Measurement of cystine in granulocytes using liquid chromatography-tandem mass spectrometry

doi:10.1016/j.clinbiochem.2007.02.005

Clinical Biochemistry

Volume 40, Issues 9–10, June 2007, Pages 692-698

https://doi.org/10.1016/j.clinbiochem.2007.02.005 Get rights and content

Abstract

Background:

Cystinosis is a rare autosomal recessive disorder characterized by an accumulation of intralysosomal cystine due to a defect in cystine transport across the lysosomal membrane. This disorder can be treated specifically using high doses of cysteamine. Accurate measurement of intracellular cystine content is necessary for the diagnosis and monitoring of treatment with cysteamine. Here we describe a new method to measure intracellular cystine. It relies on a liquid chromatography-tandem mass spectrometry assay. We compare this novel method with the cystine-binding protein assay.

Method:

Cells were isolated and lysed in the presence of N-ethylmaleimide to avoid interference from cysteine. After deproteinization, addition of stable isotope d₆ cystine and butylation, cystine was measured using an API 3000 MSMS.

Results:

The cystine assay was linear to at least 50 μmol/L. Within-run and between-run coefficients of variation were 2.9% and 5.7% respectively.

Conclusion:

It is possible to measure very low concentrations of intracellular cystine with liquid chromatography-tandem mass spectrometry. The results obtained with this novel method correlate very well with those obtained using the cystine-binding protein assay.

Introduction

Cystinosis is a rare autosomal recessive inherited disorder characterized by an accumulation of intralysosomal cystine due to a defect in cystine transport across the lysosomal membrane [1]. This results in intracellular cystine crystal formation causing irreversible damage to various organs and the kidney in particular.

Three clinical forms of cystinosis (infantile, juvenile and ocular cystinosis) have been delineated based on the severity of symptoms and the age of onset. Patients with the common infantile form of cystinosis develop renal Fanconi syndrome 3 to 6 month after birth and end-stage renal failure before the age of 10 years.

The causative gene, CTNS, maps to chromosome 17p13. CTNS encodes a 367-amino-acid protein called cystinosin which is predicted to be an integral lysosomal membrane protein with seven transmembrane domains [2], [3].

Cystinosis can be treated specifically using high doses of cysteamine. Cysteamine is an aminothiol. It reacts in a disulfide exchange reaction with cystine, producing cysteamine–cysteine disulfide. Cysteine can be transported out of the lysosome by different carriers. This results in long-term depletion of lysosomal cystine. This treatment started early in life, delays renal glomerular deterioration and improves linear growth.

Accurate measurements of intracellular cystine content are mandatory for the diagnosis as well as for the monitoring of the cysteamine treatment. Different methods have been developed to measure cystine content in leucocytes. They differ according to the type of cells used. Historically, cystine has been measured in mixed leucocytes preparations, despite the fact that it accumulates preferentially in polymorphonuclear leucocytes (PMN). Also they differ according to the methods used to measure cystine. Ion exchange chromatography or mass spectrometric and a cystine-binding protein assay have been used. The latter is highly sensitive and specific [4], [5].

To obtain the final concentration (expressed as nanomoles of half-cystine by milligrams of proteins), it is necessary to measure the amount of cystine in lysed cells and the protein content of cells debris. Both analyses are equally important.

To ensure that methods are comparable, an external quality assessment programme has been established by the European Research Network for Inherited disorders of Metabolism (ERNDIM).

Here we report on a novel method to measure intracellular cystine. It relies on a liquid chromatography-tandem mass spectrometry (MSMS) assay. We compare the results obtained using this method with data obtained using the cystine-binding protein (CBP) assay.

Section snippets

Sample collection

7–10 mL of venous blood was collected into an acid-citrate-dextrose (ACD) tube. For the monitoring of treatment with cysteamine, samples were collected 6 h after the cysteamine treatment was started. Whole blood was conserved at room temperature until the sample was prepared.

Sample preparation

PMN cells were isolated within 24 h according to guidelines from the group “cystine in white blood cells” [6]. Venous blood collected was transferred into a 15 mL conical plastic centrifuge tube containing 500 μL of 0.2 mM

Results

The mass spectrum analysis of products derived from butylation of 4 μmol of cystine shows that the reaction of esterification occurs partially. Either butyl or dibutyl ester of cystine was formed in equivalent proportions (Fig. 1).

Butylation is constant as deduced from the linearity of the standard curve (Fig. 2) when the 353.3/208.3 (dibutyl ester cystine) and the 359.3/211.3 (dibutyl ester d₆ cystine) MRM transition was used for quantification (Fig. 3). The chromatographic profile of the

Discussion

Leucocytes cystine determination is essential for the diagnosis of cystinosis and more importantly, for monitoring the effectiveness of cysteamine treatment [11]. Accurate measurement of cystine concentration is dependent on the whole biological process (appropriate sample collection, rapid blood transport, isolation of cells, cystine assay, protein assay and calculation). In 2001 guidelines, from the white cell cystine group, were proposed to achieve some standardization and validation

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Cystinosis
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B. Chadefaux-Vekemans, White cell cystine group: guideline no. 2. Polymorphonuclear leucocyte preparation. BIMDG...

There are more references available in the full text version of this article.

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Quantitative determination of D<inf>4</inf>-cystine in mice using LC-MS/MS and its application to the assessment of pharmacokinetics and bioavailability
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Cystine is the primary source material for the synthesis of glutathione. However, the pharmacokinetics and tissue distribution of cystine are largely unknown. A surrogate analyte D₄-cystine was employed to generate calibration curves for the determination of levels of D₄-cystine and endogenous cystine in mice by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Validation assessments proved the sensitivity, specificity and reproducibility of the method with a lower limit of quantification (LLOQ) of 5 ng/mL over 5–5000 ng/mL in plasma. The pharmacokinetics of D₄-cystine were evaluated after administering injections and oral solutions, both of which minimally impacted endogenous cystine levels. The absolute bioavailability of cystine was 18.6%, 15.1% and 25.6% at doses of 25, 50 and 100 mg/kg, respectively. Intravenously injected D₄-cystine resulted in dramatically high plasma levels with reduced levels in the brain and liver. Intragastrically administered D₄-cystine resulted in high levels in the plasma and stomach with relatively low levels in the lung, kidney, heart and brain.
Inherited disorders of lysosomal membrane transporters
2020, Biochimica et Biophysica Acta - Biomembranes
Disorders caused by defects in lysosomal membrane transporters form a distinct subgroup of lysosomal storage disorders (LSDs). To date, defects in only 10 lysosomal membrane transporters have been associated with inherited disorders. The clinical presentations of these diseases resemble the phenotypes of other LSDs; they are heterogeneous and often present in children with neurodegenerative manifestations. However, for pathomechanistic and therapeutic studies, lysosomal membrane transport defects should be distinguished from LSDs caused by defective hydrolytic enzymes. The involved proteins differ in function, localization, and lysosomal targeting, and the diseases themselves differ in their stored material and therapeutic approaches. We provide an overview of the small group of disorders of lysosomal membrane transporters, emphasizing discovery, pathomechanism, clinical features, diagnostic methods and therapeutic aspects. We discuss common aspects of lysosomal membrane transporter defects that can provide the basis for preclinical research into these disorders.
LC-MS/MS measurement of leukocyte cystine; effect of preanalytic factors
2020, Talanta
Citation Excerpt :
The MRM transitions and the related optimized cone voltage and the collision energy for the analytes are shown in Table 1. Generally, 12% SSA is used as a protein precipitating agent to measure leukocyte cystine level [5,20–22]. In this study, 12% SSA, 12% TCA, and acetonitrile, which are commonly used as protein precipitating agents [24–27], were investigated assessing both sensitivity and matrix effects.
Cystinosis is an autosomal recessive disorder characterized by the accumulation of cystine in lysosomes, causing irreversible damage to organs, especially the kidneys. Intracellular leukocyte cystine concentrations are used to diagnose cystinosis and to monitor cysteamine treatment. The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method without derivatization capable of measuring leukocyte intracellular cystine concentrations.
During development, the effects of using three different protein precipitation agents were evaluated in terms of sensitivity and the matrix effect, with 12% trichloroacetic acid providing the highest sensitivity. The effects of different blood collection tubes were also assessed in terms of recovery, matrix effect, and protein content. Compared to other methods, our method was quicker (run time of 3 min), was linear over the range 0.078–100 μM, and had lower limits of detection (0.0192 μM) and quantification (0.0582 μM). The intra-day and inter-day reproducibility %CVs were ≤10%. and the method had excellent recovery rates (94%–106%). Other parameters including matrix selectivity, injection carryover, leukocyte lysate stability were also validated and met the acceptance criterias of European Medicines Agency (EMA) Guideline. The assay was successfully applied to quantify cystine leukocyte concentration in healthy and cystinosis patients.
Improvement of the cystine measurement in granulocytes by liquid chromatograhy-tandem mass spectrometry
2013, Clinical Biochemistry
Citation Excerpt :
It is useful for the measurement of both high (50 μmol/L) and low (100 nmol/L) concentrations of cystine, that is, for the diagnosis and follow up of Cystinosis. By comparing our HPLC-ESI-MS/MS method with the HPLC-ESI-MS/MS method previously described by Chabli et al. [6] (Table 1), we have to emphasize that the mass spectrum analysis of the di-butylated cystine presented a different product ion (Fig. 2). Therefore, the MRM transitions we use are different from those used by Chabli et al. [6].
Cystinosis is an autosomal recessive genetic disorder due to mutations in CTNS gene, which causes a defect of cystinosin, impairing the transport of free cystine out of lysosomes and causing irreversible damage to various organs, particularly the kidney. The diagnosis of Cystinosis is carried out by measuring the cystine content in leucocytes. Accurate quantification of cystine is of capital importance not only for the diagnosis, but also for monitoring the effectiveness of cysteamine treatment. Here we describe an improvement to measure cystine in granulocytes using high-pressure-liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS).
Granulocytes were isolated from heparinized blood by isopycnic centrifugation. After lysis and deproteinization, the concentration of di-butylated cystine was measured by HPLC-ESI-MS/MS, using deuterium labelled, d6-cystine, as internal standard.
The assay was linear to at least 50 μmol/L with a good precision. Within-day and between-day coefficients of variation were about 6%. The recovery was higher than 98%. Control values were clearly distinguishable from pathological levels, even if patients were under treatment. A good correlation was observed with cystine binding protein (CBP) assay, one of the most sensitive and specific methods.
This method results in good analytical performance, and is useful for diagnosis and follow up of Cystinosis. This method offers several advantages over the CBP assay: it is less expensive, easier, quicker and it does not require radioactivity. In addition, when comparing with the HPLC-ESI-MS/MS method previously described by Chabli et al. 2006, our assay exhibits more sensitivity and is faster.
Implementation of a method to quantify white blood cell cystine as a diagnostic support for cystinosis
2020, Nefrologia
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No fasting or special conditions were required for collecting the sample. Since the main anticoagulants used in the literature are heparin and acid-citrate-dextrose (ACD),7,12–14 these two anticoagulants were compared using Vacutainer® tubes containing heparin sodium or ACD solution B. Additionally, the stability of the whole blood sample was evaluated when stored at 4 °C for 8 and 24 h. A polymorphonuclear isolation protocol from whole blood was standardised based on the one described by Levtchenko et al. and the recommendations of the program by the European Research Network for evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism.7,15
Cystinosis is an inborn error of metabolism, clinically characterised by severe renal involvement and development of corneal cystine deposits, especially in the adult form of the disease. Cystinosis is a treatable condition. Therefore, an early diagnosis is necessary to start therapy. For biochemical confirmation of the condition it is necessary to quantify intracellular cystine concentrations. For this, different methods have been described with variations in cell isolation strategies and the amino acid quantification techniques used. In order to improve confirmatory biochemical diagnosis in our setting, a protocol for intraleukocitary cystine quantification was established.
A high performance liquid chromatography based method for cystine quantification in polymorphonuclear cells was implemented. Evaluation of the best anticoagulant to use and temperature stability of the sample at 4 °C were performed. In addition, we established reference values for our population.
It was determined that intraleukocitary cystine quantification must be performed in blood samples containing acid-citrate-dextrose (ACD) as anticoagulant. Samples must be processed immediately due to their poor stability even when refrigerated. Based on the results from 50 healthy individuals, the cut-off point established for our population was 0.34 nmol1/2/cystine/mg.
The adaptation performed to the cystine quantification method here presented the highest control population that has been reported in the literature so far. Our results highlight the need for making available a cystine quantification method locally and confirm the convenience for each laboratory to establish its own reference values to provide greater reliability for interpreting results.
La cistinosis es un error innato del metabolismo cuyas características clínicas incluyen compromiso renal severo, formación de cristales de cistina en la córnea, especialmente en la presentación adulta de la enfermedad. Es una enfermedad tratable por lo cual establecer el diagnostico de forma oportuna es fundamental para iniciar terapia. Para la confirmación bioquímica de la enfermedad se requiere determinar las concentraciones intracelulares de cistina, para lo cual se han reportado diferentes métodos tanto para el aislamiento de las células como las técnicas de cuantificación del aminoácido. Con el objetivo de mejorar el diagnóstico bioquímico confirmatorio en nuestro medio establecimos un protocolo de cuantificación intraleucocitaria de cistina.
Se realizó implementación de un método de cuantificación de cistina en polimorfonucleares por cromatografía líquida de alta resolución, evaluando el mejor anticoagulante a utilizar, la estabilidad de la muestra a 4 °C y estableciendo valores de referencia para nuestra población.
Se determinó que la muestra para cuantificación intraleucocitaria de cistina debe ser anticoagulada mediante adición de ácido Cítrico-Dextrosa (ACD) como anticoagulante. La muestra debe ser procesada inmediatamente dada su baja estabilidad incluso en refrigeración. Con 50 individuos sanos se estableció como punto de corte para nuestra población 0.34 nmol1/2cistina/mg.
La adaptación realizada del método de cuantificación de cistina utiliza el número más alto de muestras control hasta ahora reportado en la literatura. Nuestros resultados dan cuenta de la necesidad de implementar el método a nivel local y reafirman la conveniencia de que cada laboratorio establezca sus propios valores de referencia para proporcionar una mayor confiabilidad a la hora de interpretar los resultados.
Cystinosis
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Clinical Biochemistry

Abstract

Background:

Method:

Results:

Conclusion:

Introduction

Section snippets

Sample collection

Sample preparation

Results

Discussion

References (14)

The targeting of cystinosin to the lysosomal membrane requires a tyrosine-based signal and a novel sorting motif

J. Biol. Chem.

Binding assays for amino acids: the utilization of a cystine binding protein from Escherichia coli for the determination of acid-soluble cystine in small physiological samples

J. Biol. Chem.

Protein measurement with the folin phenol reagent

J. Biol. Chem.

Cystinosis: a disorder of lysosomal membrane transport

Cystinosin, the protein defective in cystinosis, is a H+ driven lysosomal cystine transporter

EMBO J.

Cystinosis

N. Engl. J. Med.

Cited by (35)

Quantitative determination of D<inf>4</inf>-cystine in mice using LC-MS/MS and its application to the assessment of pharmacokinetics and bioavailability

Inherited disorders of lysosomal membrane transporters

LC-MS/MS measurement of leukocyte cystine; effect of preanalytic factors

Improvement of the cystine measurement in granulocytes by liquid chromatograhy-tandem mass spectrometry

Implementation of a method to quantify white blood cell cystine as a diagnostic support for cystinosis

Cystinosis