Substances In Human Plasma Research Articles

A novel high-throughput quantitative method for the determination of per- and poly-fluoroalkyl substances in human plasma based on UHPLC-Q/Orbitrap HRMS coupled with isotope internal standard

A novel method for the quantitative analysis of 56 per- and polyfluoroalkyl substances (PFASs) in human plasma was established on the basis of ultrahigh performance liquid chromatography tandem quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q/Orbitrap HRMS) in combination with accurate customized mass databases and isotopic internal standards. A streamlined, high-throughput, and high-recovery (RE) sample pretreatment method was developed. The method’s performance was evaluated in terms of linearity, limit of quantification, RE, repeatability, reproducibility, and matrix effect. The proposed method was applied in the simultaneous analysis of 56 PFASs in human plasma, and its results demonstrated high sensitivity, accuracy, and precision. The optimized method was implemented to analyze PFASs in 135 plasma samples, and 12 components were detected. The comparative analysis of the results from 135 plasma samples with domestic and international studies revealed elevated contents of PFOA, PFOS, PFBA, and PFTrDA, the moderate amounts of PFHxS, PFUdA, PFBS, and PFHpS, and the low concentrations of PFNA and PFDA. Notably, GenX was detected in human plasma for the first time. This finding suggests that the study region is contaminated with this substance. Correlation analysis revealed a strong relationship among PFNA, PFDA, and PFUdA, implying that these substances may have similar exposure sources.

Integration of ultrashort-chain compounds into the biomonitoring of per- and polyfluorinated substances in human plasma and serum

Ultrashort-chain (USC) per- and polyfluoroalkyl substances (PFAS) are small and very polar compounds with carbon chain lengths of shorter than C4. Their ubiquitous and high levels of occurrence in environmental aquatic systems have raised significant concern in conjunction with long-chain PFAS contamination. Measuring USC PFAS in blood can not only monitor human exposure but also serves as a valuable tool for studying the potential risks associated with USC PFAS exposure. The high polarity of USC PFAS poses a challenge to current analytical practices based on the reversed-phase liquid chromatography, primarily due to insufficient chromatographic retention. In this study, a simple and reliable workflow was developed for the simultaneous analysis of C1 to C10 perfluoroalkyl carboxylic and sulfonic acids, along with four alternative PFAS, in human plasma and serum. The chromatographic analysis was conducted using a polar-embedded reversed-phase LC column. Fetal bovine serum (FBS) was chosen for method validation due to its absence of all analytes, except for trifluoroacetic acid (TFA). Subsequently, a TFA isotope, 13C-TFA, was employed as a surrogate to assess the method accuracy for TFA in FBS. A single-step sample preparation procedure, conducted by mixing 100 µL of FBS with 200 µL of methanol, was demonstrated to be effective for the accurate and precise analysis of fortified FBS samples. Ten isotopes of C3 to C10 PFAS, serving as extracted internal standards, were added to the samples at 1 ppb to validate the accuracy of the entire workflow. Calibration standards were prepared in reverse osmosis water due to its cleanliness for all analytes. Phosphate-buffered saline was incorporated into the calibration standard solution to achieve similar chromatographic performance between standard and sample solutions. The calibration ranges varied among different analytes, spanning from 0.05 – 40 ppb, 0.1 – 40 ppb, 0.25 – 40 ppb, and 0.5 – 40 ppb. Method accuracy and precision were evaluated at three fortification levels, ranging from 0.4 to 30 ppb. All analytes and extracted internal standards exhibited recovery values within 20 % of the nominal concentration across all fortification levels. Satisfactory method precision was demonstrated with%RSD values <12 %. The validated method was applied to measure PFAS in NIST 1950 and 1957 standard reference human plasma and serum, affirming the established workflow is suitable for accurate quantification of C1 to C10 PFAS in human plasma and serum matrices.

Optimization for the analysis of 42 per- and polyfluorinated substances in human plasma: A high-throughput method for epidemiological studies

There is an increasing awareness about the presence of per- and polyfluoroalkyl substances (PFAS) in many environmental and biological compartments, including human biofluids and tissues. However, the increase of PFAS replacements, including alternatives with shorter chain or less bioaccumulative potential, has broaden the exposure and the need for wider identification procedures. Moreover, the low volumes available for human blood or plasma, and the high number of samples needed to assess adequately epidemiologic studies, require particularly fast, reproducible and, if possible, miniaturized protocols. Therefore, accurate and robust analytical methods are still needed to quantify the PFAS's burden in humans and to understand potential health risks. In this study, we have developed and validated the analysis of 42 PFAS in human plasma by means of a Captiva 96-well micro extraction plate and a LC-q-Orbitrap. For the optimization of the analytical workflow, three extraction/clean-up methods were tested, and the selected one was validated using spiked artificial and bovine plasma at four concentration levels. The final method showed high absolute recoveries for the 42 PFAS, ranging from 52% to 130%, instrumental detection limits between 0.001–0.6 ng mL−1, overall good precision (CV < 20% for most of the PFAS) and a low uncertainty (< 30% of relative expanded deviation, k = 2). The method was further validated both with the NIST plasma Standard Reference Material 1950, showing that the accuracy of the provided results was between 63%-101%, and by the proficiency test arranged by the Arctic Monitoring Assessment Program (AMAP, 2022) obtaining satisfactory results within 95% confidence interval of the assigned value.

A sensitive and robust method for the simultaneous determination of thirty-three legacy and emerging per- and polyfluoroalkyl substances in human plasma and serum.

Legacy and emerging per- and polyfluoroalkyl substances (PFAS) have attracted growing attention due to their potential adverse effects on humans. We developed a method to simultaneously determine thirty-three PFAS (legacy PFAS, precursors, and alternatives) in human plasma and serum using solid phase extraction coupled to ultra-performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS). The method yielded good linearity (>0.995) and excellent limits of detection (LODs) (0.0005~0.012ngmL-1 in plasma and 0.002~0.016ngmL-1 in serum). The relative recoveries ranged from 80.1 to 116%, with intra- and inter-day precision less than 14.3%. The robustness of this method has been tested continuously for 10months (coefficients of variation <14.9%). Our method was successfully applied to the PFAS analysis of 42 real human plasma and serum samples collected from women. The proposed method is attractive for the biomonitoring of multi-class PFAS in human health risk assessment and epidemiological studies.

Screening of new psychoactive substances in human plasma by magnetic solid phase extraction and LC-QTOF-MS

The emergence of new psychoactive substances (NPSs) is an increasing challenge in forensic toxicology. There are many extraction methods in use to isolate NPSs in biological fluids, including protein precipitation (PPT), liquid-liquid extraction (LLE), and solid phase extraction (SPE). However, there is a need to develop an effective extraction method with a short extraction time and low consumption of solvent. To meet these requirements, magnetic solid phase extraction (m-SPE) was attempted to isolate 40 NPSs in human plasma in this study. Forty NPSs (13 synthetic cannabinoids, 13 phenethylamines, 4 tryptamines, 4 other substances, 3 aminoindanes, 2 piperazines, 1 phencyclidine-type substance) were spiked in plasma and analyzed by m-SPE using COOH-functionalized multi-walled carbon nanotubes with magnetic nanoparticles (COOH-mMWCNTs). A liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) method was used for screening and identification of 40 target compounds. Method validation including limits of detection, recovery, matrix effect, and precision was performed for all 40 -target compounds. The limits of detection (LOD) of the 40 analytes were between 0.002 and 0.084 mg/L. Extraction recovery ranged from 36.9% to 110.6% (average 87%). Matrix effects ranged from –29.0% (ion suppression) to 9.8% (ion enhancement). Both intra- and inter-day precision values were less than 27.5% (RSD%). The accurate mass of QTOF-MS enabled the identification of analytes by exact monoisotopic mass and isotopic pattern. m-SPE was applied to extract 40 NPSs, and revealed less time-consuming and laborious than conventional SPE. This method proved to be an advantageous procedure to extract NPSs from biological fluids.

Magnetic Solid-Phase Extraction of Drugs and Pesticides from Human Plasma Using COOH-mMWCNTs.

Carbon nanotubes (CNTs) are useful for extracting chemical compounds due to their properties, such as surface area and the potential for chemical modification. Especially the formation of CNTs with carboxylic acid functional group makes them disperse in water-based samples and have strong interaction forces with cationizable analytes. Based on these features, carboxylic acid functionalized multi-walled CNTs (COOH-MWCNTs) have been used as extraction sorbents. CNT can also be gathered using an external magnet by forming complex with iron oxide (Fe3O4) magnetic nanoparticles (MNPs). In this study, COOH-MWCNTs with MNPs were subjected to magnetic solid-phase extraction (mSPE) in order to extract the targeted substances such as diphenhydramine, doxylamine, tramadol, escitalopram, zolpidem, diphenamid, paclobutrazol, hexaconazole, cyproconazole and mepronil from human plasma samples. The following five factors were optimized: (i) the ratio of COOH-MWCNTs to MNPs as a sorbent from 1:1 to 1:4; (ii) sorbent amount starting from 12.5 to 75%; (iii) sample pH tested pH2 to pH10 with 1N hydrochloride and 1N sodium hydroxide; (iv) agitating time from 0 to 4min and (v) elution solvent. Limit of detection of 10 targeted substances in human plasma were in the range of 0.1-0.4mg/L. The recovery of targeted substances (except diphenamid) in human plasma was 73.06-110.28% for intra-day and 83.00-107.70% for inter-day and the precision (relative standard deviation, %) in human plasma was 0.3-13.3% for intra-day and 2.9-15.6% for inter-day. The method was applied to nine authentic biological samples from overdose patients in the emergency room of Chungnam National University Hospital. The performance of mSPE was compared with the liquid-liquid extraction method using ethyl acetate. The results showed that the newly developed method in this study can be used for screening analysis in forensic and clinical toxicology.

Development and Validation of Atazanavir and Ritonavir Determination in Human Plasma by HPLC-MS Method

Introduction . HIV infection is one of the most relevant diseases from a medical, epidemiological and social point of view. Timely diagnosis, detection and control of the disease, adequate prescription of antiretroviral therapy can sufficiently reduce the viral load on the patient's body, reduce the risk of transmission of infection. Currently, combinations of various antiretroviral drugs are increasingly being prescribed as therapy. One of the most important is combination of atazanavir and ritonavir. The most important stage for the study of pharmacokinetics, studies of comparative pharmacokinetics and bioequivalence is the development of an analytical method that allows you to determine the investigated substances in human plasma. There are currently no published methods for the determination of atazanavir and ritonavir in human plasma using high performance liquid chromatography with mass selective detection using a single quadrupole mass detector. In this article presents the development and validation of a method for the determination of atazanavir and ritonavir in blood plasma after sample preparation by the method of protein precipitation. Aim . The aim of the study is to develop a method for the quantitative determination of atazanavir and ritonavir in human plasma by HPLC with mass spectrometric detection for performing the analytical part of pharmacokinetic studies. Materials and methods . Determination of atazanavir and ritonavir in human plasma by HPLC with mass spectrometric detection. A sample was prepared using protein deposition. Results and discussion . The method was validated of selectivity, matrix effect, calibration curve, accuracy, precision, limit of quantification, carry-over effect and sample stability. Conclusion . The method of the determination of atazanavir and ritonavir in human plasma was developed and validated by HPLC-MS. The analytical range of the was 50.0–10000.0 ng/mL in plasma for atazanavir and 10.0–2500.0 ng/mL in plasma for ritonavir. Method could be applied to determination of atazanavir and ritonavir in plasma for PK and BE studies.

Development and Validation of a Rapid Solid-Phase Extraction: Ultrafast Liquid Chromatographic Method for the Estimation of Azithromycin and Its Major Related Substances in Human Plasma and Dosage Forms Using a Novel Polyfunctional Silyl Reagent-Bonded Core–Shell Column

In this study, a rapid reversed-phase liquid chromatography method for the determination of azithromycin and its related substances was developed and validated on a novel polyfunctional silyl reagent [hexanhexamethyloctadecyltetrasiloxane (HMODTS–C18)] bonded core–shell RP C-18 column (100 × 4.6 mm, 2.6 µm) as per ICH guidelines. A binary gradient elution programme with a mixture of solvent A (phosphate buffer, pH 8.9) and solvent B (ACN: MeOH, 3:1) as mobile phase, at a flow rate of 1.5 mL min−1 and detection at 210 nm was finally optimized for the study. The validation results showed that the method to be specific, selective, highly sensible (0.004 mg mL−1), precise (% RSD ≤ 10), linear and accurate in a concentration range of 0.004–0.032 mg mL−1. Simple SPE method was performed for extraction and checked for repeatability. The result showed that the method is precise (%RSD < 2). The validated chromatographic method was applied to the solid-phase extracted samples of azithromycin and its four major related substances. Also, docking study was carried out using AutoDock Tools to find out the binding affinities, number of hydrogen bonds and residues involved in hydrogen bonds for azithromycin and its four major related impurities with the human plasma proteins. The results confirmed the applicability of the proposed method on the extracted human plasma samples. Finally, the study demonstrates that the impurities of azithromycin have strong binding affinities with the plasma proteins compared to that of azithromycin and half life of these impurities may be higher than azithromycin which can cause major health risk on repeated doses of azithromycin.

A UPLC/DAD method for simultaneous determination of empagliflozin and three related substances in spiked human plasma

A simple, rapid and sensitive ultrahigh performance liquid chromatographic method was developed for the determination of the anti-diabetic drug: empagliflozin (EMPA) and three related substances in spiked human plasma, using dapagliflozin (DAPA) as an internal standard and tetrahydrofuran as a plasma protein precipitating agent. The chromatographic separation was achieved on an Acquity “UPLC® BEH” C18 column (50 mm × 2.1 mm i.d, 1.7 µm particle size), and a mobile phase consisting of aqueous trifluoroacetic acid (0.1%, pH 2.5): acetonitrile (60:40, v/v) at a flow rate of 0.5 mL/min. Upon using the UPLC system, the run time could be reduced to less than 1.2 min, and the solvents consumption decreased to 0.36 mL of acetonitrile per run. The response was linear over a concentration range of 50–700 ng/mL and 40–200 ng/mL (r2 = 0.9994–0.9999) with lower limits of detection and quantification (LOD/LOQ) of 15/50, 11.5/40, 12/40 and 12.5/40 ng/mL for EMPA and the three related substances, respectively. Good accuracy was obtained with mean percentage recoveries ≥ 96.97% for the studied compounds. The method was validated according to the ICH guidelines and was found suitable for routine analysis of EMPA and its related substances in human plasma.

Associations between concentrations of perfluoroalkyl substances in human plasma and maternal, infant, and home characteristics in Winnipeg, Canada

Perfluoroalkyl substances (PFASs) are known to be toxic, bioaccumulative, and persistent. However, exposure routes and toxic effects to humans are still widely unknown. Our objectives were to evaluate potential correlations between concentrations of PFASs in maternal plasma and infant cord blood with home characteristics and developmental effects, including wheezing. The concentrations of 17 PFASs were measured in plasma from prenatal women (n = 414), postnatal women (n = 247), and cord blood (n = 50) from a subset of participants in a population-based birth cohort in Winnipeg, Manitoba, Canada, using online solid phase extraction (SPE) with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Multiple linear regression and principal component analysis (PCA) were used to evaluate possible associations with PFAS concentrations. Surveys were used to collect information regarding maternal characteristics (e.g. age, parity, duration of breastfeeding), infant characteristics (e.g. birth weight, birth length, head circumference, gestational age, and incidence of recurrent wheezing), and home characteristics (e.g. home age,carpet coverage in the most used room, presence of new furniture, or recent home renovations). PFASs in plasma were associated with maternal characteristics but not home characteristics or early childhood wheezing. PFASs were not associated with developmental effects, with the exception that perfluoroundecanoic acid (PFUA) was negatively associated with birth weight. Further studies investigating the potential influences of PFUA on birth weight are warranted.

A validated UPLC-MS/MS method to determine free and total irinotecan and its two metabolites in human plasma after intravenous administration of irinotecan hydrochloride liposome injection

Irinotecan hydrochloride liposome injection (IHLI) is a formulation of anticancer drug irinotecan hydrochloride (CPT-11) entrapped in the aqueous core of liposomes. To understand the pharmacokinetic property and evaluate the relationship between pharmacokinetics and pharmacodynamics/toxicity of IHLI, it is of prime importance to determine the concentrations of free CPT-11, total CPT-11 and its main metabolites (SN-38 and SN-38 G) in human plasma. In this paper, we developed and validated a sensitive and reliable ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to quantify the concentrations of these related substances in human plasma. Free CPT-11, SN-38 and SN-38 G in human plasma were simultaneously separated and extracted by 96-well solid phase extraction (SPE) plate, while total CPT-11 was extracted by protein precipitation (PP) method. The analytes were chromatographed on an Acquity UPLC BEH C18 column and then detected on a Xevo TQ-S tandem mass spectrometer in multiple reactions monitoring (MRM) mode using positive electrospray ionization (ESI). The UPLC-MS/MS method combined with SPE and PP techniques were fully validated in line with existing guidelines issued by regulatory agencies. In brief, all the analytes achieved a satisfactory selectivity and sensitivity in this method. The calibration curves were proved to be linear over the concentration range of 10–10000 ng/mL for total CPT-11, 0.5–1000 ng/mL for free CPT-11, 0.5–200 ng/mL for SN-38 and SN-38 G, respectively. For all the analytes, the intra- and inter-run precisions were less than 11.4% and the accuracies (in terms of RE%) were within -7.7–7.3% except for accuracies of LLOQs were within -15.8 to 7.2%. Besides, carry-over, extraction recovery, matrix effect, dilution integrity, stability and special matrices were also assessed. Finally, the method was successfully applied to a phase I clinical pharmacokinetic study of IHLI in Chinese subjects with advanced solid tumors.

Analysis of angiotensin II receptor antagonist and protein markers at microliter level plasma by LC–MS/MS

An analytical method based on a green approach is proposed for clinical analysis. The proposed procedure involves the reduction of the sample preparation steps, the amounts of reagents and organic solvents. This simple and sensitive method for the analysis of clinical drug and biomarkers in human plasma was developed using LC connected to tandem mass spectrometry (LC–MS/MS) with a nanospray ion source. In this study, the desired drug and proteins were separated on a 5 and 10 cm RP C18 nano-flow column. Undesired polar substances in human plasma were washed out by using ACN:1% FA = 20:80 (v/v) as the loading mobile phase for drug analysis and good linearity was attainable. Only a small volume of human plasma (10 μL) was utilized for the monitoring of drug plasma concentration and significant proteins under clinical studies. All the sample preparation procedures and the analytical scheme were at microliter level. This strategy would lower the consumption of reagents and organic solvents and make a contribution toward the goal of reduction of pollution from analytical methods in general.

Measurement of plasma 5-hydroxyindoleacetic acid as a possible clinical surrogate marker for the action of antivascular agents

Background: Serotonin (5HT), a naturally occurring vasoactive substance, is released from platelets into plasma under various pathological conditions. Recently, anticancer drugs that act by selectively disrupting tumour blood flow have been found to increase plasma 5HT concentrations in mice. Two such antivascular agents, flavone acetic acid (FAA) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA), have completed Phase I clinical trial and raise the important question of whether suitable surrogate markers for antivascular effects can be identified. Methods: 5HT is unstable to storage, precluding routine clinical assay, but the 5HT metabolite, 5-hydroxyindoleacetic acid (5HIAA) accumulates in plasma following 5HT release and is a more suitable marker because of its greater stability. We have developed an automated procedure for the assay of the low concentrations of 5HIAA found in humans by combining solid-phase extraction with high-performance liquid chromatography (HPLC). Results: Efficient separation of 5HIAA from possible interfering substances in human plasma, including a variety of pharmaceutical agents, was achieved on C18 columns using cetyltrimethylammonium bromide (CETAB) as an organic modifier. Adequate precision, accuracy and sensitivity were achieved by electrochemical detection (ECD) at +400 mV. Analysis of plasma from two patients treated with DMXAA in a Phase I trial demonstrated DMXAA-induced elevation of plasma 5HIAA with a time course similar to that previously described in mice. Conclusions: Measurement of changes in plasma 5HIAA provides a new approach to the monitoring of therapies with an antivascular effect. The assay is sensitive to dietary sources of 5HT, which should be minimised.

Method for the quantitative analysis of cilostazol and its metabolites in human plasma using LC/MS/MS.

An LC/MS/MS method for the simultaneous determination of cilostazol, a quinolinone derivative, and three active metabolites, OPC-13015, OPC-13213, and OPC-13217, in human plasma was developed and validated. Cilostazol, its metabolites, and the internal standard, OPC-3930 were extracted from human plasma by liquid-liquid partitioning followed by solid-phase extraction (SPE) on a Sep-Pak silica column. The eluent from the SPE column was then evaporated and the residue reconstituted in a mixture of methanol/ammonium acetate buffer (pH 6.5) (2:8 v/v). The analytes in the reconstituted solution were resolved using reversed-phase chromatography on a Supelcosil LC-18-DB HPLC column by an 17.5-min gradient elution. Cilostazol, its metabolites, and the internal standard were detected by tandem mass spectrometry with a Turbo Ionspray interface in the positive ion mode. The method was validated over a linear range of 5.0-1200.0 ng/ml for all the analytes. This method was demonstrated to be specific for the analytes of interest with no interference from endogenous substances in human plasma or from several potential concomitant medications. For cilostazol and its metabolites, the accuracy (relative recovery) of this method was between 92.1 and 106.4%, and the precision (%CV) was between 4.6 and 6.5%. During the validation, standard curve correlation coefficients equalled or exceeded 0.999 for cilostazol and its metabolites. These data demonstrate the reliability and precision of the method. The method was successfully cross-validated with an established HPLC method.

High-performance liquid chromatographic method for the determination of nelfinavir, a novel HIV-1 protease inhibitor, in human plasma

Nelfinavir mesylate, a potent and orally bioavailable inhibitor of HIV-1 protease ( K i =2 n M), has undergone Phase III clinical evaluation in a large population of HIV-positive patients. A high-performance liquid chromatography analytical method was developed to determine the pharmacokinetic parameters of the free base, nelfinavir, in these human subjects. The method involved the extraction of nelfinavir and an internal standard, 6,7-dimethyl-2,3-di-(2-pyridyl)quinoxaline, from 250 μl of human plasma with a mixture of ethyl acetate–acetonitrile (90:10, v/v). The analysis was via ultraviolet detection at 220 nm using a reversed-phase C 18 analytical column and a mobile phase consisting of 25 m M monobasic sodium phosphate buffer (adjusted to pH 3.4 with phosphoric acid)–acetonitrile (58:42, v/v) that resolved the drug and internal standard peaks from non-specific substances in human plasma. The method was validated under Good Laboratory Practice (GLP) conditions for specificity, inter- and intra-assay precision and accuracy, absolute recovery and stability. The mean recovery ranged from 92.4 to 83.0% for nelfinavir and was 95.7% for the internal standard. The method was linear over a concentration range of 0.0300 μg/ml to 10 μg/ml, with a minimum quantifiable level of 0.0500 μg/ml for nelfinavir.

Enzyme-linked immunosorbent assay for chromogranin A.

This is an enzyme-linked immunosorbent assay (ELISA) for determining chromogranin A (CGA) with use of a monoclonal antibody. CGA was isolated from bovine chromaffin granules. The analytical ELISA procedure for bovine CGA was developed and optimized. Typical standard curves ranged from 500 pg to 500 ng of CGA. We then studied human plasma CGA-immunoreactivity as measured by this assay. The curve for dilutions of human plasma paralleled the standard curve for bovine CGA. The intra-assay coefficient of variation for determination of human plasma CGA was 4.56%, indicating that reliable determinations can be performed for human plasma. However, further study revealed the presence of two CGA-immunoreactive substances in human plasma, one of which corresponds to the native CGA. The nature of the second immunoreactive substance still remains unknown. Nevertheless the measured CGA concentrations (ranging from 0.19 to 0.35 mg/L) in plasma are comparable with previously reported values.

Improved assays for hydralazine and hydralazine pyruvic acid hydrazone in human plasma

Previous determinations of hydralazine and hydralazine pyruvic acid hydrazone based on derivatization and high-performance liquid chromatography have been modified to improve the detection limits of the hydralazine assay and the selectivity of the hydralazine pyruvic acid hydrazone technique. The lower limits of determinations of hydralazine and hydralazine pyruvic acid hydrazone are 2 and 25 ng ml -1, respectively. The application of this technique to the determination of these substances in human plasma is demonstrated.

ANTI‐INFLAMMATORY AND IRRITANT EFFECTS OF A FRACTION FROM NORMAL HUMAN PLASMA

1 By the use of carrageenan-induced rat paw oedema assay the anti-inflammatory activity of a fraction isolated from normal human plasma has been measured after its intravenous, intraperitoneal and oral administration. Its effects in the dextran-induced rat paw oedema and the systemic dextran anaphylactoid reaction in the rat were also studied.2 The fraction showed marked anti-inflammatory activity in the carrageenan test after intravenous administration and a smaller but still significant activity when given intraperitoneally, but was inactive orally after the administration of a larger dose. It was active in the dextran-induced paw oedema test but not against the anaphylactoid reaction.3 A comparison between its anti-inflammatory and irritant properties revealed no correlation when each parameter was determined in relation to dose. The fraction did not affect the blood pressure of the anaesthetized rat.4 These findings are discussed in relation to the existence of a natural anti-inflammatory substance or substances in human plasma.

A METHOD FOR MEASURING PLASMA LEVELS OF DIGITALIS GLYCOSIDES.

A method is described for measuring plasma levels of cardiac glycosides, utilizing their inhibitory effect on the rubidium-86 uptake of human red cells. As little as 0.05 mµg. of digoxin/ml. of plasma produces detectable inhibition, but due to the variable effect of other substances in human plasma the practical limit of the method is 1.0 mµg./ml., about 20 times as sensitive as the duck embryo heart or the C 14 -labeled technics. Patients on digitalis leaf were found to have much higher circulating levels than patients on digoxin (average value 7.5 mµg./ ml. of digoxin equivalent for digitalis leaf, 0.05 mµg./ml. for digoxin). Patients toxic on digoxin averaged 6 mµg./ml. Of patients not taking digitalis, 7 per cent had significant levels of digitalis-like substances in their plasma; these were mostly individuals with metastatic carcinoma, severe fluid and electrolyte disturbances, or heart failure. No drug (other than cardiac glycosides) gave consistent high levels by this test.

Effects of electrical stimulation of the brain of the concentration of adrenaline-like substances in human plasma.

Cerebral electrostimulation of subconvulsive intensity causes an increase in the concentration of adrenaline-like substances in the blood (Weil-Malherbe and Bone, 1952b). This increase becomes more pronounced as the pulse repetition rate is raised from 200 pulses per second (p.p.s.) to 500 p.p.s., in contrast to the motor, sensory, and respiratory effects which diminish under these conditions (Montagu, 1955). The increasing response to the higher repetition rates showed no sign of reaching a peak in these experiments, except in one case, and since it was not possible with the existing apparatus to exceed 500 p.p.s., the optimal conditions for eliciting the response remained undetermined. With the aid of a more versatile stimulator the adrenaline-frequency relationship has subsequently been studied more extensively, and the results are presented in this article.∗