Fluorometric determination of N-terminal prolyl dipeptides, proline and hydroxyproline in human serum by pre-column high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsufonyl chloride.

A highly sensitive HPLC method for the determination of prolyl dipeptides, Pro and Hyp in serum was developed. After deproteinization of serum and pretreatment with o-phthalaldehyde, the analytes were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 degrees C for 10 min. The fluorescent derivatives of prolyl dipeptides, Pro and Hyp, were separated on tandem reversed-phase columns by a gradient elution at 55 degrees C and detected by fluorescence measured at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides were 2-5 fmol/injection (S/N=3). Pro-Hyp, Pro-Gly and Pro-Pro were identified as serum prolyl dipeptides. The within-day and between-day relative standard deviations were 1.5-7.9 and 2.4-10.8%, respectively. The recoveries were in the range of 90.8-97.3%. The concentrations of Pro-Hyp, Pro-Gly, Pro-Pro, Pro and Hyp in normal human serum (n = 10) were 0.64+/-0.35, 0.078+/-0.047, 0.022+/-0.016, 177.0+/-43.0 and 11.1+/-3.5 microM, respectively. The concentrations of Pro-Hyp and Pro-Pro in serum of a patient with bone metastases of prostatic cancer were about three times and 50 times, respectively, higher than those in normal human serum.

Development of a miniature undulation pump for the distributed artificial heart.

Research of the distributed artificial heart is important not only to acquire the means of individual organ perfusion but also to clarify the characteristics of the organ and the mechanism of blood distribution. To investigate the distributed artificial heart, the miniature undulation pump was developed. The outer diameter and the thickness of the developed pump were 38 mm and 11 mm, respectively. The priming volume of the pump was 3.2 ml. The total size including the motor unit was 38 mm in diameter and 32 mm in length. The total weight was 67.5 g. The total volume was 27.5 ml. The pump was driven with pulse width modulation by using a 1 chip motor controller. More than 5 L/min of continuous output could be obtained. The results showed that the developed miniature undulation pump system had enough performance for individual organ perfusion.

Using one rotary blood pump to produce separate pulsatile circulations in the upper and lower halves of the body.

Separate systemic circulations with pulsatile flow were obtained using 1 rotary blood pump as a left ventricular assist device. The outlet of the pump was divided into 2 conduits, 1 connected to the upper half of the body and the other connected to the lower half. An electric actuator that clamped the 2 outlet conduits alternately provided pulsatile flows. An in vitro experiment showed that the pulsatility phases of the upper and lower halves of the body were complementary with pulsatile flow, and an in vivo experiment showed that controlled flow distributions of continuous flows could be obtained.

Results of animal experiments using an undulation pump total artificial heart: analysis of 10 day and 19 day survival.

An undulation pump is a special rotary blood pump in which rotation of a brushless DC motor is transformed to an undulating motion by a disc in the pump housing attached by means of a special link mechanism. In the blood pump, a closed line between the disc and housing moves from the inlet to the outlet by this undulating disc motion, which sucks and pushes the blood from the inlet to the outlet. Because the same phenomena occurs at both sides of the disc, a continuous flow is obtained when the motor rotational speed is constant. The pump flow pattern can be easily changed from continuous flow to pulsatile flow by controlling the motor drive current pattern. A seal membrane made of segmented polyurethane protects the blood from invading the link mechanism as well as the motor. UPTAH is fabricated with two undulation pumps and two brushless DC motors. Its size is 75 mm in diameter and 80 mm long, and it has one of the great advantage of no compliance chamber required in the system. UPTAHs were implanted under cardiopulmonary bypass (CPB) into the chest cavities of 16 goats, each weighing between 41 and 72 kg. No anticoagulant and antiplatelet agent was used after the surgery. The left atrial pressure was automatically controlled to prevent its elevation and sucking of the atrial wall into the atrial cuff. The following results were obtained: (1) UPTAHs fit well into all the goats; (2) the longest survival was 19.8 days, the cause of death was bleeding from the aortic anastomosis; (3) No thrombus was observed in the blood pump despite no anticoagulant use. Hemolysis depended upon the length of CPB during surgery. When CPB time was within 2 hours, hemolysis level returned to baseline within a few days of the surgery. UPTAH is a promising implantable TAH, because of its small size and easy controllability.

Analysis of hemodynamic response with 1/R control on biventricular bypass goat.

A conductance and arterial pressure based method (1/R control) to determine the cardiac output (CO) of a total artificial heart (TAH) was developed to provide a central nervous system with control over the output of TAH. In order to clarify the deference in hemodynamic response between natural heart and 1/R control, biventricular bypass was introduced in the goat. After 2 pneumatically driven sac-type blood pumps were connected to the natural heart, the pulmonary artery was totally clamped to acquire 100% right heart bypass, and the ascending aorta was stenosed to acquire about 60 to 90% left heart bypass; 1/R control was performed substituting the output of the right artificial heart for the CO. The results demonstrated that stable control could be achieved. A discrepancy was often seen between the pulse rate (PR) of the artificial heart and the heart rate (HR) in absolute value. However, the relative changes of PR were quite similar to that of HR for the most part, indicating that the responses of 1/R control were the duplication of natural cardiac responses in normal daily activity.

Development of haemoglobin subtypes and extramedullary haematopoiesis in young rats. Effects of hypercapnic and hypoxic environment.

The influence of repeated hypoxia on the development of haemoglobin (Hb) subtypes and on extramedullary haematopoiesis (EMH) was investigated in young Wistar rats of different ages. The rats were exposed to hypercapnic/hypoxic and to "simple" hypoxic conditions. The results obtained were compared to those of an untreated age-matched control group. Different globin chains were measured using HPLC and time-of-flight (TOF) mass analysis. The number of EMH cells was evaluated by cell counting. By determining the proportions of alpha- and beta-chains, fetal, neonatal and mature types of globin chain composition could be differentiated. The beta-2 chain levels were significantly higher in hypercapnic/hypoxic environments than in the controls and simple hypoxic environments. The numbers of EMH cells in the two groups subjected to hypercapnia/hypoxia decreased significantly more slowly compared to the controls and simple hypoxia groups. Therefore, the development of Hb subtypes and the EMH activity in rats were influenced by both repeated hypercapnia and hypoxia.

Implantation of the undulation pump total artificial heart in the goat.

The undulation pump total artificial heart (UPTAH) was developed by using small-size continuous-flow displacement-type blood pumps (undulation pump). To clarify and improve the problems accompanied by the implantation in the chest, 14 animal experiments were performed on goats weighing 41.3-79.2 kg. The UPTAH could be implanted in the chest of all goats and was driven with a modulation pulsatile mode. The first problem was the atrial suction effect. This problem could be prevented to some extent by developing the soft disk and by improving atrial cuffs. An automatic detection and releasing of the atrial suction effect was also tried. The next problem was acute lung edema accompanied by the postural change of the animal. Development of the automatic control of left atrial pressure could prevent this problem. Small blood leakage from a pinpoint hole in the seal membrane was the next problem. Improvement of the manufacturing procedure of the membrane prevented this. With these improvements, a 10 day survival could be obtained with this unique implantable total artificial heart.

Present status of the total artificial heart at the University of Tokyo.

At the University of Tokyo, various types of total artificial heart (TAH) systems have been studied since 1959. At the present time, 2 types of implantable TAH have been developed. One is an undulation pump TAH (UPTAH) and the other is a flow transformed pulsatile TAH (FTPTAH). Using the UPTAH, 14 cases of implantation were performed in goats and 10 days' survival obtained. The new type of FTPTAH is under a prototype study. To prevent ring thrombus, a polyurethane membrane valve, a jellyfish valve, has been developed. The longest in vivo experiences with this valve in the TAH blood pump have been 312 days in the left side blood pump and 414 days in the right side blood pump. Conductance and arterial pressure based control (1/R control) can realize the physiological control of the TAH. Using 1/R control, 532 days of survival could be obtained in a goat with a paracorporeal TAH. The technique required to apply this control method to a implantable TAH is under development. We have proposed a new 5 year research project of the implantable TAH entitled "Comprehensive Basic Research on the Development of a Japanese Original Implantable Total Artificial Heart" to The Ministry of Welfare.

Physiological control of a total artificial heart: conductance- and arterial pressure-based control.

To obtain a physiological response by a total artificial heart (TAH), while eliminating the hemodynamic abnormalities commonly observed with its use, we proposed the use of a conductance- and arterial pressure-based method (1/R control) to determine TAH cardiac output. In this study, we endeavored to make use of a variable more closely tied to central nervous system (CNS) efferents, systemic conductance, to provide the CNS with more direct control over the output of the TAH. The control equation that calculates the target cardiac output of the TAH was constructed on the basis of measurement of blood pressures and TAH flow. The 1/R control method was tested in TAH-recipient goats with an automatic method by using a microcomputer. In 1/R control animals, the typical TAH pathologies, such as mild arterial hypertension and substantial systemic venous hypertension, did not occur. Cardiac output varied according to daily activity level and exercise in a manner similar to that observed in natural heart goats. These results indicate that we have determined a control method for the TAH that avoids hemodynamic abnormalities exhibited by other TAH control systems and that exhibits physiological responses to exercise and daily activities under the conditions tested. The stability of the control and the complete lack of inappropriate excursions in cardiac output is suggestive of CNS involvement in stabilizing the system.

Development of the undulation pump total artificial heart.

The undulation pump is a small size continuous flow displacement type blood pump that has been developed for an artificial heart. Using undulation pumps, 2 types of implantable total artificial hearts (TAHs), the undulation pump TAH (UPTAH) type 1 (UPTAH 1) and UPTAH type 2 (UPTAH 2) were developed. Both UPTAHs were designed to be small enough to implant into the chest of a goat, the experimental animal. UPTAH 1 could be reduced in size to 75 mm in diameter and 78 mm in length. The weight was 520 g. UPTAH 2 could be reduced in size to 75 mm in diameter and 80 mm in length. The weight was 650 g. UPTAH 2 could be tested in an animal experiment using an adult female goat weighing 52.3 kg. The UPTAH 2 could be implanted successfully into the goat's chest with a good fit. The goat stood after the surgery and extubation and survived for 3 h and 40 min; thus, the potential of the UPTAH for a practical implantable TAH was demonstrated.

Use of a catecholamine sensor in the control of an artificial heart system.

An electrochemical sensor system to allow real-time measurement and feedback of catecholamine concentrations was developed for use in the control of artificial hearts. Electrochemical analyses were carried out using a carbon fiber working electrode, an Ag-AgCl reference electrode, and a potentiostat. The operating parameters of the pneumatically-driven artificial heart system were altered in accordance with the algorithm for changes in the catecholamine concentration. The minimum detectable concentrations of both adrenaline and noradrenaline in a mock circulatory system using a phosphate-buffered solution were approximately 1-2 ng/ml (10(-8) mol/L). An artificial heart control system utilizing this set-up performed satisfactorily without delay, although sensor sensitivity decreased when placed in goat plasma instead of a phosphate-buffered solution, due to the adsorption of various substances such as plasma proteins onto the electrodes. This study demonstrated the future feasibility of a feedback control system for artificial hearts using catecholamine concentrations.

Basic study to develop the undulation pump for practical use: antithrombogenicity, hemolysis, and flow patterns inside the pump.

The undulation pump (formerly called the precessional displacement pump) is a continuous flow displacement-type blood pump that is being developed as an implantable total artificial heart. A new undulation pump was developed for chronic use and was examined with animal experiment and flow visualization studies. In the animal experiment using a left ventricular bypass in goats, severe hemolysis occurred. After driving for 12 h, thrombus formation inside the pump was found. In the flow visualization studies, the flow pattern showed that the flow inside the pump was a very complicated turbulent flow. Improvement of hemolysis and thrombus formation is important to realize implantable total artificial hearts using undulation pumps.

Development of a new apparatus to observe microcirculation chronically in continuous flow blood pump research.

To observe microcirculation chronically is an important key to the successful evaluation of the continuous flow blood pump. In this study, we succeeded in developing a new apparatus by which microcirculation could be observed chronically without a microscope in a conscious animal. The apparatus utilizes a charge coupled device (CCD). A thin living tissue, such as mesentery, is put directly on a highly integrated CCD and is lit up through the tissue with a light-emitting diode (LED). The vascular nets in the tissue are projected onto the CCD like a contact photograph, which is then sent to a television screen and which can be used to analyze their motion and function. A 0.5-inch CCD having 250,000 pixels was used in this study. The cover glass of the CCD was removed, and a fiber optic plate was fixed onto the surface of the CCD for the tissue to be able to contact with the apparatus surface without clearance. The CCD as well as the LED were molded with epoxy resin for electrical insulation. The apparatus was 35 mm wide and 12 mm high with a micro stand for an LED, which can be easily implanted into an animal. The apparatus was implanted into a rabbit for 12 h. The configuration of arterioles and venules, tens of micrometers in diameter, and their motions in subcutaneous tissue could be observed.

Child abuse and neglect in Japan: coin-operated-locker babies.

The coin-operated-locker baby is a type of child abuse that may be unique to Japan. The term refers to newborns who are placed, while alive or dead, in coin-operated lockers. This practice has been decreased by specific measures. It is likely that social and economic variables in Japan account for differences in the frequency and types of child abuse cases when compared to American or European societies as social conditions in Japan change the reported incidence of child abuse may increase in the future. Recently, the government of Osaka organized a group specifically designed to deal with the detection and protection of abused and neglected children. In 1993, they published a manual on how to deal with child abuse, but the Japanese judicial administration still uses old laws for abuse cases. The development of new laws is occurring in parts of the country now. This paper introduces the present status of coping with child abuse and neglect in Japan.

Can total artificial heart animals control their TAH by themselves? One year survival of a TAH goat using a new automatic control method (1/R control).

A total artificial heart (TAH) goat survived for 360 days on the new automatic control method (1/R control), in which the cardiac output of the TAH can be controlled through the cardiovascular center by making it function by reflecting the beta-adrenergic reaction in peripheral vascular resistance. This is thought to be the only long-term, real-time, measurable parameter by which information on the activity of the cardiovascular center can be received directly by the TAH system. In this goat, the hemodynamic parameters (RAP, AoP, and so forth) were kept within physiologic limits when control was stable, and the cardiac output was automatically increased in response to exercise, not unlike that in the natural heart. There were no abnormal blood chemical or hormone data except at end stage. Based on these results 1/R can be considered a physiologic control method for a TAH.

A new apparatus for chronic observation of the microcirculation in situ to evaluate artificial organ performance.

Chronic study of the peripheral circulation and metabolism is very important in evaluation of artificial organ performance. However, there has been no way to observe the microcirculation in situ, chronically and continuously, without restriction. In this study, the authors developed a new apparatus that could be implanted and connected to an artificial organ that would allow continuous observation of the microcirculation while the subject is awake. The apparatus uses a charge coupled device (CCD) under a new principle: thin living tissue, such as mesentery, is put directly on a highly integrated CCD and transilluminated with a light emitting diode (LED). The vascular nets in the tissue are projected onto the CCD like a contact photograph, which is sent to a television screen and can be analyzed for motion and function. A 0.5 inch CCD with 25K pixels was used in this study. The cover glass of the CCD was removed so the tissue would be able to directly contact the CCD surface. The CCD, as well as LED, were molded with hard polyurethane as electrical insulation. The apparatus is 35 mm in diameter and 10 mm high with a micro stand for lighting with the LED, which is easy to implant in a goat or calf. The resolution of this apparatus was tested by putting a micro scale on the CCD surface. Several tenths of micrometers could be seen. In an animal experiment with a rabbit, configurations of arterioles and venules and their motions could be observed continuously for a night until the electrical insulation was broken. This method might be a strong weapon in artificial organs research.

Fabrication of a jellyfish valve for use in an artificial heart.

For a valve to be fabricated seamlessly into an artificial heart (AH) blood pump, a jellyfish valve has been developed, in which a thin membrane is fixed at the center of a valve seat having several spokes to protect against prolapse of the membrane. The valve is superior in performances to a Björk-Shiley valve, and reveals good blood compatibility. The valve would be very useful not only for AH animal study, but for future clinical use in infants to adults. Several institutions are already trying the valve. In this paper, the fabrication of the jellyfish valve is introduced, and in vitro and in vivo results summarized. A computer aided design (CAD) system was developed to cut a male wax mold of the valve seat. The input parameters to the CAD are diameter, height, thickness of rim, number of spokes, width and thickness of spokes, etc. Jellyfish valves with diameters of 4 to 27 mm have already been fabricated for many types of AHs and assist pumps.

The second and third model of the flow transformed pulsatile total artificial heart.

For the purpose of future total implantation, a new pulsatile total artificial heart, a flow transformed pulsatile total artificial heart (FTPTAH), in which the continuous flow from a single centrifugal pump (CFP) was converted to pulsatile flow by switching two three-way valves that could alternately perfuse the systemic and pulmonary circulation, was proposed, and the data from the prototype model were reported. As the next step, the second model, in which a CFP and a spool valve (SV) driven with a solenoid were fabricated in one piece, was made and tested in a mock circulatory system. The system could send 4.7 L/min of pulsatile output alternately to the pulmonary artery and aorta, with 30 and 100 mmHg afterload, respectively, at 3000 rpm CFP. However, three problems were encountered: the output was not enough, mixture or inversion of venous and arterial blood in the CFP would occur, and heat generation at the solenoid was very severe. To solve these problems, a third model was designed in the current study. To increase pump output, hydrodynamic analysis was performed. The SV was divided into inlet and outlet to control the blood mixture or inversion. To suppress heat generation, each SV was driven back and forth by two solenoids, one on each side of the SV. The model revealed satisfactory results in a mock circulatory system.

Blood compatibility of the jellyfish valve without anticoagulant.

The blood compatibility of the jellyfish valve was studied. Artificial heart (AH) blood pumps incorporating jellyfish valves were connected to 18 goats as total artificial hearts (TAHs) and pumped for 1 to 125 days without anticoagulant or antiplatelet drugs. No thrombus was formed on the valve membrane or around the valve seat. Scanning electron microscopy showed almost no platelet deposition or microfibrin clot formation on the valve membrane, including its central region; the spokes of the valve seat were also free from platelet and microfibrin clots. No calcification was observed during these tests, and plasma free hemoglobin was between 2 and 7 mg/dl. The jellyfish valve revealed good blood compatibility, even without anticoagulant use.

A new pulsatile total artificial heart using a single centrifugal pump.

A new pulsatile total artificial heart (TAH) system, combining a single centrifugal pump (CFP) with two three-way valves, was developed. One port of each three-way valve was connected to the inlet and outlet of a CFP, respectively. The other two ports of each valve ware connected to the right and left atrium, pulmonary artery, and aorta. The CFP can perfuse the pulmonary and systemic circulation alternately with pulsatile flow. A prototype system composed of a Sarns' CFP and solenoid valves was connected to a mock circulatory system resulting in 1) a pulsatile TAH that could be produced with a single CFP, 2) 5 L/min of pulsatile output with a normal flow wave form that can be obtained alternately on the right and left side by switching the solenoid valves, and 3) flow balance between the left and the right that could be controlled easily by the length of switching duration. This new system could be miniaturized and is feasible for a totally implantable TAH.