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Articles |
1
Laboratory of Paediatrics and Neurology, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
a Author for correspondence. Fax 31-243618900; e-mail H.Blom{at}ckslkn.azn.nl
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Cells were sonicated in the presence of excess N-ethylmaleimide to prevent oxidation of cysteine to cystine and disulfide exchange reactions of cystine with available sulfhydryl moieties. Cystine was measured as cysteine after reduction with sodium borohydride and derivatization with monobromobimane, followed by separation with automated HPLC and fluorescence detection.
Results: The assay was linear to 200 µmol/L cysteine. Within-run and day-to-day (total) imprecision (CV) was <5%, and the detection limit was 0.3 µmol/L. Added cysteine, up to 200 µmol/L, was completely removed, and recovery of added cystine was 69–86%. Cystine was stable for at least 2 months in leukocytes frozen in liquid nitrogen and stored at -80 °C
Conclusions: Oxidation of cysteine to cystine and disulfide exchange reactions of cystine with sulfhydryl moieties are prevented by N-ethylmaleimide. The detection limit for the determination of cystine is adequate to measure cystine in leukocytes and cultured fibroblasts for diagnosis of cystinosis and monitoring treatment with cysteamine.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For reliable measurements of cystine in cells, three problems must be overcome: (a) The cysteine content of the cytosol, which exceeds the cystine concentration in lysosomes 15 to 100 times (as shown in this study), must be eliminated [for example, by N-ethylmaleimide (NEM)]1 before it can oxidize to cystine. (b) Disulfide exchange reactions of cystine with other sulfhydryl groups can lead to the loss of cystine and, therefore, must be prevented by elimination of the sulfhydryl groups with, e.g., NEM. (c) The method for cystine determination must have a detection limit low enough to not only enable the diagnosis of cystinosis but in particular to monitor the cystine-lowering effect of cysteamine.
Cystine may be measured by ion-exchange amino acid analysis (3), but for monitoring therapy this method falls short because of its high limit of detection; hence, a cystine-binding protein assay usually is used (4). We developed a sensitive and reproducible method for measuring the cystine in isolated mixed leukocytes and cultured fibroblasts without the use of radioactive chemicals. Free cysteine and other sulfhydryl groups in the cytosol are bound to NEM before reduction of cystine to cysteine with sodium borohydride (NaBH4) and derivatization of the thiol function of cysteine with monobromobimane, yielding a highly fluorescent derivative. The cysteine derivative is separated from other thiol-containing derivatives by HPLC and quantified by fluorescence detection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mixed leukocytes were isolated by adding 10 mL of freshly drawn blood to 2 mL of cold dextran solution (50 g/L dextran T500, 15 g/L EDTA, 7 g/L NaCl, pH 7.4), followed by gentle mixing. After at least 1 h on ice, the upper solution was diluted with an equal amount of phosphate-buffered saline (PBS), pH 7.4, and centrifuged at 600g for 10 min at 4 °C in a Sorvall RC5B plus. The pellet was suspended in 1 mL of cold PBS, and 3 mL of cold water was added for hypotonic lysis of red cells. After exactly 1.5 min on ice, 1 mL of 36 g/L NaCl solution was added and mixed, followed by centrifugation at 600g for 10 min at 4 °C. The cells were washed with 5 mL of cold PBS and centrifuged again. Finally, the pellet was suspended in 0.5 mL of cold PBS, and 50 µL was used for a differential count of the mixed cells. The remaining suspension was transferred to a 1.5-mL screw-cap Eppendorf tube and centrifuged in an Eppendorf centrifuge 5402 at 1000g for 5 min at 4 °C. The pellet was frozen quickly in liquid N2 and stored at -80 °C until determination.
Fibroblasts were cultured in EMEM with Earl’s salts and nonessential amino acids, supplemented with 100 mL/L fetal calf serum, 100 kilounits/L penicillin, and 100 mg/L streptomycin at pH 7.1. The cells were incubated at 37 °C, 100% humidity and under 5% CO2 in a 175 cm2 flask. After reaching confluency the cells were harvested by trypsinization and washed three times with cold PBS. The pellet was frozen quickly in liquid N2 and stored at -80 °C until determination.
Cell extracts were prepared by suspending the frozen pellets of
leukocytes or fibroblasts in 400 µL of NEM solution (1 mmol/L NEM in
10 mmol/L sodium phosphate buffer, pH 7.2, prepared just before use) on
ice. The cells were sonicated on ice three times for 10 s with
20-s cooling intervals. The suspension was centrifuged at
15000g for 10 min at 4 °C in an Eppendorf centrifuge
5402. The protein concentration in the supernatant was determined by
the method of Lowry et al. (5), and the cystine
concentration was measured. The principle of this method is shown in
Fig. 1
.
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The cystine determination was based on the method of Fiskerstrand et
al. (6), with some modifications (7), which
describes the determination of the thiols cysteine, cysteinylglycine,
glutathione, and homocysteine in plasma and urine. All steps are
performed by a programmable sample processor (Gilson model 232 BIO,
dilutor 401) attached to a ternary HPLC pump SP8800 (Thermo Separation)
and a Linear LC 304 fluorometer with Millennium data acquisition (Ver.
2.15.2; Millipore). The reaction vials contained 15 µL of 1 mmol/L
L-homocysteine to eliminate excess NEM. To this
reaction vial, 30 µL of cell supernatant, 30 µL of
NaBH4 solution (4 mol/L in 0.1 mol/L NaOH and 300
mL/L dimethyl sulfoxide), 10 µL of dithioerythritol (1.67 mmol/L in 2
mmol/L EDTA), and 30 µL of 1-octanol (to avoid foam formation) were
added in one step. Twenty microliters of 1.8 mol/L HCl was added, and
after a 1.5-min incubation at room temperature, 0.1 mL of 1.6 mol/L
N-ethylmorpholine and 0.4 mL of water were added to stop the
reduction reaction. After the addition of 20 µL of 6.6 mmol/L
monobromobimane (in acetonitrile) and thorough mixing, an incubation
for 3 min at room temperature completed the derivatization. The
reaction was stopped with 40 µL of concentrated acetic acid. After
mixing, 20 µL of this reaction mixture was injected on a
reversed-phase C18 column (LC-18, 15 cm x
4.6 mm i.d., 3 µm bead size, with a 2-cm pelliguard precolumn;
Supelco). After a 4-min equilibration of the column with pH 3.9 buffer
(30 mmol/L ammonium nitrate, 40 mmol/L ammonium formate, and 4 mmol/L
tetrabutylammonium hydrogen sulfate), the thiol derivatives were eluted
with a 7-min 8.7–11.1% acetonitrile gradient (flow rate, 1 mL/min)
and detected at 364 nm excitation and 474 nm emission (Fig. 2
). The
measured amount of cysteine was divided by 2 to obtain the original
cystine concentration. To check the overall procedure, a solution of
cystine (25 µmol/L in 10 mmol/L sodium phosphate buffer, pH 7.2),
with and without 1 mmol/L NEM, and a freshly prepared solution of
cysteine (100 µmol/L in 10 mmol/L sodium phosphate buffer, pH 7.2),
with and without 1 mmol/L NEM, were routinely added to a series of
samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cysteine and cystine in cells
The cysteine concentrations in homogenates of leukocytes and
fibroblasts of two controls with and without NEM are shown in Table 1
. The total cysteine and cystine concentrations are expressed as
µmol/L cysteine and µmol cysteine/mg protein. The cell extracts
were prepared in 10 mmol/L sodium phosphate buffer, pH 7.2, with and
without 1 mmol/L NEM. Thus, the cysteine assayed in homogenates with
added NEM originates from cystine. In the cytosol of leukocytes, the
cysteine concentration was ~100-fold higher than the cystine
concentration. In the fibroblasts, this difference was ~10-fold. The
cysteine concentration of the leukocytes was approximately fivefold
higher than that of the fibroblasts, whereas the cystine concentration
of both cell types was comparable. The measured cysteine concentrations
were between 0.5 and 110 µmol/L, which was within the linear range of
our determination.
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performance of the assay, recovery, and chromatography
The cysteine assay was linear up to 200 µmol/L cysteine or 100
µmol/L cystine. Our detection limit was 0.3 µmol/L cysteine. The
intra- and interrun CV was <5% (using a solution of 12.5 µmol/L
cystine). The cystine concentration was determined in leukocytes and
cultured fibroblasts of two pooled controls and a buffer solution with
and without 6.25 µmol/L cystine after addition of the NEM/phosphate
buffer. The recovery was 86% for the leukocyte extract, 69% for the
fibroblast extract, and 83% for the buffer solution with added
cystine. The overall procedure check led to the complete elimination of
the added cysteine (100 µmol/L), whereas the added cystine (25
µmol/L) was unaffected by NEM.
Fig. 2
shows the typical HPLC elution patterns after injection of a
leukocyte extract containing 17.0 µmol/L cystine from a cystinosis
patient without cysteamine treatment, a control containing 0.38
µmol/L cystine, and a fibroblasts extract of a cystinosis patient
with 2.0 µmol/L cystine.
treatment of the cells
The influence of three different methods of freezing and storing
of isolated leukocytes on the cystine concentration of a control was
studied. The lowest cystine concentration of 0.045 nmol/mg protein was
obtained by the freezing of leukocytes in liquid
N2 and storage at -80 °C. The cystine
concentration in a sample frozen and stored at -80 °C was 0.066
nmol cystine/mg protein, whereas the cystine concentration in a sample
frozen and stored at -20 °C was 0.442 nmol cystine/mg protein,
which is a considerable increase in cystine content attributable to
oxidation of cysteine.
The influence on the cystine concentration of the duration of storage at -80 °C in the isolated leukocytes of two controls frozen in liquid N2 was also examined. Cystine was measured immediately after isolation with and without freezing the leukocytes in liquid N2 and after storage at -80 °C for 1 and 2 weeks and 1 and 2 months. In all samples, no influence of freezing and storage on the cystine concentration was observed, indicating that the cystine concentration in isolated leukocytes frozen immediately in liquid N2 and stored at -80 °C is stable for up to 2 months.
The cystine concentrations in leukocytes of three treated patients to
which the NEM/sodium phosphate buffer was added before and after
thawing of the frozen pellets are shown in Table 2
. NEM addition after the cells were thawed produced a 12- to
15-fold increase in cystine concentration compared with cells that were
thawed in the presence of NEM.
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controls and patients
In leukocytes, the cystine concentration of 23 controls (ages,
21–50 years) were 0.04–0.13 nmol cystine/mg protein (mean ± SD,
0.076 ± 0.027 nmol cystine/mg protein). Eighteen patients (ages,
9 months to 36 years) during cysteamine treatment had values of
0.08–0.67 nmol cystine/mg protein, and 1 patient (age, 2 years) before
treatment had 2.43 nmol cystine/mg protein. The cystine concentrations
in cultured fibroblasts of nine controls were 0–0.17 nmol cystine/mg
protein (mean ± SD, 0.104 ± 0.069 nmol cystine/mg protein).
The cystine concentrations in cultured fibroblasts of seven cystinosis
patients were 1.60, 1.78, 2.89, 2.25, 2.23, 2.63, and 3.69 nmol
cystine/mg protein.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Because of the great importance
of this NEM reaction, we checked the reliability of each series of
samples by using cystine (25 µmol/L) and cysteine (100 µmol/L)
solutions with and without added NEM as controls for the procedure.
Thus, under the conditions we applied, all intracellular cysteine is
removed and the cystine can be adequately measured. Theoretically, our
method will also detect cysteine present in mixed cysteine-disulfides
in the cytosol. However, in cells, virtually all low-molecular weight
compounds with a thiol function, such as glutathione and cysteine, are
present in their reduced state. Furthermore, our reference values are
in the same range or even lower than those determined by other methods
(1).
To prevent oxidation of intracellular cysteine, careful treatment of
the isolated cells is also important. Table 2
demonstrates that NEM
must be added to frozen cells, so that released intracellular SH groups
can react immediately with NEM during thawing. In addition, freezing in
liquid N2 and storing the isolated cells at
-80 °C are the proper treatment to prevent unwanted oxidation of
the intracellular SH groups. Storage at -80 °C for at least 2
months will not affect the cystine concentration. Thus, isolated cells
of (treated) patients can be shipped at -80 °C (dry ice) from one
hospital to another for cystine determination.
The low detection limit of 0.3 µmol/L cysteine enables us to measure the low cystine concentrations in the leukocytes and fibroblasts of controls. The small range of the control SD values is an indication of the reliability of our method. The detection limit is adequate for monitoring the effectiveness of cysteamine treatment of cystinosis patients and enables the determination of an appropriate cysteamine dose.
The cystine concentrations found in fibroblasts and leukocytes of controls and the patients are comparable but somewhat lower than the values found in the literature (1), which might be attributable to our recovery of 69–86% cystine from the cell pellets.
An advantage of this HPLC method is also found in time savings: the derivation and elution of a sample is performed within 20 min and is fully automated. Furthermore, the use of radioactive cystine, which is required in the cystine-binding protein assay (4), is avoided; thus our method can be applied in general laboratories.
We have presented a reliable new method of cystine determination in cell extracts of mixed leukocytes and cultured fibroblasts. Its usefulness in diagnosing cystinosis and monitoring treatment by cysteamine is demonstrated. The method is fast and can be automated with high reproducibility and reliability.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  E. Levtchenko, A. de Graaf-Hess, M. Wilmer, L. van den Heuvel, L. Monnens, and H. Blom Altered status of glutathione and its metabolites in cystinotic cells Nephrol. Dial. Transplant., September 1, 2005; 20(9): 1828 - 1832. [Abstract] [Full Text] [PDF]
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 E. Levtchenko, A. de Graaf-Hess, M. Wilmer, L. van den Heuvel, L. Monnens, and H. Blom Comparison of Cystine Determination in Mixed Leukocytes vs Polymorphonuclear Leukocytes for Diagnosis of Cystinosis and Monitoring of Cysteamine Therapy Clin. Chem., September 1, 2004; 50(9): 1686 - 1688. [Full Text] [PDF]
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J. M. Geelen, L. A. H. Monnens, and E. N. Levtchenko Follow-up and treatment of adults with cystinosis in the Netherlands Nephrol. Dial. Transplant., October 1, 2002; 17(10): 1766 - 1770. [Abstract] [Full Text] [PDF]
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