Inhaled nitric oxide does not enhance lipid peroxidation in patients with acute respiratory distress syndrome.

OBJECTIVE: To investigate whether inhaled nitric oxide (NO) enhances pulmonary lipid peroxidation as indicated by arterial blood levels of malondialdehyde, hexanal, and pentanal in patients with acute respiratory distress syndrome (ARDS). DESIGN: Prospective, nonrandomized, controlled trial. SETTING: Surgical intensive care unit in a university hospital. PATIENTS: Twenty-five patients with ARDS, as defined by the American-European Consensus Conference, and a PaO2/FIO2 < or = 170 mm Hg were enrolled in the study. Four healthy subjects were studied as controls. INTERVENTIONS: On enrollment of the patients in the study, a dose-response test with increasing concentrations of inhaled NO (0, 2, 10, 40, 0 ppm) was performed. Patients who showed an increase of >20% in PaO2 were designated as responders and all others as nonresponders. In responders, this dose-response test was followed by 24 hrs of continuous treatment with inhaled NO at the best NO concentration determined during the dose-response test, whereas nonresponders received standard care. For healthy volunteers, the dose-response test took the form of spontaneous breathing of the same NO concentrations. MEASUREMENTS AND MAIN RESULTS: Eighteen patients (72%) showed an increase of >20% in PaO2 during the dose-response test. This significant improvement in arterial oxygenation in responders led to a significant reduction in FIO2 (responders, 0.73 +/- 0.05 vs. nonresponders, 0.89 +/- 0.05) after 24 hrs of therapy. On enrollment, arterial blood concentrations of malondialdehyde, hexanal, and pentanal were significantly higher than those of healthy volunteers. In addition, arterial concentrations of hexanal and pentanal exceeded mixed venous levels two- to ten-fold. Inhalation of NO did not significantly alter these blood concentrations either during the dose response test or during 24 hrs of therapy. CONCLUSIONS: In patients with ARDS, malondialdehyde, hexanal, and pentanal were significantly elevated, indicating lipid peroxidation. Lipid peroxidation was not further affected by inhalation of NO.

Is n-pentane really an index of lipid peroxidation in humans and animals? A methodological reevaluation.

Volatile hydrocarbons such as ethane and n-pentane are known to originate from peroxidation of polyunsaturated fatty acids in membrane lipids and they are accepted as a sensitive and direct index of lipid peroxidation both in vitro and in vivo. Until now, an appropriate and commonly accepted method for the analysis of volatile hydrocarbons in exhalation air has not been described. We therefore developed a methodology for routine application in humans that is based on cryofocusing in combination with gas chromatography and is adaptable to mass spectrometry. The samples may be stored in stainless steel bombs up to 3 weeks, and sample volumes necessary to analysis are variable and can be adapted to analytical requirements. The interference by water and carbon dioxide, always present in excess, is strongly reduced. Mass spectroscopic analysis of exhalation air in human control subjects demonstrates, however, the presence of isoprene as the major constituent hitherto identified as n-pentane. The commonly used columns fail to separate n-pentane and isoprene. Based upon studies of the diverse methodologies reported in literature, it must be assumed that the reported responses of the gas chromatographic "n-pentane" peak in exhalation air of humans and animals, hitherto identified exclusively by authentic reference gases, are actually responses to isoprene or, at least, a mixture of both n-pentane and isoprene.

Fatal liver failure in 16 children with valproate therapy.

The data of 16 children who died while receiving valproate (VPA) therapy in West Germany were analyzed. Five were normally developed, 5 were receiving VPA-monotherapy, and only 2 patients were aged less than 3 years. The first clinical symptoms of impending hepatotoxicity usually included nausea, vomiting, and apathy; pathologic laboratory tests reflected liver failure. Liver histology revealed microvesicular steatosis, cell necrosis, and bile duct proliferation of varying degree. An abnormal metabolite, 4-ene-VPA, was detected in all examined patients (six of six) and persisted after drug withdrawal. The pathogenesis of fatal liver failure during VPA treatment remains unknown. World-wide, approximately 100 fatalities have been reported in relation to VPA treatment. More than 90% were aged less than 20 years, 95% developed their first symptoms within the first 6 months of treatment, and 16 were treated with VPA alone. Since it is difficult precisely to define a group at risk for fatalities with VPA, careful clinical and laboratory monitoring with a special focus on vomiting and apathy, liver enzymes, and coagulation tests seem mandatory during the first 6 months after introduction of VPA. Taking into account the considerable number of fatalities during VPA treatment, the indication for its use requires careful reevaluation.

[Fatal hepatic failure in a normally developed 5-year-old boy caused by VPA monotherapy]. / Tödliches Leberversagen bei einem altersgemäss entwickelten fünf Jahre alten Jungen unter VPA-Monotherapie.

A five-year-old, normally developed boy who had been healthy except for an absence epilepsy prior to valproate (VPA) treatment died 16 weeks after the introduction of VPA-monotherapy due to liver failure and intractable bleeding disorder. This case emphasizes that the restriction of VPA-therapy to children of more than two years of age, on monotherapy, and without evidence of other diseases or retardation does not exclude fatal complications. Until today world-wide about 100 patients have died during VPA-treatment.

[Hepatotoxicity of valproic acid treatment]. / Hepatotoxizität unter Valproinsäurebehandlung.

Hepatotoxicity of valproic acid (VPA) is reported in a psychomotor retarded boy. A four-month antiepileptic therapy was followed by a combined VPA/phenytoin treatment where-upon, two months later, the patient died in a hepatic coma. In the final state the metabolic pattern of VPA was still found within the normal range with the exception of the two abnormal metabolites 4-en-VPA) (2-propyl-4-pentenoic acid) and 4.4'-dien-VPA (2-(2-propenyl-4-pentenoic acid) detected only in low concentration. The amino acid pattern in plasma was characterized by a decrease or branched chain amino acids by 50% in contrast to the increase of the aromatic amino acids. Methionine, however, was in the normal range. Due to lack of a biochemical parameter indicating a possibly irreversible development of VPA induced hepatotoxicity stopping of VPA therapy should be obligatory if abnormal unsaturated VPA metabolites are detected or if dien-VPA/3, a normal metabolic compound, is increased above the normal range. L-carnitine treatment is recommended as a prophylactic therapy.

Biotin status of epileptics.

Microbiologically determined plasma biotin levels in 404 epileptics under long-term treatment with anticonvulsants were markedly lower than in 112 controls (p less than 0.0005). Patients with partial epilepsy had lower biotin levels and higher average daily intake of AC than those with generalized epilepsy. Epileptics treated with valproate sodium in monotherapy showed considerably higher biotin levels than epileptics with monotherapy of primidone (PRM), carbamazepine (CBZ), phenytoin (PHT) or phenobarbital (PB). The group of epileptics with high average daily dose of anticonvulsants had lower biotin levels than the group with low dose. In three patients with newly recognized epilepsy biotin levels were normal before starting anticonvulsant medication, increased during the first week and fell under the starting level in the following weeks. Four epileptics treated with PHT, PB, PRM or CBZ had an increased urinary excretion of organic acids, as found in patients with a deficiency of biotin-dependent carboxylases. In 37 epileptics undergoing long-term treatment plasma lactate concentrations were determined; they had a higher mean concentration than that found in controls. Our results suggest, that the lowering of biotin in epileptics is caused by intake of anticonvulsants and has a biochemical effect in these patients. It is discussed, whether this could be a factor in the mode of action of anticonvulsants.

Five doubly unsaturated metabolites of valproic acid in urine and plasma of patients on valproic acid therapy.

The urine and plasma of epileptic patients receiving therapeutic doses of valproic acid (2-propyl- pentanoic acid) was found to contain five doubly unsaturated metabolites of valproic acid, which were identified as their trimethylsilyl derivatives by GC/MS. A series of reference substances was synthesized but only two of them were identical with native metabolites: 2(2-propenyl)-4-pentenoic acid (= 4.4'-diene) and E-2-propyl-2.4- pentadienoic acid (E-2.4-diene). The mass-spectra of the five native metabolites are given. Preliminary quantitative data obtained from four groups of patients indicate increased formation of doubly unsaturated metabolites when valproic acid-induced side-effects are present, and in cases of fatal hepatic failure. The 4.4'-diene has hitherto been found only in fatal cases with hepatic injury. Quantitative data are presented as % of the sum of valproic acid plus all its detected metabolites.

Excretion of organic acids associated with biotin deficiency in chronic anticonvulsant therapy.

Urinary organic acids, known to be elevated in children with biotin deficiency, were determined in 7 epileptics under long-term therapy with anticonvulsants and in three controls. Four patients administered phenytoin, primidone, phenobarbital, or carbamazepine, alone or in combination, had reduced plasma biotin levels (less than 250 ng/l) and an elevated excretion of certain organic acids indicating a possibly decreased activity of propionyl CoA carboxylase (3-OH-propionate, methylcitrate) and 3-methylcrotonyl CoA carboxylase (3-methylcrotonate and the glycine conjugate, 3-OH-isovalerate). Two epileptics receiving sodium valproate alone had normal circulating biotin levels and no changes in level of the investigated urinary acids were found. These findings indicate that the reduced biotin levels seen in epileptics receiving other anticonvulsants than sodium valproate lead to an elevated excretion of certain organic acids in urine.

Experimental observations on peritoneal transport in rabbits.

Peritoneal transfer kinetics of substances differing in molecular size and lipophilic properties were studied in anuric adult rabbits with ligated ureters. In conscious animals, the dialysate/plasma concentration ratios of creatinine (Cr) and inulin (In) rose exponentially up to 200 min of dwelling. Peritoneal CIn was 0.24 ml/min/kg, and CCr was 0.10 ml/min/kg of body wt. The transfer rate for dipropyl acetic acid (VPA) was higher than for its more lipophilic analogue dibutyl acetic acid (DBA); the apparent equilibrium of the dialysate/plasma concentration ratio for VPA was 0.3 to 0.6 and 0.15 to 0.20 for DBA. Correspondingly, the peritoneal CVPA was higher (0.08 to 0.16 ml/min/kg) than CDBA (0.04 to 0.05 ml/min/kg); peritoneal clearances were 8% vs. 1.5% of the plasma clearances. The addition of nitroglycerin, dopamine, isoprenalin, fenoterol, and nitroprusside sodium to the dialysate did not increase significantly the peritoneal CCr and CIn during 30-min cycles. In conclusion, the peritoneal transport kinetics of creatinine, inulin, and protein are qualitatively similar to clinical data, but of different magnitude. The efficiency of peritoneal dialysis depends on the lipophilic characteristics of the substance to be transferred. The vasoactive drugs studied seem not to be promising for increasing the efficiency of peritoneal transport.

Abnormal metabolism of valproic acid in fatal hepatic failure.

A 7-year-old boy developed a severe unilateral grand mal seizure at the age of 5 years (phenobarbitone therapy); 1.5 years later valproate (2-propylpentanoic acid, VPA) was added to the therapy. After a seizure-free period of 3 months the patient died from hepatic failure resembling Reye syndrome. Several plasma and urine samples from the final stage before and during peritoneal dialysis were analyzed by GC/MS. The predominant feature was the abnormally increased formation of both 3 mono- and 4 double unsaturated metabolites of VPA amounting in plasma to 58%-71% of the sum of VPA plus all analyzed metabolites (controls maximal 15%) and in urine to 34%-61% (controls maximal 10%). The beta-oxidation pathway of VPA was shown to be suppressed (lack of 3-keto-VPA), whereas metabolites from the omega-oxidation pathway could still be measured (urinary 5-OH-VPA plus 2-propylglutaric acid ca. 1.6%, controls more than 10%). 4-en-VPA (2-propyl-4-pentenoic acid) (5%-21% in plasma) and 4,4'-dien-VPA (2(2-propenyl)-4-pentenoic acid) (4%-7%) have been found as abnormal unsaturated metabolites not detectable in controls. Additional typical findings were the high excretion of adipic acid, suberic acid, and 4-octen-1,8-dicarboxylic acid demonstrating the enhanced capacity of omega-oxidation in fatty acid oxidation.

Determination of glomerular function in advanced renal failure.

In 15 children with advanced chronic renal failure, glomerular filtration rate was determined by different methods. Inulin clearance correlated well with the mean of creatinine and urea clearance, and also with 51-chromium edetic acid (EDTA) clearance measured over 24 hours. The absolute values of creatinine clearance and of 51Cr-EDTA clearance measured up to 8 hours were higher than inulin clearance. In advanced renal failure both the 51Cr-EDTA clearance measured over 24 hours, and the mean of creatinine and urea clearance, provide acceptable estimates of true glomerular filtration rate.

Interlaboratory variability of valproic acid determinations.

19 pooled plasma specimens were sent as unknowns to 13 participating research laboratories. The interlaboratory variability between the results was very high. Only 4 out of 13 laboratories had 6--12% of their results within the 95% confidence limit for each sample. The precision of repeated determinations was fairly good. 6 out of 10 participating laboratories had a coefficient of variation of less then 5%. The reproducibility and the agreement between the different procedures for quantitative analysis of dipropylacetate is similar to that reported for other major antiepileptic drugs.

Determination of deuterium-labeled phenylalanine and tyrosine in human plasma with high pressure liquid chromatography and mass spectrometry.

A method is presented for the recovery of deuterated phenylalanine and tyrosine from human plasma. Phenylthiohydantoine derivatives are formed (Edman reaction) which are separated and isolated by high pressure liquid chromatography. The relative concentration of the deuterated amino acid is determined by mass spectrometry. The results obtained from a healthy person after oral loading with 40% monodeuterated L-phenylalanine are presented. The method appears to be suitable for in vivo studies of phenylalanine metabolism in humans.

[Point of micropreparative high pressure liquid chromatography and thin-layer chromatography for the identification of indole compounds in human plasma (author's transl)]. / Kombinierte Anwendung der mikropräparativen Hochdruck-Flüssigkeitschromatographie und der Dünnschichtchromatographie beim Nachweis von Indolmetaboliten im menschlichen Plasma

Results are presented on the analysis of indolic metabolites of tryptophan in human plasma, using high-pressure liquid chromatography and thin-layer chromatography. Dichloromethane/ethanol extracts of denaturated plasma were analysed. Thin-layer chromatography proved to be more advantageous for the analysis of this class compounds because it is possible to use a specific staining reagent (4-dimethylaminobenzaldehyde). The advantage of high-pressure liquid chromatography lies in the rapid isolation and purification of unknown compounds for identification in an off-line method. This application is demonstrated with the isolation of N-acetyltryptophan from human plasma. Preliminary results are presented on the plasma concentration of indole-3-lactic acid, indole-3-acetic acid and N-acetyltryptophan in healthy persons, phenylketonurics, and uremic patients.

Indolic tryptophan metabolism in uraemia.

Tryptophan and some indolic metabolites were studied in urine, plasma and dialysate of uraemic patients using thin-layer- and high-pressure liquid chromatography. Some new metabolites: indole-3-carboxylic acid, indole-3-carbaldehyde, indolelactic acid and N-acetyltryptophan were detected. Levels of the latter two metabolites in urine, dialysate and especially plasma suggest increased transamination of tryptophan and a possible defect in renal amino acid acylase in uraemia.

Quantitative determination of cortisol in human plasma by high-pressure liquid chromatography.

A method is described for the determination of cortisol in human plasma by high-pressure liquid chromatography. The simplified extraction procedure makes the method applicable to routine clinical assays. Partition chromatography is carried out on a Zorbax-Sil column with the eluent system dichloromethane-ethanol-water. A 78% recovery was obtained for cortisol. The detection limit is 1 mug per 100 ml in 1 ml of plasma. Cortisol values were determined in samples from a random selection of patients.