Comparative in vitro studies on liposomal formulations of amphotericin B and its derivative,N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME)

N-Methyl-N-D-fructosyl amphotericin B methyl ester (MFAME) is a semisynthetic derivative of the antifungal antibiotic amphotericin B (AMB). In contrast to the parent antibiotic, the derivative is characterised by low toxicity to mammalian cells and good solubility in water of its salts. Comparative studies on biological properties of free MFAME, AMB and their liposomal formulations were performed. To obtain liposomal forms, the antibiotics were incorporated into small unilamellar vesicles composed of dimyristoyl phosphatidylcholine (DMPC) and DMPC:cholesterol or ergosterol, 8:2 molar ratio. The effectivity of the liposomal and free forms of AMB and MFAME were compared by determination of fungistatic and fungicidal activity against Candida albicans ATCC 10261, potassium release from erythrocytes, and haemolysis. The results obtained indicate that in contrast to AMB, incorporation of MFAME into liposomes did not further improve its selective toxicity. Studies on the antagonistic effect of ergosterol and cholesterol on the antifungal activity of the antibiotics indicated that sterol interference was definitely less pronounced in the case of MFAME than in the case of AMB.     

Dendrimer versus linear conjugate: Influence of polymeric architecture on the delivery and anticancer effect of paclitaxel

The relative difference in polymeric architectures of dendrimer and linear bis(poly(ethylene glycol)) (PEG) polymer in conjugation with paclitaxel has been described. Paclitaxel, a poorly soluble anticancer drug, was covalently conjugated with PAMAM G4 hydroxyl-terminated dendrimer and bis(PEG) polymer for the potential enhancement of drug solubility and cytotoxicity. Both conjugates were characterized by 1NMR, HPLC, and MALDI/TOF. In addition, molecular conformations of dendrimer, bis(PEG), paclitaxel, and its polymeric conjugates were studied by molecular modeling. Hydrolysis of the ester bond in the conjugate was analyzed by HPLC using esterase hydrolyzing enzyme. In vitro cytotoxicity of dendrimer, bis(PEG), paclitaxel, and polymeric conjugates containing paclitaxel was evaluated using A2780 human ovarian carcinoma cells. Cytotoxicity increased by 10-fold with PAMAM dendrimer-succinic acid-paclitaxel conjugate when compared with free nonconjugated drug. Data obtained indicate that the nanosized dendritic polymer conjugates can be used with good success as anticancer drug carriers.     

Update on Amphotericin B Pharmacology and Dosing for Common Systemic Mycoses

Amphotericin B (AMB) has been used for nearly five decades in the treatment of life-threatening mycoses. While triazoles and echinocandins have largely supplanted the routine use of AMB for common Candida and Aspergillus infections, this prototypical broad-spectrum agent or its lipid formulations are still preferred by many clinicians as the initial empiric antifungal therapy for severely immunosuppressed patients, and remain the preferred treatment in combination with 5-fluorocytosine for cryptococcal meningioencephalitis. This article reviews progress over the last decade in understanding the pharmacology and clinical dosing of AMB formulations for common systemic mycoses, including invasive candidiasis, cryptococcosis, aspergillosis and mucormycosis.     

Investigation of parameters influencing incorporation,retention and cellular cytotoxicity in liposomal formulations of poorly soluble camptothecin

Abstract

Improving tumor delivery of lipophilic drugs through identifying advanced drug carrier systems with efficient carrier potency is of high importance. We have performed an investigative approach to identify parameters that affect liposomes’ ability to effectively deliver lipophilic camptothecin (CPT) to target cells. CPT is a potent anticancer drug, but its undesired physiological properties are impairing its therapeutic use. In this study, we have identified parameters influencing incorporation and retention of lipophilic CPT in liposomes, evaluating the effect of lipid composition, lipid chemical structure (head and tail group variations, polymer inclusion), zeta potential and anisotropy. Polyethyleneglycol (PEG) surface decoration was included to avoid liposome fusing and increase the potential for prolonged in vivo circulation time. The in vitro effect of the different carrier formulations on cell cytotoxicity was compared and the effect of active targeting of one of the formulations was evaluated. We found that a combination of liposome surface charge, lipid headgroup and carbon chain unsaturation affect CPT incorporation. Retention in liposomes was highly dependent on the liposomal surroundings and liposome zeta potential. Inclusion of lipid tethered PEG provided stability and prevented liposome fusing. PEGylation negatively affected CPT incorporation while improving retention. In vitro cell culture testing demonstrated that all formulations increased CPT potency compared to free CPT, while cationic formulations proved significantly more toxic to cancer cells that healthy cells. Finally, antibody mediated targeting of one liposome formulation further enhanced the selectivity towards targeted cancer cells, rendering normal cells fully viable after 1 hour exposure to targeted liposomes.     

Modulation of polyene antibiotics self-association by ions from the Hofmeister series

The toxicity of the antifungal polyene antibiotic amphotericin B (AMB) has been related to its low solubility, more specifically to a self-associated form termed toxic aggregate. In addition, AMB in aqueous medium gives rise to concentration, ionic strength, and time-dependent polydisperse systems. For this reason different approaches, including the use of several lipid aggregates, have been used in attempts to improve the drug's solubility and increase its therapeutic index. In this context, understanding AMB's self-association properties should help in the preparation of less toxic formulations. Ions from the Hofmeister series alter water properties: while kosmotropes (water structure makers-sulfate, citrate, phosphate) decrease solute solubility, chaotropes (water structure breakers-perchlorate, thiocyanate, trichloroacetate, and the neutral molecule urea) have opposite effects. This work reports a study of the effect of Hofmeister ions and urea on the self-aggregation of AMB and some of its derivatives. Optical absorption and circular dichroism spectra were used to monitor monomeric and aggregated antibiotic. While kosmotropes increased aggregation in a concentration-dependent manner, the opposite was observed for chaotropes. It is shown, for the first time, that thiocyanate and trichloroacetate can induce complete AMB monomerization. The understanding of these processes at the physicochemical and molecular levels and the possibility of modulating the aggregation state of AMB and its derivatives should contribute to elucidate the mechanisms of action and toxicity of this widely used antibiotic and to develop more efficient and less toxic preparations.     

Decorporation of Iron Metal Using Dialdehyde Cellulose-Deferoxamine Microcarrier

Deferoxamine iron chelator has a limited therapeutic index due to rapid clearance from blood and possesses dose-limiting toxicity. Therefore, an intravenous deferoxamine delivery system based on dialdehyde cellulose (DAC) polymer was developed and its efficacy and toxicity were tested in iron-overloaded animals. The amino groups of deferoxamine were conjugated to free aldehyde moieties of dialdehyde cellulose via Schiff base reaction to form dialdehyde cellulose-deferoxamine (DAC-DFO) conjugate and characterized by Fourier transform infrared spectrophotometer, scanning electron microscope, and X-ray diffraction. The toxicity of prepared formulation was analyzed by XTT cell viability assay and LD50 study in mice. The change in serum iron levels, after intravenous administration of formulation, was observed in iron-overloaded rats. The DAC-DFO conjugate was tagged with Tc-99m to study the blood kinetics and observe change in blood circulation time. DAC-DFO conjugate was dispersible in water at concentration ～75 mg/ml. In vitro cytotoxicity assay and LD₅₀ study in mice indicated significantly enhanced safety of covalently bound deferoxamine (at >1000 mg/kg body weight compared to free drug at ～270 mg/kg dose). A preliminary scintigraphy imaging and blood clearance study, with technetium-99m, indicated prolonged circulation of conjugated DFO in rabbit blood. A single dose of formulation injected into iron overloaded animals was found to maintain the normal serum iron levels until 10 days. The polymeric conjugate was effective in maintaining normal serum iron levels until 10 days at a dose of 100 mg/kg body weight.     

Utility of Physiologically Based Modeling and Preclinical In Vitro/In Vivo Data to Mitigate Positive Food Effect in a BCS Class 2 Compound

Physiologically based pharmacokinetic (PBPK) modeling has become a useful tool to estimate the performance of orally administrated drugs. Here, we described multiple in silico/in vitro/in vivo tools to support formulation development toward mitigating the positive food effect of NVS123, a weak base with a pH-dependent and limited solubility. Administered orally with high-fat meal, NVS123 formulated as dry filled capsules displayed a positive food effects in humans. Three alternative formulations were developed and assessed in in vitro and in vivo preclinical and/or clinical studies. By integrating preclinical in vitro and in vivo data, the PBPK model successfully estimated the magnitude of food effects and the predicted values were within ±30% of the observed results. A model-guided parameter sensitivity analysis illustrated that enhanced solubility and longer precipitation times under fed condition were the main reason for enhanced NVS123's exposure in presence of food. Eventually, exposure after an amorphous formulation was found to be not significantly altered because of remarkably enhanced intestinal solubility and reduced precipitation. Gastroplus population simulations also suggested that the amorphous formulation is promising in mitigating a clinically significant food effect. Overall, these efforts supported the rationale of clinical investigation of the new formulation, and more importantly, highlighted a practical application of PBPK modeling solving issues of undesirable food effects in weakly basic compounds based on preclinical in vitro/in vivo data.     

A群脑膜炎球菌多糖-破伤风类毒素结合物中游离多糖测定方法的建立

本文建立一种测定A群脑膜炎球菌多糖－破伤风类毒素（PS－TT）结合物中游离多糖的方法。用80％乙醇使PS－TT结合物沉淀,再用60％乙醇洗涤沉淀使未结合多糖溶解,使与TT结合的和未结合的多糖分离并分别测定。该方法可有效分离结合的与未结合的多糖并分别测定,结合物中外加多糖可定量回收。本方法适用于测定A群脑膜炎球菌多糖－破伤风类毒素结合物中游离多糖含量。     

Peroral Amphotericin B Polymer Nanoparticles Lead to Comparable or Superior In Vivo Antifungal Activity to That of Intravenous Ambisome? or Fungizone?

Background

Despite advances in the treatment, the morbidity and mortality rate associated with invasive aspergillosis remains unacceptably high (70–90%) in immunocompromised patients. Amphotericin B (AMB), a polyene antibiotic with broad spectrum antifungal activity appears to be a choice of treatment but is available only as an intravenous formulation; development of an oral formulation would be beneficial as well as economical.

Methodology

Poly(lactide-co-glycolode) (PLGA) nanoparticles encapsulating AMB (AMB-NPs) were developed for oral administration. The AMB-NPs were 113±20 nm in size with ∼70% entrapment efficiency at 30% AMB w/w of polymer. The in vivo therapeutic efficacy of oral AMB-NPs was evaluated in neutropenic murine models of disseminated and invasive pulmonary aspergillosis. AMB-NPs exhibited comparable or superior efficacy to that of Ambisome® or Fungizone™ administered parenterally indicating potential of NPs as carrier for oral delivery.

Conclusions

The present investigation describes an efficient way of producing AMB-NPs with higher AMB pay-load and entrapment efficiency employing DMSO as solvent and ethanol as non-solvent. The developed oral formulation was highly efficacious in murine models of disseminated aspergillosis as well as an invasive pulmonary aspergillosis, which is refractory to treatment with IP Fungizone™and responds only modestly to AmBisome®.     

Combined Use of Crystalline Sodium Salt and Polymeric Precipitation Inhibitors to Improve Pharmacokinetic Profile of Ibuprofen through Supersaturation

To maximize the pharmacological effect of a pain reliever such as ibuprofen, early onset of action is critical. Unfortunately, the acidic nature of ibuprofen minimizes the amount of drug that can be solubilized under gastric conditions and would be available for immediate absorption upon entry into the intestine. Although the sodium salt of ibuprofen has higher solubility, rapid conversion from the salt to the poorly soluble free acid phase occurs under gastric conditions. Therefore, the combination of the highly soluble sodium salt form of ibuprofen with polymers was evaluated as an approach to prolong supersaturation of ibuprofen during the disproportionation of the salt. Binary combinations of ibuprofen sodium with polymers resulted in the identification of several formulations that demonstrated high degrees and extended durations of supersaturation during in vitro dissolution experiments. These formulations included HPMC, polyvinyl pyrrolidone-vinyl acetate copolymer (PVP-VA64), methylcellulose (MC), and hydroxypropyl cellulose (HPC). The in vitro supersaturation observed with these ibuprofen-polymer formulations translated to an increase in C_max and an earlier T_max for the PVP-VA64, MC, and HPC formulations relative to ibuprofen only controls when administered orally to rats under fasted conditions. Based on these observations, combining ibuprofen sodium with polymers such as PVP-VA64, MC, or HPC is a viable formulation approach to prolong supersaturation in the stomach and enable an optimized pharmacokinetic profile in vivo where rapid onset of action is desired.     

Ditosylate Salt of Itraconazole and Dissolution Enhancement Using Cyclodextrins

Salt formation has been a promising approach for improving the solubility of poorly soluble acidic and basic drugs. The aim of the present study was to prepare the salt form of itraconazole (ITZ), a hydrophobic drug to improve the solubility and hence dissolution performance. Itraconazolium ditolenesulfonate salt (ITZDITOS) was synthesized from ITZ using acid addition reaction with p-toluenesulfonic acid. Salt characterization was performed using ¹H NMR, mass spectrometry, Fourier transform infrared spectroscopy, differential scanning calorimetry, and X-ray diffraction. The particle size and morphology was studied using dynamic light scattering technique and scanning electron microscopy, respectively. The solubility of the salt in water and various pharmaceutical solvents was found multifold than ITZ. The dissolution study exhibited 5.5-fold greater percentage release value in 3 h of ITZDITOS (44.53%) as compared with ITZ (8.54%). Results of in vitro antifungal studies using broth microdilution technique indicate that ITZDITOS possessed similar antifungal profile as that of ITZ when tested against four fungal pathogens. Furthermore, the physical mixtures of ITZDITOS with two cyclodextrins, β-cyclodextrin (β-CD), and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) were prepared in different molar ratios and were evaluated for in vitro release. It was observed that in only 30 min of dissolution study, about 74 and 81% of drug was released from 1:3 molar ratios of ITZDITOS with β-CD and ITZDITOS with HP-β-CD, respectively, which was distinctly higher than the drug released from ITZ commercial capsules (70%). The findings warrant further preclinical and clinical studies on ITZDITOS so that it can be established as an alternative to ITZ for developing oral formulations.KEY WORDS: antifungal, BCS class II, dissolution rate, insoluble, itraconazole     

Design of Micelle Nanocontainers Based on PDMAEMA-b-PCL-b-PDMAEMA Triblock Copolymers for the Encapsulation of Amphotericin B

The clinical application of amphotericin B (AmB), a broad spectrum antifungal agent, is limited by its poor solubility in aqueous medium and also by its proven renal toxicity. In this work, AmB was encapsulated in micelles obtained from the self-assembly of PDMAEMA-b-PCL-b-PDMAEMA triblock copolymers. The amount of encapsulated AmB depended on the copolymer composition, and short blocks of polycaprolactone (PCL) and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) showed better performance. All the studied formulations exhibited a controlled release of AmB along 150 h. The formulations presented reduced hemotoxicity while maintaining antifungal activities against Candida albicans, Candida krusei, and Candida glabrata comparable with free AmB. A reduction on the hemotoxicity was found to be due to the slow release and subsequent low aggregation achieved with the use of polymer micelle nanocontainers.KEY WORDS: amphotericin B, antifungal agent, hemotoxicity, micelles     

Effect of Different Physical Factors on efficacy of Thevetia Peruviana leaf extract and bio-formulations

Plant extract possess various secondary metabolites which are antifungal in nature and can be used as a safer alternative to the synthetic fungicides. As we all know that the chemical fungicides are harmful not only for humans but also for animals, other vegetation and for complete ecosystem. To overcome this problem, we have to focused on another alternative which are biologically libel and nonhazardous also. In the present study, herbal formulation was prepared in various combination ratios with Thevetia peruviana leaf extracts, cow dung and neem oil cake. The major aim of this short study is to check the stability of the said plant extracts and prepared herbal formulation on various physical factors like heat, temperature, pH, sunlight and storage etc. The extracts and herbal formulations were exposed to varying conditions of the parameters selected for a precise time period, and then observing the effect as a function of change in the crude extract activity, herbal formulation activity and change minimum inhibitory concentration of plant extract against the Alternaria solani. Control set of MIC, and extract free medium were maintained for comparison in each set of experiment against Alternaria solani. Results suggested that efficacy of leaf extracts and different formulations was not affected by wet heat up to 100 °C while slight reduction in antifungal activity of the plant extract and herbal formulations were observed with dry heat at 100 °C. In addition, slight reduction in activity of extract and herbal formulations was observed with change in pH. However antifungal activity of plant extract as well as herbal formulations, remain unaffected at alkaline pH (pH 9) and neutral pH (pH7). Storage for 6 and 12 months had no negative effect on extract and herbal formulation efficacy and the antifungal activity was observed similar to freshly prepared extract activity. The present study concluded that the plant disease or plant pathogens can be controlled by plant extract and plant based bioformulations by increasing the shelf life with some little changes in the physical parameters such as light, temperature, pH and storage.     

Multifunctional human serum albumin-therapeutic nucleotide conjugate with redox and pH-sensitive drug release mechanism for cancer theranostics

We report on the synthesis and properties of a new multimodal theranostic conjugate based on an anticancer fluorinated nucleotide conjugated with a dual-labeled albumin. A fluorine-labeled homocysteine thiolactone has been used as functional handle to synthesize the fluorinated albumin and couple it with a chemotherapeutic agent 5-trifluoromethyl-2′-deoxyuridine 5′-monophosphate (pTFT). The conjugate allows for direct optical and ¹⁹F magnetic resonance cancer imaging and release of the drug upon addition of glutathione. Interestingly, the pTFT release from albumin conjugate could only be promoted by the increased acidity (pH 5.4). The in vitro study and primary in vivo investigations showed stronger antitumor activity than free pTFT.     

Acyclovir-Polyethylene Glycol 6000 Binary Dispersions: Mechanistic Insights

The dissolution and subsequent oral bioavailability of acyclovir (ACY) is limited by its poor aqueous solubility. An attempt has been made in this work to provide mechanistic insights into the solubility enhancement and dissolution of ACY by using the water-soluble carrier polyethylene glycol 6000 (PEG6000). Solid dispersions with varying ratios of the drug (ACY) and carrier (PEG6000) were prepared and evaluated by phase solubility, in vitro release studies, kinetic analysis, in situ perfusion, and in vitro permeation studies. Solid state characterization was done by powder X-ray diffraction (XRD), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR) analysis, and surface morphology was assessed by polarizing microscopic image analysis, scanning electron microscopy, atomic force microscopy, and nuclear magnetic resonance analysis. Thermodynamic parameters indicated the solubilization effect of the carrier. The aqueous solubility and dissolution of ACY was found to be higher in all samples. The findings of XRD, DSC, FTIR and NMR analysis confirmed the formation of solid solution, crystallinity reduction, and the absence of interaction between the drug and carrier. SEM and AFM analysis reports ratified the particle size reduction and change in the surface morphology in samples. The permeation coefficient and amount of ACY diffused were higher in samples in comparison to pure ACY. Stability was found to be higher in dispersions. The results suggest that the study findings provided clear mechanical insights into the solubility and dissolution enhancement of ACY in PEG6000, and such findings could lay the platform for resolving the poor aqueous solubility issues in formulation development.     

In vitro activity of the lipopeptide PAL-Lys-Lys-NH2, alone and in combination with antifungal agents, against clinical isolates of Candida spp

Candida albicans is known to be the organism most often associated with serious fungal infection, but other Candida spp. are emerging as clinical pathogens associated with opportunistic infections. Among antimycotic treatments, increasing attention is currently given to anti-infective drugs based upon naturally occurring peptides, such as the short lipopeptide palmitoyl PAL-Lys-Lys-NH2 (PAL). The aim of this study is to evaluate the activity of this peptide compared to the traditional antifungal agents Fluconazole (FLU), amphotericin B (AMB) and caspofungin (CAS) on Candida spp. 24 clinical isolates of Candida spp. were tested against PAL_, FLU, AMB and CAS using in vitro susceptibility tests, time killing and checkerboard assay. All of the drugs studied showed good activity against clinical isolates of candida; in particular CAS and AMB which have MICs value lower than PAL and FLU. Moreover we observed synergistic interactions for PAL/FLU (81.25%), PAL/AMB (75%) and particularly for PAL/CAS (87.5). We think that our results are interesting since synergy between PAL and CAS might be useful in clinic trails to treat invasive fungal infections.     

Pyrazolo[3,4-d]pyrimidines-loaded human serum albumin (HSA) nanoparticles: Preparation,characterization and cytotoxicity evaluation against neuroblastoma cell line

Pyrazolo[3,4-d]pyrimidine derivatives 1–5, active as c-Src inhibitors, have been selected to be formulated as drug-loaded human serum albumin (HSA) nanoparticles, with the aim of improving their solubility and pharmacokinetic properties. The present study includes the optimization of a desolvation method-based procedure for preparing HSA nanoparticles. First, characterization by HPLC-MS and Dynamic Light Scattering (DLS) showed a good entrapment efficacy, a controllable particle size (between 100 and 200 nm) and an optimal stability over time, confirmed by an in vitro drug release assay. Then, 1–4 and the corresponding NPs were tested for their antiproliferative activity against neuroblastoma SH-SY5Y cell line. Notably, 3-NPs and 4-NPs were identified as the most promising formulation showing a profitable balance of stability, small size and a similar activity compared to the free drugs in cell-based assays. In addition, albumin formulations increase the solubility of pyrazolo[3,4-d]pyrimidine avoiding the use of DMSO as solubilizing agent.     

Comparative studies on cell stimulatory, permeabilizing and toxic effects induced in sensitive and multidrug resistant fungal strains by amphotericin B (AMB) and N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME)

N-Methyl-N-D-fructosyl-amphotericin B methyl ester (MFAME) is a new derivative of amphotericin B, which is characterised by low toxicity to mammalian cells and good solubility in water of its salts. The antifungal activity and effects of MFAME towards Candida albicans and Saccharomyces cerevisiae multidrug resistant MDR(+) and sensitive MDR(-) strains was compared with those of parent compound. The results obtained indicate that MDR(+) S. cerevisiae was sensitive to MFAME as well as to AMB. MFAME exhibited the same effects on fungal cells studied as parent antibiotic. The two antibiotics, depending on the dose applied induced cell stimulation, K+ efflux, and/or had a toxic effect.     

In Vitro and In Vivo Antitumor Activity of a Novel pH-Activated Polymeric Drug Delivery System for Doxorubicin

Background

Conventional chemotherapy agent such as doxorubicin (DOX) is of limited clinical use because of its inherently low selectivity, which can lead to systemic toxicity in normal healthy tissue.

Methods

A pH stimuli-sensitive conjugate based on polyethylene glycol (PEG) with covalently attachment doxorubicin via hydrazone bond (PEG-hyd-DOX) was prepared for tumor targeting delivery system. While PEG-DOX conjugates via amid bond (PEG-ami-DOX) was synthesized as control.

Results

The synthetic conjugates were confirmed by proton nuclear magnetic resonance (NMR) spectroscopy, the release profile of DOX from PEG-hyd-DOX was acid-liable for the hydrazone linkage between DOX and PEG, led to different intracellular uptake route; intracellular accumulation of PEG-hyd-DOX was higher than PEG-ami-DOX due to its pH-triggered profile, and thereby more cytotoxicity against MCF-7, MDA-MB-231 (breast cancer models) and HepG2 (hepatocellular carcinoma model) cell lines. Following the in vitro results, we xenografted MDA-MB-231 cell onto SCID mice, PEG-hyd-DOX showed stronger antitumor efficacy than free DOX and was tumor-targeting.

Conclusions

Results from these in vivo experiments were consistent with our in vitro results; suggested this pH-triggered PEG-hyd-DOX conjugate could target DOX to tumor tissues and release free drugs by acidic tumor environment, which would be potent in antitumor drug delivery.     

Plasma concentrations of p- and m-hydroxyphenylacetic acid and phenylacetic acid in humans : Gas chromatographic—high-resolution mass spectrometric analysis

Conjugated and unconjugated phenylacetic acid and m- and p-hydroxyphenylacetic acid have been determined in the plasma of normal, healthy subjects after fasting, consumption of a meal and ingestion of deuterium-labelled amine precursors, by high-resolution gas chromatography—high-resolution mass spectrometry with selected ion monitoring of their trifluoroethyl-pentafluoropropionyl derivatives.We observed that all three conjugated acids are higher in fasting than in non-fasting subjects, and unconjugated phenylacetic acid was lower. Ingestion of deuterium-labelled amine precursors resulted in the appearance in the blood of the correspondingly labelled acids, a peak in the concentrations being reached about 1 h after consumption. Conjugated and unconjugated acids as expected increased following the consumption of a meal.Unconjugated phenylacetic acid was significantly higher in females than in males. Most values tended to increase with age, with male unconjugated and conjugated m-hydroxyphenylacetic acid and female conjugated phenylacetic and m-hydroxyphenylacetic acids increasing significantly.