A novel pyrroline-5-carboxylic acid and acetoacetic acid adduct in hyperprolinaemia type II Valerie Walker a, * , Graham A. Mills b , John M. Mellor c , G. John Langley c , R. Duncan Farrant d a Department of Chemical Pathology, Southampton General Hospital, Level D, Mail Point 6, Tremona Road, Southampton, SO16 6YD, UK b School of Pharmacy and Biomedical Sciences, University of Portsmouth, White Swan Road, Portsmouth, PO1 2DT, UK c Department of Chemistry, University of Southampton, Highfield, Southampton, SO17 1BJ, UK d Physical Sciences, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK Received 4 September 2002; received in revised form 15 January 2003; accepted 17 January 2003 Abstract 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 jC, 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. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Pyrroline-5-carboxylic acid; Hyperprolinaemia type II; Acetoacetic acid; Claisen condensation; Proline metabolism 1. Introduction Hyperprolinaemia type II (OMIM 239510) is a rare inherited autosomal recessive disorder caused by a deficiency of D 1 -pyrroline-5-carboxylate (P5C) dehy- drogenase (EC 1.5.1.12) (Fig. 1). This leads to a 10- to 15-fold increase in plasma proline, accumulation of P5C and increased urinary proline excretion [1]. Clinical presentation is with convulsions in childhood, usually precipitated by infection. Between episodes, children are generally well. Most adults enjoy normal health [2]. 0009-8981/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0009-8981(03)00077-9 * Corresponding author. Tel.: +44-23-8079-6419; fax: +44-23- 8070-6339. E-mail address: Valerie.walker@suht.swest.nhs.uk (V. Walker). www.elsevier.com/locate/clinchim Clinica Chimica Acta 331 (2003) 7 – 17