Core Unit Proteomics, Institute of Toxicology, Hannover Medical School, 30625 Hannover, Germany.
Molecules. 2021 Nov 19;26(22):7003. doi: 10.3390/molecules26227003.
Carbon dioxide (CO) and carbonates, which are widely distributed in nature, are constituents of inorganic and organic matter and are essential in vegetable and animal organisms. CO is the principal greenhouse gas in the atmosphere. In human blood, CO/HCO is an important buffering system. Inorganic nitrate (ONO) and nitrite (ONO) are major metabolites and abundant reservoirs of nitric oxide (NO), an endogenous multifunctional signaling molecule. Carbonic anhydrase (CA) is involved in the reabsorption of nitrite and nitrate from the primary urine. The measurement of nitrate and nitrite in biological samples is of particular importance. The derivatization of nitrate and nitrite in biological samples alongside their N-labeled analogs, which serve as internal standards, is a prerequisite for their analysis by gas chromatography-mass spectrometry (GC-MS). A suitable derivatization reagent is pentafluorobenzyl bromide (PFB-Br). Nitrate and nitrite are converted in aqueous acetone to PFB-ONO and PFB-NO, respectively. PFB-Br is also useful for the GC-MS analysis of carbonate/bicarbonate. This is of particular importance in conditions of pharmacological CA inhibition, for instance by acetazolamide, which is accompanied by elevated concomitant excretion of nitrate, nitrite and bicarbonate, as well as by urine alkalization. We performed a series of experiments with exogenous bicarbonate (NaHCO) added to human urine samples (range, 0 to 100 mM), as well as with endogenous bicarbonate resulting from the inhibition of CA activity in healthy subjects before and after ingestion of pharmacological acetazolamide. Our results indicate that bicarbonate enhances the derivatization of nitrate with PFB-Br. In contrast, bicarbonate decreases the derivatization of nitrite with PFB-Br. Bicarbonate is not a catalyst, but it enhances PFB-ONO formation and inhibits PFB-NO formation in a concentration-dependent manner. The effects of bicarbonate are likely to result from its reaction with PFB-Br to generate PFB-OCOOH. Nitrate reacts with concomitantly produced PFB-OCOOH to form PFB-ONO in addition to the direct reaction of nitrate with PFB-Br. By contrast, nitrite does not react with PFB-OCOOH to form PFB-NO. Sample acidification by small volumes of 20 wt.% aqueous acetic acid abolishes the effects of exogenous and endogenous bicarbonate on nitrite measurement.
二氧化碳（CO）和碳酸盐在自然界中广泛存在，是无机和有机物质的组成部分，也是动植物机体所必需的。CO 是大气中的主要温室气体。在人血中，CO/HCO 是一种重要的缓冲系统。无机硝酸盐（ONO）和亚硝酸盐（ONO）是一氧化氮（NO）的主要代谢产物和丰富的储存库，NO 是一种内源性多功能信号分子。碳酸酐酶（CA）参与从初级尿中重吸收亚硝酸盐和硝酸盐。因此，测定生物样本中的硝酸盐和亚硝酸盐尤为重要。在气相色谱-质谱联用分析（GC-MS）中，必须对生物样本中的硝酸盐和亚硝酸盐及其 N 标记的类似物（作为内标）进行衍生化。一种合适的衍生化试剂是五氟苄基溴（PFB-Br）。在丙酮水溶液中，硝酸盐和亚硝酸盐分别转化为 PFB-ONO 和 PFB-NO。PFB-Br 也可用于 GC-MS 分析碳酸盐/重碳酸盐。在药理学 CA 抑制的情况下，如使用乙酰唑胺，这会伴随着同时排泄升高的硝酸盐、亚硝酸盐和重碳酸盐以及尿液碱化，这一点尤其重要。我们在添加外源性重碳酸盐（NaHCO）的人尿样本中（范围为 0 至 100 mM）以及在健康受试者摄入药理学乙酰唑胺前后抑制 CA 活性后内源性重碳酸盐的情况下进行了一系列实验。我们的结果表明，重碳酸盐增强了 PFB-Br 对硝酸盐的衍生化作用。相反，重碳酸盐会降低 PFB-Br 对亚硝酸盐的衍生化作用。重碳酸盐不是催化剂，但它以浓度依赖的方式增强 PFB-ONO 的形成并抑制 PFB-NO 的形成。重碳酸盐的作用可能是由于其与 PFB-Br 反应生成 PFB-OCOOH。硝酸盐与同时产生的 PFB-OCOOH 反应形成 PFB-ONO，除了硝酸盐与 PFB-Br 的直接反应外。相比之下，亚硝酸盐不会与 PFB-OCOOH 反应形成 PFB-NO。通过加入少量 20wt%的乙酸溶液酸化样品，可以消除外源性和内源性重碳酸盐对亚硝酸盐测量的影响。
