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Technical Briefs |
1 Department of Laboratory Medicine, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860-8556, Japan
2 Suiyukai Clinic, 676-1 Kuzumoto-Cho, Kashihara, Nara 634-0007, Japan
3 Tsukuba Research Laboratories, Tokuyama Dental Corporation, 40 Wadai, Tsukuba City, Ibaraki 300-4247, Japan
aauthor for correspondence: fax 81-96-373-5282, e-mail uji{at}gpo.kumamoto-u.ac.jp
Homocysteine (Hcy) is a sulfur-containing nonprotein amino acid derived from the metabolism of methionine. Plasma Hcy is an independent risk factor for atherosclerosis (1)(2)(3)(4)(5).
The cyclic internal lactone form of Hcy, homocysteine thiolactone, may be the critical molecular form in the pathogenesis of atherosclerosis. In cultured cells, homocysteine thiolactone is synthesized through the reaction of methionyl-tRNA synthase (6)(7). This enzyme reacts with free amino groups of proteins to produce protein-bound homocystamide (7)(8)(9). The toxicity of homocysteinylation to the cell has been demonstrated in both in vitro and in vivo experiments. Lysyl oxidase, which catalyzes the posttranslational modification essential to the pathogenesis of connective tissue matrices, is irreversibly inactivated by homocysteine thiolactone (10).
Homocysteinylation increases the internalization of LDL by macrophages (11). Furthermore, homocysteinylated LDL elicits an autoimmune response (12). Homocysteine thiolactone hydrolase is present in human plasma and is identical to paraoxonase (13). Homocysteine thiolactone hydrolase may hydrolyze homocysteine thiolactone to Hcy and thereby prevent the homocysteinylation of proteins. In the absence of a suitable assay system for homocysteine thiolactone, however, no detailed study is available on homocysteine thiolactone or Hcy-related proteins. We have developed and evaluated an assay system for measuring protein-bound homocystamide in plasma and used it in samples from healthy adults and hemodialysis patients. Here we report the results of that study.
We obtained plasma samples from 20 healthy volunteers (12 males, 8 females; age range, 29–38 years) and 15 nondiabetic hemodialysis patients [7 males and 8 females; mean (SD) age, 49 (7) years] treated at the Suiyukai Clinic (Nara, Japan) after receiving their informed consent for this study. The patients were on hemodialysis using bicarbonate dialysate with systemic heparinization two or three times week a week. Blood was collected by venipuncture at the start of hemodialysis.
Human serum albumin (HSA) and D,L-Hcy were purchased from Sigma Chemical Co. Hydrochloric acid, trifluoroacetic acid (TFA), and HPLC-grade acetonitrile were purchased from Wako Pure Chemicals Co. Ltd. Triethylamine was purchased from Pierce Chemical Co, and 4-fluoro-7-sulfamoyl-benzofurazan (ABD-F) was purchased from Dojindo Laboratories. Microplate Enzyme Immunoassay reagent sets for total Hcy were purchased from Bio-Rad Laboratories. All other materials used were of analytical grade. The ABD-Hcy calibrator was prepared according to the method proposed by Toyo’oka and Imai (14) as follows: 3 mL of 100 µmol/L D,L-Hcy was mixed with 1 mL of 90 mmol/L ABD-F in acetonitrile, buffered to pH 8–9 with triethylamine, and incubated at 60 °C for 20 min. Synthesized Hcy-HSA was prepared according to Naruszewics et al. (11). HSA (50 mg; 750 µmol/L) was reacted with 24 µmol/L Hcy in 5 mL of phosphate-buffered saline (pH 6.8), incubated at room temperature for 1 h, and filtered. Amino acid analysis was performed (data not shown), and the results confirmed that, under these conditions, 
1 g/L Hcy-HSA was synthesized, with nine molecules of Hcy combining with one molecule of HSA.
HPLC was performed using a 600E multisolvent delivery system equipped with a 470 scanning fluorescence detector (both from Waters Associates). The reversed-phase LiChrosorb 100 RP-18 column (4.6 x 250 mm) was from Merck Japan, and the Inertsil ODS-2 column (4.6 x 250 mm) was from GL Science Inc. A PD-10 gel-filtration column (Amersham Pharmacia Biotech), equilibrated with 20 mL of 100 mmol/L borate buffer (pH 8.2) containing 5 g/L SDS and 2 mmol/L EDTA (buffer A), was used to separate the protein-bound Hcy from the low-molecular weight forms. A 1-mL plasma sample was loaded on the column, and the column was then washed with 2 mL of buffer A to elute the protein-bound Hcy fraction. We then mixed 0.1 mL of this fraction with 0.8 mL of buffer A, followed by 0.1 mL of 40 mmol/L ABD-F in dimethylformamide and 0.01 mL of 500 mmol/L tri-n-butylphosphine in dimethylformamide, and incubated the mixture at 60 °C for 30 min according to the method proposed by Toyo’oka and Imai (14) and Treuheit and Kirley (15). With this procedure, it was possible to dissociate the S–S bond between Hcy and the protein by reduction and to label the -SH group simultaneously. In this step, S–S protein-bound Hcy was converted to its free form, which was then removed by a second gel-filtration step similar to the first step, and protein-bound homocystamide was collected.
We dissociated a 2-mL fraction of this protein-bound homocystamide by hydrolysis with 2 mL of 6 mol/L HCl, sealed under reduced pressure in a test tube, at 110 °C for 20 h, and then evaporated the mixture to dryness. This sample was dissolved in 2 mL of distilled water and then evaporated. This step was repeated two more times. The hydrolysis product was dissolved in 0.5 mL of 0.9 g/L TFA and filtered through a 0.22 µm centrifugal type filter. A 100-µL aliquot of the resulting sample was separated by reversed-phase HPLC using LiChrosorb 100 RP-18 (4.6 x 250 mm) and Inertsil ODS (4.6 x 250 mm) columns. The HPLC conditions for both the LiChrosorb 100 RP-18 and Inertsil ODS columns were as follows: eluent A, 0.9 g/L TFA; eluent B, 800 mL/L acetonitrile containing 0.75 g/L TFA. The flow rate was 1 mL/min, and elution was with a linear gradient from 0% to 28% B over 25 min. Detection was with a fluorescence detector (emission wavelength, 380 nm; excitation wavelength, 500 nm).
The tHcy (free Hcy and S–S protein-bound Hcy) concentration in plasma was determined using the Microplate Enzyme Immunoassay Homocysteine reagent set (Bio-Rad) according to the manufacturer’s instructions. The peak for the ABD-Hcy calibrator was detected at 29 min (Fig. 1A
), but the ABD derivatives of the samples showed several fluorescent peaks eluting from the LiChrosorb 100 RP-18 column after hydrolysis (Fig. 1B
). We therefore collected the peak fraction with a retention time of 29 min and introduced this fraction into an Inertsil ODS reversed-phase column (4.6 x 250 mm; Fig 1C
), using similar HPLC conditions. As shown in Fig. 1C
, the peak for ABD-labeled protein-bound homocystamide in the sample eluted at 29 min. This peak was confirmed as ABD-Hcy by comparison with the retention time for the ABD-Hcy calibrator under various HPLC conditions (data not shown) and by amino acid analysis using the PICO-TAG amino acid analysis method (Waters) performed according to the manufacturer’s instructions.
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For the precise determination of protein-bound homocystamide by our proposed method, conventional Hcy (free Hcy and protein-bound Hcy via S–S bonds) needs to be removed before the acid hydrolysis step. The removal of the nonprotein-bound form of Hcy in the first gel-filtration step of our analytical procedure was assessed by the recovery of 0.1–500 mmol/L Hcy added to plasma. Less than 5% of the added Hcy was recovered. We then evaluated the elimination of nonprotein-bound ABD-Hcy, which was converted from S–S protein-bound Hcy in the second gel-filtration step. After the first gel-filtration step of the analytical procedure, ABD-Hcy calibrator at various concentrations (0.005–1.0 mmol/L) was added to plasma samples, and recoveries were determined. The results showed that <8% of ABD-Hcy had been recovered.
Calibration curves with synthetic ABD-Hcy in pooled plasma were linear to 3 µmol/L. The within-run CV (pooled plasma) was 10% at 0.79 µmol/L. The recovery for 0.01–0.1 µmol/L synthetic Hcy-HSA added to plasma was 97–107%. The concentrations of tHcy and protein-bound homocystamide and the molar ratios of protein-bound homocystamide to tHcy in plasma are shown in Table 1
.
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In plasma samples from 20 healthy adults (ages, 29–38 years), the mean (SD) concentrations were 13.9 (5.8) µmol/L for tHcy and 0.51 (0.11) µmol/L for protein-bound homocystamide.
The mean (SD) molar ratio of protein-bound homocystamide to tHcy was 0.042 (0.015), or 4.2% of tHcy. In 15 hemodialysis patients, the mean (SD) concentrations were 47.2 (26.1) µmol/L for tHcy and 0.74 (0.20) µmol/L for protein-bound homocystamide. The mean (SD) molar ratio of protein-bound homocystamide to tHcy was 0.02 (0.010), or 2.0% of tHcy.
In the circulatory system, Hcy is generally present in two forms: a free form in which the cysteine disulfide bond is included, and a protein form, the latter representing 70–80% of tHcy (3)(16)(17). On the assumption that the binding of Hcy to protein is exclusively by S–S bonds, we determined the plasma concentration of tHcy using reduced plasma samples as follows: the plasma samples were reduced to cleave the S–S bond and then deproteinized, and free Hcy was detected. However, protein-bound homocystamide could not be measured by this conventional method because the peptide bond is refractory to chemical reduction. Protein-bound homocystamide would be removed in the deproteinization step. Acid hydrolysis of plasma samples was needed to break the peptide bond linking homocystamide and protein. Because acid hydrolysis destroys Cys and Hcy with free -SH groups, we protected the -SH groups by reductive alkylation using ABD-F. ABD adducts are stable in strong acid and at high temperatures. They also exhibit fluorescence (14), which allowed us to detect trace amounts of ABD-Hcy.
In this study, we could confirm the presence of a third plasma Hcy fraction, a peptide-bound type, by hydrolysis after removal of the protein-binding fraction containing the S–S bonds as well as the free form by gel filtration. Homocysteine thiolactone, a bioactive form of Hcy, has an intramolecular ester component that is likely to bind to proteins by a peptide bond under physiologic conditions (7)(8)(9). Taking note of this chemical characteristic of homocysteine thiolactone, we believe that the peptide fraction of Hcy identified in this study is derived from homocysteine thiolactone. Another point of interest in this study was the reduced fraction of peptide-bound Hcy in the plasma of hemodialysis patients. The peptide fraction containing protein-bound homocystamide accounted for 4.2% of tHcy in healthy adults and 2.0% in hemodialysis patients. Whether the rate of conversion of Hcy to homocysteine thiolactone is decreased or the decomposition of homocysteine thiolactone is enhanced in the uremic state is of great interest. Other clinical applications of the proposed method will be reported elsewhere in the near future.
Acknowledgments
Additional information on the proposed method is available on the Clinical Chemistry Online web site (http://www.clinchem.org/content/vol48/issue6).
References
The following articles in journals at HighWire Press have cited this article:
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  A. Undas, M. Kolarz, G. Kopec, R. Glowacki, E. Placzkiewicz-Jankowska, and W. Tracz Autoantibodies against N-homocysteinylated proteins in patients on long-term haemodialysis Nephrol. Dial. Transplant., June 1, 2007; 22(6): 1685 - 1689. [Abstract] [Full Text] [PDF]
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 G. Chwatko, G. H. J. Boers, K. A. Strauss, D. M. Shih, and H. Jakubowski Mutations in methylenetetrahydrofolate reductase or cystathionine {beta}-syntase gene, or a high-methionine diet, increase homocysteine thiolactone levels in humans and mice FASEB J, June 1, 2007; 21(8): 1707 - 1713. [Abstract] [Full Text] [PDF]
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 H. Jakubowski Pathophysiological Consequences of Homocysteine Excess J. Nutr., June 1, 2006; 136(6): 1741S - 1749S. [Abstract] [Full Text] [PDF]
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 R. Glowacki and H. Jakubowski Cross-talk between Cys34 and Lysine Residues in Human Serum Albumin Revealed by N-Homocysteinylation J. Biol. Chem., March 19, 2004; 279(12): 10864 - 10871. [Abstract] [Full Text] [PDF]
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