Bilirubin-IXalpha and -IXbeta pigments, coproporphyrins and bile acids in meconium and stools from full-term and preterm neonates during the first month of life.

UNLABELLED: Individual bilirubin pigments in the excreta were quantitated by newly developed methods. In meconium, bilirubin-IXbeta predominated, whereas bilirubin-IXgamma and -IXdelta remained undetectable. The daily excretion of bilirubin-IXalpha plus -IXbeta was 0.03-1.00 and 0.04-2.00 micromoles kg(-1) of birthweight in preterm and full-term infants, respectively. The ratio of bilirubin-IXalpha to -IXbeta in meconium was 0.25 +/- 0.34, 0.32 +/- 0.30 and 0.46 +/- 0.55 in newborns of gestational ages below 30, from 31 to 36 and above 36 wk, respectively. The predominance of bilirubin-IXbeta disappeared within the first week in those with gestational age >31 wk but more slowly in the very preterm group. The ratio of monoconjugated to diconjugated bilirubin-IXalpha was 4 to 5 in full-term infants, whereas this ratio was only reached after 1 mo in preterm infants. The ratio of glucuronide or glucoside to xyloside varied widely, independent of gestational age. No correlation between faecal UCB-IXalpha and beta-glucuronidase was observed. The daily coproporphyrin excretion fell from a median of 500 microg on day 1 to below 20 microg from day 7 onwards; this decrease correlated with that of bilirubin-IXbeta. The daily 3alpha-hydroxylated bile acid loss in the excreta was two- to fivefold higher than in the adult; this, together with the higher neonatal serum levels (12-90 nmoles ml(-1)), indicates an immature intestinal reabsorption and an enhanced bile acid synthesis. CONCLUSION: Both zinc coproporphyrin and bilirubin-lXbeta are characteristic compounds of human meconium, diconjugated bilirubin-IXalpha is low or absent in meconium of very preterm infants, and faecal and serum bile acids are high.

Intestinal colonization leading to fecal urobilinoid excretion may play a role in the pathogenesis of neonatal jaundice.

BACKGROUND: Neonatal hyperbilirubinemia remains of concern because of the potential danger for the central nervous system. Because urobilinogen is a nontoxic derivative of bilirubin, the current study was conducted to examine the fecal excretion of urobilinoids and bilirubin in healthy newborns and infants, as well as their intestinal bacteria capable of reducing bilirubin, to assess a possible relation to serum bilirubin levels during the first weeks of life. METHODS: Bilirubin pigments, urobilinoids, and porphyrins were measured in stools of infants during the first week (group A, n = 60) and between the second week and the first 6 months of life (group B, n = 64). Microbiologic analysis of stools was performed in selected cases and bilirubin-converting activity of isolated bacteria was determined in vitro. RESULTS: Urobilinoids were detectable in stools of 57% of the neonates at day 5, but not before. However, fecal urobilinoid production on that day was only a fraction of that observed in adults (0.07 vs. 0.7-3.6 mg/kg per day), whereas at week 6 it increased significantly to an average of 0.9 mg/kg per day. Microbiologic analysis of neonatal stools revealed two novel bacterial strains of Clostridium perfringens and Clostridium difficile capable of reducing bilirubin to urobilinoids. CONCLUSIONS: Urobilinoids can be detected in stools of 57% of newborns at day 5 after delivery. However, the urobilinoid production during the first week of life is quantitatively insufficient to contribute significantly to the removal of bilirubin. Enhancement of the microbial conversion of bilirubin could decrease the intestinal concentration of bilirubin and may decrease the degree or enhance the removal of neonatal hyperbilirubinemia.

[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.

Intestinal excretion of unconjugated bilirubin in man and rats with inherited unconjugated hyperbilirubinemia.

Patients with Crigler-Najjar syndrome and Gunn rats cannot form bilirubin glucuronides owing to a lack of bilirubin UDP-glucuronosyltransferase activity. Because increased serum and tissue bilirubin levels remain constant, an alternative excretory route has to substitute for this deficiency. Gunn rats excrete in bile only 2-13% of the bilirubins eliminated in Wistar rats. In contrast, the biliary excretion rate of urobilinogen in Gunn and Wistar rats is comparable. The sum of bilirubins and urobilinogen excreted in the bile of Gunn rats amounts to 10-30% of pigments excreted in Wistar rats. Despite this low biliary excretion, the intestinal content and fecal excretion of bile pigments in Gunn and Wistar rats were similar. These data support an extrabiliary entrance of unconjugated bilirubin into the intestine. Additional proof for this was found in that the intestinal lumen of Gunn rats still contains a high amount of bilirubins and urobilinogen after 3 d of external biliary drainage. A similar procedure in Wistar rats resulted in the complete disappearance of bile pigments from the intestine. The direct transmural transport of bilirubin from blood to all parts of the intestinal lumen was demonstrated by injecting 14C-bilirubin i.v. into Gunn rats with isolated parts of small and large intestine. In Crigler-Najjar and Gilbert's syndrome patients, the biliary excretion of bile pigments has previously been shown to be strongly reduced. Their stools, however, contained approximately the same amount of bile pigments as in normal subjects. Although only traces of unconjugated bilirubin were detected in the stool of normal persons (4 +/- 3% of total bile pigments), higher amounts were found in patients with Crigler-Najjar disease (20 +/- 12&). These results suggest a direct intestinal permeation of unconjugated bilirubin in severe unconjugated hyperbilirubinemia both in man and rats.

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.

[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.

[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.

[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.

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)

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.

[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)

[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.

[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.

[A new model for testing substances affecting the synthesis of heme]. / Nový model pro testování látek ovlivnujících syntézu hemu.

A new model for experimental studies of substances influencing porphyrin metabolism has been created. The model is formed by yeast Saccharomyces cerevisiae (RIBM-75) grown semiaerobically. The main advantages of this model include simple evocation of porphyria (intracellular accumulation of porphyrins in semiaerobic conditions) and direct measurement of experimental values (intracellular and extracellular concentrations of porphyrins). The porphyrinostatic effects of drugs can be assessed on the basis of sum of experimental values. The ratio of experimental criteria enables us to compare the influence of drugs on porphyrin permeation across the cellular membrane. Antimalarials, chloroquine and pyrimethamine, used or tested for therapy of chronic liver porphyria, have been tested on the model. The experiments showed that both chloroquine and pyrimethamine inhibited porphyrin synthesis. This effect can represent the proper therapeutical action of both drugs, which has not been known so far. Chloroquine releases the intracellular porphyrins in yeast similarly as it does in hepatocytes. However, pyrimethamine causes intracellular accumulation of porphyrins booth in yeast and hepatocytes.

[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.

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.

Porphyrinostatic effect of isonicotinic acid hydrazide on porphyric chick embryo and yeast.

1. The common feature of "inducible" forms of hepatic porphyria is an increased activity of delta-amino-levulinate/ALA/synthase. 2. As a potential therapeutic procedure, isonicotinic acid hydrazide/INH/as inhibitor of this enzyme, was applied and found to show a porphyrinostatic effect, depending on its concentration, both in yeast and porphyric chick embryo models. 3. Moreover, pyridoxalphosphate/PDX/eliminated the porphyrinostatic effect of INH, depending again on its concentration. 4. Histochemical tests did not demonstrate any significant functional or morphological changes in the chick embryo hepatocytes following INH administration.

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.