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Detection and Quantification of Ammonia as the Ammonium Cation in Human Saliva by (1)H NMR: A Promising Probe for Health Status Monitoring, with Special Reference to Cancer

Ammonia (NH(3)) has been shown to be a key biomarker for a wide variety of diseases, such as hepatic and chronic kidney diseases (CKD), and cancers. It also has relevance to the oral health research area, and, hence, its determination in appropriate biofluids and tissues is of much importance. Howev...

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Autores principales: Bhogadia, Mohammed, Edgar, Mark, Hunwin, Kayleigh, Page, Georgina, Grootveld, Martin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10383521/
https://www.ncbi.nlm.nih.gov/pubmed/37512499
http://dx.doi.org/10.3390/metabo13070792
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author Bhogadia, Mohammed
Edgar, Mark
Hunwin, Kayleigh
Page, Georgina
Grootveld, Martin
author_facet Bhogadia, Mohammed
Edgar, Mark
Hunwin, Kayleigh
Page, Georgina
Grootveld, Martin
author_sort Bhogadia, Mohammed
collection PubMed
description Ammonia (NH(3)) has been shown to be a key biomarker for a wide variety of diseases, such as hepatic and chronic kidney diseases (CKD), and cancers. It also has relevance to the oral health research area, and, hence, its determination in appropriate biofluids and tissues is of much importance. However, since it contains exchangeable >N-H protons, its analysis via (1)H NMR spectroscopy, which is a widely employed technique in untargeted metabolomic studies, is rendered complicated. In this study, we focused on the (1)H NMR analysis of this biomarker in less invasively collected human saliva samples, and we successfully identified and quantified it as ammonium cation (NH(4)(+)) in post-collection acidulated forms of this biofluid using both the standard calibration curve and standard addition method (SAM) approaches. For this purpose, n = 27 whole mouth saliva (WMS) samples were provided by healthy human participants, and all donors were required to follow a fasting/oral environment abstention period of 8 h prior to collection. Following acidification (pH 2.00), diluted WMS supernatant samples treated with 10% (v/v) D(2)O underwent (1)H NMR analysis (600 MHz). The acquired results demonstrated that NH(4)(+) can be reliably determined in these supernatants via integration of the central line of its characteristic 1:1:1 intensity triplet resonance (complete spectral range δ = 6.97–7.21 ppm). Experiments performed also demonstrated that any urease-catalysed NH(3) generation occurring post-sampling in WMS samples did not affect the results acquired during the usual timespan of laboratory processing required prior to analysis. Further experiments demonstrated that oral mouth-rinsing episodes conducted prior to sample collection, as reported in previous studies, gave rise to major decreases in salivary NH(4)(+) levels thereafter, which renormalised to only 50–60% of their basal control concentrations at the 180-min post-rinsing time point. Therefore, the WMS sample collection method employed significantly affected the absolute levels of this analyte. The LLOD was 60 μmol/L with 128 scans. The mean ± SD salivary NH(4)(+) concentration of WMS supernatants was 11.4 ± 4.5 mmol/L. The potential extension of these analytical strategies to the screening of other metabolites with exchangeable (1)H nuclei is discussed, as is their relevance to the monitoring of human disorders involving the excessive generation and/or uptake of cellular/tissue material, or altered homeostasis, in NH(3).
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spelling pubmed-103835212023-07-30 Detection and Quantification of Ammonia as the Ammonium Cation in Human Saliva by (1)H NMR: A Promising Probe for Health Status Monitoring, with Special Reference to Cancer Bhogadia, Mohammed Edgar, Mark Hunwin, Kayleigh Page, Georgina Grootveld, Martin Metabolites Article Ammonia (NH(3)) has been shown to be a key biomarker for a wide variety of diseases, such as hepatic and chronic kidney diseases (CKD), and cancers. It also has relevance to the oral health research area, and, hence, its determination in appropriate biofluids and tissues is of much importance. However, since it contains exchangeable >N-H protons, its analysis via (1)H NMR spectroscopy, which is a widely employed technique in untargeted metabolomic studies, is rendered complicated. In this study, we focused on the (1)H NMR analysis of this biomarker in less invasively collected human saliva samples, and we successfully identified and quantified it as ammonium cation (NH(4)(+)) in post-collection acidulated forms of this biofluid using both the standard calibration curve and standard addition method (SAM) approaches. For this purpose, n = 27 whole mouth saliva (WMS) samples were provided by healthy human participants, and all donors were required to follow a fasting/oral environment abstention period of 8 h prior to collection. Following acidification (pH 2.00), diluted WMS supernatant samples treated with 10% (v/v) D(2)O underwent (1)H NMR analysis (600 MHz). The acquired results demonstrated that NH(4)(+) can be reliably determined in these supernatants via integration of the central line of its characteristic 1:1:1 intensity triplet resonance (complete spectral range δ = 6.97–7.21 ppm). Experiments performed also demonstrated that any urease-catalysed NH(3) generation occurring post-sampling in WMS samples did not affect the results acquired during the usual timespan of laboratory processing required prior to analysis. Further experiments demonstrated that oral mouth-rinsing episodes conducted prior to sample collection, as reported in previous studies, gave rise to major decreases in salivary NH(4)(+) levels thereafter, which renormalised to only 50–60% of their basal control concentrations at the 180-min post-rinsing time point. Therefore, the WMS sample collection method employed significantly affected the absolute levels of this analyte. The LLOD was 60 μmol/L with 128 scans. The mean ± SD salivary NH(4)(+) concentration of WMS supernatants was 11.4 ± 4.5 mmol/L. The potential extension of these analytical strategies to the screening of other metabolites with exchangeable (1)H nuclei is discussed, as is their relevance to the monitoring of human disorders involving the excessive generation and/or uptake of cellular/tissue material, or altered homeostasis, in NH(3). MDPI 2023-06-26 /pmc/articles/PMC10383521/ /pubmed/37512499 http://dx.doi.org/10.3390/metabo13070792 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Bhogadia, Mohammed
Edgar, Mark
Hunwin, Kayleigh
Page, Georgina
Grootveld, Martin
Detection and Quantification of Ammonia as the Ammonium Cation in Human Saliva by (1)H NMR: A Promising Probe for Health Status Monitoring, with Special Reference to Cancer
title Detection and Quantification of Ammonia as the Ammonium Cation in Human Saliva by (1)H NMR: A Promising Probe for Health Status Monitoring, with Special Reference to Cancer
title_full Detection and Quantification of Ammonia as the Ammonium Cation in Human Saliva by (1)H NMR: A Promising Probe for Health Status Monitoring, with Special Reference to Cancer
title_fullStr Detection and Quantification of Ammonia as the Ammonium Cation in Human Saliva by (1)H NMR: A Promising Probe for Health Status Monitoring, with Special Reference to Cancer
title_full_unstemmed Detection and Quantification of Ammonia as the Ammonium Cation in Human Saliva by (1)H NMR: A Promising Probe for Health Status Monitoring, with Special Reference to Cancer
title_short Detection and Quantification of Ammonia as the Ammonium Cation in Human Saliva by (1)H NMR: A Promising Probe for Health Status Monitoring, with Special Reference to Cancer
title_sort detection and quantification of ammonia as the ammonium cation in human saliva by (1)h nmr: a promising probe for health status monitoring, with special reference to cancer
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10383521/
https://www.ncbi.nlm.nih.gov/pubmed/37512499
http://dx.doi.org/10.3390/metabo13070792
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