Measurement of surgical stress in anaesthetized children.

BACKGROUND: The surgical stress index (SSI), derived from a combination of heart rate (HR) and photoplethysmographic amplitude (PPGA) time series, is a novel method for continuous monitoring of intraoperative stress and has been validated in adults. The applicability of SSI and its constituents to monitoring children has not been previously evaluated. METHODS: In this controlled trial, 22 anaesthetized patients, aged 4-17 yr, undergoing strabismus surgery were randomized into two groups, Group LL and Group BSS. Patients in Group LL received topical conjunctival anaesthesia with a 1:1 mixture of lidocaine 2% and levobupivacaine 0.75%, and patients in Group BSS received balanced salt solution. RESULTS: Endotracheal intubation (n=22) increased median (range) SSI from 39.2 (22.6-55.6) to 53.6 (35.8-63.3) (P<0.001), decreased PPGA from 5.62 (2.79-9.69) to 5.27 (2.59-7.54)% (P=0.001), and increased the difference of response entropy (RE) and state entropy (SE) of frontal biopotentials (RE-SE) from 3.1 (0.06-9.1) to 5.7 (0.6-9.4) (P=0.01). Conventional haemodynamic variables also increased, median (range) HR from 72.9 (56.7-113.8) to 84.2 (60.4-124.8) beats min(-1) (P<0.001), and systolic non-invasive arterial pressure (S-NIBP) from 87 (78-143) to 103 (79-125) (P=0.007). When 3 min baseline before surgery was compared with 12 min of surgery, median (range) SSI increased from 43.3 (31.2-58.0) to 49.9 (39.3-57.2) (P=0.042) vs from 46.6 (26.8-57.8) to 52.1 (31.7-60.1) (P=0.024) and PPGA decreased from 6.60 (3.10-8.24) to 5.80 (3.03-7.65)% (P<0.001) vs from 5.51 (3.25-9.84) to 5.06 (3.08-8.99)% (P=0.042), in Groups LL and BSS, respectively, but SSI or other indicators did not differ significantly between the groups. CONCLUSIONS: SSI, PPGA, HR, NIBP, RE, and RE-SE detect autonomic responses to nociceptive stimuli in anaesthetized children undergoing strabismus surgery.

Multiple deletions of mitochondrial DNA in several tissues of a patient with severe retarded depression and familial progressive external ophthalmoplegia.

Multiple deletions of mitochondrial DNA (mtDNA) have recently been reported in familial progressive external ophthalmoplegia (PEO), in a case of progressive encephalomyopathy, and in inherited recurrent myoglobinuria. The inheritance of familial PEO has been autosomal dominant, which indicates that a mutation in an unknown nuclear gene results in several mtDNA deletions of different sizes in these patients. We report a patient with autosomal dominant PEO, whose major clinical symptom, however, was severe retarded depression. The morphological analyses of the tissue samples derived from autopsy showed various abnormalities in the mitochondria in all the tissues studied. The activities of the respiratory chain enzymes encoded by mtDNA were remarkably reduced in the skeletal muscle. The mtDNA analyses confirmed that besides myopathy, this patient had a multisystem disorder with widespread distribution of multiple deletions of mtDNA. The highest percentage of mutated mtDNA was found in the brain, skeletal muscle and the heart, the relative quantity of mutated mtDNA correlating to the severity of the clinical symptoms.

The pattern of retinal ganglion cell dysfunction in Leber hereditary optic neuropathy.

Leber inherited optic neuropathy (LHON) is characterized by subacute bilateral loss of central vision due to dysfunction and loss of retinal ganglion cells (RGCs). Comprehensive visual electrophysiological investigations (including pattern reversal visual evoked potentials, pattern electroretinography and the photopic negative response) performed on 13 patients with acute and chronic LHON indicate early impairment of RGC cell body function and severe axonal dysfunction. Temporal, spatial and chromatic psychophysical tests performed on 7 patients with acute LHON and 4 patients with chronic LHON suggest severe involvement or loss of the midget, parasol and bistratified RGCs associated with all three principal visual pathways.

The plasma membrane potential of human neutrophils. Role of ion channels and the sodium/potassium pump.

Calcium-depleted human neutrophils are depolarised when suspended in calcium-free media containing sodium ions, and are repolarised by extracellular replenishment of Ca2+. The depolarisation is due to a high inward sodium current, which is blocked by calcium and by several other divalent cations, but not by barium. Addition of calcium results in a rise in the cytosolic concentration from approx. 20 nM to the resting level of approx. 130 nM. Calcium influx is strongly accelerated by a voltage-gated calcium channel. This channel might be responsible for the depolarising Na+ current in the absence of divalent cations. In the polarised state the neutrophil membrane has a high intrinsic permeability to K+, which may be low or absent in the depolarised state. Generation of membrane potential from the depolarised state is mainly due to the electrogenic sodium/potassium pump. However, the resting potential of about -75 mV is maintained primarily by the K+ conductance, and only to a small extent by the sodium/potassium pump.

Disorders associated with multiple deletions of mitochondrial DNA.

Multiple deletions of mitochondrial DNA (mtDNA) have recently been described in a number of patients with neurological disorders. Most cases have been clinically characterized by autosomal dominant inheritance, adult onset, and a slowly progressive course with external ophthalmoplegia and muscle weakness. Some patients have had evidence of central or peripheral nervous system involvement or episodes of myoglobinuria. Muscle biopsy findings include ragged-red fibres (RRF), muscle fibres with absent COX-activity and abundant abnormal mitochondria with paracrystalline inclusions. Biochemically, a generalized reduction in the activities of mtDNA-encoded enzymes is observed in skeletal muscle. Southern blotting or PCR analysis reveal multiple populations of deleted mtDNA. The deletions occur at multiple sites between the replication initiation sites, involving a large portion of mtDNA, and most deletions seem to be flanked by direct sequence repeats, shown to be "hot spots" in the case of single large deletions. Apparently, a defect in a nuclear gene results in multiple deletions of mtDNA. Both clinical, genetic and molecular genetic observations indicate heterogeneity of this new disease category, apparently based on a disturbance in the "cross-talk" between the nuclear and the mitochondrial genomes.

Studies on the proton-translocating NADH:ubiquinone oxidoreductases of mitochondria and Escherichia coli using the inhibitor 1,10-phenanthroline.

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is uncompetitively inhibited by 1,10-phenanthroline (OP). EPR spectroscopy of submitochondrial particles indicates that OP, similarly to rotenone, inhibits electron transfer between the Fe-S clusters of complex I and the ubiquinone pool. The proton-translocating NADH dehydrogenase (NDH1) of E. coli is more sensitive to OP than is NDH1 of Paracoccus. EPR spectroscopy of membranous E. coli NDH1 shows that two slow- and one fast-relaxing Fe-S clusters become detectable upon reduction by NADH in the presence of OP. However, none of them resembles the mitochondrial cluster 2.

Electron transfer properties of NADH:ubiquinone reductase in the ND1/3460 and the ND4/11778 mutations of the Leber hereditary optic neuroretinopathy (LHON).

We report the electron transfer properties of the NADH:ubiquinone oxidoreductase complex of the respiratory chain (Complex I) in mitochondria of cells derived from LHON patients with two different mutations in mitochondrial DNA (mtDNA). The mutations occur in the mtDNA genes coding for the ND1 and ND4 subunits of Complex I. The ND1/3460 mutation exhibits 80% reduction in rotenone-sensitive and ubiquinone-dependent electron transfer activity, whereas the proximal NADH dehydrogenase activity of the Complex is unaffected. This is in accordance with the proposal that the ND1 subunit interacts with rotenone and ubiquinone. In contrast, the ND4/11778 mutation had no effect on electron transfer activity of the Complex in inner mitochondrial membrane preparations; also Km for NADH and NADH dehydrogenase activity were unaffected. However, in isolated mitochondria with the ND4 mutation, the rate of oxidation of NAD-linked substrates, but not of succinate, was significantly decreased. This suggests that the ND4 subunit might be involved in specific aggregation of NADH-dependent dehydrogenases and Complex I, which may result in fast ('solid state') electron transfer from the former to the latter.

Mutations in subunit 6 of the F1F0-ATP synthase cause two entirely different diseases.

A lowered efficiency of oxidative phosphorylation was recently found in a Leber hereditary optic neuropathy (LHON) proband carrying a mutation in the mtDNA gene for subunit 6 of the membrane-bound F0 segment of the F1F0-ATP synthase [9]. This phenotype was transferred to cytoplasmic hybrid cells together with the mutation, proving its functional significance. Increasing the respiratory rate in the mitochondria from this mutant raised the ATP/2e- ratio back to normal values. A different mutation in the same mtDNA gene has been found in patients with the NARP syndrome [10]. Although the ATP/2e- ratio is also decreased in this mutant, in this case an increase in the respiratory rate could not compensate for it. Whilst both mutations affect subunit 6 of the proton-translocating F0 segment, the LHON mutation induces a proton leak whereas the NARP mutation blocks proton translocation. Hence, the latter will have much more destructive metabolic consequences in agreement with the large clinical differences between the two diseases.

Autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA: clinical, biochemical, and molecular genetic features of the 10q-linked disease.

Autosomal dominant progressive external ophthalmoplegia (adPEO) is a mitochondrial disease characterized by accumulation of multiple large deletions of mtDNA in patients' tissues. We previously showed that the disease is genetically heterogeneous by assigning two nuclear loci predisposing to mtDNA deletions: one on chromosome 10q 23.3-24.3 in a Finnish family and one on 3p 14.1-21.2 in three Italian families. To reveal any locus-specific disease features, we report here the clinical, biochemical, and molecular genetic characteristics of the 10q-linked disease in the single family reported to date. All seven patients and four asymptomatic subjects had ragged-red fibers and multiple deletions of mtDNA in their muscle. Ptosis and external ophthalmoplegia were the major clinical findings, and depression or avoidant personality traits were frequently, but not consistently, present in the subjects carrying mutant mtDNA. In six of the subjects with mutant mtDNA, the activities of the respiratory chain complexes I or IV, or both, were below or within the low normal range. Two autopsy studies revealed the characteristic distribution of mutant mtDNA in these patients: highest proportion of mutant mtDNA is found in different parts of the brain, followed by the skeletal and ocular muscle, and the heart.

Pathology of skeletal muscle and impaired respiratory chain function in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency with the G1528C mutation.

Lactic acidosis and mitochondrial abnormalities have been reported in long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency. We studied muscle morphology and the respiratory chain function in ten patients with LCHAD deficiency and the G1528C mutation. In eight cases the light microscopy of muscle specimens showed fatty infiltration and fibre degeneration. The degenerated fibres appeared as ragged red fibres in four cases. Electron microscopy revealed enlarged mitochondria often with swollen appearance in four out of seven patients. The number of mitochondria had also increased. Complex I associated enzyme activities in muscle mitochondria were decreased in five out of seven patients, and in three of them Complex II or II + III associated activities were also affected. We suggest that the reason for respiratory chain dysfunction and structural changes of mitochondria is the accumulation of toxic intermediates of fatty acid beta-oxidation in mitochondria. Because these changes may confound the differential diagnostics between LCHAD deficiency and respiratory chain defects, awareness of their frequency is important.

ATP, phosphocreatine and lactate in exercising muscle in mitochondrial disease and McArdle's disease.

We studied exercise-induced changes in the adenosine triphosphate (ATP), phosphocreatine (PCr), and lactate levels in the skeletal muscle of mitochondrial patients and patients with McArdle's disease. Needle muscle biopsy specimens for biochemical measurement were obtained before and immediately after maximal short-term bicycle exercise test from 12 patients suffering from autosomal dominant and recessive forms of progressive external ophthalmoplegia and multiple deletions of mitochondrial DNA (adPEO, arPEO, respectively), five patients with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) 3243 A-->G point mutation, and four patients with McArdle's disease. Muscle ATP and PCr levels at rest or after exercise did not differ significantly from those of the controls in any patient group. In patients with mitochondrial disease, muscle lactate tended to be lower at rest and increase more during exercise than in controls, the most remarkable rise being measured in patients with adPEO with generalized muscle symptoms and in patients with MELAS point mutation. In McArdle patients, the muscle lactate level decreased during exercise. No correlation was found between the muscle ATP and PCr levels and the respiratory chain enzyme activity.

Palmitate oxidation in muscle mitochondria of patients with the juvenile form of neuronal ceroid-lipofuscinosis.

The finding that the intracellular storage material in juvenile neuronal ceroid lipofuscinosis (JNCL) consists of the subunit c of ATP synthase prompted us to study energy conservation in JNCL patients. The activities of respiratory chain enzymes in isolated muscle mitochondria from 8 JNCL cases were normal, but oxidation of palmitate was reduced in 6 patients. The degree of reduction was related to the age of the patients. None of the patients had clinical symptoms or laboratory findings of impaired energy conservation, which suggest that the reduced palmitate oxidation was not associated with a major defect in fatty acid oxidation.

Neuroradiologic findings in children with mitochondrial disorders.

PURPOSE: We report the neuroradiologic findings in 25 children with various mitochondrial diseases. METHODS: Twenty-two children with a mitochondrial disorder had MR imaging of the brain and three children had CT studies. In all cases, the diagnosis was based on examination of muscle morphology, analysis of oxygen consumption and respiratory chain enzyme activity in isolated muscle mitochondria, and analysis of rearrangements of the mitochondrial DNA. RESULTS: Fifteen patients were found to have the classical syndromes of mitochondrial diseases. Four children had Kearns-Sayre syndrome, but only one had the typical neuroradiologic findings of basal ganglia and brain stem lesions, T2 hyperintensity of the cerebral white matter, and cerebellar atrophy; the others had nonspecific or normal findings. Eight patients had Leigh syndrome, and all showed changes in the putamina. Involvement of the caudate nuclei, globus pallidi, thalami, and brain stem was common, and diffuse supratentorial white matter T2 hyperintensity was seen in two of these patients. Three patients had mitochondrial encephalopathy with lactic acidosis and strokelike episodes (MELAS), with infarctlike lesions that did not correspond to the vascular territories. Ten children with complex I or IV deficiencies and abnormal muscle morphology had nonspecific imaging findings, such as atrophy and abnormal or delayed myelination. One patient with combined complex I and IV deficiency had extensive white matter changes. None of the patients with clinical encephalopathy had normal findings. CONCLUSION: MR imaging is helpful in the diagnosis of the classical mitochondrial diseases; however, nonspecific findings are common.

Diagnosis of fatal infantile defects of the mitochondrial respiratory chain: age dependence and postmortem analysis of enzyme activities.

We studied two diagnostic aspects of fatal infantile defects of the mitochondrial respiratory chain: the age dependence of muscle mitochondrial enzyme activities and the reliability of diagnosis from autopsy samples. In morphologically normal quadriceps muscle samples of 46 children between the ages of 3 days and 15 years, activities of complex I plus III (NADH:cytochrome c oxidoreductase) and complex II plus III (succinate:cytochrome c oxidoreductase) increased 2-fold during the first three years of life, while that of complex II (succinate dehydrogenase), complex IV (cytochrome c oxidase), and citrate synthase did not show significant correlation with age. We suggest that these changes are related to age and stress the importance of strictly age-matched controls when diagnosing a mitochondrial disease of early childhood. The value of autopsy samples in diagnostic studies was evaluated by comparing mitochondrial enzyme activities in quadriceps muscle from autopsies and from surgical biopsies. In quadriceps muscle mitochondria, all the enzyme activities studied remained stable for at least 3 h after death. Using age-matched controls and autopsy samples, we diagnosed a respiratory chain enzyme deficiency in two infants, and the defects were confirmed in cultured skin fibroblasts.

Diphenyleneiodonium inhibits reduction of iron-sulfur clusters in the mitochondrial NADH-ubiquinone oxidoreductase (Complex I).

Diphenyleneiodonium (DPI) inhibits the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) on the substrate side of the Fe-S clusters. In the inhibited NADH-supplemented state all of the Fe-S clusters are oxidized, whereas the reduced minus oxidized difference spectrum of the protein-bound FMN can be visualized. It is characterized by troughs at 370 and 450 nm and a small increase of absorbance in the 500-700-nm region. DPI probably reacts irreversibly with FMN, because oxidation of FMN is blocked even after its extraction from the enzyme. Inhibition requires preincubation of enzyme in the presence of NADH and DPI. The lower the NADH/NAD+ ratio or the pH, or the higher the NAD+/DPI ratio, the more DPI is required for inhibition. NAD+ and DPI apparently compete for a common site. Both ubiquinone and dichlorophenolindophenol reductase activities are fully blocked by DPI, whereas the ferricyanide reductase activity is inhibited by 75%. Similar results were found with Complex I and two rotenone-insensitive preparations, subcomplex I lambda and the flavoprotein fraction. DPI also inhibits NADH oxidation by bacterial NADH-ubiquinone oxidoreductase-1 (NDH-1) in membranes of Paracoccus denitrificans and Escherichia coli.

Severe hyperprostaglandin E syndrome with hyperthyroidism--studies of pathogenetic mechanisms.

Hyperprostaglandin E syndrome is a rare disease usually presenting with renal symptoms such as polyuria, polyhydramnios, hypercalciuria, hypokalaemia, and recurrent episodes of extreme fever, diarrhoea, and convulsions. We report a severe variant of this syndrome with obvious pain and prostaglandin E2 (PGE2)-stimulated hyperthyroidism, an association not previously described. Urinary excretion of PGE2 and its metabolite 7 alpha-hydroxy-5,11-diketotetranorprosta-1,16-dioic acid were markedly increased above normal levels (to 53.3 and 1895 ng/h per 1.73 m2, respectively). We studied oxidative capacity of peroxisomes and mitochondria, the sites where PGE2 oxidation takes place. A generalized mitochondrial disease could be ruled out and no deficiency was found in liver peroxisomal oxidases. The basic pathology of hyperprostaglandin E syndrome remains unsolved.

Congenital hypoplastic anemia, diabetes, and severe renal tubular dysfunction associated with a mitochondrial DNA deletion.

Mitochondrial DNA (mtDNA) deletion is associated with a variety of clinical entities. In addition to progressive external ophthalmoplegia and Kearns-Sayre syndrome, mtDNA deletions have been demonstrated in Pearson's syndrome. We report an mtDNA deletion in an infant with a variant of Pearson's syndrome. Not only does she have congenital anemia, severe tubulopathy, and exocrine pancreas insufficiency, but she also has diabetes and cerebral atrophy. However, there are no signs of gut or liver involvement. Bone marrow improved while new tissues were involved, thus showing variability in progression of the disease. Decreased respiratory chain enzyme activities were demonstrated in muscle, and an mtDNA deletion was demonstrated in muscle, kidney, leukocytes, and fibroblasts.