MERRF-like phenotype associated with a rare mitochondrial trnaile mutation (m.4284 G>A).

Nearly all patients affected by myoclonic epilepsy with ragged-red fibres (MERRF) harbour a mutation in the mitochondrial transfer RNALys gene. We report on an 8-year-old girl with clinical and diagnostic features of MERRF. After excluding one of the common mutations associated with MERRF, a complete sequence analysis of the mitochondrial genome revealed an m.4284 G>A mutation in the mitochondrial transfer RNAIle gene. This mutation has only once been described in a family with variable clinical symptoms, but has not yet been linked to MERRF. This case extends the mutational spectrum associated with the MERRF phenotype, and demonstrates the importance of performing a comprehensive mutational analysis in patients with suspected mitochondrial disease when common mutations have been ruled out.

Leigh disease with brainstem involvement in complex I deficiency due to assembly factor NDUFAF2 defect.

Mitochondrial NADH: ubiquinone oxidoreductase (complex I) deficiency accounts for most defects in mitochondrial oxidative phosphorylation. Pathogenic mutations have been described in all 7 mitochondrial and 12 of the 38 nuclear encoded subunits as well as in assembly factors by interfering with the building of the mature enzyme complex within the inner mitochondrial membrane. We now describe a male patient with a novel homozygous stop mutation in the NDUFAF2 gene. The boy presented with severe apnoea and nystagmus. MRI showed brainstem lesions without involvement of basal ganglia and thalamus, plasma lactate was normal or close to normal. He died after a fulminate course within 2 months after the first crisis. Neuropathology verified Leigh disease. We give a synopsis with other reported patients. Within the clinical spectrum of Leigh disease, patients with mutations in NDUFAF2 present with a distinct clinical pattern with predominantly brainstem involvement on MRI. The diagnosis should not be missed in spite of the normal lactate and lack of thalamus and basal ganglia changes on brain MRI.

Evaluation of saliva as a specimen for monitoring zidovudine therapy in HIV-infected patients.

To facilitate studies of the pharmacokinetic properties of zidovudine, the relationship between plasma and salivary concentrations of the drug was studied, after oral dosage, in 10 HIV-infected patients. Zidovudine concentrations were determined in plasma, unstimulated mixed saliva and citric-acid-stimulated mixed saliva over a period of 3 1/2 hours by high-performance liquid chromatography. Correlation coefficients were r = 0.97 (P less than 0.0001) for stimulated saliva compared with plasma and r = 0.89 (P less than 0.0001) for unstimulated saliva, with average values in unstimulated saliva being 113.8 +/- 44.6% in plasma and 67.8 +/- 25.4% in stimulated saliva. Stimulated saliva values found to be 70% of the total reflected the concentration of the unbound drug in plasma. Except for a shorter half-life time (t1/2) in saliva, pharmacokinetic parameters showed a good correlation in the three types of specimen. These findings and the convenience of sample collection suggest that citric-acid-stimulated saliva might be an appropriate specimen for monitoring zidovudine therapy.

Liposomal doxorubicin in the treatment of advanced AIDS-related Kaposi sarcoma.

Neither single-agent therapy nor any combination treatment has been satisfactory enough to be regarded as standard in systemic advanced Kaposi sarcoma. In an attempt to achieve high efficacy in combination with low toxicity, we used a new liposomal formulation of doxorubicin. Pharmacologic data had established a long plasma half-life, an increased accumulation in tumor tissue, and a decrease in uptake by tissues such as liver, spleen, and bone marrow. In a phase I/II open-label, dose-escalating trial 40 male AIDS patients with advanced Kaposi sarcoma were enrolled to receive intravenous "stealth" liposomal doxorubicin biweekly at doses of 10 mg/m2 (n = 10), 20 mg/m2 (n = 27), and 40 mg/m2 (n = 3). The median CD4 count at baseline was 25/microL. After six cycles (12 weeks), 39 patients were evaluable. Three patients (7.5%) showed a complete response, which was histologically confirmed. A partial response was documented in 33 patients (85%). Stable disease was observed in three patients (7.5%). During a median treatment duration of 25 weeks, four patients developed stomatitis (10%), and four patients (10%) experienced alopecia. The most frequent hematologic toxicity was neutropenia. Grade 4 neutropenia was seen in 42.5%, and grade 3 toxicity was seen in 30%. Toxicity was dose-dependent and more frequent in the 40 mg/m2 stratum. During a median observation period of 25 weeks, opportunistic infections occurred in 57.5% of the patient population. We conclude that liposomal doxorubicin at dose levels of 10 and 20 mg/m2 is safe and effective for treatment of advanced Kaposi sarcoma in AIDS. A controlled trial comparing liposomal doxorubicin to conventional combination therapy is underway.

Energy metabolism in graded perinatal asphyxia of the rat.

Although information on energy metabolism during hypoxemic-ischemic states is abundant, data on perinatal asphyxia (PA) are limited. As results from hypoxia-ischemia cannot be directly extrapolated to PA, a clinical entity characterized by acidosis, hypoxemia and hypercapnia, we decided to use a rat model of graded PA during delivery. Cesarean section was performed at the 21st day of gestation and the pups, still in the uterus horns, were asphyxiated from 0 to 20 minutes. In this model survival decreases with the length of asphyxia. Early changes of energy-rich phosphates in brain, heart and kidney were determined by HPLC. ATP and phosphocreatine gradually decreased with the length of asphyxia, with highest ATP depletion rate occurring in the kidney. ATP: brain 1.39 +/- 0.71 (0 min) to 0.06 microM/g wwt (20 min); heart 4.73 +/- 0.34 (0 min) to 1.08 +/- 0.47 (20 min); kidney 1.62 +/- 0.11 (0 min) to 0.02 +/- 0.02 (20 min). Phosphocreatine: brain 1.65 +/- 0.68 (0 min) to 0.51 +/- 0.45 microM/g (20 min); heart 6.98 +/- 0.38 (0 min) to 6.17 +/- 1.07 (20 min); kidney 8.23 +/- 0.86 (0 min) to 3.76 +/- 0.54 (20 min). We present data on energy derangement in a rat model of PA, closely resembling the clinical situation, showing that energy depletion precedes cell damage and death.

Brain fatty acids in perinatal asphyxia.

In hypoxic or ischemic states the release of fatty acids is proposed to have several harmful effects on brain structure and function. We therefore decided to study brain FFA in a simple, clinically related animal model resembling intrauterine perinatal asphyxia (PA). Cerebral blood flow (CBF), brain fatty acids (C14:0, C16:1, C16:0, C18:1, C1 8:0, sigma C), plasma glucose, lactate, beta-hydroxybutyrate (beta-OHB), non-esterified fatty acids (NEFA) and insulin were determined in PA and compared to the normoxic state. Brain C 14:0 FFA were not significantly different from normoxic rats. Brain FFA C 16:0 were comparable between groups but significantly decreased at 20 min of PA. C 18:0 FFA showed a trend to increase with the length of PA reaching significance at 10 min of asphyxia only and were declining at 20 min, however, not significantly. Brain C 16:1 and C 18:1 FFA concentrations were comparable between groups. The parameters cerebral blood flow, glucose and lactate showed a stepwise and significant increase with the length of PA, whereas beta-HOB, NEFA and insulin showed no changes. CBF, glucose and lactate showed a strong association whereas other parameters failed to correlate with each other. Only inconsistent trends of increased brain FFA were found and the association between brain glucose and brain FFA could be ruled out. Although CBF was manifold and significantly elevated in PA, brain FFA pattern suggests that the increase of CBF is obviously not mediated by brain FFA. We conclude that FFA may not be involved in the early phase-pathogenesis of PA.

[Comparative investigation of the efficacy of two nucleocapsid analogs in FIV infected cats]. / Vergleichende Untersuchung über die Wirksamkeit zweier Nukleosidanaloga bei FIV-infizierten Katzen.

Antiviral activity of the acyclic nucleoside phosphonates 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and 9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) against feline immunodeficiency virus (FIV) was investigated in field cats. The study was designed as a placebo-controlled double-blind study enrolling 27 FIV infected cats. Nine cats received PMEA at a dosage of 10 mg/kg body weight, nine cats received FPMPA at a dosage of 25 mg/kg body weight. A variety of parameters were established to evaluate the therapeutic efficacy of the compounds. The improvement was monitored by different aspects: clinical status, index of Karnofsky modified for the cat, laboratory parameters, immunological parameters, surrogate markers, and a virological parameter. Concerning the clinical and immunological parameters cats of both treatment groups disclosed a relevant improvement. Efficacy of antiviral treatment was a little bit higher in the cats treated with PMEA than in the animals injected with FPMPA. However, side effects were also more prominent in the PMEA treated group.

Multiple acyl-CoA-dehydrogenase deficiency (MADD)--a novel mutation of electron-transferring-flavoprotein dehydrogenase ETFDH.

This is the case of a 41 year old man, suffering general weakness and elevated liver enzymes, sensitive to a treatment with riboflavin and coenzyme Q(10). Tandem mass spectroscopy and molecular analysis reveal a multiple acyl-CoA-dehydrogenase deficiency (MADD) with two novel heterozygote missense mutations of the EFTDH gene.

Heart rate variability, hemostatic and acute inflammatory blood parameters in healthy adults after short-term exposure to welding fume.

The present study aimed to investigate, whether short-term experimental exposure to high levels of welding fumes would be capable of exerting acute effects in healthy subjects. Specifically, we assessed cardiovascular function in terms of heart rate variability (HRV) as well as the concentrations of inflammatory mediators and hemostatic proteins in blood as outcome measures. Twenty subjects without a history of airway and cardiovascular diseases were exposed to either control air or welding fume for 1 h on 2 separate days under standardized conditions. The median concentration of the alveolar particle fraction during welding was 3.5 mg/m(3 )(quartiles: 1.4-6.3 mg/m(3); range 1.0-25.3 mg/m(3)). Five hours later a panel of clinical assessments was performed, including HRV measurement and drawing of blood samples. There were no changes in symptom ratings or lung function after welding fume exposure. Exposures did also not differ regarding effects on time- and frequency-domain parameters of HRV. Similarly, blood leukocyte numbers, cell differentials and the blood levels of fibrinogen, C-reactive protein, antithrombin III, factor VIII, von Willebrand factor, ristocetin cofactor, sICAM-1, tumor necrosis factor alpha, interleukin 6, interleukin 8 and epithelial neutrophil activating peptide 78 were not altered by welding fume inhalation. However, there was a significant fall in the level of endothelin-1 (P < 0.01). In conclusion, the data did not indicate effects of clinical significance of a short-term high-level exposure to welding fumes on HRV or a set of blood hemostatic and acute inflammatory parameters in healthy subjects. The small but statistically significant effect on endothelin levels demonstrated that measurable effects could be elicited even in these individuals. Overall, welding fumes are not likely to exert acute cardiovascular effects in healthy individuals.

Determination of amino acid tissue concentrations by microdialysis: method evaluation and relation to plasma values.

Microdialysis is an in vivo technique to monitor tissue concentrations of low molecular weight substances by means of a continuously perfused artificial capillary with a semipermeable membrane placed into the region of interest. The suitability of microdialysis to determine tissue concentrations of amino acids was evaluated in vitro by placing the catheter into Ringer buffer or into a plasma protein (50g/l) solution containing 32 different amino acids (150 micromol/l each). All amino acids tested crossed freely the microdialysis membrane with recoveries close to 100%. Microdialysis fluid was sampled from subcutaneous tissue of five newborns and amino acid content analysed. Total and non protein bound amino acids were determined in the patients plasma by acid precipitation or ultrafiltration, respectively. Mean subcutaneous tissue concentrations were lower as compared to plasma for taurine, serine, alanine, aspartate, glutamate and ornithine and higher for valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and arginine, indicating net uptake or release of amino acids from subcutaneous tissue. Thus, microdialysis offers a convenient and minimal invasive way to study tissue amino acid composition and appears to be a promising analytical tool for the study of amino acid metabolism in vivo.

Glucose monitoring with long-term subcutaneous microdialysis in neonates.

BACKGROUND: Microdialysis is a new approach for continuous monitoring of small molecules in the extracellular space, and hypoglycemia is a common problem in neonatal intensive care. The objective of this study was to evaluate subcutaneous microdialysis for long-term glucose monitoring in neonatal intensive care. We determined the relative recovery of the microdialysis system in vitro and in vivo, the stability of the relative recovery in vivo during long-term microdialysis, and the correlation between blood and dialysate concentrations of glucose and urea. Furthermore, we evaluated the sensitivity and specificy of subcutaneous microdialysis for the diagnosis of hypoglycemia. PATIENT AND METHODS: Thirteen infants (10 neonates) with gestational ages of 30.2 to 45.6 weeks were investigated by microdialysis of subcutaneous adipose tissue and blood sampling. Subcutaneous microdialysis was performed for a median (range) duration of 9 (4-16) days. RESULTS: The application was safe, even in extremely low birth weight infants (<1000 g) with scanty subcutaneous adipose tissue. The mean +/- standard deviation of the relative recovery in vitro was 101 +/- 3% for glucose and 100 +/- 2% for urea. Using urea as the internal standard, the mean relative recovery in vivo was 96.4 +/- 12.7% at the beginning and remained constant up to 16 days. The correlation between microdialysate and blood was significant for glucose (r = 0.88) and urea (r = 0.98). Subcutaneous microdialysis allowed the detection of asymptomatic hypoglycemias. The diagnostic sensitivity of a dialysate glucose

Determination of endothelin-1 immunoreactivity in plasma, cerebrospinal fluid and urine.

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide apparently involved in a number of vascular diseases in man. Nonetheless, its determination in biological samples is difficult, and data on plasma or urine concentrations are controversial. We investigated different sample preparation procedures as well as different radioimmunoassays for their influence on ET-1 measurement. Recovery of ET-1 depended on the extraction procedure, the type and size of the extraction columns and on the biological matrix itself. Incomplete removal of matrix components by the extraction leads to the formation of particulate matter in the evaporated eluate. When dissolved in assay buffer, ET-1 was found to be absorbed to and only partly released from these particulates, so that it was not accessible for measurement in a radioimmunoassay. This was the case for all extraction procedures investigated except for that involving acetic acid. HPLC analysis of spiked samples revealed that ET-1 is in part degraded during extraction, most probably to Meth-sulphoxide ET-1. Dilution curves of synthetic pure ET-1 standards from different suppliers, prepared either in plasma with subsequent extraction or in assay buffer of the respective radioimmunoassay, resulted in considerable differences in the measured values for ET-1-immunoreactivity. Every radioimmunoassay tested had a specific pattern of recognizing different synthetic ET-1 standards. In conclusion, the measurement of ET-1-immunoreactivity is strongly influenced by the experimental conditions of sample preparation as well as by the radioimmunoassay employed.

Simple high-performance liquid chromatographic method for the detection of phenylpropionylglycine in urine as a diagnostic tool in inherited medium-chain acyl-coenzyme A dehydrogenase deficiency.

Deficiency of medium-chain acyl-CoA dehydrogenase is a frequent and treatable metabolic defect, which can be diagnosed by detection of phenylpropionylglycine in urine after an oral load of phenylpropionic acid. We studied the determination of phenylpropionylglycine in urine by isocratic ion-exclusion chromatography on a cation-exchange column using water-sulphuric acid (pH values between 2 and 4) as mobile phase. Phenylpropionylglycine, phenylpropionic acid and hippuric acid exhibited high retention factors with only a slight decline at increasing solvent pH. This resulted in a good separation from interfering substances after direct injection of urine. We hypothesize that pi-pi interactions between the aromatic carbonic acids and the ion-exchange resin are responsible for the strong retention on the stationary phase. We conclude that, even in asymptomatic patients, determination of phenylpropionylglycine in urine after a phenylpropionic acid load by ion-exclusion chromatography is a rapid and reliable diagnostic tool for the detection of medium-chain acyl-CoA dehydrogenase deficiency.

Determination of total homocysteine in human plasma by isocratic high-performance liquid chromatography.

A simple, sensitive and precise isocratic HPLC method for the determination of total homocysteine in human plasma is described. The thiol compounds were liberated from plasma proteins by reduction with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic marker, 7-fluoro-benzo-2-oxa-1,3-diazole-4-sulphonate. The derivatives were separated isocratically within 7 min by reversed-phase HPLC using a Superspher 100 RP-18 column as stationary phase. By using this approach more than 200 samples a day can be assayed for total homocysteine. The method was linear up to 100 mumol/l and proved to be sensitive with a detection limit of 0.1 mumol/l and the lowest limit of reliable quantification of 0.5 mumol/l for homocysteine in buffer. Intra- and inter-assay coefficients of variation were both < 4% at a concentration of 10 mumol/l homocysteine. Similar results were obtained for homocysteine concentrations between 0.5 and 100 mumol/l. The analytical recovery for these concentrations ranged from 94.9 to 117.0%. As compared to other protocols published so far, this modified method is less complicated but equally sensitive and reproducible and allows a rapid determination of total homocysteine and cysteine in human plasma under routine conditions.

Absorption and elimination kinetics of zidovudine in the cerebrospinal fluid in HIV-1-infected patients.

Current knowledge of zidovudine (ZDV) levels in human cerebrospinal fluid (CSF) is limited to single sample determination and extrapolation to time after administration. Longitudinal studies have not been performed. Pharmacokinetic parameters of ZDV in CSF were determined in six HIV-1-infected patients. CSF samples were collected by an intraspinal catheter over a period of 6 hours after a single intravenous (IV) dose of ZDV (2.5 mg/kg). ZDV concentrations were measured by high-performance liquid chromatography (HPLC). ZDV was cleared rapidly from plasma, with a mean terminal elimination half-life (t 1/2) of 75.5 +/- 4.9 minutes. ZDV penetrated slowly into the CSF, reaching maximal concentration (Cmax) 2 hours after the start of the infusion in all patients. ZDV was cleared from the CSF with a mean t 1/2 of 187.6 +/- 69.3 minutes. Mean Cmax in the CSF was 1.3 +/- 1.2 micromol/l (17% of that of plasma), and mean area under the concentration time curve (AUC) was 358 +/- 200 micromol x minutes/l (75% of that of plasma). There was a significant correlation between plasma and CSF for Cmax (r = 0.88, p = .009) and AUC (r = 0.89, p = .014). Calculated trough levels in CSF for a 12-hour dosing interval were 0.090 +/- 0.065 micromol/l and thus about twice the 50% inhibitory concentration (IC50) of susceptible HIV strains. The CSF-plasma ratio of ZDV increased in a nearly linear fashion with time after drug administration. Thus, ZDV has a distinct pharmacokinetic profile in CSF compared with other compartments of the body.

Mitochondrial and cytosolic branched-chain amino acid transaminases from yeast, homologs of the myc oncogene-regulated Eca39 protein.

We have isolated a high copy suppressor of a temperature-sensitive mutation in ATM1, which codes for an ABC transporter of Saccharomyces cerevisiae mitochondria. The suppressor, termed BAT1, encodes a protein of 393 amino acid residues with an NH2-terminal extension that directs Bat1p to the mitochondrial matrix. A highly homologous protein, Bat2p, of 376 amino acid residues was found in the cytosol. Both Bat proteins show striking similarity to the mammalian protein Eca39, which is one of the few known targets of the myc oncogene. Deletion of a single BAT gene did not impair growth of yeast cells. In contrast, deletion of both genes resulted in an auxotrophy for branched-chain amino acids (Ile, Leu, and Val) and in a severe growth reduction on glucose-containing media, even after supply of these amino acids. Mitochondria and cytosol isolated from bat1 and bat2 deletion mutants, respectively, contained largely reduced activities for the conversion of branched-chain 2-ketoacids to their corresponding amino acids. Thus, the Bat proteins represent the first known isoforms of yeast branched-chain amino acid transaminases. The severe growth defect of the double deletion mutant observed even in the presence of branched-chain amino acids suggests that the Bat proteins, in addition to the supply of these amino acids, perform another important function in the cell.

Endothelin-1 is elevated in the cerebrospinal fluid of HIV-infected patients with encephalopathy.

In a cross-sectional, non-randomized, prospective study in an outpatient clinic a possible relationship between the cerebrospinal fluid (CSF) concentrations of the potent vasoconstrictor peptide endothelin-1 (ET-1) and prevalence and degree of HIV-encephalopathy was studied. Forty-eight CSF samples from HIV-infected patients undergoing lumbar punction for diagnostic reasons were investigated for ET-1 concentrations. In 37 patients ET-1 was also measured in plasma. Patients were investigated clinically and staged with respect to HIV encephalopathy. Patients with arterial hypertension, diabetes or acute opportunistic infections were excluded from the study. In the remaining, 18 of the CSF samples were from patients with normal neurological findings (grade 0-0.5), whereas 30 were from patients with HIV encephalopathy (grade 1-3). The mean CSF ET-1 concentration was significantly elevated (P = 0.001) in patients with HIV encephalopathy (1.97 +/- 2.33 pmol/l) as compared to those patients without encephalopathy (0.57 +/- 0.67 pmol/l). Moreover, there was a significant correlation between ET-1 CSF concentrations and the degree of HIV encephalopathy (r = 0.49, P < 0.001). In addition, there was a significant correlation between ET-1 levels in the CSF and the IgG serum to CSF ratio. However, we found no correlation between HIV encephalopathy and neither CSF total protein, IgG, albumin or the serum to CSF ratio of IgG or albumin. In conclusion, we could demonstrate a close relationship between CSF ET-1 concentrations and the degree of HIV encephalopathy. Thus, by virtue of its long-lasting and potent vasoconstrictor activity ET-1 might contribute to the pathogenesis of HIV encephalopathy.