June Ayling, Ph.D.

June Ayling, Ph.D.Professor Emerita
USA Department of Pharmacology


Research Interests

My laboratory explores the chemical, biochemical, and clinical aspects of two critical cofactors: tetrahydrofolate plus its one-carbon derivatives and tetrahydrobiopterin. The latter can be biosynthesized by animals, whereas the former cannot and is, therefore, a vitamin. Both are based on the pteridine nitrogen heterocycle and share some common attributes. Tetrahydrobipoterin (BH4) is the cofactor for the three aromatic amino acid (phenylalanine, tyrosine and tryptophan) hydroxylases and nitric oxide synthase. We were able to determine that the natural configuration used by these enzymes is the 6S-diastereoisomer. Further work demonstrated the many ways in which the structure of the dihydroxypropyl side chain of BH4 imparts important regulatory mechanisms to the hydroxylases. Moreover, we found that the minimum structural requirement for cofactor activity surprisingly does not require a complete pteridine moiety; the simpler 2,4,5-triamino-pyrimidin-4-one allowed a slow single turnover of phenylalanine hydroxylase, but was destroyed while incorporating an atom from 18O2. Moreover, a pyrimidodiazepine analog also turned out be a cofactor but which was irreversibly cleaved through opening of its seven-member ring. These observation are yet to be rationalized by a comprehensive understanding of the underlying mechanism of hydroxylase activation of molecular oxygen, which is the rate limiting step in amine neurotransmitter (dopamine and serotonin) biosynthesis.

In the early 1930’s Lucy Wills, a British physician in India, ascertained that the anemia of her poor pregnant patients was due to a deficiency of some unknown nutrient that could be supplied by consuming liver or yeast. This factor was first isolated from four tons of spinach leaves and named folic acid in 1941 by Esmond Snell, my PhD advisor.

A compound thought to be folic acid was first synthesized by Lederle in 1945 as a substitute for the liver extracts used for treating anemia. However, it was not for several years that it was discovered that the naturally occurring folates were almost entirely more reduced forms, predominately tetrahydrofolates. None the less, since folic acid was effective against anemia, it was assumed to be equal to the natural folates and has been used in supplements for decades. The work of my laboratory on the reduced pteridines led us to investigate possible differences. To obtain meaningful quantities of the natural (or unnatural) 6-position diastereoisomers of the tetrahydrofolates and other tetrahyropterins, we developed the first and still only stereospecific total synthesis. Certain chirally pure tetrahydrofolates can also be produced by repeated fractional crystallization, but this approach is limited to a few species

We compared the clinical properties of folic acid with 5-methyl-6(S)-tetrahydrofolate (5‑MTHF), the most abundant folate in the blood and many animal tissues. Not only is oral 5‑MTHF more rapidly absorbed from the GI tract of human subjects, much less of this is excreted into the urine than folic acid especially at doses above 0.91 micromoles (=0.4 mg of folic acid). This is due in large part to the extremely slow conversion of folic acid into reduced folates in the liver. In humans the rate of conversion of folic acid per gram of tissue by dihydrofolate reductase is 50 times slower and more variable in human than rat liver. The kidneys preferentially excrete the still unmetabolized folic acid into the urine. For many reasons our laboratory proposed that the natural folates, 5‑MTHF in particular, would be advantageous when incorporated in multivitamin supplements. This led to the development of the most advanced prenatal vitamins and also treatments for amelioration of hyper-homocysteinemia associated with cardiovascular disease and stroke. Pharmacokinetic advantages of 5‑MTHF over folic acid, especially at high dose, were discovered that can help to decrease the risk of birth defects such as of the heart and spina bifida in women who learn of their pregnancy soon after conception. A simple oral regimen of 5‑MTHF can make a deficient women folate replete in just two days. The same regimen using folic acid does not uniformly yield the same result. Thus, although low dose folic acid is adequate for slowly boosting folate levels in the months prior to conception, 5-MTHF is better for rapid repletion during early pregnancy for the many women who fail to follow the recommended prenatal nutritional advice.

Folic acid has been reported to cause lesions in DNA when irradiated with ultraviolet light. This damage, which includes double strand breaks, is particularly troublesome since they are difficult to repair with high fidelity. Double strand breaks are a major cause of skin cancer in those exposed to too much sun or tanning beds. We investigated whether 5‑MTHF would induce similar damage. Using supercoiled plasmid DNA we observed that 5-MTHF, unlike folic acid, did not produce nicks. Serendipitously, we also found that including 5-MTHF along with folic acid defeated the damage done by the latter! This was seen with photosensitizers other than folic acid, even those with a visible action spectrum such as rose bengal. Studying the effects of O2 concentration and azide showed that 5-MTHF acts as a diffusion limited quencher of excited photosensitizers and scavenger of singlet molecular oxygen. We then measured the levels of 5‑MTHF in human skin and found that not only is this in an especially high percentage abundance in the epidermis, but that folate replete individuals have levels likely capable of protection against solar radiation. Thus, if light skin pigmentation (or insufficiently applied sunscreen) allows penetration of some UV, 5‑MTHF can act as a second level of defense against DNA damage by endogenous photosensitizers. Those with low skin folate levels (which we have shown is linked to the folate concentration in plasma) can be at elevated risk. Our laboratory has developed a technology to quickly restore epidermal folate levels that can be used soon before expected sun or other UV exposure and be synergistic with sunscreens.