Investigation of the intrinsic cannabinoid activity of hemp-derived and semisynthetic cannabinoids with ß-arrestin2 recruitment assays-and how this matters for the harm potential of seized drugs.

Cultivation of industrial low-Δ9-tetrahydrocannabinol (Δ9-THC) hemp has created an oversupply of cannabidiol (CBD)-rich products. The fact that phytocannabinoids, including CBD, can be used as precursors to synthetically produce a range of THC variants-potentially located in a legal loophole-has led to a diversification of cannabis recreational drug markets. 'Hemp-compliant', 'hemp-derived' and 'semisynthetic' cannabinoid products are emerging and being advertised as (legal) alternatives for Δ9-THC. This study included a large panel (n = 30) of THC isomers, homologs, and analogs that might be derived via semisynthetic procedures. As a proxy for the abuse potential of these compounds, we assessed their potential to activate the CB1 cannabinoid receptor with a ß-arrestin2 recruitment bioassay (picomolar-micromolar concentrations). Multiple THC homologs (tetrahydrocannabihexol, THCH; tetrahydrocannabiphorol, THCP; tetrahydrocannabinol-C8, THC-C8) and THC analogs (hexahydrocannabinol, HHC; hexahydrocannabiphorol, HHCP) were identified that showed higher potential for CB1 activation than Δ9-THC, based on either higher efficacy (Emax) or higher potency (EC50). Structure-activity relationships were assessed for Δ9-THC and Δ8-THC homologs encompassing elongated alkyl chains. Additionally, stereoisomer-specific differences in CB1 activity were established for various THC isomers (Δ7-THC, Δ10-THC) and analogs (HHC, HHCP). Evaluation of the relative abundance of 9(S)-HHC and 9(R)-HHC epimers in seized drug material revealed varying epimeric compositions between batches. Increased abundance of the less active 9(S)-HHC epimer empirically resulted in decreased potency, but sustained efficacy for the resulting diastereomeric mixture. In conclusion, monitoring of semisynthetic cannabinoids is encouraged as the dosing and the relative composition of stereoisomers can impact the harm potential of these drugs, relative to Δ9-THC products.

Standardisation and harmonisation of thyroid-stimulating hormone measurements: historical, current, and future perspectives.

Thyroid-stimulating hormone (TSH) is an important clinical marker in the diagnosis and management of thyroid disease. TSH measurements are reported in milli-International Units per Litre (mIU/L), traceable to a World Health Organisation (WHO) reference material. There is a wide variety of commercial immunoassays for TSH measurements available, which have historically been poorly harmonised due to a lack of commutability of the WHO reference materials with patient samples. This led to the recent development of a serum-based reference panel for TSH, traceable to the WHO reference material, available via the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC), aimed at harmonisation of TSH immunoassays. This report describes recent developments in the TSH reference system, including establishment of the 4th WHO International Standard for TSH, and aims to clarify the relationship between the available reference materials and their intended uses. This 4th WHO IS is widely available and defines the unit of TSH activity, therefore its continued existence is of paramount importance, however it continues to show a lack of commutability with patient in many TSH immunoassays. This makes the C-STFT TSH panel, albeit available in restricted numbers, a critical resource to ensure better TSH assay harmonisation.

In vitro cannabinoid receptor activity, metabolism, and detection in seized samples of CH-PIATA, a new indole-3-acetamide synthetic cannabinoid.

The rapidly evolving synthetic cannabinoid receptor agonist (SCRA) market poses significant challenges for forensic scientists. Since the enactment of a generic ban in China, a variety of new compounds have emerged capable of evading the legislation by carrying new structural features. One recent example of a SCRA with new linker and head moieties is CH-PIATA (CH-PIACA, CHX-PIATA, CHX-PIACA). CH-PIATA bears an additional methylene spacer in the linker moiety between the indole core and the traditional carbonyl component of the linker. This study describes detections in 2022 of this new SCRA in the United States, Belgium, and Scottish prisons. CH-PIATA was detected once in a seized powder by Belgian customs and 12 times in Scottish prisons in infused papers or resin. The metabolites of CH-PIATA were investigated via in vitro human liver microsome (HLM) incubations and eight metabolites were identified, dominated by oxidative biotransformations. A blood sample from the United States was confirmed to contain a mixture of SCRAs including CH-PIATA via presence of the parent and at least five of the metabolites identified from HLM incubations. Furthermore, this paper evaluates the intrinsic in vitro cannabinoid 1 and 2 (CB1 and CB2) receptor activation potential of CH-PIATA reference material and the powder seized by Belgian customs by means of ß-arrestin 2 recruitment assays. Both the reference and the seized powder showed a weak activity at both CB receptors with signs of antagonism found. Based on these results, the expected harm potential of this newly emerging substance remains limited.

Pharmacokinetics of gamma-hydroxybutyric acid in 6-week-old swine (Sus scrofa domesticus) after intravenous and oral administration.

Sedative as well as protective effects during hypoxia have been described for gamma-hydroxybutyric acid (GHB). Six swine (Sus scrofa domesticus) of 6 weeks old were administered NaGHB at a dose of 500 mg/kg intravenously (IV) and 500 and 750 mg/kg orally (PO) in a triple cross-over design. Repeated blood sampling was performed to allow pharmacokinetic analysis of GHB. Whole blood concentration at time point 0 after IV administration was 1727.21 ± 280.73 µg/mL, with a volume of distribution of 339.45 ± 51.41 mL/kg and clearance of 164.94 ± 47.05 mL/(kg h). The mean peak plasma concentrations after PO administration were 326.57 ± 36.70 and 488.01 ± 154.62 µg/mL for 500 mg/kg and 750 mg/kg, respectively. These were recorded at 1.42 ± 0.72 and 1.58 ± 0.58 h after PO dose for GHB 500 mg/kg and 750 mg/kg, respectively. The elimination half-life for IV and PO 500 mg/kg and PO 750 mg/kg dose was respectively 1.33 ± 0.30, 1.16 ± 0.31 and 1.11 ± 0.33 h. The bioavailability (F) for PO administration was 45%. No clinical adverse effects were observed after PO administration. Deep sleep was seen in one animal after IV administration, other animals showed head pressing and ataxia.

Thyroid Stimulating Hormone and Thyroid Hormones (Triiodothyronine and Thyroxine): An American Thyroid Association-Commissioned Review of Current Clinical and Laboratory Status.

Background: Despite being the most performed laboratory endocrine investigation, the optimum use of thyroid tests (thyrotropin [TSH] and thyroid hormone [TH] measurement) is open to question and the interpretation of the results from these tests can be ambiguous. The American Thyroid Association (ATA) with its expertise support the endeavor of the U.S. Centers for Disease Control (CDC) and the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) to improve and maintain standardization and harmonization of thyroid testing. ATA mandated an international interdisciplinary working group panel to survey the status of thyroid testing by reviewing the recent literature to revise or update the criteria as needed in mutual agreement and to inform clinical care. Summary: This review represents the conclusions on the clinical use of current routine TSH and TH (thyroxine [T4] and triiodothyronine [T3]) assays, taking into account geographic differences in disease prevalence and clinical and laboratory practice among writing members. The interaction between physiological, pathophysiological, and pharmacological factors and thyroid assays can affect their measurements and confound result interpretation. These factors need to be considered in the clinical context of the patient for appropriate test ordering and result interpretation. Despite significant advances in laboratory methods over the past 50 years, routine thyroid assays remain susceptible to idiosyncratic analytical interference that may produce spurious results. Improved standardization needs to be demonstrated through ongoing international efforts before results from different assays can be considered equivalent. Emerging technology (e.g., mass spectrometry) shows promise for improved analytical performance, but more evidence of its clinical utility and improved throughput is required before it can be considered for routine use. Close clinical-laboratory collaboration is encouraged to overcome and avoid the pitfalls in thyroid testing as well as resolve clinically discrepant results. The evidence base supporting the conclusions of this review is summarized in four detailed online technical supplements. Conclusions: Over the past five decades, testing for TSH, T4, and T3 has evolved from manual radioisotopic immunoassays to nonisotopic multiplexed immunometric assays using highly automated equipment. Despite these technical advances, physicians and laboratorians performing these analyses must understand limitations of these methods to properly order tests and interpret results.

Practical considerations for accurate determination of free thyroxine by equilibrium dialysis.

Background: Free thyroxine (FT4) measurement is one of the most requested tests in patient care for diagnosing and treating thyroid-related illnesses. Equilibrium dialysis (ED) is considered the "gold standard" for FT4 measurement; however, several factors have a profound effect on the reliability of FT4 assays and require special consideration. Methods: In the current study, we focused on evaluating critical factors that could contribute to reporting errors, such as adsorption of thyroxine (T4) to labware surfaces, stability of serum samples, stock solutions, and calibrator storage conditions, as well as the solvents used to prepare T4 solutions. Results: The adsorption of T4 in ethanolic solutions and dialysates to labware surfaces can be reduced with the careful selection of pipette tips, test tubes, and 96-well plates. Adding pH modifiers to neat T4 solutions can improve its stability. FT4 in serum samples remains stable after exposure to four freeze-thaw cycles, 5 °C for 18-20 h, or -70 °C for a minimum of three years. Conclusion: The presented study has demonstrated that the loss of analyte due to pre-analytical and analytical factors during operation of the FT4 reference measurement procedure (RMP) can be minimized by careful selection of all labware for sample preparation. It was found that the accuracy and imprecision of FT4 assays can be influenced by different types of dialysis devices, but acceptable alternatives to ED membranes were identified. This study demonstrates approaches to establish a FT4 method that is independent from specific suppliers and addresses critical pre-analytical and analytical factors important for FT4 measurements.

Development of an equilibrium dialysis ID-UPLC-MS/MS candidate reference measurement procedure for free thyroxine in human serum.

BACKGROUND: Accurate and reliable measurement of human serum free thyroxine (FT4) is critical for the diagnosis and treatment of thyroid diseases. However, concerns have been raised regarding the performance of FT4 measurements in patient care. Centers for Disease Control and Prevention Clinical Standardization Programs (CDC-CSP) address these concerns by creating a FT4 standardization program to standardize FT4 measurements. The study aims to develop a highly accurate and precise candidate Reference Measurement Procedure (cRMP), as one key component of CDC-CSP, for standardization of FT4 measurements. METHODS: Serum FT4 was separated from protein-bound thyroxine with equilibrium dialysis (ED) following the recommended conditions in the Clinical and Laboratory Standards Institute C45-A guideline and the published RMP [20,21,23]. FT4 in dialysate was directly quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. Gravimetric measurements of specimens and calibrator solutions, calibrator bracketing, isotope dilution, enhanced chromatographic resolution, and T4 specific mass transitions were used to ensure the accuracy, precision, and specificity of the cRMP. RESULTS: The described cRMP agreed well with the established RMP and two other cRMPs in an interlaboratory comparison study. The mean biases of each method to the overall laboratory mean were within ±2.5%. The intra-day, inter-day, and total imprecision for the cRMP were within 4.4%. The limit of detection was 0.90 pmol/L, which was sufficiently sensitive to determine FT4 for patients with hypothyroidism. The structural analogs of T4 and endogenous components in dialysate did not interfere with the measurements. CONCLUSION: Our ED-LC-MS/MS cRMP provides high accuracy, precision, specificity, and sensitivity for FT4 measurement. The cRMP can serve as a higher-order standard for establishing measurement traceability and provide an accuracy base for the standardization of FT4 assays.

Description and validation of an equilibrium dialysis ID-LC-MS/MS candidate reference measurement procedure for free thyroxine in human serum.

OBJECTIVES: Free thyroxine (FT4) in serum is routinely measured in clinical practice to diagnose and monitor thyroid disease. Due to its concentration in picomolar range and the delicate equilibrium of free and protein-bound T4, accurate measurement is challenging. As a consequence, large inter-method differences in FT4 results exists. Optimal method design and standardization of the FT4 measurement is therefore necessary. The IFCC Working Group for Standardization of Thyroid Function Tests proposed a reference system with a conventional reference measurement procedure (cRMP) for FT4 in serum. In this study, we describe our FT4 candidate cRMP and its validation in clinical samples. METHODS: This candidate cRMP is based on equilibrium dialysis (ED) combined with determination of T4 with an isotope-dilution liquid chromatography tandem mass-spectrometry (ID-LC-MS/MS) procedure and was developed according to the endorsed conventions. Its accuracy, reliability, and comparability was investigated using human sera. RESULTS: It was shown that the candidate cRMP adhered to the conventions and its accuracy, precision, and robustness were adequate in serum of healthy volunteers. CONCLUSIONS: Our candidate cRMP measures FT4 accurately and performs well in serum matrix.

Liquid chromatography-tandem mass spectrometry for therapeutic drug monitoring of immunosuppressants and creatinine from a single dried blood spot using the Capitainer® qDBS device.

In recent years, a lot of attention has been given to a more patient-centric therapeutic drug monitoring (TDM) of immunosuppressant drugs (tacrolimus, sirolimus, everolimus and cyclosporin A) by the use of microsampling techniques. By adopting Dried Blood Spots (DBS) after a finger prick, instead of conventional venous blood draws, follow-up can (partially) be established from patients' homes. Despite the many advantages of DBS, one of the major disadvantages associated with this technique is the well described hematocrit (hct) effect. In order to overcome the hct area bias, different strategies have been proposed, amongst which the use of dried blood sampling techniques based on the volumetric collection of blood. The aim of this study was to evaluate the use of the Capitainer® qDBS (quantitative Dried Blood Spot) device for the combined TDM of four immunosuppressants and creatinine from a single qDBS. The set-up of an adequate sample preparation allowing both immunosuppressants and creatinine quantification was one of the key challenges in the method development due to device-specific interferences. Liquid chromatography tandem-mass spectrometry methods for the quantification of tacrolimus, sirolimus, everolimus, cyclosporin A and creatinine from qDBS (10 µL) were developed and validated based on international guidelines, also taking into account DBS-specific parameters. The methods proved to be accurate and reproducible, with absolute biases below 10% and within-run CVs (%) below 8% over a calibration range from 1 to 50 ng/mL for tacrolimus, sirolimus and everolimus, 20-1500 ng/mL for cyclosporin A, and 15-700 µmol/L for creatinine. Reproducible (CV < 15%) IS-compensated relative recovery values were obtained, showing no hematocrit-dependence (compared to a hct of 0.37), except for cyclosporin A at higher hct values. Application on venous blood left-over patient samples showed good agreement between the results of Capitainer® qDBS and whole blood with 98% (47/48), 93% (41/44), 89% (41/46), 88% (38/43) and 89% (116/131) of the samples lying within 20% of the whole blood result for tacrolimus, sirolimus, everolimus, cyclosporin A and plasma/serum for creatinine, respectively. For creatinine a blood/plasma ratio of 0.85 was found and used to convert qDBS results to plasma/serum results. As a next step, capillary finger prick samples will need to demonstrate the clinical applicability of the method in a real life setting.

Cov2MS: An Automated and Quantitative Matrix-Independent Assay for Mass Spectrometric Measurement of SARS-CoV-2 Nucleocapsid Protein.

The pandemic readiness toolbox needs to be extended, targeting different biomolecules, using orthogonal experimental set-ups. Here, we build on our Cov-MS effort using LC-MS, adding SISCAPA technology to enrich proteotypic peptides of the SARS-CoV-2 nucleocapsid (N) protein from trypsin-digested patient samples. The Cov2MS assay is compatible with most matrices including nasopharyngeal swabs, saliva, and plasma and has increased sensitivity into the attomole range, a 1000-fold improvement compared to direct detection in a matrix. A strong positive correlation was observed with qPCR detection beyond a quantification cycle of 30-31, the level where no live virus can be cultured. The automatable sample preparation and reduced LC dependency allow analysis of up to 500 samples per day per instrument. Importantly, peptide enrichment allows detection of the N protein in pooled samples without sensitivity loss. Easily multiplexed, we detect variants and propose targets for Influenza A and B detection. Thus, the Cov2MS assay can be adapted to test for many different pathogens in pooled samples, providing longitudinal epidemiological monitoring of large numbers of pathogens within a population as an early warning system.

A new cannabinoid receptor 1 selective agonist evading the 2021 "China ban": ADB-FUBIATA.

Following the class-wide ban of synthetic cannabinoid receptor agonists (SCRAs) in China, SCRAs carrying new core and linker structures, aimed at circumventing the recent Chinese generic legislation, have appeared on the recreational drug market. A very recent example is (S)-2-(2-(1-(4-fluorobenzyl)-1H-indol-3-yl)acetamido)-3,3-dimethylbutanamide (ADB-FUBIATA), which is structurally closely related to the potent SCRA ADB-FUBICA, but carries an additional methylene in the linker region of the molecule. ADB-FUBIATA has recently been identified in seized materials in China, Russia, the United States, and also Belgium; however, its pharmacological characteristics were unknown. The aim of this study was to evaluate the intrinsic cannabinoid receptor (hCB1 and hCB2 ) activation potential of this previously unknown substance via two distinct yet similar in vitro ß-arrestin2 recruitment assays, based on the NanoLuc Binary Technology®. At CB1 , a potency of 635 nM (EC50 ) was found, with an efficacy (Emax ) of 141% relative to the reference compound CP55,940. On the other hand, ADB-FUBIATA had almost no activity at CB2 , indicative of a clear CB1 selectivity. Interestingly, this activation pattern differs markedly from that observed for ADB-FUBICA, which was previously found to be potent and efficacious at both cannabinoid receptors. Additionally, the bioassays were applied to a seized powder containing ADB-FUBIATA, as analytically confirmed by high-performance liquid chromatography coupled to diode-array detection (HLPC-DAD), gas chromatography coupled to mass spectrometry (GC-MS), liquid chromatography couple to time-of-flight mass spectrometry (LC-QTOF-MS), Fourier transform infrared spectroscopy (FTIR), and nuclear magnetic resonance (NMR). The EC50 and Emax values obtained for this powder were very similar to those of the ADB-FUBIATA analytical standard, suggesting a high purity of the powder, although analytical techniques did reveal that the sample was not entirely pure.

Cannabinoid receptor activation potential of the next generation, generic ban evading OXIZID synthetic cannabinoid receptor agonists.

In recent years, several nations have implemented various measures to control the surge of new synthetic cannabinoid receptor agonists (SCRAs) entering the recreational drug market. In July 2021, China put into effect a new generic legislation, banning SCRAs containing one of seven general core scaffolds. However, this has driven manufacturers towards the synthesis of SCRAs with alternative core structures, exemplified by the recent emergence of "OXIZID SCRAs." Here, using in vitro ß-arrestin2 recruitment assays, we report on the CB1 and CB2 potency and efficacy of five members of this new class of SCRAs: BZO-HEXOXIZID, BZO-POXIZID, 5-fluoro BZO-POXIZID, BZO-4en-POXIZID, and BZO-CHMOXIZID. All compounds behaved as full agonists at CB1 and partial agonists at CB2 . Potencies ranged from 84.6 to 721 nM at CB1 and 2.21 to 25.9 nM at CB2 . Shortening the n-hexyl tail to a pentyl tail enhanced activity at both receptors. Fluorination of this pentyl analog did not yield a higher receptor activation potential, whereas an unsaturated tail resulted in decreased potency and efficacy at CB1 . The cyclohexyl methyl analog BZO-CHMOXIZID was the most potent compound at both receptors, with EC50 values of 84.6 and 2.21 nM at CB1 and CB2 , respectively. Evaluation of the activity of a seized powder containing BZO-4en-POXIZID suggested a high purity, in line with high-performance liquid chromatography coupled to diode-array detection (HPLC-DAD), gas chromatography coupled to mass spectrometry (GC-MS), liquid chromatography coupled to time-of-flight mass spectrometry (LC-QTOF-MS), and Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) analysis. Furthermore, all tested compounds showed a preference for CB2 , except for BZO-POXIZID. Overall, these findings inform public health officials, law enforcement agencies, and clinicians on these newly emerging SCRAs.

Set-up of a population-based model to verify alcohol abstinence via monitoring of the direct alcohol marker phosphatidylethanol 16:0/18:1.

BACKGROUND AND AIMS: Phosphatidylethanol 16:0/18:1 (PEth) is a biomarker for alcohol intake. It has a half-life of 7.9 days. Chronic alcohol consumption causes high PEth values. It can take weeks before PEth values fall below the decision limit for 'alcohol abstinence'. Our aim was to validate whether alcohol abstinence can be determined based on two consecutive PEth results above the decision limit. DESIGN: Observational study. SETTING: Belgium, February 2019. The study was linked to a social initiative in Belgium, 'Tournée Minérale'. PARTICIPANTS: Adults (aged > 18 years, n = 796) with varying drinking habits who self-reportedly refrained from alcohol consumption during the study. MEASUREMENTS: A validated liquid chromatography-tandem mass spectrometry method was used to quantify PEth in participants' dried blood samples, collected at three time-points via remote fingerprick-based self-sampling. FINDINGS: A population-based algorithm to evaluate abstinence based on 95% prediction limits was developed by fitting a linear mixed-effect model to discern patterns in PEth elimination over time. It took intra- and inter-individual variability into consideration. The algorithm was included in a two-step decision tree, assessing whether (i) PEth values fell within the prediction interval and (ii) the slope between two PEth values was consistent with no alcohol consumption. Data for 74 participants reporting no alcohol intake during the study were used for validation. With a detection limit of 'four units spread over 14 days', the sensitivity and specificity of the decision tree was 89%. CONCLUSIONS: Claims of alcohol abstinence can be verified using a two-step decision tree for phosphatidylethanol 16:0/18:1 values, even when those values are above the limit for 'alcohol abstinence'.

Optimizing Available Tools for Achieving Result Standardization: Value Added by Joint Committee on Traceability in Laboratory Medicine (JCTLM).

BACKGROUND: The JCTLM created a Task Force on Reference Measurement System Implementation (TF-RMSI) to provide guidance on metrological traceability implementation for the in vitro diagnostics (IVD) community. CONTENT: TF-RMSI investigated the reference measurement systems (RMS) for 13 common measurands by applying the following procedural steps: (a) extracting data from the JCTLM database of available certified reference materials (CRMs) and reference measurement procedures (RMPs); (b) describing the RMS to which each recruited CRM or RMP belongs; (c) identifying the intended use of the CRMs, and, if used as a common calibrator for IVD measuring systems and/or trueness assessment of field methods was included, checking the CRM's certificate for information about commutability with clinical samples; and (d) checking if the CRM or RMP measurement uncertainty (MU) has the potential to be small enough to avoid significantly affecting the analytical performance specifications (APS) for MU of clinical sample results when the MU from the IVD calibrator and from the end-user measuring system were combined. SUMMARY: We produced a synopsis of JCTLM-listed higher-order CRMs and RMPs for the selected measurands, including their main characteristics for implementing traceability and fulfilling (or not) the APS for suitable MU. Results showed that traceability to higher-order references can be established by IVD manufacturers within the defined APS for most of the 13 selected measurands. However, some measurands do not yet have suitable CRMs for use as common calibrators. For these measurands, splitting clinical samples with a laboratory performing the RMP may provide a practical alternative for establishing a calibration hierarchy.

Cov-MS: A Community-Based Template Assay for Mass-Spectrometry-Based Protein Detection in SARS-CoV-2 Patients.

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.

Porphyrins produced by acneic Cutibacterium acnes strains activate the inflammasome by inducing K+ leakage.

Some Cutibacterium acnes subgroups dominate on healthy skin, whereas others are frequently acne associated. Here we provide mechanistic insights into this difference, using an anaerobic keratinocyte-sebocyte-C. acnes co-culture model. An acneic C. acnes strain as well as its porphyrins activates NRLP3 inflammasome assembly, whereas this was not observed with a non-acneic strain. Low levels of intracellular K+ in keratinocytes stimulated with extracted porphyrins or infected with the acneic strain were observed, identifying porphyrin-induced K+ leakage as trigger for inflammasome activation. Using a panel of C. acnes strains, we found that porphyrin production and IL-1ß release are correlated and are higher in acneic strains. This demonstrates that the latter produce more porphyrins, which interact with the keratinocyte cell membrane, leading to K+ leakage, NLRP3 inflammasome activation, and IL-1ß release and provides an explanation for the observation that some C. acnes strains are associated with healthy skin, whereas others dominate in acneic skin.