Sonoluminescence and acoustic emission spectra at different stages of cavitation zone development.

The way in which a cavitation zone develops in a focused pulsed ultrasound field is studied in this work. Sonoluminescence (SL), total hydrophone output and cavitation noise spectra have been recorded across a gradual, smooth increase in applied voltage. It is shown that the cavitation zone passes through a number of stages of evolution, according to increasing ultrasound intensity, decreasing pulse period and increasing ultrasound pulse duration. Sonoluminescence is absent in the first phase and the hydrophone output spectra consists of a main line with two or three harmonics whose intensity is much lower than that of the main (fundamental) line. The second stage sees the onset of SL whose intensity increases smoothly and is accompanied by the appearance of higher harmonics and subharmonics in the cavitation noise spectra. In some cases, the wide-band (WBN) component can be seen in noise spectra during the final part of the second stage. In the third stage, SL intensity increases significantly and often quite sharply, while WBN intensity increases in the same manner. This is accompanied by a synchronous increase in the absorption of ultrasound by the cavitation zone, which is manifested in a sharp decrease in the hydrophone output. In the fourth stage, both SL and WBN intensities tend to decrease despite the increased voltage applied to the transducer. Furthermore, the fundamental line tends to decrease in strength as well, despite the increasing ultrasound intensity. The obtained results clearly identify the different stages of cavitation zone development using cavitation noise spectra analyses. We then hypothesize that three of the above stages may be responsible for three known types of ultrasound action on biological cells: damping viability, reversible cell damage (sonoporation) and irreversible damage/cytotoxicity.

Anaesthetic efficacy of bupivacaine 2-hydroxypropyl-ß-cyclodextrin for dental anaesthesia after inferior alveolar nerve block in rats.

Bupivacaine is a long-acting local anaesthetic that is widely used in medicine and dentistry. The duration and intensity of its sensory blockade in animal models is increased by its inclusion in complexes with cyclodextrins. The aim of the present study was to evaluate the anaesthetic efficacy of bupivacaine 2-hydroxypropyl-ß-cyclodextrin (HPßCD) inclusion complex for dental anaesthesia after inferior alveolar nerve block in rats. Thirty rats were each given an injection close to the mandibular foramen of 0.2ml of one of the following formulations: 0.5% bupivacaine alone; 0.5% bupivacaine with 1:200,000 epinephrine; and 0.5% bupivacaine-HPßCD inclusion complex (bupivacaine-HPßCD). The other sides were used as controls, with either 0.9% saline or anaesthetic-free HPßCD solution being injected. The onset, success, and duration of pulpal anaesthesia were assessed by electrical stimulation ("pulp tester") on inferior molars. Results were analysed using ANOVA (Tukey), log rank, and chi square tests (α=5%). There were no differences among the formulations in onset of anaesthesia (p=0.59) or between the bupivacaine plus epinephrine and bupivacaine plus HPßCD in duration of anaesthesia, but bupivacaine plus epinephrine gave significantly higher values than bupivacaine alone (p=0.007). Bupivacaine plus epinephrine was a better anaesthetic than bupivacaine alone (p=0.02), while Bupi-HPßCD gave intermediate results, and therefore did not differ significantly from the other 2 groups (p=0.18 with bupivacaine alone; and p=0.44 with bupivacaine plus epinephrine). The bupivacaine-HPßCD complex showed similar anaesthetic properties to those of bupivacaine with epinephrine.

Innovative formulations for the controlled and site-specific delivery of antiinflammatory drugs.

Pharmaceutical technology has introduced a promising pathway in the future of medicine in particular nanotechnological innovations have provided the opportunity to design and develop efficient drug delivery systems able to target and treat several diseases, including those mediated by inflammation. The engineering of drug delivery systems can be used to target tissues involved in the pathology under treatment, to avoid early drug biological environmental degradation and to modulate drug pharmacokinetics. Glucocorticoids and non-steroidal anti-inflammatory drugs are the most commonly prescribed drug categories worldwide for the treatment of disorders associated with inflammation. Although glucocorticoids can be highly effective in treating inflammation, their systemic application is limited due to the high incidence of serious adverse effects, mainly in long-term treatment. Non-steroidal anti-inflammatory drugs are a heterogeneous group of compounds and most of them have unfavorable pharmacokinetics and pharmacodynamics, leading to adverse effects, such as gastrointestinal disorders. Therefore, the need for drug delivery systems for long term administration of anti-inflammatory drugs with a well-controlled release profile is evident. The aim of this review is to assess innovative colloidal drugs carriers, in particular liposomes and nanoparticles, with special focus on site-specific delivery for particularly problematic tissues such as the gastrointestinal tract, joints and eyes.

Cholesteryl butyrate solid lipid nanoparticles inhibit the adhesion and migration of colon cancer cells.

BACKGROUND AND PURPOSE Cholesteryl butyrate solid lipid nanoparticles (cholbut SLN) provide a delivery system for the anti-cancer drug butyrate. These SLN inhibit the adhesion of polymorphonuclear cells to the endothelium and may act as anti-inflammatory agents. As cancer cell adhesion to endothelium is crucial for metastasis dissemination, here we have evaluated the effect of cholbut SLN on adhesion and migration of cancer cells. EXPERIMENTAL APPROACH Cholbut SLN was incubated with a number of cancer cell lines or human umbilical vein endothelial cells (HUVEC) and adhesion was quantified by a computerized micro-imaging system. Migration was detected by the scratch 'wound-healing' assay and the Boyden chamber invasion assay. Expression of ERK and p38 MAPK was analysed by Western blot. Expression of the mRNA for E-cadherin and claudin-1 was measured by RT-PCR. KEY RESULTS Cholbut SLN inhibited HUVEC adhesiveness to cancer cell lines derived from human colon-rectum, breast, prostate cancers and melanoma. The effect was concentration and time-dependent and exerted on both cancer cells and HUVEC. Moreover, these SLN inhibited migration of cancer cells and substantially down-modulated ERK and p38 phosphorylation. The anti-adhesive effect was additive to that induced by the triggering of B7h, which is another stimulus inhibiting both ERK and p38 phosphorylation, and cell adhesiveness. Furthermore, cholbut SLN induced E-cadherin and inhibited claudin-1 expression in HUVEC. CONCLUSION AND IMPLICATIONS These results suggest that cholbut SLN could act as an anti-metastastic agent and they add a new mechanism to the anti-tumour activity of this multifaceted preparation of butyrate.

High energy shock waves and 5-aminolevulinic for sonodynamic therapy: effects in a syngeneic model of colon cancer.

The cytotoxic effect of the natural porphyrin precursor 5-aminolevulinic acid (ALA) exposed to high energy shock waves (HESW) was investigated in vitro on DHD/K12/TRb rat colon cancer cells and in vivo on a syngeneic colon cancer model. In vitro, viable cell growth was determined by trypan blue exclusion assay and cell death was investigated by flow cytometry. ALA (50 µg/ml) and HESW (E1, EFD = 0.22 mJ/mm², 1000 shots or E2, EFD = 0.88 mJ/mm², 500 shots) showed a significant reduction of cancer cell proliferation at day 3 compared to cells exposed to ALA (p < 0.01) or HESW (p < 0.001) alone. An enhancement of necrotic and apoptotic cells was observed after combined treatment at day 1 with ALA and HESW E1 (a 3.1 and 6.4 fold increase vs ALA alone) or E2 (a 3.4 and 5.3 fold increase vs ALA alone). In vivo, apoptosis detection was carried out by TUNEL assay, the pro-apoptotic gene Bad and Bcl-2 mRNA expression was evaluated by quantitative SYBR Green real time RT-PCR and cleavage of poly(ADP-ribose)-polymerase (PARP) was investigated by Western Blotting. An enhancement of apoptosis was observed in tumour tissues after the combined treatment at day 1 with ALA (375 mg/kg i.v.) and HESW (E2) compared to that of ALA exposure alone with improved apoptotic index (a 2.0 fold increase), Bad enhanced mRNA expression (p < 0.01), Bcl-2 decreased mRNA expression (p < 0.05) and increased PARP cleavage. The interaction between HESW and ALA is then effective in inducing apoptosis on a syngeneic colon cancer model.

A survey of dioxins (PCDDs and PCDFs) and dioxin-like PCBs in sheep and goat milk from Campania, Italy.

In 2008, after the crisis of buffalo dairy fields in Campania, Italy, an assessment of the contamination of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) was also necessary for other animal species bred in the region. The contents of PCDDs, polychlorinated dibenzofurans (PCDFs), and dl-PCBs were determined by high-resolution gas chromatography/mass spectrometry (HR-GC/MS) (according to USEPA method 1613) in 69 sheep and goat milk samples from 63 farms. In eleven samples from six sheep farms, the PCDD/Fs levels exceeded the maximum limit of 3.0 pg g(-1) fat established by the European Commission, in particular the concentrations ranged between 3.89 and 12.90 pg g(-1) fat. Statistical treatment of the results for the congener profiles of the non-compliant and compliant samples has been used to identify the sources of contamination.

Levels and congener profiles of polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and dioxin-like polychlorinated biphenyls in cow's milk collected in Campania, Italy.

Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and certain dioxin-like polychlorinated biphenyls (dl-PCBs) are a family of chemically-related lipophilic compounds characterized by similar toxicity. Due to their properties they are universally distributed in the environment and classified as persistent organic pollutants (POPs). From most of studies carried out to evaluate human dietary intake, milk and dairy products result as a major contributors of PCDD/Fs uptake. Of course the main source of milk contamination is animal feeds. Lactating ruminants, cows included, transfer these compounds to the food chain by ingestion of contaminated vegetables or soil. Their resistance to degradation and a high lipophilicity means that PCDD/Fs and dl-PCBs may be accumulated into fat tissues from which they are transferred to milk during lactation period. Seventy-nine cows milk samples, collected in the monitoring plan 2008, were analyzed for PCDD/Fs and dl-PCBs. Eleven milk samples were non-compliant corresponding to five breeding livestock located in Caserta province. The distribution of PCDD/Fs and dl-PCBs congeners in these samples was examined in order to determine the likely sources of dioxins. The results show that the congener profile is characterized by a prevalence of PCDFs in respect of PCDDs, that represents the typical pattern of thermal origin contamination.

Prospective study of the effects of concomitant medications on thiopurine metabolism in inflammatory bowel disease.

BACKGROUND: Thiopurines are increasingly used in the treatment of inflammatory bowel disease (IBD), being the most common immunosuppressive therapy; however, potentially harmful interactions between thiopurines and other drugs (especially 5-aminosalicylic acid, 5-ASA) were described. AIM: To explore potential interactions between thiopurines and concomitant medications. METHODS: A total of 183 consecutive IBD patients were enrolled. Clinical characteristics and concomitant medications were recorded. Thiopurine metabolism was analysed with thiopurine S-methyl transferase (TPMT) genetic variants and enzyme activity assays. Comparisons were carried out with stratification of patients according to clinical characteristics and active treatments. RESULTS: Based on TPMT genetics, 95% IBD patients were wild-type homozygous, the remaining being heterozygous. Median TPMT activity was 24.9 U/Hgb g (IQR 20.7-29.5). No difference in TPMT activity was noted according to 5-ASA exposure. IBD patients on thiopurines had higher TPMT activity levels, but no dose-effect was evident. No difference in TPMT activity was observed in 41 (63%) patients co-treated with 5-ASA. In patients on active thiopurines also, 6-TGN and 6-MMP levels were evaluated and no significant difference was observed based on co-medication. TPMT activity was independently associated only with thiopurines dose (P = 0.016). CONCLUSIONS: Our data suggest the absence of significant interactions between thiopurines and 5-ASA.

Determination of ibuprofen, ketoprofen, diclofenac and phenylbutazone in bovine milk by gas chromatography-tandem mass spectrometry.

A method has been developed to analyse for ibuprofen (IBP), ketoprofen (KPF), diclofenac (DCF) and phenylbutazone (PBZ) residues in bovine milk. Milk samples were extracted with acetonitrile and sample extracts were purified on Isolute C(18) solid-phase extraction cartridges. Aliquots were analysed by gas chromatography-tandem mass spectrometry (GC-MS/MS). The method was validated in bovine milk, according to the criteria defined in Commission Decision 2002/657/EC. The decision limits (CCalpha were 0.59, 2.69, 0.90 and 0.70 ng ml(-1), respectively, for IBP, KPF, DCF and PBZ, and detection capabilities (CCbeta) of 1.01, 4.58, 1.54 and 1.19 ng ml(-1), respectively, were obtained. The measurement uncertainty of the method was 17.8%, 80.9%, 28.2% and 20.2% for IBP, KPF, DCF and PBZ, respectively. Fortifying bovine milk samples (n = 18) in three separate assays show the accuracy of the method to be between 104% and 112%. The precision of the method, expressed as relative standard deviations for the within-laboratory reproducibility at the three levels of fortification (5, 7.5 and 10 ng ml(-1)) was less than 8% for IBP, DCF and PBZ, respectively. Poor precision was obtained for KPF with a relative standard deviation of 28%.

Cytotoxicity of anticancer drugs incorporated in solid lipid nanoparticles on HT-29 colorectal cancer cell line.

Solid lipid nanoparticles (SLN) carrying cholesteryl butyrate (chol-but), doxorubicin and paclitaxel had previously been developed, and the antiproliferative effect of SLN formulations versus conventional drug formulations was here evaluated on HT-29 cells. The 50% inhibitory concentration (IC(50) values were interpolated from growth curves obtained by trypan blue exclusion assay. In vitro cytotoxicity of SLN carrying chol-but (IC(50 72 h) 0.3 +/- 0.03 mM vs >0.6 mM) and doxorubicin (IC(50 72 h) 81.87 +/- 4.11 vs 126.57 +/- 0.72 nM) was higher than that of conventional drug formulations. Intracellular doxorubicin was double after 24 h exposure to loaded SLN versus the conventional drug formulation, at the highest concentration evaluated by flow cytometry. In vitro cytotoxicities of paclitaxel-loaded SLN and conventional drug formulation (IC(50 72 h) 37.36 +/- 6.41 vs 33.43 +/-1.17 nM) were similar. Moreover, the combination of low concentrations of chol-but SLN (0.1-0.2 mM) and doxorubicin (1.72 nM) or paclitaxel (1.17 nM) exerted a greater-than-additive antiproliferative effect at 24 h exposure, while the combination of Na-but and doxorubicin or paclitaxel did not. These preliminary in vitro results suggest that SLN could be proposed as alternative drug delivery system.

Determination of disodium clodronate in human plasma and urine using gas-chromatography-nitrogen-phosphorous detections: validation and application in pharmacokinetic study.

We present a specific method for the determination of disodium clodronate in human plasma and urine using a gas-chromatographic system with nitrogen phosphorus detector (NPD). The compound was extracted from plasma and urine samples by an anion-exchange resin and derivatizated with bistrimethylsilyltrifluoroacetamide (BSTFA). Sodium bromobisphosphonate was used as internal standard. The calibration curves were linear in both plasma and urine, with a regression coefficient r > 0.9975 in plasma and r > 0.9977 in urine. The limit of quantitation was 0.3 microg/ml in plasma and 0.5 microg/ml in urine. The method was validated by intra-day assays at three concentration levels. During the study we carried out inter-day assays to confirm the feasibility of the method. The precision in plasma at 0.5, 15, and 45 microg/ml was 12.4, 0.2, and 6.5% (n = 40), respectively; in urine at 0.8, 8, and 40 microg/ml it was 8.6, 6.4, and 9.3% (n = 40), respectively. The method was accurate and reproducible, and was successfully applied to determine the pharmacokinetic parameters of clodronate in healthy volunteers after intravenous infusion and intramuscular injection of 200 mg of the compound. The Cmax after intravenous infusion and intramuscular injection was 16.1 and 12.8 microg/ml, respectively. AUC(0-48 h) after infusion administration and intramuscular injection was 44.2 +/- 18.0 and 47.5 +/- 12.4 h microg/ml, respectively. The elimination half-life in both administrations was 6.31 +/- 2.7 h.

Long-term analgesic effect of clodronate in rodents.

Several studies have shown that treatment with bisphosphonates can reduce the pain associated with different painful diseases. In a previous study we demonstrated that in mice two bisphosponates, clodronate and pamidronate, had an antinociceptive effect under acute conditions not related to bone processes, after in vein (iv) or intracerebroventricular (icv) injection. The present study tested the time-dependent antinociceptive action of clodronate and pamidronate in comparison with that of acetylsalicylic acid (ASA) and morphine after iv and icv injection using the tail-flick test in acute and chronic treatment. The effects of clodronate on other measures of animal behaviour were also evaluated. In the tail-flick test, administration of clodronate iv produced an antinociceptive effect that was greater than that of ASA and statistically significant up to 16 h; pamidronate iv showed a significant antinociceptive effect for only 6 h. Clodronate and pamidronate icv showed an increase in tail-flick latency time that was significant and lasted for 16 and 6 h, respectively, while morphine produced an antinociceptive effect for 24 h. In the test we found significant differences between male and female mice in the latency time values but not in the length of the analgesic effect. In the chronic treatment paradigm, clodronate produced a significant increase of the tail-flick latency after the first injection. The analgesic effect increased up to 50% after 5 days of treatment. Significant analgesic effects were still present after 3, 7, and 14 days from the end of treatment. Clodronate did not produce any significant behavioural effects in the Rota-rod test, pentobarbital-induced sleeping time, and locomotor activity cage. These data indicate that clodronate presents a central and peripheral prolonged antinociceptive effect, without any behavioural side effects.

Dexibuprofen (S+-isomer ibuprofen) reduces gastric damage and improves analgesic and antiinflammatory effects in rodents.

UNLABELLED: We determined the analgesic and antiinflammatory actions and the related acute mucosal gastric damage from the active S(+)-isomer ibuprofen (dexibuprofen), in comparison with those of the standard racemic formulation of ibuprofen in rodents. The antinociception was evaluated by hot-plate and tail-flick methods after IV and oral (PO) administration in mice and after PO administration in rats. S(+)-Ibuprofen was at least twice more potent than the ibuprofen racemic formulation. The antiinflammatory action of the test compound, assessed with the abdominal constriction test in mice (IV and PO) and with hind paw edema in rats (IV and PO), was found to be significantly more potent than that of ibuprofen after IV treatment in mice and PO administration in rats. Moreover, the test compound caused significantly less mucosal gastric damage than the racemic formulation administered at identical doses (50 mg/kg PO in rats). In conclusion, the S(+)-ibuprofen isomer was found to be more potent than the racemic formulation in analgesic and antiinflammatory tests and presented fewer gastric toxic effects. On the basis of the results of this work, we suggest that the administration of chemical entities, such as R(-)-ibuprofen, should be avoided if they are not essential for the anticipated therapeutic activity. IMPLICATIONS: Ibuprofen is a nonsteroidal antiinflammatory drug often prescribed as a racemic formulation. We studied the analgesic and antiinflammatory effects of the active S(+)-isomer. The S(+)-ibuprofen was found to be more potent than the racemic formulation and produced less acute gastric damage.

Rotational constraint in posterior-stabilized total knee prostheses.

Rotational stresses from box-post impingement have been implicated in the loosening of posterior-stabilized total knee prostheses. A bench model was constructed to assess the forces generated by tibiofemoral rotation. Rotational torque under load was measured in two different posteriorstabilized total knee prostheses using an axial-torsion load cell at 0 degrees, 20 degrees, and 40 degrees flexion over 20 degrees internal and external rotation. The Sigma posterior-stabilized prosthesis generated little torque through 5 degrees internal and external rotation. An increase in torque then occurred because of box-post impingement, generating peak torques of 17 to 18 N-m at 12 degrees to 14 degrees rotation. The bench model produced the same deformation of the polyethylene post as seen on retrieved specimens. The Scorpio posterior-stabilized prosthesis had a relatively continuous rise in generated torque from tibiofemoral conformity. Box-post impingement did not occur resulting in 32% lower torque between 12 degrees and 14 degrees rotation. Peak rotational torques of 15 to 16 N-m were reached at 19 degrees to 20 degrees rotation. Tibiofemoral conformity is the primary source of rotational constraint. Box-post impingement can be a source of additional rotational constraint. Depending on specific design features, small changes in relative tibiofemoral component rotation can more than double the generated torque. Axial rotation of the knee in vivo can generate substantial torque. Relative tibiofemoral rotational position is an important factor influencing component function and fixation.

Simultaneous HPLC determination of 5-fluorouracil and its metabolites in plasma of cancer patients.

5-Fluorouracil (5-Fu) is a commonly used anticancer agent for treatment of solid tumours. Certain studies have reported conflicting results between individual plasma concentration levels and toxicity or therapeutic effects. For this reasons some authors proposed to evaluate the plasma levels of 5-Fu metabolites 5-fluorouridine, 5-fluoro-2'-deoxyuridine and 5-fluoro-5,6-dihydro-uracil. The aim of the present work is to develop and validate a new HPLC method simultaneously determining 5-fluorouracil and its three metabolites, to be used to study the plasma levels, therapeutic effects and toxicity in cancer patients. The analytes were separated on a 4.6 x 250 mm ODS1 (5 micro m) not end-capped column, operating at room temperature. Elution was performed under isocratic conditions, employing a 1.5 mM K(3)PO(4) mobile phase (pH 5). 5-Bromo-5,6-dihydro-uracil was used as internal standard. The limits of quantitation were 0.5 micro g/mL for 5-fluorouracil, 1 micro g/mL for 5-fluoro-5,6-dihydro-uracil, 3 micro g/mL for 5-fluoro-2'-deoxyuridine and 5-fluorouridine; the stability, recovery, linearity, accuracy and specificity of the compounds were evaluated according to the criteria widely accepted. Using this method we measured plasma samples of 18 cancer patients treated with folinic acid (100 mg/m(2)) by intravenous administration, followed by an i.v. bolus of 5-Fu (400 mg/m(2)). The concentration levels of 5-fluorouracil and for 5-fluoro-5,6-dihydro-uracil were detectable in all the subjects while 5-fluorouridine and 5-fluoro-2'-deoxyuridine were present only in eight patients.

Distribution of polychlorinated biphenyls in contaminated swine tissue.

In 1999 an accidental contamination of feed occurred in Belgium. This incident lead Authorities to increase monitoring levels of polychlorinated biphenyls (PCB) in food of animal origin. In our Department 220 samples of swine tissues, meat and adipose tissue, were analysed. The quantitation of PCB was made from the sum of 7 congeners obtained by gas chromatography with an electron capture detector. Confirmatory analysis was performed by gas chromatography/mass spectrometry. The PCBs in the majority of samples were close to the method limit of detection and only a few samples had PCB concentrations > the limit of quantitation. In those tissues the average concentration of PCBs was 0.035 mg/kg. The legal limit of 0.200 mg PCB/kg established by the European Union was exceeded by only 1 sample, a smoked ham from Belgium. This sample showed the presence of all selected congeners. We report the profile of the PCB congeners in this sample.

Effect of pen size on behavioral, endocrine, and immune responses of water buffalo (Bubalus bubalis) calves.

Female water buffalo (Bubalus bubalis) calves (n = 28) aged 7 to 10 d were divided into four groups of seven animals each to examine the effects of space allowance (Group A: 2.6 indoor m2 + 2.0 outdoor m2/calf; Group B: 2.6 indoor m2/calf; Group C: 1.5 indoor m2/calf; Group D: 1.0 indoor m2/calf) on behavioral, endocrine, and immune variables for a period of 60 d. Animals were offered 7 L/d of a commercial acidified milk substitute. The calves averaged 45.9 kg initially and 92.4 kg finally. The behavior observations were conducted 7 d after grouping and fortnightly thereafter. At wk 4 and 8, the phytohemagglutinin (PHA) skin test was performed to induce aspecific delayed hypersensitivity. At wk. 1 and 3, calves were injected i.m. with keyhole limpet hemocyanin. Antibody titers were determined at weekly intervals for 7 wk. Calves in pens with greater space allowance (Groups A and B) were less active than Groups C and D (P<.001). The latter groups were also observed feeding more often at wk 7 (P<.01). Calves provided with an outdoor paddock spent less time standing than Groups C and D (P<.01), and lay with a greater number of outstretched legs (P<.001). Groups C and D showed a lower reaction to PHA in both skin tests than did Groups A and B (P<.001 and P<.05, respectively). Group A showed an antibody response consistently higher than groups B, C, and D (P<.01, P<.05, and P<.05, respectively). At the end of the experimental period, the calves were subjected to an isolation test lasting 10 min. Group D showed a longer duration of movement with respect to Groups A and B (P<.01); animals from Group C walked more than did Group A (P<.05). Cortisol concentration evaluated 0, 10, 45, 90, 150, and 225 min after separation from the group was higher in Groups C and D than in Groups A and B (P<.01). For all animals, the highest cortisol level was observed immediately after the isolation test (P<.001). Space restriction resulted in evidence of stress in the animals as shown by alterations in a number of physiological responses. However, the use of small groups of only seven animals per pen may have affected their reactions to space restriction. It is possible that using larger groups could change these conclusions.