Homocysteine Disulphides and Vascular Disease

55 Disease Markers 27 (2009) 55–61 DOI 10.3233/DMA-2009-0649 IOS Press Homocysteine disulphides and vascular disease Mauro Iulianoa , Gaetano De Tommaso a and Raffaele Ragone b,∗ a b Dipartimento di Chimica, Universit à Federico II, Naples, Italy Dipartimento delle Scienze Biologiche, Universit à Federico II, Naples, Italy The final draft of this manuscript was ready on occasion of the retirement of Professor Liberato Ciavatta, who has been an inspiration to a generation of students. M.I., G.D.T. and R.R. wish to dedicate this paper to his integrity and rigor in scientific research. Abstract. The total plasma concentration of homocysteine is a marker of this amino acid’s atherogenic potential. However, the homocysteine pool exists almost entirely as oxidized homocysteine equivalents (OHcyE), composed of homocystine and cysteinehomocysteine disulphides (20–30%), and protein-bound disulphide (70–80%). We have noticed that the total concentration of OHcyE in injured coronary artery tissue is higher than the aqueous solubility of homocystine (∼1.4–1.5 × 10−3 mol kg−1 versus ∼0.6 mol kg−1 ). Based on the measurement of the solubility of homocystine in a plasma-mimetic condition (0.17 mol kg−1 NaCl at 37◦ C), we have estimated that OHcyE may really reach their saturation limit in the vascular tissue (0.93–1.02 × 10−3 mol kg−1 ), above which their deposition as solid phase may occur. This means that significant leakage of intracellular fluid can promote OHcyE crystallization in tissue fluids, which may serve to initiate inflammation. We speculate that deposition of OHcyE crystals could damage blood vessels and act as a primer of homocysteine-triggered inflammation, thus being along the causal pathway that leads to vascular dysfunction. Keywords: Atherosclerosis, cardiovascular disease, homocysteine, homocystine solubility, risk factors, vascular dysfunction Abbreviations: OHcyE, oxidized homocysteine equivalents; DPP, differential pulse polarography 1. Introduction Demethylation of the essential amino acid methionine produces the thiolic non-proteinogenic amino acid homocysteine [1]. Mild to moderate elevations of blood homocysteine have been associated with high risk of coronary heart disease and other vascular alterations [2–4]. Even though a causal role of homocysteine in cardiovascular disease remains to be established [5–7], it is believed that homocysteine excess may damage vascular tissue, so that blood ho∗ Corresponding author: Raffaele Ragone, Dipartimento delle Scienze Biologiche, Università Federico II, via Mezzocannone 16, 80134 Naples, Italy. Tel.: +39 081 253 6682; Fax: +39 199 707 0716582; E-mail: . mocysteine is currently considered as an independent index of vascular risk [8–10]. Mechanisms underlying homocysteine-associated vascular injury are under investigation [11,12]. Till now, almost all homocysteine-concerned research has assumed that elevation of the total plasma concentration of this amino acid (free and protein-bound) is a marker of its atherogenic potential. However, free homocysteine comprises three distinct fractions, i.e., reduced homocysteine, homocysteine-homocysteine homo-disulphide (homocystine) and cysteine-homocysteine hetero-disulphide. Since the reduced form barely amounts to 1–2% of the body’s total homocysteine, the homocysteine pool exists almost entirely as homocystine and cysteinehomocysteine (20–30%), also referred to as ‘oxidized homocysteine equivalents’ (OHcyE), and protein- ISSN 0278-0240/09/$17.00  2009 – IOS Press and the authors. All rights reserved 56 M. Iuliano et al. / Homocysteine disulphides and vascular disease bound disulphide (70–80%) [13,14]. It is also worth considering that the total plasma concentration of cysteine, a proteinogenic amino acid that is the lower structural homologue of homocysteine because of one less –CH2 – group in the side-chain, is 20- to 30-fold higher than that of homocysteine, and the concentration of reduced cysteine is ∼70-fold higher than that of reduced homocysteine (5.0 ± 3.6 versus 0.07 ± 0.02 µmol/L, mean ± s.d.) [15]. Nevertheless, there is no evidence that cysteine causes vascular damage, but a toxic effect of homocysteine on endothelial cells has been widely claimed [12,16–18], possibly through thrombosispromoting inflammatory pathways [19]. Recently, it has been pointed out that homocystine is the upper structural homologue of cystine (the homodisulphide of cysteine) [20], which is known to be scarcely soluble [21] and therefore capable to form kidney stones in genetically-disposed patients [22]. By analogy, it has been hypothesized [20] that vascular injury could be mechanically primed by the formation of homocystine crystals in the bloodstream, which could transiently grow after methionine intake and then dissolve during the time lag needed to reach basal conditions. In spite of the fact that, likely for the above reason, homocystine precipitates have never been found in endothelial cells or other tissues, it is worth mentioning that, in a population of patients with heart disease, levels of OHcyE were close to 1.4–1.5 × 10 −3 mol kg−1 , which is ∼15 fold higher than in normal coronary artery [23] (see Discussion for more details). At a glance, this elevation corresponds to a concentration of OHcyE higher than the aqueous solubility of homocystine at room temperature (unpublished data from our lab), and is affected by a statistical variability significantly lower than that observed in plasma. Indeed, the saturation concentration of OHcyE in the occluded coronary artery tissue can be reasonably placed between ∼5 × 10−4 and ∼0.9–1.0 × 10 −3 mol kg−1 at the plasma ionic strength, depending on the relative abundance of cysteine-homocysteine and homocystine (see Discussion for more details). The equilibrium relationship involving cystine, homocystine and mixed disulphide under physiological concentrations of cysteine and homocysteine has been previously investigated [14,24,25]. Although intracellular water also contains proteins, lipids, polysaccharides, and other species that may influence phase transitions, prevention and treatment of solid deposition in the human body, such as crystals of xanthine, uric acid and urates, cystine, oxalates, etc, are based on the understanding of the physicochemi- cal properties underlying the precipitation of the substances involved [26]. Pursuing this idea, we have measured the solubility of homocystine in aqueous sodium chloride solutions at physiological temperature. 2. Materials and methods 2.1. Chemicals Purissimum grade (
99.0%) DL-homocystine [meso -4,4’-dithio-bis(2-aminobutanoic acid)] was purchased from Fluka. Ultra-pure (
99.99%) sodium chloride and sodium azide from Baker and Aldrich, respectively, were dried at 120 ◦C and stored in a dryer before use. Surfactant and perchloric acid solutions were prepared by diluting Triton X-100 as obtained from LKB Bromma and purum p.a. HClO 4 from Merck, respectively. 2.2. Solubi (...truncated)