Serum samples provide higher sensitivity for biomarker discovery studies. Due to the presence of spurious amount of sarcosine in vacutainer EDTA tubes, plasma EDTA is not suitable for studies requiring accurate quantification of sarcosine.
Volumetric absorptive microsampling (VAMS) is a novel approach that allows single-drop (10 μL) blood collection. Integration of VAMS with mass spectrometry (MS)-based untargeted metabolomics is an attractive solution for both human and animal studies. However, to boost the use of VAMS in metabolomics, key pre-analytical questions need to be addressed. Therefore, in this work, we integrated VAMS in a MS-based untargeted metabolomics workflow and investigated pre-analytical strategies such as sample extraction procedures and metabolome stability at different storage conditions. We first evaluated the best extraction procedure for the polar metabolome and found that the highest number and amount of metabolites were recovered upon extraction with acetonitrile/water (70:30). In contrast, basic conditions (pH 9) resulted in divergent metabolite profiles mainly resulting from the extraction of intracellular metabolites originating from red blood cells. In addition, the prolonged storage of blood samples at room temperature caused significant changes in metabolome composition, but once the VAMS devices were stored at − 80 °C, the metabolome remained stable for up to 6 months. The time used for drying the sample did also affect the metabolome. In fact, some metabolites were rapidly degraded or accumulated in the sample during the first 48 h at room temperature, indicating that a longer drying step will significantly change the concentration in the sample.
KeywordsMetabolomics; Volumetric absorptive microsampling; Mass spectrometry ✉ Giuseppe Paglia beppepaglia@gmail.com.
Compliance with ethical standardsThis study was performed in accordance with the ethical standards. The local ethics committee (Comitato etico del comprensorio sanitario di Bolzano) approved the study, and all participants provided written informed consent.
Adenine phosphoribosyltransferase (APRT) deficiency is a hereditary disorder that leads to excessive urinary excretion of 2,8-dihydroxyadenine (DHA), causing nephrolithiasis and chronic kidney disease. Treatment with allopurinol or febuxostat reduces DHA production and attenuates the renal manifestations. Assessment of DHA crystalluria by urine microscopy is used for therapeutic monitoring, but lacks sensitivity. We report a high-throughput assay based on ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS) for quantification of urinary DHA.
The UPLC–MS/MS assay was optimized by a chemometric approach for absolute quantification of DHA, utilizing isotopically labeled DHA as an internal standard. Experimental screening was conducted with D-optimal design and optimization of the DHA response was performed with central composite face design and related to the peak area of DHA using partial least square regression. Acceptable precision and accuracy of the DHA concentration were obtained over a calibration range of 100 to 5000 ng/mL on three different days. The intra- and inter-day accuracy and precision coefficients of variation were well within ±15% for quality control samples analyzed in replicates of six at three concentration levels. Absolute quantification of DHA in urine samples from patients with APRT deficiency was achieved wihtin 6.5 min. Measurement of DHA in 24 h urine samples from three patients with APRT deficiency, diluted 1:15 (v/v) with 10 mM ammonium hydroxide (NH4OH), yielded a concentration of 3021, 5860 and 10563 ng/mL and 24 h excretion of 816, 1327 and 1649 mg, respectively. A rapid and robust UPLC–MS/MS assay for absolute quantification of DHA in urine was successfully developed. We believe this method will greatly facilitate diagnosis and management of patients with APRT deficiency.
Metabolomics in human serum samples provide a snapshot of the current metabolic state of an individuum. Metabolite concentrations are influenced by both genetic and environmental factors. Concentrations of certain metabolites can further depend on age, sex, menopause, and diet of study participants. A better understanding of these relationships is pivotal for the planning of metabolomics studies involving human subjects and interpretation of their results. We generated one of the largest single-site targeted metabolomics data sets consisting of 175 quantified metabolites in 6872 study participants. We identified metabolites significantly associated with age, sex, body mass index, diet, and menopausal status. While most of our results agree with previous large-scale studies, we also found novel associations including serotonin as a sex and BMI-related metabolite and sarcosine and C2 carnitine showing significantly higher concentrations in post-menopausal women. Finally, we observed strong associations between higher consumption of food items and certain metabolites, mostly phosphatidylcholines and lysophosphatidylcholines. Most, and the strongest, relationships were found for habitual meat intake while no significant relationships were found for most fruits, vegetables, and grain products. Summarizing, our results reconfirm findings from previous population-based studies on an independent cohort. Together, these findings will ultimately enable the consolidation of sets of metabolites which are related to age, sex, BMI, and menopause as well as to participants’ diet.
The aim of this study was to obtain a longitudinal evaluation of the exposure to chlorpyrifos (CP) and chlorpyrifos-methyl (CPM) in agricultural workers in South Tyrol and in a residential group living in the same area. CP and CPM are widely used pesticides in agriculture. Biological monitoring of CP and CPM exposure in humans can be achieved by analyzing urinary levels of 3,5,6-trichloro-2-pyridinol (TCPy). TCPy a metabolite of CP and CPM which is produced by a two-step metabolic transformation. Between May 14th, 2014 and March 16th, 2015 we conducted a longitudinal study on 28 farmers actively working in spray pesticide treatment and 43 non-farmers living in the same agricultural area of South Tyrol (Italy). Urine samples were collected at two time points: during the pesticide treatment period and in a temporally distant season that should guarantee metabolite clearance. We developed and validated a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the determination of urinary TCPy levels. During the treatment season, both farmers and residents showed higher TCPy levels (median = 6.8 and 6.73 ug/g creatinine, respectively) than during the non-treatment season (median = 2.54 and 3.22 ug/g creatinine, respectively), suggesting a similar effect of the pesticide spraying on both groups. However, the observed TCPy levels resulted in a daily CP and CPM intake well below the limits recommended by FAO/WHO. During the non-treatment season, non-farmers showed higher TCPy levels values than farmers, suggesting the existence of TCPy of other unmeasured sources of exposure not considered in this study. This suggests that, for a comprehensive evaluation of the risks associated with TCPy exposure, additional sources should be identified in addition to CP and CPM pesticides.
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