Sterilization effect of atmospheric plasma on Escherichia coli and Bacillus subtilis endospores

Aims:  Escherichia coli and Bacillus subtilis spores were treated with an atmospheric plasma mixture created by the ionization of helium and oxygen to investigate the inactivation efficiency of a low-temperature plasma below 70°C.
Methods and results:  An electrical discharge plasma was produced at a radio frequency (RF) of 13·56 MHz, connected to a perforated circular electrode with a discharge spacing of 1–15 mm. The discharge gas was helium with 0–2% oxygen. For the plasma treatment, a dried E. coli cell or B. subtilis endospore suspension on a cover-glass was exposed to oxygen downstream of the plasma from holes in an RF-powered electrode. The sterilization effect of the RF plasma was highest with 0·2% oxygen, corresponding to the maximum production of oxygen radicals.
Conclusions:  Oxygen radicals generated by RF plasma are effective for the destruction of bacterial cells and endospores.
Significance and Impact of the study:  Low-temperature atmospheric plasma can be used for the disinfection of diverse objects, especially for the inactivation of bacterial endospores.     

Cold atmospheric air plasma sterilization against spores and other microorganisms of clinical interest

Physical cold atmospheric surface microdischarge (SMD) plasma operating in ambient air has promising properties for the sterilization of sensitive medical devices where conventional methods are not applicable. Furthermore, SMD plasma could revolutionize the field of disinfection at health care facilities. The antimicrobial effects on Gram-negative and Gram-positive bacteria of clinical relevance, as well as the fungus Candida albicans, were tested. Thirty seconds of plasma treatment led to a 4 to 6 log(10) CFU reduction on agar plates. C. albicans was the hardest to inactivate. The sterilizing effect on standard bioindicators (bacterial endospores) was evaluated on dry test specimens that were wrapped in Tyvek coupons. The experimental D(23)(°)(C) values for Bacillus subtilis, Bacillus pumilus, Bacillus atrophaeus, and Geobacillus stearothermophilus were determined as 0.3 min, 0.5 min, 0.6 min, and 0.9 min, respectively. These decimal reduction times (D values) are distinctly lower than D values obtained with other reference methods. Importantly, the high inactivation rate was independent of the material of the test specimen. Possible inactivation mechanisms for relevant microorganisms are briefly discussed, emphasizing the important role of neutral reactive plasma species and pointing to recent diagnostic methods that will contribute to a better understanding of the strong biocidal effect of SMD air plasma.     

Role of superoxide in the germination of Bacillus anthracis endospores

The spore forming Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, has achieved notoriety due to its use as a bioterror agent. In the environment, B. anthracis exists as a dormant endospore. Germination of endospores during their internalization within the myeloid phagocyte, and the ability of those endospores to survive exposure to antibacterial killing mechanisms such as superoxide (O(2)*-, is a key initial event in the infective process. We report herein that endospores exposed to fluxes of O(2)*- typically found in stimulated phagocytes had no effect on viability. Further endospores of the Sterne strain of B. anthracis were found to scavenge O(2)*-, which may enhance the ability of the bacterium to survive within the hostile environment of the phagolysosome. Most intriguing was the observation that endospore germination was stimulated by a flux of O(2)*- as low as 1 microM/min. Data presented herein suggest that B. anthracis may co-opt O(2)*- which is produced by stimulated myeloid phagocytes and is an essential element of host immunity, as a necessary step in productive infection of the host.     

How much ozone bactericidal activity is compromised by plasma components?

Aims:  Evaluation of bactericidal effect of different concentrations of ozone when used (a) as a gas, or (b) dissolved in saline. The addition of hydrogen peroxide or 4-hydroxynonenal dissolved in saline was also tested, as well as the effect of human plasma.
Methods and Results:  Staphylococcus aureus , methicillin-resistant Staph. aureus (MRSA), and Pseudomonas aeruginosa , suspended in their culture media were tested. While all bacteria suspended in protein-free saline were killed at high ozone concentrations, they survived when as little as 5% human plasma was present. Hydrogen peroxide was 100-fold less active than ozone and needed to remain in contact with bacteria for at least 60 min. 4-hydroxynonenal (2 μmol l⁻¹) was inhibitory for proliferation of both Staph. aureus and MRSA, but not for Ps. aeruginosa .
Conclusions:  Ozone and the cascade of its derivative products are potent bactericidal agents, but even small amounts of human plasma, hence of hydro- and liposoluble antioxidants, in bacterial suspensions inhibit oxidation and protect bacteria.
Significance and Impact of the Study:  Any substantial in vivo cytocidal effect of ozone and its derivatives can be excluded. On the other hand, topical and continuous action of various ozone preparations remains valuable in a variety of skin and mucosal infections.     

Inactivation of Escherichia coli K12 Using Atmospheric Gas Plasma Produced from Humidified Working Gas

The bactericidal effect of atmospheric gas plasma (AGP) on Escherichia coli K12 cells in 0.9% [w/v] sodium chloride was investigated under normal atmospheric pressure. A plasma‐generating unit was supplied with working gas (air) at 25 °C and a relative humidity (RH) of 0, 14, 32, 43, 50, 60 or 70%. Gas plasma was generated using radio frequency discharge (20 kHz) under atmospheric pressure, and blown onto the surface of an E. coli cell suspension. Seven log‐cycles of cells were completely inactivated within 15 minutes of the start of AGP treatments using working gas at 32, 43, 50 or 60% RH. AGP at 14% RH inactivated only 2 log‐cycles of cells, but no inactivation was observed when air at 0% or 70% RH was used. The inactivation curves were biphasic and the rate constants for both stages were closely related to the RH of the working gas. The rate of cell inactivation was at its maximum at an RH of 43%. The bactericidal effect of the AGP treatment was not a result of ozone generation, however, the moisture content of the working gas was a significant factor in ensuring that E. coli K12 inactivation occurred. The interpretation of these data was that chemical species generated from the water molecules in the working gas were bactericidal in their effects.     

Bactericidal effect of solar water disinfection under real sunlight conditions

Batch solar disinfection (SODIS) inactivation kinetics are reported for suspensions in water of Campylobacter jejuni, Yersinia enterocolitica, enteropathogenic Escherichia coli, Staphylococcus epidermidis, and endospores of Bacillus subtilis, exposed to strong natural sunlight in Spain and Bolivia. The exposure time required for complete inactivation (at least 4-log-unit reduction and below the limit of detection, 17 CFU/ml) under conditions of strong natural sunlight (maximum global irradiance, approximately 1,050 W m(-2) +/- 10 W m(-2)) was as follows: C. jejuni, 20 min; S. epidermidis, 45 min; enteropathogenic E. coli, 90 min; Y. enterocolitica, 150 min. Following incomplete inactivation of B. subtilis endospores after the first day, reexposure of these samples on the following day found that 4% (standard error, 3%) of the endospores remained viable after a cumulative exposure time of 16 h of strong natural sunlight. SODIS is shown to be effective against the vegetative cells of a number of emerging waterborne pathogens; however, bacterial species which are spore forming may survive this intervention process.     

Novel soluble epoxide hydrolase inhibitor protects mitochondrial function following stress

Epoxyeicosatrienoic acids (EETs) are active metabolites of arachidonic acid that are inactivated by soluble epoxide hydrolase enzyme (sEH) to dihydroxyeicosatrienoic acid. EETs are known to render cardioprotection against ischemia reperfusion (IR) injury by maintaining mitochondrial function. We investigated the effect of a novel sEH inhibitor (sEHi) in limiting IR injury. Mouse hearts were perfused in Langendorff mode for 40 min and subjected to 20 min of global no-flow ischemia followed by 40 min of reperfusion. Hearts were perfused with 0.0, 0.1, 1.0 and 10.0 μmol·L(-1) of the sEHi N-(2-chloro-4-methanesulfonyl-benzyl)-6-(2,2,2-trifluoro-ethoxy)-nicotinamide (BI00611953). Inhibition of sEH by BI00611953 significantly improved postischemic left-ventricular-developed pressure and reduced infarct size following IR compared with control hearts, and similar to hearts perfused with 11,12-EETs (1 μmol·L(-1)) and sEH(-/-) mice. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 10 μmol·L(-1)), or the plasma membrane K(ATP) channels (pmK(ATP)) inhibitor (glibenclamide, 10 μmol·L(-1)) abolished the improved recovery by BI00611953 (1 μmol·L(-1)). Mechanistic studies in H9c2 cells demonstrated that BI0611953 decreased ROS generation, caspase-3 activity, proteasome activity, increased HIF-1∝ DNA binding, and delayed the loss of mitochondrial membrane potential (ΔΨ(m)) caused by anoxia-reoxygenation. Together, our data demonstrate that the novel sEHi BI00611953, a nicotinamide-based compound, provides significant cardioprotection against ischemia reperfusion injury.     

Bovicin HC5 reduces thermal resistance of Alicyclobacillus acidoterrestris in acidic mango pulp

Aims:  To test the effect of bovicin HC5 against vegetative cells and endospores of Alicyclobacillus acidoterrestris DSMZ 2498 in synthetic media and in acidic mango pulp.
Methods and Results:  Alicyclobacillus acidoterrestris was grown in synthetic medium at 40°C and pH 4·0. The effect on vegetative cells was assayed by adding bovicin HC5 to synthetic medium (40–160 AU ml⁻¹) or to mango pulp (100 AU ml⁻¹) at various pH values and determining the effect on growth (OD_600nm) and viable cell number, respectively. The effect of bovicin HC5 on spore germination and thermal sensitivity of A. acidoterrestris was tested in mango pulp (pH 4·0) containing 80 AU ml⁻¹ of bovicin HC5. Bovicin HC5 was bactericidal against vegetative cells of A. acidoterrestris at different pH values and showed sporicidal activity against endospores of this bacterium. When spores of A. acidoterrestris were heat treated in the presence of bovicin HC5, D -values decreased 77% to 95% compared to untreated controls at temperatures ranging from 80 to 95°C.
Conclusion:  Bovicin HC5 was bactericidal and sporicidal against A. acidoterrestrsi DSMZ 2498.
Significance and Impact of the Study:  These results indicated that bovicin HC5 has potential to prevent spoilage of acidic fruit juices by thermocidophilic spore-forming bacteria.     

Production of greenhouse tomatoes (Lycopersicon esculentum) using Nannotrigona perilampoides, Bombus impatiens and mechanical vibration (Hym.: Apoidea)

Importation of exotic bumblebees for greenhouse pollination may be restricted in México, thus making it necessary to evaluate the potential of native species as pollinators in enclosures. We studied the foraging activity and fruit production of tomato using one colony of Nannotrigona perilampoides (NP) and one colony of Bombus impatiens (BI) in greenhouses with ≈1000 plants. Mechanical vibration (MV) was included as a test treatment. The foraging activity was measured as the number of flowers visited within 5 min, the time spent on a flower collecting pollen, the number of visits that a flower received and the duration of a foraging trip. BI collected pollen more rapidly, visited more flowers within 5 min and did more visits per flower when compared with NP that also lasted longer in their trips. Significant correlations were found between environmental variables and the number of bees entering the hive and the number of bees on the flowers. For NP, the highest correlation was found for light intensity whilst in BI a negative effect of environmental temperature was detected. Regarding the quantity of fruit, BI resulted in higher fruit set when compared with NP, but the latter performed similarly to MV. However, the weight of the fruit and seed number was significantly higher for BI when compared with NP, and this was higher than for MV. Our results demonstrate that at the densities of tomato plants tested, one colony of BI was more efficient pollinator when compared with NP. We suggest that pollination efficiency of NP could have been limited by a reduced number of foragers on the plants at a given time and their limited flight range when compared with BI. Therefore, it will be necessary to evaluate if increasing densities of colonies of NP could improve tomato yield in tropical greenhouses.     

Cold atmospheric plasma jet effects on V79-4 cells

The effects of cold plasmas are due to charged particles, reactive oxygen species (ROS), reactive nitrogen species (RNS), UV photons, and intense electric field. In order to obtain a more efficient action on mammalian cells (useful for cancer therapy), we used in our studies chemically activated cold plasma (He and O2 gas mixture). V79-4 cells were exposed to plasma jet for different time periods (30, 60, 90, 120 and 150s), using different combinations of helium and oxygen inputs (He:2.5l/min + 02:12.5ml/min; He:2.51/min + O2:25ml/min; He:2.51/min + O2:37.5 ml/min). Using MTT test we demonstrated that plasma jet induced cell viability decrease in all cases. The effect of chemically activated cold plasma--apoptosis or necrosis--depends on gas mixture and treatment period. Taking into account that ROS density in cell microenvironment is related to O2 percent in the gas mixture and treatment period, we can presume that cell death is due to ROS produced in plasma jet.     

等离子体射流灭活液体中铜绿假单胞菌的研究

【目的】测定等离子射流对铜绿假单胞菌(Pseudomonas aeruginosa)的灭活效果,探究低温等离子体射流的杀菌机理。【方法】采用平板计数法测定等离子体射流的杀菌效果,荧光显微镜、透射电镜观察等离子体作用后菌体结构的变化,蛋白浓度测定和SDS-PAGE电泳检测菌液上清液中可溶性蛋白的泄漏量。【结果】等离子体射流处理铜绿假单胞菌菌液5 min,杀灭率可达到99.9%以上。透射电镜观察可见细菌菌体结构发生改变,细胞壁、细胞膜损伤破裂,细胞内容物泄露。进一步对处理铜绿假单胞菌上清液中的蛋白质含量变化进行检测,结果显示随着处理时间的增加,上清液中蛋白质含量持续增加,在2 min时达到最大值。【结论】等离子体射流可以通过破坏细胞结构造成细胞质泄露,使其丧失正常的细胞功能,从而达到快速有效地杀灭铜绿假单胞菌的效果。     

The use of flow cytometry to study the germination of Bacillus cereus endospores.

BACKGROUND: At present the study of endospore germination is conducted using microbiological methods which are slow and yield data based on the means of large heterogeneous populations. Flow cytometry (FCM) offers the potential to rapidly quantify and identify germination and outgrowth events for large numbers of individual endospores. METHODS: Standard methods were employed to arrest the germination of Bacillus cereus endospores at defined stages. Endospores were then stained with SYTO 9 alone or carboxyfluorescein diacetate (CFDA) together with Hoechst 33342 and analysed using FCM. Comparisons were made between FCM as a method to measure germination rate and standard microbiological techniques. RESULTS: Germinating endospores displayed increases in permeability to SYTO 9 and hydrolysis of CFDA compared with controls. Statistically significant correlations were found between the standard plate count method and both FCM methods for measuring the percentage of germinating and outgrowing endospores up to 75 min after addition of germinant. CONCLUSIONS: Using FCM, the percentage of germinating or outgrowing endospores at various time points during germination and/or outgrowth can be quantified. FCM with CFDA/Hoechst 33342 staining may be used to estimate overall germination rate, whereas FCM with SYTO 9 staining may be used to quantify ungerminated, germinating and outgrowing endospores.     

Bactericidal and sporicidal performance of a polymer-encapsulated chlorine dioxide-coated surface

Aims:  To investigate the physical characteristics and the bactericidal and sporicidal potential of a polymer-encapsulated ClO₂ coating.
Methods and Results:  An antimicrobial coating based on polymer-encapsulated ClO₂ was developed. A low viscosity, water/oil/water double emulsion coating was formulated for easy on-site application. Escherichia coli , Pseudomonas aeruginosa , Bacillus subtilis and Staphylococcus aureus were applied onto the coating to study the bactericidal capabilities of the coating. The bactericidal performance of the coating increased when the contact time with the tested bacteria increased. Over 99% of the E. coli , Ps. aeruginosa , B. subtilis were killed with a contact time of 30 min. Although endospores of B. subtilis are more resistant, about 75% of the spores were killed after 72 h on the coating. Moreover, a sustained release of gaseous ClO₂ was achieved to maintain about 90% removal of B. subtilis with a 10-min contact time during a 28-day study period. The coating also exhibits antiadhesive properties against bacteria.
Conclusions:  A polymer-encapsulated ClO₂ coating with sustained release of ClO₂ and promising bactericidal and sporicidal features was tested for 28 days.
Significance and Impact of the Study:  This study provides a new direction for developing polymer-encapsulated ClO₂ coatings that possess persistent bactericidal and sporicidal properties.     

Porphyrin derivatives as photosensitizers for the inactivation of Bacillus cereus endospores

Aims:  In this study, we propose (i) to study the photodynamic inactivation (PDI) efficiency of neutral and cationic porphyrin derivatives, (ii) to characterize the kinetics of the inactivation process using Bacillus cereus as a model endospore-producing bacterium and (iii) to conclude on the applicability of porphyrin derivatives in the inactivation of bacterial endospores.
Methods and Results:  The study of PDI of Bacillus cereus endospores, taken as model-endospores, using porphyrin derivatives differing in the number of positive charges and in the meso-substituent groups, showed that neutral, monocationic and dicationic porphyrins are quite ineffective, in contrast with the tri- and tetra-cationic molecules. The most effective porphyrin is a tricationic porphyrin with a meso-pentafluorophenyl group. With this photosensitizer (PS), at 0·5  μ mol l⁻¹, a reduction of 3·5 log units occurs after only 4 min of irradiation. None of the porphyrin derivatives showed toxicity in the absence of light.
Conclusions:  Some porphyrin derivatives are efficient PSs for the inactivation of bacterial endospores and should be considered in further studies. Small modifications in the substituent groups, in addition to charge, significantly improve the effectiveness of the molecule as a PS for endospore inactivation.
Significance and Impact of the Study:  Tetrapyrrolic macrocycles should be regarded as worthy to explore for the PDI of spore-producing gram-positive bacteria. The development of molecules, more selective and effective, emerges as a new objective.     

Production of Photons in the Bactericidal Effect of Transient Electric Arcs in Aqueous Systems

The radiation in the visible and ultraviolet regions from submerged, transient electrical arcs was measured with a K(3)Fe(C(2)O(4))(3) chemical actinometer and was compared to the bactericidal effect obtained with the same electrical arrangements. Photon production and bactericidal effect were obtained at lower voltages with a smaller electrode separation than with a wider one. At higher voltages, both increased with wider electrode separations. The voltages at maximal photon production efficiency coincided with those of maximal bactericidal efficiency. However, the same photon radiation produced by different electrical arrangements did not always yield the same bactericidal effect in the small discharge vessel usually employed. In a larger discharge vessel, the bactericidal effect was closely correlated with the photon production. The efficiency of photon production by transient arcs was smaller than that of germicidal mercury lamps, particularly with respect to wavelengths of great bactericidal activity. The mechanisms of inactivation and their use for practical disinfection purposes are discussed.     

The effects of carbon dioxide anesthesia and anoxia on rapid cold-hardening and chill coma recovery in Drosophila melanogaster

Carbon dioxide gas is used as an insect anesthetic in many laboratories, despite recent studies which have shown that CO(2) can alter behavior and fitness. We examine the effects of CO(2) and anoxia (N(2)) on cold tolerance, measuring the rapid cold-hardening (RCH) response and chill coma recovery in Drosophila melanogaster. Short exposures to CO(2) or N(2) do not significantly affect RCH, but 60 min of exposure negates RCH. Exposure to CO(2) anesthesia increases chill coma recovery time, but this effect disappears if the flies are given 90 min recovery in air before chill coma induction. Flies treated with N(2) show a similar pattern, but require significantly longer chill coma recovery times even after 90 min of recovery from anoxia. Our results suggest that CO(2) anesthesia is an acceptable way to manipulate flies before cold tolerance experiments (when using RCH or chill coma recovery as a measure), provided exposure duration is minimized and recovery is permitted before chill coma induction. However, we recommend that exposure to N(2) not be used as a method of anesthesia for chill coma studies.     

Effect of muramyl dipeptide analog on Salmonella enteritidis infection in beige mice with Chediak-Higashi syndrome

Beige mutant (bg/bg) mice with Chediak-Higashi syndrome (CHS) were much more sensitive to virulent Salmonella enteritidis No. 11 strain than parental C57 BL/6 (+/+) or heterozygous (bg/+) mice, and they had weaker bactericidal activity against the organisms. Muramyl dipeptide (MDP) and N alpha-(N-acetyl-muramyl-L-alanyl-D-isoglutamyl)-N epsilon-stearoyl-L-lysine [MDP-Lys(L18)], a synthetic derivative of MDP, failed to confer any protection against the infection, but the MDPs showed some ability to stimulate the bactericidal activity in the peritoneal cavities and spleens of these mice. The bactericidal effect of MDP-Lys(L18) was dose-dependent, and the greatest effect was seen when it had been injected 24 hr before the infection. Multiple injections of MDP were much more beneficial than a single injection. Previous injection of N2,O2'-dibutyryl guanosine 3' : 5'-cyclic monophosphate (DB-cGMP) improved the impaired bactericidal capacity in beige mice, but the simultaneous injection of N6,O2-dibutyryl adenosine 3' : 5'-cyclic monophosphate (DB-cAMP) with DB-cGMP abolished the effect of DB-cGMP. The augmentation of bactericidal capacity by MDP-Lys(L18) was not affected by the injection of either DB-cGMP or DB-cAMP, suggesting that the effect of the MDPs was not related directly to cyclic nucleotide regulation in beige mice.     

Water disinfection with the hydrogen peroxide-ascorbic acid-copper (II) system.

Treatment of secondary effluents with hydrogen peroxide (10 mg/liter)-ascorbic acid (10 mg/liter)-Cu2+ (0.5 mg/liter) for 60 min resulted in around 99% reduction of the initial plate count. Hydrogen peroxide could be replaced by other peroxygen compounds; ascorbic acid could be replaced by other reducing agents, of which sodium sulfite and ethanol were the most effective. Cu2+, however, could not be replaced by other metal ions without loss of bactericidal efficiency of the ternary combination. Enterobacteriaceae, total and fecal coliforms, staphylococci, and micrococci were reduced by 99.0 to 99.9%. Group D streptococci aerobic spores were reduced by 80 and 15%, respectively. Clostridium perfringens, yeasts, and molds were not killed by the disinfectant combinations. The effect of pH was only minor in the range from 6 to 7.5. At a higher pH value the bactericidal effects tended to decrease. The hydrogen peroxide-ascorbic acid-Cu2+ combination made it possible to obtain 99% reduction within 30 min. When using the hydrogen peroxide-sodium sulfite-Cu2+ or the hydrogen peroxide-ethanol-Cu2+ combinations, 60 min of contact time was necessary to obtain 99% reduction of the initial plate count. Cu2+ combined to an intermediate product of the ascorbic acid autoxidation is the toxic agent, and its penetration into the cell is promoted by hydrogen peroxide.     

Effect of N2-He-O2 on decompression outcome in rats after variable time-at-depth dives

No study of decompression sickness has examined both variable gas mixtures and variable time at depth to the point of statistical significance. This investigation examined the effect of N2-He-O2 on decompression outcome in rats after variable time-at-depth dives. Unanesthetized male albino rats were subjected to one of two series of simulated dives: 1) N2-He-O2 dives (20.9% O2) at 175 feet of seawater fsw) and 2) N2-O2 dives (variable percentage of O2; depths from 141 to 207 fsw). Time at depth ranged from 10 to 120 min; rats were then decompressed within 10 s to surface pressure. The probability of decompression sickness (severe bends symptoms or death) was analyzed with a Hill equation model, with parameters for gas potency and equilibrium time for the three gases and weight of the animal. Relative potencies for the three gases were of similar magnitude for bends and statistically different for death in ascending order: O2 less than He less than N2. Estimated gas uptake rates were different. N2 took three to four times as long as He to reach full effect; the rate of O2 appeared to be considerably shorter than that of N2 or He. The large influence of O2 on decompression outcome questions the simplistic view that O2 cannot contribute to the decompression requirement.     

Simulated first-phase insulin release using Humulin or insulin analog HIM2 is associated with prolonged improvement in postprandial glycemia

We examined the extent to which priming the liver with a pulse of Humulin or the insulin analog hexyl-insulin monoconjugate 2 (HIM2) reduces postprandial hyperglycemia. Somatostatin (0.5 microg.kg(-1).min(-1)) was given with basal intraportal insulin and glucagon for 4.5 h into three groups of 42-h-fasted conscious dogs. From 0-5 min, group 1 (BI, n = 6) received saline, group 2 (HI, n = 6) received a Humulin pulse (10 mU.kg(-1).min(-1)), and group 3 (HIM2, n = 6) received a HIM2 pulse (10 mU.kg(-1).min(-1)). Duodenal glucose was infused (5.0 mg.kg(-1).min(-1)) from 15 to 270 min. Arterial insulin in BI remained basal (6 +/- 1 microU/ml) and peaked at 52 +/- 15 (HI) and 164 +/- 44 microU/ml (HIM2) and returned to baseline by 30 and 60 min, respectively. Arterial plasma glucose plateaued at 265 +/- 20, 214 +/- 15, and 193 +/- 14 mg/dl in BI, HI, and HIM2. Glucose absorption was similar in all groups. Significant net hepatic glucose uptake occurred at 85, 55, and 25 min in BI, HI, and HIM2, respectively. Nonhepatic glucose clearance at 270 min differed among groups (BI, HI, HIM2): 0.62 +/- 0.11, 0.76 +/- 0.26, and 1.61 +/- 0.29 ml.kg(-1).min(-1) (P < 0.05). A brief (5-min) insulin pulse improved postprandial glycemia, stimulating hepatic glucose uptake and prolonging enhancement of nonhepatic glucose clearance. HIM2 was more effective than Humulin, perhaps because its lowered clearance caused higher levels at the liver and periphery and its biological activity was not reduced proportionally to its decreased clearance.