Relationship between biliary lipid and protoporphyrin secretion; potential role of mdr2 P-glycoprotein in hepatobiliary organic anion transport.

BACKGROUND/AIMS: Erythropoietic protoporphyria, caused by ferrochelatase deficiency, leads to protoporphyrin accumulation in the liver. Therapeutic attempts to increase the secretion of this hydrophobic organic anion into bile are hampered by a lack of understanding of the secretory mechanism(s) involved. We have investigated biliary secretion of protoporphyrin in rats and mice, primarily targeted on the role of biliary lipids in this process. METHODS: Gel permeation chromatography was applied to investigate the association of porphyrins with lipid fractions in bile. Secretion of endogenous porphyrins was studied in (GY mutant) rats and mdr2 P-glycoprotein deficient mice, under conditions of widely varying biliary lipid secretion rates. RESULTS: Gel permeation chromatography revealed that, in native human and rat bile, protoporphyrin associated with cholesterol/phospholipid vesicles upon elution with bile salt-free buffer. In contrast, the more hydrophilic coproporphyrin isomers I and III were found only in bile salt/organic anion hybrid particles and smaller aggregates. Interruption of the enterohepatic circulation in normal Wistar rat resulted in parallel decrease of endogenous protoporphyrin-, lipid-, and bile salt secretion, but did not alter the secretion of coproporphyrin I and III. Uncoupling of lipid- from bile salt secretion by sulfated taurolithocholate resulted in impaired secretion into bile of protoporphyrin only. Conversely, secretion of coproporphyrin I and III, but not that of protoporphyrin, was impaired in mutant Groningen Yellow rats with defective ATP-dependent hepatobiliary organic anion transport. In mice homozygous for a disruption of the mdr2 P-glycoprotein gene, resulting in complete absence of phospholipids in bile and strongly reduced cholesterol output, secretion of protoporphyrin was reduced by 90%, whereas that of coproporphyrin I and III was affected to a much lesser extent. CONCLUSIONS: Our data demonstrate a close association between protoporphyrin and lipid secretion into bile, indicating that these processes are, at least functioning coupled. This finding implicates a role of mdr2 P-glycoprotein activity in hepatobiliary removal of the hydrophobic organic anion protoporphyrin. Hence, it may be speculated that protoporphyrin secretion can be influenced by drugs, diet or other means that affect biliary lipid secretion.

Evaluation and clinical application of the Enterotest for the determination of human biliary porphyrin composition.

The Enterotest string test is an easy and non-invasive method for sampling duodenal fluid, which has been successfully used for the analysis of duodenal microflora, as well as biliary bile acid and lipid composition. The method was evaluated for determination of porphyrins in duodenal bile in normal subjects and subjects with porphyria, following cholecystokinin induced gall bladder contraction; it is known that analysis of biliary porphyrins is more discriminatory for the diagnosis of asymptomatic porphyria than their analysis in faeces or urine. Moreover, serial analysis of bile from patients with erythropoietic protoporphyria may help in establishing their ability to secrete protoporphyrin in bile and to assess effects of treatment. The binding of various porphyrins to Enterotest strings was investigated by incubating pieces of the string in different human bile samples with low to very high porphyrin concentrations, followed by HPLC analysis of porphyrins both in the native bile and in extracts obtained from the strings. No differences between porphyrin composition in native bile and extracts were observed. Duodenal fluid obtained by means of the Enterotest from volunteers not receiving cholecystokinin showed large variations in porphyrin patterns not resembling those of native bile. Mesoporphyrin, a secondary porphyrin derived from protoporphyrin by bacteria, was often detectable. These data indicate that the duodenal content without cholecystokinin injection does not reflect biliary porphyrin composition. The presence of mesoporphyrin in the whole intestinal tract, but not in serum and bile, suggests that there is no enterohepatic circulation of secondary porphyrins. There was close agreement between the porphyrin ratios found with the standard duodenal intubation technique and the Enterotest, performed simultaneously in one healthy volunteer after induction of gall bladder contraction by cholecystokinin. From these experiments, it was concluded that fluid adsorbed to the Enterotest string after gall-bladder contraction can be used to determine biliary porphyrin composition. Since duodenal bile is diluted gall bladder bile, variable porphyrin concentrations were found when applying the Enterotest in combination with cholecystokinin in the same subject on successive days. However, porphyrin ratios, such as the protoporphyrin to coproporphyrin I ratio, were relatively constant. In subjects with symptomatic variegate porphyria, the Enterotest showed highly aberrant porphyrin patterns, with increased protoporphyrin to coproporphyrin I ratios and, in addition, the presence of some unknown porphyrins. A deviating biliary protoporphyrin/coproporphyrin I ratio in one patient appeared to be a useful diagnostic index for the presence of latent variegate porphyria (or variegate porphyria in remission).(ABSTRACT TRUNCATED AT 400 WORDS)

Determination of porphyrins in bile using high performance liquid chromatography and some clinical applications.

A simple and fast HPLC method for the determination of porphyrins in bile without extraction is described. Porphyrins were determined in bile from control subjects and from patients after orthotopic liver transplantation, including three patients with erythropoietic protoporphyria. It was found that: 1) coproporphyrin I is the predominant porphyrin in bile of controls, accompanied by some coproporphyrin III and protoporphyrin, whereas protoporphyrin mostly but not always is the predominant porphyrin in the bile of erythropoietic protoporphyria patients. In two of the three erythropoietic protoporphyria patients, the bile contained a hundred times more protoporphyrin than that of non-porphyric orthotopic liver transplantation patients. The third erythropoietic protoporphyria patient remained cholestatic and was unable to excrete sufficient amounts of protoporphyrin. 2) All investigated bile samples contained no secondary porphyrins derived from protoporphyrin, i.e. no deutero-, pempto-, or mesoporphyrin. Even when extracts of bile and serum were concentrated fifty to a hundred times, no traces of deutero-, pempto- and mesoporphyrin were detected. This complete absence of secondary porphyrins suggests that an enterohepatic circulation of dicarboxylic porphyrins from the distal gastrointestinal tract does not exist. 3) The HPLC chromatograms contain peaks from unknown compounds. No correlation between porphyrins and these compounds was found. Porphyrin profiles were followed in the bile of some orthotopic liver transplantation patients. Three episodes are recognizable. During the first three days after orthotopic liver transplantation there is a very high coproporphyrin excretion. There is then a lag of one to three weeks, in which no or very low porphyrin concentrations are detectable, followed by the restoration of normal biliary porphyrin patterns.

Hereditary tyrosinaemia type I: a long-term study of the relationship between the urinary excretions of succinylacetone and delta-aminolevulinic acid.

Patients with hereditary tyrosinaemia type I (HT) excrete large amounts of succinylacetone (SA) in urine. Owing to structural resemblance of SA to delta-aminolevulinic acid (ALA), SA inhibits the second enzyme in the pathway for haeme biosynthesis, porphobilinogen synthase, resulting in increased urinary ALA excretion. We investigated the relationship between urinary SA and ALA excretions of two patients with different forms of HT (late-infantile and juvenile). In both patients the urinary SA and ALA excretions showed a more or less inverse correlation. The patient with the early-infantile form of HT had a relatively greater increase in urinary SA and ALA excretions in comparison to the patient with the juvenile form of HT. A possible explanation for this unexpected inverse correlation between the urinary excretion of SA and ALA might be a lack of intramitochondrial glycine, a substrate for delta-aminolevulinic acid synthesis. It has been reported previously that high concentrations of SA reversibly and competitively inhibit the transport of glycine through membranes.

Pharmacokinetics of parenteral 13-cis-retinoic acid formulations in rats.

The pharmacokinetics of three 13-cis-retinoic acid formulations were studied after intraperitoneal (ip) administration to rats. Rats were given ip injections of 2.5 mg of 13-cis-retinoic acid per 360 g of body weight; the drug was administered as an alkaline solution, suspended in corn oil, or as a mixture with polysorbate 80. The alkaline solution was also given intravenously (iv) via the tail vein as a control. The mean elimination rate constant, calculated from data from iv administration, was 0.72 +/- 0.088 h-1 (r = 0.988). The peak concentration in plasma and the time to reach this maximum were 14 mg/L and 0.5 h, 22 mg/L and 2 h, and 10 mg/L and 1 h for the drug administered as an alkaline solution, suspended in corn oil, and as a mixture with polysorbate 80, respectively. The areas under the concentration-time curve (concentration in plasma versus time) were 34.9 +/- 8.78 mg.h/L for the iv dose and 34.1 +/- 9.97, 62.4 +/- 32.3, and 25.9 +/- 12.0 mg.h/L for the ip doses of alkaline solution, suspension in oil, and mixture with polysorbate 80, respectively. Because of the rapid increase of concentration in plasma, which is identical to that of the iv profile, and the ease of its handling and preparation, the ip administered alkaline solution is the preferable formulation.

Familial increased serum intestinal alkaline phosphatase: a new variant associated with Gilbert's syndrome.

Investigation of mild, inherited increased serum alkaline phosphatase activity partially combined with Gilbert's syndrome in one family showed, apart from a normal liver fraction, an intestinal isoenzyme pattern and an extra band in the agar electrophoresis. Analysis by agarose electrophoresis before and after incubation of neuraminidase showed that the extra fraction was an intestinal variant isoenzyme. The precise genetic background of the two disorders in this family could not be determined from the available data. Abnormal activities of (regular) intestinal alkaline phosphatase isoenzyme caused the increase in serum alkaline phosphatase in the absence of disease.

A retrospective study of a patient with homozygous form of acute intermittent porphyria.

In 1964 a child with an exceptional form of porphyria was described; she excreted persistently excessive amounts of delta-aminolaevulinic acid, porphobilinogen and uroporphyrin in her urine from early childhood. The biochemical profile resembled that of acute intermittent porphyria (AIP). The child died at the age of 8 years. Reinvestigation of some urine samples by HPLC revealed differences in comparison with urines of other patients with AIP. The clinical picture characterized by porencephaly and severe retardation in development was completely different from that of AIP. Her mother suffered from AIP but the father never had attacks. Investigations on blood and urine samples of the father showed that he also was affected. Due to the early onset in the index patient, its persistent character, and the fact that both parents are affected we postulate retrospectively to have diagnosed a case of homozygous or a double heterozygous AIP, hitherto undescribed.

Identification of the mutations in the parents of a patient with a putative compound heterozygosity for acute intermittent porphyria.

The molecular abnormalities responsible for acute intermittent porphyria were investigated in both parents of a girl who was retrospectively diagnosed as having a homozygous form of the disease. The mutations in the parents are different from each other and both of them correspond to previously identified G to A changes in the coding part of the porphobilinogen deaminase mRNA. These point mutations lead to the presence of a catalytically-defective but immunologically-reactive enzyme. Our results support the conclusion that the propositus girl may represent the first case of compound heterozygosity for acute intermittent porphyria alleles.

Enterobacteriaceae suppression by three different oral doses of polymyxin E in human volunteers.

Polymyxin E is frequently used as an oral drug for flora suppression of the gastrointestinal canal. The suppression effect is dose dependent because polymyxin E is moderately inactivated by faecal and food compounds. Three oral polymyxin E doses (150, 300, 600 mg daily) were given to six volunteers for 6 days. The Enterobacteriaceae suppression effect was compared by means of the suppression index i.e. ratio of total number of faecal samples free of Enterobacteriaceae to the total number of faecal samples. The impact on the indigenous (mostly anaerobic) flora was measured in four ways: (i) beta-aspartylglycine content; (ii) volatile fatty acid pattern; (iii) yeast overgrowth and (iv) Streptococcus faecalis decrease. Enterobacteriaceae suppression was most successful during 600 mg oral polymyxin E (suppression indices during 150, 300 and 600 mg were 0.32, 0.55 and 0.89 respectively). None of the four markers of indigenous flora alterations were positive. However, using this dosage half of the volunteers suffered rather severe gastrointestinal side-effects. Oral polymyxin E in a dosage of minimum 600 mg daily seems to possess the ideal properties of a flora suppression agent, if the gastrointestinal side-effects could be mitigated.

Patterns of porphyrin excretion in feces as determined by liquid chromatography; reference values and the effect of flora suppression.

While determining reference values for porphyrins in feces as measured by liquid chromatography, we observed strong fluctuations in fecal porphyrin contents. To explain these fluctuations, we selectively suppressed the intestinal flora of healthy persons. Suppression of aerobic flora had no effect on fecal porphyrin excretions, whereas suppression of anaerobic flora completely inhibited the transformation of protoporphyrin to pempto- and deuteroporphyrin for as long as five days after stopping medication. During this latter, the conversion to mesoporphyrin was clearly increased in one person and in others partly affected or decreased. During complete suppression of flora for prolonged periods, the production of proto- and coproporphyrins was decreased and deutero-, pempto-, and mesoporphyrins were absent. We conclude that the nature of fecal porphyrins is mostly affected by action of anaerobic bacteria, different kinds of bacteria having different effects. Some, like aerobic Gram-negative bacteria, have little or no effect on porphyrins; some cause production of mesoporphyrin; some promote a conversion to pempto- and deuteroporphyrin; and some mainly cause production of copro- and protoporphyrin. We give examples in which normal to slightly increased excretions of fecal porphyrin do not exclude a diagnosis of porphyria, and relatively high concentrations do not confirm one.

Hepatic disease in erythropoietic protoporphyria.

A standardized hepatological investigation was performed in 9 unselected patients with erythropoietic protoporphyria (EPP). The aim of this study was to detect early liver involvement due to EPP and to determine the significance of several diagnostic procedures. Scintigraphy revealed slight enlargement of liver and spleen in all cases. Light microscopic examination of liver tissue in 7 patients showed protoporphyrin deposition in 4 and signs of fibrosis in 3 cases. Cirrhosis was not found. Electron microscopical examination (EM) of all 7 cases was negative and needle-shaped crystals were not found. Therefore we regard EM of little diagnostic value in the detection of early liver involvement. The literature on fatal and asymptomatic cases of liver involvement in EPP is discussed with emphasis on possible predictive and provocative factors. A proposal for EPP patients intending to reduce possible risk factors is made.