The role of pH on the thermodynamics and kinetics of muscle biochemistry: an in vivo study by (31)P-MRS in patients with myo-phosphorylase deficiency.

In this study we assessed ΔG'(ATP) hydrolysis, cytosolic [ADP], and the rate of phosphocreatine recovery using Phosphorus Magnetic Resonance Spectroscopy in the calf muscle of a group of patients affected by glycogen myo-phosphorylase deficiency (McArdle disease). The goal was to ascertain whether and to what extent the deficit of the glycogenolytic pathway would affect the muscle energy balance. A typical feature of this pathology is the lack of intracellular acidosis. Therefore we posed the question of whether, in the absence of pH decrease, the rate of phosphocreatine recovery depends on the amount of phosphocreatine consumed during exercise. Results showed that at the end of exercise both [ADP] and ΔG'(ATP) of patients were significantly higher than those of matched control groups reaching comparable levels of phosphocreatine concentration. Furthermore, in these patients we found that the rate of phosphocreatine recovery is not influenced by the amount of phosphocreatine consumed during exercise. These outcomes provide experimental evidence that: i) the intracellular acidification occurring in exercising skeletal muscle is a protective factor for the energy consumption; and ii) the influence of pH on the phosphocreatine recovery rate is at least in part related to the kinetic mechanisms of mitochondrial creatine kinase enzyme.

Clinical and neuroimaging evidence of interictal cerebellar dysfunction in FHM2.

We used multimodal magnetic resonance (MR) techniques [brain diffusion-weighted magnetic resonance imaging, diffusion-weighted imaging (DWI), proton MR spectroscopy (MRS), (1)H-MRS; and skeletal muscle phosphorous MRS, (31)P-MRS] to investigate interictal brain microstructural changes and tissue energy metabolism in four women with genetically determined familial hemiplegic migraine type 2 (FHM2), belonging to two unrelated families, compared with 10 healthy women. Brain DWI revealed a significant increase of the apparent diffusion coefficient median values in the vermis and cerebellar hemispheres of FHM2 patients, preceding in two subjects the onset of interictal cerebellar deficits. (31)P-MRS revealed defective energy metabolism in skeletal muscle of FHM2 patients, while brain (1)H-MRS showed a mild pathological increase in lactate in the lateral ventricles of one patient and a mild reduction of cortical N-acetyl-aspartate to creatine ratio in another one. Our MRS results showed that a multisystem energy metabolism defect in FHM2 is associated with microstructural cerebellar changes detected by DWI, even before the onset of cerebellar symptoms.

Pitfalls and advantages of different strategies for the absolute quantification of N-acetyl aspartate, creatine and choline in white and grey matter by 1H-MRS.

This study extensively investigates different strategies for the absolute quantitation of N-acetyl aspartate, creatine and choline in white and grey matter by (1)H-MRS at 1.5 T. The main focus of this study was to reliably estimate metabolite concentrations while reducing the scan time, which remains as one of the main problems in clinical MRS. Absolute quantitation was based on the water-unsuppressed concentration as the internal standard. We compared strategies based on various experimental protocols and post-processing strategies. Data were obtained from 30 control subjects using a PRESS sequence at several TE to estimate the transverse relaxation time, T(2), of the metabolites. Quantitation was performed with the algorithm QUEST using two different metabolite signal basis sets: a whole-metabolite basis set (WhoM) and a basis set in which the singlet signals were split from the coupled signals (MSM). The basis sets were simulated in vivo for each TE used. Metabolites' T(2)s were then determined by fitting the estimated signal amplitudes of the metabolites obtained at different TEs. Then the absolute concentrations (mM) of the metabolites were assessed for each subject using the estimated signal amplitudes and either the mean estimated relaxation times of all subjects (mean protocol, MP) or the T(2) estimated from the spectra derived from the same subject (individual protocol, IP). Results showed that MP represents a less time-consuming alternative to IP in the quantitation of brain metabolites by (1)H-MRS in both grey and white matter, with a comparable accuracy when performed by MSM. It was also shown that the acquisition time might be further reduced by using a variant of MP, although with reduced accuracy. In this variant, only one water-suppressed and one water-unsuppressed spectra were acquired, drastically reducing the duration of the entire MRS examination. However, statistical analysis highlights the reduced accuracy of MP when performed using WhoM, particularly at longer echo times.

DNA synthesis and interaction between controlled feeding schedules and partial hepatectomy in rats.

The rate of DNA synthesis has been measured during liver regeneration in rats adapted to a controlled feeding schedule. The results show two different phenomena in the regulation of DNA synthesis. The first is the appearance of a peak of DNA synthesis following the operation itself and independent of the time of the day; the second one is the presence of constant diurnal variations in the rate of DNA synthesis in response to the partial hepatectomy and following the stimulus or stimuli of the controlled feeding schedule.

The 13042G --> A/ND5 mutation in mtDNA is pathogenic and can be associated also with a prevalent ocular phenotype.

BACKGROUND: Overlapping phenotypes including LHON, MELAS, and Leigh syndrome have recently been associated with numerous mtDNA point mutations in the ND5 gene of complex I, now considered a mutational hot spot. OBJECTIVE: To identify the mtDNA defect in a family with a prevalent ocular phenotype, including LHON-like optic neuropathy, retinopathy, and cataract, but characterised also by strokes, early deaths, and miscarriages on the maternal line. RESULTS: Sequencing of the entire mitochondrial genome from the proband's muscle DNA identified the heteroplasmic 13042G-->A transition, which was previously described only once in a patient with a different mitochondrial disease. This mutation fulfils the major pathogenic criteria, inducing an amino acid change (A236T) at an invariant position in a highly conserved domain of the ND5 gene. Phosphorus magnetic resonance spectroscopy in the proband disclosed an in vivo brain and skeletal muscle energy metabolism deficit. CONCLUSIONS: These findings conclusively establish the pathogenic role of the 13042G-->A mutation and underscore its variable clinical expression.

The mono-exponential pattern of phosphocreatine recovery after muscle exercise is a particular case of a more complex behaviour.

A mathematical model is proposed showing that the mono-exponential recovery of phosphocreatine (PCr) after exercise is an approximation of a more complex pattern, which is identified by a second-order differential equation. The model predicts the possibility of three different patterns of PCr recovery: bi-exponential, oscillatory damped, and critically damped; the mono-exponential pattern being a particular case of the functions which are solutions of the differential equation. The model was tested on a sample of recovery data from 50 volunteers, checking whether the recovery patterns predicted by the model lead to a significant improvement of fit (IF) compared with the mono-exponential pattern. Results show that the IF is linked to pH. Bi-exponential solutions showed an IF in the pH range 6.65-6.85, and the oscillatory solutions at pH>6.9. Critically damped solutions displayed a poor IF. Oscillation frequencies found in the oscillatory recoveries increase at increasing pH. These results show that pH has a pivotal role on the pattern of PCr recovery and implications on the regulation of oxidative phosphorylation are discussed.

Failure of muscle energy metabolism in a patient with adenylosuccinate lyase deficiency. An in vivo study by phosphorus NMR spectroscopy.

31P-nuclear magnetic resonance spectroscopy was used to investigate in vivo the energy metabolism of the calf muscle in a 10-year-old patient with adenylosuccinate lyase deficiency and severe psychomotor retardation. The patient showed a markedly reduced PCr/P(i) molar ratio, known to well represent the cytosolic phosphorylation potential, due to low PCr and high P(i) content in resting muscle. Moreover, intracellular ATP concentration was significantly lower than in the control group both at rest and at the end of post-exercise recovery. The rate of patient's PCr recovery after an exercise in ischaemic conditions was also out of the reference range, suggesting a reduced ability of mitochondria to respond to metabolic needs.

Functional modification of liver form-B RNA polymerase activity by a protein fraction from rats accustomed to controlled feeding schedules.

A protein fraction extracted from the liver of rats accustomed to eating during the first 8 h of a daily 12 h dark period is able to increase homologous form-B RNA polymerase activity. When the protein factor is added to RNA polymerase extracted at the end of the daily fasted period (0.9:OOh) the sensitivity of enzyme to ionic strength is modified, and resembles that of the enzyme extracted during the eating period (15:00 h) either using liver deproteinized DNA or chromatin as template. The chromatin-bound RNA polymerase activity is also modified in the presence of the factor, showing a modification of enzyme sensitivity to ionic strength similar to the one induced by food intake.

1,25-Dihydroxycholecalciferol receptor regulation in hormonally induced differentiation of mouse mammary gland in culture.

Mammary explants from pregnant mouse cultured in the presence of insulin, cortisol, and PRL express their differentiated function by producing milk proteins such as casein. Sucrose density gradient analysis of high salt extract of cultured explants showed the presence of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3 specific binding activity sedimenting at 3.3 S. Specific 1,25-(OH)2D3 binding activity in mammary explants varied as a function of the hormonal milieu in culture. The highest activity was found in explants cultured in the presence of insulin, cortisol, and PRL, whereas only a marginal activity was present in explants cultured with insulin. The comparison of 1,25-(OH)2D3 binding activity in explants cultured with insulin and with the three hormone combination by Scatchard plot analysis indicated that the synergistic actions of insulin, cortisol, and PRL increased the number of 1,25-(OH)2D3 specific binding proteins as much as 6-fold; however, the affinity constant of the binding protein was relatively constant in the two systems. Time course studies showed that the increase in 1,25-(OH)2D3 binding activity was detectable as early as 12 h after the addition of cortisol and PRL to insulin-primed explants. The increase was inhibited by the presence of actinomycin D and cycloheximide, suggesting that the hormonal stimulation of 1,25-(OH)2D3 binding activity involves both transcriptional and translational process. These results indicate that insulin, cortisol, and PRL stimulate the specific 1,25-(OH)2D3 binding activity during the induction of mammary functional differentiation when milk protein synthesis takes place.

Low brain intracellular free magnesium in mitochondrial cytopathies.

The authors studied, by in vivo phosphorus magnetic resonance spectroscopy (31P-MRS), the occipital lobes of 19 patients with mitochondrial cytopathies to clarify the functional relation between energy metabolism and concentration of cytosolic free magnesium. All patients displayed defective mitochondrial respiration with low phosphocreatine concentration [PCr] and high inorganic phosphate concentration [Pi] and [ADP]. Cytosolic free [Mg2+] and the readily available free energy (defined as the actual free energy released by the exoergonic reaction of ATP hydrolysis, i.e., deltaG(ATPhyd)) were abnormally low in all patients. Nine patients were treated with coenzyme Q10 (CoQ), which improved the efficiency of the respiratory chain, as shown by an increased [PCr], decreased [Pi] and [ADP], and increased availability of free energy (more negative value of deltaG(ATPhyd)). Treatment with CoQ also increased cytosolic free [Mg2+] in all treated patients. The authors findings demonstrate low brain free [Mg2+] in our patients and indicate that it resulted from failure of the respiratory chain. Free Mg2+ contributes to the absolute value of deltaG(ATPhyd). The results also are consistent with the view that cytosolic [Mg2+] is regulated in the intact brain cell to equilibrate, at least in part, any changes in rapidly available free energy.

Defective brain energy metabolism shown by in vivo 31P MR spectroscopy in 28 patients with mitochondrial cytopathies.

We studied brain energy metabolism by phosphorus magnetic resonance spectroscopy (31P MRS) in 28 patients with mitochondrial cytopathies, and 20 normal control subjects. Fourteen patients had myopathy alone, six had only mild brain symptoms, and eight showed different degrees of brain involvement. Brain 31P MRS showed a low phosphocreatine content in all patients, accompanied by a high inorganic phosphate in 14 of 28 patients. The average value of the Pi concentration in the patient group was significantly (p = 0.009) different from the control group. The cytosolic pH was normal. From these data were derived a high concentration of ADP (calculated from the creatine kinase equilibrium), a high percent value of V/Vmax for ATP biosynthesis, and a low phosphorylation potential, all features showing a derangement of brain energy metabolism, in all patients with mitochondrial cytopathies. 31P MRS proved to be sensitive enough to disclose a deficit of mitochondrial functionality not only in the affected patients, but also in those without clinically evident brain symptoms.

Isolation of a cDNA clone containing a sequence complementary to the intestinal calcium-binding protein of the chick.

The present work describes the construction of a cDNA library in pBR322 plasmid from an mRNA population enriched for the intestinal calcium-binding protein (CaBP) mRNA of the chick. We report the isolation of one recombinant clone containing a vitamin D-regulated sequence, which is complementary to part of the CaBP mRNA. Northern blot hybridization experiments allowed us to identify a 1900 nucleotide RNA species as the CaBP mRNA.

The use of phosphorus magnetic resonance spectroscopy to study in vivo the effect of coenzyme Q10 treatment in retinitis pigmentosa.

Phosphorus magnetic resonance spectroscopy (31P-MRS) has emerged as a noninvasive reliable tool for in vivo study of human tissue bioenergetics. It detects and quantifies some phosphorylated compounds present in millimolar concentration inside the cell, including ATP, phosphocreatine (PCr) and inorganic phosphate (Pi). By 31P-MRS we studied brain and skeletal muscle energy metabolism of three patients with retinitis pigmentosa before and after oral coenzyme Q10 (CoQ10) (100 mg/day). Before treatment we found a low PCr content in the brains of all patients, accompanied by a high [Pi] and high [ADP]. In two of three patients CoQ10 treatment resulted in a larger brain energy reserve mainly shown by an increased [PCr]. Abnormal muscle mitochondrial function was found only in one patient as shown by a reduced rate of PCr resynthesis after exercise. In this patient CoQ10 treatment resulted in an increased rate of PCr resynthesis. Our observations indicate that CoQ10 can improve mitochondrial functionality in the brain and skeletal muscle of patients with retinitis pigmentosa.

Phosphorus magnetic resonance spectroscopy in cluster headache.

We performed in vivo MR spectroscopy phosphorus (31P-MRS) on the brain and skeletal muscles of 14 patients affected with cluster headache (CH). We examined patients in interictal periods, and also examined nine of them during the cluster period, although not during the attack. Brain 31P-MRS showed reduced phosphocreatine (PCr) levels, an increased ADP concentration (calculated from the creatine kinase equilibrium), a reduced phosphorylation potential, and a high relative rate of ATP biosynthesis (V/Vmax %). The inorganic phosphate (P(i)) content was increased during the cluster period. Ten of 13 patients also showed a slow rate of PCr recovery in muscle after the exercise. 31P-MRS in CH patients showed abnormalities of brain and skeletal muscle energy metabolism comparable with those seen in various types of migraine, thus leading us to suggest a similarity in biochemical pathogenic mechanisms between CH and migraine.

Abnormal brain and muscle energy metabolism shown by 31P magnetic resonance spectroscopy in patients affected by migraine with aura.

We studied brain and muscle energy metabolism by phosphorus 31 magnetic resonance spectroscopy (31P-MRS) in 12 patients affected by migraine with aura (classic migraine) in interictal periods. Brain 31P-MRS disclosed a low phosphocreatine content in all patients, accompanied by high adenosine diphosphate concentration, a high percentage of V/Vmax (adenosine triphosphate), and a low phosphorylation potential--features showing an unstable state of metabolism in classic migraine. Abnormal muscle mitochondrial function, in the absence of clinical signs of muscle impairment, was present in nine of the 12 patients examined.

Leber's hereditary optic neuropathy: genetic, biochemical, and phosphorus magnetic resonance spectroscopy study in an Italian family.

Three siblings of a family affected with Leber's hereditary optic neuropathy (LHON) showed a mitochondrial DNA mutation at position 11778. The lactate response to a standardized effort was increased in only one case. Muscle biopsies and biochemistry of muscle and platelet mitochondrial enzymes were normal. All patients showed an altered energy metabolism during exercise and during recovery after exercise on phosphorus 31-magnetic resonance spectroscopy (31P-MRS) of muscle. Brain 31P-MRS showed a decreased energy reserve (decreased PCr/Pi ratio) in all patients. 31P-MRS noninvasively demonstrated an altered mitochondrial energy metabolism in muscle and, for the first time, in the brains of LHON patients.

Defective brain and muscle energy metabolism shown by in vivo 31P magnetic resonance spectroscopy in nonaffected carriers of 11778 mtDNA mutation.

In vivo phosphorus magnetic resonance spectroscopy (31P-MRS) showed defective brain and muscle energy metabolism in three affected siblings in a family with Leber's hereditary optic neuropathy (LHON) with the 11778 mtDNA mutation. We studied 14 nonaffected members of the same pedigree by 31P-MRS and molecular genetics. Nine of 14 individuals studied had the 11778 mtDNA mutation, with various degrees of heteroplasmy. A decreased brain energy reserve, as shown by low phosphocreatine content and phosphorylation potential and high [ADP], was present in eight of these nine subjects with the 11778 mutation. A low rate of postexercise phosphocreatine recovery in muscle was present in six of the nine mutated individuals. Normal MRS findings in the brain of one and the muscle of three carriers were accompanied by a low percentage of mutated mtDNA. All subjects without mutation had normal brain and muscle MRS. 31P-MRS disclosed defective bioenergetics in the brain or muscle or both of all asymptomatic carriers studied from our pedigree.

Aspects of human bioenergetics as studied in vivo by magnetic resonance spectroscopy.

We outline the relevant capabilities of in vivo phosphorus MR spectroscopy by discussing some aspects of normal human biochemistry as studied by this technique. The transport of inorganic phosphate from cytosol into mitochondria in the human skeletal muscle was studied by exploiting a new experimental protocol. We found that Pi was transported into mitochondria in the absence of ATP biosynthesis and in the presence of a pH gradient. The control of CoQ on the efficiency of oxidative phosphorylation in the skeletal muscle and brain was studied by administering CoQ to patients with mitochondrial cytopathies due to known enzyme defects. Before CoQ we had detected a relevant reduction of mitochondrial functionality in the skeletal muscle as shown by the reduced rate of phosphocreatine recovery from exercise, and in the occipital lobes by reduced [phosphocreatine] and a high [ADP] and [Pi]. After CoQ all brain variables were remarkably improved. Treatment with CoQ also improved the rate of muscle phosphocreatine recovery from exercise. Our in vivo findings support the hypothesis that the concentration of CoQ rather than the rate of its lateral diffusion in the mitochondrial membrane controls the efficiency of oxidative phosphorylation. Other experiments were undertaken to clarify the functional relationship between cytosolic free [Mg2+] and cell bioenergetics in the intact human brain. In the same group of patients with mitochondrial cytopathies we found decreased delta G of ATP hydrolysis and low cytosolic free [Mg2+]. Treatment with CoQ resulted in improved brain bioenergetics and increased free [Mg2+]. These findings strongly indicate that decreased free magnesium was secondary to defective mitochondrial respiration, and support the hypothesis that cytosolic free [Mg2+] is regulated in the intact brain cell to equilibrate, at least in part, any changes in rapidly available free energy.

Muscle phosphoglycerate mutase (PGAM) deficiency in the first Caucasian patient: biochemistry, muscle culture and 31P-MR spectroscopy.

Muscle phosphoglycerate mutase (PGAM) deficiency has been so far identified in only six patients, five of these being African Americans. We report the results of clinical, morphological, biochemical, muscle culture and 31P-MR spectroscopy studies in the first Caucasian patient with muscle PGAM deficiency. A 23-year-old man had a 10-year history of cramps after physical exertion with one episode of pigmenturia. Neurological examination and EMG study were normal. ECG and echocardiography revealed hypertrophy of the interventricular septum and slight dilation of the left chambers of the heart. Muscle biopsy revealed increased glycogen content and some accumulation of mitochondria. Muscle PGAM activity was markedly decreased (6.5% and 9.7% of control value in two different biopsies). Citrate synthase and other mitochondrial respiratory chain enzyme activities were much higher than normal. In contrast to the marked decrease of PGAM activity observed in muscle biopsy, total enzyme activity in the patient's aneural muscle culture was normal, being represented exclusively by BB isoenzyme. The deficiency of PGAM-MM isoenzyme was reproduced in the patient's innervated muscle culture. Muscle 31P-MR spectroscopy showed accumulation of phosphomonoesters only on fast "glycolytic" exercise. On "aerobic" exercise, Vmax, calculated from the work-energy cost transfer function, showed an increase consistent with the morphological and biochemical evidence of mitochondrial proliferation. This might represent a sort of compensatory aerobic effort in an attempt to restore muscle power.

Lipoic (thioctic) acid increases brain energy availability and skeletal muscle performance as shown by in vivo 31P-MRS in a patient with mitochondrial cytopathy.

A woman affected by chronic progressive external ophthalmoplegia and muscle mitochondrial DNA deletion was studied by phosphorus magnetic resonance spectroscopy (31P-MRS) prior to and after 1 and 7 months of treatment with oral lipoic acid. Before treatment a decreased phosphocreatine (PCr) content was found in the occipital lobes, accompanied by normal inorganic phosphate (Pi) level and cytosolic pH. Based on these findings, we found a high cytosolic adenosine diphosphate concentration [ADP] and high relative rate of energy metabolism together with a low phosphorylation potential. Muscle MRS showed an abnormal work-energy cost transfer function and a low rate of PCr recovery during the post-exercise period. All of these findings indicated a deficit of mitochondrial function in both brain and muscle. Treatment with 600 mg lipoic acid daily for 1 month resulted in a 55% increase of brain [PCr], 72% increase of phosphorylation potential, and a decrease of calculated [ADP] and rate of energy metabolism. After 7 months of treatment MRS data and mitochondrial function had improved further. Treatment with lipoate also led to a 64% increase in the initial slope of the work-energy cost transfer function in the working calf muscle and worsened the rate of PCr resynthesis during recovery. The patient reported subjective improvement of general conditions and muscle performance after therapy. Our results indicate that treatment with lipoate caused a relevant increase in levels of energy available in brain and skeletal muscle during exercise.