Cingulate circuits are associated with escalation of heroin use and naloxone-induced increases in heroin self-administration.

Opioid use disorder (OUD) is defined as a compulsion to seek and take opioids, loss of control over intake and the development of a negative emotional state when access to opioids is denied. Using functional magnetic resonance imaging (fMRI) data in a rat model of OUD, we demonstrate that the escalation of heroin self-administration (SA) and the increased heroin SA following an injection of an opioid receptor antagonist (naloxone) are associated with changes in distinct brain circuits, centered on the cingulate cortex (Cg). Here, SA escalation score was negatively associated with changes in resting state functional connectivity (rsFC) between the Cg and the dorsal striatum. Conversely, increased heroin SA following naloxone injection, was associated with increased connectivity between the Cg and the extended amygdala and hypothalamus. Naloxone-induced increased SA was also positively associated with changes in the amplitude of low frequency fluctuations within the Cg, a measure of spontaneous neuronal activity. Characterizing the distinct brain circuit and behavior changes associated with different facets of addiction increases our understanding of OUD and may provide insight into addiction prevention and treatment.

Comparative Genome Analysis and Global Phylogeny of the Toxin Variant Clostridium difficile PCR Ribotype 017 Reveals the Evolution of Two Independent Sublineages.

The diarrheal pathogen Clostridium difficile consists of at least six distinct evolutionary lineages. The RT017 lineage is anomalous, as strains only express toxin B, compared to strains from other lineages that produce toxins A and B and, occasionally, binary toxin. Historically, RT017 initially was reported in Asia but now has been reported worldwide. We used whole-genome sequencing and phylogenetic analysis to investigate the patterns of global spread and population structure of 277 RT017 isolates from animal and human origins from six continents, isolated between 1990 and 2013. We reveal two distinct evenly split sublineages (SL1 and SL2) of C. difficile RT017 that contain multiple independent clonal expansions. All 24 animal isolates were contained within SL1 along with human isolates, suggesting potential transmission between animals and humans. Genetic analyses revealed an overrepresentation of antibiotic resistance genes. Phylogeographic analyses show a North American origin for RT017, as has been found for the recently emerged epidemic RT027 lineage. Despite having only one toxin, RT017 strains have evolved in parallel from at least two independent sources and can readily transmit between continents.

Arrangement and number of clustered regularly interspaced short palindromic repeat spacers are associated with erythromycin susceptibility in emm12, emm75 and emm92 of group A streptococcus.

Clustered regularly interspaced short palindromic repeats (CRISPR) are composed of numerous repeat-spacer units and are considered a prokaryotic defence system against foreign nucleic acids. Since antibiotic-resistant genes are frequently encoded in foreign nucleic acids, the aim of this study was to test whether erythromycin susceptibility in group A streptococcus (Streptococcus pyogenes) is associated with characteristics of CRISPR elements. Erythromycin susceptibility of 330 isolates collected between 1997 and 2003 was analysed. Among 29 emm types, emm12, emm75 and emm92 showed significant changes in erythromycin-resistance rates. By sequencing the spacers from two CRISPR loci, spacer contents in emm12, emm75 and emm92 strains were associated with erythromycin susceptibility. Strains with fewer spacers were more resistant to erythromycin. Moreover, in emm4 strains, which showed no significant change in their annual erythromycin-resistance rate, CRISPR type and number of spacers were not correlated with erythromycin susceptibility. These results highlight a novel association between CRISPR spacer content and erythromycin susceptibility in group A streptococcus.

A comparative study of clinical Aeromonas dhakensis and Aeromonas hydrophila isolates in southern Taiwan: A. dhakensis is more predominant and virulent.

Aeromonas dhakensis, often phenotypically identified as Aeromonas hydrophila, is an important human pathogen. The present study aimed to compare the clinical and biological features of A. dhakensis and A. hydrophila isolates from human wounds. A total of 80 Aeromonas wound isolates collected between January 2004 and April 2011 were analysed. The species was identified by the DNA sequence matching of rpoD and gyrB (or rpoB if necessary). Most of the Aeromonas isolates were identified as A. dhakensis (37, 46.3%), and 13 (16.3%) as A. hydrophila. Both species alone can cause severe skin and soft-tissue infections. More A. dhakensis isolates were found in wounds exposed to environmental water (32.4% vs 0%, p 0.042). More biofilm formation was noted among A. dhakensis isolates (mean optical density at 570 nm, 1.23 ± 0.09 vs 0.78 ± 0.21, p 0.03). The MICs of ceftriaxone, imipenem and gentamicin for A. dhakensis isolates were higher (p <0.0001, <0.04, and <0.01, respectively). The survival rates of Caenorhabditis elegans co-incubated with A. dhakensis from day 1 to day 3 were lower than those of worms infected with A. hydrophila in liquid toxicity assays (all p values <0.01). Isolates of A. dhakensis exhibited more cytotoxicity, as measured by the released leucocyte lactate dehydrogenase levels in human normal skin fibroblast cell lines (29.6 ± 1.2% vs 20.6 ± 0.6%, p <0.0001). The cytotoxin gene ast was primarily present in A. hydrophila isolates (100% vs 2.7%, p <0.0001). In summary, A. dhakensis is the predominant species among Aeromonas wound isolates, and more virulent than A. hydrophila.

In vitro and in vivo therapeutics of ß-thujaplicin on LPS-induced inflammation in macrophages and septic shock in mice.

ß-thujaplicin, an active constituent from Chamaecyparis obtusa, has been shown to have acaricidal and antimicrobial effects. Very few studies have focused on the potential of the anti-inflammatory effect of ß-thujaplicin. Moreover, its capability of inhibiting inflammatory mediators e.g. TNF-a gene transcription, nitric oxide (NO) and prostaglandin E2, remains unknown. Besides those molecular mechanisms behind the anti-inflammatory effect of ß-thujaplicin, solid proof of its effectiveness in vivo has not yet been studied. In our study, in vitro effects of ß thujaplicin were verified on RAW 264.7 macrophages which were stimulated by LPS. Indomethacin was used as a positive control. The inducible NO production after stimulation was measured by Griess reagent. PGE2, IL-6 and TNF-α were measured by ELISA methods. Protein expressions of iNOS, COX2, and NF-κB were evaluated by Western blotting. Septic ICR mice were administered 20 mg/kg of LPS and then the mortality rate was monitored. Within the concentration range which was devoid of cytotoxicty, ß-thujaplicin exhibited a clear dose-dependent inhibition on LPS-induced NO production. Furthermore, ß-thujaplicin inhibited LPS-induced PGE2, IL-6, and TNF-α production as well as iNOS, COX2, and NF- κB protein expression more substantially potent than indomethacin. In agreement with the in vitro study, ß-thujaplicin was shown to be effective in vivo for inhibiting LPS-induced NO and TNF-α production and a significant decrease in mortality rate of mice suffering from septic shock was observed. This study demonstrates the potential of ß-thujaplicin in treatment of inflammation and sepsis. These effects occur through an efficient blockage of TNF-α and iNOS production. ß-thujaplicin efficacy is comparable to that of indomethacin thus it can be a substitution but bear less depletion of PGE2, making this compound very promising in clinical applications. ß-thujaplicin, an active constituent from Chamaecyparis obtusa, has been shown to have acaricidal and antimicrobial effects. Very few studies have focused on the potential of the anti-inflammatory effect of ß-thujaplicin. Moreover, its capability of inhibiting inflammatory mediators e.g. TNF-alpha gene transcription, nitric oxide (NO) and prostaglandin E2, remains unknown. Besides those molecular mechanisms behind the anti-inflammatory effect of ß-thujaplicin, solid proof of its effectiveness in vivo has not yet been studied. In our study, in vitro effects of ß-thujaplicin were verified on RAW 264.7 macrophages which were stimulated by LPS. Indomethacin was used as a positive control. The inducible NO production after stimulation was measured by Griess reagent. PGE2, IL-6 and TNF-alpha were measured by ELISA methods. Protein expressions of iNOS, COX2, and NF-kB were evaluated by Western blotting. Septic ICR mice were administered 20 mg/kg of LPS and then the mortality rate was monitored. Within the concentration range which was devoid of cytotoxicty, ß-thujaplicin exhibited a clear dose-dependent inhibition on LPS-induced NO production. Furthermore, ß-thujaplicin inhibited LPS-induced PGE2, IL-6, and TNF-alpha production as well as iNOS, COX2, and NF-kB protein expression more substantially potent than indomethacin. In agreement with the in vitro study, ß-thujaplicin was shown to be effective in vivo for inhibiting LPS-induced NO and TNF-alpha production and a significant decrease in mortality rate of mice suffering from septic shock was observed. This study demonstrates the potential of ß-thujaplicin in treatment of inflammation and sepsis. These effects occur through an efficient blockage of TNF-alpha and iNOS production. ß-thujaplicin efficacy is comparable to that of indomethacin thus it can be a substitution but bear less depletion of PGE2, making this compound very promising in clinical applications.

Narrowband ultraviolet B phototherapy for patients with refractory uraemic pruritus: a randomized controlled trial.

BACKGROUND: Pruritus is very common in uraemic patients, but the treatment remains challenging. Studies regarding narrowband ultraviolet B (NB-UVB) phototherapy for uraemic pruritus are rare. OBJECTIVES: To investigate whether or not NB-UVB phototherapy is an effective treatment for uraemic pruritus. METHODS: We conducted a single-blind, randomized, controlled trial for patients with refractory uraemic pruritus. The treatment group received NB-UVB phototherapy three times per week for 6 weeks. The dose of NB-UVB started from 210 mJ cm(-2) and was increased by 10% each time. The control group received time-matched exposures to long-wave UVA radiation. A visual analogue scale (VAS) score was evaluated weekly for pruritus intensity for 12 weeks. The characteristics of pruritus were also assessed by a questionnaire at baseline and after 6 weeks of phototherapy. RESULTS: Both the NB-UVB and control groups had significant and comparable improvement in the pruritus intensity VAS scores during the period of phototherapy and follow-up. Compared with the control group, the NB-UVB group showed a significant improvement in the involved body surface area affected by pruritus (P = 0·006), but not in sleep quality. More detailed regression and estimating analysis revealed that the patients in the NB-UVB group had lower pruritus intensity scores at week 6, week 10 and week 12. This may indicate a beneficial difference at certain time points, but the effect seems marginal. CONCLUSIONS: NB-UVB phototherapy does not show a significant effect in reducing pruritus intensity compared with a control group for refractory uraemic pruritus. Further studies are warranted.

Toll-like receptor 2 deficiency improves insulin sensitivity and hepatic insulin signalling in the mouse.

AIMS/HYPOTHESIS: Substantial evidence suggests a link between elevated inflammation and development of insulin resistance. Toll-like receptor 2 (TLR2) recognises a large number of lipid-containing molecules and transduces inflammatory signalling in a variety of cell types, including insulin-responsive cells. Considering the contribution of the fatty acid composition in TLR2-depedent signalling, we hypothesised that the inflammatory signals transduced by TLR2 contribute to insulin resistance. METHODS: Mice deficient in TLR2 were used to investigate the in vivo roles of TLR2 in initiating and maintaining inflammation-associated insulin resistance and energy homeostasis. RESULTS: We first recapitulated the observation with elevated expression of TLR2 and inflammatory cytokines in white adipose tissue and liver of ob/ob mice. Aged or high-fat-fed TLR2-deficient mice were protected from obesity and adipocyte hypertrophy compared with wild-type mice. Moreover, mice lacking TLR2 exhibited improved glucose tolerance and insulin sensitivity regardless of feeding them regular chow or a high-fat diet. This is accompanied by reductions in expression of inflammatory cytokines and activation of extracellular signal-regulated kinase (ERK) in a liver-specific manner. The attenuated hepatic inflammatory cytokine expression and related signalling are correlated with increased insulin action specifically in the liver in TLR2-deficient mice, reflected by increased insulin-stimulated protein kinase B (Akt) phosphorylation and IRS1 tyrosine phosphorylation and increased insulin-suppressed hepatocyte glucose production. CONCLUSIONS/INTERPRETATION: The absence of TLR2 attenuates local inflammatory cytokine expression and related signalling and increases insulin action specifically in the liver. Thus, our work has identified TLR2 as a key mediator of hepatic inflammation-related signalling and insulin resistance.

Exposure to traffic exhausts and oxidative DNA damage.

AIMS: To assess the relations between exposure to traffic exhausts and indicators of oxidative DNA damage among highway toll station workers. METHODS: Cross-sectional study of 47 female highway toll station workers exposed to traffic exhausts and 27 female office workers as a reference group. Exposure assessment was based on average and cumulative traffic density and a biomarker of exposure, urinary 1-hydroxypyrene-glucuronide (1-OHPG). Urinary 8-hydroxydeoxyguanosine (8-OHdG) was used as a biomarker of oxidative DNA damage. Plasma nitric oxide (NO) was measured as an indicator of oxidative stress related to traffic exhaust exposure. RESULTS: The mean concentration of urinary 8-OHdG was substantially higher among the exposed non-smokers (13.6 microg/g creatinine) compared with the reference non-smokers (7.3 microg/g creatinine; difference 6.3, 95% CI 3.0 to 9.6). The mean concentration of NO among the exposed (48.0 micromol/l) was also higher compared with the reference non-smokers (37.6 micromol/l; difference 10.4, 95% CI -0.4 to 21.2). In linear regression adjusting for confounding, a change in log(8-OHdG) was statistically significantly related to a unit change in log(1-OHPG) (beta = 0.372, 95% CI 0.081 to 0.663). CONCLUSIONS: Results indicate that exposure to traffic exhausts increases oxidative DNA damage. Urinary 8-OHdG is a promising biomarker of traffic exhaust induced oxidative stress.

A comparison on the emission of polycyclic aromatic hydrocarbons and their corresponding carcinogenic potencies from a vehicle engine using leaded and lead-free gasoline.

Our objective in this study was to assess the effect of using two kinds of lead-free gasoline [including 92-lead-free gasoline (92-LFG) and 95-lead-free gasoline (95-LFG), rated according to their octane levels] to replace the use of premium leaded gasoline (PLG) on the emissions of polycyclic aromatic hydrocarbons (PAHs) and their corresponding benzo[a]pyrene equivalent (BaP(eq)) amounts from the gasoline-powered engine. The results show that the three gasoline fuels originally contained similar total PAHs and total BaP(eq) contents; however, we found significant differences in the engine exhausts in both contents. The above results suggest that PAHs originally contained in the gasoline fuel did not affect the PAH emissions in the engine exhausts. The emission factors of both total PAHs and total BaP(eq) obtained from the three gasoline fuels shared the same trend: 95-LFG > PLG > 92-LFG. The above result suggests that when PLG was replaced by 95-LFG, the emissions would increase in both total PAHs and total BaP(eq), but when replaced by 92-LFG would lead to the decreased emissions of both contents. By taking emission factors and their corresponding annual gasoline consumption rates into account, we found that both total PAH and total BaP(eq) emissions increased from 1994 to 1999. However, the annual increasing rates in total BaP(eq) emissions were slightly higher than the corresponding increasing rates in total PAHs.

Health-risk assessment for workers exposed to polycyclic aromatic hydrocarbons (PAHs) in a carbon black manufacturing industry.

This study was established to assess workers' health-risks posed by PAHs exposures via both routes of inhalation and dermal contact. Personal inhalation exposure sampling was conducted on eight wet pelletizing workers and 22 packaging workers, by using a sampling train comprising an IOM personal inhalable aerosol sampler followed by an XAD-2 sorbent tube. Two workers were randomly selected from both exposure groups, and dermal exposures assessed by using soft polypropylene pads attached to the skin for nine different body surface areas for each selected worker. All personal inhalation and dermal samples were analyzed for 21 polycyclic aromatic hydrocarbon (PAH) species, and then converted to benzo[a]pyrene equivalent (BaPeq) concentrations by using the list of toxic equivalent factors (TEFs) suggested by Nisbet and LaGoy [Regul Toxicol Pharmocol 16 (1992) 290]. The resultant inhalation and dermal BaPeq exposure levels were used to estimate lifetime risks for lung cancer and skin cancer by using the BaP unit risks of 7.0 x 10(-2) (microg/m3)(-1) and 37.47(mg/kg bodyweight/day)(-1), respectively. Results show the personal inhalation BaPeq exposure levels for pelletizing and packaging workers were 622 and 774 ng/m3, respectively. The corresponding lifetime lung cancer risks estimated for both exposure groups were 4.35 x 10(-2) and 5.42 x 10(-2) respectively. For dermal exposures, results show personal dermal BaPeq exposure levels for both exposure groups were 0.664 and 0.847 microg/kg per day, respectively. The corresponding estimated lifetime skin cancer risks were 1.13 x 10(-3) and 1.56 x 10(-3), respectively. Although the estimated skin cancer risks were lower than the corresponding lung cancer risks for both exposure groups, however, both were higher than the designated significant risk level (= 10(-3)) which was defined by the US Supreme Court in 1980. Considering the bioavailability of particle-bound PAHs still remains unknown, the health risks obtained from this study could be overestimated and thus require further investigation.

The involvement of glutamate in recall of the conditioned NK cell response.

The molecular mechanisms responsible for the conditioned enhancement of natural killer (NK) cell activity were investigated. The primary goal of the study was to examine the roles of glutamate and gamma-aminobutyric acid (GABA) in recall of the conditioned NK cell response. Both neurochemical blocking assay and high performance liquid chromatography (HPLC) technique were used in the study. Results from the neurochemical blocking assay demonstrated that glutamate but not GABA was required in recall of the conditioned NK cell response. NMDA but not the kainate/AMPA receptors, are believed to be involved. The levels of glutamate that were released and/or taken up also appeared to be critical in that interruption of glutamate release and/or uptake blocked the conditioned NK cell response. Results from the HPLC analysis, however, did not show any significant difference in the glutamate content between the conditioned and control brains.

A study of workers' exposures to the inhalable and 'total' aerosol fractions in the primary nickel production industry using mannequins to simulate personal sampling.

This paper describes a study that was carried out at work sites in the primary nickel production industry to investigate the difference between inhalable and 'total' aerosol exposures by using the mannequin sampling method, and to compare the results with those from an earlier study where actual workers' personal exposures were assessed in the same way. Experiments were carried out at 21 work sites located in mining, milling, smelting, and refining works of two primary nickel production companies. During sampling, mannequins were used to simulate the physical presence of workers and the 'exposures' of these were obtained for strategic positions at selected work sites. The orientations of each mannequin with respect to the wind were rotated through 90 degrees every hour in order to simulate the approximate orientation-averaging corresponding to actual workers. Two samplers were placed side-by-side on each mannequin: the Institute of Occupational Medicine (IOM) inhalable aerosol sampler, and the 37-mm plastic cassette widely used as a personal sampler for 'total' aerosol. Each collected sample was analyzed to obtain both overall dust and overall nickel content. A total of 116 such sample pairs were collected. The results show that inhalable aerosol exposure levels-for both overall dust and for total nickel content-were consistently and significantly higher than the corresponding total aerosol exposure levels. Weighted least squares linear regression yielded (inhalable/'total') aerosol ratios ranging from 1.38 to 3.90 and 1.20 to 4.01, respectively, for overall dust and for total nickel content for different work sites. Comparison of these results with those from the earlier study of actual workers' personal exposures were in good agreement for most of the work sites studies. However, the actual intensities of exposure using the mannequin sampling method were consistently lower than those obtained from actual workers' personal sampling in our earlier study. The consistency of the (inhalable/'total') ratios between mannequin and actual personal sampling strongly suggests that the characteristics of the aerosol sampled by the two methods, most notably the particle size distribution, were the same. This in turn suggests that mannequin sampling can be useful in occupational hygiene for determining such properties of personal workers' exposures. It certainly provides a useful and cost effective method for determining factors at work sites in individual industry settings by which to examine the impact of changing exposure assessment from one based on 'total' aerosol to the recommended new approach based on inhalable aerosol.

Heterotrimeric G-protein and signal transduction in the nematode-trapping fungus Arthrobotrys dactyloides.

The fungus Arthrobotrys dactyloides produces specialized constricting rings to trap and then consume nematodes. The signal transduction pathway involved in the nematode-trapping process was examined. Mastoparan, an activator of G-protein, had a stimulatory effect on the inflation of ring cells, whereas a G-protein inhibitor, pertussis toxin, prevented ring-cell expansion. The 40-kDa G alpha of heterotrimeric G-proteins was specifically ADP-ribosylated by pertussis toxin. Using an antibody specific to the 35-kDa subunit G beta, we showed that immunogold-labeled G beta was more concentrated in ring cells than in the hyphae. In the absence of nematodes, the rings could be inflated by either pressurizing the culture in a syringe, raising intracellular Ca2+ concentrations, or adding warm water. We used these methods to reveal differences in responses to antagonists. The results support a model in which the pressure exerted by a nematode on the ring activates G-proteins in the ring cells. The activation leads to an increase in cytoplasmic Ca2+, activation of calmodulin, and finally the opening of water channels. The ring cells expand to constrict the ring and thus immobilize the nematode.

On-line, continuous and automatic monitoring of extracellular malondialdehyde concentration in anesthetized rat brain cortex.

An assay for in vivo, continuous and automatic monitoring of extracellular malondialdehyde concentrations in anesthetized rat brain cortex was developed. This method involved the use of microdialysis perfusion, on-line derivatization and on-line high-performance liquid chromatographic analysis. Microdialysate from an implanted microdialysis probe was on-line reacted with thiobarbituric acid at 80 degrees C for 10 min prior to on-line collection and automatic injection into a HPLC system equipped with a fluorescence detector. This method gave a linear response between the concentrations of the malondialdehyde in the microdialysates and the TEP solution where the microdialysis probe was placed. This method was used to observe the increased extracellular malondialdehyde production following elevated extracellular glutamate levels, which were achieved by perfusion of L-trans-pyrrolidine-2,4-dicarboxylate, a competitive inhibitor of glutamate uptake transporter.

Diethylmaleate decreased ascorbic acid release induced by cerebral ischemia in cerebral cortex of the anesthetized rat.

The effect of diethylmaleate administration on ascorbic acid release following cerebral ischemia was investigated in anesthetized rat brain cortex. Cerebral ischemia, induced by ligating bilateral common carotid arteries and unilateral middle cerebral artery, significantly increased the extracellular ascorbic acid levels. Diethylmaleate (4 mmoles/kg, i.p.), which has been shown in earlier studies to decrease the ischemia-induced glutamate release, significantly reduced the ischemia-induced ascorbic acid release. The ischemia-induced ascorbic acid release was unaffected by perfusing NMDA receptor antagonist MK 801 (75 microM). Additionally, elevated extracellular glutamate levels, achieved by either externally applied glutamate solutions or by perfusing L-trans-pyrrolidine-2,4-dicarboxylate (PDC) (31.4 mM and 15.7 mM) to inhibit the glutamate uptake transporter, also significantly increased the extracellular ascorbic acid levels. These results suggested that ascorbic acid release in cerebral ischemia might be related to the elevated extracellular glutamate levels, which occurs following cerebral ischemia.

Circadian variations in plasma neutral and basic amino acid concentrations in young men on an ordinary Taiwanese diet.

BACKGROUND AND PURPOSE: Plasma levels of circulating free amino acids reflect the net status of protein breakdown and synthesis, and may be linked to various disease states. We studied circadian variations in plasma concentrations of neutral and basic amino acids during a 24-hour period in healthy young men who consumed ordinary Taiwanese test meals. METHODS: Ten subjects ingested the test diet (protein intake, 1.5 g.kg-1.d-1) which was offered in three meals and two light snacks during the day. Thirteen heparinized blood samples were collected from each subject to analyze plasma amino acid concentrations during the experimental period, at 1- to 3-hour intervals. RESULTS: The plasma concentrations of all neutral amino acids, including the large neutral amino acids (leucine, isoleucine, valine, tryptophan, tyrosine, and phenylalanine) and methionine, as well as the small neutral amino acids (glycine, serine, threonine, and proline) and the basic amino acids (histidine, arginine, lysine), varied significantly as a function of the time of day (p < 0.001). Except for glycine and proline, all of the neutral amino acids exhibited a marked evening elevation after dinner, with the highest plasma concentration at 23:00. Proline showed peak concentrations at 09:00, while glycine and the basic amino acids exhibited peak concentrations at 21:00. Most of the plasma amino acids exhibited the lowest concentrations at 12:00. CONCLUSION: Plasma neutral and basic amino acid concentrations exhibited significant circadian variations. The present study also provided the mean fasting plasma levels of amino acids in healthy young men consuming an ordinary Taiwanese diet.

Circadian variations in plasma and erythrocyte glutamate concentrations in adult men consuming a diet with and without added monosodium glutamate.

This study evaluated the effect of monosodium glutamate (MSG) ingestion as a component of the diet on the 24-h variations in plasma and whole-blood glutamate (GLU) concentrations in healthy adult men. In the first arm of the study, subjects were given test meals without added MSG for 3 d. Protein and energy intakes of the subjects were 1.5 g and 40 kcal/(kg body weight.d), respectively. On d 3, blood samples were collected over the 24-h period. One week later, the same protocol was repeated, except that 100 mg/(kg body weight.d) MSG was added to the meals (15, 40 and 45 mg/kg body weight to breakfast, lunch and dinner, respectively). Both plasma and whole-blood samples were analyzed for free amino acids. Unlike large neutral amino acids, which experienced high peak plasma concentrations at 2100-2300 h, the circadian variations in plasma GLU concentrations were small, varying between 33 and 48 micromol/L on days in which no MSG was fed, and between 32 and 53 micromol/L on days in which MSG was added to the meals. In both trials, plasma GLU concentration increased (P < 0.01) after lunch and dinner, and decreased early in the morning (P < 0.05). Calculated erythrocyte GLU concentrations varied between 500 and 640 micromol/L, with or without MSG addition to the meals. The rather low plasma GLU concentrations over the 24-h period, despite high dietary intake of MSG, indicate that dietary MSG is metabolized very rapidly.