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Comparison of liquid chromatography-tandem mass spectrometry and sandwich ELISA for determination of keratan sulfate in plasma and urine.

ABSTRACT: BACKGROUND AND AIM:Mucopolysaccharidosis IVA (MPS IVA) leads to skeletal dysplasia through excessive storage of chondroitin-6-sulfate and keratan sulfate (KS). KS is synthesized mainly in cartilage and released into circulation, making it a critical biomarker for MPS IVA to evaluate clinical course and effectiveness of therapies. Therefore, an accurate and sensitive method is required to measure KS levels. MATERIAL AND METHODS:Using sandwich ELISA and liquid chromatography tandem mass spectrometry (LC/MS/MS) assays, we measured KS levels in blood and urine from MPS IVA patients and healthy controls to evaluate comparability of results. Blood (patients, n = 110; controls, n = 364) and urine (patients, n = 103; controls, n = 326) specimens were obtained. RESULTS:Plasma and urine KS measurements in patients were age-dependent and higher than age-matched controls. We observed a moderate correlation (r = 0.666; P < 0.001) between urine KS measurements and a weak correlation (r = 0.333; P = 0.002) between plasma KS measurements by ELISA and LC/MS/MS methods in patients. No correlation was found between plasma KS measurements in controls. The difference between KS measurements assayed by LC/MS/MS and ELISA was greater in controls than in patients. A moderate correlation between blood and urine KS measurements in the same individual was observed. CONCLUSION:These findings indicate that both methods to measure blood and urine KS are suitable for diagnosis, monitoring therapies, and longitudinal assessment of the disease course in MPS IVA, but the LC/MS/MS method measures over 10 times more KS present in body fluids.

SUBMITTER: Hintze JP

PROVIDER: S-EPMC3140273 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Comparison of liquid chromatography-tandem mass spectrometry and sandwich ELISA for determination of keratan sulfate in plasma and urine.

Hintze Jonathan P JP Tomatsu Shunji S Fujii Tadashi T Montaño Adriana M AM Yamaguchi Seiji S Suzuki Yasuyuki Y Fukushi Masaru M Ishimaru Takeshi T Orii Tadao T

Biomarker insights 20110627

<h4>Background and aim</h4>Mucopolysaccharidosis IVA (MPS IVA) leads to skeletal dysplasia through excessive storage of chondroitin-6-sulfate and keratan sulfate (KS). KS is synthesized mainly in cartilage and released into circulation, making it a critical biomarker for MPS IVA to evaluate clinical course and effectiveness of therapies. Therefore, an accurate and sensitive method is required to measure KS levels.<h4>Material and methods</h4>Using sandwich ELISA and liquid chromatography tandem ...[more]

PMID: 21792275

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Project description:UNLABELLED: BACKGROUND: Anabolic androgenic steroids, such as stanozolol, are typically misused by athletes during preparation for competition. Out-of-competition testing presents a unique challenge in the current anti-doping detection system owing to logistic reasons. Analysing hair for the presence of a prohibited drug offers a feasible solution for covering the wider window in out-of-competition testing. To assist in vivo studies aiming to establish a relationship between drug levels detected in hair, urine and blood, sensitive methods for the determination of stanozolol and its major metabolite 3'-hydroxystanozolol were developed in pigmented hair, urine and serum, using brown Norway rats as a model system and liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: For method development, spiked drug free rat hair, blood and urine samples were used. The newly developed method was then applied to hair, urine and serum samples from five brown Norway rats after treatment (intraperitoneal) with stanozolol for six consecutive days at 5.0?mg/kg/day. The assay for each matrix was linear within the quantification range with determination coefficient (r2) values above 0.995. The respective assay was capable of detecting 0.125?pg/mg stanozolol and 0.25?pg/mg 3'-hydroxystanozolol with 50?mg hair; 0.063?ng/mL stanozolol and 0.125?ng/mL 3'-hydroxystanozolol with 100 ?L of urine or serum. The accuracy, precision and extraction recoveries of the assays were satisfactory for the detection of both compounds in all three matrices. The average concentrations of stanozolol and 3'-hydroxystanozolol, were as follows: hair?=?70.18?±?22.32?pg/mg and 13.01?±?3.43?pg/mg; urine?=?4.34?±?6.54?ng/mL and 9.39?±?7.42?ng/mL; serum?=?7.75?±?3.58?ng/mL and 7.16?±?1.97?ng/mL, respectively. CONCLUSIONS: The developed methods are sensitive, specific and reproducible for the determination of stanozolol and 3'-hydroxystanozolol in rat hair, urine and serum. These methods can be used for in vivo studies further investigating stanozolol metabolism, but also could be extended for doping testing. Owing to the complementary nature of these tests, with urine and serum giving information on recent drug use and hair providing retrospective information on habitual use, it is suggested that blood or urine tests could accompany hair analysis and thus avoid false doping results.

Dataset Information

Comparison of liquid chromatography-tandem mass spectrometry and sandwich ELISA for determination of keratan sulfate in plasma and urine.

Publications

Comparison of liquid chromatography-tandem mass spectrometry and sandwich ELISA for determination of keratan sulfate in plasma and urine.

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