Dynamic behaviour of the soft palate during nasal positive pressure ventilation under anaesthesia and paralysis: comparison between patients with and without obstructive sleep-disordered breathing.

BACKGROUND: Difficult mask ventilation is common and is known to be associated with sleep-disordered breathing (SDB). It is our hypothesis that the incidence of expiratory retropalatal (RP) airway closure (primary outcome) during nasal positive pressure ventilation (PPV) is more frequent in patients with SDB (apnea hypopnea index ≥5 h-1) than non-SDB subjects. METHODS: The severity of SDB was assessed before surgery using a portable sleep monitor. In anaesthetized and paralysed patients with (n=11) and without SDB (n=9), we observed the behaviour of the RP airway endoscopically during nasal PPV with the mouth closed and determined the dynamic RP closing pressure, which was defined as the highest airway pressure above which the RP airway closure was reversed. The static RP closing pressure was obtained during cessation of mechanical ventilation in patients with dynamic RP closure during nasal PPV. RESULTS: The expiratory RP airway closure accompanied by expiratory flow limitation occurred more frequently in SDB patients (9/11, 82%) than in non-SDB subjects (2/9, 22%; exact logistic regression analysis: P=0.022, odds ratio 3.6, 95% confidence interval 1.1-15.4). Receiver operating characteristic curve analyses indicated AHI >10h-1 and presence of habitual snoring as clinically useful predictors for the occurrence of RP closure during PPV. Dynamic RP closing pressure was greater than the static RP closing pressure by approximately 4-5 cm H2O. CONCLUSIONS: Valve-like dynamic RP closure that limits expiratory flow during nasal PPV occurs more frequently in SDB patients.

The impact of childhood abuse history, domestic violence and mental health symptoms on parenting behaviour among mothers in Japan.

BACKGROUND: To assess the impact of childhood abuse history, domestic violence experiences and mental health symptoms on the parenting behaviour of mothers in Japan who have separated from violent husbands or partners. METHODS: A self-administered questionnaire survey was conducted on a sample of mothers (n = 304) and their children (n = 498) residing in 83 mother-child homes in Japan. The survey assessed the mothers' childhood abuse history (physical, psychological and sexual abuse and neglect history), domestic violence experiences, current mental health symptoms (dissociative, depressive and traumatic symptoms) and parenting behaviours after moving into the homes to separate from a violent husband or partner. RESULTS: The mothers' childhood abuse history and experience of domestic violence were not associated with their not playing with their children. In contrast, the mothers' dissociative and depressive symptoms were significantly associated with not playing with their children. Although there was no association between the mothers' total childhood abuse history and not praising their children, their childhood physical abuse history was significantly associated with their not praising their children. The dissociative and depressive symptoms were also associated with no praise. Interestingly, the experience of domestic violence showed an inverse association with no praise. CONCLUSIONS: Mental health symptoms, more specifically dissociative and depressive symptoms, are associated with a decrease in parenting quality. Mothers who were physically abused as children are less likely to praise their own children, independent of maternal mental health symptoms. In contrast, mothers who experienced domestic violence but subsequently separated from their violent husbands or partners are more likely to praise their children. The treatment of mental health symptoms, particularly dissociative and depressive symptoms, therapy for childhood abuse history and separation from violent husbands or partners might be effective ways to enhance the quality of parenting in Japan.

Vascular pharmacology of a novel cannabinoid-like compound, 3-(5-dimethylcarbamoyl-pent-1-enyl)-N-(2-hydroxy-1-methyl-ethyl)benzamide (VSN16) in the rat.

BACKGROUND AND PURPOSE: A putative novel cannabinoid receptor mediates vasorelaxation to anandamide and abnormal-cannabidiol and is blocked by O-1918 and by high concentrations of rimonabant. This study investigates VSN16, a novel water-soluble agonist, as a vasorelaxant potentially acting at non-CB1, non-CB2 cannabinoid receptors in the vasculature. EXPERIMENTAL APPROACH: VSN16 and some analogues were synthesized and assayed for vasodilator activity in the rat third generation mesenteric artery using wire myography. Also carried out with VSN16 were haemodynamic studies in conscious rats and binding studies to CB1 receptors of rat cerebellum. KEY RESULTS: VSN16 relaxed mesenteric arteries in an endothelium-dependent manner. The vasorelaxation was antagonized by high concentrations of the classical cannabinoid antagonists, rimonabant and AM 251, as well as by O-1918, an antagonist at the abnormal-cannabidiol receptor but not at CB1 or CB2 receptors. It did not affect [3H]CP55,940 binding to CB1 receptors in rat cerebellum. The vasorelaxation was not pertussis toxin-sensitive but was reduced by inhibition of nitric oxide synthesis, Ca(2+)-sensitive K+ channels (KCa) and TRPV1 receptors. In conscious rats VSN16 transiently increased blood pressure and caused a longer-lasting increase in mesenteric vascular conductance. Structure-activity studies on vasorelaxation showed a stringent interaction with the target receptor. CONCLUSIONS AND IMPLICATIONS: VSN16 is an agonist at a novel cannabinoid receptor of the vasculature. It acts on the endothelium to release nitric oxide and activate KCa and TRPV1. As it is water-soluble it might be useful in bringing about peripheral cannabinoid-like effects without accompanying central or severe cardiovascular responses.

Sound spectrogram analysis in patients receiving Kawahara's surgical voice restoration for advanced carcinoma of the hypopharynx and cervical esophagus.

Loss of voice after total laryngectomy is a major concern that has prompted much effort to develop methods of surgical voice restoration. In 1992, Kawahara described a tracheocolic shunt for voice restoration using a revascularized ileocolic graft. With this method, aspiration through the phonatory shunt is prevented by the ileocolic valve (Bauhin's valve), while vocalization is allowed. The purpose of this study was to use sound spectrogram analysis to evaluate voices surgically restored in that way. Between 2002 and 2005, 10 consecutive patients underwent laryngopharyngoesophagectomy with Kawahara's surgical voice restoration for advanced carcinoma of the hypopharynx and/or cervical esophagus at Akita University Hospital, Japan. We then used sound spectrography to analyze and compare the voices of patients receiving Kawahara's voice restoration, against healthy volunteers and patients who underwent the same surgery without voice restoration and spoke using an electronic larynx. We also evaluated the intelligibility of conversation and performed a listening test. The sound spectrograms showed that when produced by the electronic larynx, consonant sounds and voice frequencies below 300 Hz were not clearly recognized. By contrast, in patients who received Kawahara's surgical voice restoration, consonant and vowel sounds at frequencies above and below 300 Hz were clearly recognized. Although conversation was intelligible with both Kawahara's surgical voice restoration and the electronic device, listeners judged the voice produced by the former to be superior. Thus, the voice produced by Kawahara's surgical voice restoration is superior to that produced by the electronic larynx.

Extravascular lung water measured using single transpulmonary thermodilution reflects perioperative pulmonary edema induced by esophagectomy.

Pulmonary edema is the most frequent postoperative complication following esophagectomy for thoracic esophageal cancer. We enrolled 23 patients who underwent esophagectomy with extended lymph node dissection for thoracic esophageal cancer in a prospective observational clinical trial. We used the PiCCO device to measure extravascular lung water with the aim of determining whether it correlates with the respiratory index and whether it is predictive of pulmonary complications. Based on constant criteria, the tracheal tubes of 11 patients were removed on the morning of postoperative day 1 (extubation group), while 12 patients remained intubated (intubation group). These two groups significantly differed in that all patients in the extubation group recovered without any pulmonary complications, whereas 4 patients (33%) in the intubation group developed pulmonary complications. The extravascular lung water measured using PiCCO correlated significantly with the respiratory index. In the intubation group, both extravascular lung water and respiratory index were elevated 12 h after surgery and were even higher 24 h after surgery. The extravascular lung water measured using PiCCO reflects the level of postoperative pulmonary edema and predicts the pulmonary complications induced by esophagectomy with extended lymph node dissection.

Roles of cyclooxygenase 2 and microsomal prostaglandin E synthase 1 in rat acid reflux oesophagitis.

BACKGROUND: Although prostaglandin E2 (PGE2), cyclooxygenase 2 (COX-2), and microsomal prostaglandin E synthase 1 (mPGES-1) are known to play a role in various inflammatory events, their roles in the pathogenesis of gastro-oesophageal reflux disease are not known. AIMS: We examined the dynamics of COX-1, COX-2, mPGES-1, mPGES-2, cytosolic PGES (cPGES), and PGE2 synthetic activity in rat acid reflux oesophagitis and the effects of COX-2 inhibitors on the severity of oesophagitis. METHODS: Acid reflux oesophagitis was induced by ligating the transitional region between the forestomach and the glandular portion and wrapping the duodenum near the pylorus. Rats were killed on day 3 (acute phase) or day 21 (chronic phase) after induction of oesophagitis. RESULTS: Expression of COX-2 and mPGES-1 was markedly increased in oesophagitis while modest changes in COX-1, cPGES, and mPGES-2 expression were observed. COX-2 and mPGES-1 were colocalised in epithelial cells of the basal layer, as well as inflammatory and mesenchymal cells in the lamina propria and submucosa. COX-2 inhibitors significantly reduced the severity of chronic oesophagitis but did not affect acute oesophageal lesions. COX-2 inhibitors significantly inhibited the increase in PGE2 synthesis in oesophageal lesions on both days 3 and 21. Epithelial proliferation was significantly increased in the basal layer on day 21. Inflammatory cells and epithelial cells of the basal layer exhibited reactions for EP4 in oesophagitis. CONCLUSION: PGE2 derived from COX-2 and mPGES-1 plays a significant role in the pathogenesis of chronic acid reflux oesophagitis, and possibly in basal hyperplasia and persistent inflammatory cell infiltration.

Carbonate-containing hydroxyapatite derived from calcium tripolyphosphate gel with urea.

Carbonate containing hydroxyapatite (CO3HAp) is one of the candidate materials as a bioresorbable bone substitute. In the present work, CO3HAp was efficiently prepared by a hydrothermal treatment of calcium tripolyphosphate gel with urea at 140 degrees C for 24 h. Chemical potential plots of the CO3HAp for estimation of its dissolution behavior suggested that the CO3HAp is more soluble than hydroxyapatite (HAp) and is as soluble as octacalcium phosphate (OCP) and/or beta -tricalcium phosphate (TCP). This material is expected to be applied to bioresorbable materials such as bone fillers.

Pulsed Doppler tissue imaging for the assessment of myocardial viability: comparison with 99mTc sestamibi perfusion imaging.

The aim of the present study was to examine whether Doppler tissue imaging demonstrated comparable diagnostic performance for the detection of viable myocardium compared to myocardial perfusion imaging with Tc hexakis-2-methoxyisobutylisonitrile (MIBI). We studied 30 patients with old myocardial infarction who underwent percutaneous transluminal coronary angioplasty (PTCA). Myocardial single photon emission computed tomography (SPECT) with Tc-MIBI and two-dimensional echocardiography were carried out within 7 days before PTCA. We measured regional Tc-MIBI uptake for each myocardial segment from SPECT and peak systolic velocity and a ratio of regional pre-ejection period to regional ejection time (PEP/ET) from pulsed Doppler tissue imaging. Biplane left ventriculography was performed before interventional procedures and repeated 3 months after PTCA. Myocardial viability was determined when wall motion was improved at least one grade after PTCA. The peak systolic velocity was positively correlated with regional Tc-MIBI uptake (R =0.59, P<0.01). The PEP/ET demonstrated inverse correlation with Tc-MIBI uptake ( R=-0.59, P<0.01). Peak systolic velocity of viable segments was higher than that of non-viable segments ( P<0.05). The PEP/ET was lower in viable segments than in non-viable segments ( P<0.05). Peak systolic velocity and PEP/ET demonstrated high diagnostic accuracy for detecting viable myocardium compared with Tc-MIBI perfusion imaging (80% and 79% vs 90%). These data indicate that measurements of regional peak systolic velocity and PEP/ET by Doppler tissue imaging are useful for evaluating myocardial viability quantitatively and provide helpful information for a clinical judgment in an interventional strategy.

[Indication for surgical and microwave coagulation therapy for multiple (> or = 5) bilobar liver metastases from colorectal cancer].

We analyzed methods and clinicopathological factors for multiple (> or = 5) bilobar liver metastases (H3) from colorectal cancer and evaluated the indication of surgical and microwave coagulation therapy (MCT) for them. Twenty-four patients with H3 underwent surgical therapy and were divided into two groups. Group a: 9 patients with a prognosis of more than 700 days. Group b: the remaining 15 patients. There was no significant difference in prognosis between those receiving MCT and resection + MCT as a surgical therapy. The number and maximum diameter of tumors tended to be smaller in Group a. The number of tumors was less than or equal to 9 and the maximum size of the tumors was 38 mm. Moreover, the tumor could be controlled by MCT alone if the tumor size was less than 30 mm. MCT is a useful therapy for these cases and the indication for surgical therapy may depend on the number and maximum size of tumors.

[Comparison of microwave coagulation using a new type electrode with radiofrequency ablation in the liver of living animals].

In the present study, we compared microwave coagulation using a new type of electrode, a teflon-coated electrode that was developed in order to increase the area of coagulation, with radiofrequency ablation using a Radionics Cool-tip electrode inserted into the pig liver. Two Landrace male pig were put under general anesthesia. A microwave electrode (insulated area: 6 mm, teflon-coated electrode 16 G) and a radiofreqency (RF) electrode (Cool-tip RF single electrode 17 G) were passed through the surface of the livers of the pig. A thermometer was placed 1 cm from the tip of the electrode in order to measure the changes in the temperature of the area surrounding the electrode. In this study, the microwave setting was 80 W, and the RF pulse was set automatically. The coagulated and ablated areas of the liver were measured after 2.5, 5, and 10 minutes of energy delivery (n = 4). The diameter of the coagulated area of the liver following 2.5, 5 and 10 minutes of microwave exposure was 23.5 +/- 4.8 mm, 29.5 +/- 5.2 mm and 32.5 +/- 6.4 mm, respectively. On the other hand, the diameter of the ablated area of the liver following 2.5, 5 and 10 minutes of RF exposure was 18.5 +/- 4.1 mm, 24 +/- 7.8 mm and 28 +/- 4.9 mm, respectively. The mean temperature of the liver 1 cm from the microwave and RF electrodes (measured time: 2 minutes) was 69.6 degrees C and 56.3 degrees C. respectively (n = 12). Thus, the temperature of the area surrounding the microwave electrode was significantly higher than the temperature of the area surrounding the RF electrode (p = 0.0065). The teflon-coated microwave electrode achieved superior results to the Radionics Cool-tip electrode with respect to the diameter of the coagulated area and the temperature of the area in which the electrode was inserted, at the specified times.

Reverse redistribution of 99m Tc-sestamibi after direct percutaneous transluminal coronary angioplasty in acute myocardial infarction: relationship with wall motion and functional response to dobutamine stimulation.

Reverse redistribution (RR) of 99mTc-sestamibi is observed after direct percutaneous transluminal coronary angioplasty (PTCA) in acute myocardial infarction (AMI). The purpose of this study was to clarify the functional characteristics of myocardial segments with RR after direct PTCA in AMI. Thirty patients with AMI who had undergone direct PTCA were examined. Myocardial perfusion tomography with 99mTc-sestamibi and low dose dobutamine echocardiography were performed within 2 weeks of the onset. The 99mTc-sestamibi images were obtained 1 and 3 h after tracer administration. The left ventricle was divided into nine segments, and regional 99mTc-sestamibi uptake and clearance were quantitatively evaluated in each segment. RR was defined as a decrease in 99mTc-sestamibi uptake of >10% on 3 h delayed images compared with the 1 h early images. The left ventricle in the echocardiographic images was also divided into nine segments corresponding to the scintigraphic images, and regional wall motion was assessed in the resting condition as the baseline and during dobutamine administration (5-10 microg x kg(-1) x min(-1)). Out of a total of 270 myocardial segments, 111 segments were perfused by the culprit coronary artery and were defined as ischaemic segments. There were 25 segments with RR and 86 segments without RR in the ischaemic myocardium. Enhanced clearance of 99mTc-sestamibi was observed in ischaemic segments with RR (P<0.001). Echocardiography demonstrated that 24 out of 25 segments with RR and 61 out of 86 segments without RR had wall motion abnormalities. Dobutamine infusion improved wall motion in 20 (83%) of the 24 dysfunctional segments with RR and 33 (54%) of the 61 dysfunctional segments without RR (P<0.02). These findings suggest that RR indicates reversible functional abnormalities associated with preserved contractile reserve in response to dobutamine. The early and delayed imaging of 99mTc-sestamibi provides useful information regarding the residual viability of the dysfunctional myocardium in AMI patients.

Proliferative activity of the parathyroid cells in rats with secondary hyperparathyroidism.

Proliferative kinetics of parathyroid cells in secondary hyperparathyroidism (HPT) are still unknown. We examined the histopathological changes and proliferative activity of parathyroid cells in spontaneously hypercholesterolemic (SHC) rats that exhibit secondary HPT and in normal Sprague Dawley (SD) rats from 3 weeks to 32 weeks of age. Proliferative activity [proliferating cell nuclear antigen(PCNA) labeling index], evaluated by means of immunohistochemical examination of PCNA, declined in SD rats with age from 10.8% at 3 weeks of age to 0.15% at 32 weeks of age. In SHC rats, a PCNA labeling index of 11.6% declined to 3.12% at 14 weeks of age and rebounded to 6.15% at 26 weeks of age. Parathyroid glands increased in size as determined by the maximum cross-sectional area, but in SHC rats, the increase was significantly greater, paralleling the progression of renal dysfunction, and at 32 weeks they were almost three times larger than in SD rats. Parathyroid hormone (PTH) levels in SHC rats also rose sharply after 20 weeks and reached 611 pg/ml at 32 weeks, while PTH in SD rats remained unchanged at approximately 110 pg/ml. This study showed that in the course of developing HPT in SHC rats, there is a large increase in the size of the parathyroid gland, a concomitant increase in PTH levels, and a PCNA labeling index that is higher than in normal SD rats.

Prednisolone suppresses ischemia-reperfusion injury of the rat liver by reducing cytokine production and calpain mu activation.

BACKGROUND: We investigated the effects of prednisolone on cytokine production and calpain mu activation during hepatic ischemia-reperfusion (IR) injury. METHODS: The hilar area of the left lateral and median lobes of rat liver was clamped for 60 min. Prednisolone was administered at 1.0, 3.0, or 10 mg/kg at 30 min before ischemia. In addition to biochemical and microscopic analyses, IL-beta and TNF-alpha production was evaluated by RT-PCR. Calpain mu activation and talin degradation were determined by Western blotting, using specific antibodies. RESULTS: In the control and prednisolone (1.0 mg/kg) groups, serum AST and ALT levels were elevated, and cell membrane bleb formation was observed after 2 h of reperfusion. Moreover, calpain mu activation, talin degradation, and overexpression of IL-beta and TNF-alpha mRNAs were detected. Infusion of prednisolone at 3.0 or 10 mg/kg significantly suppressed biochemical and microscopic changes. At 10 mg/kg, prednisolone markedly suppressed IL-beta and TNF-alpha transcription and calpain mu activation and talin degradation, consistent with the improved 7-day survival after total hepatic ischemia (75% vs. 25% in control group, P = 0.039). CONCLUSIONS: Cytoprotective effect of prednisolone in hepatic IR injury was closely associated with suppression of IL-beta/TNF-alpha production and calpain mu activation.

Carboxyl group of residue Asp647 as possible proton donor in catalytic reaction of alpha-glucosidase from Schizosaccharomyces pombe.

cDNA encoding Schizosaccharomyces pombe alpha-glucosidase was cloned from a library constructed from mRNA of the fission yeast, and expressed in Saccharomyces cerevisiae. The cDNA, 4176 bp in length, included a single ORF composed of 2910 bp encoding a polypeptide of 969 amino-acid residues with M(r) 106 138. The deduced amino-acid sequence showed a high homology to those of alpha-glucosidases from molds, plants and mammals. Therefore, the enzyme was categorized into the alpha-glucosidase family II. By site-directed mutagenesis, Asp481, Glu484 and Asp647 residues were confirmed to be essential in the catalytic reaction. The carboxyl group (-COOH) of the Asp647 residue was for the first time shown to be the most likely proton donor acting as the acid catalyst in the alpha-glucosidase of family II. Studies with the chemical modifier conduritol B epoxide suggested that the carboxylate group (-COO-) of the Asp481 residue was the catalytic nucleophile, although the role of the Glu484 residue remains obscure.

Early stage of urolithiasis formation in experimental hyperparathyroidism.

PURPOSE: We have previously noted marked acceleration in the proliferative activity of parathyroid cells in rats with spontaneous hypercholesterolemia and secondary hyperparathyroidism. Using this proliferative potential we investigated whether transplantation of these enlarged parathyroids into normal rats would induce hyperparathyroidism and renal stones. MATERIALS AND METHODS: We used 26-week-old male rats with spontaneous hypercholesterolemia as donors, and 5-week-old normal male Sprague-Dawley rats and rats with spontaneous hypercholesterolemia as recipients. Enlarged parathyroid glands were transplanted into group 1--Sprague-Dawley rats with no treatment, group 2--Sprague-Dawley rats that received FK-506 as an immuno-suppressor, group 3--rats with spontaneous hypercholesterolemia rats that underwent parathyroidectomy plus FK-506 administration and group 4--Sprague-Dawley rats that underwent parathyroidectomy plus FK-506 administration. Parathyroidectomy was performed in recipients before transplantation to ensure a low calcium condition. RESULTS: Grafts were rejected within 11 and 15 weeks in groups 1 and 2, respectively. In group 3, 78% of the grafts were successful even after 19 weeks. In group 4 graft survival was 30% at 15 weeks with complete rejection at 19 weeks. In group 3 gradually elevated serum parathyroid hormone was observed as well as stone plaques containing calcium oxalate and calcium phosphate in renal tubules located mainly in the corticomedullary junction. An increased number of plaques was associated with higher parathyroid hormone. CONCLUSIONS: Our study shows that transplanted parathyroid glands function with an immunosuppressive agent and the maintenance of hypocalcemic conditions, and they secrete sufficient parathyroid hormone to demonstrate hyperparathyroidism. Plaque in these kidneys indicates an early stage of urolithiasis caused by hyperparathyroidism.

Immunohistochemical expression of P-glycoprotein in the rat urinary bladder and the effect of verapamil on intravesical chemotherapy.

BACKGROUND: The expression of P-glycoprotein (Pgp) is thought to be common in bladder epithelium and the multidrug resistance mediated by Pgp must be considered to improve the efficacy of chemotherapy for bladder tumors. METHODS: The expression of Pgp in normal and tumor tissue of the rat urinary bladder was first examined immunohistochemically. The effect of verapamil, an expected modulator of Pgp, on intravesical chemotherapy of the rats was then investigated. RESULTS: Pgp was immunohistochemically detected in normal epithelium and in tumor tissue of the rat urinary bladder. In those normal and tumor-bearing bladders, verapamil promoted the uptake of intravesically instilled pirarubicin, but the efflux of intracellular accumulated pirarubicin was observed subsequently in both conditions with and without verapamil. The drug concentration decreased more rapidly in the verapamil group than in the control group. CONCLUSIONS: Verapamil is thought to be useful in promoting uptake of intravesically instilled pirarubicin, but it did not appear to be so efficient at limiting the efflux of intracellular accumulated pirarubicin.

[A case of multiple sclerosis with recurrent episodes of psychiatric symptoms].

We reported a 19-year-old woman with multiple sclerosis (MS) with recurrent episodes of psychiatric symptoms at the age of 10 and 19, as the first and 11th relapse, respectively. Her first episode of MS resembled acute encephalitis at the age of 9 and she relapsed 12 times. The psychiatric symptoms were not mood disorders but altered states of consciousness, such as the delirous state. She was treated with high doses of intravenous methylpredonisolone and her symptoms were almost resolved, but her anxiety neurosis remained. Her MRI showed demyelination in the white matter of the frontal lobe and the hippocampus, which suggested that psychiatric symptoms of MS would be related to the frontal lobe lesion.

Purification and identification of the essential ionizable groups of honeybee, Apis mellifera L., trehalase.

Trehalase (EC 3.2.1.28) of the bound type was purified as an electrophoretically homogeneous protein from adult honeybees by fractionation with ammonium sulfate, hydrophobic chromatography, and DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, butyl-Toyopearl 650M, and p-aminophenyl beta-glucoside Sepharose 4B column chromatographies. The enzyme preparation was confirmed to be a monomeric protein containing 3.1% carbohydrate. The molecular weight was estimated to be approximately 69,000, and the optimum pH was 6.7. The Michaelis constant (Km) was 0.66 mM, and the molecular activity (k0) was 86.2 s(-1). The enzyme was an "inverting" type which produced beta-glucose from alpha, alpha-trehalose. Dependence of the V and Km values on pH gave values for the ionization constants, pKe1 and pKe2, of essential ionizable groups 1 and 2 of the free enzyme of 5.3 and 8.5, respectively. When the dielectric constant of the reaction mixture was decreased, pKe1, and pKe2 were shifted to higher values of + 0.2 and + 0.5 pH unit, respectively. The ionization heat (deltaH) of ionizable group 1 was estimated to be + 1.8 kcal/mol, and the deltaH value of group 2 was + 1.5 kcal/mol. These findings strongly support the notion that the essential ionizable groups of honeybee trehalase are two kinds of carboxyl groups, one being a dissociated type (-COO(-), ionizable group 1) and the other a protonated type (-COOH, ionizable group 2), although the pKe2 value is high.