins through enzymatic reactions) activity was also higher in cells treated with NtBHA than in untreated cells. NtBHA accumulates within the mitochondria at 10 to 15 fold the extra-cellular concentration and is maintained in a reduced form by mitochondrial NADH (like R-Lipoic Acid).

Delays cellular senescence

IMR -90 cells (human lung diploid fibroblasts) serve as an in vitro model for studying aging at a cellular level. Normal cells irreversibly lose replicative capacity as a function of population doublings (PDs), or due to DNA damage, leading ultimately to senescence. NtBHA effectively delayed senescence at concentrations as low as 10 microM compared with 200 microM PBN to produce a similar effect.

NtBHA has delayed both natural senescence-associated changes in mitochondria and induced cellular senescence by maintaining cells that were provided suboptimal levels of growth factors such as insulin, thyroid hormones and steroids.

Proven benefits in vivo

Old rats have reduced ambulatory abilities and lower food consumption as compared to young rats. It was found that feeding NtBHA to old rats improved ambulatory activity and reversed age-related decreases in food consumption, without affecting body weight. The respiratory control ratio of mitochondria from liver of old rats improved after feeding them NtBHA. These findings suggest that NtBHA improved mitochondrial function and efficiency in vivo.

The age-dependent increase in proteins with thiol-mixed disulfides was significantly lower in old rats treated with NtBHA. NtBHA was effective only in old rats; no significant effect was observed in young rats and produced no observable side effects.

Anti-Glycation effects

Dr Alan Hipkiss discovered that NtBHA, along with amino guanidine and kinetin delayed senescence in cultured cells. Interestingly, PBN shares this ability, but not the classical antioxidants such as BHT, Vitamin C, Vitamin E or N-acetyl cysteine. The effective agents resemble carnosine in that they seem to react with glycoxidised proteins, as well as functioning as antioxidants. These findings suggest that pluripotency (acting by several interrelated but distinct mechanisms) may be necessary for their effective "anti-aging activity."

Is NtBHA the active component of the anti-aging, spin trap PBN?

Dr Bruce Ames ' research group at UC Berkeley believes that the therapeutic benefits of PBN can be explained by its in vivo hydrolysis to NtBHA. The Ames team has experimented extensively with PBN, which led them to test NtBHA.

• PBN (alpha phenyl-tert-butylnitrone) is an experimental anti-aging compound that is being extensively researched. It has been proven to extend the mean life span of experimental animals, reduce or prevent brain damage from stroke, and lowers the susceptibility to further strokes.
• PBN also reversed oxidative damage within areas of the brain known to be important for cognitive function (see PBN Overview and Spin Traps for more details).
• PBN has also been shown to reduce systemic and neuro-inflammatory processes implicated in Alzheimer's disease and all the chronic degenerative diseases of aging.

When PBN is orally administered it is broken down in gastric acid to benzaldehyde and NtBHA. Oral doses of PBN are poorly absorbed (an estimated 20-30% was bio-available) due to the instability of the PBN molecule in the acid environment of the stomach. This led Dr. Ames and his colleagues to speculate that the benefits of PBN are due to its hydrolysis product, NtBHA. They have published research contending NtBHA is the biologically active portion of the PBN molecule.

An alternative explanation for the relationship between NtBHA and PBN

Dr John Carney and Dr Robert Floyd , two of the primary PBN researchers, have a different explanation for the efficacy of this compound. For years, PBN was thought to exert its effects by spin-trapping free radicals. In the last few years Floyd and Carney have shown that there are many antioxidants that are better at scavenging free radicals and at much lower doses than are used in PBN experiments. Yet PBN has numerous benefits unachievable with these antioxidants. Very sophisticated experiments have shown that PBN (like R-Lipoic Acid) works by altering signal transduction pathways that alter genetic expression of pro-inflammatory cytokines.

A major weakness in the Ames hypothesis comes from the drug metabolite studies, which show that injected PBN is primarily metabolized and excreted as 4-hydroxy-PBN. Only a trace of benzoic acid was detected (from hydrolysis and subsequent oxidation of benzaldehyde). This proves that the PBN molecule is intact in vivo and not hydrolyzed to NtBHA, suggesting two different mechanisms are at work, and are dependent on the route of administration.

Using PBN and TBHA may be synergistic

Since both PBN and NtBHA are known to penetrate the mitochondrial membrane and alter numerous enzyme systems, it is likely that the common feature of both is their ability to alter the redox properties of the cells, which in turn trigger or suppress gene transcription factors such as NF-kB. This also suggests that pluripotency and utilization of both PBN and NtBHA may lead to a synergistic effect, leading to a more effective means of controlling age-related changes.

Other N-Hydroxylamines

N-Benzyl hydroxylamine and N-methyl hydroxylamine are compounds unrelated to PBN, but were also effective in delaying cellular senescence. All the N-hydroxylamines tested significantly decreased the endogenous production of oxidants. The acceleration of senescence induced by hydrogen peroxide was reversed by the N-hydroxylamines. DNA damage was also decreased significantly following treatment with N-hydroxylamines. The N-hydroxylamines delay age-dependent changes in mitochondria and prevent reduction of cytochrome C (FeIII) by superoxide radical and reverse an age-dependent decay of mitochondrial aconitase by apparently reacting with the superoxide radical.

Note

N-tert-butyl hydroxylamine is sold as a research chemical only. It is not intended for human consumption.

References

The use of N-t-butyl hydroxylamine for radioprotection in cultured cells and mice. Lee JH, Kim IS, Park JW. Carcinogenesis. 2004 Aug;25(8):1435-42. Epub 2004 Mar 11.

Iron accumulation during cellular senescence. Killilea DW, Wong SL, Cahaya HS, Atamna H, Ames BN. Ann N Y Acad Sci. 2004 Jun;1019:365-7.

N-t-Butyl hydroxylamine is an antioxidant that reverses age-related changes in mitochondria in vivo and in vitro. Atamna, H., Robinson, C., Ingersoll, R., Elliott, H., Ames, B. N. FASEB J. 2001 Oct;15 (12):2196-204.

On the "struggle between chemistry and biology during aging"--implications for DNA repair, apoptosis and proteolysis, and a novel route of intervention. Hipkiss AR. Biogerontology 2001; 2(3):173-8

Delaying brain mitochondrial decay and aging with mitochondrial antioxidants and metabolites. Liu J, Atamna H, Kuratsune H, Ames BN. Ann N Y Acad Sci 2002; Apr;959:133-66

N-t-butyl hydroxylamine, a hydrolysis product of alpha-phenyl-N-t-butyl nitrone, is more potent in delaying senescence in human lung fibroblasts. Atamna H, Paler-Martinez A, Ames BN. J Biol Chem 2000; Mar 10;275(10):6741-8

Mitochondrial Decay in Aging: Reversal through Supplementation of Acetyl-l-Carnitine and PBN. Hagen, TM; Wehr, CM; Ames, B. Annals of the NY Academy of Science 1998; 854: 214-223

Delaying Aging With Mitochondrial Micronutrients and Antioxidants Ames, BN; Liu, J; Atamna H, The Scientific World Journal 2001; 1: 81-82SR

Oxidative DNA Damage and Senescence of Human Diploid Fibroblast Cells Chen, Q; Fischer, A; Reagan, J; Yan, LJ; Ames, BN,Proc Natl Acad Sci USA 1995; Vol 92, 4337-4341

Personal communication, Dean W, October 2002

Nitrone-based free radical traps as neuroprotective agents in cerebral ischaemia and other pathologies. Hensley, K; Carney, JM; Stewart, CA, Tabatabaie, T; Pye, Q, Floyd, RA Int Rev Neurobiol 1997; 40: 299-317