Acta haemat. 53: 219-225 (1975)
Cellular and Subcellular Localization of Heme and Hemopexin in the Rabbit H. H eng L iem , M ehdi T avassoli and U rsula M uller-E berhard Departments of Biochemistry and Hematology, Scripps Clinic and Research Foundation, and the Department of Pediatrics, University of California at San Diego, La Jolla, Calif.
Key Words. Albumin • Autoradiography • Heme metabolism • Hemopexin • Li ver ultrastructure
Hemopexin, a serum /(-glycoprotein, serves as a carrier of heme in the blood [12], It binds heme with a much greater avidity than albumin [16], Heme is transported to the liver, where it is degraded mainly to bilirubin [14]. Previous work from our laboratory [8] has shown that the hemehemopexin complex is removed exclusively by hepatocytes and not by the reticuloendothelial elements of the liver (Kupffer cells). In the present re port, we have extended our investigations to the subcellular localization of 3H-heme and 125I-hemopexin. We have furthermore shown that aggre gated hemopexin is taken up by the reticuloendothelial system throughout the body. In addition, we have shown that lî5I-albumin is not removed by the hepatocytes during the process of plasma heme clearance.
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 12:32:41 AM
Abstract. The cellular distribution of intravenously administered 3H-heme, ,25Ihemopexin and ,25I-albumin was studied in rabbits. Radioautography of the tissue slides of several organs was examined by light and electron microscopy. All experi ments were terminated 60 min after injection of the isotope-labeled materials. 3Hheme and native monomeric ,2!I-hemopexin (heme saturated) were found to be asso ciated with the endoplasmic reticulum and microbodies of the hepatocytes. Aggre gated hemopexin was also taken up by macrophages of lung alveoli, splenic pulp, interstitial cells of the kidney and Kupffer cells. l2!I-albumin (heme saturated) could not be located in liver, spleen, bone marrow, lung, or kidney tissues.
220
H eng L iem /T avassoli/M uller -E berhard
Animals. New Zealand white rabbits, 0.8-1.1 kg, were purchased from Rancho de Conejo, Calif. Several days prior to the experiment, Lugol’s solution was added to the drinking water. 3H-heme preparation. Heme was labeled in vitro with 3H-aminolevulinic acid as previously described [11]; the specific activity was 4X107 dpm/mg. Heme solutions were freshly prepared and 1 ml serum was admixed. Approximately 65 fig 3H-heme were injected. Purification and isotope-labeling of proteins. Rabbit hemopexin was purified from rabbits induced with lead acetate according to a previously described method [3]. Rabbit albumin was purified from plasma of healthy adult rabbits by prepara tive zone electrophoresis employing Pevikon as supportive medium [7]. Both prote ins were tested for purity by immunoelectrophoresis and polyacrylamide gel electro phoresis [3]. Purified rabbit hemopexin and albumin were labeled with ,25I according to M c C onahey and D ixon [6]. The specific activity of the proteins was 7X108 dpm/mg protein. Rabbit scrum, 0.2 ml, was added to each aliquot of isotope-labeled protein and the mixture was centrifuged at 105,000 g for 2 h. This step was omitted when partly aggregated protein was used [9]. The amount of >25I-hemopexin injected was 3.8X108 dpm; that of albumin was 1.7X109 dpm. More than 97°/o of the iso tope was protein-bound when tested either by precipitation with 95°/o ethanol or with monospecific antibodies. Experimental procedure. Six rabbits were used. In two of them hemolysis was produced by administration of phenylhydrazine s.c. dissolved in phosphate-buffered saline, pH 7.4, and administered (10 mg/kg body weight) every other day times five. Their hematocrit was 42 and 45°/o before treatment and 27 and 30% preceding the experiment, at which time intense reticulocytosis was present. Two animals were in jected with either 125I-hemopexin or 3H-heme mixed with 1 ml serum. Two animals served as controls, i.e. they were not treated with phenylhydrazine, but were given either isotope-labeled heme or heme-saturated hemopexin. Two additional animals, also untreated, received either ,25l-methemalbumin or partly aggregated 12!I-hemehemopexin as previously described [9]. 125I-hemopexin was incubated with 0.4 mg hematin, an amount enough to saturate all circulating hemopexin, and ,25I-albumin with 4 mg hematin for 30 min at room temperature before injection. 1 h after injection of 3H-heme or iodinated protein, the rabbits were sacrificed under anesthesia with sodium pentobarbital (Valley Veterinary Supply, North Hol lywood, Calif.), 40 mg/kg body weight. Immediately following exsanguination through the abdominal aorta, liver, lung, kidney, spleen, and bone marrow were re moved. Small sections were taken for microscopic studies and autoradiography. Microscopic and autoradiographic studies. For light microscopy, the tissues were fixed in 10% phosphate-buffered formaline and embedded in paraffin. Sections were obtained at 5 and processed for radioautography using NTB-2 (Kodak nuclear emulion) [15]. The exposure time varied from 2 to 8 weeks. Sections were then lightly stained with hematoxylin and examined for localization of silver grains in the tissues. Several slides were studied for each exposure period.
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 12:32:41 AM
Materials and Methods
Localization of Heme and Hemopexin
221
For electron microscopy, small pieces of tissue were fixed in 3%> phosphate-buf fered glutaraldehyde (pH 7.4) at 4 °C. The tissue was then rinsed in buffer, postfixed in l°/o cold 0 s 0 4 similarly buffered. After dehydration in graded alcohol, it was embedded in epoxy resin. Ultrathin sections in the range of silver were obtained with a Dupont diamond knife and placed on a Formvar-coated grid and processed for radioautography [15] using Ilford 3 emulsion (Essex, England) after an exposure time varying from 2 to 8 weeks. After each exposure period, several grids were stained with uranyl acetate and lead citrate and studied in a Hitachi HU 11-A elec tron microscope for subceilular localization of radioactivity [15].
In a previous report, we had demonstrated that intravenously adminis tered 3H-heme and 125I-hemopexin are removed by hepatocytes [8]. The evidence was obtained by examining radioautographic tissue slides of several organs with the light microscope. A tracer amount of 3H-heme was given as hematin, cyanmethemoglobin, methemalbumin or heme-hemopexin. Whatever pigment was injected, the heme was associated with liver parenchymal but not with Kupffer cells within 15-120 min of injection. In the same study, tracer amounts of 125I-rabbit hemopexin were demon strated to gain access to the hepatocyte. The results of the present study confirm this conclusion. In contrast, 125I-albumin, administered as methemalbumin, was not found in any organ studied. This supports our recent suggestion that all hepatic 125I-albumin could be attributed to the plasma content of this org an [4] and is in agreement with results described by B issell et al. [1]. Even when a large quantity of hematin was given (12.5 mg/kg heme), the amount of 125I-albumin found in the liver did not increase [4]. By contrast, in the experiment in which partly aggregated hemopexin was given, silver grains were discernible both in hepatocytes and Kupffer cells (fig. 1). Silver grains were also seen in the interstitial tissue of the lung and the kidney and in the cordal compartment of the splenic red pulp. This distribution pattern suggests that the aggregated hemopexin was removed by the reticuloendothelial system. In this context, it is of in terest to note that intravenously administered hemoglobin [13] and hapto globin-hemoglobin were found in cells of the reticuloendothelial system [17]. Uptake of aggregated protein molecules may have been the cause of engulfment of these pigments. B issell et al. [1] located both entities only in hepatocytes.
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 12:32:41 AM
Results and Discussion
222
H eng L iem /T avassoli/M uller-E berhard
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Cellular and Subcellular Localization of Heme and Hemopexin in the Rabbit H. H eng L iem , M ehdi T avassoli and U rsula M uller-E berhard Departments of Biochemistry and Hematology, Scripps Clinic and Research Foundation, and the Department of Pediatrics, University of California at San Diego, La Jolla, Calif.
Key Words. Albumin • Autoradiography • Heme metabolism • Hemopexin • Li ver ultrastructure
Hemopexin, a serum /(-glycoprotein, serves as a carrier of heme in the blood [12], It binds heme with a much greater avidity than albumin [16], Heme is transported to the liver, where it is degraded mainly to bilirubin [14]. Previous work from our laboratory [8] has shown that the hemehemopexin complex is removed exclusively by hepatocytes and not by the reticuloendothelial elements of the liver (Kupffer cells). In the present re port, we have extended our investigations to the subcellular localization of 3H-heme and 125I-hemopexin. We have furthermore shown that aggre gated hemopexin is taken up by the reticuloendothelial system throughout the body. In addition, we have shown that lî5I-albumin is not removed by the hepatocytes during the process of plasma heme clearance.
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 12:32:41 AM
Abstract. The cellular distribution of intravenously administered 3H-heme, ,25Ihemopexin and ,25I-albumin was studied in rabbits. Radioautography of the tissue slides of several organs was examined by light and electron microscopy. All experi ments were terminated 60 min after injection of the isotope-labeled materials. 3Hheme and native monomeric ,2!I-hemopexin (heme saturated) were found to be asso ciated with the endoplasmic reticulum and microbodies of the hepatocytes. Aggre gated hemopexin was also taken up by macrophages of lung alveoli, splenic pulp, interstitial cells of the kidney and Kupffer cells. l2!I-albumin (heme saturated) could not be located in liver, spleen, bone marrow, lung, or kidney tissues.
220
H eng L iem /T avassoli/M uller -E berhard
Animals. New Zealand white rabbits, 0.8-1.1 kg, were purchased from Rancho de Conejo, Calif. Several days prior to the experiment, Lugol’s solution was added to the drinking water. 3H-heme preparation. Heme was labeled in vitro with 3H-aminolevulinic acid as previously described [11]; the specific activity was 4X107 dpm/mg. Heme solutions were freshly prepared and 1 ml serum was admixed. Approximately 65 fig 3H-heme were injected. Purification and isotope-labeling of proteins. Rabbit hemopexin was purified from rabbits induced with lead acetate according to a previously described method [3]. Rabbit albumin was purified from plasma of healthy adult rabbits by prepara tive zone electrophoresis employing Pevikon as supportive medium [7]. Both prote ins were tested for purity by immunoelectrophoresis and polyacrylamide gel electro phoresis [3]. Purified rabbit hemopexin and albumin were labeled with ,25I according to M c C onahey and D ixon [6]. The specific activity of the proteins was 7X108 dpm/mg protein. Rabbit scrum, 0.2 ml, was added to each aliquot of isotope-labeled protein and the mixture was centrifuged at 105,000 g for 2 h. This step was omitted when partly aggregated protein was used [9]. The amount of >25I-hemopexin injected was 3.8X108 dpm; that of albumin was 1.7X109 dpm. More than 97°/o of the iso tope was protein-bound when tested either by precipitation with 95°/o ethanol or with monospecific antibodies. Experimental procedure. Six rabbits were used. In two of them hemolysis was produced by administration of phenylhydrazine s.c. dissolved in phosphate-buffered saline, pH 7.4, and administered (10 mg/kg body weight) every other day times five. Their hematocrit was 42 and 45°/o before treatment and 27 and 30% preceding the experiment, at which time intense reticulocytosis was present. Two animals were in jected with either 125I-hemopexin or 3H-heme mixed with 1 ml serum. Two animals served as controls, i.e. they were not treated with phenylhydrazine, but were given either isotope-labeled heme or heme-saturated hemopexin. Two additional animals, also untreated, received either ,25l-methemalbumin or partly aggregated 12!I-hemehemopexin as previously described [9]. 125I-hemopexin was incubated with 0.4 mg hematin, an amount enough to saturate all circulating hemopexin, and ,25I-albumin with 4 mg hematin for 30 min at room temperature before injection. 1 h after injection of 3H-heme or iodinated protein, the rabbits were sacrificed under anesthesia with sodium pentobarbital (Valley Veterinary Supply, North Hol lywood, Calif.), 40 mg/kg body weight. Immediately following exsanguination through the abdominal aorta, liver, lung, kidney, spleen, and bone marrow were re moved. Small sections were taken for microscopic studies and autoradiography. Microscopic and autoradiographic studies. For light microscopy, the tissues were fixed in 10% phosphate-buffered formaline and embedded in paraffin. Sections were obtained at 5 and processed for radioautography using NTB-2 (Kodak nuclear emulion) [15]. The exposure time varied from 2 to 8 weeks. Sections were then lightly stained with hematoxylin and examined for localization of silver grains in the tissues. Several slides were studied for each exposure period.
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 12:32:41 AM
Materials and Methods
Localization of Heme and Hemopexin
221
For electron microscopy, small pieces of tissue were fixed in 3%> phosphate-buf fered glutaraldehyde (pH 7.4) at 4 °C. The tissue was then rinsed in buffer, postfixed in l°/o cold 0 s 0 4 similarly buffered. After dehydration in graded alcohol, it was embedded in epoxy resin. Ultrathin sections in the range of silver were obtained with a Dupont diamond knife and placed on a Formvar-coated grid and processed for radioautography [15] using Ilford 3 emulsion (Essex, England) after an exposure time varying from 2 to 8 weeks. After each exposure period, several grids were stained with uranyl acetate and lead citrate and studied in a Hitachi HU 11-A elec tron microscope for subceilular localization of radioactivity [15].
In a previous report, we had demonstrated that intravenously adminis tered 3H-heme and 125I-hemopexin are removed by hepatocytes [8]. The evidence was obtained by examining radioautographic tissue slides of several organs with the light microscope. A tracer amount of 3H-heme was given as hematin, cyanmethemoglobin, methemalbumin or heme-hemopexin. Whatever pigment was injected, the heme was associated with liver parenchymal but not with Kupffer cells within 15-120 min of injection. In the same study, tracer amounts of 125I-rabbit hemopexin were demon strated to gain access to the hepatocyte. The results of the present study confirm this conclusion. In contrast, 125I-albumin, administered as methemalbumin, was not found in any organ studied. This supports our recent suggestion that all hepatic 125I-albumin could be attributed to the plasma content of this org an [4] and is in agreement with results described by B issell et al. [1]. Even when a large quantity of hematin was given (12.5 mg/kg heme), the amount of 125I-albumin found in the liver did not increase [4]. By contrast, in the experiment in which partly aggregated hemopexin was given, silver grains were discernible both in hepatocytes and Kupffer cells (fig. 1). Silver grains were also seen in the interstitial tissue of the lung and the kidney and in the cordal compartment of the splenic red pulp. This distribution pattern suggests that the aggregated hemopexin was removed by the reticuloendothelial system. In this context, it is of in terest to note that intravenously administered hemoglobin [13] and hapto globin-hemoglobin were found in cells of the reticuloendothelial system [17]. Uptake of aggregated protein molecules may have been the cause of engulfment of these pigments. B issell et al. [1] located both entities only in hepatocytes.
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/11/2019 12:32:41 AM
Results and Discussion
222
H eng L iem /T avassoli/M uller-E berhard
.
0
A
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