Yoshio Shimizu

Microassay system for newborn screening for phenylketonuria, maple syrup urine disease, homocystinuria, histidinemia and galactosemia with use of a fluorometric microplate reader

Abstract Microfluorometry for measuring phenylalanine, branched-chain amino acids, homocysteine and histidine in dried blood spots is presented as a new mass screening method for phenylketonuria, maple syrup urine disease, homocystinuria and histidinemia, respectively. Simple and rapid determinations of the metabolites are achieved by adapting each of the optimum fluorogenic reactions to a microplate scale followed by using a highly sensitive microplate reader. Each assay for several hundreds of samples can be completed within 3 h, with fully calculated results by an on-line microcomputer. The proposed system, both with simple procedure and quantitative results, is useful for the multiple routine screening for the four aminoacidopathies, in addition to galactosemia as we reported earlier [20]. (1) The usage of a microplate system for routine microplate screening might be able to open a new style of mass-screening. (2) Measurement of homocysteine seems to be more desirable than measuring methionine in order to detect classic homocystinuria, and to enable detection of the remethylatine defects. However, the recovery procedure of homocysteine needs to be improved; furthermore, the measuring of three branched-chain amino acids will be better than determining only leucine for detecting a mild form of maple syrup urine disease or other conditions which increase the levels of these amino acids. The manner in which we measure phenylalanine in our method is more sensitive than others using the microplate system.

Effective site of bronchodilation by antiasthma drugs in subjects with asthma

We studied the effective sites of airway response to atropine and fenoterol aerosols and to the intravenous injection of aminophylline in patients with stable and spontaneous asthma, by the simultaneous assessment of respiratory resistance (Rrs) and anatomic dead space (VD). Central airway response was determined by VD, and overall response was determined by Rrs. Peripheral airway response was inferred from Rrs when the change in VD was slight. Atropine (4 mg/ml) or fenoterol (0.4 mg/ml) was continuously inhaled during tidal breathing for 5 minutes. Inhalation of both atropine and fenoterol increased Grs (reciprocal of Rrs) (p less than 0.01) with a simultaneous increase in VD (p less than 0.01) in the patients with stable and spontaneous asthma. Fenoterol increased Grs more than did atropine at an equivalent increase in VD in patients with spontaneous asthma (p less than 0.05). Intravenous injection of aminophylline (250 mg) had no effect on either Grs or VD in patients with stable asthma, but it significantly increased Grs (p less than 0.01) without change in VD in patients with spontaneous asthma. These results suggest that the predominant sites of bronchodilation induced by inhaled atropine are the central airways, that those sites induced by intravenous injection of aminophylline are the peripheral airways, and that inhaled fenoterol dilates both the central and peripheral airways in subjects with asthma. Differences among clinically used bronchodilators on the effective sites may be considered in the treatment of bronchial asthma.

A comparison of ST segment deviation and calculated solid angle during acute regional ischemia in the isolated canine heart at precordial, epicardial and intramyocardial lead surfaces

Although solid angle analysis has been considered to be reasonable for explaining the distribution of ST segment deviation following ischemia, it has not been tested fully, especially for ST segment changes in various sites at different lead surfaces. Thus, we investigated the applicability of solid angle theory to the mechanism of ischemic ST segment deviation at intramyocardial, epicardial and precordial leads. We used seven isolated, coronary perfused, isovolumic contracting canine hearts in a homogeneous cylindrical volume conductor. ST segment potentials from 246 electrodes were continuously measured during left circumflex coronary artery occlusion for five minutes. The ischemic boundary was obtained from a postmortem angiography, and the solid angle subtended by the ischemic boundary was calculated at every electrode site. Despite the difference between epicardial and precordial ST segment potential distributions, there was a high correlation between measured ST segment potential and calculated solid angle at epicardial (r = 0.86 +/- 0.05, 0.77-0.93), precordial (r = 0.93 +/- 0.05, 0.84-0.99), and intramyocardial leads (r = 0.95 +/- 0.03, 0.91-0.99). We conclude that solid angle analysis can be used to approximate the distribution of ischemic ST segment deviation at different lead surfaces in acute ischemia.

Pressure-length loop in the ischemic segment during left circumflex coronary artery stenosis and its modification by afterload reducing in excised perfused canine hearts

SummaryBy using excised perfused heart preparations, we investigated the regional myocardial functions in the presence of a flow-limiting coronary stenosis of the left circumflex coronary artery (LCX) (approximately a 50% flow reduction of pre-ischemic control), as well as global cardiac functions during afterload reducing, while keeping left ventricular end-diastolic pressure (LVEDP) and heart rate constant. After inducing the LCX stenosis, cardiac output (CO), peak left ventricular pressure (peak LVP) and stroke work (SW) decreased from pre-ischemic control values, i.e., 81.1±3.2%, p<0.005, 88.1±3.8%, p<0.02 and 72.2±5.7%, p<0.005, respectively (n=7), whereas pressure-length (P-L) loop areas changed as follows; ischemic control values of the left anterior descending coronary artery (LAD) and LCX regions were 96.6±6.0%, n.s. and 72.6±9.0% of pre-ischemic control, p<0.02, respectively.Following afterload reducing with LCX stenosis, CO increased gradually, while the ischemic regional function started to further aggravate, and the initial point of further ischemic aggravation obtained in this experiment occurred at 63.5±6.9 mm Hg of mean aortic pressure (AoP). These results suggested that the increase of total cardiac function such as CO following afterload reducing was probably induced at the expense of aggravated regional ischemia. Therefore it was concluded that the treatment of ischemic myocardium by reducing afterload pressure should be done very carefully.

Direct-Writing Recorder of the Frequency Dependence of Dynamic Compliance Analyzed from One Cycle of Breathing and Pulmonary Resistance: Effect of Fenoterol on Asthmatic Subjects

We developed a new method of direct-writing recording of the frequency dependence of dynamic compliance analysed from one cycle of breathing and pulmonary resistance. Both pulmonary resistance (RL) and frequency dependence of dynamic compliance (Cdyn.f) are calculated by Fourier-series analysis of flow and transpulmonary pressure in a single cycle of breathing. RL was obtained from fundamental harmonics. Cdyn.f was calculated from 1st and 2nd harmonics and estimated by the ratio of Cdyn at 0.5 Hz to Cdyn at zero frequency, C0.5/C0. We used fenoterol aerosol which contained 0.2 mg of fenoterol with one puff of aerosol. Two puffs of fenoterol aerosol were used in each subject and followed changes of RL, C0 and C0.5 as long as 30 min. After fenoterol inhalation RL decreased considerably and C0.5/C0 cont [C0.5/(control C0)] increased. With time, while RL was kept stable, C0.5/C0 cont increased further. We suggest that fenoterol has a potent effect on the small airways which is enhanced with time.