BACKGROUND: From investigations of a child with hyperprolinaemia type II, we demonstrated in vitro that pyridoxal phosphate forms a novel adduct with a proline metabolite, pyrroline-5-carboxylic acid, through Claisen condensation. Studies indicated that this was a previously unsuspected generic reaction of aldehydes and some ketones. We have subsequently found the acetoacetic acid adduct in both plasma and urine from the affected child. METHODS: Mixtures of acetoacetic acid and pyrroline-5-carboxylic acid were co-incubated at pH 7.4 and 37 degrees C, dried, or extracted and dried, derivatised and analysed by gas chromatography/mass spectrometry (GC/MS). Urine and plasma from the child were analysed. RESULTS: Fourteen new peaks were found in derivatised pyrroline-5-carboxylic acid/acetoacetic acid co-incubates. From accurate molecular mass data, the four largest peaks were probably diastereoisomers of tri-trimethylsilyl (tri-TMS) derivatives of alcohol adducts formed by Claisen condensation. Eight other peaks were mono- and di-trimethylsilyl derivatives of the adduct and a decarboxylated product. The adduct was demonstrated unequivocally in the child's acute urine and traces in plasma. CONCLUSIONS: Pyrroline-5-carboxylic acid forms an adduct with acetoacetic acid, which was present in urine of a sick child with hyperprolinaemia type II. Evidence suggests it formed in vivo. The biological significance of this novel reaction of aldehydes and ketones merits investigation.
BACKGROUND: From investigations of a child with hyperprolinaemia type II, we demonstrated in vitro that pyridoxal phosphate forms a novel adduct with a proline metabolite, pyrroline-5-carboxylic acid, through Claisen condensation. Studies indicated that this was a previously unsuspected generic reaction of aldehydes and some ketones. We have subsequently found the acetoacetic acid adduct in both plasma and urine from the affected child. METHODS: Mixtures of acetoacetic acid and pyrroline-5-carboxylic acid were co-incubated at pH 7.4 and 37 degrees C, dried, or extracted and dried, derivatised and analysed by gas chromatography/mass spectrometry (GC/MS). Urine and plasma from the child were analysed. RESULTS: Fourteen new peaks were found in derivatised pyrroline-5-carboxylic acid/acetoacetic acid co-incubates. From accurate molecular mass data, the four largest peaks were probably diastereoisomers of tri-trimethylsilyl (tri-TMS) derivatives of alcohol adducts formed by Claisen condensation. Eight other peaks were mono- and di-trimethylsilyl derivatives of the adduct and a decarboxylated product. The adduct was demonstrated unequivocally in the child's acute urine and traces in plasma. CONCLUSIONS:Pyrroline-5-carboxylic acid forms an adduct with acetoacetic acid, which was present in urine of a sick child with hyperprolinaemia type II. Evidence suggests it formed in vivo. The biological significance of this novel reaction of aldehydes and ketones merits investigation.
Authors: Jona Merx; Rianne E van Outersterp; Udo F H Engelke; Veronique Hendriks; Ron A Wevers; Marleen C D G Huigen; Huub W A H Waterval; Irene M L W Körver-Keularts; Jasmin Mecinović; Floris P J T Rutjes; Jos Oomens; Karlien L M Coene; Jonathan Martens; Thomas J Boltje Journal: Commun Biol Date: 2022-09-21