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Technical Briefs |
1
Clinical Chemistry,
2
Neurology,
3
Nephrology, and
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Internal Medicine, University Hospital, S-221 85 Lund, Sweden;
a author for correspondence: fax 46-46189114, e-mail anders.andersson{at}klinkem.lu.se
Plasma thiols have been the object of growing interest because numerous studies have indicated that even a mild degree of hyperhomocysteinemia is associated with an increased risk of developing occlusive vascular diseases (1)(2)(3)(4)(5). However, the mechanism behind the vascular injuries is still unknown. Studies of the possible pathogenetic mechanism of increased plasma homocysteine concentrations are difficult because little is known about the mechanism for the formation of different homocysteine species in vivo.
We recently published a method that measures reduced and total fractions of homocysteine, cysteine, glutathione, and cysteinylglycine(6). In a preliminary study, we found that patients suffering from stroke have hyperhomocysteinemia, whereas their reduced homocysteine was within the health-related reference interval(7). We hypothesized that the increased concentrations of the oxidized forms of homocysteine in plasma were attributable to a hyperoxidative state in the plasma. We also observed (8) that patients suffering from renal failure had concentrations of reduced homocysteine within the reference interval despite increased total homocysteine. The redox state of homocysteine in plasma may be influenced by other thiols (9), such as glutathione, which is involved in maintaining the intracellular thiols in reduced form.
In the present study, we therefore investigated the relationships
between homocysteine, cysteine, and glutathione in 29 healthy subjects
and 15 patients with renal or liver failure. We used a newly developed
preparation procedure, especially designed to minimize several known
pitfalls that frequently influence plasma glutathione determinations.
Increased hemolysis during sample collection causes falsely increased
plasma glutathione measurements because of the high glutathione
contents in the blood cells. Plasma glutathione also decreases with
time because of the activity of 
-glutamyltransferase in
plasma. Furthermore, reduced glutathione disappears within minutes in
cell-free plasma because of oxidation(7)(10)(11)(12). In the present study, we also
determined thiols in whole blood and hemoglobin in plasma.
Specimens were collected from nine anuric hemodialysis patients (seven men and two women; mean age, 76 years; range, 55–79 years). The renal diagnoses were polycystic kidney disease (n = 2), diabetic nephropathy (n = 2), glomerulonephritis (n = 2), nephrosclerosis (n = 1), bilateral nephrectomy attributable to cancer (n = 1), and light chain nephropathy (n = 1). No patient had clinical signs of heart failure or respiratory insufficiency, and there was no laboratory evidence of liver dysfunction in any of the cases. The subjects received daily supplementation with 5 mg of folic acid and 5 mg of pyridoxine.
Specimens were also collected from six male patients with liver disease (mean age, 56 years; range, 28–74 years). All of the patients had advanced liver disease with cirrhosis, one because of autoimmune hepatitis, and the others because of alcohol. None of the patients had pulmonary diseases or impaired renal function.
After the exclusion of two individuals because of increased sample collection hemolysis (353 and 2670 mg hemoglobin/L plasma), 29 apparently healthy individuals (14 men and 15 women; mean age, 64 years; range, 41–87 years) participated in the study. Their ages did not differ significantly from the age of the patients with renal or liver failure. Eight apparently healthy individuals participated in the comparison of the present method with the previously described method(6).
Blood was drawn (with a tourniquet applied) into an EDTA-Vacutainer Tube® that had been prechilled in ice water. Immediately (within 10 s) after sample collection, the tube was placed in ice water and chilled for 6 min. During the cooling period, 50 µL of blood was mixed with 450 µL of 33 g/L sulfosalicylic acid and placed at -70 °C for later analysis of reduced thiols in whole blood. After the cooling period, another aliquot of blood was centrifuged for 3 min at 10 000g, and 1.2 mL of the plasma was transferred to a preprepared tube containing 0.3 mL of 150 g/L sulfosalicylic acid, 20 mmol/L L-serine, and 20 mmol/L sodium tetraborate. The acidified plasma was mixed and stored at -70 °C and analyzed within 4 days. The remaining plasma was stored at -70 °C for later determination of hemoglobin in plasma by derivative spectroscopy (13).
To determine total thiols in plasma, 500 µL of thoroughly mixed acidified plasma was neutralized with 1 mol/L NaOH to pH 7–8, mixed with 75 µL of 0.1 mol/L dithiothreitol, and incubated at room temperature. After 15 min, the solution was acidified with HCl (1 mol/L) and centrifuged for 3 min at 10 000g. The supernatant was applied to HPLC as described previously (6). The remaining acidified plasma was centrifuged for 3 min at 10 000g. The supernatant was used for the determination of reduced thiols as described previously (6). For the determination of reduced thiols in whole blood, the tube with acidified blood was thawed and centrifuged for 3 min at 10 000g, and 20 µL of the supernatant was applied to the HPLC.
The Student independent t-test was used to test whether differences between the groups of patients and the healthy individuals were significant. Linear regression analysis was used to obtain correlation coefficients.
To study the influence of -glutamyltransferase on the measured
glutathione concentration, we assayed plasma samples (n = 8),
using a preparation containing serine/borate as described above and a
preparation without serine/borate according to the previous method(6). Plasma samples were mixed with the preparations 2
h after the sample collections and assayed the same day by HPLC. Under
these conditions, the mean total glutathione measured with the original
method was 53% lower than the total glutathione measured with the
modified method (P <0.001), whereas the other thiols
showed similar values when assayed with the different methods.
The concentrations of plasma hemoglobin (29–168 mg/L) in all 44 individuals did not show any significant correlation (r = 0.24; P = 0.15) to reduced plasma glutathione. When the samples were divided into one group with low (29–74 mg/L; n = 22) and one with high plasma hemoglobin (76–168 mg/L; n = 22), the plasma concentration of reduced glutathione did not differ (P = 0.11). Therefore, we consider that hemolysis corresponding to a plasma hemoglobin concentration <170 mg/L had no significant influence on the plasma concentration of glutathione.
Reported plasma concentrations in healthy subjects showed large variability, 0.34–31.7 µmol/L, when total glutathione was assayed by use of HPLC(6)(7)(12)(14)(15)(16)(17)(18) or colorimetric methods (19)(20) and when reduced glutathione, 1.9–6.9 µmol/L, was assayed by HPLC(7)(12)(14)(17)(21)(22). Our modified preparation procedure improves the reliability of the method, and the values obtained for reduced and total thiols are similar to those that we (6)(7) and others(17) have observed previously in healthy individuals.
Patients with renal failure showed decreased plasma concentrations of
reduced cysteine and reduced homocysteine but increased concentrations
of the total forms of these thiols compared with the healthy
individuals (Table 1A
). Both the decreased concentration of reduced plasma
homocysteine and the increased concentration of total plasma
homocysteine could be attributed to increased prooxidant activity in
plasma from these patients, which might lead to a rapid oxidation of
reduced homocysteine to disulfides. Homocysteine bound to plasma
proteins through disulfide bonds is not as metabolically available as
homocysteine in low-molecular weight disulfides or in its reduced form
and therefore accumulates in the circulation (23). Another
possible explanation for the decreased concentration of reduced plasma
homocysteine is a lowered intracellular production of reduced
homocysteine because of a more efficient metabolism of homocysteine
caused by vitamin supplementation. All investigated thiols showed
decreased reduced/total ratios in patients with renal failure compared
with the healthy individuals (Table 1
), which supports previous
findings of an impaired redox status in plasma from these patients(8)(22)(23)(24)(25)(26)(27).
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Patients with liver failure showed significantly lower concentrations
of reduced glutathione in whole blood (Table 1B
) and in plasma (Table 1A
) compared with healthy individuals.
The total plasma homocysteine concentration was increased, whereas the
concentrations of total glutathione and total cysteine in plasma were
not significantly changed. The reduced/total ratios of thiols in plasma
were significantly decreased for glutathione and homocysteine (Table 1A
). The decreased whole blood concentration of reduced glutathione in
this study and in others (28)(29) might
be attributable to lower hepatic production of glutathione in liver
disease. The decreased reduced/total ratios and the increased total
homocysteine concentration in plasma may indicate an increased
oxidative activity, which previously has been reported to be present in
plasma from patients with liver disease (30).
In the healthy individuals, reduced glutathione in whole blood showed a correlation with reduced cysteine in blood (r = 0.50; P <0.01) and in plasma (r = 0.36; P = 0.05), but no correlation to any other analyte or ratio. The total plasma concentration of glutathione correlated (r = 0.84; P <0.001) with reduced plasma glutathione, but neither total nor reduced plasma glutathione correlated with any form of homocysteine, cysteine, or their ratios. These findings show that glutathione does not influence the redox status of homocysteine or cysteine and suggest that glutathione is not an exclusive reductant in plasma. The concentration of reduced cysteine in whole blood showed a correlation with reduced plasma cysteine (r = 0.66; P <0.001), reduced plasma homocysteine (r = 0.44; P <0.05), and with the reduced/total ratios for plasma cysteine (r = 0.60; P <0.001) and for plasma homocysteine (r = 0.47; P <0.01). Furthermore, reduced plasma cysteine correlated to reduced homocysteine (r = 0.83; P <0.001). The reduced/total ratio of plasma cysteine correlated with that of homocysteine (r = 0.90; P <0.001), with the reduced form of homocysteine in plasma (r = 0.65; P <0.001), and inversely with the total forms of cysteine (r = -0.65; P <0.001) and homocysteine (r = -0.47; P <0.01) in plasma. These findings suggest that reduced cysteine plays a role, at least partly, in the maintenance of the redox status in plasma.
References
The following articles in journals at HighWire Press have cited this article:
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  S. Li and A. R. Whorton Functional characterization of two S-nitroso-L-cysteine transporters, which mediate movement of NO equivalents into vascular cells Am J Physiol Cell Physiol, April 1, 2007; 292(4): C1263 - C1271. [Abstract] [Full Text] [PDF]
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 T. Seres, R. G. Knickelbein, J. B. Warshaw, and R. B. Johnston Jr. The Phagocytosis-Associated Respiratory Burst in Human Monocytes Is Associated with Increased Uptake of Glutathione J. Immunol., September 15, 2000; 165(6): 3333 - 3340. [Abstract] [Full Text] [PDF]
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