Sites of formation of the serum proteins transferrin and hemopexin.

Sites of synthesis of hemopexin and transferrin were determined by culturing various tissues of rabbits and monkeys in the presence of labeled amino acids. Labeling of the serum proteins was examined by means of autoradiographs of immunoelectrophoretic patterns as well as by precipitation in the test tubes employing immunospecific antisera. Good correlation was seen between the results obtained by the two different methods. The liver was found to be the only site of many tissues studied that synthesized hemopexin. Transferrin production was observed in the liver, submaxillary gland, lactating mammary gland, testis, and ovary.

Studies on the efflux of heme from biological membranes.

It is unknown how heme is distributed intracellularly from its site of synthesis in the mitochondria to other organelles. In previous work (Biochemistry 23, 3715, 1984) the transfer of heme from lipid bilayers to soluble proteins had been found to be independent of the recipient proteins' affinity for heme. Here, we investigated whether proteins are involved in the transfer of heme from biological membranes into aqueous media. We followed the release of 14C-labeled heme, from mitochondria preloaded with the heme, to BSA and found that only about 28%, of the heme was extracted on the first wash. After the third wash 35-50% of the heme that had been partitioned into the membranes was extracted. Fourth and fifth washes with BSA or a cytosolic heme-binding protein (HBP, also known as liver fatty acid binding protein) removed only insignificant amounts of 14C-labeled heme. Similarly, a large portion of the preloaded 14C-labeled heme could not be extracted from a variety of isolated membranes (inner and outer mitochondrial membranes, plasma membranes of liver cells, kidney cortex cells and erythrocyte membranes). By contrast, essentially all [14C]palmitate preloaded in biological membranes and all 14C-labeled heme preloaded in synthetic membranes was released to albumin (Biochemistry 23, 3715, 1984). These observations suggest that, in general, heme associates with membrane components which can be distinguished into two compartments. One compartment releases its heme spontaneously, while another compartment binds heme so tightly that a specific process has to be evoked for its release.

Effect of porphyrinogenic agents on protein synthesis and bilirubin formation by the isolated perfused rat liver.

We established an isolated rat liver perfusion system for the study of heme catabolism. The liver of rats fasted for 48 h is perfused with an erythrocyte-free medium. Ultrastructural analysis shows integrity of all subcellular organelles with the exception of minor alterations in the rough endoplasmic reticulum. The perfused liver synthesizes serum proteins at a constant rate for 5 h. Albumin is secreted at a mean rate of 17 +/- 2 mg/h per 100 g liver, hemopexin at 5.0 +/- 0.7, haptoglobin at 3.2 +/- 0.6 and transferrin at 5.1 +/- 0.8 mg/h per 100 g liver. The mean ratio of ATP : ADP is 3.5 +/- 0.1, and that of lactate: pyruvate 27 +/- 6. The rate of conversion of heme into bilirubin is comparable to that reported for in vivo studies. A minimal effect on protein synthesis is observed after administration of the porphyrinogenic agents, allylisopropylacetamide (AIA) and 3,5-diethoxycarbonyl-1,4 dihydrocollidine (DDC). Pretreatment of the rats with the iron chelator, Desferal, causes a 3-4-fold increase in hemopexin but not in albumin and transferrin synthesis. A striking 2-3-fold enhancement of bile bilirubin production follows treatment with DDC and Desferal, but not with AIA. The amount of bilirubin formed from heme added to the perfusate is reduced by AIA and DDC and enhanced by Desferal treatment. It is proposed that unavailability of iron in a certain hepatic tissue pool causes protoporphyrin IX accumulation which may serve as an alternate source for bilirubin production.

Transfer of heme from heme-albumin to hemopexin.

Exchange of heme in vitro between two heme-binding serum proteins, albumin and hemopexin, was examined spectrophotometrically. Hemopexin, albumin and heme in molar ratios of 1 : 70 : 1 were incubated at 22 degrees C, pH 7.3. The heme was added as free heme, heme-hemopexin or methemalbumin. Due to the high affinity of hemopexin for heme, Kd near 10(-13) M, only negligible amounts of heme were transferred from hemopexin to albumin in 48 h. However, more than 80% of heme was transferred from methemalbumin to hemopexin within 24 h. Heme added to a 1 : 70 mixture of the apo-proteins is initially bound by albumin; but more than 90% is bound by hemopexin in 24 h. Addition of dithionite causes nearly all of the heme present, whether added as free heme or methemalbumin, to associate with hemopexin in 15 min. Albumin thus appears to have a much lower affinity for ferro- than for ferri-heme. Results obtained from similar experiments with human serum and human serum made hemopexin-free by immunoadsorption fully corroborate those obtained with mixtures of purified albumin and hemopexin. These observations suggest that the rate-limiting step in the heme transport function of hemopexin is the formation of the heme-hemopexin complex, rather than the uptake of the complex by the liver.

Effects of hexachlorobenzene and iron loading on rat liver mitochondria.

The effects of hexachlorobenzene treatment and simultaneous iron-overload on the iron and porphyrin content of rat liver and rat liver mitochondria have been examined. In order to assess damages to the mitochondrial membrane occurring with these treatments, the content of malondialdehyde and selected functional properties of mitochondria were compared with those from control animals. Prolonged intake of hexachlorobenzene (8 weeks) resulted in a strikingly increased level of porphyrins together with a moderate increase in iron concentration. Simultaneous administration of hexachlorobenzene and iron-dextran caused the porphyrin level to reach 25% of the amount induced by hexachlorobenzene alone. The iron concentrations in liver as well as in liver mitochondria are also decreased under these conditions, as compared to the effect of iron-dextran. In contrast, the effects of hexachlorobenzene combined with iron-dextran on mitochondrial oxidative phosphorylation and malondialdehyde content are greater than those of either hexachlorobenzene or iron-dextran. These data suggest that porphyrin accumulation per se causes little deleterious effect and that both agents administered together act synergistically in causing damage to the mitochondrial membrane.

Griseofulvin causing hyperhemopexinemia and hepatic proliferation in mice: an in vivo and in vitro study.

Concentrations of hemopexin, a porphyrin-binding serum protein synthesized exclusively in the liver, increased significantly and concomitantly with levels of erythrocyte and liver protoporphyrin and coproporphyrin in mice made porphyric with 1% griseofulvin in the feed. Liver weights of porphyric mice increased remarkably. In comparison with controls, the ratio of the weight of normal to porphyric livers was at 10 days 1:2.1, at 21 days 1:2.8, and at 46 days 1:3.8. This increase in liver size was accompanied by increased cell division. The hepatic hyperplastic tissue fragments survived in vitro for several weeks and could be subcultured. The cultured cells, like those of the original liver, showed intense protoporphyrin fluorescence in the cytoplasm.

Initial rate of sodium taurocholate uptake in isolated elutriated hepatocytes from untreated and phenobarbital-treated rats.

The initial rate of sodium taurocholate uptake was measured in rat hepatocytes separated by centrifugal elutriation into five cell fractions whose difference in size was verified by flow cytometry. The hepatocytes were prepared from untreated and phenobarbital-treated rats. For untreated animals, the initial rate of taurocholate uptake at concentrations of 5 or 50 microM was the same for hepatocytes prior to fractionation and for each of the five elutriated fractions. Treatment of the animals with phenobarbital was associated with a significant increase in hepatocyte size in all fractions and caused a significant increase in the initial uptake rate. The extent of the rate increase in hepatocytes prior to fractionation was similar to that observed for each of the five hepatocyte subpopulations. Our observation indicates that phenobarbital causes a significant increase in the initial rate of sodium taurocholate uptake and suggests that large and small hepatocytes possess no inherent differences controlling the initial uptake process.

Heterogeneous distribution of drug metabolism in elutriated rat hepatocytes.

Centrifugal elutriation was used to separate isolated rat hepatocytes into five fractions containing cells of different sizes. These fractions were compared with regard to cell size, morphology and function. Analyzed by flow cytometry, the small cells were found to be enriched in fraction 1 and the large cells in fraction 5. Evaluation by light and electron microscopy indicated that the fractions contained single hepatocytes of normal structure. The cytochrome P-450 content and the 7-ethoxycoumarin O-deethylase activity were assessed in hepatocytes from untreated animals, those treated with phenobarbital, and those treated with phenobarbital plus allylisopropylacetamide. In both untreated and phenobarbital-treated animals, cytochrome P-450 content and 7-ethoxycoumarin O-deethylase activity rose significantly from fraction 1 to fraction 5. The P-450 content gradually rose up to 2-fold. The enzyme activity rose 5-fold, and it increased steeply between fractions 2 and 3. The cytochrome P-450 content in phenobarbital-plus-allylisopropylacetamide-treated animals was decreased in all fractions but more extensively in fraction 5 than in fraction 1.

Evidence that variation among untreated rabbits in hepatic progesterone 21-hydroxylase activity is indicative of enzyme heterogeneity rather than a transient inductive effect.

Previous work has shown that the 21-hydroxylation of progesterone in the hepatic microsomal fraction of outbred NZW rabbits varies over a tenfold range. In contrast, the 16-hydroxylase activity is relatively constant and is not correlated to the activity of the 21-hydroxylase. The distribution of the 21-hydroxylase activity is roughly bimodal with about one-third of the animals (21-H) exhibiting 21-hydroxylase activity exceeding 1 nmol/min/mg microsomal protein, whereas the remainder (21-L) generally exhibit an activity that is less than 1 nmol/min/mg protein. To determine if this was due to a transient phenomenon, liver punch biopsies were collected from 28 rabbits at intervals of approximately three weeks for at least three serial samples. The 21- and 16-hydroxylase activities were determined in the postmitochondrial fractions of these biopsy samples. A substantial variability in both 21- and 16-hydroxylase activities was observed for serial biopsy samples from individual rabbits. The variation of the 16-hydroxylase activity paralleled, however, that of the 21-hydroxylase, thus suggesting that the variation between biopsy samples for individual rabbits was due to factors such as contamination with blood and connective tissue, which would affect both activities equally. Rabbits, therefore, were phenotyped as 21-H or 21-L on the basis of the 21/16-hydroxylase ratio. The mean ratio was 3.2 +/- 1.2 and 0.8 +/- 0.3 for rabbits phenotyped as 21-H and 21-L, respectively. Similar values for this ratio were obtained for the other group of rabbits phenotyped as 21-H and 21-L, 3.8 +/- 1.6 and 0.5 +/- 0.2, respectively, following the isolation of microsomes from whole liver homogenates. The ratio of 21/16-hydroxylase activity was found to be relatively constant for biopsy samples obtained from the same animal over the course of this study, thus indicating that the elevated 21-hydroxylase activity is not a transient phenomenon.

Catabolism of homologous and heterologous hemopexin in the rat and uptake of hemopexin by isolated perfused rat liver.

Iodinated hemopexin (Hx) from three different species, rabbit, human and rat, was injected into rats and its clearance from the plasma measured. Rabbit, human and rat apo-Hx were cleared from the plasma with a T 1/2 of 20--31 h, 31--32 h, and 48--60 h respectively. Heme injection (10 mg/kg) after equilibration of the protein immediately accelerates elimination of the hemopexins of all three species (T 1/2 of 4.5 to 10 h). This indicates that the two heterologous hemopexins maintain their function in heme transport. The T 1/2 of rabbit and human Hx return to pre-heme injection values 16 to 20 hours after the injection of heme. For rat Hx, however, the T 1/2, which was 54 +/- 3.5 h before heme injection, was reduced to 25 +/- 1.3 h 20 hours after heme injection. Administration of 1.2--1.3 mg of protoporphyrin IX or uroporphyrin III, after equilibration of iodinated rat Hx, did not change the T 1/2 of the protein, whereas the same amount of coproporphyrin III significantly reduced its T 1/2 from 54 +/- 3.5 to 37 +/- 0.4 h. In addition, the uptake of rat apo-Hx, heme-Hx and albumin by rat liver tissue was measured in an isolated liver perfusion system using radioiodinated proteins screened in vivo. The uptake of apo-Hx by the liver after 2 h (46.8 ml/100 g) was less than that of heme-Hx (67.3 ml/100 g). The amount of apo-Hx and heme-Hx associated with the liver, relative to that circulating in the perfusate, was greater than that of albumin (12.1 ml/100 g). These results are considered to represent selective uptake of Hx by the liver induced by its interaction with heme.

Hepatic uptake, intracellular accumulation and biliary secretion of 5-methyltetrahydrofolate.

Transport of the reduced folate coenzyme, 5-methyltetrahydrofolate, from plasma to bile was studied in the isolated perfused rat liver. The system was inhibited by metabolic poisons, pteroylglutamate, 10-formylfolate, and amethopterin. The coenzyme was concentrated in hepatic parenchyma 1.3 fold over the concentration in the medium. After an initial delay of approximately 20 min the biliary secretion of the coenzyme nearly paralleled the hepatic uptake, and the coenzyme was concentrated in bile 15-19 times above the perfusion medium. The transport process was saturable with a Kt value of 0.45 mM and Vmax of 0.66 mumoles/hr/g liver. Chromatography of the bile revealed minimal biotransformation of the secreted coenzyme. In experiments in vivo 5-methyltetrahydrofolate was absorbed readily from the intestine, as demonstrated by the urinary excretion of 32-40% of the dose within 48 hr after peroral administration. These studies indicate that hepatic uptake and biliary secretion of 5-methyltetrahydrofolate occurs by an energy-dependent, carrier-mediated process in which the coenzyme accumulates in the liver, is secreted into bile against a high concentration gradient and undergoes enterohepatic circulation.

Secretion of haem by hepatic parenchymal cells.

Hepatic parenchymal cells in primary culture, and also the intact perfused liver, secrete newly synthesized haem into extracellular fluids. In cultures incubated with the haem precursor delta-amino[4-14C]laevulinate, labelled haem was formed at a linear rate for at least 8 h, and 10-20% of the total labelled haem was present in the culture medium. The appearance of labelled extracellular haem was proportional both to the concentration of labelled precursor offered to the cells and to the time of incubation. Similar results were obtained when [2-14C]glycine was added as haem precursor. Studies with the isolated perfused liver indicated that newly synthesized haem is secreted also by the intact liver. Approximately equal amounts of haem appeared in the bile and in perfusate. The findings are discussed in relation to the pathogenesis of symptoms in the hereditary hepatic porphyrias.

Cellular and subcellular localization of heme and hemopexin in the rabbit.

The cellular distribution of intravenously administered 3H-heme, 125I-hemopexin and 125I-albumin was studied in rabbits. Radioautography of the tissue slides of several organs was examined by light and electron microscopy. All experiments were terminated 60 min after injection of the isotope-labeled materials. 3H-heme and native monomeric 125I-hemopexin (heme saturated) were found to be associated with the endoplasmic reticulum and microbodies of the hepatocytes. Aggregated hemopexin was also taken up by macrophages of lung alveoli, splenic pulp, interstitial cells of the kidney and Kupffer cells. 125I-albumin (heme saturated) could not be located in liver, spleen, bone marrow, lung, or kidney tissues.

The effect in vivo and in vitro of allylisopropylacetamide on the content of hepatic microsomal cytochrome P-450 2 of phenobarbital treated rabbits.

Rabbits treated with phenobarbital were given a single injection of allylisopropylacetamide (AIA) s.c. and/or heme i.v. Hepatic microsomes were isolated 1, 5 and 24 hours post injection and the microsomal contents of both total cytochrome P-450 chromophore, and the protein moiety of P-450 2 were determined by spectrophotometric and immunochemical methods respectively. AIA caused the levels of total P-450 chromophore and of P-450 2 protein to decline to 30% of the control values at 5 hours post-injection. Concurrent administration of heme with AIA diminished the decrease in the total microsomal content of P-450 chromophore but not in that of P-450 2 protein. These findings suggest that the destruction of the heme prosthetic group of P-450 by suicide substrates such as AIA may lead to an enhanced degradation of the apo-P-450.