The introduction of new paramagnetic shift reagents in the nuclear magnetic resonance (NMR) method has made it possible to distinguish intra- and extracellular ions in tissues or organs in vitro. We measured the intra- and extracellular 23Na and 1H in vivo in the gerbil brain and skeletal muscle by NMR spectroscopy employing the shift reagent, dysprosium triethylenetetraminehexaacetate (Dy[TTHA]3-). Without Dy(TTHA)3-, the 23Na and 1H signals were seen only as single peaks, but gradual intravenous infusion of Dy(TTHA)3- separated these signals into two peaks, respectively. The unshifted peaks reflected the intracellular 23Na and 1H signals, while the shifted peaks reflected the extracellular signals. In the brain spectra, an additional small peak, which represented intravascular signals, was detected and its intensity increased after injection of papaverine hydrochloride. The present method is advantageous over the microelectrode technique because of its nondestructiveness and its capability for obtaining intra- and extracellular volume information from measurements of the 1H spectra, the peaks of which reflect the intra- and extracellular water amounts. The intracellular Na+ increase associating with increased cellular volume after ouabain in the muscle was clearly visualized by this method. The technique is clearly of use for physiological and pathophysiological studies of organs.

Summary:The relationships among CBF, cerebral en ergy metabolism, Na + pump activity, and electrocortico grams (ECoG) following graded hypotension were studied in 48 gerbils. Energy metabolism and Na+ pump activity were estimated by in vivo 31p and 23Na nuclear magnetic resonance (NMR) spectroscopy, and CBF was determined by [14C]iodoantipyrine methods at the end of the experiments. The CBF measured in normotensive an imals was 0.51 ± 0.07 ml/g brain/min. Following graded hypotension, no 31p spectral change was observed until CBF fell to 0.21-0.27 ml/g brain/min, at which level the intracellular pH began to decrease in association with Address correspondence and reprint requests to Dr. Hiroaki Naritomi at Cerebral Circulation Laboratory, National Cardio vascular Center, 5-7-1 Fujishiro-dai, Suita, Osaka 565, Japan.Abbreviations used: ECoG, electrocorticogram; NMR, nu clear magnetic resonance; PCr, phosphocreatine; pHi, intracel lular pH; Pi, inorganic phosphate. 16in association with inorganic phosphate (Pi) elevation. At this level, ECoG became isoelectric, although no adeno sine triphosphate (ATP) change yet resulted. At a flow level of 0.12-0.14 ml/g brain/min, ATP began to decrease gradually. At 0.04-0.05 ml/g brain/min, PCr and ATP vir tually disappeared, and the 23Na signal intensity suddenly changed. The present study demonstrated flow thresholds for the development of tissue acidosis, PCr-Pi changes, and ATP reduction. It appears that functional suppression occurs prior to ATP changes, whereas Na + pump failure results after ATP depletion. Key Words: CBF thresholds for cerebral ischemia-Cerebral energy metabolism-Intracellular pH-Na+ pump activity31p and 23Na nuclear magnetic resonance spectroscopy.pump failure differ considerably. The former lies somewhere between 0. 15 and 0.20 mllg brain/min (Sharbrough et aI. , 1973; Trojaborg and Boysen, 1973; Heiss et aI. , 1976; Astrup et aI. , 1977; Bran ston et aI. , 1977), whereas the latter is at approxi mately 0. 06-0. 08 ml/g brain/min (Astrup et aI., 1977; Branston et aI., 1977). The former may repre sent the flow threshold at which mild energy distur bance begins, whereas the latter may represent the threshold for energy exhaustion. However, few studies have yet been undertaken on the flow-en ergy relationship, and the above mentioned as sumptions remain to be confirmed.In vivo nuclear magnetic resonance (NMR) spec troscopy is a potentially useful tool for examining the physiological and biochemical natures of organs noninvasively. With this method, it is possible to estimate not only changes of high-energy phos phate, but also those of intracellular pH (pHi) or

Two recombinant mutants of porcine kidney D-amino acid oxidase [EC 1.4.3.3, DAO], in which Tyr(228) and His(307) are replaced with Phe and Leu, respectively, have been expressed in Escherichia coli and purified to apparent homogeneity. The molecular size and amino-terminal sequence of the two mutants were the same as those of the native DAO. Kinetic analysis revealed that the Michaelis constants of the Phe-228 and Leu-307 mutants for D-alanine were 71- and 10-fold and the inhibition constants for benzoate, a potent competitive inhibitor, were 1,189- and 18-fold greater than those of the native DAO, respectively. The maximum velocities of the Phe-228 and Leu-307 mutants were 66 and 58% that of the native DAO. The kinetically estimated dissociation constant of the Leu-307 mutant for FAD was 28-fold greater than that of the native DAO, whereas the value of the Phe-228 mutant was comparable to that of the native DAO. The Leu-307 mutant and the recombinant wild-type DAO were inactivated by D-propargylglycine (D-PG), a suicide substrate. However, the Phe-228 mutant was resistant to the inactivation. Absorption peaks of the Phe-228 mutant were blue-shifted about 10 nm from the corresponding peaks of the wild-type DAO, and the oxidized form was fully reduced by D-alanine without appearance of the purple intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

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