Determination of creatinine-related molecules in saliva by reversed-phase liquid chromatography with tandem mass spectrometry and the evaluation of hemodialysis in chronic kidney disease patients
Graphical abstract
Introduction
The kidneys play a critical role in maintaining metabolic homeostasis in a living body. The renal hypofunction causes various diseases such as cardiovascular onset [1], [2], [3]. Therefore, the detection of renal hypofunction is highly desired. A few subjective symptoms are observed during the deterioration of the renal function. For instance, the serum concentrations of creatine (Cr) and creatinine (Cre) are used for the determination of the renal function. The concentration of blood urea nitrogen (BUN) and Cre generally increase after a serious kidney problem. Therefore, a more accurate glomerular filtration rate (GFR) or approximation of creatinine clearance (Ccr) is measured when a renal disease is suspected [4], [5]. Consequently, the determination of Cr and Cre is a key step for the evaluation of the renal function.
Chronic kidney disease (CKD) refers to conditions in which the renal function is irreversibly reduced by a variety of chronic diseases such as diabetes and hypertension. Since deterioration of the kidney function is a high risk factor for cardiovascular disease, the condition has been attracting attention in recent years. The diagnosis of CKD is commonly done by creatinine-based estimated GFR (eGFR), which is calculated from the patient's serum Cre. However, the use of blood is not always suitable due to the invasive, hygienic and infection problems during its sample collection and handling. The sampling is usually performed by the medical staff, such as a doctor and nurse, while self-sampling is difficult. In contrast, saliva is relatively clean and the samples can be quickly and noninvasively collected and easily stored [6], [7]. Therefore, we focused on saliva as an alternative to blood and as a new diagnostic material. Because saliva is derived from plasma, the components are believed to reflect the blood levels [8], [9], [10].
The Cre is produced by the non-enzymatic dehydration of Cr which is derived from the enzyme reaction of arginine (Arg) and glycine (Fig. 1). The determination of these molecules is usually performed by HPLC after derivatization with a suitable reagent such as 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) [11]. The separation efficiency and detection sensitivity increase due to the introduction of a highly sensitive fluorescence moiety and the increased hydrophobicity based on the derivatization. Although the labeling method is excellent for the determination of Arg and Cr, the procedure is time-consuming. Furthermore, the labeling of Cre, which has no reactive functional group in its structure, is generally difficult. The Cre in blood and urine has been successfully determined by several methods such as ELISA [12], colorimetry using a molecularly imprinted polymer (MIP) membrane [13], LC-UV [14], LC-MS and GC–MS [15]. A review paper related to a Cre biosensor has also been published by Mohabbati-Kalejahi et al. [16]. With respect to the saliva analysis, Xing et al. [17] reported the determination of Cre by capillary electrophoresis with electrochemical detection (CE-ED). However, the simultaneous determination of Arg, Cr and Cre in saliva has not been reported till now. Based on these backgrounds, the simultaneous determination of Arg, Cr and Cre in the saliva of CKD patients was performed by UPLC-ESI-MS/MS together with the saliva of healthy volunteers. Furthermore, the evaluation of the hemodialysis of CKD patients was carried out by the determination of Arg, Cr and Cre before and after the dialysis.
Section snippets
Materials and chemicals
Creatine (Cr), creatinine (Cre), and arginine (Arg) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Arginine-2,3,3,4,4,5,5-d7 (Arg-d7), creatine-methyl-d3 (Cr-d3), and creatinine-methyl-d3 (Cre-d3), used as the internal standard (I.S.), were purchased from C/D/N Isotopes (Quebec, Canada). Ammonium acetate (AA) of analytical grade was from Wako Pure Chemicals (Osaka, Japan). Acetonitrile (ACN), methanol (MeOH) and formic acid (FA) of LC-MS grade were obtained from Kanto Chemicals (Tokyo,
Separation and detection of Cre and the related compounds
Cre is enzymatically produced from the reaction of Arg and glycine via Cr in the body (Fig. 1). The separation of these compounds, which are highly hydrophilic and low-molecular mass, is generally difficult by reversed-phase liquid chromatography. Thus, the simultaneous separation was first attempted using 6 different types of columns and various elution solvents. The BEH C18, ODS-CX15 and HS-F5 columns were used for reversed-phase (RP) chromatography, whereas the Amide-80, ZIC-HILIC and
Conclusion
The simultaneous determination of Arg, Cr and Cre in saliva by UPLC-ESI-MS/MS was reported in this paper. The present UPLC-MS/MS method is fast and sensitive for the determination of Arg, Cr and Cre. Furthermore, the procedure is simple and requires no time-consuming step such as derivatization [11]. The proposed method using saliva is useful for the evaluation of the renal function in CKD patients, because the proposed noninvasive and quantitative determination method only requires a small
Acknowledgments
This study was supported in part by a Grant-in-Aid for Scientific Research (C) 15K07895 from the Japan Society for the Promotion of Science (JSPS).
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