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.

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.

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.

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.

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 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.

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.

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.

Use of saliva specimens for monitoring indinavir therapy in human immunodeficiency virus-infected patients.

Indinavir concentrations were determined in plasma and saliva over a random period of 4 h. On average, levels in saliva were 70% +/- 38% of the corresponding levels in plasma. These findings suggest that saliva might serve as an appropriate specimen for monitoring of plasma indinavir levels in patients treated with indinavir.

The biochemical metabolite screen in the Munich ENU Mouse Mutagenesis Project: determination of amino acids and acylcarnitines by tandem mass spectrometry.

BACKGROUND: Gene mutations often result in altered protein expression and, in turn, lead to changes in metabolite levels in one or more distinct biochemical pathways. Traditional analytical methods for metabolite determination are usually time consuming, expensive, and, thus, not suitable for high throughput analysis. However, recent developments in electrospray-tandem-mass-spectrometry allow comprehensive metabolite scanning from very small amounts of blood with high speed, cost effectiveness, and accuracy. METHODS: A blood spot from a filter paper equivalent to 3 microl of blood was punched out and transferred to a 96-well microtiter plate. After addition of a set of 14 stable isotope-labeled internal standards, amino acids and acylcarnitines were extracted with methanol. The dried residue was derivatized with butanolic hydrochloric acid and subjected to MSMS analysis. RESULTS: Acyl-carnitines were all determined by a precursor ion scan of 85 Da. Neutral loss scanning of 102 Da was suitable for the quantitation of threonine, serine, proline, histidine, alanine, aspartic acid, glutamic acid, methionine, tyrosine, phenylalanine, isoleucine/leucine and valine. Glycine was detected by a loss of a 56-Da fragment, whereas a 119-Da loss was suitable for the measurement of citrulline, ornithine, arginine, and lysine. Specific problems encountered: owing to their identical molecular weight, isoleucine and leucine could not be quantitated separately, and, owing to their instability, glutamine and asparagine were found to be decarboxylated to their respective acids. Determination was linear over the concentration range tested (20 to 1000 micromol/L), and intraassay and interassay coefficients of variation were in the range of 10-15%. CONCLUSION: ESI-MSMS proved to be a highly sensitive, linear, and sufficiently precise method for the quantitative determination of amino acids and acylcarnitines in mouse blood, allowing large-scale screening applications when speed and cost effectiveness are mandatory.

Megalin knockout mice as an animal model of low molecular weight proteinuria.

Megalin is an endocytic receptor expressed on the luminal surface of the renal proximal tubules. The receptor is believed to play an important role in the tubular uptake of macromolecules filtered through the glomerulus. To elucidate the role of megalin in vivo and to identify its endogenous ligands, we analyzed the proximal tubular function in mice genetically deficient for the receptor. We demonstrate that megalin-deficient mice exhibit a tubular resorption deficiency and excrete low molecular weight plasma proteins in the urine (low molecular weight proteinuria). Proteins excreted include small plasma proteins that carry lipophilic compounds including vitamin D-binding protein, retinol-binding protein, alpha(1)-microglobulin and odorant-binding protein. Megalin binds these proteins and mediates their cellular uptake. Urinary loss of carrier proteins in megalin-deficient mice results in concomitant loss of lipophilic vitamins bound to the carriers. Similar to megalin knockout mice, patients with low molecular weight proteinuria as in Fanconi syndrome are also shown to excrete vitamin/carrier complexes. Thus, these results identify a crucial role of the proximal tubule in retrieval of filtered vitamin/carrier complexes and the central role played by megalin in this process.

Lack of absorption of didanosine after rectal administration in human immunodeficiency virus-infected patients.

The feasibility of rectal administration of didanosine (DDI) was studied in six human immunodeficiency virus-infected patients. After oral intake of a DDI solution (100 mg/m2 of body surface area) combined with an antacid (Maalox), pharmacokinetic parametric values were in accordance with previously published data; the mean +/- standard deviation for terminal half-life was 59.5 +/- 15.0 min, that for peak concentration was 5.2 +/- 3.9 mumol/liter, and that for the area under the time-concentration curve (AUC) was 494 +/- 412 min.mumol/liter. After rectal administration of a similarly prepared DDI solution (100 mg/m2 of body surface area), plasma DDI levels were below the detection limit (0.1 mumol/liter) at all time points in five of the six patients, and in the remaining patient the AUC after rectal application was only 5% of that after oral administration. We conclude that oral administration of DDI cannot be easily replaced by rectal application.

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.

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.

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.