G. K. Radda

31P NMR saturation transfer measurements of the steady state rates of creatine kinase and ATP synthetase in the rat brain

There are two theories concerning the function of phosphocreatine (PCr) in cellular energy metabolism. In one PCr is seen as a reservoir, functioning to maintain constant cytoplasmic ATP concentrations. The alternate theory postulates that PCr acts as an energy shuttle between the site of ATP production in the mitochondria and the site of utilization in the cytoplasm (reviews [ 1,2]). Both theories require that the exchange flux in the creatine kinase (CPK) reaction be considerably greater than the rate of ATP hydrolysis. This is necessary in order to stabilize ATP concentrations during transitions in workload, or during periods when ATP synthesis is impaired, such as ischaemia. Calculations of cytoplasmic phosphorylation potential [3] and measurements of intracellular pH [4] have been based on the assumption that the CPK reaction is near equilibrium. It is now possible to test this assumption in vivo using the technique of 31P NMR saturation transfer; the flux through the CPK reaction was 5 times the steady state rate of ATP turnover in the Langendorff perfused rat heart 151. The total extractable activities of CPK in the rat brain and heart are similar; 500 pmol . g-’ . min-’ in heart [6] and 200 pmol . g-’ min-’ in brain [7], measured at 25’C. The mitochondrial form of the enzyme accounts for 30-40% of the total activity in the heart [l] and 5% in brain [7]. However, mitochondrial CPK activity in both organs is very similar on a per gram mitochondrial protein basis [8]. Despite these similarities, the metabolic response to a decrease in the efficiency of cellular energy transduction,

EXCESSIVE INTRACELLULAR ACIDOSIS OF SKELETAL MUSCLE ON EXERCISE IN A PATIENT WITH A POST-VIRAL EXHAUSTION/FATIGUE SYNDROME: A 31P Nuclear Magnetic Resonance Study

A patient with prolonged post-viral exhaustion and excessive fatigue was examined by 31P nuclear magnetic resonance. During exercise, muscles of the forearm demonstrated abnormally early intracellular acidosis for the exercise performed. This was out of proportion to the associated changes in high-energy phosphates. This may represent excessive lactic acid formation resulting from a disorder of metabolic regulation. The metabolic abnormality in this patient could not have been demonstrated by traditional diagnostic techniques.

Application of 31P NMR to model and biological membrane systems.

Proton, deuterium and carbon magnetic resonance techniques have been widely used to study the hydrocarbon chain region of model bilayer systems and biological membranes (for a review see [l]). More recently, phosphorus magnetic resonance (31P NMR) has been introduced to study the polar headgroup region of membranes [2-71. In this communication we show how 31P NMR can define an order parameter for the phosphate headgroup in model and biological membranes. In addition, in oriented systems the 31P NMR spectrum can provide information on the orientation of the membrane.

A 31P-nuclear magnetic resonance study of skeletal muscle metabolism in rats depleted of creatine with the analogue β-guanidinopropionic acid

Abstract Rats were fed a diet containing 1% β-guanidinopropionic acid (GPA) for 6–10 weeks to deplete their skeletal muscle of creatine. 31 P-NMR was used to monitor metabolic changes in the gastrocnemius muscle at rest, during stimulated steady-state isometric contraction at 4 Hz and during recovery from stimulation. In resting muscles, the [creatine phosphate] was reduced to 10% (2.8 μmol·g −1 ) and the [ATP] to 50% (3.3 μmol·g −1 ) of those found in rats fed a control diet. The concentration of the phosphorylated form of the analogue (PGPA) was 23 μmol·g −1 . There was no significant difference in muscle performance or in the relative changes in the [ATP] during stimulation. Intracellular pH decreased rapidly on stimulation and recovered during the stimulation period to near resting values in both groups. In control rats, the initial decrease in pH was greater and the time to recovery was longer than in GPA-fed rats. The rate at which PGPA supplied energy to the contracting muscle (0.027 mM·s −1 ) was insignificant relative to the minimum estimated rate of ATP turnover (1 mM·s −1 ). The rate of PGPA resynthesis during recovery (0.018 mM·s −1 ) is enzyme-limited and provides an independent estimate of creatine kinase flux during this period (18.9 mM·s −1 ). The creatine kinase flux (creatine phosphate → ATP) in the resting muscle of GPA-fed rats was 12-fold less than in control animals, 1.3 vs. 15.7 mM·s −1 . These results demonstrate that neither the [creatine phosphate] nor the activity of creatine kinase is critical for aerobic metabolism. Skeletal muscle appears to adapt to a diminished creatine pool by enhancing its aerobic capacity.

Regulation of creatine kinase during steady-state isometric twitch contraction in rat skeletal muscle

In vivo 31P-NMR saturation transfer measurements of the creatine kinase exchange flux in the direction creatine phosphate----ATP were made in the gastrocnemius muscle of rats at rest and during steady-state isometric twitch contraction at frequencies from 0.25 to 2 Hz. There was no correlation between creatine kinase exchange flux and either free [ADP] or oxygen consumption, both of which increase with stimulation frequency. The flux was found to be nearly constant over all conditions at about 16 mM X s-1, 10-times greater than the highest estimated ATP turnover in this study. The kinetic properties of skeletal muscle creatine kinase in vivo are similar to, but not completely predictable from, the equilibrium exchange fluxes measured on the isolated enzyme. These results are not consistent with strong functional coupling between ATP synthesis and mitochondrial creatine kinase.

Creatine kinase kinetics, ATP turnover, and cardiac performance in hearts depleted of creatine with the substrate analogue β-guanidinopropionic acid

Rats were fed a diet containing 1% of the creatine substrate analogue beta-guanidinopropionic acid for 6-10 weeks. 31P-NMR investigation of isolated, glucose-perfused working hearts showed a 90% reduction in [phosphocreatine] from 22.2 to 2.5 mumol/g dry wt in guanidinopropionic acid-fed animals but no change in [Pi], [ATP], or intracellular pH. The unidirectional exchange flux in the creatine kinase reaction (direction phosphocreatine----ATP) was measured by saturation transfer NMR in hearts working against a perfusion pressure of 70 cm of water. This exchange was 10 mumol/g dry wt per s in control hearts and decreased 4-fold to 2.5-2.8 mumol/g dry wt per s in hearts from guanidinopropionic acid-fed animals. Oxygen consumption and cardiac performance were measured in parallel experiments at two perfusion pressures, 70 and 140 cm. No significant differences were observed in oxygen uptake or in any of the performance criteria between hearts from control and guanidinopropionic acid-fed rats at either workload. Assuming an ADP:O ratio of 3, the oxygen consumption measurements correspond to ATP turnover rates of 4.2-7.8 mumol/g dry per s. These rates are 1.5-3-times greater than the rate of the phosphocreatine----ATP exchange in hearts from guanidinopropionic acid-fed rats. These data suggest that phosphocreatine cannot be an obligate intermediate of energy transduction in the heart.

A mitochondrial encephalomyopathy. A combined 31P magnetic resonance and biochemical investigation.

A 15-year-old girl presented with recurrent encephalopathic episodes, epilepsy, myopathy and chronic lactic acidosis. A muscle biopsy revealed the presence of ragged red fibres and mitochondria with paracrystalline inclusions. Biochemical studies on freshly isolated skeletal muscle mitochondria demonstrated a deficiency of NADH-CoQ reductase activity. Investigation of her gastrocnemius muscle at rest by phosphorus nuclear magnetic resonance displayed a reduced phosphocreatine concentration with elevated levels of inorganic phosphate and ADP. Abnormalities were also apparent in her brain spectrum. It is therefore possible that the mitochondrial defect present in skeletal muscle is also being expressed in the brain.

Total ion content of skeletal and cardiac muscle in the mdx mouse dystrophy: Ca2+ is elevated at all ages

The mdx mouse has been shown to have a gene defect at the locus which is homologous to that which is defective in Duchenne muscular dystrophy and they both lack dystrophin, the protein product of this defective gene. The exact cause of myofibre necrosis in DMD is not known but there is evidence to support a causal relationship between elevated calcium and tissue necrosis. Since the mdx mouse exhibits age-dependent changes in the proportion of tissue necrosis, we have measured total ion content (Ca2+, Na+, K+, and Mg2+) in the heart and skeletal muscle of animals at different ages to determine if ionic changes correlate with reported periods of necrosis. Total calcium is elevated throughout the ages studied (10 days, 30 days and 254-347 days) in both tissues and does not correlate with necrosis, although it appears that pre-necrotic tissues do not exhibit such a wide variation in calcium content as is observed in tissues from older animals. These changes are discussed with reference to the other ions measured and to the regulation of intracellular calcium.

Excitatory amino acid synthesis in hypoxic brain slices: does alanine act as a substrate for glutamate production in hypoxia?

Abstract: Excitatory amino acids are an important cause of cell death in the hypoxic and ischaemic brain. Neuronal glutamate stores are depleted rapidly in hypoxia, but alanine production rises under such conditions and has been suggested to be a potential precursor of glutamate. To test this hypothesis, we have investigated amino acid metabolism using 13C NMR with superfused guinea pig cortical slices subjected to varying degrees of hypoxia. During severe hypoxia, brain slices metabolising 5 mM [2‐13C]pyruvate exported [2‐13C]alanine into the superfusion fluid. The metabolic fate of alanine during normoxia and hypoxia was tested by superfusion of brain slices with 10 mM glucose and 2 mM [2‐13C, 15N]alanine. Metabolism of exogenous alanine leads to the release of aspartate into the superfusion fluid. The pattern of labelling of aspartate indicated that it was synthesised via the glial‐specific enzyme pyruvate carboxylase. 13C‐labelled glutamate was produced with both normoxia and hypoxia, but concentrations were 30‐fold lower than for labelled aspartate. Thus, although substantial amounts of glutamate are not synthesised from alanine in hypoxia, there is significant production of aspartate, which also may have deleterious effects as an excitatory amino acid.

An in vivo and in vitro H-magnetic resonance spectroscopy study of mdx mouse brain: abnormal development or neural necrosis?

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder primarily affecting young boys, often causing mental retardation in addition to the well-known progressive muscular weakness. Normal dystrophin expression is lacking in skeletal muscle and the central nervous system (CNS) of both DMD children and the mdx mouse model. The underlying biochemical lesion causing mental impairment in DMD is unknown. 1H-magnetic resonance spectroscopy (1H-MRS) detects choline-containing compounds, creatine and N-acetyl aspartate (NAA) in vivo. NAA is commonly used as a chemical marker for neurons, and a decline in NAA is thought to correlate with neuronal loss. Control mice were compared to mdx using a combination of in vivo and in vitro 1H-MRS methods to determine whether neural necrosis or developmental abnormalities occur in dystrophic brain. NAA levels were normal in mdx brain compared to controls suggesting minor, if any, neuronal necrosis in dystrophic brain. In contrast, choline compounds and myo-inositol levels were increased, indicative of gliosis or developmental abnormalities in dystrophic brain.