ORIGINAL ARTICLE Homocysteine metabolism in peripheral blood mononuclear cells: evidence for cystathionine beta-synthase activity in resting state Monika Katko • Erzsebet Zavaczki • Viktoria Jeney • Gyorgy Paragh • Jozsef Balla • Zsuzsa Varga Received: 13 April 2011 / Accepted: 8 September 2011 / Published online: 22 September 2011 Ó Springer-Verlag 2011 Abstract Activated peripheral blood mononuclear cells (PBMC) release homocysteine and possess cystathionine b-synthase (CBS) activity; however, it was thought that there is no CBS in resting state. Previously, we found that nickel decreased intracellular homocysteine concentration in un-stimulated (e.g. resting) PBMC, suggesting that resting PBMC might also have active homocysteine metabolism. Here, we demonstrated that un-stimulated PBMC synthesize (incorporate L-[methyl- 14 C]methionine to DNA, lipids and proteins), release (increase extracellular homocysteine), and metabolize homocysteine. Intracellular homocysteine concentration varied with incubation time, depending on extracellular concentrations of methionine, homocysteine, and glutathione. Methionine synthase activity was constant and independent of thiol concentra- tions. In Western blot, CBS protein was clearly identified in freshly isolated PBMC. CBS protein level and activity increased with incubation time, upon stimulation, and similar to intracellular homocysteine, depending on intra- and extracellular homocysteine and glutathione concen- trations. According to our knowledge, this is the first evidence that certifies homocysteine metabolism and regulatory role of CBS activity to keep balanced intracel- lular homocysteine level in resting PBMC. Homocysteine, released by PBMC, in turn can modulate its functions contributing to the development of hyperhomocysteinemia- induced diseases. Keywords PBMC Á Cystathionine b-synthase Á Intracellular homocysteine Á Extracellular homocysteine Á Methionine Á Glutathione Introduction Homocysteine is a non-essential, sulfur-containing amino acid. It is synthesized from methionine via multi step process (Fig. 1). First, methionine receives an adenosine group from ATP, a reaction catalyzed by S-adenosylme- thionine synthase (MAT), to give S-adenosylmethionine (SAM). SAM then transfers the methyl group to acceptor molecules by methyltransferases (MT)—involving DNA methyltransferase—and forms S-adenosylhomocysteine (SAH). SAH is then hydrolyzed by S-adenosylhomocys- teine hydrolase (SAHH) to yield homocysteine. Homo- cysteine is metabolized by two primary fates; it can convert via Vitamin B 12 dependent methionine synthase (MS) in folate cycle to methionine, or it can be irreversibly removed in transsulfuration pathway. One key enzyme in the transsulfuration pathway is the cystathionine b-syn- thase (CBS), which catalyses condensation of homocyste- ine and serine to cystathionine. This reaction uses Vitamin B 6 as cofactor. Cystathionine c-lyase then converts this amino acid to cysteine, ammonia, and a-ketobutyrate. Cysteine is a substrate of glutathione (GSH) synthesis whose availability is the main determinant of cellular GSH synthesis. The third remethylation pathway using betaine as methylation agent for homocysteine is restricted to liver and kidney (Delgado-Reyes et al. 2001) (Fig. 1). The main organ of homocysteine formation is the liver. Deficiencies of vitamins, e.g., folate, Vitamin B 6 , or B 12 can lead to elevated homocysteine levels (Chanarin et al. 1985; Morrow and Barness 1972). Hyperhomocysteinemia M. Katko Á E. Zavaczki Á V. Jeney Á G. Paragh Á J. Balla Á Z. Varga (&) First Department of Medicine, Medical and Health Science Center, University of Debrecen, Nagyerdei krt. 98, H-4012 Debrecen, P.O. Box 19, Hungary e-mail: vargazs@internal.med.unideb.hu; zsuzsa.vargadr@gmail.com 123 Amino Acids (2012) 43:317–326 DOI 10.1007/s00726-011-1080-2