Isolation, structural determination, and biological activity of 6 alpha-hydroxytaxol, the principal human metabolite of taxol.

The principal biotransformation product of taxol was found to be identical for human hepatic microsomes, human liver slices, and patient bile samples. We have isolated this metabolite from the bile of a patient given taxol, and we report its structure and its cytotoxicity relative to taxol. The NMR and SIMS data presented here indicate that, in humans, taxol is regiospecifically hydroxylated at the 6-position on the taxane ring and that this hydroxyl is stereospecifically placed trans to the hydroxyl at position 7, yielding 6 alpha-hydroxytaxol. This metabolite is apparently not formed in rats. Tests of the growth inhibition potential of 6 alpha-hydroxytaxol versus taxol in two human tumor cell lines showed that the metabolite was approximately 30-fold less cytotoxic than taxol. Thus the cytochrome P-450-mediated biotransformation of taxol to 6 alpha-hydroxytaxol can be classified as a detoxification reaction.

The protonated 4,4'-diaminotriphenylmethyl cation as a reagent and cyclohexanone as an absorbant for sulphur dioxide determination.

Protonated 4,4'-diaminotriphenylmethyl cation, DATM, has been investigated as a colorimetric reagent for the determination of sulphur dioxide. The bisulphite addition compound with formaldehyde alkylates a primary arylamine group in the protonated reagent to re-establish resonance, and hence produces a colour that is proportional in intensity to the concentration of either bisulphite or formaldehyde (the other being in excess). The large bathochromic spectral shift results in a negligible blank correction. Cyclohexanone in aqueous solution has been found to stabilize bisulphite solutions for up to 6 hr. The stabilization by cyclohexanone has been compared with stabilization by tetrachloromercurate(II). The Beer-Lambert law is obeyed with good precision for both sulphite and formaldehyde determination.

13C nuclear magnetic resonance study of the pyruvate dehydrogenase-catalyzed acetylation of dihydrolipoamide.

The dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex catalyzes a reversible reaction between acetyl-CoA and dihydrolipoamide that results in the formation of S-acetyldihydrolipoamide. We have used 13C nuclear magnetic resonance to investigate this reaction using exogenous forms of dihydrolipoamide in place of the protein-bound substrate. With substrate levels of dihydrolipoamide and enzymatically generated [1-13C]acetyl-CoA, both 6-S-[1-13C]acetyl- and 8-S-[1-13C]acetyldihydrolipoamide were formed in the transacetylation reaction and both species participated in the reverse reaction to yield [1-13C]acetyl-CoA and free dihydrolipoamide. The 8-S-acetyl derivative was the principal product. It is suggested that acetylation of both the 6- and 8-thiols of dihydrolipoamide results as a consequence of intramolecular migration following acetylation at a single site. After longer periods of reaction, some 6,8-S,S-[1-13C]diacetyldihydrolipoamide also accumulated. We have also found that [1-13C]acetyl-CoA reacts slowly with dihydrolipoamide in a nonenzymatic reaction to yield the two monoacetylated and some diacetylated derivative. In the reverse reaction catalyzed by the dihydrolipoyl transacetylase, it was clear that monoacetyl derivatives were depleted much more rapidly than the diacetyl derivatives, although we could not quantitate the change in the low concentration of the diacetyl derivative.