Potassium-based algorithm allows correction for the hematocrit bias in quantitative analysis of caffeine and its major metabolite in dried blood spots

Although dried blood spot (DBS) sampling is increasingly receiving interest as a potential alternative to traditional blood sampling, the impact of hematocrit (Hct) on DBS results is limiting its final breakthrough in routine bioanalysis. To predict the Hct of a given DBS, potassium (K+) proved to be a reliable marker. The aim of this study was to evaluate whether application of an algorithm, based upon predicted Hct or K+ concentrations as such, allowed correction for the Hct bias. Using validated LC-MS/MS methods, caffeine, chosen as a model compound, was determined in whole blood and corresponding DBS samples with a broad Hct range (0.18-0.47). A reference subset (n = 50) was used to generate an algorithm based on K+ concentrations in DBS. Application of the developed algorithm on an independent test set (n = 50) alleviated the assay bias, especially at lower Hct values. Before correction, differences between DBS and whole blood concentrations ranged from -29.1 to 21.1 %. The mean difference, as obtained by Bland-Altman comparison, was -6.6 % (95 % confidence interval (CI), -9.7 to -3.4 %). After application of the algorithm, differences between corrected and whole blood concentrations lay between -19.9 and 13.9 % with a mean difference of -2.1 % (95 % CI, -4.5 to 0.3 %). The same algorithm was applied to a separate compound, paraxanthine, which was determined in 103 samples (Hct range, 0.17-0.47), yielding similar results. In conclusion, a K+-based algorithm allows correction for the Hct bias in the quantitative analysis of caffeine and its metabolite paraxanthine

New regulatory molecules in growth and invasion of melanoma and breast cancer: cadherins, heregulin and follistatin

Kanker is vaak het resultaat van een meerstapsproces, waarbij normale cellen de eigenschap verworven hebben te overleven, ongebreideld te delen en naburig weefsel te invaderen, wat kan leiden tot metastase. In deze studie hebben we onderzocht welke rol groeifactor- systemen en cel-celadhesiemoleculen kunnen spelen in melanoom en borstkanker. Een cruciale factor in het proces van carcinogenese is de wederzijdse communicatie tussen de tumor en de micro-omgeving die tot stand kan worden gebracht door oplosbare factoren. Op basis van de identificatie van twee glycoprotes die vrijgesteld werden door melanoomcellen, karakteriseerden we twee nieuwe groeifactor- systemen in humaan melanoom die beide kunnen bijdragen tot maligne progressie. Het gebruik van massaspectrometrie als een biochemische benadering leidde tot de identificatie van follistatine als een factor die werd vrijgesteld door verscheidene melanoomcellijnen. Vermits we zagen dat follistatine melanocyten kon beschermen tegen de groeiremmende en pro-apoptotische effecten van activine (een transformerende groeifactor-b-achtige ligand), kan secretie ervan mogelijk bijdragen tot het overleven van getransformeerde melanocytische cellen. Uitgaand van een biologische benadering vonden we dat een door melanoom geconditioneerd medium activering van een 185 kD prote kon induceren in een testcellijn. Deze activiteit kon toegeschreven worden aan de aanwezigheid van hereguline, een epidermale groeifactor-achtige ligand voor receptor tyrosine kinases. De daaropvolgende evaluatie van het hereguline-humaan epidermale groeifactor receptorsysteem in een set van melanoomcellijnen toonde de aanwezigheid van meerdere ontregelingen aan, gaande van autocriene activering tot ligandongevoeligheid te wijten aan de afwezigheid van (functionele) receptoren. Wanneer hereguline getest werd in een in vitro model van invasie en aggregatie, gebruik makend van een borstkankercellijn met een functioneel deficiꭴ cel-celadhesiecomplex, vonden we dat invasie werd tegengegaan, terwijl aggregatie bevorderd werd. Cruciaal voor cel-celcontacten tussen epitheliale cellen zijn cadherines, calcium-afhankelijke cel-celadhesie-moleculen die betrokken zijn in de regeling van de beweeglijkheid, groei, differentiatie en overleving van cellen. E(epitheliaal)-cadherine, het prototype van deze familie, bezit een goed beschreven invasie-onderdrukkende functie. Functionele inactivering van E-cadherine kan ontstaan op zowel genomisch, transcriptioneel, als post-transcriptioneel niveau. Een voorbeeld van dit laatste is de proteolytische klieving van E-cadherine, met een vrijstelling van zijn ectodomein als resultaat, waarbij we vaststelden dat dit ectodomein invasie bevorderde en aggregatie tegenwerkte. In tegenstelling tot E-cadherine, dat reeds intensief werd bestudeerd in kanker, is er slechts weinig gekend over de rol van het sterk verwante P-cadherine in kanker. Wij stelden vast dat P-cadherine, afhankelijk van de context, zowel als een invasie-onderdrukker als een invasie-promotor kan functioneren. Melanoomcellen verliezen P-cadherine tijdens progressie. Het herstel van deze expressie resulteerde in een verbeterde cel-celadhesie en een opheffing van hun invasief vermogen. In borstkanker daarentegen, correleert de opregulatie van P-cadherine met oestrogeenreceptor negativiteit en zwakke overleving. We legden een rechtstreekse link tussen P-cadherine expressie en het gebrek aan oestrogeenreceptorsignalering en vonden dat P-cadherine, via zijn intracellulair membraan-proximaal domein, invasie bevorderde in borstkankercellen. Cancer is often the result of a multi-step process in which normal cells have acquired the ability to survive and proliferate limitlessly and can invade the surrounding tissue, which may lead to metastasis. In this study, we addressed the role that growth factor systems and cell-cell adhesion molecules may play in melanoma and breast cancer. Crucial in the process of carcinogenesis is the mutual cross-talk between the tumor and the micro-environment, an interaction which may be exerted by soluble factors. Based on the identification of two glycoproteins that were secreted by melanoma cells, we characterized two new growth factor systems in human melanoma. Both of these may contribute to malignant progression. The use of mass spectrometry as a biochemical approach led to the identification of follistatin as a factor secreted by several melanoma cell lines. Since we found that follistatin can protect melanocytes against the growth-inhibitory and pro-apoptotic effects of activin (a transforming growth factor-?-like ligand), its secretion may promote the survival of transformed melanocytic cells. Using a biological approach as a starting point, we found that a melanoma conditioned medium can induce activation of a 185kD protein in a test cell line. This activity was attributed to the presence of heregulin, an epidermal growth factor-like ligand for receptor tyrosine kinases. Subsequent evaluation of the heregulin-human epidermal growth factor receptor system in a panel of melanoma cell lines revealed the presence of multiple deregulations. These included both autocrine activation and ligand insensitivity owing to the absence of (functional) receptors. When heregulin was tested in an in vitro model of invasion and aggregation, using a breast cancer cell line with a functionally deficient cell-cell adhesion complex, we found that invasion is counteracted, whereas aggregation is promoted. Crucial for cell-cell contacts between epithelial cells are cadherins, calcium-dependent cell-cell adhesion molecules that are involved in the regulation of cell motility, growth, differentiation and survival. E(epithelial)-cadherin, the prototype member of this family, has a well-described invasion-suppressor function. Functional inactivation of E-cadherin may occur at the genomic, transcriptional, and post-transcriptional level. An example of the latter is proteolytic cleavage of E-cadherin, resulting in release of its ectodomain, which we found to be capable of promoting invasion and counteracting aggregation. In contrast to E-cadherin, which has been extensively studied in cancer, little is known about the role of the highly related P-cadherin in cancer. We found that P-cadherin may either function as an invasion-suppressor or as an invasion promoter, depending on the context. Melanoma cells lose P-cadherin expression during progression. Reconstituting this expression led to improved cell-cell adhesion and counteraction of invasion. In breast cancer, in contrast, upregulation of P-cadherin correlates with estrogen receptor negativity and poor survival. We established a direct link between P-cadherin expression and the lack of estrogen receptor signaling and found that P-cadherin, via its intracellular membrane-proximal domain, promotes invasion of breast cancer cells

Why dried blood spots are an ideal tool for CYP1A2 phenotyping

Background and Objective: Dried blood spot (DBS) sampling has gained wide interest in bioanalysis during the last decade and has already been successfully applied in pharmacokinetic and phenotyping studies. However, all of the available phenotyping studies used small datasets and did not include a systematic evaluation of DBS-specific parameters. The latter is important since several of these factors still challenge the breakthrough of DBS in routine practice. In this study, caffeine and paraxanthine are determined in capillary DBS, venous DBS, whole blood and plasma for cytochrome P450 (CYP) 1A2 phenotyping. The aim of this study was to explore the usefulness of DBS as a tool for CYP1A2 phenotyping. Methods: A CYP1A2 phenotyping study was conducted in 73 healthy volunteers who received a 150 mg oral dose of caffeine. Six hours post-administration, caffeine and paraxanthine concentrations and paraxanthine:caffeine molar concentration ratios, i.e., the actual CYP1A2 phenotyping indices, were determined in capillary DBS (obtained by non-volumetric application, direct from the fingertip), venous DBS, whole blood, and plasma. Furthermore, the impact of DBS-specific parameters, including hematocrit, volume spotted, and punch location, was evaluated. Results: Concentrations of caffeine and paraxanthine in capillary DBS were, respectively, on average 12.7 and 13.8 % lower than those in venous DBS and 31.5 and 33.1 % lower than those in plasma. While these differences were statistically significant (p = 0.053). This ratio also alleviated the impact of hematocrit and volume spotted. Conclusions: Using the largest DBS-based phenotyping study to date, we have demonstrated that CYP1A2 phenotyping in capillary DBS is a valid and convenient alternative for the classical plasma-based approach. Additionally, we have provided an objective basis as to why DBS are an ideal tool for CYP1A2 phenotyping

Determination of gamma-hydroxybutyric acid in dried blood spots using a simple GC-MS method with direct 'on spot' derivatization

The objective of this study was the development of an accurate and sensitive method for the determination of gamma-hydroxybutyric acid (GHB) in dried whole blood samples using a GC-MS method. The complete procedure was optimized, with special attention for the sample pre-treatment, and validated. Therefore, dried blood spots (DBS) of only 50 µl were prepared and, after addition of internal standard GHB-d6, directly derivatized using 100 µl of a freshly prepared mixture of trifluoroacetic acid anhydride (TFAA) and heptafluorobutanol (HFB-OH) (2:1). The derivatized extract was injected into a gas chromatograph coupled to a mass spectrometer (GC-MS), operating in the electron impact mode (EI), with a total run time of 12.3 min. Method validation included the evaluation of linearity, precision, accuracy, sensitivity, selectivity and stability. A weighting factor of 1/x2 was chosen and acceptable intra-batch precision, inter-batch precision and accuracy were seen. The linear calibration curve ranged from 2 to 100 µg/ml, with a limit of detection of 1 µg/ml. Our procedure, utilizing the novel approach of direct “on spot” derivatization, followed by analysis with GC-MS, proved to be reliable, fast and applicable in routine toxicology

Activity-based detection of consumption of synthetic cannabinoids in authentic urine samples using a stable cannabinoid reporter system

Synthetic cannabinoids (SCs) continue to be the largest group of new psychoactive substances (NPS) monitored by the European Monitoring Center of Drugs and Drugs of Abuse (EMCDDA). The identification and subsequent prohibition of single SCs has driven clandestine chemists to produce analogues of increasing structural diversity, intended to evade legislation. That structural diversity, combined with the mostly unknown metabolic profiles of these new SCs, poses a big challenge for the conventional targeted analytical assays, as it is difficult to screen for "unknown" compounds. Therefore, an alternative screening method, not directly based on the structure but on the activity of the SC, may offer a solution for this problem. We generated stable CB1 and CB2 receptor activation assays based on functional complementation of a split NanoLuc luciferase and used these to test an expanded set of recent SCs (UR-144, XLR-11, and their thermal degradation products; AB-CHMINACA and ADB-CHMINACA) and their major phase I metabolites. By doing so, we demonstrate that several major metabolites of these SCs retain their activity at the cannabinoid receptors. These active metabolites may prolong the parent compound's psychotropic and physiological effects and may contribute to the toxicity profile. Utility of the generated stable cell systems as a first -line screening tool for SCs in urine was also demonstrated using a relatively large set of authentic urine samples. Our data indicate that the stable CB reporter assays detect CB receptor, activation by extracts of urine in which SCs (or their metabolites) are present at low- or subnanomolar (ng/mL) level. Hence, the developed assays do not only allow activity profiling of SCs and their metabolites, it may also serve as a screening tool, complementing targeted and untargeted analytical assays and preceding analytical (mass spectrometry based) confirmation

A novel nanobody-based bio-assay using functional complementation of a split nanoluciferase to monitor Mu- opioid receptor activation

The Mu opioid receptor (MOR) has been the subject of intense research over the past decades, especially in the field of analgesic therapeutics. It is the primary target for both clinical and recreational opioids. Recently, camelid-derived nanobodies have received significant attention due to their applicability in stabilizing the crystal structure of activated MOR, via specific recognition of and binding to the active receptor conformation. In the present study, we developed and applied a novel bio-assay to monitor MOR activation, utilizing intracellular expression of one such nanobody, Nb39. The principle of functional complementation of a split nanoluciferase was used to assess recruitment of Nb39 to MOR, following activation by a set of five synthetic opioids. The obtained pharmacological parameters—negative logarithm of EC50 (pEC50, as a measure of potency) and maximal response provoked by a ligand (Emax, as a measure of efficacy; relative to hydromorphone)—were compared with those obtained using a G protein recruitment assay, in which a mini-Gi protein (engineered GTPase domain of Gαi subunit) is recruited to activated MOR. Similar EC50 but distinct Emax values were obtained with both bio-assays, with lower Emax values for the Nbbased bio-assay. Both bio-assays may assist to gain better insight into activation of the MOR

Volumetric absorptive microsampling as an alternative tool for therapeutic drug monitoring of first-generation anti-epileptic drugs

Dosage adjustment of anti-epileptic drugs by therapeutic drug monitoring (TDM) is very useful, especially for the first-generation anti-epileptic drugs (AEDs). Microsampling-the collection of small volumes of blood-is increasingly considered a valuable alternative to conventional venous sampling for TDM. Volumetric absorptive microsampling (VAMS) allows accurate and precise collection of a fixed volume of blood, eliminating the volumetric blood hematocrit bias coupled to conventional dried blood spot collection. The aim of this study was to develop and validate an LC-MS/MS method for the determination and quantification of four anti-epileptic drugs (carbamazepine, valproic acid, phenobarbital, and phenytoin) and one active metabolite (carbamazepine-10,11-epoxide) in samples collected by VAMS. The method was fully validated based on international guidelines. Precision (%RSD) was below 10%, while, with a single exception, accuracy (%bias) met the acceptance criteria. Neither carry-over nor unacceptable interferences were observed, the method being able to distinguish between the isomers oxcarbazepine and carbamazepine-10,11-epoxide. All compounds were stable in VAMS samples for at least 1 month when stored at room temperature, 4 A degrees C, and - 20 A degrees C and for at least 1 week when stored at 60 A degrees C. Internal standard-corrected matrix effects were below 10%, with %RSDs below 4%. High (> 85%) recovery values were obtained and the effect of the hematocrit on the recovery was overall limited. Successful application on external quality control materials and on left-over patient samples demonstrated the validity and applicability of the developed procedure

Feasibility of following up gamma-hydroxybutyric acid concentrations in sodium oxybate (Xyrem®)-treated narcoleptic patients using dried blood spot sampling at home : an exploratory study

Background: Gamma-hydroxybutyric acid (GHB), well known as a party drug, especially in Europe, is also legally used (sodium oxybate, Xyrem (R)) to treat a rare sleep disorder, narcolepsy with cataplexy. This exploratory study was set up to measure GHB concentrations in dried blood spots (DBS) collected by narcoleptic patients treated with sodium oxybate. Intra- and inter-individual variation in clinical effects following sodium oxybate administration has been reported. The use of DBS as a sampling technique, which is stated to be easy and convenient, may provide a better insight into GHB concentrations following sodium oxybate intake in a real-life setting. Objective The aim was twofold: evaluation of the applicability of a recently developed DBS-based gas chromatography mass spectrometry (GC MS) method, and of the feasibility of the sampling technique in an ambulant setting. Methods: Seven narcoleptic patients being treated with sodium oxybate at the Department for Respiratory Diseases of Ghent University Hospital were asked to collect DBS approximately 20 min after the first sodium oxybate (Xyrem (R); UCB Pharma Ltd, Brussels, Belgium) intake on a maximum of 7 consecutive days. Using an automatic lancet, patients pricked their fingertip and, after wiping off the first drop of blood, subsequent drops were collected on a DBS card. The DBS cards were sent to the laboratory by regular mail and, before analysis, were visually inspected to record DBS quality (large enough, symmetrically spread on the filter paper with even colouration on both sides of the filter paper). Results: Of the seven patients, three patients succeeded to collect five series of DBS, one patient decided to cease participation because of nausea, one was lost during follow-up and two patients started falling asleep almost immediately after the intake of sodium oxybate. Analysing the DBS in duplicate resulted in acceptable within-DBS card precision. DBS with acceptable quality were obtained by patients without supervision. Conclusion: Our results demonstrate the acceptable precision of the complete procedure, from sampling at home to quantitative analysis in the laboratory. Given the intra-and inter-individual variability in clinical effects seen with sodium oxybate, the easy adaptation of DBS sampling opens the possibility of following up GHB concentrations in patients in real-life settings in future studies

A novel, nondestructive, dried blood spot-based hematocrit prediction method using noncontact diffuse reflectance spectroscopy

Dried blood spot (DBS) sampling is recognized as a valuable alternative sampling strategy both in research and in clinical routine. Although many advantages are associated with DBS sampling, its more widespread use is hampered by several issues, of which the hematocrit effect on DBS-based quantitation remains undoubtedly the most widely discussed one. Previously, we developed a method to derive the approximate hematocrit from a nonvolumetrically applied DBS based on its potassium content. Although this method yielded good results and was straightforward to perform, it was also destructive and required sample preparation. Therefore, we now developed a nondestructive method which allows to predict the hematocrit of a DBS based on its hemoglobin content, measured via noncontact diffuse reflectance spectroscopy. The developed method was thoroughly validated. A linear calibration curve was established after log/log transformation. The bias, intraday and interday imprecision of quality controls at three hematocrit levels and at the lower and upper limit of quantitation (0.20 and 0.67, respectively) were less than 11%. In addition, the influence of storage and the volume spotted was evaluated, as well as DBS homogeneity. Application of the method to venous DBSs prepared from whole blood patient samples (n = 233) revealed a good correlation between the actual and the predicted hematocrit. Limits of agreement obtained after Bland and Altman analysis were -0.076 and. +0.018. Incurred sample reanalysis demonstrated good method reproducibility. In conclusion, mere scanning of a DBS suffices to derive its approximate hematocrit, one of the most important variables in DBS analysis