Change of serum L-tryptophan levels following the development and recovery of acute puromycin aminonucleoside nephrosis in rats

Summary It is known that total L-tryptophan (Trp) levels decrease with a decrease in albumin-bound Trp levels and an increase in free Trp levels in the plasma or serum of nephrotic children. We, therefore, examined the change of serum Trp levels following the development and recovery of acute nephrosis in 6-week-old male Wistar rats injected once with puromycin aminonucleoside (100mg/kg body weight) and checked the levels of 16 amino acids including Trp in the serum and the levels of Trp in the liver, kidney, and urine under nephrotic conditions. In this study, the development and recovery of nephrosis were checked by the changes of levels of urinary protein and serum protein and albumin. Total serum Trp and albumin-bound serum Trp levels decreased with the development of nephrosis and these decreased levels returned to the normal level with its recovery. In contrast, free serum Trp levels increased with the development of nephrosis and this increased level returned to the normal level with its recovery. In the serum of nephrotic rats, the decrease of albumin-bound Trp levels and the increase of free Trp levels were well consistent with a decrease in albumin levels and an increase in the level of non-esterified fatty acids which are known to weaken the binding of Trp to albumin and among 16 amino acids studied, only Trp showed a significant change in its levels. Trp levels increased in the liver and kidney but not in the urine under nephrotic conditions. These results indicate that the change of serum Trp levels should be closely related to the condition of nephrosis and that although serum Trp is lost under nephrotic conditions, the lost serum Trp is accumulated in the liver and kidney.     

Tryptophan metabolism,disposition and utilization in pregnancy

Tryptophan (Trp) requirements in pregnancy are several-fold: (1) the need for increased protein synthesis by mother and for fetal growth and development; (2) serotonin (5-HT) for signalling pathways; (3) kynurenic acid (KA) for neuronal protection; (4) quinolinic acid (QA) for NAD⁺ synthesis (5) other kynurenines (Ks) for suppressing fetal rejection. These goals could not be achieved if maternal plasma [Trp] is depleted. Although plasma total (free + albumin-bound) Trp is decreased in pregnancy, free Trp is elevated. The above requirements are best expressed in terms of a Trp utilization concept. Briefly, Trp is utilized as follows: (1) In early and mid-pregnancy, emphasis is on increased maternal Trp availability to meet the demand for protein synthesis and fetal development, most probably mediated by maternal liver Trp 2,3-dioxygenase (TDO) inhibition by progesterone and oestrogens. (2) In mid- and late pregnancy, Trp availability is maintained and enhanced by the release of albumin-bound Trp by albumin depletion and non-esterified fatty acid (NEFA) elevation, leading to increased flux of Trp down the K pathway to elevate immunosuppressive Ks. An excessive release of free Trp could undermine pregnancy by abolishing T-cell suppression by Ks. Detailed assessment of parameters of Trp metabolism and disposition and related measures (free and total Trp, albumin, NEFA, K and its metabolites and pro- and anti-inflammatory cytokines in maternal blood and, where appropriate, placental and fetal material) in normal and abnormal pregnancies may establish missing gaps in our knowledge of the Trp status in pregnancy and help identify appropriate intervention strategies.     

The tissue and subcellular distribution of [3H]dolichol in the rat

Following the intravenous injection of nanomolar amounts of [³H]dolichol into rats, the radioactivity rapidly appeared in the high-density lipoprotein fraction of the plasma and circulated with a half-life of about 9 h. A fraction of the injected activity was excreted in the feces, presumably through the bile, but evidence was obtained that little oxidative breakdown of dolichol occurred. All tissues assayed acquired radioactivity, but the liver attained the highest specific activity and the largest percentage of the total radioactive dolichol. Subcellular fractionation of the liver revealed that mitochondrial preparations contained the bulk of the labeled dolichol at all times tested up to 40 h after injection. Disruption of the mitochondrial structure by two different techniques permitted the isolation of inner and outer membrane fractions and it was found that the [³H]dolichol was concentrated in the outer membrane fraction. The significance of these findings is discussed.     

Characterization of the L-tryptophan transport system in the liver of growing rats

In order to characterize the system of L-tryptophan (TRP) transport into liver during the growing period of 10 to 42 days, the changes of tryptophan 2,3-dioxygenase (TDO) activity, levels of serum, liver, brain, and muscle TRP, and the rate and mode of TRP uptake into isolated hepatocytes were examined in male Wistar rats. Liver TDO activity increased rapidly at 16 days of age. A marked and rapid decrease in free serum TRP level occurred before weanling, while a small decrease in total serum TRP level was found after weanling. The change of liver TRP level was similar to that of free serum TRP level and correlated well. There was a significant inverse correlation between liver TDO activity and either free serum TRP level or liver TRP level. A rapid change in TRP level did not occur in brain and muscle during the growing period. The concentrations of brain and muscle TRP correlated better with those of total serum TRP than with those of free serum TRP. The rate of TRP uptake into hepatocytes isolated from rats aged 10 days was lower than that from rats aged 21 and 42 days. The former hepatocytes were lacking in a high-affinity saturable transport component for TRP uptake which was present in the latter ones. The present results indicate that a great change in the system of TRP transport into liver occurs in growing rats, and that in suckling rats a high level of free serum TRP contributes to the efficient transport of the amino acid into the liver.     

Design of compounds having enhanced tumour uptake,using serum albumin as a carrier—part II. In vivo studies

In the present in vivo study the uptake kinetics of radioiodinated albumin were determined in normal organs, and tumours of rats using sequential scintigraphy. Rat serum (RSA) was radioiodinated either directly at a tyrosine residue (d-RSA), or indirectly at a residualizing marker tagged to the albumin (rm-RSA). These labelling procedures did not alter the kinetics of labelled albumin, as shown by blood disappearance curves. Directly labelled albumin was shown to have tumour uptake. Residualizing markers like tyramine-cellobiose (TCB), tyramine-deoxysorbitol (TDS) and aminonaphthaltyrimide-deoxysorbitol (ANTDS) are metabolically inert. After the intracellular degradation of the albumin carrier the TCB-, TDS- and ATNDS-residues accumulate in the lysosomes, particularly those of tumour cells. It was able to be demonstrated that residualizing-marker tagged albumin-bound radioactivity was five times higher after 72 h than the tumour radioactivity after use of directly labelled RSA. These data found support when whole-body retention of directly labelled RSA, and residualizing marker-RSAs, were determined. After 72 h, 60% of ¹³¹I bound to RSA directly had been excreted, compared to only 25% of the activity attached indirectly to RSA with a residualizing marker. Whole-body autoradiography of rats injected with directly labelled RSA, or residualizing marker-RSA, support these results. Most of the radioactivity of directly labelled RSA was excreted within 24 h, whereas labelled residualizing marker-RSAs were also stored in tumour and liver tissue. ANTDS bound to RSA allows fluorescence microscopy. Cryosections of tumours from rats preinjected 10 min and 24 h with ANTDS-RSA before dissection, demonstrated that the fluorescence is localized on and in tumour cells. This indicates that cellular uptake of the marker takes place. Fluorescence was not observed in muscle tissue. This appears to suggest that the albumin uptake is greater in tumours than in normal tissue, and that it is metabolized in the tumour cells.     

Contribution of serum albumin to the transport of orally administered L-tryptophan into liver of rats with L-tryptophan depletion

Summary The role of serum albumin in the transport of orally administered L-tryptophan (Trp) into rat tissues was examined using analbuminemic and Sprague-Dawley (SD) rats with and without a-methyl-DL-tryptophan (AMT)-induced Trp depletion. Trp was orally administered to rats 16h after AMT or 0.85% NaCl administration, when liver tryptophan 2,3-dioxygenase and protein synthetic activities in AMT-treated rats were similar to those of 0.85% NaCl-treated rats. After oral Trp administration, regardless of the presence or absence of Trp depletion, free serum Trp concentrations were similar in the analbuminemic and SD rats, while total serum Trp concentrations were lower in analbuminemic rats than in SD rats. Although liver, brain, and muscle Trp concentrations after oral Trp administration under Trp depletion were lower in analbuminemic rats than in SD rats, the ratio of the liver Trp concentration in analbuminemic rats to that in SD rats was smaller than that of the brain or muscle Trp concentration. These results suggest that variations in serum albumin levels could affect the transport of orally administered Trp into the liver of rats with Trp depletion.     

Albumin secreted by rat liver bypasses Golgi apparatus cisternae

Albumin was isolated immunologically from various subcellular fractions from livers of adult male rats receiving an intraperitoneal injection of [³H]leucine to investigate the kinetics and pathway of subcellular transfer of newly synthesized albumin during secretion. At appropriate time intervals, livers were excised and fractionated into endoplasmic reticulum and Golgi apparatus. Golgi apparatus were further subfractionated into cisternae and secretory vesicles. In endoplasmic reticulum fractions, labeled albumin appeared within 7.5 min of injection of isotope, followed by a rapid decline in specific activity. Albumin in Golgi apparatus was labeled and concentrated in secretory vesicles over 25 min. The radioactivity in albumin per mg total protein was highest in secretory vesicles and insignificant in the cisternal fraction. Labeled albumin was present in serum by 30 min and radioactivity in serum albumin reached a plateau within 60–90 min after injection of isotope. Results provide evidence for the migration of albumin from its site of synthesis on endoplasmic reticulum membrane-bound polyribosomes to its site of secretion into the circulation via the Golgi apparatus. The pathway of albumin transport to secretory vesicles is suggested to involve peripheral elemenst of the Golgi apparatus. Secretory vesicle formation and maturation required 20 to 30 min for completion, via a mechanism whereby the inner spaces of the central saccules may be bypassed.     

Biological evidence for a precursor protein of serum albumin.

Radioactive leucine was injected into the portal vein of rats followed after 15 seconds by a 180 fold excess of nonradioactive leucine. An albumin-like protein in the liver became highly labelled within 15 minutes after injection. After 150 minutes, the radioactivity in the albumin-like protein had decreased to one tenth. In the serum, radioactively labelled albumin started to appear after 15 minutes and increased there-after up to 150 minutes after injection. Radioactivity in albumin within the liver remained constant at a low level. These results suggest that the albumin-like protein is a biological precursor protein of serum albumin, i.e. a proalbumin.     

The effect of cobalt on the uptake of plasma iron by the liver.

1. After an intraperitoneal injection of 59Fe the recovery of radioactivity in the liver, but not in other tissues, was increased in cobalt-pretreated rats. There was no proportional increase in the radioactivity recovered from liver haem. 2. Rats were injected intravenously with serum containing protein-bound 59Fe and 125I-labelled albumin as a marker. At various times after injection the specific radioactivities of iron in plasma and of non-haem iron in liver were determined; corrections were applied for the content of plasma in samples of liver. In cobalt-pretreated rats there was a more rapid loss of 59Fe radioactivity from the plasma and a corresponding increase in the uptake of 59Fe into liver non-haem iron. 3. The results are discussed in relation to the possible sites of action of cobalt, and the possibility is considered that only a fraction of the liver non-haem iron may be involved.     

Uptake and degdradation of formaldehyde-treated 125I-labeled human serum albumin in rat liver cells in vivo and in vitro

The uptake of formaldehyde-treated ¹²⁵I-labelled human serum albumin in rat hepatocytes and nonparenchymal liver cells was measured in vivo and in vitro. Isolated liver cells were prepared by treating the perfused liver with collagenase. Purified hepatocytes and nonparenchymal cells were obtained by differential centrifugation. Human serum albumin was found to be taken up exclusively or almost exclusively by nonparenchymal cells in vitro and in vivo (after intravenous injection). The maximal rate of human serum albumin-uptake in vitro was comparable to that in vivo. Nonparenchymal cells degraded human serum albumin in vitro as indicated by release of trichloroacetic acid-soluble radioactivity. Degradation started about 20–30 min after addition of human serum albumin to cells and rate of degradation was proportional to rate of uptake. Human serum albumin-degradation could be studied without interference of concurrent uptake by separating cells that had been preincubated with human serum albumin from the medium and then reincubating them with human serum albumin-free medium. The lag phase before human serum albumin-degradation starts and the inhibitory effect of chloroquine on degradation indicate that human serum albumin is degraded in lysosomes. The data obtained show that enzymatically prepared nonparenchymal liver cells retain their endocytic activity in vitro. Denatured human serum albumin should be useful both as a marker for rat liver macrophages and for the study of intracellular proteolysis in these cells.     

Transfer of esterified cholesterol from serum lipoproteins to the liver.

The fate of cholesteryl esters of the serum lipoproteins was studied in intact rats and in isolated perfused rat livers. The lipoproteins of fasting rat serum were labeled in vitro with [3H]cholesteryl oleate. Following intravenous injection, it was found that the majority of the radioactive ester was rapidly taken up by the liver where hydrolysis of the ester bond occurred. At 5 min, 58% of the injected material was recovered in the liver, 85% of which was still in the ester form, while at 30 min only 22% of the liver radioactivity was in cholesteryl esters. There was very little difference in the rate at which radioactivity was taken up from the different lipoprotein classes. Similar phenomena were observed in the perfused liver, but it was found that although the radioactive esters were being taken up, there was no change in the concentrations of free or esterified cholesterol in the perfusing medium, indicating that the lipoprotein cholesteryl ester was gaining access to the liver through an exchange of molecules. After uptake, cell fractionation experiments showed that the plasma membranes had the greatest relative amounts of radioactivity, suggesting that this is the site of exchange. Small amounts of radioactivity were recovered in the bile, demonstrating that serum lipoproteins can serve as precursors of at least some of the bile steroids.     

Dopamine β-hydroxylase: Determination of half-life and appearance in lymph fluid after IV injection of 125I-DBH into rats

A 4 day half-life of dopamine beta-hydroxylase (DBH) was determined for rats injected IV with ¹²⁵I-rat DBH from the slow exponential component of radioactivity appearing in plasma, urine, feces and combined urine and feces. Half-life estimates for ¹²⁵I-rat DBH injected IV into WKY and SHR animals did not differ from Sprague Dawley (Zivic Miller) rats. Radioactivity declined in parallel in plasma, urine and feces following IV ¹²⁵I-rat DBH administration and each radioactivity falloff curve could be resolved into two components. The slow phase of the decline of radioactivity excreted into urine and feces from which DBH half-life was calculated occurred between 5 and 25 days after ¹²⁵I-rat DBH injection. The early fast phase which is associated with distribution of the exogenous protein in body fluids and tissues continued for approximately the first 140 hr after DBH injection. The distribution characteristics of IV administered active bovine DBH and ¹²⁵I-rat DBH into the lymphatic system were examined. After active bovine DBH or ¹²⁵I-rat DBH was injected IV into rats, active DBH or radioactivity, respectively, appeared in lymph fluid (thoracic duct) within 20 min; reached peak concentrations within 90 min, and thereafter, declined in parallel with the plasma concentration. The concentration of radioactivity in plasma and lymph fluid were found to be unequal at 9 hr but were equivalent 68–75 hrs after IV injection of ¹²⁵I-rat DBH. Based on the amount of active DBH or radioactivity which accumulates in lymph fluid it is clear that'a substantial amount (> 50%) of the DBH in blood circulates through the lymphatic channels. Analysis of parallel experiments with labelled serum albumin indicate that use of these methods to study plasma proteins do provide sensitive measures of biological half-life and lymphatic distribution characteristics. Specifically for DBH, the results of our study suggest that DBH normally circulates in plasma and lymph fluid with a biological half-life of 4 days.     

Synthesis of antithrombin III and alpha-1-antitrypsin by the perfused rat liver

Livers isolated from control or turpentine-injected rats were perfused for 3 h with human red cells suspended in Krebs-Henseleit solution containing bovine serum albumin, dextran, glucose, heparin, cortisol, insulin, a mixture of 20 amino acids and [³H]leucine. Changes in the concentrations of antithrombin III and α-1-antitrypsin were evaluated by rocket immunoelectrophoresis using specific antisera, and incorporation of the ³H radioactivity into the total protein, albumin, antithrombin III and α-1-antitrypsin in the perfusate was measured. The results indicate that both antithrombin III and α-1-antitrypsin are synthesized in the liver. Local inflammation induced in the liver donors moderately stimulated the synthesis of α-1-antitrypsin but it affected only marginally that of antithrombin III.     

Dehydroepiandrosterone sulphate in rat brain: incorporation from blood and metabolism in vivo

The incorporation of [7-³H]dehydroepiandrosterone[³⁵S]sulphate into brain tissue elements from the circulatory system and its metabolic fate in the brain were studied in developing rats. Approximately 0.037 % of [³H] and 0.023% of [³⁵S] were incorporated into the brain within 15 min after the intracardiac injection of the labelled steroid. More than one-half of the incorporated [³H] was recovered as free steroid, whereas the rest was recovered as sulphate. The ³H/³⁵S ratio in the sulphate fraction suggested that the sulphate entered the brain with the sulphate linkage intact. Upon intracerebral injection of the double-labelled steroid, approximately 6 per cent of the radioactivity was recovered in the brain at 30 min after the injection and 1 per cent was recovered at 1 h after the injection. Of the remaining radioactivity recovered from the brain, 5 per cent was found in the free steroid fraction, probably formed by hydrolysis of the sulphate; 90 per cent was in the sulphate ester fraction; and the rest was in the fraction of more polar compounds. To identify the metabolites, [4-¹⁴C]dehydroepiandrosterone sulphate was injected into the rat brain. Significant amounts of radioactivity were found in androstenediol sulphate, which was isolated from the brain. This compound was apparently derived from dehydroepiandrosterone sulphate by reduction of the 17-keto group to a 17β-hydroxyl group without prior hydrolysis. There was suggestive evidence that free androstenediol was also formed in the brain in this experiment.     

Metabolic interactions between animal cells through permeable intercellular junctions.

The isolated perfused rat liver was used to study the degradation of ¹²⁵I-labelled protein supplied in the perfusion medium. Formaldehyde-denatured proteins (human serum albumin, bovine serum albumin and especially rat liver phosphoenolpyruvate carboxykinase (GTP)) were taken up by the liver and degraded at high rates. Native human serum albumin was not degraded at significant rates by the perfused liver, while native phosphoenolpyruvate carboxykinase (GTP) was catabolised at about one-fourth the rate of the denatured enzyme. The degradation rate of denatured human serum albumin increased markedly as protein was added up to 0.7 mg, and more gradually with further increases in added protein. The biphasic nature of concentration dependence probably reflects the contribution of different cell types in the liver. Autoradiographic examination of serial biopsies taken during perfusion of the liver with formaldehyde-denatured, ¹²⁵I-labelled bovine serum albumin showed that at the cellular level the radioactivity was located predominantly in Kupffer and other non-parenchymal cells; and at the subcellular level the radioactivity was largely in endocytic vesicles, lysosomes and occasionally in the sinusoidal spaces. No significant radioactivity was found associated with other cytoplasmic organelles or the nucleus. It is concluded that lysosomes of the non-parenchymal cells are primarily responsible for the degradation of denatured extracellular protein that enters the liver.     

Synthesis of N-acetylneuraminic acid and of CMP-N-acetylneuraminic acid in the rat liver cell.

Adult male rats, under starving and normal conditions, were injected intravenously with N-acetyl[3H]mannosamine and after various time intervals the specific radioactivities of free N-acetylneuraminic acid (NeuAc) and CMP-N-acetylneuraminic acid were determined in the liver. The specific radioactivity of free NeuAc was high even within 20s after injection; the maximum was reached between 7 and 10 min. The specific radioactivity of CMP-NeuAc showed a lag phase of approx. 1 min. Thereafter it increased quickly and rose above the specific radioactivity of free NeuAc, reaching a maximum about 20 min after injection. These results point to a channelling of the newly synthesized NeuAc molecules into a special compartment, from which they are preferentially used by the enzyme CMP-sialic acid synthetase. It is suggested that the cytosolic enzyme N-acetylneuraminic acid 9-phosphate phosphatase is working in concert with the nuclear localized enzyme CMP-N-acetylneuraminic acid synthetase. Incorporation of radioactive sialic acid into sialoglycoproteins in liver occurred 2 min after injection, and after 10 min bound radioactivity began to appear in the circulation, indicating a transport time of 8 min of sialoglycoproteins from the point of attachment of sialic acid to the point of excretion.     

Heterolysosome formation in mouse liver

When formaldehyde-treated ¹³¹I-albumin was injected into mice, the total liver radioactivity did not change significantly from 5 minutes to 60 minutes after injection. There was a progressive increase with time in the amount of radioactivity associated with liver particles which could be released by osmotic shock; the quantity of material tightly bound to particles, but not releasable by osmotic shock, did not change. At five minutes after injection the liver particles did not release acid-soluble radioactivity into the medium when incubated at 37°. These particles contain the injected protein in osmotically releasable form not associated with proteolytic enzymes and therefore correspond to phagosomes. At 10, 30 or 60 minutes after injection, the particles degraded the protein at similar rates but the activity ceased after 90 minutes incubation when only 50 to 60% of the osmotically releasable material was hydrolyzed. This cessation of activity was shown to be due to a thermal disruption of the particles during incubation.     

Evidence for reverse cholesterol transport in vivo from liver endothelial cells to parenchymal cells and bile by high-density lipoprotein.

Acetylated low-density lipoprotein (acetyl-LDL), biologically labelled in the cholesterol moiety of cholesteryl oleate, was injected into control and oestrogen-treated rats. The serum clearance, the distribution among the various lipoproteins, the hepatic localization and the biliary secretion of the [3H]cholesterol moiety were determined at various times after injection. In order to monitor the intrahepatic metabolism of the cholesterol esters of acetyl-LDL in vivo, the liver was subdivided into parenchymal, endothelial and Kupffer cells by a low-temperature cell-isolation procedure. In both control and oestrogen-treated rats, acetyl-LDL is rapidly cleared from the circulation, mainly by the liver endothelial cells. Subsequently, the cholesterol esters are hydrolysed, and within 1 h after injection, about 60% of the cell- associated cholesterol is released. The [3H]cholesterol is mainly recovered in the high-density lipoprotein (HDL) range of the serum of control rats, while low levels of radioactivity are detected in serum of oestrogen-treated rats. In control rats cholesterol is transported from endothelial cells to parenchymal cells (reverse cholesterol transport), where it is converted into bile acids and secreted into bile. The data thus provide evidence that HDL can serve as acceptors for cholesterol from endothelial cells in vivo, whereby efficient delivery to the parenchymal cells and bile is assured. In oestrogen-treated rats the radioactivity from the endothelial cells is released with similar kinetics as in control rats. However, only a small percentage of radioactivity is found in the HDL fraction and an increased uptake of radioactivity in Kupffer cells is observed. The secretion of radioactivity into bile is greatly delayed in oestrogen-treated rats. It is concluded that, in the absence of extracellular lipoproteins, endothelial cells can still release cholesterol, although for efficient transport to liver parenchymal cells and bile, HDL is indispensable.     

Immunoprecipitation is inappropriate for the isolation of radiochemically pure albumin from tissues

Rats were given intravenous injections of l-[1-¹⁴C]leucine. Twelve minutes after injection, testes, kidneys, livers, and hepatomas were excised rapidly from one group of animals bearing Morris hepatoma 5123tc. From a second group of rats, the blood was removed 90 min after injection. Tissue extracts and serum were divided into three portions each, and albumin was isolated from each of the three portions by one of three different methods.The three different isolation procedures were the following: (A) pretreatment of the tissue extracts and serum with bovine serum albumin and its specific antiserum and subsequent immunoprecipitation of the rat serum albumin, (B) direct immunoprecipitation followed by dissolving the precipitated rat serum albumin in acid/ethanol, precipitation with ether, and ion-exchange chromatography of the redissolved albumin on CM-cellulose, and (C) a modification of a procedure published previously including fractionation with trichloroacetic acid, ethanol, ether, and ammonium sulfate, chromatography on Sephadex G-100 and DEAE-cellulose, and preparative disc electrophoresis in polyacrylamide gel at pH 10.3 and pH 2.7.With method (A), radiochemically pure albumin can be obtained only from serum. Even though testis and kidney do not synthesize albumin, albumin preparations isolated by this procedure from these organs contain significant amounts of radioactivity. Specific radioactivities measured in albumin prepared by method (A) from the four tissues examined are 5–19 times larger than those in preparations isolated by method (C). Thus, method (A) is inappropriate for the isolation of radiochemically pure albumin from the tissues studied.Procedure (B) is sufficient to obtain radiochemically pure albumin from the serum as well as from the other tissues examined except liver. With liver, this method yields an albumin preparation containing 53% more radioactivity than does albumin isolated with method (C).Method (C) is the only procedure yielding radiochemically pure albumin from all sources, including liver. In liver and hepatoma, the properties of the radioactive impurities are very similar to the properties of albumin itself. Therefore, several purification steps and a careful analysis of the distribution of radioactivity among the albumin fractions after chromatographies and electrophoreses are necessary to separate radioactive impurities from the albumin from homogenates of these two sources.     

Radiolabeled products in rat liver and serum after administration of antibody—amide—DTPA—indium-111

Anti-human serum albumin antibody (Ab) was used as a model antibody. Ab was conjugated with DTPA using cyclic DTPA dianhydride reaction and radiolabeled with ¹¹¹In. The labeled Ab was purified by affinity chromatography. Size exclusion HPLC of this product showed 62% of ¹¹¹In bound to monomeric Ab and 38% of the activity bound to antibody oligomers with molecular weights ranging from 300,000 to 450,000. The labeled antibody preparation was injected into the tail vein of rats. The radioactive substances in serum and the supernatant from liver homogenates were analyzed for molecular weight and immunoreactivity. Size exclusion HPLC of the serum samples indicated that the monomeric and dimeric Abs disappeared from the serum at a similar rate over a 48 h period. In addition, a new radioactive substance with an estimated molecular weight of 35,000 appeared in the serum. The immunoreactive fraction of the circulating ¹¹¹In substances decreased slowly, somewhat proportional to the appearance of the metabolite. On the other hand, the immunoreactivity of the ¹¹¹In substances in the supernatant from the liver homogenate decreased rapidly and no appreciable immunoreactivity was observed after 48 h. The labeled antibody was catabolized very rapidly in the liver and the major activity in the supernatant was associated with a small molecular weight metabolite which had a HPLC retention time identical to that of DTPA-¹¹¹In. The second metabolite had an estimated molecular weight of 35,000. No radioactivity was associated with transferrin.