A fluorescent residualizing label for studies on protein uptake and catabolism in vivo and in vitro.

Residualizing labels are tracers which remain in lysosomes after uptake and catabolism of the carrier protein and have been especially useful for studies on the sites of plasma protein degradation. Thus far these labels have contained radioactive reporters such as 3H or 125I. In the present paper we describe a fluorescent residualizing label, NN-dilactitol-N'-fluoresceinylethylenediamine (DLF). Modification of asialofetuin (ASF) or rat serum albumin (RSA) with DLF affected neither their normal kinetics of clearance from the rat circulation nor their normal tissue sites of uptake and degradation. After injection of DLF-ASF, fluorescent degradation products were recovered nearly quantitatively in liver and retained with a half-life of about 2 days. Fluorescent degradation products from DLF-RSA were recovered in skin and muscle, and were localized in fibroblasts by fluorescence microscopy. These results confirm previous studies with radioactive residualizing labels in which fibroblasts in peripheral tissues were identified as primary sites of albumin degradation. Fluorescent catabolites also accumulated in fibroblasts incubated with DLF-RSA in vitro, and residualized with a half-life of about 2 days. Overall, the data establish that DLF functions efficiently as a fluorescent residualizing label both in vivo and in vitro. The advantages of fluorescent, compared with radioactive, residualizing labels should make them valuable tools for studies on protein uptake and catabolism in biological systems.     

The sites of catabolism of murine monomeric IgA

The tissue sites of monomeric IgA (mIgA) catabolism were determined in a BALB/c mouse model. Mouse mIgA myeloma proteins were labeled either by direct iodination or by coupling the residualizing label, dilactitol-125I-tyramine (125I-DLT) to the proteins; catabolites from protein labeled with 125I-DLT accumulate at the site of protein degradation, allowing identification of the tissue and cellular sites involved in catabolism of the protein. The circulating half-lives of 125I- and 125I-DLT-mIgA were the same. The distribution of radioactivity in tissues was measured at 1, 3, 24, and 96 h after iv. injection of 125I-DLT-labeled mIgA, dimeric IgA (dIgA), IgG, or mouse serum albumin. The greatest uptake of 125I-DLT-mIgA was attributable to the liver. This organ accounted for more internal catabolism of mIgA than all other tissues combined. In contrast, 125I-DLT-IgG was catabolized equally in skin, muscle, and liver. These data indicate that, in mice, the liver is the major site of mIgA catabolism. To determine the cell types involved, collagenase digestion was used to isolate parenchymal and non-parenchymal cells from perfused liver of animals injected with 125-DLT-mIgA. Most of the radioactivity was associated with the hepatocyte fraction, even though both cell types showed uptake of 125I-DLT-mIgA. Inhibition studies, with asialofetuin and mouse IgA demonstrated that the uptake of mIgA by liver cells was mediated primarily by the asialoglycoprotein receptor.     

Intracellular processing of residualizing labels in different cell types in vitro.

In previous autoradiographic studies on the sites of catabolism of rat serum albumin (RSA) in the rat, fibroblasts in skin and muscle were shown to accumulate degradation product from RSA labeled with the residualizing label dilactitol-125I-tyramine (125I-DLT) (Strobel et al., 1986 J. Biol. Chem., 261:7989-7994). Residualizing labels remain at the cellular site of degradation of the carrier protein because of their size, hydrophilicity, and resistance to lysosomal hydrolases. This study was designed to evaluate whether fibroblasts might retain labeled degradation products more efficiently than other cell types. The uptake of 125I-DLT-RSA and release of its degradation products and of a second non-biodegradable probe, fluorescein isothiocyanate (FITC)-dextran, were studied in fibroblasts, endothelial cells, and macrophages, all cell types previously implicated in the catabolism of albumin in vivo. The rates of uptake of labeled protein and dextran were comparable in all cell types and consistent with fluid phase endocytosis. The rate of release of both intact protein (30-35% of total radioactivity released) and radioactively labeled degradation products followed similar kinetics and had half-lives ranging from 26 to 37 hr. The rate of release of FITC-dextran was slower than that of radioactivity, with a half-life of 42-125 hr. Thus, although there were differences between the rates of release of the fluorescent and radioactive materials in vitro, there were no significant differences in the disposition of protein-derived catabolites among these three cell types.     

Loci of catabolism of beta-very low density lipoprotein in vivo delineated with a residualizing label, 125I-dilactitol tyramine

beta-Very low density lipoprotein (beta-VLDL) may be a major atherogenic lipoprotein, and knowledge of the sites of its catabolism should facilitate elucidation of mechanisms important in the regulation of its plasma concentrations. In this study, catabolic sites of beta-VLDL have been delineated in normolipidemic rabbits with a novel, radioiodinated, residualizing label, 125I-dilactitol tyramine (125I-DLT). Comparative studies of beta-VLDL and low density lipoprotein catabolism were performed with 125I-DLT conjugated to each lipoprotein and with lipoproteins iodine-labeled conventionally. Conjugation did not alter size distributions or charge characteristics of lipoprotein particles. The overall processing (binding and degradation) of lipoproteins by cultured rabbit skin fibroblasts was not influenced by 125I-DLT derivatization, suggesting that attachment of the label did not influence cell receptor-lipoprotein interactions. Furthermore, although degradation products of 125I-lipoproteins leaked out of the cells and into the medium, the degradation products of 125I-DLT lipoproteins were retained by the cells. The principal catabolic site of beta-VLDL in normolipidemic rabbits was found to be the liver with 54 +/- 4% of injected 125I retained in this organ 24 h after injection of 125I-DLT-beta-VLDL. When catabolism was normalized to tissue weight, the liver and adrenals were found to be approximately equally active in the metabolism of beta-VLDL. In agreement with results of other studies with residualizing labels, the principal organ of catabolism of 125I-DLT-LDL in vivo was the liver. The adrenals were the most highly catabolizing organ when results were normalized for tissue weight. The quantitative differences observed in the tissue distributions of injected 125I-DLT-beta-VLDL and 125I-DLT-low density lipoprotein suggested that a significant proportion of beta-VLDL is removed by tissues before conversion to low density lipoprotein.     

Identification of fibroblasts as a major site of albumin catabolism in peripheral tissues

Rat serum albumin has been labeled with dilactitol-125I-tyramine, (125I-DLT) a radioactive tracer which remains entrapped within lysosomes following cellular uptake and degradation of the carrier protein. Similar kinetics of clearance from the rat circulation were observed for albumin labeled conventionally with 125I or 125I-DLT-albumin, both proteins having circulating half-lives of approximately 2.2 days. In contrast, the recovery of whole body radioactivity had half-lives of approximately 2.2 and 5.1 days, respectively, for the two protein preparations, indicating substantial retention of degradation products derived from catabolism of 125I-DLT-albumin. Measurement of total and acid-soluble radioactivity in tissues 2 or 4 days after injection of 125I-DLT-albumin revealed that skin and muscle accounted for the largest fraction (50-60%) of degradation products in the body. Fibroblasts were identified by autoradiography as the major cell type containing radioactive degradation products in skin and muscle. Fibroblasts were isolated from skin by collagenase digestion, followed by density gradient centrifugation. The amount of acid-soluble radioactivity recovered in these cells was in excellent agreement with that predicted based on acid precipitation of solubilized whole skin preparations. These studies demonstrate for the first time that fibroblasts are a major cell type involved in the degradation of albumin in vivo.     

Phospholamban is a good substrate for cyclic GMP-dependent protein kinase in vitro, but not in intact cardiac or smooth muscle.

Protein residualizing labels facilitate localization of tissue sites of protein catabolism and the quantification of protein accumulation because of their prolonged intracellular retention of protein accumulation because of their prolonged intracellular retention times. Radioiodinated residualizing labels have been used to define the metabolism of a wide variety of proteins, but this has necessitated destructive analysis. Here we describe the implementation and validation of a novel 19F-containing residualizing label for protein, NN-dilactitol-3,5-bis(trifluoromethyl)benzylamine (DLBA), that permits the non-invasive assessment of protein accumulation and catabolism by n.m.r. spectroscopy in vivo. DLBA comprises a reporter molecule containing six equivalent 19F atoms. 19F is strongly n.m.r.-active, has 100% natural abundance, and is present in minimal background concentrations in soft tissues. We validated the use of DLBA as a protein-labelling compound by coupling to asialofetuin (ASF), a protein that is recognized exclusively by hepatic tissue via a saturable receptor-mediated process. Coupling of DLBA to ASF by reductive amination had no effect on the physiological receptor-mediated uptake of the protein in rat liver in vivo. The 19F-n.m.r. spectrum of DLBA exhibited a single peak that was subject to a small chemical-shift change and broadening after coupling to ASF. Pronase digestion of DLBA-ASF was performed to simulate intracellular degradation products, and resulted in a narrower set of resonances, with chemical shifts intermediate between those of uncoupled DLBA and DLBA-ASF. Intravenous administration of DLBA-ASF to rats followed by quantification of 19F in homogenates of liver tissue indicated that the half-life of residence time of degradation products from DLBA-ASF in liver was approx. 2 days. This intracellular half-life was comparable with that described for similar residualizing labels that contain radioiodide as a reporter. Similar results for the half-life of retention were obtained non-destructively and non-invasively in situ with the use of a whole-body radio-frequency antenna to acquire sequential spectra over 80 h after intravenous administration of DLBA-ASF. Quantification of these spectra demonstrated an initial accumulation of DLBA-ASF in liver followed by an expected gradual loss of 19F-labelled degradation products. The approach developed offers promise for the sequential and longitudinal characterization of metabolism of specific proteins in individual experimental animals and ultimately in human subjects.     

125I-glycoconjugate labels for identifying sites of protein catabolism in vivo: effect of structure and chemistry of coupling to protein on label entrapment in cells after protein degradation

Residualizing radioactive labels are designed to remain entrapped within cells following degradation of a carrier protein, and have been used for identification of the tissue and cellular sites of plasma protein catabolism. In this study we describe a convenient synthesis and purification of a series of 125I-labeled glycoconjugates, and an evaluation of their efficiency of retention in liver following degradation of a model carrier protein, asialofetuin. Glycoconjugates were prepared in 65-90% yield by reductive amination of reducing sugars with aromatic amines using NaBH3CN. The products were purified in a single ion-exchange chromatographic step, and then labeled with 125I. The derivatives prepared were mono-and disubstituted lactitol-,cellobiitol-and glucitol-[125I]tyramine and lactitol-[125I]tyrosine. 125I-Glycoconjugates were coupled to asialofetuin using either cyanuric chloride or, for lactose-containing labels, by treatment with galactose oxidase followed by reductive amination with NaBH3CN. Attachment of labels by either procedure did not affect the normal rapid clearance of asialofetuin from the rat circulation nor its uptake and degradation in liver lysosomes. Leakage of 125I-labeled degradation products from cells was measured by following the kinetics of loss of whole-body radioactivity. We observed that degradation products from larger, disubstituted glycoconjugates were retained more efficiently than those from smaller and monosubstituted derivatives, and that glycoconjugates coupled to protein via reductive amination were retained in the body more efficiently than those coupled by cyanuric chloride. Overall, dilactitol-[125I]tyramine coupled to protein by reductive amination was entrapped most efficiently in liver.     

Differences between the catabolism and tumour distribution of intact monoclonal antibody (791T/36) and its Fab/c fragment in mice with tumour xenografts revealed by the use of a residualizing radiolabel (dilactitol-125I-tyramine) and autoradiography

Summary Radioiodine-labelled 791T/36 monoclonal antibody (mAb) and its Fab/c fragment, consisting of one Fab arm and the Fc portion, have identical whole-body survival curves in BALB/c mice (t1/2 = 3.75 days). Therefore, these two forms of this antibody provide a suitable model for studying the role of valency in the targeting efficiency of antibodies to tumours in vivo. 791/T36 antibody and its Fab/c fragment were labelled either by direct iodination using the iodogen method (¹²⁵I) or by dilactitol-¹²⁵I-tyramine (¹²⁵I-DLT), a residualizing label, which accumulates in the cells involved in degradation of the carrier protein. In tumour-bearing nude mice, the percentage of injected dose of mAb or Fab/c fragment reaching the specific 791T tumour was similar, and these proteins appeared to be catabolized at a similar rate in this tissue. mAb, but not the Fab/c fragment, was found to be very actively catabolized by the liver and spleen of tumour-bearing mice compared to control nude mice, this probably resulting from clearance of immune complexes. This effect was most pronounced when the mAb was labelled with¹²⁵I-DLT, the percentage of injected dose of mAb reaching the spleen and liver being higher than the percentage of injected dose reaching the tumour. This effect was not seen with the Fab/c fragment. Autoradiographic studies on tumour sections, which exhibit antigenic sites throughout the tumour mass, showed that the Fab/c fragment was already homogeneously distributed in the tumour 12 h after injection whereas the whole antibody was mainly localized at the periphery of the tumour. Those results suggest a binding site barrier effect. Overall, these results indicate that the highest valency and affinity may not be the optimal choice for mAb to be used for therapeutic purposes.     

O-(4-Diazo-3,5-di[125I]iodobenzoyl)sucrose, a novel radioactive label for determining organ sites of catabolism of plasma proteins.

A method is described for radiolabelling proteins with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose (DD125IBS). When proteins so labelled were degraded within lysosomes, the radioactive fragments were largely retained within the organelle. High specific radioactivities were obtained without changing the properties of the protein. The validity of the method was demonstrated in vivo in rats using the short-lived protein lactate dehydrogenase, isoenzyme M4, and the long-lived protein bovine serum albumin. Derivatization with DD125IBS did not alter the clearance of either protein. Uptake of DD125IBS-labelled lactate dehydrogenase, isoenzyme M4, by liver and spleen of rats was determined. Radioactivity in these tissues increased up to about 2 h after injection (at this time the protein has been almost completely cleared from the blood) and subsequently declined with a half-life of approx. 20 h. After differential fractionation of liver, radioactivity was largely found in the mitochondrial and lysosomal fraction. The results of these studies establish that DD125IBS covalently coupled to plasma proteins should be a useful radioactive tracer for identifying the tissue and cellular sites of catabolism of relatively long-lived circulating proteins.     

Characterization of Na+-dependent phosphate uptake in cultured kidney cells (JTC-12) from monkey.

O-(4-Diazo-3-[125I]iodobenzoyl)sucrose ([125I]DIBS), a novel labelling compound specifically designed to study the catabolic sites of serum proteins [De Jong, Bouma, & Gruber (1981) Biochem. J. 198, 45-51], was applied to study the tissue sites of degradation of serum lipoproteins. [125I]DIBS-labelled apolipoproteins (apo) E and A-I, added in tracer amounts to rat serum, associate with high-density lipoproteins (HDL) just like conventionally iodinated apo E and A-I. No difference is observed between the serum decays of chromatographically isolated [125I]DIBS-labelled and conventionally iodinated HDL labelled specifically in either apo E or apo A-I. When these specifically labelled HDLs are injected into fasted rats, a substantial [125I]DIBS-dependent 125I accumulation occurs in the kidneys and in the liver. No [125I]DIBS-dependent accumulation is observed in the kidneys after injection of labelled asialofetuin or human low-density lipoprotein. It is concluded that the kidneys and the liver are important sites of catabolism of rat HDL apo E and A-I.     

Uptake and degradation of vitamin D binding protein and vitamin D binding protein-actin complex in vivo in the rat.

We have labelled the rat vitamin D binding protein (DBP), DBP-actin and rat albumin with 125I-tyramine-cellobiose (125I-TC). In contrast with traditional 125I-labelling techniques where degraded radioactive metabolites are released into plasma, the 125I-TC moiety is trapped intracellularly in the tissues, where the degradation of the labelled proteins takes place. By using this labelling method, the catabolism of proteins can be studied in vivo. In this study we have used this labelling technique to compare the tissue uptake and degradation of DBP, DBP-actin and albumin in the rat. DBP-actin was cleared from plasma at a considerably faster rate than DBP. After intravenous injection of labelled DBP-actin complex, 48% of the radioactive dose was recovered in the liver after 30 min, compared with 14% when labelled DBP was administered. Only small amounts of DBP-actin complex were recovered in the kidneys. In contrast with the results obtained with DBP-actin complex, liver and kidneys contributed about equally in the uptake and degradation of DBP determined 24 h after the injection. When labelled DBP was compared with labelled albumin, the amount of radioactivity taken up by the liver and kidneys by 24 h after the injection was 2 and 5 times higher respectively. In conclusion, liver and kidneys are the major organs for catabolism of DBP in the rat. Furthermore, binding of actin to DBP enhances the clearance of DBP from circulation as well as its uptake by the liver.     

Turnover of endogenous, microinjected, and sequestered protein in Xenopus oocytes

Pulse-labeled oocyte proteins were found to have a maximum average half-life of 73 h. In general, larger peptides underwent degradation at a faster rate than smaller peptides. In this respect, oocytes are similar to most other cells. Microinjected ¹²⁵I-labeled bovine serum albumin (BSA) was degraded over a 40 h period with a half-life of 20–30 h, regardless of the method of protein labeling, culture medium employed, size of oocyte microinjected, or hormonal history of the oocyte. The last two results, if applicable to oocyte proteins in general, imply that protein catabolism is constant throughout the later stages of oogenesis and that growth is primarily regulated by a stimulation of anabolism. Individual proteins microinjected into oocytes undergo rates of degradation consistent with turnover rates obtained in other systems. Sequestered ¹²⁵I-labeled BSA is only partially (40%) degraded, which indicates that, unlike microinjected ¹²⁵I-labeled BSA, it has access to a cytoplasmic compartment (yolk platelets?) within which it is relatively stable.     

Plasma clearance and liver metabolism of native and asialo-human transcortin in the rat.

Plasma kinetics and liver metabolism of iodinated human corticosteroid-binding protein have been studied in ovariectomized female rats. 125I-labeled human corticosteroid-binding globulin prepared by a modified chloramine T reaction was shown to be physically intact and biologically active. Intravenously injected 125I-labeled human corticosteroid-binding globulin was shown to give a complex clearance pattern from the plasma, with half-lives of 7.5 and 51 min. Estrogen injections had no effect on plasma clearance rate. Direct involvement of liver plasma membrane receptors for asialoglycoproteins in 125I-labeled human corticosteroid-binding globulin metabolism was demonstrated in vivo and in vitro using asialofetuin as a competitive inhibitor. 125I labeled human asialo-corticosteroid-binding globulin was cleared from the plasma with a half-life of less than 1 min, while the simultaneous injection of 5 mg asialofetuin maintained the circulating plasma lebels. Asialofetuin also slowed the clearance of intact 125I-labeled human corticosteroid-binding globulin from the plasma (t1/2 = 90 min). Binding of 125I-labeled human asialo-corticosteroid-binding globulin to rat liver plasma membranes in vitro was inhibited in a dose-dependent manner by asialofetuin, but not by intact human corticosteroid-binding globulin or fetuin. 125I-labeled human corticosteroid-binding globulin did not bind significantly to the membranes. It is concluded that human corticosteroid-binding globulin clearance from rat plasma is rapid and that the carbohydrate moiety of human corticosteroid-binding globulin is involved in its clearance and catabolism by the liver.     

Determination of the tissue sites responsible for the catabolism of large high density lipoprotein in the African green monkey

In vivo multicompartmental modeling of the turnover of HDL subfractions has suggested that HDL containing four molecules of apoA-I per particle and no other apolipoproteins (large LpA-I) are terminal particles in plasma. We hypothesized that these terminal particles were the end product of HDL metabolism and, as such, would be cleared preferentially by the liver. Thus, the purpose of this study was to determine: 1) the tissue sites of catabolism of large LpA-I in African green monkeys, and 2) whether saturated versus n;-6 polyunsaturated dietary fat affected tissue accumulation. Large LpA-I were isolated, without ultracentrifugation, by size exclusion and immunoaffinity chromatography and radiolabeled with either the residualizing compound, (125)I-labeled tyramine cellobiose (TC), or with (131)I. After injection into recipient animals, the plasma die-away of the radiolabels was followed for 12 or 24 h, after which the animals were killed and tissues were collected for determining radiolabel sites of catabolism. The plasma die-away of the (125)I-labeled TC-LpA-I and (131)I-labeled LpA-I doses was similar suggesting that the TC radiolabeling did not modify the metabolism of the large LpA-I dose. The liver, adrenal, kidney, and spleen had the greatest accumulation of large LpA-I degradation products on a per gram tissue basis. On a whole organ basis, the liver was the major site of large LpA-I degradation in both the 12-h (15.4 +/- 0.3% of injected dose) and 24-h (9.1 +/- 0.6% of injected dose) catabolic studies. The kidney, compared to the liver, had less uptake of large LpA-I radioactivity in either study (1.3 +/- 0.4% and 1.2 +/- 0.3% of injected dose). There was no apparent influence of dietary fat type on the tissue accumulation of large LpA-I. We conclude that the liver is the primary site of catabolism of large LpA-I in the African green monkey.     

Purification of residualizing glycoconjugate labels for protein by reversed-phase high-pressure liquid chromatography

Residualizing labels are radioactive or fluorescent tracers used for identifying the tissue and cellular sites in which circulating proteins are catabolized in the body. When attached to protein the labels do not affect normal mechanisms of protein catabolism, but remain at the cellular site of protein uptake, after the carrier protein itself is degraded to diffusible catabolites. Until recently these labels consisted of biologically indigestible carbohydrates attached to a radioactive reporter molecule. In this report we describe the synthesis and purification of a new fluorescent residualizing label, N,N-dilactitol-N'-fluoresceinyl-ethylenediamine. The label is prepared by first derivatizing ethylenediamine 1:1 with fluorescein isothiocyanate and then coupling lactose to the remaining primary amino group by reductive amination. A rapid one step purification of this and other glycoconjugate labels by reversed-phase high-pressure liquid chromatography is described.     

Quantitative assessment of lipoprotein metabolism by positron emission tomography with an 18F-containing residualizing label

Residualizing labels for proteins are designed to remain entrapped within cells following uptake and degradation of the carrier protein. In the present work we report the synthesis of a novel residualizing label, N-lactitol-S-([¹⁸F]fluorophenacyl)-cysteamine ([¹⁸F]LCSH, and its use for quantifying the accumulation of low density lipoprotein in tissues in vivo by positron emission tomography (PET). The retention of degradation products in tissues from lipoprotein or from other rapidly catabolized protein pharmaceuticals tagged with [¹⁸F]LCSH reduces leakage of tracer into the plasma compartment. Thus, residualizing labels provide a valuable tool for enhancing signal-to-noise ratios, even during the relatively short interval of PET studies.     

Lysosomal degradation of ribonuclease A and ribonuclease S-protein microinjected into the cytosol of human fibroblasts

We have analyzed the subcellular localization of 125I-labeled ribonuclease A and ribonuclease S-protein (residues 21-124) after erythrocyte-mediated microinjection into confluent cultures of IMR-90 human lung fibroblasts. Microinjected cells were fractionated by two consecutive Percoll gradients, and the distribution of radioactive ribonuclease A and S-protein was compared to patterns for known enzyme markers. Ribonuclease A is localized in the cytosol immediately after microinjection, but thereafter a portion of the microinjected enzyme is associated with lysosomes. We obtained similar results for ribonuclease S-protein except extensive association with a nonlysosomal intracellular structure is also evident. The effects of ammonium chloride on proteolysis indicate that ribonuclease A and ribonuclease S-protein are degraded at least in part by lysosomal pathways. Degradation of long-lived cellular proteins is inhibited by 17% in the presence of serum and by 35% in the absence of serum. The effects of ammonium chloride on catabolism of microinjected proteins are more variable. Inhibition in the presence and absence of serum ranged between 43 and 64% for both ribonuclease A and ribonuclease S-protein. To quantitatively assess the role of lysosomal and cytosolic pathways in the degradation of microinjected proteins, we have tagged proteins with the inert trisaccharide, [3H] raffinose. The radioactive degradation products of such proteins are completely retained within lysosomes since the lysosomal membrane is impermeable to [3H] raffinose coupled to lysine or small peptides. These studies show that ribonuclease A and S-protein are degraded almost entirely by lysosomes while bovine serum albumin is degraded principally in the cytosol. A mixture of rat liver cytosolic proteins is degraded approximately 60% in the cytosol and 40% by lysosomes confirming that both lysosomal and nonlysosomal pathways of proteolysis are important in confluent human fibroblasts.     

Tissue sites of degradation of apoprotein A-I in the rat

The tissue sites of degradation of apoprotein A-I were determined in the rat in vivo using a newly developed tracer of protein catabolism, an adduct of 125I-tyramine and cellobiose. This methodology takes advantage of the fact that when a protein labeled with 125I-tyramine-cellobiose is taken up and degraded, the radiolabeled ligand remains trapped intracellularly. Thus, radio-iodine accumulation in a tissue acts as a cumulative measure of protein degradation in that tissue. In the present studies, apoprotein AI (apo-A-I) was labeled with tyramine-cellobiose (TC). The TC-labeled apo-A-I was then reassociated with high density lipoprotein (HDL) in vivo by injection into donor animals. After 30 min, serum from donor animals was recovered and then injected into recipient rats. TC-labeled apo-A-I in the donor serum was shown to be exclusively associated with HDL. The fractional catabolic rate of 125I-TC-apo-A-I was not significantly different from that of conventionally labeled apo-A-I. The kidney was the major site of degradation, accounting for 39% of the total. The liver was responsible for 26% of apo-A-I catabolism, 96% of which occurred in hepatocytes. The kidney was also the most active organ of catabolism/g of wet weight. The tissues next most active/g of wet weight were ovary and adrenal, a finding that is compatible with a special role of HDL in the rat for delivery of cholesterol for steroidogenesis. Immunofluorescence studies of frozen sections of rat kidney demonstrated the presence of apo-A-I on the brush-border and in apical granules of proximal tubule epithelial cells. Preliminary studies using HDL labeled both with 125I-TC-apo-A-I and [3H]cholesteryl ethers again demonstrated high rates of renal uptake of apo-A-I but less than 1% of total ether uptake. It is postulated that the high activity of kidney was not due to uptake of intact HDL particles, but rather, due to glomerular filtration and tubular reabsorption of free apo-A-I.     

In vivo clearance of low density lipoprotein in pigeons occurs by a receptor-like mechanism that is not down-regulated by cholesterol feeding

The contribution of receptor-dependent and receptor-independent mechanisms for low density lipoprotein (LDL) clearance in vivo was determined in White Carneau and Show Racer pigeons fed either cholesterol free or cholesterol containing diets. The methylation of pigeon LDL resulted in the inhibition of recognition by the LDL receptor which allowed its use as a tracer of receptor-independent clearance. The fractional catabolic rate (FCR) of radiolabeled LDL in 20 control pigeons (means +/- S.E., 0.277 +/- 0.013 pools/h) was approximately seven times faster than for methylated LDL indicating that 86% of the total LDL clearance occurred by a receptor-mediated process. Total LDL clearance was reduced by 27% (FCR = 0.202 +/- 0.012 pools/h) in 14 cholesterol-fed pigeons, but receptor-mediated mechanisms were still responsible for 80% of the total LDL clearance. LDL uptake by individual tissues was measured using the residualizing label 125I-tyramine cellobiose. The liver was the primary site of LDL clearance in both control and cholesterol-fed birds. LDL receptors were active in every tissue examined and accounted for over 85% of the LDL clearance in the liver and over 90% in the adrenal gland. Consistent with the whole body LDL clearance findings, cholesterol-feeding did not significantly reduce receptor-mediated clearance of 125I-tyramine cellobiose-LDL by the liver or any of the other tissues. Hepatic sterol synthesis, however, was reduced by greater than 90% in cholesterol-fed animals. These data are consistent with the conclusion that LDL clearance in vivo in pigeons is mediated primarily by an LDL receptor-like mechanism that shows little down-regulation with hypercholesterolemia even though cholesterol synthesis is efficiently down-regulated.     

Identification of the sites of albumin catabolism in the rat

Rat serum albumin was labeled with [G-³H]raffinose, a nondegradable radioactive tracer which has been shown to accumulate in cells following protein degradation (Van Zile et al., 1979, J. Biol. Chem.254, 3547–3553). Stoichiometric labeling of albumin with [³H]raffinose did not affect either its circulating half-life or its equilibration into extravascular spaces. The tissue distribution of acid-soluble radioactivity was followed for up to 96 h after intracardial injection of labeled protein. At 18, 48, and 96 h postinjection muscle and hide accounted for the highest proportion (40–60%) of catabolized dose of albumin in the body. Additional small percentages of radioactive degradation products were recovered in liver, kidney, and other visceral organs. The data provide the first direct evidence that the major fraction of albumin catabolism in the rat occurs in muscle and hide.