TY - JOUR

T1 - Simultaneous GC-ECNICI-MS measurement of nitrite, nitrate and creatinine in human urine and plasma in clinical settings

AU - Hanff, Erik

AU - Lützow, Moritz

AU - Kayacelebi, Arslan Arinc

AU - Finkel, Armin

AU - Maassen, Mirja

AU - Yanchev, Georgi Radoslavov

AU - Haghikia, Arash

AU - Bavendiek, Udo

AU - Buck, Anna

AU - Lücke, Thomas

AU - Maassen, Norbert

AU - Tsikas, Dimitrios

N1 - Publisher Copyright: © 2016 Elsevier B.V.

PY - 2017/3/15

Y1 - 2017/3/15

N2 - Creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous substances. Nitrite (ONO−) and nitrate (ONO2−) are metabolites of nitric oxide (NO), a signalling molecule with multiple biological functions. Under certain and standardized conditions, the concentration of nitrate in the urine is a suitable measure of whole body NO synthesis. The urinary nitrate-to-nitrite molar ratio (UNOxR) may indicate nitrite-dependent renal carbonic anhydrase (CA) activity. In clinical studies, urine is commonly collected by spontaneous micturition. In those cases the nitrate and nitrite excretion must be corrected for creatinine excretion. Pentafluorobenzyl (PFB) bromide (PFB-Br) is a useful derivatization reagent of numerous inorganic and organic compounds, including urinary nitrite, nitrate and creatinine, for highly sensitive and specific quantitation by GC–MS. Here, we report on the simultaneous PFB-Br derivatization (60 min, 50 °C) of ONO−, O15NO−, ONO2−, O15NO2−, creatinine (do-Crea) and [methylo-2H3]creatinine (d3-Crea) in acetonic dilutions of native human urine and plasma samples (4:1, v/v) and their simultaneous quantification by GC–MS as PFBNO2, PFB15NO2, PFBONO2, PFBO15NO2, do-Crea-PFB and d3-Crea-PFB, respectively. Electron capture negative-ion chemical ionization (ECNICI) of these derivatives generates anions due to [M−PFB]−, i.e., the starting analytes. Quantification is performed by selected-ion monitoring (SIM) of m/z 46 (ONO−), m/z 47 (O15NO−), m/z 62 (ONO2−), m/z 63 (O15NO2−), m/z 112 (do-Crea), and m/z 115 (d3-Crea). Retention times were 2.97 min for PFB-ONO2/PFB-O15NO2, 3.1 min for PFB-NO2/PFB-15NO2, and 6.7 min for do-Crea-PFB/d3-Crea-PFB. We used this method to investigate the effects of long-term oral NaNO3 or NaCl (serving as placebo) supplementation (each 0.1 mmol/kg body weight per day for 3 weeks) on creatinine excretion and UNOxR in 17 healthy young men. Compared to NaCl (n = 8), NaNO3 (n = 9) supplementation increased UNOxR (1709 ± 355 vs. 369 ± 77, P < 0.05). Creatinine excretion did not differ between the groups (6.67 ± 1.34 mM vs. 5.72 ± 1.27 mM, P = 0.57). The method is also applicable to human plasma. In 78 adults patients newly diagnosed for cerebrovascular disease (CVD), there was a close correlation (r = 0.9833) between the creatinine concentrations measured in plasma by GC-ECNICI-MS and those measured in serum by an enzymatic assay. Creatinine-corrected plasma nitrate and nitrite concentrations (P = 0.035 and P = 0.004, respectively) but not their concentrations (P = 0.68 and P = 0.40, respectively) differ between male (n = 54) and female (n = 24) CVD patients. No such differences were found between preterm newborn boys (n = 25) and girls (n = 22). Like in urine, circulating creatinine may be useful to correct for gender-specific differences in plasma nitrite and nitrate in adults. Chronic NaNO3 supplementation to healthy young men does not affect renal CA-dependent nitrite excretion or creatinine synthesis and excretion.

AB - Creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous substances. Nitrite (ONO−) and nitrate (ONO2−) are metabolites of nitric oxide (NO), a signalling molecule with multiple biological functions. Under certain and standardized conditions, the concentration of nitrate in the urine is a suitable measure of whole body NO synthesis. The urinary nitrate-to-nitrite molar ratio (UNOxR) may indicate nitrite-dependent renal carbonic anhydrase (CA) activity. In clinical studies, urine is commonly collected by spontaneous micturition. In those cases the nitrate and nitrite excretion must be corrected for creatinine excretion. Pentafluorobenzyl (PFB) bromide (PFB-Br) is a useful derivatization reagent of numerous inorganic and organic compounds, including urinary nitrite, nitrate and creatinine, for highly sensitive and specific quantitation by GC–MS. Here, we report on the simultaneous PFB-Br derivatization (60 min, 50 °C) of ONO−, O15NO−, ONO2−, O15NO2−, creatinine (do-Crea) and [methylo-2H3]creatinine (d3-Crea) in acetonic dilutions of native human urine and plasma samples (4:1, v/v) and their simultaneous quantification by GC–MS as PFBNO2, PFB15NO2, PFBONO2, PFBO15NO2, do-Crea-PFB and d3-Crea-PFB, respectively. Electron capture negative-ion chemical ionization (ECNICI) of these derivatives generates anions due to [M−PFB]−, i.e., the starting analytes. Quantification is performed by selected-ion monitoring (SIM) of m/z 46 (ONO−), m/z 47 (O15NO−), m/z 62 (ONO2−), m/z 63 (O15NO2−), m/z 112 (do-Crea), and m/z 115 (d3-Crea). Retention times were 2.97 min for PFB-ONO2/PFB-O15NO2, 3.1 min for PFB-NO2/PFB-15NO2, and 6.7 min for do-Crea-PFB/d3-Crea-PFB. We used this method to investigate the effects of long-term oral NaNO3 or NaCl (serving as placebo) supplementation (each 0.1 mmol/kg body weight per day for 3 weeks) on creatinine excretion and UNOxR in 17 healthy young men. Compared to NaCl (n = 8), NaNO3 (n = 9) supplementation increased UNOxR (1709 ± 355 vs. 369 ± 77, P < 0.05). Creatinine excretion did not differ between the groups (6.67 ± 1.34 mM vs. 5.72 ± 1.27 mM, P = 0.57). The method is also applicable to human plasma. In 78 adults patients newly diagnosed for cerebrovascular disease (CVD), there was a close correlation (r = 0.9833) between the creatinine concentrations measured in plasma by GC-ECNICI-MS and those measured in serum by an enzymatic assay. Creatinine-corrected plasma nitrate and nitrite concentrations (P = 0.035 and P = 0.004, respectively) but not their concentrations (P = 0.68 and P = 0.40, respectively) differ between male (n = 54) and female (n = 24) CVD patients. No such differences were found between preterm newborn boys (n = 25) and girls (n = 22). Like in urine, circulating creatinine may be useful to correct for gender-specific differences in plasma nitrite and nitrate in adults. Chronic NaNO3 supplementation to healthy young men does not affect renal CA-dependent nitrite excretion or creatinine synthesis and excretion.

KW - Clinical studies

KW - Creatinine

KW - Nitric oxide

KW - Quantification

KW - Renal carbonic anhydrase

KW - Supplementation

UR - http://www.scopus.com/inward/record.url?scp=84961869733&partnerID=8YFLogxK

U2 - 10.1016/j.jchromb.2016.03.034

DO - 10.1016/j.jchromb.2016.03.034

M3 - Article

C2 - 27052124

AN - SCOPUS:84961869733

VL - 1047

SP - 207

EP - 214

JO - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

SN - 1570-0232

ER -