Exocytosis as a possible mechanism for the secretion of coproporphyrin from a chloroquine-treated yeast.

Chloroquine, which is known to be concentrated in intracellular acid vesicles, stimulates the release of porphyrins from yeast. Experiments with normal baker's yeast Saccharomyces cerevisiae and the sec-1 mutant, with suppressed exocytosis, showed that the release of porphyrins was stimulated more in normal yeast than it was in the mutant. It is suggested that chloroquine stimulates the exocytosis of porphyrins.

Quantitation of urobilinogen in feces, urine, bile and serum by direct spectrophotometry of zinc complex.

Previous methods to quantitate urobilinogen lack precision due to either incomplete reduction of urobilin or to losses of pigment before the use of Ehrlich's aldehyde reaction or due to pigment precipitation, as occurs in Schlesinger's fluorescent assay. The present procedure modifies the latter assay to obviate described problems as it is based on direct spectrophotometry (or spectrofluorometry) of a zinc complex of urobilin in dimethylsulfoxide. The sample is extracted with dimethylsulfoxide to increase recovery of urobilinogen from samples of various origin (feces, urine, bile, serum etc.) and to prevent the precipitation of proteins. After oxidation of urobilinogen with iodine, the concentration of the resulting urobilin is directly determined from the absorption (or fluorescent) spectrum. High sensitivity and high specificity for the procedure result from the high value of absorption coefficient and by the characteristic absorption spectrum of zinc complex of urobilin, respectively. Within-day and day-to-day coefficients of variation of stool and bile samples range from 1.6 to 9.2%. The smallest concentration of urobilinogen measurable by spectrophotometry is approximately 0.5 mumol/l, by fluorometry it is 0.25 mumol/l. The recovery varies from 82.2 to 93.8% depending on re-extraction of the sample. The method is linear in the range of 1 to 35 mumol/l and of 0.5 to 17.5 mumol/l for spectrophotometric and fluorescent determinations, respectively. The results obtained with the present method correlated well with Ehrlich's determination (r2 = 0.912), but are approximately two-fold higher. Storage of the samples at -20 degrees C or extraction with dimethylsulfoxide prior to storage are good ways for sample preservation. Twenty stool samples from healthy adults were determined.(ABSTRACT TRUNCATED AT 250 WORDS)

[Experimental study of the action of antimalarial agents with respect to therapy in porphyria. Combined effect of chloroquine and pyrimethamine]. / Experimentální studium mechanismu úcinku antimalarik se zretelem k terapii porfyrické choroby. Kombinovaný úcinek chlorochinu a pyrimetaminu.

On an experimental model created from semiaerobically cultivated yeast cells of Caccharomyces cerevisiae (RIMB-75) the authors tested the effect of antimalarics used or tested for treatment of symptomatic liver porphyria. Chloroquine and pyrimethamine inhibited the synthesis of porphyrins, whereby pyrimethamine was more effective. Chloroquine released moreover intracellular porphyrins, contrary to pyrimethamine, which caused their intracellular cumulation. An equimolar combination of the two preparations preserved the inhibitory action of pyrimethamine, and the intracellular porphyrin content was reduced. Based on experimental results the suggested combination of chloroquine and pyrimethamine was successfully tested in clinical work.

[A case of recurrent alimentary lead poisoning]. / Prípad opakované alimentární otravy olovem.

The authors describe a case of lead poisoning in a family (father, mother, son, daughter). The condition was at first diagnosed as acute hepatic porphyria. The correct diagnosis was made on the basis of increased urinary excretion of delta-aminolevulinic acid and coproporphyrin and on the basis of the revealed reversibly inhibited activity of delta-aminolevulinic acid dehydratase in red blood cells. The source of intoxication was the use of red lead pigment instead of dried red pepper.

[Trimethoprim in the therapy of symptomatic liver porphyria]. / Trimetoprim v lécbe symptomatické jaterní porfyrie.

Administration of antimalaria drugs to patients with symptomatic hepatic porphyria (porphyria cutanea tarda) at the First Medical Clinic made it possible due to previous experimental studies, to influence the porphyrim metabolism of yeasts. The effect of trimetoprim was investigated in clinical work in 12 hitherto not treated patients with the manifest form of symptomatic hepatic porphyria. The group comprised 9 men, mean age 56.4 years, and 3 women mean age 43.6 years. During the two-year clinical study dermatological symptoms of the disease became milder or receded. Concurrently there was a significant regression of porphyrinuria. In the whole group porphyrinuria declined to 11% of the original values. The porphyrin content of hepatic tissue declined considerably after two years treatment. In the group as a whole to 40% of the original values. Trimetoprim is another drug which influences porphyrin metabolism. The authors did not detect any undesirable side-effects of trimetoprim treatment. The effect of trimetoprim on clinical and biochemical parameters of the disease is less marked than the effect of chloroquine. This new treatment can be used in patients resistant to chloroquine or in combination with other anti-malaria drugs.

[Pharmacokinetics and pharmacodynamics of furosemide in liver cirrhosis]. / Farmakokinetika a farmakodynamika furosemidu u jaterní cirhózy.

12 patients with cirrhosis of the liver (6 with ascites) and 6 control persons were given 80 mg furosemide intravenously and were followed up for pharmacokinetic, renal and haemodynamic effects. The patients with ascitic cirrhosis (AC) and the control persons were found to have the same plasma furosemide concentrations; however, the AC patients excreted significantly less furosemide into the urine in an 8-hour period than the control persons, which was due especially to lower urine furosemide excretion during the first hour after application in comparison with the control group (75 +/- 6.8 vs. 100.8 +/- 8.8 mumol/h, p less than 0.05). During the first post-furosemide hour, the AC patients had a significantly lower diuresis (13.3 +/- 2.4 vs. 23.9 +/- 1.1 ml/min, p less than 0.01) as well as natriuresis (1367.4 +/- 771.4 vs. 3242 +/- 137.5 mumol/min, p less than 0.01) than the control group and a significantly lower excretion fraction of sodium than the patients with live cirrhosis without ascites (6.5 +/- 0.8 vs. 10.7 +/- 1.5%, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

[The first case of Crigler-Najjar syndrome in the Czech Republic]. / První prípad Criglerova-Najjarova syndromu v Ceské republice.

BACKGROUND: Crigler-Najjar syndrome is a rare disease due to a congenital deficiency of bilirubin UDP glucuronosyl transferase in the liver tissue. It is characterised by high levels of unconjugated bilirubin in plasma through the whole life. The aim of the study was to confirm the clinical diagnosis of the first Crigler-Najjar syndrome case in our country. METHODS AND RESULTS: 34 years old Gypsy women was admitted to our GI clinic for clinical examination before scheduled cholecystectomy. The high plasmatic level of unconjugated bilirubin was found and therefore the diagnosis of Crigler-Najjar syndrome was anticipated. The diagnosis was based on the chromatographic analysis of biliary bile pigments. The amount of diconjugates was considerable decreased. In addition, the molecular analysis of DNA isolated from peripheral blood leukocyte was performed to confirm our conclusions. Our patients was found to be homozygous for a nucleotide shift in the unique exon of bilirubin UDP glucuronosyl transferase 1, substituting guanine into an adenine at position 211.

[Self-medication of the common cold]. / Samoterapie symptomu nemoci z nachlazení.

Self-medication can be useful in the multisymptomatic management of the common cold and other preferentially non-febrile flu-like symptoms, especially as at present multicomponent remedies are available, which may make self-medication significantly easier with consequent better compliance of adult patients. Children, on the contrary, are not suitable acceptants of self-medication mediated by their parents because the sickness exhibits in children nearly exclusively febrile progress. The procedure of the therapy to be really effective and safe must be necessarily concentrated into pharmacies, where safety and efficacy of therapy are ensured by the pharmacist as the last link of contact between the patient and the drug. Any other distribution of the OTC drugs is therefore strictly unacceptable. Patients suffering from the common cold usually exhibit simultaneously various symptoms of the disease. These different symptoms are, however, experienced during the day with different intensity. During the day and often also in the course of the labour process there is a need to concentrate on the most bothersome symptoms, notably the relief of the nasal congestion with the exploration of the safest and most effective drug, such as pseudoephedrine. In addition, it is necessary to administer an effective nonsteroidal anti-inflammatory drug, preferably ibuprofen, which provides relief from symptoms caused by release of inflammatory mediators. In the night time there is a need of a multisymptom therapy which may relieve cough and any other disturbing cold symptoms to allow satisfactory beneficial, continuous, and refreshing sleep.

Urobilinogen-i is a major derivative of bilirubin in bile of homozygous Gunn rats.

Gunn rats lack bilirubin UDP-glycosyltransferases, but diazo-negative derivatives of bilirubin have been described in their bile. In order to investigate this alternative disposal of bilirubin, crude bile samples from Gunn and Wistar rats were directly analysed by h.p.l.c. Besides bilirubin (in Gunn rats) or its glycosides (in Wistar rats), two major compounds were detected. A yellow one corresponded to the previously documented vitamin B-2 and was equally prominent in Gunn rats or Wistar-rat bile. The other compound was colourless, but on standing in contact with air it was spontaneously oxidized to a pinkish-yellow pigment. It was far more prominent in Gunn-rat bile. Analysis of bile obtained after intravenous injection of [14C]bilirubin to Gunn rats demonstrated that this compound was highly labelled. Freezing and thawing of the bile resulted in the formation of a series of diazo-negative derivatives, demonstrating that the original compound was quite labile. Spectral (adsorption and fluorescent) and chromatographic (h.p.l.c., t.l.c. and paper chromatography) analysis of the oxidized form of the labelled compound allowed its identification as urobilin-i. The colourless compound secreted in bile was urobilinogen-i. Administration of neomycin and bacitracin to Gunn rats or gut resection suppressed the biliary excretion of urobilinogen and thus confirmed its intestinal origin. Urobilinogen seems thus to represent the major bilirubin derivative present in Gunn-rat bile. Its breakdown products might represent the so-far-unidentified diazo-negative polar bilirubin derivatives. Since only a small amount of bilirubin is present in Gunn-rat bile, the urobilinogen formed in the intestinal lumen seems to be derived from bilirubin reaching the gut via routes other than the biliary one.

A model for testing compounds influencing porphyrin synthesis.

The yeast Saccharomyces cerevisiae cultivated semi-anaerobically in a synthetic medium was used as a model to establish (a) total porphyrin synthesis, (b) ratio of intracellular to extracellular porphyrin concentrations. The antimalarials used for the therapy of porphyria cutanea tarda, chloroquine and pyrimethamine, reduced the total synthesis of porphyrins, pyrimethamine being more effective than chloroquine, like in porphyric patients. Both drugs exerted an antagonistic influence on the release of porphyrins from cells. Chloroquine reduced the concentration ratio of porphyrins while pyrimethamine increased it, apparently through inhibition of permeation of porphyrins. Combined treatment with the two compounds may hold promise for the therapy of porphyria cutanea tarda.

Fasting-related hyperbilirubinemia in rats: the effect of decreased intestinal motility.

BACKGROUND & AIMS: Fasting increases serum bilirubin levels in both humans and rats. Because the pathogenesis of fasting hyperbilirubinemia is not fully understood, the effect of fasting on disposition of bile pigments was investigated in rats. METHODS: Bilirubin and urobilinogen were determined in excreta, bile, plasma, and liver tissues of fasted Gunn and Wistar rats. RESULTS: Fasting increased the intestinal transit time of Wistar rats. As a result, the fecal output of bile pigments was decreased by food deprivation. In contrast, the intestinal content of total bile pigments was augmented in both Wistar and Gunn rats. This finding was paralleled by the increase of serum bilirubin concentration in both rat strains. A similar increment of serum bilirubin levels was observed after injection of bilirubin into the cecum of Wistar rats. Furthermore, biliary efflux of bilirubin in Wistar rats was increased after 48 hours of fasting. Intubation of nonabsorbable bulk to fasted Wistar rats prevented the increase of serum bilirubin levels during a 48-hour period of food deprivation. CONCLUSIONS: Fasting decreases intestinal motility and elimination of bile pigments. Accumulation of bilirubin in the intestine during fasting allows enhanced enterohepatic circulation and results in an increased reflux to plasma. This seems to be a major factor involved in fasting-induced hyperbilirubinemia.

Effect of chloroquine on membrane permeability in yeast--release of cellular coproporphyrin.

1. The influx and efflux of labelled substances with and without chloroquine was studied in yeast cells. 2. The uptake of delta-aminolevulinic acid by Saccharomyces cerevisiae is characterized by a KT of 3-4 mM and Jmax of 1.0-1.2 mumol min-1 g dry weight-1. 3. A method for loading yeast with labelled coproporphyrin is suggested. 4. The uptake of sorbitol and coproporphyrin was slightly stimulated, while the uptake of 6-deoxyglucose was slightly, that of 2-aminoisobutyric acid and leucine strongly inhibited by chloroquine. 5. The efflux of coproporphyrin, 2-aminoisobutyric acid and sorbitol was stimulated while that of leucine was not influenced by chloroquine. 6. The result showed that chloroquine influenced directly but nonspecifically the membrane permeability, apparently mainly that of the vacuolar membrane.

Solid phase extraction and isocratic separation of urinary porphyrins by HPLC.

A method for determining urine porphyrins by HPLC is described. In the preliminary step, porphyrins are purified in high yields and concentrated by low pressure reverse-phase chromatography on C18 (octadecylsilane bonded silica) cartridge. Porphyrins are stable for 10 days after adsorption on C18 cartridge. The separation of porphyrin esters is performed on an aminopropyl-bonded silica column with an eluting system containing n-heptane and ethyl acetate. The system enables rapid isocratic separation of porphyrin methyl esters with high selectivity. The simplicity and reproducibility of the whole procedure allows its application to the routine analysis of urinary porphyrins in the clinical laboratory.

[Inhibition of porphyrin synthesis by isonicotinic acid hydrazide in models simulating porphyria]. / Inhibice syntézy porfyrinu hydrazidem kyseliny isonikotinové na modelech simulujících porfyrii.

Increased activity of haem synthesis key enzyme is a common phenomenon in hepatic porphyrias. Synthase activity of delta-aminolevulinic acid is extensively increased in acute hepatic porphyrias or it can be by means of feedback moderately secondarily increased in their chronic forms. Isonicotinic acid hydrazide (INH) is a competitive inhibitor of delta-aminolevulinic acid synthase as it combines with enzyme co-factor, pyridoxalphosphate (PDX) to form hydrazone. The INH porphyrinstatic effects were verified by testing two experimental models. The yeast model exhibited 5-fold inhibition at 10 mM INH concentration in medium. The chicken germ model after a 10 microM INH dose exhibited 82% inhibition. This inhibition can be eliminated by PDX administration to reach the original porphyrin synthesis at equimolar INH and PDX concentrations. Histological study of transversal chicken liver sections proved that 1 microM INH administration does not result in disturbance of chicken hepatocyte integration. The suggested preparation has the advantage of being commonly manufactured and approved to be used in human medicine.

[Determination of paracetamol in the blood using high-performance liquid chromatography]. / Stanovení paracetamolu v séru metodou vysokoúcinné kapalinové chromatografie.

The paper reports the determination of paracetamol in the serum by means of high-performance liquid chromatography. Prior to analysis, the sample is purified by deproteination with perchloric acid. The analysis is carried out isocratically on the reverse phase (SEPARON SIX C18, 5 microns, 150 x x 3.7 mm). The mobile phase consists of 40% methanol in 0.4% phosphoric acid. The detection is performed at 254 nm. The calibration curve is linear in the region 6.62-331 mumol.l-1 (1.0-50.0 micrograms.ml-1) with 95% reproducibility. The sensitivity of detection is 1.3 mumol.l-1 (0.2 microgram.ml-1). The suitability of the method was checked by processing 300 serum samples.

The influence of chloroquine on the formation of porphyrins by yeasts.

A single two-compartment model suitable for studying the production and elimination of porphyrins from cells was prepared. Chloroquine with increasing concentrations:-- Inhibits the total production of porphyrins. Reduces intracellular concentration of porphyrins. Increases transversal permeation of porphyrins through the cellular membrane.