Hepatic Pigment Research Articles

Hepatic pigmentation with photosensitivity in corriedale sheep

This datasheet on hepatic pigmentation with photosensitivity in corriedale sheep covers Identity, Hosts/Species Affected, Diagnosis.

Effects of Thermal Stress on Hepatic Melanomacrophages of Eupemphix nattereri (Anura)

Melanomacrophages are the pigmented cells present in the hematopoietic organs. Besides melanin, hemosiderin and lipofuscin are also observed in the melanomacrophages. For the liver, however, numerous studies relate these cells to immunological and metabolic functions. Therefore, the aim of this study is to evaluate the hepatic metabolism by quantifying melanin, hemosiderin and lipofuscin in the anuran Eupemphix nattereri submitted to varying thermal conditions. E. nattereri adult males were separated into three groups, as follows: (i) five animals in the control group were kept at room temperature (27°C); (ii) 30 animals were submitted to hyperthermic (35.1°C); and (iii) 30 to hypothermic (18.9°) conditions. In each experiment, the animals were analyzed and separated into two different treatments: (1) immediately after undergoing the stress; and, (2) after recovering from the stress caused by the stimulus, at three distinct times (12 hr, 24 hr, and 48 hr). Both hyperthermia and hypothermia decreased hepatic pigmentation after thermal stress. The recovered animals of both experimental treatments showed as much pigmentation as the control animals. Thermal stress alters hemosiderin and lipofuscin as well, which may be related to liver function catabolism. In conclusion, liver pigmentation decreased due to temperature variation and duration of thermal stimulation to which the animals were exposed. The increase in temperature rather than hypothermia led to more drastic physiological disorders. In this study, we observed that thermal stress for a short period compromises the morphology and liver function, as observed by the changing pigmentation of melanomacrophages. These analyses can be used as biomarkers of environmental effects.

Correlation Between Hepatic Lipogranuloma Pigmentation and Stage of Cronic Liver Disease in Dogs

Introduction: Hepatic lipogranulomas are poorly defined lesions and have an undetermined significance and etiology. They are often found in the hepatic parenchyma of dogs, and are associated with previous hepatocyte death and circulatory disorders. Aims: The aim of the present study was to determine the correlation between the pigmentation and localization of hepatic lipogranulomas and the evolutive stage of chronic liver disease in dogs. Materials and methods: The study was conducted on a total of 25 liver samples obtained after necropsy from dogs with chronic liver disease (cirrhosis, lobular dissecting hepatitis, chronic hepatitis) with or without portosystemic shunts. Sections were stained with hematoxylin-eosin method for morphological diagnosis and for the identification of lipogranulomas. For characterization of intracytoplasmic pigments special histochemical methods were used (Perl’s reaction, PAS reaction, Oil red, Hall method, Rodamine stain). Immunohistochemical exam using antibodies against CD 68, and fluorescence microscopy was also performed. Results: In the present study we described two types of hepatic lipogranulomas: diffuse septal and intralobular well delimited. The grade of pigmentation and chemical composition of intracytoplasmic pigments changed over time, if in the initial phases lipogranulomas were diffuse, less pigmented and pigments were predominantly represented by ceroid, in later stages (with portosystemic shunts) lipogranulomas were heavily pigmented, pigments being represented primarily by hemosiderin. Conclusion: In the present study we showed that pigmentation of hepatic lipogranulomas is more intense in the late stages of chronic liver disease, when portal hypertension with portosystemic shunts occurs and the predominant pigment changes from ceroid to hemosiderin.

Responses of melanocytes and melanomacrophages of Eupemphix nattereri (Anura: Leiuperidae) to Nle4, D-Phe7-α-melanocyte stimulating hormone and lipopolysaccharides

Melanocytes are found in various organs of ectothermic animals, playing a protective role against bacteria and free radicals. It is known that pigment cells from hematopoietic organs have immune functions. However, the role of visceral melanocytes is not well understood. Cutaneous melanocytes are responsive to α-melanocyte stimulating hormone (α-MSH), which is associated with the dispersion of melanin granules within melanocytes. α-MSH has also been reported to inhibit most forms of inflammatory responses by decreasing the pro-inflammatory cytokines and neutrophil migration. The present study evaluated the influence of an α-MSH analog (Nle4, D-Phe7-α-MSH) and lipopolysaccharides (LPS) from Escherichia coli on the liver and testicular tissues of the anuran Eupemphix nattereri. The tested hypotheses were: (i) the pigmented area will increase following hormone and LPS administration, (ii) pre-treatment with α-MSH will decrease the number of mast cells, and (iii) the hormone will have protective effects against LPS-induced responses. We found that hormone administration did not change hepatic pigmentation, but increased testicular pigmentation. Testicular pigmentation quickly increased after LPS administration, whereas there was a late response in the liver. The response of enhanced pigmentation was delayed and the number of mast cells decreased in animals previously treated with the α-MSH analog when compared to the LPS group. Hemosiderin and lipofuscin were found in melanomacrophages, but not in testicular melanocytes. Although both the liver and the testes of E. nattereri have pigmented cells, these are distinct in morphology, embryonic origin, and pigmentary substances. These differences may be responsible for the different responses of these cells to the α-MSH analog and LPS administration.

Intracellular pigment epithelium-derived factor contributes to triglyceride degradation

Pigment epithelium-derived factor is well known as a secreted glycoprotein with multiple functions, such as anti-angiogenic, neuroprotective and anti-tumor activities. However, its intracellular role remains unknown. The present study was performed to demonstrate the intracellular function of pigment epithelium-derived factor on triglyceride degradation. Hepatic pigment epithelium-derived factor levels increased at the early stage and subsequently decreased after 16 weeks in high-fat-diet-fed mice compared to those in chow-fed mice. Similarly, oleic acid led to long-term downregulation of pigment epithelium-derived factor in HepG2 cells. Endogenous pigment epithelium-derived factor was an intracellular protein with cytoplasmic distribution in hepatocytes by immunostaining. Exogenous FITC-labeled pigment epithelium-derived factor could be absorbed into hepatocytes. Both signal peptide deletion and full-length pigment epithelium-derived factor transfection HeLa cells and hepatocytes promoted triglyceride degradation. Intracellular pigment epithelium-derived factor co-immunoprecipitated with adipose triglyceride lipase and promoted triglyceride degradation in an adipose triglyceride lipase-dependent manner. Additionally, pigment epithelium-derived factor bound to the C-terminal of adipose triglyceride lipase (aa268–504) and adipose triglyceride lipase-G0/G1 switch gene-2 complex simultaneously, which facilitated adipose triglyceride lipase-G0/G1 switch gene-2 translocation onto lipid droplet using bimolecular fluorescence complementation assay. Moreover, knockdown of endogenous pigment epithelium-derived factor in hepatocytes diminished triglyceride degradation. Taken together, these results indicate that hepatic pigment epithelium-derived factor was decreased in obese mice accompanied with hepatic steatosis. Intracellular pigment epithelium-derived factor binds to and facilitates adipose triglyceride lipase translocation onto lipid droplet, which promotes triglyceride degradation. These findings suggest that a decreased level of hepatic pigment epithelium-derived factor may contribute to hepatic steatosis in obesity.

Effect of anluohuaxian tablet combined with γ-IFN on schistosomal liver fibrosis

The therapeutic effects of anluohuaxian tablet combined with gamma-IFN on schistosomal liver fibrosis and its mechanism were studied in a murine model and clinical cases of schistosomal liver fibrosis. Fifty Kunming mice were randomly divided into 5 groups: normal control group, infection control group, anluohuaxian tablet-treated group, gamma-IFN-treated group and combined treatment (anluohuaian tablet+gamma-IFN) group. Pathologic changes in liver, including hepatic pigmentation and the size of schistosomal egg granuloma, were observed by HE staining after treatment for 8 weeks. The expression of the type I and collagen III, and TIMP-1 was detected by immunohistochemistry. TGF-beta1 mRNA expression was examined by real-time fluorescent quantitative PCR. Sixty patients with schistosomal liver fibrosis were divided into treatment group and control group. The patients in treatment group were treated with anluohuaxian tablet in combination with gamma-IFN for 6 months. Before and after treatment, the changes of symptoms and signs, liver function, serum liver fibrosis indexes and imaging indexes were observed. The results showed that as compared with infection control group, all forms of treatments relieved the hepatic pathological injury with apparently diminished size of schistosomal egg nodules and decreased percentage of pigmentation (P<0.05). Furthermore, the expression of collagen I and III, TIMP-1, and TGF-beta1 mRNA in combined treatment group was significantly decreased as compared with anluohuaxian tablet-treated and gamma-IFN-treated groups (P<0.05). In the clinical observation, the serum liver fibrosis indexes, the portal vein width as well as the spleen thickness was significantly reduced in treatment group as compared with control group (P<0.05). It was concluded that the combined use of anluohuaxian tablet with gamma-IFN in schistosomal liver fibrosis could protect liver function, alleviate liver fibrosis, and could be used as a choice in treating patients with schiatosomal liver fibrosis.

Ethanol Exposure Depletes Hepatic Pigment Epithelium-Derived Factor, a Novel Lipid Regulator

Ethanol abuse can lead to hepatic steatosis and evolve into cirrhosis and hepatocellular carcinoma. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein that is expressed by hepatocytes. Proteomic, experimental, and clinical studies implicate PEDF's role in lipid regulation. Because matrix metalloproteinase (MMP)-2/9 activity regulates PEDF levels, we investigated whether PEDF degradation by MMPs has a permissive role in ethanol-induced hepatic steatosis. PEDF levels were examined in liver biopsy specimens from patients with ethanol-induced steatosis. Hepatic PEDF levels and MMP activity were assessed in 2 animal models of ethanol feeding (rats on an alcohol-containing liquid diet and mice given intragastric infusion of ethanol). The consequences of PEDF depletion in the liver were examined in PEDF-null mice. Liver biopsy samples from patients with ethanol-induced steatosis had reduced PEDF levels, compared with normal liver samples. Ethanol-fed animals had histologic steatosis and increased liver triglyceride content (P< .05), as well as reduced levels of hepatic PEDF and increased MMP-2/9 activity. Ethanol-exposed hepatic lysates degraded PEDF in a MMP-2/9-dependent manner, and liver sections demonstrated abundant MMP-2/9 activity in situ. Addition of recombinant PEDF to PEDF-null hepatocytes, reduced their triglyceride content. Ethanol exposure leads to marked loss of hepatic PEDF in human livers and in 2 animal models of ethanol feeding. Loss of PEDF contributes to the accumulation of lipids in ethanol-induced hepatic steatosis.

Hepatic pigment cells of Proteidae (Amphibia, Urodela): A comparative histochemical and ultrastructural study

AbstractAmphibian liver pigment cells, also known as Kupffer cells, contain various amounts of melanin, haemosiderin and lipofuscin. We used different histochemical and ultrastructural methods to analyse and compare the level of hepatic pigmentation and the structure of hepatic pigment cells in the livers of three representatives of the family Proteidae; two subspecies of the hypogean Proteus anguinus (depigmented Proteus a. anguinus and pigmented Proteus a. parkelj) and the epygean Necturus maculosus. Our analysis revealed differences at histological and ultrastructural level. While the percentage of the pigmented area and ultrastructural characteristics are similar in both subspecies of P. anguinus, great differences occur in the amount and structure of the pigment cells between P. anguinus and N. maculosus. Pigment cells are more numerous and compose larger pigmented clusters in P. anguinus. They are structurally more heterogeneous and contain a larger amount of haemosiderin when compared to N. maculosus. Our results confirm a high degree of variation in hepatic pigmentation among different amphibian species. Because many factors influence the level of hepatic pigmentation in poikilotherms, differences among species from different habitats and also among individuals of the same species are expected but are not easily explained. We propose two possible explanations for the large amount of iron present in Proteus anguinus: (1) accumulation of pigments due to the very low metabolic rate and extended lifetime; (2) large iron storage capacity as an adaptation to a low and discontinuous food supply in caves.

Melatonin, melanogenesis, and hypoxic stress in the newt, Triturus carnifex.

Groups of 6 specimens each of the newt Triturus carnifex were treated with melatonin to see if the hormone inhibited melanogenesis in the Kupffer cells of the liver (melanomacrophages), a process markedly stimulated by hypoxia. A dose of 500 microg/g in 27% ethanol, injected intraperitoneally, induced loss of consciousness and tetany of all the skeletal muscles, which on the contrary appeared relaxed in animals pre-anesthetised by immersion in chlorbutol at 0.2%. Anesthetised specimens injected with melatonin showed a significantly lower increase in hepatic pigmentation after acute hypoxia, a condition attained by sealing each specimen in a 620 mL respiratory chamber with water containing 1.1 ppm of oxygen for the time needed to consume it all (about two hours). If hypoxia is reached gradually, beginning with 8 ppm of oxygen (normoxic condition), the increase in hepatic pigmentation after melatonin injection does not differ significantly from that of non-hormone treated specimens: thus melatonin does not seem to play a direct part in controlling hepatic melanogenesis. Instead, the hormone induces significant increase in oxygen consumption, marked general steatosis of the liver and the almost total disappearance of glycogen. Intraperitoneal injection of 500 microg/g of melatonin in anesthetised animals exposed to the air (normoxic) also causes severe steatosis and an unexpected increase in the hepatic deposits of melanin, as after hypoxic treatment. A dose of 100 ng/g in 1% ethanol, ineffective when injected intraperitoneally, also induces these effects if injected directly into the arterial blood-stream through the conus arteriosus, thus avoiding the hepatic filter. The phenomena observed appear to be induced by a powerful endocrine mechanism that provokes metabolic hypoxia by consuming all the available ATP for synthesizing fat. A less intense form of steatosis can also be observed in animals subjected to hypoxia but without prior hormone treatment, indicating that a natural process triggered by hypoxic stress is pushed to the extreme by exogenous melatonin: the hormone changes the entire energy metabolism of the organism so that it can survive for a long time under adverse environmental conditions.

Changing concepts of haemochromatosis.

In the 110 years since its original description haemochromatosis has been variously considered to be a form of alcoholic cirrhosis, a result of diabetes and an inherited metabolic defect of iron metabolism (Figure 1). The association between cirrhosis, hepatic pigment deposits and diabetes mellitis was recognized first by Troisier M. (1871) “la cirrhose pigmentaire dans le diabete sucre”, Hanot (1886) suggested the term “bronze diabetes” for the disorder because he believed that the diabetes was of primary importance causing both the cirrhosis and skin pigmentation. It was von Recklinghausen (1889) who proposed the term haemochromatosis because he believed that the pigment came from the blood. Although strictly a misnomer this term has been used ever since to describe the syndrome of iron loading associated with tissue injury.

Hepatoid adenocarcinoma of the renal pelvis producing alpha-fetoprotein of hepatic type and bile pigment

A right renal pelvic mass in a 72-year-old man was resected. The histologic appearance of the tumor was a mixture of tubular adenocarcinoma cells and hepatoid neoplastic cells, and there was a resemblance to hepatoid adenocarcinoma. The intraoperative level of serum alpha-fetoprotein (AFP) was calculated to be 2246 ng/ml, and the postoperative level ranges from 183.6 to 285.6 ng/ml. Lectin binding assays showed that the serum AFP was the hepatic carcinoma type. In a hepatoid portion, an iron-negative, brown to green pigment was positive for bile. Alpha-fetoprotein was immunohistochemically evident in the neoplastic cells. In addition to the hepatic differentiation, the tumor had differentiated into intestinal absorptive or pancreatobiliary tract cells, as deduced from the frequent presence of spicular bodies, a unique light microscopic feature equivalent to microvilli with an actin core. The hepatoid adenocarcinoma is a distinct type of AFP-producing carcinoma present in the organs with epithelium of endodermal origin. Hepatoid adenocarcinoma in the renal pelvis may arise from a metaplasia of neoplastic mesonephric cells into endodermal cells.

Hepatic handling of photoirradiated bilirubin a study in isolated perfused Wistar rat liver

Conjugation has been considered the rate-limiting step for bilirubin hepatic transport, and bypass of this metabolic step could explain why photobilirubins can be rapidly cleared by the liver. In this paper we assessed whether photoirradiation may enhance the bilirubin overall hepatic transport in the isolated perfused Wistar rat liver, a model possessing intact transport and conjugating systems. Bilirubin was administered as a bolus so as to reach a perfusate concentration of approximately 10 microM (bilirubin/albumin molar ratio 1:17). Perfusate light exposure (0.56.10(15) quanta s-1 cm-2) yielded 7-10% of configurational photoisomers, which were further identified as (4Z,15E/4E,15Z)-bilirubin IX alpha. Under such conditions, the perfusate removal rate was increased by 39% over that from dark conditions. Likewise, biliary excretion, estimated as total bilirubin recovery at 60 min, was also increased (+48%). This later improvement was mainly produced at the expense of unconjugated bilirubin, which most likely derived from its configurational photoisomers that, once excreted into bile, readily re-isomerized to the parent compound. In addition, this increment was partially due to a delayed improvement of monoglucuronide pigment excretion. The calculated hepatic pigment content was significantly higher under light conditions. A direct assessment of hepatic content of different bilirubin moieties at 20 min after bilirubin administration confirmed that such an increment was fully accounted for by unconjugated pigment. Our finding that hepatic pigment content rose (despite a higher biliary excretion) when the bilirubin was irradiated suggests a higher net uptake of photoisomers than native pigment. This observation, and the finding that bilirubin photoisomers were usually excreted without undergoing conjugation even if the metabolic system is active, contribute to explain the greater appearance of unconjugated bilirubin in Wistar rat bile under light exposure.

Toxicity Studies of Intravenous Vitamin E in Newborn Rabbits

This study was designed to investigate the toxicity of intravenously administered alpha-tocopherol, alpha-tocopheryl acetate, and a polysorbate vehicle similar to that used in a commercial preparation of alpha-tocopheryl acetate intended for intravenous administration. Cesarean-delivered newborn rabbits fed intravenously were administered 100 mg of either alpha-tocopherol or alpha-tocopheryl acetate in a polysorbate vehicle, or the vehicle only, or no treatment for 6 or 7 days. Two intravenous diets were employed which differed in nutritional content and were termed low energy (LE) and high energy (HE). High concentrations of alpha-tocopherol were present in the tissues of all pups that received either treatment with alpha-tocopherol or alpha-tocopheryl acetate in the polysorbate vehicle, irrespective of the nutritional regimen. Pups in all treatment and control groups which received the LE diet had hepatic centrilobular degeneration, necrosis and pigment accumulation, which was attributed to malnutrition. No additional toxicities could be attributed to the vitamin E or polysorbate treatments. Administration of the HE diet eliminated the nutrition-related centrilobular degeneration, and revealed treatment-related liver changes. HE pups treated with alpha-tocopheryl acetate in the polysorbate vehicle had microscopic evidence of mild bile stasis and had elevated serum bilirubin. Minimal lipidosis or fatty change in the liver was observed only in alpha-tocopherol and alpha-tocopheryl acetate-treated pups. Lipidosis in the spleen was moderate in the alpha-tocopherol group and minimal in the alpha-tocopheryl acetate group. Lipidosis also occurred in the adrenal gland primarily in the alpha-tocopheryl acetate group.(ABSTRACT TRUNCATED AT 250 WORDS)

8 The clinical manifestations of chronic iron overload

Primary or genetic haemochromatosis is an inherited disease characterized by an inappropriate degree of iron absorption with accumulation of excessive amounts of tissue iron. Parenchymal iron accumulation results in the typical clinical features of the disease including hepatic cirrhosis, diabetes, testicular atrophy and skin pigmentation. The disease is inherited in an autosomal recessive manner. The gene for the disease has not been identified but is tightly linked to the A locus of the histocompatibility complex on chromosome 6. The approximate homozygote frequency in Caucasians is 0.3% with an equal sex ratio. Excessive body iron stores have been described in a number of other conditions, particularly alcoholic liver disease. There is increasing evidence that many of these individuals are in fact also suffering from genetic haemochromatosis. Diagnostic tests including serum iron, transferrin saturation, serum ferritin and liver iron concentration make it possible to detect sufferers of the disease. Screening relatives of affected individuals with these tests allows a diagnosis to be made before permanent tissue damage has occurred. Removal of excess iron stores by repeated phlebotomy is the primary treatment. If iron is removed before significant tissue damage has occurred, the complications and natural course of the disease will be prevented provided reaccumulation of iron does not occur. Excessive iron accumulation with resultant organ damage also occurs in anaemias associated with ineffective erythropoiesis and after excessive parenteral iron administration or repeated blood transfusions. Similar clinical features may be seen. Chelation therapy is the mainstay of treatment in these cases where long-term venesection is not possible.

2,2-Dialkyl-1,2-dihydroquinolines: cytochrome P-450 catalyzed N-alkylporphyrin formation, ferrochelatase inhibition, and induction of 5-aminolevulinic acid synthase activity.

Incubation of 2,4-diethyl-1,2-dihydro-2-methylquinoline (DMDQ) with hepatic microsomes from rats pretreated with phenobarbital, 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile, or dexamethasone results in minor loss of the cytochrome P-450 chromophore and accumulation of a hepatic pigment. The hepatic pigment consists of the four regioisomers of N-ethylprotoporphyrin IX and minor amounts of the corresponding N-methyl regioisomers. Exposure of chick embryo liver cells to DMDQ results in inhibition of their ferrochelatase activity, induction of their 5-aminolevulinic acid synthase activity, and accumulation of protoporphyrin IX. 1,2-Dihydro-2,2,4-trimethylquinoline (TMDQ) causes negligible loss of cytochrome P-450 in rat liver microsomes but in vivo still produces the four N-methylprotoporphyrin IX regioisomers in low yield. Furthermore, it inhibits ferrochelatase activity, elevates 5-aminolevulinic acid synthase activity, and causes protoporphyrin IX accumulation in cultured chick embryo hepatocytes. One-electron oxidation of the 2,2-dialkyl-1,2-dihydroquinolines to radical cations is postulated to result in N-alkylation of the prosthetic heme group of cytochrome P-450. The N-alkylprotoporphyrins IX thus formed are potent inhibitors of ferrochelatase. Inhibition of ferrochelatase causes the induction of 5-aminolevulinic acid synthase and the accumulation of protoporphyrin IX. Heme alkylation and ferrochelatase inhibition may be generally associated with substrates that are subject to cytochrome P-450 mediated oxidative extrusion of alkyl radicals.

Hereditary chronic conjugated hyperbilirubinemia in mutant rats caused by defective hepatic anion transport.

A mutant rat strain is described with autosomal recessive conjugated hyperbilirubinemia. Transport of conjugated bilirubin and tetrabromosulfophthalein from liver to bile is severely impaired whereas uptake of organic anions from plasma to liver is normal. During the first 10 days of life, serum bilirubin levels are 147 +/- 11 mumoles per liter with 68.7% diconjugates and 27.9% monoconjugates. In adult rats, serum bilirubin is 33 +/- 8 mumoles per liter with 81.8% diconjugates and 12.1% monoconjugates vs. 0.3 +/- 0.1 mumole per liter unconjugated bilirubin in normal adult rats. Bile acid metabolism is only mildly affected. In young rats, serum bile acid levels are normal. In adult rats, bile acid levels are elevated to 49 +/- 11 mumoles per liter vs. 10 +/- 6 mumoles per liter in normal rats. The bile flow in mutant rats is reduced to about 50%. This might be caused by a reduction of the bile acid-independent bile fraction. Liver marker enzyme activities in mutant rat serum are normal. Liver morphology is also normal. Total urinary coproporphyrin excretion is not elevated but urinary coproporphyrin isomer I excretion is increased, a pattern like that in Dubin-Johnson syndrome in humans. However, unlike Dubin-Johnson syndrome, the mutant rats do not have the characteristic black hepatic pigment. These rats provide a unique model to study mechanisms of bile formation and cholestasis.

A preliminary report on hepatic pigmentation in Falkland Islands sheep.

Pigmentation of hepatocytes and Kupffer's cells with lipofuscin and melanin-like pigment is described. The occurrence of pigmented reticulo-endothelial cells in the lungs and lymphoid tissue was also noted. No discernible impairment of function accompanied the pigmentation.

N-Methylprotoporphyrin IX: chemical synthesis and identification as the green pigment produced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine treatment.

The hepatic pigment accumulated in 3,5-diethoxycarbonyl-1,4-dihydrocollidine-treated rats, which has been reported to inhibit ferrochelatase, has been isolated and purified. The pigment has been resolved into one major, one minor, and two trace components, all of which appear to be isomeric porphyrins. The major fraction has been unambiguously identified by spectroscopic methods as the isomer of N-methylprotoporphyrin IX (isolated as the dimethyl ester) in which vinyl-substituted pyrrole ring A is methylated. The minor product appears to be an isomer of the same porphyrin with the N-methyl group on propionic acid-substituted ring C, and the trace components have the same high-pressure liquid chromatography retention times as the other two possible isomers of the porphyrin. The four isomers of N-methylprotoporphyrin IX have been chemically synthesized, independently characterized, and used to confirm the structures of the biologically products.

On the Nature and Excretion of the Hepatic Pigment in the Dubin-Johnson Syndrome

A dark hepatic pigment occurs in the Dubin-Johnson Syndrome (DJS) and it has been suggested but not proved that it is melanin. if acute viral hepatitis (AVH) complicates DJS, the pigment disappears from the liver and reappears after recovery. The unusually dark urine seen in a DJS patient with AVH suggested urine as the possible site of pigment excretion. We have used a sensitive new method for detecting melanin to investigate these problems. The method is based on the fact that melanin is an unusually stable free radical whose electron spin resonance (ESR) spectrum is characteristically altered by visible light, pH changes, zinc(II) and paramagnetic trace elements. Liver and urine specimens were examined in (a) a DJS patient during and after AVH, (b) patients with uncomplicated DJS, (c) patients with AVH without DJS, (d) patients with other types of hepatitis, and (e) patients with apparently normal livers. The ESR spectra indicated the absence of melanin in all specimens. A stable free radical with unusual properties was found in the liver of patients with uncomplicated DJS. This was absent from the liver of a DJS patient studied during acute hepatitis but was present in his urine. It disappeared from his urine during and after recovery from hepatitis. We conclude that the hepatic pigment in DJS is: (a) not melanin; (b) excreted in the urine during acute hepatitis; (c) a stable free radical; and (d) not present in the liver or urine in any of the other diseases that were studied.

Self-catalyzed destruction of cytochrome P-450: covalent binding of ethynyl sterols to prosthetic heme.

The hepatic pigment accumulated as a consequence of the self-catalyzed destruction of cytochrome P-450 by norethisterone (17-hydroxy-19-nor-17 alpha-pregn-4-en-20-yn-3-one), after acidic methylation and purification, consists of two virtually identical, probably isomeric, porphyrins. Radiolabeled norethisterone is incorporated into both porphyrin products. The major of the two porphyrins exhibits a mass spectrometric molecular ion exactly equivalent to the sum of norethisterone and dimethylprotoporphyrin IX, less two hydrogen atoms: unequivocably demonstrating covalent association of the sterol with this porphyrin in a 1:1 ratio. Cytochrome P-450 is therefore destroyed by self-catalyzed addition of norethisterone to its heme prosthetic group. Cytochrome P-450 is also destroyed by norgestrel (13-ethyl-17-hydroxyl-18, 19-dinor-17 alpha-pregn-4-en-20-yn-3-one) and by 1-ethynylcyclohexanol but not by 17-hydroxy-19-nor-17alpha-pregn-4,20-dien-3-one. The destructive potential is thus clearly a property of the propargylic alcohol function. A mechanism involving enzymatic oxidation of the triple bond is postulated.