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Factors affecting S-homocysteinylation of LDL apoprotein B.

Increased plasma homocysteine (Hcy) [3] is an independent risk factor for vascular disease in humans (1, 2). Although the exact mechanism of Hcy toxicity is unknown, several mechanisms have been investigated that may explain its role in atherosclerosis pathogenesis (3, 4), including endothelial injury, reduction of vascular nitric oxide (NO) production and bioavailability, mitotic effect on smooth muscle cells, influence on leukocyte behavior and hemostasis, and oxidative modification of LDL. Recent study results have suggested that Hcy-induced vascular damage could be a result of Hcy-thiolactone (HcyT), an Hcy-reactive product formed in several cell types as a result of editing reactions of some aminoacyl-tRNA synthetase (5, 6). The synthesis of HcyT is directly proportional to the plasma Hcy:methionine ratio (6). Because HcyT binds protein lysyl residues by amide linkage, individual proteins are homocysteinylated at rates proportional to their lysine contents. Jakubowski found that protein N-homocysteinylation occurs even at HcyT concentrations as low as 10 nmol/L, which is a physiologic concentration (7). N-homocysteinylation leads to protein damage consisting of multimerization and precipitation of extensively modified proteins. Model enzymes, such as methionyl-tRNA synthetase and trypsin, are inactivated by N-homocysteinylation (7). The interaction between HcyT and LDL causes LDL aggregation and higher uptake of N-homocysteinylated LDL (Hcy-LDL) by cultured macrophages (8). Ferretti et al. (9) theorized that N-homocysteinylated LDLs are internalized by membrane receptors with intracellular release of Hcy after hydrolytic degradation. Even at low concentrations (up to 10 [micro]mol/ L), Hcy is cytotoxic and induces cell injury and oxidative damage in cultured cells, with generation of reactive oxidative species, formation of superoxide and hydrogen peroxide, and increased lipid peroxidation product concentrations (10, 11). LDL incubation with 100 /[micro]mol/L HcyT for 2 h was reported to cause the N-homocysteinylation of ~10% of apoB100 lysyl residues (9). Assuming a relative molecular mass (Mr) of -500 000 for apoB and ~200 lysyl residues per apoB molecule (12), ~20 new -SH groups are introduced in the sequence of apoprotein, thus tripling the number of "free sulfydryl" groups of native protein, reported to be 9 (13). The term free sulfydryl, however, used to define sulfydryl groups of proteins that are not involved in intramolecular bridges, may be improper in this context. We demonstrated (14,15) that in vivo low-Mr thiols such as Hcy, cysteine (Cys), cysteinylglycine (CysGly), glutamylcysteine, and glutathione link these "free" -SH groups by disulfide bonds. Therefore lipoproteins may be N-homocysteinylated by HcyT but also S-homocysteinylated by Hcy. Increased total plasma Hcy concentrations lead to increased concentrations of HcyT that reacts with lysine proteins, leading to increased lipoprotein N-homocysteinylation, which increases the number of -SH sites for S-homocysteinylation. Other plasma LMW thiols compete for the same sites, however, depending on their concentration. Therefore, the interaction between physiologic plasma thiols and apoprotein may be related to the balance among the different thiols. No data have been reported on variables affecting the concentrations of Hcy bound to LDL by disulfide linkage. To understand the mechanisms involved in the S-homocysteinylation of LDL, considered a risk factor for vascular disease (16), we measured total plasma thiol and LDL-bound thiol concentrations in a healthy population and investigated the relationships between total and apoprotein-bound thiols.

Materials and Methods


We purchased Hcy, Cys, oxidized CysGly, reduced Cys-Gly, glutathione, glutamylcysteine, [Na.sub.3]P[O.sub.4] x 12 [H.sub.2]O, [H.sub.3]B[O.sub.3], KBr, NaCl, [Na.sub.2]EDTA, N-methyl-D-glucamine, dimethyl sulfoxide, NN-dymethilphormamide, tri-n-butylphosphine, NaOH, 5-iodoacetamidofluorescein, and 5-sulfosalicylic acid (SSA) from Sigma; and chloroform, methanol, and acetonitrile from Carlo Erba. We used membrane filters (0.45 [micro]m) obtained from Millipore to filter all buffer solutions before capillary electrophoresis analysis. We used a Sephadex PD-10 column purchased from Amersham-Pharmacia.


We selected a total of 104 healthy persons of both sexes (44 men and 60 women, ages 13-79 years) who were recruited consecutively from the Laboratorio Generale di Medicina di Base, ASL 1 Sassari. All participants gave informed consent for participation in the study and for personal data management according to Italian laws. As confirmed by medical interviews, participants were not receiving dietary supplements of vitamin [B.sub.6], B12, folate, or drug therapy. Hypercholesterolemia and hyperhomocysteinemia were not exclusion criteria, because the aim of this study was to investigate the possible relationships between lipoprotein metabolism and thiols for both within reference interval and increased cholesterol and/or Hcy concentrations.


We collected blood into tubes containing EDTA. Plasma was prepared by centrifugation at 2000g for 10 min at 4[degrees]C. LDLs were isolated by ultracentrifugation according to methods described by Himber et al. (17) and McDowell et al. (18). Briefly, we added 0.9 mL of plasma to a centrifugation tube containing KBr (0.4451 g), adjusting the density of plasma to 1.300 g/L. This mixture was then overlaid with 2.1 mL of 150 mmol/L NaCl and centrifuged at 541 000g for 2 h at 4[degrees]C. LDL, VLDL, and HDL orange bands were recovered, and LDL was mixed (ratio, 1/1) with a solution containing KBr and EDTA 1% (density, 1.063 g/L) and centrifuged at 541000g for 2 h at 4[degrees]C.


We precipitated 200 [micro]g of LDL apoprotein with SSA (final concentration, 7.5%) and then centrifuged it at 2000g for 5 min. The protein pellet was washed twice with 500 [micro]L SSA 5% and once with 1 mL of acetonitrile/water (70/30) to remove SSA residue that could interfere with subsequent steps. After centrifugation, we discarded 950 [micro]L of supernatant and dried the apoprotein under decreased pressure in a Concentrator 5301 for 30 min at 60[degrees]C. After dissolving apoprotein in 200 [micro]L of 50 mmol/L NaOH at 60[degrees]C for 30 min, we reduced disulfide bonds by incubation with 20 [micro]L of 100 mL/L tri-n-butylphosphine in N,N-dymethilphormamide for 10 min, added 900 [micro]L of acetonitrile, vortex-mixed and centrifuged the solution at 2000g for 5 min, and then dried 1 mL of supernatant under decreased pressure at 60[degrees]C for 4 h. Dry samples were resuspended with 100 [micro]L of derivatization medium (0.08 mmol/L 5-iodoacetamidofluorescein, 25 mmol/L sodium phosphate buffer, pH 12.5). After 15 min at room temperature, derivatized samples were diluted 40 times in water and analyzed by capillary electrophoresis as previously described (15). Briefly, we used a 75-[micro]m i.d. and 57-cm-long (50 cm to the detection window) uncoated fused-silica capillary and performed analysis by applying 30 nL of sample under nitrogen pressure (0.5 psi for 5 s) in a mixture of 30 mmol/L sodium phosphate, 33 mmol/L boric acid, and 75 mmol/L N-methyl-D-glucamine, pH 11.3. The separating conditions (18 kV, 165 [micro]A at normal polarity) were reached in 20 s and were held at a constant voltage for 15 min. Separations were carried out at 40[degrees]C and monitored at 488 run excitation and 520 run emission wavelength.


We measured total plasma thiols by capillary electrophoresis laser-induced detection as described by Zinellu et al. (19); apoB-100 protein content of the LDL fractions by the Lowry method; and total cholesterol, HDL cholesterol, and triglycerides by the enzymatic colorimetric method with commercial reagent sets (Roche Diagnostics).


The main biochemical variables of the 104 healthy volunteers analyzed as a whole and according to sex are shown in Table 1. As expected for a healthy population, 10% of the individuals enrolled in the study were hyperhomo-cysteinemic (Hcy, >15 [micro]mol/L). Men showed lower concentrations of HDL cholesterol and significantly higher concentrations of total plasma CysGly and Hcy than women. The sum of LMW thiols bound to apoprotein was higher in men than in women. In particular, men had significantly higher concentrations of apoB-CysGly, apoB-Hcy, and apoB-Cys. The distribution of plasma and LDL-bound thiols is shown in Fig. 1. Percentage values were calculated from the data reported in Table 1. The distribution of total plasma thiols differed substantially from that of LDL-bound thiols; in particular, the percentage of CysGly bound to apoB was higher than the percentage of the same thiol in plasma, and GSH concentrations were higher in the LDL fraction than in plasma. On the other hand, concentrations of the remaining thiols were significantly lower in lipoproteins than in plasma, and for apoprotein thiol distribution, only the apoB-bound Hcy percentage differed significantly in men vs women (2.70 vs 2.93%; P <0.01 data not shown). In Table 2, the concentrations and the relative amounts of plasma thiols bound to LDL are shown for all participants and according to sex. In 1 L of plasma, 0.522 [micro]mol of thiols (0.193% of total plasma thiols) are bound to LDL, The concentrations of apoB-bound Hcy are significantly lower in women than in men (0.0136 vs 0.0184 [micro]mol/L; P <0.01). The percentage values of thiols bound to LDL did not differ in women and men. Pearson correlation between Hcy concentrations contained in the LDL fraction and factors that could affect the amount of lipoproteins-bound Hcy (as CysGly, Hcy, Cys, GSH, G1uCys, total cholesterol, LDL, HDL and age) suggest that only total plasma Hcy is related to apoB-Hcy concentrations (P <0.0001). Using the stepwise multiple linear regression with LDL-Hcy as the dependent variable and sex, Cys, Hcy, CysGly, LDL, and HDL as independent variables, we found that total Hcy was the most important determinant of LDL-bound Hcy (t-test, 7.979; P <0.0001) but also a positive association with LDL (t-test, 2.068; P <0.041) and sex (t-test, 3.410; P <0.001) was found, whereas Cys (t-test, -1.986; P <0.046) and, mainly, CysGly (t-test, -4.003; P <0.0001) were negatively associated with apoB-Hcy concentrations.


Protein-mixed disulfide formation as a functional response to oxidative modification under pathologic or physiologic conditions is an important process in living organisms and takes place in all biological compartments. Plasma S-thiolated proteins have been detected in healthy humans, in patients with cardiovascular diseases, and in several cell types after oxidant exposure (20). Although increased protein S-thiolation is considered an in vivo marker of oxidative injury caused by noxious agents (21,22), the importance of this index in clinical studies has not been well documented. Thiols in plasma are mainly linked to albumin, but interactions between Hcy and ceruloplasmin, fibrin, and transthyretin have also been reported (23-25). These Hcy-mediated posttranslational modifications may have important functional consequences. In vitro studies have shown that homocysteinylation of the Cys9 residue of annexin II, the endothelial cell surface docking protein for tissue plasminogen activator, inhibits binding (26). Hcy also binds the fibrinbinding domain of plasma fibronectin in vitro, inhibiting its ability to bind fibrin (24), and binds factor Va in vitro, making it resistant to inactivation by activated protein C (27). Homocysteinylation appears to activate latent elastolytic metalloproteinase pro-MMP-2 by disulfide bond formation with the "Cys switch" on the propeptide (28). As with any other chemical reaction, the extent of Hcy protein derivative formation is dependent on time and concentration. The longer the exposure duration and the higher the concentrations of Hcy, the greater the biochemical damage inflicted. Furthermore, if the attacked molecules are long lived and the derivation reactions are irreversible, the harmful effects will be cumulative and clinical consequences progressive. Recent studies have demonstrated that plasma lipoproteins are also susceptible to S-homocysteinylation (14,15). At least 9 free sulfhydryl groups (-SH) in apoB-100 primary structure have been reported that could potentially bind plasma free aminothiols (13). Because lysyl residues of apoB could react in vivo with plasma HcyT by an amide bond, the number of these apoprotein sulfhydryl groups could increase considerably, thus increasing the number of sites that may be bound by plasma aminothiols (9). Even if we did not measure protein N-homocysteinylation concentrations, a 50 g/L LDL solution can be calculated to contain ~1 [micro]mol/L of Hcy-N-apoprotein, as recently reported by Jakubowski (29). Therefore, our apoB-100 samples should also be N-homocysteinilated proportionally to plasma tHcy concentrations (29, 30). Although each nanomole of apoB contains 9 nmol of free Cys residues (taking into account only native -SH of apoprotein and not those deriving from N-homocysteinylation), our data suggest that just few of these -SH sites are S-thiolated (~0.5 nmol/nmol apoB, as described in Table 1). Thus, apoproteins show less S-thiolation than expected from their free -SH content, and S-thiolation is probably limited by the accessibility of the same -SH residues. Hence, apoprotein N-homocysteinylation appears relevant to increase of new -SH sites available for the S-thiolation, but the importance of this finding remains to be elucidated.

Given that at least 5 LMW aminothiols occur in plasma, Hcy competes with other thiols for binding of susceptible -SH sites. Our results show that increasing concentrations of plasma Hcy lead to higher S-homocysteinylation of lipoprotein in vivo. These data, calculated with Pearson correlation analysis, were confirmed by multiple regression analysis, by which we also found that plasma Cys and CysGly both seem to inhibit interaction between Hcy and apoprotein. In particular, CysGly is a more effective competitor than Cys, although CysGly was present in plasma at 1/10th the concentration of Cys. Thus CysGly, which accounts for ~12% of total thiols in plasma, was more highly represented in the LDL fraction, making up -24% of all bound thiols. Because CysGly is a redox labile metabolite, it likely reacts more avidly with free -SH groups of apoB-100, acting as a physiologic inhibitor of Hcy-LDL linkage. Although total CysGly concentrations are higher in men than women, the Hcy/CysGly ratio is lower in women [women, 0.31 (0.1); men, 0.34 (0.1)], and although the difference is not statistically relevant (P = 0.14), it may account for the lower amount of Hcy bound to apoprotein in women.

By multiple regression analysis, we also found that LDL cholesterol concentrations may influence the quantity of Hcy bound to LDL. Increasing plasma cholesterol concentrations are related to increased interactions between Hcy and LDL apoprotein. Therefore, in hypercholesterolemia, a relatively higher quantity of thiols per gram of apoprotein may be transported within the LDL fraction. Apoprotein-bound thiols could be thus internalized by endothelial cells, degraded in lysosomes, and released in the cytosol, where they could alter the intracellular redox potential or modify intracellular proteins, causing endothelial dysfunction. It is known that LDL could pass, via transcytosis, through the vascular endothelium to reach the subendothelial space. Because increased concentrations of plasma LDL increase proportionally the rate of LDL entry (31) in hypercholesterolemic individuals, increased amounts of homocysteinilated LDL could accumulate in the intima. There, modified LDL could be avidly taken up from macrophages by membrane receptor or by phagocytosis, leading to intracellular cholesterol accumulation and foam cell formation (32). Finally, the increase of LDL Hcy suggests that LDL atherogenicity may be enhanced by the modification of its chemical and biological properties, leading to endothelial vascular injury and deposition of cholesterol, lipids, and Hcy in the intima space and the development of atherosclerotic plaques. CysGly could have a protective role decreasing the interaction between LDL apoprotein and Hcy, thus decreasing the amount of Hcy transferred from plasma to endothelial and subendothelial spaces. Further studies are required to better explain the kinetic reactions between Hcy and apoB-100, the inhibitory function of CysGly in this reaction, and the role of apoprotein-bound thiols in endothelial cell function.

This study was supported by the Assessorato dell'Igiene e Sanita Regione Autonoma della Sardegna and by the Ministero dell'Istruzione, dell'Universita e della Ricerca of Italy. Language revision by Maria Antonietta Meloni is greatly appreciated.


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[1] Dipartimento di Scienze Biomediche, Cattedra di Biochimica Clinica, and

[2] Dipartimento di Scienze Fisiologiche, Biochimiche e Cellulari, University degli Studi di Sassari, Sassari, Italia.

[3] Nonstandard abbreviations: Hcy, homocysteine; HcyT, homocysteinethiolactone; Cys, cysteine; SSA, sulfosalicylic acid; CysGly, cysteinylglycine.

* Address correspondence to these authors at: Chair of Clinical Biochemistry, Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy. Fax 39-079228120; e-mail angelozinellu@ (A.Z.), (C.C.).

Received March 31, 2006; accepted August 29, 2006.

Previously published online at DOI: 10.1373/clinchem.2006.071142
Table 1. Mean biochemical parameters of studied population.

 All subjects, Men (44),
 mean (SD) mean (SD)

Age, years 54.6 (16.1) 53.3 (15.6)
Total cholesterol, mmol/L 5.271 (1.09) 5.069 (1.03)
LDL cholesterol, mmol/L 3.261 (1.02) 3.165 (0.966)
HDL cholesterol, mmol/L 1.40 (0.451) 1.31 (0.376)
Triglycerides, mmol/L 1.274 (0.612) 1.405 (0.634)
Plasma thiols
 Total LMW thiols, [micro]mol/L 275.9 (55.2) 286.3 (50.9)
 Total CysGly, [micro]mol/L 35.1 (7.0) 38.2 (8.2)
 Total Hcy, [micro]mol/L 11.05 (3.2) 12.43 (3.20)
 Total Cys, [micro]mol/L 221.7 (48.3) 227.9 (42.9)
 Total GSH, [micro]mol/L 4.88 (1.50) 4.60 (1.52)
 Total GluCys, [micro]mol/L (c) 3.11 (0.71) 3.16 (0.63)
 apoB-Thiols, nmol/[micro]mol apoB 446 (104) 494 (106)
 apoB-CysGly, nmol/[micro]mol apoB 108 (22) 116 (25)
 apoB-Hcy, nmol/[micro]mol apoB 13.7 (6.2) 16.6 (7.5)
 apoB-Cys, nmol/[micro]mol apoB 324 (92) 349 (98)
 apoB-GSH, nmol/[micro]mol apoB 9.5 (3.7) 10.2 (4.5)
 apoB-GluCys, nmol/[micro]mol apoB 2.6 (0.4) 2.6 (0.4)

 Women (60),
 mean (SD)

Age, years 56.4 (17.3)
Total cholesterol, mmol/L 5.421 (1.11)
LDL cholesterol, mmol/L 3.331 (1.05)
HDL cholesterol, mmol/L 1.57 (0.474) (a)
Triglycerides, mmol/L 1.180 (0.584)
Plasma thiols
 Total LMW thiols, [micro]mol/L 268.3 (57.4)
 Total CysGly, [micro]mol/L 32.9 (4.8) (b)
 Total Hcy, [micro]mol/L 10.04 (2.88) (b)
 Total Cys, [micro]mol/L 217.2 (51.8)
 Total GSH, [micro]mol/L 5.09 (1.46)
 Total GluCys, [micro]mol/L (c) 3.07 (0.77)
 apoB-Thiols, nmol/[micro]mol apoB 431 (95) (a)
 apoB-CysGly, nmol/[micro]mol apoB 103 (18) (a)
 apoB-Hcy, nmol/[micro]mol apoB 11.6 (4) (d)
 apoB-Cys, nmol/[micro]mol apoB 305 (88) (a)
 apoB-GSH, nmol/[micro]mol apoB 9.0 (3.0)
 apoB-GluCys, nmol/[micro]mol apoB 2.5 (0.4)

(a) P <0.05.

(b) P <0.0001.

(c) GluCys, glutamylcysteine.

(d) P <0.001.

Table 2. Distribution of plasma thiols bound to LDL
for all participants and after sorting for sex. (a)

 apoB-bound CysGly Hcy

All participants (104)
 [micro]mol/L 0.125 (0.050) 0.0156 (0.0083)
 % 0.366 (0.159) 0.147 (0.086)
Men (44)
 [micro]mol/L 0.131 (0.055) 0.0184 (0.0100)
 % 0.351 (0.136) 0.153 (0.087)
Women (60)
 [micro]mol/L 0.121 (0.047) 0.0136 (0.0063) (b)
 % 0.377 (0.175) 0.143 (0.087)

 apoB-bound Cys GSH

All participants (104)
 [micro]mol/L 0.367 (0.154) 0.0107 (0.0047)
 % 0.168 (0.075) 0.237 (0.111)
Men (44)
 [micro]mol/L 0.392 (0.168) 0.0111 (0.0048)
 % 0.173 (0.073) 0.261 (0.101)
Women (60)
 [micro]mol/L 0.352 (0.143) 0.0103 (0.0047)
 % 0.165 (0.076) 0.220 (0.116)

 apoB-bound GluCys (b) Total

All participants (104)
 [micro]mol/L 0.0029 (0.0010) 0.522 (0.203)
 % 0.098 (0.039) 0.193 (0.082)
Men (44)
 [micro]mol/L 0.0029 (0.0011) 0.555 (0.222)
 % 0.096 (0.034) 0.196 (0.078)
Women (60)
 [micro]mol/L 0.0029 (0.0011) 0.500 (0.186)
 % 0.100 (0.043) 0.191 (0.086)

(a) All values given as mean (SD).

(b) GluCys, glutamylcysteine.

(c) P <0 0.01 vs apoB-bound Hcy concentration in men.

Fig. 1. Distribution of plasma and LDL-bound thiols of all patients.


GluCys 1.14%
CysGly 12.9%
Hcy 4.02%
Cys 80.1%
GSH 1.83%


GluCys 0.58%
CysGly 24.4%
Hcy 2.98%
Cys 70.0%
GSH 2.11%

Note: Table made from pie chart.
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Title Annotation:Lipids, Lipoproteins and Cardiovascular Risk Factors
Author:Zinellu, Angelo; Zinellu, Elisabetta; Sotgia, Salvatore; Formato, Marilena; Cherchi, Gian Mario; Dei
Publication:Clinical Chemistry
Date:Nov 1, 2006
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