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Technical Briefs |
1
Institute of Biological Chemistry & Nutrition, University of Hohenheim, Garbenstrasse 30, 70593 Stuttgart, Germany
a author for correspondence: fax 49-711-4592283, e-mail kkuhn{at}uni-hohenheim.de
Cellular glutathione homeostasis depends on a complex process of precursor amino acid uptake, synthetic enzymatic capacity, and redox cycling of the oxidized tripeptide to its reduced state (1). Appropriate assessment of cellular glutathione status should include measurement of concentrations of the precursor amino acids and dipeptides involved in glutathione homeostasis.
The use of the fluorogenic reagent ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) previously has been restricted to the quantification of homocysteine in plasma and serum (2)(3)(4) as well as thiols in urine (5). For the measurement of biologically important thiols, SBD-F possesses major advantages: high reactivity with thiol compounds, a low detection limit, high stability of the derivatives, and lack of native fluorescence or fluorescent byproducts (2)(3)(6). Previously reported methods using electrochemical detection (7)(8)(9) required that samples be analyzed within 2–12 h of processing. HPLC methods using derivatization of thiols with monobromobimane (10), N-(1-pyrenyl)maleimidine, or o-phthaldialdehyde (11) were used to measure intracellular thiols in muscle biopsies and in cell cultures, respectively. Apparently, these methods are less suitable in biological matrices because the sensitivities of the N-(1-pyrenyl)maleimidine and monobromobimane methods, respectively, merely facilitate measurements of Cys and glutathione (10)(11), whereas in the isocratic o-phthaldialdehyde method serious interferences invalidate separation of overlapping peaks (11).
In the present work, we report a routinely manageable, sensitive HPLC
method for the separation of glutathione, its precursor amino acid Cys
as well as the dipeptides 
-Glu-Cys and Cys-Gly in small tissue
specimens.
Heart, liver, lungs, musculus tibialis anterior, and small intestine were removed from male Sprague–Dawley rats (190–240 g) between 0830 and 1100 under anesthetic [Narketan® (0.1 mL/100 g of body weight) and Xylazin® (0.08 mL/100 g of body weight)]. Specimens were snap frozen in liquid nitrogen within 2 min after removal. Sample preparation was carried out as quickly as possible at 4 °C to minimize changes in the glutathione redox status.
Human mucosal biopsies (4–18 mg wet weight) were taken endoscopically (Robert-Bosch Hospital, Stuttgart, Germany) by coloscopy or gastroscopy and immediately snap frozen in liquid nitrogen. Patients gave informed written consent, and the study protocol had been approved previously by the local ethics committee. The procedure was in accordance with the ethical standards as formulated in the revised Helsinki Declaration of 1983.
Each specimen (wet weight, 4–100 mg) was transferred to a Potter-Elvejhem glass homogenizer, extracted with 1–5 mL of ice-cold 60 g/L sulfosalicylic acid (SSA)-1 mmol/L EDTA, and rapidly homogenized while the protein was directly precipitated. After centrifugation (1500g for 5 min at 4 °C), aliquots of the clear supernatants were either derivatized and analyzed promptly or stored for stability evaluation at -60 °C for 1, 4, and 12 weeks, respectively. The precipitate was analyzed for the concentrations of protein-bound thiols.
Glutathione recovery was determined by adding known amounts of either reduced (GSH) or oxidized (GSSG) glutathione (final concentrations, 10–20 and 2.5–5 µmol/L, respectively) to the SSA solution used for homogenization. Pieces from the same tissue were extracted for analysis with SSA solutions with and without added glutathione.
Reduction (n-tributylphosphine; Sigma) and derivatization (SBD-F; Fluka) of free thiol compounds were performed as described previously (3). We could confirm the stability of the SBD-F derivatives for 24 h at 4 °C (autosampler temperature). A new approach was that additional measurements were performed for each sample by omitting the thiol-reducing step with n-tributylphosphine, thus yielding the fraction of reduced thiols. In the reduction of disulfide bonds, dithiothreitol has been a widely used reagent (10)(11). However, in combination with the SBD-F method, n-tributylphosphine should be the reducing agent of choice. Indeed, the reaction of dithiothreitol with SBD-F yields fluorescent byproducts that interfere with the HPLC separation and detection of the physiologic thiols.
The chromatographic system was a Merck HPLC system with an
L-7100 HPLC pump, a D-7000 interface, an L-7360 column oven (set at
20 °C), an L-7250 autosampler with a Rheodyne injection valve
(20-µL loop), and an L-7480 fluorescence detector (385 nm
excitation/515 nm emission). Data analysis was performed using Hitachi
D-7000 HPLC System Manager software. To minimize the matrix-related
errors of the original SBD-F method, we introduced a gradient elution
chromatography, which allowed greater flexibility to adapt the system
to a variety of tissue matrices: 0 min, 6% B; 8 min, 7% B; 9 min,
60% B; 14 min, 80% B; 15 min, 100% B; 22 min, 100% B; 24 min, 6%
B; 30 min, 6% B. Elute A was methanol-phosphate buffer (0.1 mol/L, pH
6; 2:98, by volume), and elute B was methanol-water (50:50, by volume).
Routinely, single-point calibration was achieved by comparison with an
external calibration solution [10 µmol/L Cys, 5 µmol/L -Glu-Cys
(Kyowa Hakko), 2.5 µmol/L Cys-Gly (Bachem), 50 µmol/L glutathione]
run every 8–10 samples. In Fig. 1
, typical chromatograms depict the separation of four thiols
from rat cardiac muscle.
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The "within-run" imprecision of the method evaluated by the
relative standard deviation (RSD) was assessed from replicate HPLC
analyses of 20 calibrators derivatized separately and was 3.2%
for Cys, 2.2% for -Glu-Cys, 2.9% for Cys-Gly, and 2.0% for
glutathione.
The "within-day" variation in tissue samples (muscle, liver,
mucosa) was assessed by the RSD from duplicate analyses on 10
consecutive days. The RSD was <2% for both free and reduced
glutathione; for the free and reduced thiols, respectively, the RSD was
2.3–2.9% and 4.6–6.6% for Cys, 4.8–10.1% and 4.9–18.6% for
-Glu-Cys, and 6.2–18.0% and 4.5–11.9% for Cys-Gly.
Calibration curves based on 12 duplicate analyses of the calibrators
(Cys, 0.2–44 µmol/L; -Glu-Cys, 0.1–22 µmol/L; Cys-Gly,
0.05–11 µmol/L; glutathione, 1–220 µmol/L) on 5 consecutive days
showed excellent linearity of the response in the range of tissue
concentrations for all thiol compounds; correlation coefficients
(r) were always 
0.999. The variations of the slopes were
2.8% for Cys, 1.5% for -Glu-Cys, 2.0% for Cys-Gly, and 1.1% for
glutathione.
The detection limits (signal-to-noise ratio of 3:1) were 0.3
pmol for Cys, 0.2 pmol for -Glu-Cys, and 0.1 pmol for Cys-Gly and
glutathione. In the present study, the modified SBD-F method
facilitated a 10- to 20-fold increase in sensitivity compared with the
monobromobimane method, thus allowing satisfactory and highly
reproducible measurements of glutathione and Cys in tissue specimens of
~5 mg, such as endoscopic intestinal biopsies, whereas for
measurements of dipeptides, 20 mg (liver, mucosa) to 60 mg (muscle) was
required. Endoscopic techniques are less invasive and time-consuming
than conventional surgical procedures, allowing rapid sample handling,
thereby minimizing ischemia-induced changes in glutathione status.
The tissue concentrations of thiol compounds are given in Table 1
. GSH ranged from 94% of free tissue glutathione in intestinal
mucosa up to 98% in the heart; the amount of protein-bound glutathione
in precipitated protein was <0.1%. Indeed, the values for tissue
distribution of glutathione are in good agreement with earlier reported
data (12). The mean recoveries of GSH and GSSG were 91–98%
of the original value except in the heart samples, where an average of
88% of the amount of GSSG added was recovered as SBD-F derivatives.
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Stability of thiol compounds during prolonged storage of the
deproteinized tissue homogenates is a prerequisite for reliable
analysis in clinical and experimental setting. Previously, it had been
shown that the glutathione content in kidney SSA homogenates was
comparable to original measurements within a 10% error after storage
at -70 °C for 12 months (13). We were able to confirm
the stability of glutathione for up to 12 weeks at -60 °C, with
concentrations of free glutathione and GSH assessed after 12 weeks
ranging from 92% to 106% of the original value. Tissue Cys
concentrations showed greater variability; they were decreased by
8–23% after storage for 12 weeks. The considerable variations
observed in the repeatedly measured concentrations of the dipeptides
-Glu-Cys and Cys-Gly suggest that their reliable assessment
necessitates immediate SBD-F derivatization.
Acknowledgments
We gratefully acknowledge financial support from Fresenius-Kabi, Bad Homburg, Germany. We thank S. Cvek and M. Wolter for excellent technical assistance, are indebted to Prof. Dr. H-C. Bode, Robert Bosch Hospital, Stuttgart, for providing us with the human mucosal biopsy specimens.
References
-glutamylglutathione and other low-molecular mass biological thiol compounds by isocratic high-performance liquid chromatography with fluorimetric detection. J Chromatogr B 1998;708:285-289.
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