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Technical Briefs |
1 Department of Laboratory Medicine, Warren Magnuson Clinical Center, NIH, Bethesda, MD 20892
aaddress correspondence to this author at: Department of Laboratory Medicine, NIH, Building 10, Room 2C-407, Bethesda, MD 20892-1508; fax 301-402-1885, e-mail ghortin{at}mail.cc.nih.gov
McCully (1) initially observed that patients with
extremely increased plasma concentrations of homocysteine (Hcy)
attributable to homocystinuria have accelerated atherosclerosis.
Subsequently, even moderate hyperhomocysteinemia became recognized as a
risk factor for atherosclerosis and thrombosis (2)(3)(4),
although the mechanism by which increased concentrations of Hcy produce
these effects is uncertain. Hcy occurs in the circulation in multiple
forms, including Hcy linked via disulfides to albumin (
70%), as a
mixed disulfide with Cys (25%), as a disulfide-linked dimer (<5%),
as the free reduced amino acid (<5%), and as a thiolactone (trace)
(5)(6). It is not clear which of these components are
physiologically active, and efforts to measure individual components
have been technically challenging because of the interconversion of
different forms during specimen processing. As a result, measurement of
total Hcy (tHcy), including all forms except the trace amount of
thiolactone, has become the standard clinical test (5)(6)(7).
The present study examined the relationships among concentrations of tHcy, plasma albumin, and total Cys (tCys; includes protein-bound, disulfide-linked forms, and reduced). Considering that albumin and Cys serve as covalent carriers of most of the Hcy in circulation, these components may affect circulating tHcy and the physiological action of Hcy. Cys has the potential for multiple interactions with Hcy because Cys is not only a covalent carrier but also a competitor for binding sites on proteins, a potential competitor for uptake into cells, and a metabolite of Hcy via the transulfuration pathway (8)(9).
tHcy and tCys in EDTA plasma were analyzed simultaneously by HPLC after
the reduction of plasma disulfides with tris(2-carboxyethyl)phosphine,
precipitation of proteins with trichloroacetic acid, derivatization
with 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), and fluorescent
detection as described previously (10) using cystamine as an
internal standard. Performance characteristics of the assay have been
described previously (10). HPLC analysis was performed on
151 unselected plasma specimens submitted to the laboratory for tHcy
determination. Patients included 75 males and 76 females (median age,
54 years; <20 years, n = 2; 20–39 years, n = 30; 40–59
years, n = 72; 60–79 years, n = 42; 
80 years, n = 4;
the age of 1 subject was unknown). Albumin was measured for 142
specimens using a Hitachi 917 analyzer with bromcresol green reagent
from Roche. Our nonparametric reference interval for albumin was 37–47
g/L. The use of specimens was approved by our Institutional Review
Board.
We anticipated that an increased quantity of the major binding protein
(albumin) might relate to increases in tHcy or tCys, but we found
little evidence for this. Neither tHcy nor tCys correlated with albumin
(range, 25–48 g/L) in plasma (Fig.
1, A and B
). It may be that the binding capacity of plasma
proteins for Hcy far exceeds normal plasma tHcy (8), and
factors controlling the interchange between protein-linked and soluble
forms of Hcy and Cys are not fully understood.
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In this study, tHcy concentration was 28-fold lower than tCys: mean
tHcy concentration, 9.0 µmol/L; range, 4.4–17.7 µmol/L (most
within reference interval); mean tCys concentration, 238 µmol/L;
range, 141–368 µmol/L). There was a highly significant (P
<0.0001) positive correlation between tHcy and tCys (Fig. 1C
). This
agrees with the observations of El-Khairy et al. (11) for
subjects with tHcy concentrations <15 µmol/L. The observed linear
relationship between tHcy and tCys is consistent with the proposition
that part of the Cys is derived from Hcy metabolism via the
transulfuration pathway in an amount proportional to the Hcy
concentration.
Subjects with tHcy <13 µmol/L, which is the upper reference limit
assigned in our laboratory, had higher tCys/tHcy ratios (mean, 29;
range, 17–40; n = 138) than subjects with tHcy
concentrations 13 µmol/L (mean ratio, 20; range, 15–25;
n = 13). There was an overall negative correlation of the
tCys/tHcy ratio with tHcy concentration (Fig. 1D
). The relationship had
substantial curvature; therefore, it could not be represented well by a
simple linear fit.
Individuals with homocystinuria attributable to defects in the transulfuration pathway provide further evidence for the importance of this pathway in the relationship between tCys and tHcy; these individuals have low plasma tCys despite severe hyperhomocysteinemia (9). The relationship between tHcy and tCys appears to be more complex than a simple precursor–product relationship, however. The linear relationship between tHcy and tCys is reported to break down at tHcy >15 µmol/L, with tCys decreasing as tHcy increases above this concentration (11). In addition, slight decreases in tCys occur after methionine or Hcy loading at times when there is transient hyperhomocysteinemia (12)(13). In these situations, there would be a progressive decline in tCys/tHcy ratios as tHcy increases. Renal clearance of amino acids may have a role in lowering tCys concentrations because both tCys and tHcy are increased in patients with renal failure (14).
As shown here and in earlier reports (10)(11), dual analysis of tHcy and tCys can be readily provided by a chromatographic method. Evaluation of the tCys/tHcy ratio potentially has several practical consequences. One consequence is that the tCys/tHcy ratio may reflect abnormalities in the function of the transulfuration pathway. It is estimated that 1% of the population is heterozygous for deficiency of cystathionine-ß-synthase, the enzyme for which homozygous deficiency leads to homocystinuria (15). tHcy is increased and tCys is decreased in heterozygous deficiency of this enzyme, and a low tCys/tHcy ratio assists in identifying heterozygotes (15). The other consequence is that the tCys/tHcy ratio may have physiologic significance in that some of the causes of hyperhomocysteinemia, such as homocystinuria (9) and renal failure (14), that are strongly associated with cardiovascular disease produce low tCys/tHcy ratios, and Cys may affect bioavailability of Hcy by serving as a carrier or competitor of Hcy. Further investigation is warranted to explore whether this ratio serves as an indicator of cardiovascular risk that supplements tHcy measurements. The third consequence is that low tCys/tHcy serves as an indicator of preanalytical errors. tCys decreases (10–20% over 1 day) if anticoagulated whole blood samples are left at room temperature, whereas tHcy increases (30–90%) (16)(17). Thus, unusually low tCys/tHcy ratios should trigger a review of whether there is a physiological explanation or a preanalytical problem such as delayed separation of plasma from blood cells and/or storage of whole blood at inappropriately high ambient temperature. Finally, evaluation of the tCys/tHcy ratio may serve as an internal quality check of each analysis. In the absence of other causes (see above), unexpectedly high or low tCys/tHcy ratios would identify samples with increased probability of analytical errors. For the 138 plasma specimens with tHcy <13 µmol/L, the nonparametric and parametric reference intervals for the tCys/tHcy ratio would be 19–42 and 17–40, respectively.
References
The following articles in journals at HighWire Press have cited this article:
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  G. L. Hortin, N. Seam, and G. T. Hoehn Bound Homocysteine, Cysteine, and Cysteinylglycine Distribution between Albumin and Globulins Clin. Chem., December 1, 2006; 52(12): 2258 - 2264. [Abstract] [Full Text] [PDF]
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 R. G. V. Smolders, K. de Meer, P. Kenemans, C. Jakobs, W. Kulik, and M. J. van der Mooren Oral Estradiol Decreases Plasma Homocysteine, Vitamin B6, and Albumin in Postmenopausal Women But Does Not Change the Whole-Body Homocysteine Remethylation and Transmethylation Flux J. Clin. Endocrinol. Metab., April 1, 2005; 90(4): 2218 - 2224. [Abstract] [Full Text] [PDF]
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Q. Han, M. Xu, L. Tang, X. Sun, N. Zhang, X. Tan, X. Tan, Y. Tan, and R. M. Hoffman Homogeneous Enzymatic Colorimetric Assay for Total Cysteine Clin. Chem., July 1, 2004; 50(7): 1229 - 1231. [Full Text] [PDF]
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