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Introduction
4-hydroxy-l-proline (hydroxyproline) is a non-proteinogenic amino acid, that has a molecular weight of 131.13 g/mol and is synthesized by post-translational hydroxylation of proline during collagen biosynthesis (Fig. 1). Investigations of physiological and pathological collagen metabolism most commonly utilize measurements of hydroxyproline in the plasma, urine and body tissue. Therefore, determination of hydroxyproline provides useful information for the diagnosis and prognosis of diseases caused by disorders of the collagen metabolism (1). Such conditions include hyperthyroidism, hyperparathyroidism, acromegaly, Paget’s disease, osteomalacia, rickets, Marfan syndrome, osteogenesis imperfecta, sclerodactyly, dermatomyositis and Cushing syndrome (2).
Fibrosis that occurs in the liver, lungs, kidneys, skin and other organs has the capacity to develop into chronic hepatitis, liver sclerosis, liver cancer, pulmonary fibrosis and glomerulonephritis (3). Therefore, preventing the development and reducing the severity of fibrosis in patients is highly important. Hydroxyproline acts as an important diagnostic indicator of the severity of fibrosis.
The measurement of hydroxyproline in plasma, urine and body tissue is possible by colorimetric methods, high-performance liquid chromatography (HPLC) and flow injection analyses (4–6). However, these methods require large sample volumes, due to their low sensitivity. In addition, HPLC requires a long separation time for each sample. In recent years, liquid chromatography-mass spectrometry (LC-MS) has been emerging as advantageous for its high sensitivity and short separation time. LC-MS has been utilized to measure low concentrations of drugs in serum and urine with contaminated substances (7–9). The present study aimed to apply the novel LC-MS method to the measurement of hydroxyproline. As an indicator of fibrosis, hydroxyproline in the liver and lung of a rat model of fibrosis was measured by LC-MS, and compared with previous results obtained by HPLC and colorimetric methods.
Materials and methods
Ethics statement
The study protocol was approved by the Ethics Committee of Harbin Medical University (Harbin, China). A standard procedure for obtaining written informed consent was included in the protocol, and was approved by the Ethics Committee of Harbin Medical University.
Reagents
Dimethylnitrosamine (DMN) and hydroxyproline were obtained from Nacalai Tesque Inc. (Kyoto, Japan). Nembutal was purchased from Dai-Nihon Pharmaceuticals Inc. (Osaka, Japan) and bleomycin was obtained from Nihon Kayaku Inc. (Tokyo, Japan). All other chemicals were of reagent grade.
Preparation of a model of pulmonary and liver fibrosis in rats
A model...