Quantification of Letrozole, Palbociclib, Ribociclib, Abemaciclib, and Metabolites in Volumetric Dried Blood Spots: Development and Validation of an LC-MS/MS Method for Therapeutic Drug Monitoring.

Therapeutic drug monitoring (TDM) may be beneficial for cyclin-dependent kinase 4/6 inhibitors (CDK4/6is), such as palbociclib, ribociclib, and abemaciclib, due to established exposure-toxicity relationships and the potential for monitoring treatment adherence. Developing a method for quantifying CDK4/6is, abemaciclib metabolites (M2, M20), and letrozole in dried blood spots (DBS) could be useful to enhance the feasibility of TDM. Thus, an optimized LC-MS/MS method was developed using the HemaXis DB10 device for volumetric (10 µL) DBS collection. Chromatographic separation was achieved using a reversed-phase XBridge BEH C18 column. Detection was performed with a triple quadrupole mass spectrometer, utilizing ESI source switching between negative and positive ionization modes and multiple reaction monitoring acquisition. Analytical validation followed FDA, EMA, and IATDMCT guidelines, demonstrating high selectivity, adequate sensitivity (LLOQ S/N ≥ 30), and linearity (r ≥ 0.997). Accuracy and precision met acceptance criteria (between-run: accuracy 95-106%, CV ≤ 10.6%). Haematocrit independence was confirmed (22-55%),with high recovery rates (81-93%) and minimal matrix effects (ME 0.9-1.1%). The stability of analytes under home-sampling conditions was also verified. Clinical validation supports DBS-based TDM as feasible, with conversion models developed for estimating plasma concentrations (the reference for TDM target values) of letrozole, abemaciclib, and its metabolites. Preliminary data for palbociclib and ribociclib are also presented.

Metabolic signatures of metabolites of the purine degradation pathway in human plasma using HILIC UHPLC-HRMS.

The metabolic disorders in the purine degradation pathway have proven to be closely associated with several human diseases. However, the etiology is not yet fully understood. Profile assay of purine intermediates and uric acid involved in the metabolic pathway can provide additional insight into the nature and severity of related diseases. Purine metabolites are endogenous chemicals with high hydrophilicity, polarity, and similar structures, thus there is a great need for a specific method to quantify them directly in biological fluids with a short running time. Herein, eight purine degradation pathway metabolites, including xanthine, hypoxanthine, guanine, xanthosine, inosine, guanosine, adenosine and uric acid, in human plasma were quantitatively measured using hydrophilic interaction chromatography-tandem high-resolution mass spectrometry (HILIC-HRMS) in a short running time of 10 min. The method was systematically validated for specificity, linearity of the calibration curve, the limit of detection, the limit of quantification, the lower limit of quantification, precision, accuracy, extraction recovery, matrix effect, and stability. The results showed that the method was linear (R2 > 0.99), accurate (the intra- and inter-day recoveries of all analytes ranged from 90.0 % to 110.0 %), and precise (the intra- and inter-day precisions were less than 6.7 % and 8.9 %, respectively) with the lower limits of quantification ranging from 3 to 10,000 ng/mL. The extraction recoveries and matrix effects were repeatable and stable. All the analytes were stable in the autosampler and could be subject to three freeze-thaw cycles. The developed method was ultimately applied to 100 plasma specimens from healthy individuals. The results showed that the concentrations of different purine metabolites varied dramatically in plasma specimens. Diet and body mass index (BMI) were the most significant factors determining purine levels, followed by drinking and sex. Age, smoking and bedtime showed a very weak correlation with purine metabolism. The findings of the present work reveal the characteristics of purine metabolism in human plasma under non-pathological conditions. The results also highlight the factors that can cause changes in purine metabolism, which are useful in developing effective treatment strategies for metabolic disorders of purines, particularly for those caused by lifestyle factors.

Therapeutic Monitoring of Palbociclib, Ribociclib, Abemaciclib, M2, M20, and Letrozole in Human Plasma: A Novel LC-MS/MS Method.

BACKGROUND: Therapeutic drug monitoring (TDM) using cyclin-dependent kinase inhibitors (CDK4/6is) is a novel approach for optimizing treatment outcomes. Currently, palbociclib, ribociclib, and abemaciclib are the available CDK4/6is and are primarily coadministered with letrozole. This study aimed to develop and validate an LC-MS/MS method for the simultaneous analysis of CDK4/6is, 2 active metabolites of abemaciclib (M2 and M20), and letrozole in human plasma for use in TDM studies. METHODS: Sample pretreatment comprised protein precipitation with methanol and dilution of the supernatant with an aqueous mobile phase. Chromatographic separation was achieved using a reversed-phase XBridge BEH C18 column (2.5 µm, 3.0 × 75 mm XP), with methanol serving as the organic mobile phase and pyrrolidine-pyrrolidinium formate (0.005:0.005 mol/L) buffer (pH 11.3) as the aqueous mobile phase. A triple quadrupole mass spectrometer was used for the detection, with the ESI source switched from negative to positive ionization mode and the acquisition performed in multiple reaction monitoring mode. RESULTS: The complete validation procedure was successfully performed in accordance with the latest regulatory guidelines. The following analytical ranges (ng/mL) were established for the tested compounds: 6-300, palbociclib and letrozole; 120-6000, ribociclib; 40-800, abemaciclib; and 20-400, M2 and M20. All results met the acceptance criteria for linearity, accuracy, precision, selectivity, sensitivity, matrix effects, and carryover. A total of 85 patient samples were analyzed, and all measured concentrations were within the validated ranges. The percent difference for the reanalyzed samples ranged from -11.2% to 7.0%. CONCLUSIONS: A simple and robust LC-MS/MS method was successfully validated for the simultaneous quantification of CDK4/6is, M2, M20, and letrozole in human plasma. The assay was found to be suitable for measuring steady-state trough concentrations of the analytes in patient samples.

A new dried blood spot LC-MS/MS method for therapeutic drug monitoring of palbociclib, ribociclib, and letrozole in patients with cancer.

Therapeutic drug monitoring (TDM) is strongly suggested to define the proper drug dosage to overcome inter- and intra-patient variability in drug exposure, which is typically observed with oral anticancer agents, such as palbociclib (PALBO), ribociclib (RIBO) and letrozole (LETRO), all approved for the treatment of HR+, HER2- locally advanced or metastatic breast cancer (BC). Optimal TDM implementation requires a blood sampling organization that can be hampered by limited availability of health and laboratory personnel. Dried Blood Spot (DBS) sampling is proposed to overcome such limitations. The aim of this work was the development of a new LC-MS/MS method to analyze DBS samples containing PALBO, RIBO, and LETRO. Analytes extraction from DBS was performed by adding a methanolic solution containing the corresponding internal standards. LC-MS/MS analysis was performed using a LC Nexera (Shimadzu) system coupled with an API 4000 QTrap (SCIEX) mass spectrometer. The chromatographic separation was performed on a Luna Omega Polar C18 column (Phenomenex). The method was applied to 38 clinical samples collected by finger prick. The influence of hematocrit and spot size, sample homogeneity, stability, and correlation between finger prick and venous DBS measurement were assessed. The analytical validation was performed according to EMA and FDA guidelines. The analytical range of the method was 1 to 250 ng/mL for PALBO, 40 to 10000 ng/mL for RIBO, and 2 to 500 ng/mL for LETRO, where linearity was assessed, obtaining mean coefficients of determination (R2) of 0.9979 for PALBO, 0.9980 for RIBO, and 0.9987 for LETRO). The LC-MS/MS method runtime was 6.6 min. Incurred sample reanalysis demonstrated reproducibility, as the percentage difference between the two quantifications was lower than 20% for 100% of PALBO, 81.8% of RIBO, and 90.9% of LETRO paired samples. Intra- and inter-day precision (CV (%)) was lower than 11.4% and intra- and inter-day accuracy was between 90.0 and 106.5%. DBS sample stability at room temperature was confirmed for 2.5 months. A positive correlation was observed between DBS and plasma concentrations for the 3 drugs, Lin's concordance correlation coefficients obtained by DBS normalization applying a selected strategy were 0.958 for PALBO, 0.957 for RIBO, and 0.963 for LETRO. In conclusion, a fast, easy, and reproducible DBS LC-MS/MS method for the simultaneous quantification of palbociclib; ribociclib and letrozole was developed to be used in clinical practice.

Elevated blood purine levels as a biomarker of seizures and epilepsy.

OBJECTIVE: There is a major unmet need for a molecular biomarker of seizures or epilepsy that lends itself to fast, affordable detection in an easy-to-use point-of-care device. Purines such as adenosine triphosphate and adenosine are potent neuromodulators released during excessive neuronal activity that are also present in biofluids. Their biomarker potential for seizures and epilepsy in peripheral blood has, however, not yet been investigated. The aim of the present study was to determine whether blood purine nucleoside measurements can serve as a biomarker for the recent occurrence of seizures and to support the diagnosis of epilepsy. METHODS: Blood purine concentrations were measured via a point-of-care diagnostic technology based on the summated electrochemical detection of adenosine and adenosine breakdown products (inosine, hypoxanthine, and xanthine; SMARTChip). Measurements of blood purine concentrations were carried out using samples from mice subjected to intra-amygdala kainic acid-induced status epilepticus and in video-electroencephalogram (EEG)-monitored adult patients with epilepsy. RESULTS: In mice, blood purine concentrations were rapidly increased approximately two- to threefold after status epilepticus (2.32 ± .40 µmol·L-1 [control] vs. 8.93 ± 1.03 µmol·L-1 [after status epilepticus]), and levels correlated with seizure burden and postseizure neurodegeneration in the hippocampus. Blood purine concentrations were also elevated in patients with video-EEG-diagnosed epilepsy (2.39 ± .34 µmol·L-1 [control, n = 13] vs. 4.35 ± .38 µmol·L-1 [epilepsy, n = 26]). SIGNIFICANCE: Our data provide proof of concept that the measurement of blood purine concentrations may offer a rapid, low-volume bedside test to support the diagnosis of seizures and epilepsy.

Testosterone replacement therapy in blood donors modulates erythrocyte metabolism and susceptibility to hemolysis in cold storage.

BACKGROUND: Red blood cells (RBCs) derived from patients who receive testosterone replacement therapy (TRT) may be considered eligible for component production and transfusion. The aim of this study was to identify testosterone-dependent changes in RBC metabolism and to evaluate its impact on susceptibility to hemolysis during cold storage. STUDY DESIGN AND METHODS: We characterized stored RBCs from two cohorts of TRT patients who were matched with control donors (no TRT) based upon sex, age, and ethnicity. We further evaluated the impact of testosterone deficiency (orchiectomy) on RBC metabolism in FVB/NJ mice. RBC metabolites were quantified by ultra-high-pressure liquid chromatography-mass spectrometry. RBC storage stability was determined in RBC units from TRT and controls by quantifying storage, osmotic, and oxidative hemolysis. RESULTS: Orchiectomy in mice was associated with significant (P < 0.05) changes in RBC metabolism as compared with intact males including increased levels of acyl-carnitines, long-chain fatty acids (eg, docosapentaenoic acids), arginine, and dopamine. Stored RBCs from TRT patients exhibited higher levels of pentose phosphate pathway metabolites, glutathione, and oxidized purines (eg, hypoxanthine), suggestive of increased activation of antioxidant pathways in this group. Further analyses indicated significant changes in free fatty acids and acyl-carnitines in response to testosterone therapies. With regard to hemolysis, TRT was associated with enhanced susceptibility to osmotic hemolysis. Correlation analyses identified acyl-carnitines as significant modifiers of RBC predisposition to osmotic and oxidative hemolysis. CONCLUSIONS: These observations provide new insights into testosterone-mediated changes in RBC metabolome and biology that may impact the storage capacity and posttransfusion efficacy of RBCs from TRT donors.

Severe rhabdomyolysis induced by possible drug-drug interaction between Ribociclib and Simvastatin.

INTRODUCTION: Drug-drug interactions with cyclin-dependent kinases inhibitors 4 and 6 (CDK4/6) are known and should be taken into account. CASE REPORT: A 68-year-old woman, on prior Simvastatin therapy, developed severe rhabdomyolysis after three weeks of Ribociclib initiation. She showed general weakness with mobility problems and was admitted to our hospital. MANAGEMENT AND OUTCOME: Ribociclib and Simvastatin were discontinued and the patient received intensive intravenous hydration. She finally recovered her mobility after two weeks. DISCUSSION: We hypothesize that Simvastatin induced rhabdomyolysis by possible interaction with Ribociclib. Ribociclib is a strong inhibitor of CYP 3A4 and a potential inhibitor of OATP1B1 membrane transporter. Simvastatin plasma concentration may reach toxic levels due to Ribociclib inhibition. To assess the relevance of our hypothesis, we used the Drug Interaction Scale. With a total score of 7, the interaction is considered as "probable." Because of the high risk of severe rhabdomyolysis, the concomitant use of Simvastatin with Ribociclib should be avoided or otherwise careful monitoring of creatine kinase is warranted.

Validated HPLC-UV method for simultaneous quantification of phosphatidylinositol 3-kinase inhibitors, copanlisib, duvelisib and idelalisib, in rat plasma: Application to a pharmacokinetic study in rats.

Phosphatidylinositol 3-kinase (PI3K) inhibitors are a novel class of anticancer drugs that are approved to treat various malignancies. We report the development and validation of a HPLC method for the simultaneous quantitation of three PI3K inhibitors, namely copanlisib, duvelisib and idelalisib, in rat plasma as per the regulatory guidelines of the United States Food and Drug Administration. The method involves extraction of copanlisib, duvelisib and idelalisib along with an internal standard (IS; filgotinib) from rat plasma (100 µL) using a liquid-liquid extraction process. The chromatographic separation of the analytes was achieved using step-wise gradient elution on a Hypersil Gold C18 column. The UV detection wavelength was set at λmax = 280 nm. Copanlisib, duvelisib, idelalisib and the IS eluted at 7.16, 12.6, 11.9 and 9.86 min, respectively, with a total run time of 15 min. The calibration curve ranged from 50 to 5000 ng/mL for all the analytes. Inter- and intra-day precision and accuracy, stability studies, dilution integrity and incurred sample reanalysis were investigated for all three analytes, and the results met the acceptance criteria. The validated HPLC method was successfully applied to a pharmacokinetic study in rats.

Walnut Consumption, Plasma Metabolomics, and Risk of Type 2 Diabetes and Cardiovascular Disease.

BACKGROUND: Walnut consumption is associated with lower risk of type 2 diabetes (T2D) and cardiovascular disease (CVD). However, it is unknown whether plasma metabolites related to walnut consumption are also associated with lower risk of cardiometabolic diseases. OBJECTIVES: The study aimed to identify plasma metabolites associated with walnut consumption and evaluate the prospective associations between the identified profile and risk of T2D and CVD. METHODS: The discovery population included 1833 participants at high cardiovascular risk from the PREvención con DIeta MEDiterránea (PREDIMED) study with available metabolomics data at baseline. The study population included 57% women (baseline mean BMI (in kg/m2): 29.9; mean age: 67 y). A total of 1522 participants also had available metabolomics data at year 1 and were used as the internal validation population. Plasma metabolomics analyses were performed using LC-MS. Cross-sectional associations between 385 known metabolites and walnut consumption were assessed using elastic net continuous regression analysis. A 10-cross-validation (CV) procedure was used, and Pearson correlation coefficients were assessed between metabolite weighted models and self-reported walnut consumption in each pair of training-validation data sets within the discovery population. We further estimated the prospective associations between the identified metabolite profile and incident T2D and CVD using multivariable Cox regression models. RESULTS: A total of 19 metabolites were significantly associated with walnut consumption, including lipids, purines, acylcarnitines, and amino acids. Ten-CV Pearson correlation coefficients between self-reported walnut consumption and the plasma metabolite profile were 0.16 (95% CI: 0.11, 0.20) in the discovery population and 0.15 (95% CI: 0.10, 0.20) in the validation population. The metabolite profile was inversely associated with T2D incidence (HR per 1 SD: 0.83; 95% CI: 0.71, 0.97; P = 0.02). For CVD incidence, the HR per 1-SD was 0.71 (95% CI: 0.60, 0.85; P < 0.001). CONCLUSIONS: A metabolite profile including 19 metabolites was associated with walnut consumption and with a lower risk of incident T2D and CVD in a Mediterranean population at high cardiovascular risk.

Pharmacokinetics, Safety, and Tolerability of Single- and Multiple-Dose Once-Daily Baricitinib in Healthy Chinese Subjects: A Randomized Placebo-Controlled Study.

The objective of this phase 1 study was to evaluate the pharmacokinetics, safety, and tolerability of baricitinib after single and multiple doses in healthy Chinese adults. Eligible subjects received a once-daily dose of baricitinib 2, 4, or 10 mg or placebo on day 1 (single dose) and days 4 through 10 for 7 consecutive days (multiple doses). Plasma pharmacokinetic samples were collected up to 48 hours after dosing on days 1 and 10, with predose samples collected before dosing on day 1 and days 4 through 10. Safety and tolerability were also assessed. Baricitinib was rapidly absorbed, reaching peak plasma concentrations within 0.5 to 1 hour (median). Plasma concentrations declined rapidly following the attainment of peak concentrations, with a mean terminal half-life of 5.7 to 7.3 hours. Steady-state plasma concentrations of baricitinib were achieved after the second day of once-daily dosing, with minimal accumulation of baricitinib in plasma (up to 10% increase in area under the plasma concentration-time curve). Single- and multiple-dose mean values for area under the plasma concentration-time curve from time zero to infinity and maximum plasma concentration appeared to increase in an approximately dose-proportional manner across the dose range. Single and multiple oral doses of once-daily baricitinib up to 10 mg were well tolerated by healthy Chinese subjects.

Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for characterizing caffeine, methylliberine, and theacrine pharmacokinetics in humans.

Coffea liberica possesses stimulant properties without accumulating the methylxanthine caffeine. The basis for this peculiar observation is that methylurates (e.g., theacrine and methylliberine) have replaced caffeine. The stimulant properties of methylurates, alone and in combination with caffeine, have recently been investigated. However, human pharmacokinetics and LC-MS/MS methods for simultaneous measurement of methylxanthines and methylurates are lacking. To address this deficiency, we conducted a pharmacokinetic study in which subjects (n = 12) were orally administered caffeine (150 mg), methylliberine (Dynamine™, 100 mg), and theacrine (TeaCrine®, 50 mg) followed by blood sampling over 24 h. Liquid-liquid extraction of plasma samples containing purine alkaloids and internal standard (13C-Caffeine) were analyzed using a C18 reversed-phase column and gradient elution (acetonitrile and water, both containing 0.1% formic acid). A Waters Xevo TQ-S tandem mass spectrometer (positive mode) was used to detect caffeine, methylliberine, theacrine, and IS transitions of m/z 195.11 â†’ 138.01, 225.12 â†’ 168.02, 225.12 â†’ 167.95, and 198.1 â†’ 140.07, respectively. The method was validated for precision, accuracy, selectivity, and linearity and was successfully applied to characterize the oral pharmacokinetics of caffeine, methylliberine, and theacrine in human plasma. Successful development and application of LC-MS/MS-based methods such as ours for the simultaneous measurement of methylxanthines and methylurates are essential for the characterization of potential pharmacokinetic and pharmacodynamic interactions.

Simultaneous quantification of palbociclib, ribociclib and letrozole in human plasma by a new LC-MS/MS method for clinical application.

A novel LC-MS/MS method was developed for the quantification of the new cyclin dependent kinase inhibitors (CDKIs) palbociclib and ribociclib and the aromatase inhibitor letrozole used in combinatory regimen. The proposed method is appropriate to be applied in clinical practice due to the simple and fast sample preparation based on protein precipitation, the low amount of patient sample necessary for the analysis (10 µL) and the total run time of 6.5 min. It was fully validated according to FDA and EMA guidelines on bioanalytical method validation. The linearity was assessed (R2 within 0.9992-0.9983) over the concentration ranges of 0.3-250 ng/mL for palbociclib, 10-10000 ng/mL for ribociclib and 0.5-500 ng/mL for letrozole that properly cover the therapeutic plasma concentrations. A specific strategy was implemented to reduce the carryover phenomenon, formerly known for these CDKIs. This method was applied to quantify the Cmin of palbociclib, ribociclib and letrozole in plasma samples from patients enrolled in a clinical study. The same set of study samples was analysed twice in separate runs to assess the reproducibility of the method by means of the incurred samples reanalysis. The results corroborated the reliability of the analyte concentrations obtained with the bioanalytical method, already proved by the validation process. The percentage differences were always within ±10% for all the analytes and the R2 of the correlation graph between the two quantifications was equal to 0.9994.

Effect of phytic acid on postprandial serum uric acid level in healthy volunteers: a randomized, double-blind, crossover study.

Phytic acid, a constituent of various plants, has been related to health benefits. Phytic acid has been shown to inhibit purine nucleotide metabolism in vitro and suppress elevation of plasma uric acid levels after purine administration in animal models. This study investigated the effect of phytic acid on postprandial serum uric acid (SUA) in humans. This randomized, double-blind, crossover design study included 48 healthy subjects with normal fasting SUA. Subjects consumed a control drink and a phytic acid drink with purine-rich food, and serum and urine uric acid levels were measured for 360 min after purine loading. Phytic acid lowered the incremental area under the curve (0-360 min) and incremental maximum concentration of SUA after purine loading (p < 0.05); tended to lower cumulative urinary uric acid excretion (0-360 min) after purine loading (p < 0.10); and suppressed postprandial SUA in this clinical study. Altogether, our findings suggest that phytic acid may play a beneficial role in controlling postprandial SUA.

Metabolic Tools for Identification of New Mutations of Enzymes Engaged in Purine Synthesis Leading to Neurological Impairment.

The cellular pool of purines is maintained by de novo purine synthesis (DNPS), recycling and degradation. Mutations in genes encoding DNPS enzymes cause their substrates to accumulate, which has detrimental effects on cellular division and organism development, potentially leading to neurological impairments. Unspecified neurological symptoms observed in many patients could not be elucidated even by modern techniques. It is presumable that some of these problems are induced by dysfunctions in DNPS enzymes. Therefore, we determined the concentrations of dephosphorylated DNPS intermediates by LC-MS/MS as markers of yet unpublished mutations in PFAS and PAICS genes connected with dysfunctions of carboxylase/phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS) or phosphoribosylformylglycinamidine synthase (PFAS). We determined the criteria for normal values of metabolites and investigated 1,447 samples of urine and 365 dried blood spots of patients suffering from various forms of neurological impairment. We detected slightly elevated aminoimidazole riboside (AIr) concentrations in three urine samples and a highly elevated 5-formamidoimidazole-4-carboxamide riboside (FGAr) concentration in one urine sample. The accumulation of AIr or FGAr in body fluids can indicate PAICS or PFAS deficiency, respectively, which would be new disorders of DNPS caused by mutations in the appropriate genes. Measurement of DNPS intermediates in patients with neurological symptoms can uncover the cause of serious cellular and functional impairments that are otherwise inaccessible to detection. Further genetic and molecular analysis of these patients should establish the causal mutations for prenatal diagnosis, genetic consultation, and reinforce the DNPS pathway as a therapeutic target.

Purine metabolism in sprint- vs endurance-trained athletes aged 20‒90 years.

Purine metabolism is crucial for efficient ATP resynthesis during exercise. The aim of this study was to assess the effect of lifelong exercise training on blood purine metabolites in ageing humans at rest and after exhausting exercise. Plasma concentrations of hypoxanthine (Hx), xanthine (X), uric acid (UA) and the activity of erythrocyte hypoxanthine-guanine phosphoribosyl transferase (HGPRT) were measured in 55 sprinters (SP, 20‒90 years), 91 endurance runners (ER, 20‒81 years) and 61 untrained participants (UT, 21‒69 years). SP had significantly lower levels of plasma purine metabolites and higher erythrocyte HGPRT activity than ER and UT. In all three groups, plasma purine levels (except UA in UT) significantly increased with age (1.8‒44.0% per decade). HGPRT activity increased in SP and ER (0.5‒1.0%), while it remained unchanged in UT. Hx and X concentrations increased faster with age than UA and HGPRT levels. In summary, plasma purine concentration increases with age, representing the depletion of skeletal muscle adenine nucleotide (AdN) pool. In highly-trained athletes, this disadvantageous effect is compensated by an increase in HGPRT activity, supporting the salvage pathway of the AdN pool restoration. Such a mechanism is absent in untrained individuals. Lifelong exercise, especially speed-power training, limits the age-related purine metabolism deterioration.

Rejuvenation of RBCs: validation of a manufacturing method suitable for clinical use.

BACKGROUND: Rejuvenation of stored red blood cells (RBCs) increases levels of adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) to those of fresh cells. This study aimed to optimize and validate the US-approved process to a UK setting for manufacture and issue of rejuvenated RBCs for a multicenter randomized controlled clinical trial in cardiac surgery. STUDY DESIGN AND METHODS: Rejuvenation of leukoreduced RBC units involved adding a solution containing pyruvate, inosine, phosphate, and adenine (Rejuvesol, Zimmer Biomet), warming at 37°C for 60 minutes, then "manual" washing with saline adenine glucose mannitol solution. A laboratory study was conducted on six pools of ABO/D-matched units made the day after donation. On Days 7, 21, and 28 of 4 ± 2°C storage, one unit per pool was rejuvenated and measured over 96 hours for volume, hematocrit, hemolysis, ATP, 2,3-DPG, supernatant potassium, lactate, and purines added (inosine) or produced (hypoxanthine) by rejuvenation. Subsequently, an operational validation (two phases of 32 units each) was undertaken, with results from the first informing a trial component specification applied to the second. Rejuvenation effects were also tested on crossmatch reactivity and RBC antigen profiles. RESULTS: Rejuvenation raised 2,3-DPG to, and ATP above, levels of fresh cells. The final component had potassium and hemolysis values below those of standard storage Days 7 and 21, respectively, containing 1.2% exogenous inosine and 500 to 1900 µmoles/unit of hypoxanthine. The second operational validation met compliance to the trial component specification. Rejuvenation did not adversely affect crossmatch reactivity or RBC antigen profiles. CONCLUSION: The validated rejuvenation process operates within defined quality limits, preserving RBC immunophenotypes, enabling manufacture for clinical trials.

Quantification of serum purine metabolites for distinguishing patients with hepatitis B from hepatocellular carcinoma.

Aim: In order to differential diagnosis of chronic hepatitis B (HBV-I) and hepatocellular carcinoma (HCC), a UPLC-MS/MS method for measuring purine metabolites was developed. Methodology & results: serum samples from 26 HBV-I and 35 HCC patients were collected. Ten purine metabolites were simultaneously quantified by UPLC-MS/MS with tubercidin and uric acid-1,3-15N2 as internal standards. The method was validated to meet the requirements of clinical sample analysis. A logistic equation was established for differential diagnosis of HBV-I and HCC by combination of xanthosine and guanine with the area under the receiver operating characteristic curve of 0.885. Conclusion: Guanine and xanthosine are intermediates in the metabolism of purine, which play an important role in gene synthesis, and metabolism regulation. The alteration of serum purine metabolite may contribute to differential diagnosis of HBV-I and HCC.

Cupric ion functionalized polydopamine coated magnetic microspheres as solid-phase adsorbent for the extraction of purines in plasma.

In this paper, Cu2+ functionalized Fe3O4@polydopamine core-shell (Fe3O4@PDA@Cu2+) magnetic microspheres were prepared by the chelation between Cu2+ and catechol of polydopamine surface. The synthetic magnetic adsorbent was characterized by Fourier transform infrared spectroscopy, energy dispersive X-ray spectroscopy, vibrating sample magnetometry, thermogravimetric analysis, scanning electron microscope and transmission electron microscopy. Four purines include guanine, adenine, hypoxanthine and xanthine were selected as model compounds to evaluate the applicability of this adsorbent. Several parameters that effected the extraction efficiency, such as extraction time, adsorbent amount, solution pH, ionic strength, eluent type, concentration of eluent and eluent time, were investigated. Under the optimized conditions, good linearity was obtained with correlation coefficients between 0.9983 and 0.9999 for the four analytes, and their LOD and LOQ were 0.42-2.15 ng/mL and 1.41-6.50 ng/mL, respectively. Meanwhile, the RSDs of intra-day and inter-day precision were in the range of 1.43%-5.55% and 4.56%-7.01%, respectively. The spiked recoveries of four purines in real sample were 70.01%-102.42%, indicating this proposed method might have potential applications for the analysis of purines in real samples. In addition, the developed method was used to monitor the concentrations of adenine in rat plasma at different time points after intragastric administration.

Metabolic disorder of amino acids, fatty acids and purines reflects the decreases in oocyte quality and potential in sows.

Oocyte quality is closely related to female fertility. Nevertheless, core nutritional metabolites influencing oocyte quality are unclear. Herein, comprehensive metabolomics analysis of follicular fluid, serum, and urine from low reproductive performance (LRP) and normal reproductive performance (NRP) sows was conducted. Twenty-seven, fourteen and sixteen metabolites (involved in metabolism of amino acids, fatty acids, purine and pyrimidine) were altered in follicular fluid, serum and urine, respectively, in LRP compared with NRP sows, and could decrease oocyte quality and developmental potential, ultimately leading to low fertility. Deoxyinosine, guanidine acetate, thymidine, 5,6-epoxy-eicosatrienoic acid, carnosine, docosahexaenoic acid and carbamoyl phosphate in follicular fluid, cysteine, carnitine, serotonin, hypoxanthine, valine and arginine in serum, as well as carnitine, phenyl glycine, N-acetyl glutamine, propionyl carnitine and choline in urine could be selected as diagnostic markers to indicate oocyte quality. Consistent with metabolomics data, we confirmed changes in concentrations of fatty acids and amino acids in follicular fluid. Targeting purine metabolism, elevating levels of deoxyinosine in in-vitro maturation medium of porcine oocyte significantly promoted the blastocyst rate. Collectively, this study provided new information of potential targets for predicting oocyte quality and developmental potential, and may help with strategies for early diagnosis or therapeutic/dietary intervention in improving reproductive outcomes.

Point-of-care measurements reveal release of purines into venous blood of stroke patients.

Stroke is a leading cause of death and disability. Here, we examine whether point-of-care measurement of the purines, adenosine, inosine and hypoxanthine, which are downstream metabolites of ATP, has potential to assist the diagnosis of stroke. In a prospective observational study, patients who were suspected of having had a stroke, within 4.5 h of symptom onset and still displaying focal neurological symptoms at admission, were recruited. Clinical research staff in the Emergency Departments of two hospitals used a prototype biosensor array, SMARTCap, to measure the purines in the venous blood of stroke patients and healthy controls. In controls, the baseline purines were 7.1 ± (SD) 4.2 µM (n = 52), while in stroke patients, they were 11.6 ± 8.9 µM (n = 76). Using the National Institutes for Stoke Scale (NIHSS) to band the severity of stroke, we found that minor, moderate and severe strokes all gave significant elevation of blood purines above the controls. The purine levels fall over 24 h. This was most marked for patients with haemorrhagic strokes (5.1 ± 3.6 µM, n = 9 after 24 h). The purine levels measured on admission show a significant correlation with the volume of affected brain tissue determined by medical imaging in patients who had not received thrombolysis or mechanical thrombectomy. ClinicalTrials.gov Identifier: NCT02308605.