Psychobiological stress response to a lung cancer diagnosis: a prospective study of patients in Iceland and Sweden.

BACKGROUND: A diagnostic work-up leading to a lung cancer diagnosis is a severely stressful experience that may impact tumor progression. Yet, prospective data are scarce on psychological and biological components of stress at the time of lung cancer diagnosis. The aim of this study was to assess pre-to-post diagnosis change in psychological distress and urinary excretion of catecholamines in patients with suspected lung cancer. METHODS: Participants were 167 patients within the LUCASS study, recruited at referral for suspected lung cancer to University Hospitals in Iceland and Sweden. Patients completed questionnaires on perceived distress (Hospital Anxiety and Depression Scale, HADS) before and after diagnosis of lung cancer or a non-malignant origin. A subpopulation of 85 patients also provided overnight urine for catecholamine analysis before and at a median of 24 days after diagnosis but before treatment. RESULTS: A lung cancer diagnosis was confirmed in 123 (73.7%) patients, with a mean age of 70.1 years. Patients diagnosed with lung cancer experienced a post-diagnosis increase in psychological distress (p = 0.010), while patients with non-malignant lung pathology showed a reduction in distress (p = 0.070). Both urinary epinephrine (p = 0.001) and norepinephrine (p = 0.032) levels were higher before the diagnosis among patients eventually diagnosed with lung cancer compared to those with non-malignant lung pathology. We observed indications of associations between pre-to-post diagnosis changes in perceived distress and changes in urinary catecholamine levels. CONCLUSION: Receiving a lung cancer diagnosis is associated with an increase in psychological distress, while elevated catecholamine levels are evident already before lung cancer diagnosis.

Influence of collection tubes during quantitative targeted metabolomics studies in human blood samples.

BACKGROUND: Plasma and serum are the most widely used matrices in clinical studies. However, some variability in absolute concentrations of metabolites are likely to be observed in these collection tubes matrices. METHODS: We analyzed 189 metabolites using the same protocol for quantitative targeted metabolomics (LC-MS/MS AbsoluteIDQ p180 Kit Biocrates) in three types of samples, serum, plasma EDTA and citrate, of 80 subjects from the Cooperative Health Research In South Tyrol cohort (40 healthy elderly and 40 healthy young). RESULTS: The concentration levels were higher in serum than citrate and EDTA, in particular for amino acids and biogenic amines. The average Pearson's correlation coefficients were however always higher than 0.7 for these two classes of metabolites. We could also demonstrate that blank EDTA vacutainer tubes contain a significant amount of sarcosine. Finally, we compared the metabolome of young people against elderly subjects and found that the highest number of metabolites significantly changing with age was detected in serum. CONCLUSION: 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.

Pre-analytic evaluation of volumetric absorptive microsampling and integration in a mass spectrometry-based metabolomics workflow.

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. Graphical abstract Volumetric absorptive microsampling (VAMS) is a novel technology that allows single-drop blood collection and, in combination with mass spectrometry (MS)-based untargeted metabolomics, represents an attractive solution for both human and animal studies. 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. The latter revealed that prolonged storage of blood samples at room temperature caused significant changes in metabolome composition, but if VAMS devices were stored at - 80 °C, the metabolome remained stable for up to 6 months.

Quantitative UPLC-MS/MS assay of urinary 2,8-dihydroxyadenine for diagnosis and management of adenine phosphoribosyltransferase deficiency.

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 5000ng/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 6.5min. Measurement of DHA in 24h urine samples from three patients with APRT deficiency, diluted 1:15 (v/v) with 10mM ammonium hydroxide (NH4OH), yielded a concentration of 3021, 5860 and 10563ng/mL and 24h excretion of 816, 1327 and 1649mg, 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.

Cyclodextrin solubilization of carbonic anhydrase inhibitor drugs: formulation of dorzolamide eye drop microparticle suspension.

Topically applied carbonic anhydrase inhibitors (CAIs) are commonly used to treat glaucoma. However, their short duration of action requiring multiple daily dosing can hamper patient compliance. The aim of this study was to develop novel aqueous CAI eye drop formulation containing self-assembled drug/cyclodextrin (D/CD) microparticles that enhance and prolong drug delivery to the eye. Phase-solubility of each drug tested (i.e. methazolamide, brinzolamide and dorzolamide HCl) was determined in either pure water or an aqueous eye drop medium. The pH was adjusted to maximize the fraction of unionized drug. Dorzolamide had the highest affinity for γ-cyclodextrin (γCD) and, thus, was selected for further investigation. Hydroxypropyl methylcellulose (HPMC) was the most effective polymer tested for stabilization of the dorzolamide/γCD complexes and gave the highest mucoadhesion at 0.5% w/v concentration. Thus, the dorzolamide eye drop vehicle containing γCD (18% w/v) and HPMC (0.5% w/v) was developed. The physicochemical properties of this formulation complied with the specifications of the eye drop suspension monograph of the European Pharmacopoeia. The in vivo testing of the formulation showed that the drug was delivered to the aqueous humor in rabbits for at least 24h with the maximum drug concentration at 4h. Furthermore, this formulation delivered the drug to the posterior segment of the eye after topical administration. These results indicate that this CAI eye drop formulation has the potential of being developed into a once-a-day product.