A new method of preparing TRH derivative-loaded poly(dl-lactide-coglycolide) microspheres based on a solid solution system.

We investigated a new method of preparing peptide-loaded poly(dl-lactide-co-glycolide) microspheres with high encapsulation efficiency, low initial burst, and long-term sustained release by dissolving a peptide in a polymer by applying a solid solution system to the preparation of an oil phase. Solid solutions were prepared by dissolving a polymer (poly(dl-lactide-co-glycolide)) and a peptide (TRH derivative) in mixed solvents and then evaporating the solvents. Microspheres were prepared by an O/W emulsion solvent evaporation method, using the solution of the solid solution in dichloromethane as an oil phase. The state of the peptide in the solid solution and in the microspheres was evaluated by X-ray diffraction analysis. Release of the peptide from the microspheres was evaluated by an in vitro drug release test. Observation of the oil phase, X-ray diffraction analysis, and DSC analysis revealed that the peptides were dispersed in a molecular state in the solid solution and in microspheres with peptide loading of up to 15%. Encapsulation efficiency was over 90% for microspheres with peptide loading of up to 15%. The release of the peptide from the microspheres lasted over 21 days at least with the limited initial burst in vitro. High encapsulation efficiency, low initial burst, and long-term sustained release can be accomplished with microspheres prepared by a method based on a solid solution system.

Pharmacokinetic behaviour of cisplatin in peritoneal fluid after intraperitoneal administration of cisplatin-loaded microspheres.

The objective of this study was to establish a pharmacokinetic model for the estimation of unchanged cis-dichlorodiammine-platinum (II) (CDDP) concentration in peritoneal fluid after intraperitoneal administration of cisplatin-loaded microspheres (CDDP-MS) and to elucidate the accuracy of this model by comparisons between actual and simulated values after intraperitoneal administration of CDDP-MS. We developed a method enabling the precise and quick assessment of the drug concentration in the peritoneal cavity. The pharmacokinetic parameters obtained after intravenous bolus injection at a dose of 2 mg kg(-1) were total body clearance (1026 mL h(-1) kg(-1)), elimination rate constant (3.24 h(-1)) and distribution volume of systemic circulation (316.7 mL kg(-1)). After an intraperitoneal bolus injection at a dose of 5 mg kg(-1), the absorption rate constant from the peritoneal cavity (3.64 h(-1)) and the distribution volume of the peritoneal cavity (13.5 mL kg(-1)) were determined. The protein-binding rate constant in ascites was 0.58 h(-1). Using these pharmacokinetic parameters, we established a pharmacokinetic model consisting of two compartments. Administration of CDDP-MS at a dose of 10 mg kg(-1), which released CDDP over 7 days in-vitro, yielded sustained concentrations of unchanged CDDP (1-2 mg mL(-1)) in the peritoneal cavity that persisted for 7 days, and that were predictable by applying the in-vitro dissolution profile to the pharmacokinetic model. The findings obtained from this study are useful for understanding the basic pharmacokinetic characteristics of unchanged CDDP in the peritoneal cavity and may also be important in the development of optimized CDDP-MS formulations.

Evaluation of in vivo release characteristics of protein-loaded biodegradable microspheres in rats and severe combined immunodeficiency disease mice.

The in vivo release characteristics of protein-loaded biodegradable microspheres were examined in normal rats and severe combined immunodeficiency disease (SCID) mice. Bovine-derived superoxide dismutase (bSOD), encapsulated into microspheres comprised of poly(D,L-lactic-co-glycolic acid) and poly(D,L-lactic acid), was used as a model protein. Three types of bSOD-loaded microspheres with different release profiles were subcutaneously administered to normal rats. Anti-bSOD antibodies were first detected at day 9 after the administration of these microspheres, which was independent of their release profiles in vitro. A typical formulation with a sigmoidal release profile was subcutaneously administered to SCID mice. No immunoreaction was observed. The plasma bSOD concentration-time profile, determined by an enzyme-linked immunosorbent assay, well reflected the in vitro release profile of the formulation. The disappearance profile of active bSOD from the administration site also partly corresponded to both profiles. However, the relative bioavailability calculated with a profile on the subcutaneous injection of bSOD solution was 40.7%. These results indicated possible instability of bSOD released gradually at the administration site. The results of the present study using SCID mice would be suitable for discussing the in vitro-in vivo correlation of protein release from microspheres, and useful for designing long-term release formulations of protein drugs.

Preparation of gelatin microparticles by co-lyophilization with poly(ethylene glycol): characterization and application to entrapment into biodegradable microspheres.

Gelatin microparticles were prepared by co-lyophilization with poly(ethylene glycol) (PEG) as a protein micronization adjuvant. Aqueous solutions containing gelatin and PEG at various mixing ratios were freeze-dried. The lyophilizates were dispersed in methylene chloride and subjected to particle size analysis. The particle size decreased as the PEG/gelatin ratio increased. The microparticles isolated from the suspension had spherical microdomains with sizes ranging from 1 to 10 microm, which indicated that phase separation between PEG and gelatin during freezing was involved in the formation mechanism of gelatin microparticles. By using this technology, gelatin microparticles with an average size of less than 10 microm, with high purity of more than 90% and with good dispersibility could be obtained with high yield. The gelatin microparticles with average sizes from 5 to 20 microm were applied to encapsulation into biodegradable PLGA/PLA microspheres via a solid-in-oil-in-water emulsion process. The entrapment efficiency was highly dependent on the particle size and the size distribution, signifying that solid microparticles with an average diameter of less than 5 m and an maximal diameter of less than 10 microm would be required for effective encapsulation. These gelatin microparticles would be useful for studying and developing various drug delivery systems.

Applicability of various amphiphilic polymers to the modification of protein release kinetics from biodegradable reservoir-type microspheres.

The effects of various amphiphilic polymers on the kinetics of protein release from reservoir-type microspheres, prepared by a solid-in-oil-in-water emulsion-solvent evaporation method, were investigated. Bovine serum albumin (BSA), as a model protein, was firstly micronized through co-lyophilization with amphiphilic polymers, such as poly (ethylene glycol) (PEG), polyvinylpyrrolidone (PVP), and pluronic F68. This process was based on the aqueous phase separation of protein and amphiphilic polymer induced by freezing-condensation. Mixing of poly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid) (PLA) (at a ratio of 4:6) in a methylene chloride solution provided a'polymer-alloy' structure, where the preformed solid BSA microparticles were selectively distributed in the inner PLGA-rich phase. The reservoir-type microspheres obtained through this process showed high entrapment efficiencies (more than 85%) and reduced initial burst releases (less than 10%). Although PVP did not modify the BSA release profile, PEG and pluronic F68 enhanced the BSA release, with no increase of the initial burst effect, responding to their loading percentage: 3% loading of PEG or pluronic F68 resulted in typical zero-order release kinetics. The abilities of these amphiphilic polymers to modify the protein release profile could be predicted from their partitioning characteristics in the polymer-alloys and in the methylene chloride/water system.

Protein encapsulation into biodegradable microspheres by a novel S/O/W emulsion method using poly(ethylene glycol) as a protein micronization adjuvant.

A new method for preparing protein-loaded biodegradable microspheres by a process involving solid-in-oil-in-water (S/O/W) emulsion was established using poly(ethylene glycol) (PEG). In the first step, a protein solution was lyophilized with PEG, which resulted in the formation of spherical protein microparticles, less than 5 microm in diameter, dispersed in a continuous PEG phase. This process was well explained by the aqueous phase separation phenomenon induced by freezing-condensation. Since this lyophilizate could be directly dispersed in an organic phase containing biodegradable polymer by dissolving PEG with methylene chloride, a conventional in-water drying method could be adopted in the second step. Through this S/O/W emulsion process, horseradish peroxidase was effectively entrapped into monolithic-type microspheres of poly(DL-lactic-co-glycolic acid) (PLGA), without significant loss of activity. Bovine superoxide dismutase (bSOD), as another model protein, could be encapsulated into reservoir-type microspheres by the 'polymer-alloys method' using both poly(DL-lactic acid) (PLA) and PLGA. The initial release of bSOD from this reservoir-type microsphere was efficiently reduced. Further, the bSOD release kinetics could be suitably modified by adjusting the loading amounts of PEG or polymer composition. In this study, the multi-functional nature of PEG was successfully utilized in the preparation and designing of protein-loaded microspheres.

Formation and isolation of spherical fine protein microparticles through lyophilization of protein-poly(ethylene glycol) aqueous mixture.

PURPOSE: Preparation of spherical fine protein microparticles by the lyophilization of a protein-poly(ethylene glycol) (PEG) aqueous mixture was investigated. The main objective was to establish a method for preparing protein microparticles suitable for pharmaceutical production. METHODS: Aqueous solutions containing bovine serum albumin (BSA) and PEG at various mixing ratios were freeze-dried. The lyophilizates were dispersed in methylene chloride and subjected to particle size analysis. Analogous studies were performed using several model proteins. A phase diagram of the PEG-BSA aqueous system was obtained by the titration method. RESULTS: The particle size of BSA decreased as the PEG-BSA ratio increased. A bending point was observed in this relationship, at which the PEG-BSA ratio coincided with that of the critical point on the phase diagram of the PEG-BSA system. These results were explained by the freezing-induced condensation, followed by phase separation in the PEG-BSA system. CONCLUSIONS: Spherical fine protein microparticles were successfully obtained at high yield and without any activity loss under optimum conditions. This new technology could be applicable to proteins with a wide range of molecular weights, and is expected to be developed for dry powder inhalations or long-term sustained release microsphere formulations.

Potassium current suppression in patients with peripheral nerve hyperexcitability.

Acquired neuromyotonia (Isaac's syndrome) is considered to be an autoimmune disease, and the pathomechanism of nerve hyperexcitability in this syndrome is correlated with anti-voltage-gated K(+) channel (VGKC) antibodies. The patch-clamp technique was used to investigate the effects of immunoglobulins from acquired neuromyotonia patients on VGKCs and voltage-gated Na(+) channels in a human neuroblastoma cell line (NB-1). K(+) currents were suppressed in cells that had been co-cultured with acquired neuromyotonia patients' immunoglobulin for 3 days but not for 1 day. The activation and inactivation kinetics of the outward K(+) currents were not altered by these immunoglobulins, nor did the immunoglobulins significantly affect the Na(+) currents. Myokymia or myokymic discharges, with peripheral nerve hyperexcitability, also occur in various neurological disorders such as Guillain-Barré syndrome and idiopathic generalized myokymia without pseudomyotonia. Immuno-globulins from patients with these diseases suppressed K(+) but not Na(+) currents. In addition, in hKv 1.1- and 1.6-transfected CHO (Chinese hamster ovary)-K1 cells, the expressed VGKCs were suppressed by sera from acquired neuromyotonia patients without a change in gating kinetics. Our findings indicate that nerve hyperexcitability is mainly associated with the suppression of voltage-gated K(+) currents with no change in gating kinetics, and that this suppression occurs not only in acquired neuromyotonia but also in Guillain-Barré syndrome and idiopathic generalized myokymia without pseudomyotonia.

Stereoselective metabolism of bisoprolol enantiomers in dogs and humans.

To clarify the mechanism of the species difference in the metabolism of bisoprolol enantiomers, in vitro metabolic studies were performed using dog liver microsomes and human cytochrome P450 (CYP) isoforms. The O-deisopropylation of bisoprolol enantiomers showed biphasic kinetics in dog liver microsomes. The intrinsic clearance (Vmax/Km) for O-deisopropylation of R(+)-bisoprolol was higher than S(-)-isomer in both high-affinity and low-affinity components. The R/S ratio of the intrinsic clearance in high- and low-affinity components was 1.34 and 1.65, respectively. The inhibition studies in dog liver microsomes using CYP isoform-selective inhibitors indicated that the O-deisopropylation of both bisoprolol enantiomers was mediated via the CYP2D and CYP3A subfamily, and suggested that high-affinity oxidation was dependent on CYP2D. The kinds of CYP subfamilies in dogs, which contribute to the metabolism of bisoprolol enantiomers, were the same as those in humans. The intrinsic clearance for O-deisopropylation of R(+)bisoprolol by human recombinant CYP2D6 was also different from that of S(-)-enantiomers (R/S:1.50). However, unlike the dog microsomes, the intrinsic clearance by the human recombinant CYP3A4 did not show a stereoselective difference. Therefore, the species difference in the R/S ratio of metabolic clearance for the oxidation of bisoprolol enantiomers (dog > human) is mainly due to the species difference in the stereoselectivity of one of the cytochrome P450 subfamilies (CYP3A).

Pharmacokinetics and metabolism of bisoprolol enantiomers in humans.

The plasma concentrations and urinary excretions of bisoprolol enantiomers in four Japanese male healthy volunteers after a single oral administration of 20 mg of racemic bisoprolol were evaluated. The AUC(infinity) and elimination half-life of (S)-(-)-bisoprolol were slightly larger than those of (R)-(+)-bisoprolol in all subjects. The metabolic clearance of (R)-(+)-bisoprolol was significantly (P < 0.05) larger than that of (S)-(-)-bisoprolol (S/R ratio: 0.79+/-0.03), although the difference was small. In contrast, no stereoselective in vitro protein binding of bisoprolol in human plasma was found. An in vitro metabolic study using recombinant human cytochrome P450 (CYP) isoforms indicated that oxidation of both bisoprolol enantiomers was catalyzed by the two isoforms, CYP2D6 and CYP3A4. CYP2D6 metabolized bisoprolol stereoselectively (R > S), whereas the metabolism of bisoprolol by CYP3A4 was not stereoselective. The S/R ratio of the mean clearance due to renal tubular secretion was 0.68, indicating a moderate degree of stereoselective renal tubular secretion. These findings taken together suggest that the small differences in the pharmacokinetics between (S)-(-)- and (R)-(+)-bisoprolol are mainly due to the stereoselectivity in the intrinsic metabolic clearance by CYP2D6 and renal tubular secretion.

Deep venous thrombosis of the leg due to psychiatric stupor.

We report the cases of two patients with psychiatric stupor who developed venous thrombosis. A 29-year-old schizophrenic woman had been hospitalized in psychiatric institutions three times because of stupor associated with auditory hallucinations and thought blocking. These symptoms recurred and she was admitted to our hospital with deep venous thrombosis of her left leg. The other patient was a 67-year-old woman with depression. She had also suffered from insomnia. Following admission to our hospital, she developed a depressive stupor complicated by deep venous thrombosis of her left leg. Both cases were treated with sodium heparin and urokinase, and completely resolved. It is well known that dehydration, infection and decubitus ulcers are important physical complications of psychiatric stupor, but there have been few reports of deep venous thrombosis as a physical complication of stupor.

Stereoselective pharmacokinetics of bisoprolol after intravenous and oral administration in beagle dogs.

The stereoselective pharmacokinetics of bisoprolol, a highly beta 1-selective adrenoceptor blocking agent, was studied in dogs. After intravenous and oral administration of the racemate, there was a difference in the plasma concentration between S(-)- and R(+)-bisoprolol. The area under the curve (AUC) of concentration versus time of S(-)-bisoprolol was approximately 1.5 times higher than that of R(+)-bisoprolol and the elimination half-life of S(-)-bisoprolol was approximately 1.4 times longer than that of R(+)-bisoprolol. However, no differences were observed in the volume of distribution, absolute bioavailability, and renal clearance between the two enantiomers. The plasma protein binding of S(-)-bisoprolol was also the same as that of the R(-)-isomer. No chiral inversion or enantiomer-enantiomer interaction was observed, when enantiomers were solely administered via the intravenous route. The comparison of the oxidative metabolic rate of two enantiomers using dog liver microsomes demonstrated that the metabolite was more slowly formed from S(-)- than from R(+)-bisoprolol. Consequently, we concluded that the stereoselective difference in the metabolic clearance between S(-)- and R(+)-bisoprolol caused the difference in the disposition of bisoprolol enantiomers.

Acute hemodynamic improvement by thermal vasodilation in congestive heart failure.

BACKGROUND: A warm-water bath (WWB) or sauna bath (SB) has generally been considered inappropriate for patients with severe congestive heart failure (CHF). However, a comprehensive investigation of the hemodynamic effects of thermal vasodilation in CHF has not been previously undertaken. METHODS AND RESULTS: To investigate the acute hemodynamic effects of thermal vasodilation in CHF, we studied 34 patients with chronic CHF (mean age, 58 +/- 14 years). Clinical stages were New York Heart Association functional class II in 2, III in 19, and IV in 13 patients. Mean ejection fraction was 25 +/- 9%. After a Swan-Ganz catheter was inserted via the right jugular vein, the patient had a WWB for 10 minutes at 41 degrees C or an SB for 15 minutes at 60 degrees C. Blood pressure, ECG, echo-Doppler, expiration gas, and intracardiac pressures were recorded before, during, and 30 minutes after each bath. Oxygen consumption increased mildly, pulmonary arterial blood temperature increased by 1.2 degrees C, and heart rate increased by 20 to 25 beats per minute on average at the end of WWB or SB. Systolic blood pressure showed no significant change. Diastolic blood pressure decreased significantly during SB (P < .01). Cardiac and stroke indexes increased and systemic vascular resistances decreased significantly during and after WWB and SB (P < .01). Mean pulmonary artery, mean pulmonary capillary wedge, and mean right atrial pressures increased significantly during WWB (P < .05) but decreased significantly during SB (P < .05). These pressures decreased significantly from the control level after each bath (P < .01). Mitral regurgitation associated with CHF decreased during and 30 minutes after each bath. Cardiac dimensions decreased and left ventricular ejection fraction increased significantly after WWB and SB. In an additional study, plasma norepinephrine increased significantly during SB in healthy control subjects and in patients with CHF and returned to control levels by 30 minutes after SB. CONCLUSIONS: Hemodynamics improve after WWB or SB in patients with chronic CHF. This is attributable to the reduction in cardiac preload and afterload. Thus, thermal vasodilation can be applied with little risk if appropriately performed and may provide a new nonpharmacological therapy for CHF.

[Effects of hot water bath or sauna on patients with congestive heart failure: acute hemodynamic improvement by thermal vasodilation].

The acute hemodynamic effects of thermal vasodilation caused by exposure to hot water bath or sauna in chronic congestive heart failure were investigated in 32 patients (mean age 57 +/- 15 years old) with dilated cardiomyopathy (25 idiopathic and 7 ischemic). The clinical symptoms were New York Heart Association Class II in 2 patients, III in 17 and IV in 13, and the mean ejection fraction was 25 +/- 9% (9-44%). Exposure to hot water bath was for 10 minutes at 41 degrees C in a semi-sitting position, and to sauna for 15 minutes at 60 degrees C in a supine position using a special far infrared ray sauna chamber. Blood pressure, electrocardiogram, two-dimensional and Doppler echocardiograms, expiration gas, and intracardiac pressure tracings were recorded before (control), during, and 30 minutes after hot water bath or sauna. 1. The increase in oxygen consumption was only 0.3 Mets during hot water bath or sauna, and returned to the control level 30 minutes later. 2. The deep temperature in the main pulmonary artery increased by 1.0-1.2 degrees C on average at the end of hot water bath or sauna. 3. Heart rate increased significantly (p < 0.01) by 20-25/min during bathing and still increased 30 min later. 4. Systolic blood pressure did not change significantly during and after hot water bath or sauna, while, diastolic blood pressure decreased significantly during (p < 0.05) and after sauna (p < 0.01), and after hot water bath (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitive determination of bisoprolol enantiomers in plasma and urine by high-performance liquid chromatography using fluorescence detection, and application to preliminary study in humans.

A sensitive, stereoselective high-performance liquid chromatographic method with fluorescence detection for the measurement of bisoprolol enantiomers in human plasma and urine has been developed. Bisoprolol was extracted at alkaline pH with chloroform, followed by solid-phase extraction. The effluent was evaporated, and the reconstituted residue was chromatographed on a Chiralcel OD column with a mobile phase of hexane-2-propanol (10:0.9, v/v) containing 0.01% (v/v) diethylamine. Within the plasma and urine enantiomeric concentration ranges of 5-100 ng/ml and 25-1250 ng/ml, respectively, a linear relationship was obtained between the peak-height ratios and the corresponding concentrations. The limit of quantitation, defined as three times the baseline noise, was 2 ng/ml for each enantiomer in plasma. A preliminary pharmacokinetic study was undertaken in three healthy male volunteers following an oral dose of 5 mg of racemic bisoprolol. The results confirm that this assay is suitable for pharmacokinetic studies of bisoprolol enantiomers in humans following oral administration of the therapeutic dose.

A simple noninvasive measurement of stenotic mitral valve area: an alternative approach using M-mode and Doppler echocardiography.

Doppler echocardiography is a widely used noninvasive technique to examine the mitral valve area (MVA) by obtaining mitral pressure half-time (PHT) and to assess the severity of the stenosis. However, several hemodynamic factors influence the PHT and may render the PHT data inaccurate in any measurement of MVA under certain conditions. Using a simple echo-Doppler (E-D) method, we assessed the MVA in a physiological equation. The mitral flow volume (MFV) is represented by MVA x transmitral mean flow velocity (mV) x diastolic filling time (DFT). Thus, the formula can be restated as MVA (cm2) = MFV (cm3)/mV (cm/sec) x DFT (sec). We measured MFV by M-mode, and mV and DFT by continuous wave Doppler echocardiography. This formula was tested in 43 patients with isolated mitral stenosis. MVA was obtained by the PHT and E-D methods, and the data obtained were validated against the results of cardiac catheterization. The results obtained using the E-D method showed much better correlation (r = 0.82) with those of catheterization than those with the PHT method (r = 0.52). The inter- and intraobserver variabilities were checked. The results obtained with the E-D method were found to be reproducible. To further validate the accuracy of the E-D method, MVA was measured by both methods at different R-R intervals after exercise and the results were compared. The MVA obtained by the PHT method showed marked variations; whereas, that obtained by the E-D method remained nearly constant. Similarly, in a patient with atrial fibrillation, the MVA assessed by the PHT method varied from beat to beat; whereas, the fluctuations in MVA were minimal using the E-D method. We concluded that the E-D method can be reliable and clinically easily applicable for the accurate assessment of MVA.

[Pulmonary venous flow patterns in normal subjects and cardiac patients: a transesophageal echocardiographic study].

The aim of this study is, first, to analyze pulmonary venous flow velocity (PVFV) pattern in normal subjects and second, to compare it with the various diseased state. PVFV was recorded in eleven normal volunteers, five patients with lone atrial fibrillation, twenty eight patients with valvular heart diseases and six patients with cardiomyopathy using transesophageal color Doppler echocardiography by placing the sample volume at the junction of the left superior pulmonary vein and left atrium. PVFV in normal subjects demonstrated distinct four waveforms: due to atrial systole (AS wave) and diastole (AD wave), and due to ventricular systole (VS wave) and diastole (VD wave). PVFV changed with respiration in normal subjects. The peak velocity of VD wave was increased with inspiration (p less than 0.001). The ratio of velocity VS/VD was increased during expiration (p less than 0.01). The ratio of area AD + VS/VD was significantly decreased with inspiration (p less than 0.01). We feel that this is the normal variation in pulmonary venous return during respiration , influenced by changes in the venous return on the right side of the heart. In all patients with atrial fibrillation, AS and AD waves were disappeared. The negative deflection occasionally observed was due to mitral valve closure. In patients with mitral stenosis, the peak velocity of VD wave was significantly decreased compared to that of normal subjects, but it was not significantly different between the patients with mitral valve replacement and normal subjects. The peak velocity of VD wave was also correlated with pressure half time among the patients with mitral stenosis, mitral valve replacement and mitral commissurotomy. On the other hand, it was significantly increased in patients with mitral regurgitation and returned to normal level after the operation. The peak velocity of VS wave was correlated with the left atrial dimension among the patients with mitral valve diseases except these with mitral regurgitation. In patients with mitral regurgitation, the peak velocity was decreased compared to that of normal subjects and reversed flow was seen in half of the patients. Also, it was decreased in patients with dilated cardiomyopathy and increased in patients with hypertrophic cardiomyopathy. In conclusion, PVFV is influenced not only by changes of venous return with respiration but also by the left atrial size, presence or absence of MS or MR, left atrial or left ventricular systolic and diastolic function in the various diseased states.

[Mental stress echocardiography for patients with mitral valve prolapse].

Our clinical experience suggests that anxiety may provoke the augmentation of the degree of mitral valve prolapse (MVP) and change the mitral inflow velocity pattern in patients with MVP. To evaluate this systematically, we recorded 2-dimensional (2-D) and pulsed wave Doppler echocardiograms during acute mental stress in eight patients with MVP and eight age-matched normal subjects. Acute mental stress was administered by applying arithmetical task or reminding each patient of their most uncomfortable memories. Heart rate and blood pressure were significantly increased during mental stress and returned to the control level within a few minutes after its release in both groups. 2-D and Doppler echocardiograms were constantly recorded before, during and after acute mental stress. MVP was prominently augmented during mental stress in three of the eight patients. During mental stress, the mitral inflow velocity decreased in the rapid filling phase (R) and increased in the atrial filling phase (A), resulting in significant increase of the A/R in seven of the eight patients with MVP, especially in the patients associated with an increase of MVP. In normal subjects, mitral valve prolapse did not develop and the A/R was minimally increased or remained almost the same during mental stress. In conclusion, mental stress echocardiography seems to be a useful provocation test for the assessment of MVP and it is further expected to propose a lot of potential applications as a new method of stress echocardiography.