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Articles |
Departments of
1
Pathology and
2
Neurology, and
3
Department of Human Nutrition and Dietetics, University of Illinois at Chicago Medical Center, 840 South Wood Street, 201G CSB, Chicago, IL 60612.
aAddress correspondence to this author at: Quest Diagnostics, Inc., Laboratory Administration, 4225 East Fowler Ave., Tampa, FL 33617. Fax 813-978-3987; e-mail Robert.H.Williams{at}Questdiagnostics.com.
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Abstract |
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Methods: We measured aminothiols and their stability in stabilized protein-free filtrate using acidic sodium citrate (BioPool® StabilyteTM, pH 4.3) vs EDTA whole blood. Before analysis, plasma samples were also ultrafiltered to obtain a protein-free filtrate. The concentrations of total Hcy (tHcy), fHcy, and rHcy and their related aminothiols, cysteine, cysteinylglycine, and glutathione were simultaneously determined on acidic sodium-citrated blood using reversed-phase HPLC with fluorescence detection. Bound and oxidized aminothiols were calculated by difference using the concentrations of the total, free, and reduced fractions. Using this approach, we compared the redox status in newly diagnosed ischemic stroke patients (n = 20) and healthy age- and sex-matched subjects (n = 20).
Results: tHcy, tCys, tCysGly, and tGSH concentrations in whole blood with Stabilyte were stable for 8 h; the reduced fraction of each aminothiol was stable for 4 h. Recovery in the protein-free filtrate was 90–100% for all reduced thiols in acidified sodium-citrated blood. Patients with ischemic stroke had higher plasma tHcy, fHcy, bHcy, rHcy, and oxHcy (P <0.0005) and higher plasma t-, f-, r-, and oxCys (P <0.05). t-, b-, and rCysGly concentrations were lower in the stroke patients (P <0.05), as were t-, b-, and oxGSH (P <0.005).
Conclusions: Collection of blood in acidic sodium citrate (BioPool Stabilyte) permits the determination of the redox status of Hcy and its related aminothiols, which may add to the understanding of their relationship to the etiology of cerebrovascular disease.
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Introduction |
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Plasma total Hcy (tHcy) consists of various forms: a protein-bound fraction (70–80%), free oxidized (disulfide) forms (20–30%), and free reduced (or sulfhydryl) forms (<3%); disulfide forms include Hcy-Hcy disulfides (homocystine) and Hcy-Cys mixed disulfides (1)(4)(5). The rapid oxidation and redistribution of the various Hcy species do not influence tHcy in fresh plasma. Such changes can occur, however, after a delay in the centrifugation and separation of whole blood (6) or with inconsistency in the choice of an anticoagulant (7)(8)(9)(10). These preanalytical factors can produce a spurious increase of tHcy or misinterpretation of Hcy status because of the rapid oxidation of the reduced species that can occur if fractionation of the various Hcy species is to be determined as part of the overall "redox" status of plasma (4). Thus, most routine clinical studies are based on the measurement of tHcy.
Simultaneous measurement of other plasma aminothiols along with Hcy may be of clinical interest because many of them are metabolically related (11). Because aminothiol fractions associated with plasma proteins are probably not biologically active (12), the free forms (reduced and oxidized) may be more likely to play a role in the pathogenesis of disease. Increased free plasma Hcy and Cys have been reported in patients with renal failure (13) and in patients with cerebral infarction (14).
The concentrations of those aminothiol fractions evaluated in plasma redox status [reduced (r)-, free (f)-, oxidized (ox)-, and protein-bound (b)- fractions of Hcy, Cys, cysteinlyglycine (CysGly), and glutathione (GSH)] also have been analyzed to ascertain their role in vascular disease (14)(15)(16). Perturbations in the redox status have been reported in patients with homocystinuria (12), cerebral infarction (16), peripheral vascular disease (15), and renal disease (13). Methods for determining the redox status have been developed by Mansoor et al. (17) and Andersson et al. (18), but sample handling is complex and special reagents are required. Both approaches are labor-intensive and impractical for a clinical setting, especially for large-scale epidemiologic studies or clinical trials.
Without special handling, samples with anticoagulants such as heparin and EDTA are unreliable because of the rapid oxidation of the thiols. BioPool® StabilyteTM (acidic sodium citrate, pH 4.3) is reportedly effective in maintaining tHcy and other aminothiol concentrations in whole blood for up to 8 h after collection (8)(9)(10).
A strong relationship exists between ischemic stroke (19)(20) and hyperhomocysteinemia (21)(22)(23), and plasma tHcy is correlated with carotid artery intimal wall thickening (24) and extracranial carotid artery stenosis (25). The biochemical role of Hcy is unclear.
The present study is designed to determine the effectiveness of acidified citrate (Stabilyte) in maintaining the stability of the various fractions of Hcy and related aminothiols and to examine those components involved in redox status and their relationship to plasma albumin and vitamin status in ischemic stroke patients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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99%), 5-sulfosalicylic acid dihydrate, dimethyl
sulfoxide (GC grade), and acetonitrile (HPLC grade) were purchased from
Sigma-Aldrich. Hydrochloric acid, nitric acid, and ammonium hydroxide
(Optima grades), glacial acetic acid (HPLC grade), and sodium hydroxide
(ACS certified) were purchased from Fisher Chemical Company.
Monobromobimane (Thiolyte-MB®) was purchased
from Calbiochem-Novabiochem. HPLC-grade water was prepared using a
MilliQTM System (Millipore Corporation). The
analytical column [150 x 4.6 mm; Prodigy®
C-18 (ODS); 3 µm particle size] and the guard column [30 x
4.6 mm; C-18 (ODS); 5 µm particle size] were obtained from
Phenomenex, Inc. Amicon Centrifree®
micropartition filters (molecular weight cutoff, 30 000) were
purchased from Amicon Bioseparations, Millipore Corporation.
Preparation of reagents.
Reagents were prepared as described
previously (26) for total and free thiol concentrations,
with the exception of the ammonium formate–nitrate buffer. This
solvent was prepared by the addition of 3.0 mL of formic acid and 4.0
mL of nitric acid to 1900 mL of HPLC-grade water followed by the
addition of ammonium hydroxide to bring the pH to 3.65 (for total and
free thiol analysis) or 3.50 (for reduced thiol analysis). HPLC-grade
water was used to adjust the final volume to 2000 mL.
Preparation of aminothiol calibrators.
A multiconstituent
calibration solution consisting of D,L-Hcy (640 µmol/L),
L-Cys (4800 µmol/L), L-CysGly (1500
µmol/L), and GSH (600 µmol/L) was prepared by dissolving 8.7 mg of
Hcy, 58.6 mg of Cys, 26.7 mg of CysGly, and 18.4 mg of GSH in 100 mL of
0.1 mol/L hydrochloric acid containing 100 mmol/L dithioerythritol.
Six-point calibration curves were constructed by diluting the mixed
working solution to obtain the following concentrations for each
aminothiol: Hcy, 2, 4, 8, 16, 32, and 64 µmol/L; Cys, 15, 30, 60,
120, 240, and 480 µmol/L; CysGly, 4.675, 9.375, 18.75, 37.5, 75, and
150 µmol/L; and GSH, 1.875, 3.75, 7.5, 5, 30, and 60 µmol/L. This
solution was then derivatized as described below. For reduced
aminothiol determination, the multiconstituent working calibration
solution was further diluted with 0.1 mol/L hydrochloric acid
containing 100 mmol/L dithioerythritol to obtain six calibrators with
the following concentrations: Hcy, 0.125–4.0 µmol/L; Cys, 1.25–40.0
µmol/L; CysGly, 0.293–9.375 µmol/L; and GSH, 0.117–3.75 µmol/L.
The calibrators were derivatized using the procedure described below
for reduced thiol analysis. To confirm the lower and upper limits of
linearity, calibrators for total aminothiols were prepared in the same
manner as above at the following concentrations: Hcy, 0–250 µmol/L;
Cys, 0–1000 µmol/L; CysGly, 0–200 µmol/L; and GSH, 0–200
µmol/L. For reduced aminothiols, the concentrations were as follows:
Hcy, 0–50 µmol/L; Cys, 0–200 µmol/L; CysGly, 0–50 µmol/L; and
GSH, 0–5 µmol/L.
HPLC
Aminothiols were measured with a Waters LC Module-1 (comprising a
model 600 ternary solvent delivery system and a model 715 autosampler)
in conjunction with a Waters 474 fluorometer (Waters Corp.) using
excitation and emission wavelengths of 365 and 475 nm, respectively.
Column temperature was maintained at 50 °C. Solvent gradient and
chromatographic separation, integration, and quantification were
controlled by Waters Millennium 32 Chromatography Software.
subjects
Study participants.
Twenty patients recently diagnosed with
ischemic stroke at the Stroke Service of the University of Illinois at
Chicago Medical Center were enrolled in the study. Patients were not
receiving dietary supplements of vitamins B6,
B12, or folate. Blood from all patients was
obtained in a fasting state before commencement of vitamin therapy.
Control subjects.
Twenty healthy adult volunteers were
recruited as control subjects and were age- and sex-matched
approximately to the study population. Subjects were fasting and free
from all medications, including vitamin and nutritional supplements for
the past 30 days, as determined by medical interview.
Sample collection and storage for study participants and control
subjects.
Blood was collected by venipuncture from study
participants and control subjects into evacuated tubes containing
BioPool Stabilyte (0.5 mol/L acidic citrate, pH 4.3; Biopool
Laboratory). For total aminothiols, blood was centrifuged
(2000g for 10 min at 4 °C) within 30 min of collection to
obtain plasma, which was aliquoted and then stored at -70 °C and
analyzed within 1 week. For reduced aminothiols, an initial stability
study was conducted with blood collected in BioPool Stabilyte. Samples
were analyzed immediately after collection and then after storage at
-70 °C for 2 weeks. This timeframe was chosen on the basis of
previous studies using acidified protein-free plasma (13).
Results were compared with the use of a paired-sample t-test
to determine whether any significant differences occurred in reduced
aminothiols during storage over this time period.
sample analysis
Derivatization procedures.
Free aminothiols were determined by
adding plasma to Amicon Centrifree Micropartition Devices followed by
centrifugation (20 min at 2000g) to obtain ultrafiltrates. A
sample of each ultrafiltrate (30 µL) was pipetted into a 2-mL amber
glass vial and derivatized as described previously for plasma total
thiols (26). Reduced aminothiols were measured by the
addition of 210 µL of blood collected in Stabilyte to a conical
centrifuge tube, followed by 10 µL of 1.5 mol/L ethylmorpholine (pH
9.5), 10 µL of isotonic saline, and 20 µL of monobromobimane (25
mmol/L in acetonitrile). After incubation at room temperature
(20 °C) for 3 min, derivatization was stopped by the addition of 25
µL of 500 g/L sulfosalicylic acid. The precipitated samples were
vortex-mixed for 10 s and centrifuged at 10 000g for 5
min to obtain the supernatant. Before injection into the HPLC, 45 µL
of the supernatant was pipetted into amber glass vials, to which 20
µL of 5.0 mmol/L EDTA, 100 µL of 1.5 mol/L ethylmorpholine, 100
µL of HPLC-grade deionized water, and 20 µL of glacial acetic acid
were then added. Oxidized aminothiols (free aminothiols - reduced
aminothiols) and bound aminothiol concentrations (total aminothiol
- free aminothiols) were calculated by difference.
Chromatography.
Derivatized samples prepared for total and
free aminothiols (10 µL) and reduced aminothiols (20 µL) were
injected onto a Prodigy ODS analytical column (150 x 4.6 mm)
maintained at 50 °C and preconditioned with an ammonium
formate–ammonium nitrate buffer for 30 min. Aminothiols were eluted
from the column at a flow rate of 2.0 mL/min using a linear gradient of
acetonitrile (from 0–10.5% in 11 min) in the same buffer.
Vitamin analysis.
Plasma concentrations of vitamin
B12 and folate were determined by a microparticle
sandwich immunoassay using a Bayer IMMUNO-1®
Analyzer (Bayer Diagnostics); vitamin B6
(pyridoxal-5'-phosphate) was measured using a modified radioenzymatic
tyrosine decarboxylase method (27).
Albumin and creatinine analysis.
Plasma albumin and creatinine
were determined on a Beckman Synchron® CX-7
analyzer (Beckman Instruments).
Recovery study: rHcy in ultrafiltrate.
Protein-free
ultrafiltrate (25 mL) was obtained from pooled serum by ultrafiltration
using a YM-30 Amicon Millipore Ultrafiltration Membrane (molecular
weight cutoff, 30 000) and a Millipore Stirred Ultrafiltration Cell,
Model 8050 (Amicon Bioseparations) under positive pressure (<75 mmHg
nitrogen). Four-milliliter aliquots were added to BioPool Stabilyte
collection tubes, and 3-mL aliquots were added to EDTA collection tubes
(to account for differences in anticoagulant dilution). Both Stabilyte
and EDTA aliquots, along with unmodified ultrafiltrate, were used as
diluents at room temperature (18–23 °C) for the recovery study. A
multiconstituent stock solution of aminothiols was prepared using the
three aforementioned diluents at the following concentrations: Hcy and
GSH, 1.25, 2.5, 5.0, and 10.0 µmol/L; Cys, 25, 50, 100, and 200
µmol/L; and CysGly, 5.0, 10.0, 20.0, and 40.0 µmol/L. Calibrators
were immediately derivatized and analyzed for reduced aminothiols using
HPLC as described above.
Stability study: total and reduced aminothiols in whole blood.
Five paired samples of whole blood were collected from nonfasting
volunteer subjects (n = 5) into EDTA and Stabilyte. Five
whole-blood aliquots were prepared for total and reduced aminothiol
determinations at baseline and at 2-h intervals up to 8 h. Samples
were stored at room temperature (18–23 °C) until analysis was
performed for each time interval. Total and reduced aminothiols were
analyzed in duplicate as described above.
statistical analysis
For total and reduced aminothiol stability studies, a two-tailed
t-test was used to identify significant changes in results
from baseline; P <0.05 was considered significant.
Comparison of thiol fractions and other variables between the ischemic
stroke patients and control subjects was assessed by one-way ANOVA at a
significance of P <0.05. When linear transformation did not
normalize the distribution of the data, the Mann–Whitney
U-test was used to assess the data.
Analyse-It® Statistical Software (Ver. 1.44) was
used for all statistical analyses.
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Results |
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; the retention time windows for Cys, CysGly, Hcy, and GSH were
8.80 ± 0.20, 9.60 ± 0.15, 10.80 ± 0.12, and
11.25 ± 0.20 min, respectively.
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stored plasma stability study
Plasma concentrations (µmol/L) of reduced aminothiol obtained
from blood collected in Stabilyte (n = 5) and analyzed immediately
after sample collection and preparation and 2 weeks after storage at
-70 °C were compared using a paired-sample t-test
(P <0.05 was considered significant). No significant
difference was observed for rHcy (P = 0.580), rCys
(P = 0.667), rCysGly (P = 0.560), or
rGSH (P = 0.1835).
reduced thiol recovery study
The recoveries for reduced thiols in protein-free filtrates
prepared from unmodified serum and serum initially added to collection
tubes containing the anticoagulants Stabilyte and EDTA are given in
Table 1
. Depending on the initial concentration of the reduced thiol,
the recovery was 91–99.7% when Stabilyte was the anticoagulant. With
EDTA as the anticoagulant, the recovery of rHcy was 61–69% and
55–68% in the filtrate from unmodified serum. A similar recovery
pattern is also noted for the other aminothiols (Table 1
). The rapid
oxidation of the thiols was evident by the low recovery in the more
neutral pH medium (EDTA-treated filtrate, pH 5.9; untreated filtrate,
pH 7.2), whereas oxidation appeared to be prevented in the
Stabilyte-treated filtrate (pH 4.5), as evidenced by higher recovery.
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whole blood stability study
The stability of tHcy at room temperature in whole blood is shown
in Table 2
. An increase in tHcy was apparent within 2 h of collection
in EDTA-anticoagulated whole blood, whereas the blood collected in
Stabilyte showed very little change until 6 h of incubation (Table 2
). rHcy also remained stable for 4 h in whole blood collected in
Stabilyte (Table 3
). The effects of aminothiol oxidation were evident in
EDTA-anticoagulated whole blood because mean rHcy concentrations
at baseline (0.121 µmol/L; n = 5) were 35% of the
concentration shown in Stabilyte, and they approached the lower limits
of analytical detection (0.04 µmol/L) within 4 h. tCys was
stable for up to 8 h in Stabilyte (Table 2
), and rCys in Stabilyte
was stable for 4 h (Table 3
). Similar patterns were shown for
tCysGly and tGSH (Table 2
) and rCysGly and rGSH (Table 3
). All total
aminothiols in whole blood collected in Stabilyte were stable at room
temperature (18–23 °C) for up to 8 h, with the exception of
CysGly, which became significantly increased at 8 h of incubation.
All total aminothiols collected in EDTA-anticoagulated whole blood
showed significant increases at room temperature (18–23 °C) within
4 h of collection; tCysGly and tGSH were increased within 2 h
(Table 2
). A minimum stability of 4 h at room temperature
(18–23 °C) was achieved in Stabilyte for all reduced thiols;
rCysGly and rGSH were stable up to 8 h. Baseline results for
reduced thiols in EDTA were consistently and significantly lower
(P <0.001) than that of Stabilyte. Decreases among all
reduced thiols were further evident within 2 h of incubation
(Table 3
).
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plasma aminothiol components and redox status in patients with
ischemic stroke and in control subjects
All measured and calculated fractions of plasma Hcy were
significantly higher in the ischemic stroke study group (Table 4
) compared with control subjects. tCys, fCys, rCys, and oxCys
were also significantly higher in stroke patients. Lower aminothiol
fractions seen in the stroke group included tCysGly, bCysGly, rCysGly,
tGSH, bGSH, and oxGSH. No apparent differences were shown between
stroke patients and control subjects for bCys, fCysGly, oxCysGly, fGSH,
or rGSH. Among the other variables measured, only vitamin
B6 was significantly lower (P =
0.005) in the stroke group (Table 5
). No differences were seen for vitamin
B12, folate, albumin, and creatinine.
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Discussion |
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0.070–0.30 µmol/L) and its rapid oxidation after
sample collection (17). The method of Mansoor and co-workers
(12)(17) requires the collection of blood into
evacuated tubes containing monobromobimane, in which the reduced
aminothiols combine with monobromobimane to form fluorescent adducts
(12). This method also uses multiple tubes of blood to
measure the reduced, oxidized, and total aminothiol fractions, and it
requires immediate centrifugation and processing (17). The
method of Andersson and co-workers (16)(18) also
requires special handling and rapid processing of the sample, along
with the addition of sulfosalicylic acid to the separated EDTA plasma.
Collection of blood in Stabilyte permits rapid analysis of total, free,
and reduced aminothiols without the need for immediate derivatization
or sample preparation. Given the different approaches for aminothiol
analysis, our results for reduced aminothiols in healthy subjects
compare closely with those of previous studies (16)(17)(18).
Combining the data for male and female subjects, Mansoor et al.
(17) showed a mean rHcy concentration of 0.24 µmol/L
(compared with 0.18 µmol/L in our combined control group) and a mean
rCysGly concentration of 3.05 µmol/L (compared with 2.79 µmol/L in
our study). Identical or similar findings were also reported by
Andersson and co-workers (16)(18), who obtained
a rHcy concentration of 0.18 µmol/L and a rCysGly concentration of
2.40 µmol/L. Free aminothiols were also comparable to previous
studies (13)(16).
Although both anticoagulants can chelate metals (especially copper
ions) that can enhance autooxidation of aminothiols, this process is
enhanced when the pH is at or near the pK of the sulfhydryl
group (29). Given the pKa
of the sulfhydryl group of Hcy (8.66), the use of Stabilyte appears to
provide a more acidic medium for whole blood (pH 5.5–5.9) than does
EDTA (29). Willems et al. (9) have shown that
tHcy obtained from blood collected in Stabilyte has a higher plasma
baseline concentration (at time zero) than blood collected in EDTA,
regardless of storage temperature (0 °C or room temperature). In
addition, the reduced fractions of the aminothiols are highly unstable.
Andersson et al. (16) determined the mean half-lives of the
following aminothiols in nonacidified EDTA plasma incubated at 4 and
22 °C, respectively: rHcy, 44 and 14.3 min; rCys, 80 and 36.8 min;
rCysGly, 60 and 24.1 min; and rGSH, 38 and 11.7 min. After 2 h of
incubation, the reduced fractions of all aminothiols were <20% of the
baseline value (16). These investigators showed that rapid
loss of the reduced fractions from blood collected in EDTA can be
overcome by acidification of the plasma (16).
Although the exact mechanism by which Stabilyte maintains the
concentration of tHcy is unknown, Willems et al. (9) have
proposed that the enzymes in the erythrocyte involved in the metabolism
of Hcy are blocked at this low pH. The decreased pH may help to
stabilize the thiol (-SH) group and prevent or reduce the metabolism
and export of Hcy and other aminothiols from blood cells, i.e.,
erythrocytes. However, the increase in both total (Table 2
) and reduced
(Table 3
) aminothiols when Stabilyte was used compared with EDTA are
not explained by the decreased pH imparted by this anticoagulant, and
further experiments are necessary to explain this observation. The
smaller SE for each concentration of aminothiol in our study may be
attributable to increased stability of aminothiols in Stabilyte,
especially with the reduced fraction.
Unless specially treated or handled, tHcy can be spuriously increased if any delay occurs in processing samples collected in EDTA, heparin, or sodium fluoride (6)(16)(25). Previous studies have shown that samples collected in Stabilyte can maintain the tHcy concentration for up to 6 h after collection (8)(9)(10). Our findings corroborate those of Willems and co-workers (8)(9) and Salazar et al. (10) that Stabilyte is effective at maintaining the plasma concentrations of total aminothiols for at least 6 h at room temperature. In addition, we have shown that Stabilyte can also maintain whole blood rHcy and rCys concentrations up to 4 h and rCysGly and rGSH up to 8 h at room temperature (18–23 °C). Previous studies have shown that GSH is extremely susceptible to autooxidation and is rapidly oxidized in human plasma, even in the presence of EDTA (30)(31)(32). The use of Stabilyte appears to overcome this problem. No special handling during collection and storage is required. If analysis for reduced aminothiols cannot be performed immediately, plasma obtained from blood collected in Stabilyte can be stored at -70 °C for a minimum of 2 weeks without significant loss of any reduced fractions. With our method, reliable analysis of total aminothiols along with their free and reduced forms can be achieved using ultrafiltration and the same HPLC method and anticoagulant, which can stabilize all of these compounds (3)(8)(9). Direct measurements of total, free, and reduced Hcy, Cys, CysGly, and GSH permit calculation of the bound and oxidized thiol fractions, thus providing a means of assessing the overall plasma redox status.
In our patient study, total, free, and reduced Hcy and Cys
concentrations were all significantly increased in stroke patients
compared with the control group. With the exception of rCys, these
findings are consistent with those found previously in patients with
peripheral vascular disease (12) and renal disease
(13). Stroke patients also had significantly lower vitamin
B6 concentrations. Vitamin
B12 and folate concentrations, however, were not
significantly lower in stroke patients. Because albumin is the main
carrier protein for Hcy and the other thiols
(5)(33), serum concentrations of this protein
were measured to determine whether hypoalbuminemia contributed to the
increased fractions of free aminothiols seen with our patients.
However, no significant difference in albumin concentrations was noted
with our ischemic stroke patients compared with control subjects.
Although highly protein-bound medications can potentially displace Hcy
from albumin, thus causing an increase in fHcy, extensive review of
patient medical records did not identify a common drug that could have
played this role. In addition, most patients (16 of 20) were not taking
medications before hospitalization. Increased fHcy could also occur
with a rapid turnover of albumin, as seen in renal disease. However,
based on the creatinine results for our patients (Table 5
), this does
not appear evident. Competitive binding for albumin by another
aminothiol could displace Hcy, thus increasing its free concentration.
However, Smolin and Benevenga (34) have shown that Hcy
preferentially binds to albumin even in the presence of fCys. Rapid
export of fHcy and rHcy from erythrocytes offers another possible
explanation: Andersson et al. (6) have shown that the
erythrocytes contribute significantly to the plasma pool of Hcy with
the reduced fraction of Hcy likely the predominant intracellular
species (35). However, based on current knowledge of the
pathogenesis of ischemic stroke, there is no physiologic rationale for
such an explanation. We also observed significant decreases in tCysGly,
bCysGly, rCysGly, tGSH, bGSH, and oxGSH in our stroke study group. The
decreased fractions of CysGly may be related to the decrease in GSH
because GSH (Cys-Gly-Glu) is broken down to CysGly and Glu. Additional
experiments are necessary to explain why CysGly was decreased and why
only certain fractions of GSH were decreased in the stroke subjects.
The increase of rHcy with perturbation of the redox status in ischemic stroke patients is a novel finding. Increases in rHcy can affect the protein binding of other aminothiols and, thus, the overall redox status of plasma aminothiols (36). Such perturbations may affect cellular metabolism, which can play a role in the atherosclerotic process noted in vascular disease. Recent evidence suggests that disturbances in the concentrations of rHcy and rCys may affect cellular-mediated reactions that involve the oxidation of LDL, a key step in atherogenesis (37)(38)(39)(40). Although the oxidation of LDL is recognized as having a role in the atherosclerotic process of vascular disease, the mechanism is only partially understood.
Traditional determinations of tHcy by HPLC or automated immunoassays will fail to detect increases in fHcy or rHcy. Although routine measurement of fHcy and rHcy in the general population may be impractical, we believe that the use of our approach makes the analysis less labor-intensive. It has been suggested that those aminothiol fractions involved in the redox of human plasma should be included as part of any epidemiologic or mechanistic study of cardiovascular disease (4). Our results also suggest that measurement of other total plasma aminothiols along with their various fractions may be important in the overall assessment of hyperhomocysteinemia. Including the measurement of intracellular rHcy in addition to plasma rHcy in future studies has recently been recommended because rHcy is most likely the metabolically active form (41). Given the perturbation of the redox status noted in various pathologic conditions, intracellular measurements of the various aminothiol fractions, especially the reduced forms, may be more invaluable in assessing the role that aminothiols play in the atherosclerotic process of vascular disease.
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Acknowledgments |
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The following articles in journals at HighWire Press have cited this article:
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  A. B. Sobol, E. Bald, and J. Loba Fractions of Total Plasma Homocysteine in Patients with Ischemic Stroke Before the Age of 55 Years Angiology, March 1, 2005; 56(2): 201 - 209. [Abstract] [PDF]
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