Lincoln's Inn Fields, London WC2A 3PN, U.K.. " P n.m.r. has been used to study ischaemia non-invasively in a number of systems, ranging from isolated organs ...
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619th MEETING, CAMBRIDGE
Buffering capacity of muscle determined by ' H a.nd 3' P nuclear magnetic resonance spectroscopy 70
STEPHEN R. WILLIAMS,* EDWARD PROCTOR? and DAVID G . GADIAN* *Department of Physics in Relation to Surgery, and t Department of Applied Physiology and Surgical Sciences, The Royal College of Surgeons of England, 35-43 Lincoln's Inn Fields, London WC2A 3 P N , U . K . " P n.m.r. has been used to study ischaemia non-invasively in a number of systems, ranging from isolated organs to intact animals, including human muscle (for discussion and references, see Gadian, 1982). Recently techniques have also been developed for detecting lactate in vivo by ' H n.m.r. (Rothman et al., 1984a,h; Williams et al., 1985) and it has been demonstrated that metabolism can be followed using ' H and " P n.m.r. for both brain (Behar et al., 1985) and muscle (Williams et al., 1985). Here we report studies of ischaemia in the rat hind limb in which phosphocreatine breakdown and the fall in pH are determined by 3' P n.m.r. and lactate production is followed by ' H n.m.r. Quantification of the data enables the buffering capacity of the muscle to be determined. The experiments were carried out as described previously (Williams et al., 1985) using a Bruker 360AM spectrometer operating at a frequency of 360MHz for ' H . A saddleshaped coil, doubly tuned to the ' H and '' P frequencies, was placed round the thigh of an anaesthetized rat. lschaemia was induced by tightening a ligature around the top of the thigh. The ligation produced total ischaemia, with no blood flow to the muscle. I ' P spectra were recorded in 32 scans using a 30' pulse at 1.8s intervals. Lactate was detected using the spin-echo double resonance experiment (Campbell & Dobson, 1979) to 'edit' the spectrum so that the large signal from fat, which normally obscures the lactate resonance, was suppressed (Rothman et al., 1 9 8 4 ~ ;Williams e f al., 1985). Quantification of spectra in vivo is difficult, particularly when a spin-echo is used to collect the data, as one needs to know the Tz values of the resonances. Using the spin-echo double-resonance experiment the lactate signal cannot be detected at echo times much shorter than about 20ms; therefore its relaxation behaviour cannot be known at short echo times. Because of this uncertainty we have quantified the spectra in vivo by relating them to extract data. Immediately after recording a ' H spectrum in vivo the ischaemic muscle of a group of animals was freeze-clamped 1-1.5 h after ligation, by which time there was no phosphocreatine and the lactate concentration and pH were stable. After perchloric acid extraction and neutralization the ratio of creatine to lactate was determined by recording quantitative H n.m.r. spectra. In a second group of animals the total cratine + phosphocreatine concentration was determined by enzymatic assay (Bernt et al., 1974; Lamprecht et al., 1974) after freeze-clamping. Using these data to calibrate the spectra in vivo it was possible to calculate absolute lactate concentrations by comparing the heights of the lactate peak (1.32 p.p.m.) and the creatine phosphocreatine peak (3.03 p.p.m.). This is a valid procedure as the creatine + phosphocreatine peak at 3.03 p.p.m. remains at the same height throughout the experiment and the peak widths are unchanged. Total creatine + phosphocreatine was 24.9 -t 3.7 (s.D.) mmol/kg wet wt. (n = 7) and the final lactate concentration in the freeze-clamped muscles was 48.5 f 7.8 (s.D.) mmol/kg wt wt. (n = 6). Using this information lactate concentration has been plotted as a function of pH in Fig. 1. Linear regression lines were fitted to each of the seven experiments in Fig. 1 ., to give a mean slope of 63.6 f 9.1 (s.D.) mmol/pH unit per kg wet wt. and a mean intercept of
'
+
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... A
.
*
. . .. 6.5
I
' t
0.
. . . . . 1'.
:
1
1.
L
.
6.0 0
35
70
Lactate (mmol/kg wet wt.)
Fig. 1. Lactace concentration during ischaemiu as a function o f PH
The pH was determined from the chemical shift of the inorganic phosphate resonance relative to phosphocreatine from the ''P n.m.r. spectra (Gadian, 1982). The lactate concentration was calculated from the spin-echo double-resonance spectra (see the text). Each point represents one determination on one rat. Data from seven rats are included in the Figure.
7.16 0.07 (s.D.). The individual correlation coefficients varied from - 0.91 to - 0.97 with from seven to ten points in each determination. There are two independent conclusions from this work: firstly, buffering is linear over the pH range 7.0-6.2, as has been suspected on the basis of muscle homogenates (BateSmith 1938; Heisler & Piiper, 1971); secondly, the buffering capacity is significantly higher than most values previously reported for mammalian muscle which have ranged from 28 to 56 mequivalents/pH unit per kg wet wt. (see, for example, Burton, 1978). We thank the Rank Foundation, Picker International and the Wolfson Foundation for support. Bate-Smith, E. C. (1938) J. Physiol. (London) 92, 336-343 Behar. K. L., den Hollander, J. A., Petroff, 0. A. C., Hetherington, H. P., Prichard, J. W. & Shulman, R. G. (1985) J. Neurochem. 44, 1045-1055 Bernt, E., Bergmeyer, H. U. & Moellerung, H. (1974) in Methods oJ Enzymafic Analysis (Bergmeyer, H. U. & Gawehn, H., eds.), 2nd English edn., vol. 4, pp 1772-1775, Verlag Chemie, Weinheim/ Academic Press, New York and London Burton, R. F. (1978) Respir. Physiol. 33, 51-58 Campbell, I. D. & Dobson, C. M. (1979) Methods Biochem. Anal. 25, 1-133 Gadian, D. G. (1982) Nuclear Magnetic Resonance and its Application to Living Systems, Oxford University Press, Oxford Heisler, N. & Piiper, J. (1971) Respir. Physiol. 12, 169-178 Lamprecht, W., Stein, P., Heinz, F. & Weisser, H. (1974) in Methocis of Enzymatic Analysis (Bergmeyer, H. U. & Gawehn, H., eds.), 2nd English edn., vol. 4, pp. 177551779. Verlag Chemie, Weinheim/ Academic Press, New York and London Rothman, D. L., Behar, K. L., Hetherington, H. P. & Shulman, R. G. (19840) Proc. Nafl. Acad. Sci. U.S.A.81, 6330-6334
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BIOCHEMICAL SOCIETY TRANSACTIONS
Rothman, D. L., Arias Mendoza, F., Shulman, G. I. & Shulman, R. G. (19846) J . Magn. Reson. 60,430-436 Williams, S. R.. Gadban, D. G., Proctor, E., Sprague, D. B., Talbot, D. F., Young, I. R. & Brown, F. F. (1985) J . Magn. Reson. 63,
'3
40M1 I
Received 12 June 1986
C nuclear magnetic resonance studies in vivo on the metabolism of [ 1-13Clbenzoate by mutants of Pseudomonas putida
ANTHONY E. G . CASS,* DOUGLAS W. RIBBONS,* JOHN T. ROSSITER* and STEVE R. WILLIAMS? *Centrefor Biotechnology, Imperial College, London SW7 2AZ. U.K., and TDepurtment of Physics, Royal College of Surgeons, 35-43 Lincoln's Inn Fields, London WCZA 3PN, U . K . Using I3C n.m.r. spectroscopy we are examining several aspects of the metabolism of I3C-enriched hydrocarbons, phenols and aromatic acids in whole bacterial cell suspensions of wild-type and mutant strains of Pseudomonas Putida. Pseudomonads can catabolize aromatic compounds via ortho and meta ring-cleavage pathways. The best understood of the mefu ring-cleavage pathways for aromatic catabolism is that for catechol (and alkyl catechols) initially described 25 years ago in Pseudomonads (Dagley & Stopher, 1959; Dagley et al., 1960; Kojima et al., 1961) and later found to be important in a variety of Gram-positive and Gram-negative bacteria. However, there still remain queries regarding the structures of some intermediates and the relative importance of the physiological flux of metabolites through the branched pathways depicted in Fig. I . It is appropriate to establish and document the chemistry and physiology of meta ring-cleavage pathways so that a firm basis is available for understanding the gene assemblies and their regulatory constraints. The mutant of Pseudomonasputida, PpU 103, is deficient in 3,5-cyclohexa-diene- 1,2-diol- 1 -carboxylic (NAD) dehydrogenase (decarboxylating) and is known to accumulate 3 5 cyclohexa-diene- I ,2-diol-l-carboxylate (11) when fed benzoate (I). An experiment using [ I-"C]benzoate as substrate was undertaken to monitor the accumulation of (11). Cells of
Fig. 1 . meta ring-cleavage pathway ( a ) Benzoate dioxygenase; ( b ) benzoate diol dehydrogenase; ( c ) catechol 2,3-dioxygenase; ( d ) 2-hydroxymuconic scmialdehyde dehydrogenase; ( e ) 4-oxalocrotonate tautomerase; ( , f ) 4-oxalocrotonate decarboxylase; (g) hydratase; (h) aldolase. ~~
Mutant PpDl
L = : r
=
30mln
r
=
~
PpU 103 were induced with benzoate overnight, washed and resuspended in phosphate buffer (100mM, pH 7.5). [I-"C]Benzoate (0.5m1, 0.1 M) and succinate (0.1 ml, I M) were added to a lOmm diameter n.m.r. tube containing 3 ml of cell suspension. "C n.m.r. spectra were recorded at a frequency of 90.56MHz on a Bruker AM 360 spectrometer with a 7.3cm usable bore 8.5T magnet. Broadband ' H decoupling was used throughout. The bacteria were oxygenated by bubbling with 0, during the relaxation delay. The temperature was maintained at 30'C. The formation of Mutsnt ppUIO3
1
I
r
=
96mln
=
64min
Omin
I
Fig. 2.
C n.m.r. spectra of whole-cell suspensions of the mutants Pseudumonas putidu PpDI and PpUlO3 Spectra were recorded in blocks of 240 scans, at 2 s intervals. Chemical shifts in p.p.m.
1986
