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Technical Briefs |
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A/C Diagnostics, LLC, and AntiCancer, Inc., 7917 Ostrow St., San Diego, CA 92111
a author
for correspondence: fax 858-268-4175, e-mail all{at}anticancer.com
McCully (1) in 1969 first observed that plasma hyperhomocysteinemia was involved in the pathogenesis of arteriosclerosis. More studies have reported that total serum or plasma homocysteine (tHCY) is an independent risk factor for cardiovascular diseases, including arteriosclerosis, coronary artery disease, cerebrovascular disease, and myocardial infarction (2)(3)(4)(5)(6)(7). Furthermore, plasma tHCY is a strong predictor of mortality in patients with existing coronary artery disease (8).
A major basis for hyperhomocysteinemia is insufficient intake of
vitamins B6, B12, and folic
acid, which are necessary for homocysteine metabolism and which lower
the risk for homocysteine-associated diseases (9)(10)(11).
Homocystinuria can result from inborn errors, such as
deficiencies in the enzymes cystathionine ß-synthase,
methionine synthase, and methylenetetrahydrofolate reductase
(12). These patients have premature vascular disease,
50% of untreated patients have thromboembolic events, and the
mortality rate is 20% before the age of 30 (12).
Many methods have been developed to measure tHCY, such as the tHCY enzyme conversion immunoassay designed for the Abbott IMx analyzer (13), the microtiter-plate tHCY enzymatic immunoassay kit (14), and HPLC (15)(16)(17). However, these methods are relatively complex and require highly specialized equipment.
To meet the need for a widely available homocysteine assay, we have
developed and describe here a simple single-enzyme tHCY assay based on
a highly specific homocysteine 
,
-lyase (rHCYase) (18).
L-Homocystine, L-cysteine (L-CYS), L-methionine (L-MET), dithiothreitol (DTT), Triton X-100, citric acid, potassium ferricyanide, and other common reagents for the tHCY enzymatic assay were obtained from Sigma. 4-Fluoro-7-sulfobenzofurazan ammonium salt was obtained from Wako Fine Chemicals, and N,N-dibutylaniline was obtained from Acros Organics. N,N-Dibutyl phenylene diamine (DBPDA) was synthesized in our laboratory (L. Tang and Y. Tan, unpublished data). rHCYase was produced in our laboratory (18).
Blood was collected by venipuncture into Vacutainer Tubes (Becton Dickinson) containing EDTA. Samples were stored immediately at 0–4 °C. Plasma for tHCY measurements was separated within 1 h by centrifugation at 300g for 5 min at 4 °C. The tHCY is stable for 1 month at 4 °C and indefinitely at -20 or -80 °C (19)(20). To determine baseline homocysteine concentrations, blood donors fasted for 12 h.
The principle of the assay is as follows. In reaction I, rHCYase
specifically converts homocysteine to -ketobutyrate, ammonia, and
H2S. In reaction II, the
H2S combines with DBPDA to form 3,7-bis(dibutyl
amino)phenothiazine-5-ium chloride, which is highly
fluorescent.
For the reduction reaction, we used flat-bottomed 96-well cell culture cluster plates with low-evaporation lids (cat. no. 0720089; Corning). Five microliters of calibrators or plasma samples and 185 µL of assay buffer [40 mmol/L sodium phosphate buffer (pH 8.4), containing 2 mL/L Triton X-100 and 0.25 mmol/L DTT] were added to the wells. Two wells were used for each sample. Reduction was carried out at 37 °C for 30 min. This reaction was necessary to break the disulfide linkages in plasma proteins and to reduce the low-molecular weight disulfides homocystine and homocysteine-cysteine mixed disulfide.
For each sample or calibrator, we added 30 µL of rHCYase
(0.05 g/L), the equivalent to 0.1 U, in 40 mmol/L potassium
phosphate buffer containing 20 µmol/L pyridoxal phosphate to one
well. One unit of enzyme is defined as the amount that catalyzes 1
µmol of H2S per minute from homocysteine
(18). We added 30 µL of enzyme buffer to the other well to
serve as background. The enzymatic reaction was carried out at room
temperature for 3 min. The lid was left on the dish to prevent possible
loss of H2S. The enzymatic reaction was stopped
by the addition of 50 µL of chromophore reagent (20 mmol/L
DBPDA in 3 mol/L
H2SO4), followed by 30 µL
of oxidizing agent [5 mmol/L potassium ferricyanide in 10 mmol/L
sodium phosphate buffer (pH 7.2)]. The total time for these additions,
using a multichannel pipetter, was 40 s, thus minimizing any
H2S losses. The chromogenic reaction was carried
out at room temperature for 10 min. The resulting fluorescence was
measured at an excitation wavelength of 665 nm and an emission
wavelength of 690 nm in a Spectra Gemini tunable fluorescence
microtiter plate reader (Molecular Devices).
L-HCY was generated by the reduction of
L-homocystine (Sigma) with 0.25 mmol/L DTT for 30 min at
37 °C. The resulting L-HCY was used as a calibrator at
concentrations of 1–100 µmol/L. Calibrators were always run in
duplicate. Samples with 1–100 µmol/L tHCY could be measured by the
assay without additional dilution. Samples with tHCY >100 µmol/L
were diluted in normal saline. The calibration curve was linear
and repeatable between 1 and 100 µmol/L (Fig. 1
A).
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The HPLC tHCY assay used the ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) derivatization method as described by Ubbink et al. (21) and Dudman et al. (22) with some modifications. Plasma samples were reduced with tri-n-butylphosphine and were derivatized with SBD-F. An L-6200A Intelligent Pump HPLC (Hitachi) was used for the measurement of tHCY with a reversed-phase Supelcosil LC-18-DB column (25 cm x 4.8 mm; particle size, 5 µm; Supelco) at room temperature.
Plasma samples from 100 subjects and patients were analyzed with the
tHCY enzymatic assay and the HPLC assay. Each sample was analyzed in
duplicate, and the average homocysteine values and SDs were calculated.
The correlation was calculated by regression analysis and Bland-Altman
methods (23) (Fig. 1
, B and C).
The precision of this assay was evaluated in a study based on NCCLS
guideline EP5-T2 (24). Three plasma samples
containing low, medium, and high tHCY were measured. Each analysis also
included low, medium, and high L-homocystine
calibrators (14). The within- and between-assay CVs were
<5% and <10%, respectively (Table 1
).
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To study linearity, three plasma samples with homocysteine concentrations between 20 and 30 µmol/L were serially diluted one-, two-, four-, and eightfold with saline. The samples were analyzed in four replicates in a single analytical run. Mean concentrations and dilution recovery were calculated (14).
To determine the limit of quantification [defined as the lowest concentration having a CV <20% (14)], we diluted calibrators containing 25 µmol/L L-HCY with saline to obtain concentrations of 0.5, 1, 1.5, 2.0, and 2.5 µmol/L, and assayed four replicates. The limit of quantification was 1.0 µmol/L.
A series dilution of a sample with high tHCY (25 µmol/L as determined by HPLC), was measured by the tHCY enzymatic assay in duplicate. The observed homocysteine was 100% ± 20% (mean ± SD) of the expected concentration. Mean recovery of known amounts of L-HCY added to a plasma sample with a tHCY concentration of 8.24 µmol/L was 95%.
We tested for interference from L-CYS and L-MET by adding them to calibrator (20 µmol/L L-HCY solution) and plasma samples at concentrations of 0–200 µmol/L, and also by assaying the pure chemicals at the same concentrations. L-CYS had <5% interference in the concentration range tested, and L-MET had no interference. The time course of the HCY enzymatic assay was determined with L-HCY (20 µmol/L) alone, L-CYS (200 µmol/L) alone, and the mixture of L-HCY (20 µmol/L) plus L-CYS (200 µmol/L). The reaction rate for L-HCY was 21.5-fold faster than for L-CYS. There was almost no interference from L-CYS to L-HCY after 3 min of enzymatic reaction. Lack of interference from L-CYS and L-MET was also confirmed by the high correlation between the tHCY enzymatic assay and the HPLC method.
The specificity of the homocysteine enzymatic assay for L-HCY was high. The ratios for L-CYS and L-MET in the assay were 0.78% and 0.067%, respectively, with L-HCY taken as 100%.
The increasing requirement for tHCY measurement has led to the development of various tHCY assays (13)(14)(15)(16)(21)(22)(25), but none are ideal. HPLC methods (15)(16)(19)(20)(21)(22)(25) have been used as the standard evaluation for tHCY, but the disadvantages of such methods for clinical application include the limited number of samples that can be processed by HPLC and the need for appropriate HPLC instruments and skilled operators.
Shipchandler and Moore (13) and Frantzen et al. (14) recently described an enzyme conversion immunoassay for tHCY. The assay involves conversion of homocysteine to S-adenosylhomocysteine. Detection is then based on use of a monoclonal antibody that recognizes S-adenosylhomocysteine, and a fluorescent analog. This assay has high precision but is limited to specialized equipment, has >10 steps, and processes only 20 samples in 60 min, which precludes high throughout.
The present tHCY enzymatic assay is a simple, high-throughput method
with only three steps. This method can be performed with readily
available instruments. The rHCYase developed in our laboratory
(18) has high sensitivity and specificity for homocysteine.
The enzyme is not inhibited by effective reducing agents, such as DTT,
present in amounts sufficient to quantitatively reduce all homocysteine
disulfides in plasma. Our laboratory has also developed a
high-sensitivity fluorescent chromophore (DBPDA) to detect
H2S that gives a signal of 100 fluorescence
units over background for 10 µmol/L plasma tHCY.
Only 5 µL of EDTA plasma is required for a microtiter plate tHCY enzymatic fluorescence assay, which is at least 10 times less than other assays. Plasma derived from a finger prick is sufficient for the tHCY enzymatic assay. Measurement of tHCY from 45 samples can be completed in less than 1 h in a single 96-well plate. Multiple plates can be analyzed simultaneously without much extra effort, enabling thousands of samples to be measured in 1 day in one laboratory.
Acknowledgments
This research was supported in part by NIH SBIR Grant 1R43HL63263-02.
References
,-lyase. Protein Expr Purif 1998;14:267-274.[Web of Science][Medline]
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