Quality and stability of extemporaneous pyridoxal phosphate preparations used in the treatment of paediatric epilepsy.

OBJECTIVES: To assess the pyridoxal 5'-phosphate (PLP) content and stability of extemporaneous PLP liquids prepared from dietary supplements used for the treatment of vitamin B6 -dependent epilepsy. METHODS: Pyridoxal 5'-phosphate liquids were prepared in accordance with the guidelines given to patients from marketed 50 mg PLP dietary capsules and tablets. The PLP content and its stability were evaluated under conditions resembling the clinical setting using reverse phase HPLC and mass spectrometry. KEY FINDINGS: Pyridoxal 5'-phosphate content in most of the extemporaneously prepared liquids from dietary supplements was found to be different from the expected amount (~16-60 mg). Most of these PLP extemporaneous liquids were stable at room temperature (protected from light) after 24 h but unstable after 4 h when exposed to light. A key photodegradation product of PLP in water was confirmed as 4-pyridoxic acid 5'-phosphate (PAP). CONCLUSION: Pyridoxal 5'-phosphate tablets from Solgar® were found to be the most reliable product for the preparation of extemporaneous PLP liquids. This work highlighted the difference between the marketed PLP dietary supplements quality and the importance of proper storage of aqueous PLP. There is a need to develop pharmaceutical forms of PLP that ensure dose accuracy and avoid potentially unsafe impurities with the aim of enhancing safety and compliance.

Determination of quality of pyridoxal-5'-phosphate enzyme preparations by spectroscopic methods.

The present study evaluates purified aspartate transaminase (AST, EC 2.6.1.1) preparations from three commercial sources. The enzyme molecule contains pyridoxal-5'-phosphate coenzyme (PLP), which provides AST characteristic absorption spectra in the wavelength range of 300-500 nm. The coenzyme bound in the active site also shows circular dichroism (CD) spectra in the same range. Besides, AST like other proteins may be modified in vitro or in vivo by reactions with other molecules, e.g. reactive sugars, and may form fluorescent products (advanced glycation end products, AGE). Spectroscopic methods were used to assess the quality of AST preparations from three different sources, Serva, Roche, and Sigma. Absorption spectra showed that the peak 360 nm characteristic of the active PLP form of AST prevailed in the Serva and Sigma preparations, while 330 nm was the major peak in the Roche preparation. CD spectra demonstrated the major maximum at 360 nm in the Serva and Roche samples, thus suggesting the predominance of the active PLP form in both preparations. The Sigma sample showed a CD profile less characteristic of AST. Fluorescence measurements revealed formation of AGE in the case of the Roche preparation, while fluorescence of the other two preparations was low. In general, the Serva sample presented the most convenient properties of purified AST among the preparations tested. The results will be used for the selection of a commercial enzyme preparation applicable in our future spectroscopic studies of glycation of AST as a model protein and in our research of the influence of antioxidants on this process.

Does Tris-HCl effectively participate in transamination during hemoglobin pyridoxylation?

To test whether Tris is required for covalent binding of pyridoxal phosphate (PLP) to hemoglobin, we carried out the reaction in solutions of Tris homologues, carrying a blocked amine function. With the exception of Mono-Tris, these compounds permitted the synthesis of modified hemoglobins with acceptable spectral properties, P50 values, cooperativity and methemoglobin content, refuting Tris HCI participation during hemoglobin pyridoxylation.