Atherosclerosis: cell biology and lipoproteins.
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Animals↗
Biomedical subjects
A Daugherty
Publications and source records attributed to A Daugherty.
Lipoprotein oxidation as a mediator of atherogenesis: insights from pharmacological studies.
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Animals↗
The role of cholesterol accumulation in prosthetic vascular graft anastomotic intimal hyperplasia.
PURPOSE: To demonstrate that modulation of plasma cholesterol concentrations affects prosthetic vascular graft anastomotic intimal hyperplasia (AIH), aortic grafts were examined histologically and biochemically in 41 rabbits. METHODS: Twenty-seven rabbits were fed standard rabbit diet, whereas 14 were fed cholesterol-supplemented diet to induce hypercholesterolemia. RESULTS: A smooth muscle cell proliferative response, similar to AIH in humans, was seen equally at the proximal and distal anastomoses. However, surface area and thickness of AIH were significantly greater in rabbits with hypercholesterolemia. Anastomotic tissue cholesterol concentrations were fifteenfold higher in rabbits with hypercholesterolemia than in rabbits with normal cholesterol concentrations and anastomotic cholesterol concentrations were fivefold higher than in the aorta away from the graft in rabbits with hypercholesterolemia. Preferential deposition of radioiodinated dilactitol tyramine coupled to low-density lipoproteins, but not albumin, was demonstrated in anastomotic areas and grafts of rabbits with normal cholesterol concentrations as well. Surface area and thickness of AIH correlated closely with plasma and tissue cholesterol concentrations. CONCLUSIONS: Oxidized products of lipoproteins have been shown to stimulate production of growth factors that cause smooth muscle cell proliferation, migration, and synthetic function. It is likely they play an important part in prosthetic vascular graft AIH, similar to their role in atherogenesis.
Anastomosis, Surgical↗
Probucol reduces the cellularity of aortic intimal thickening at anastomotic regions adjacent to prosthetic grafts in cholesterol-fed rabbits.
Intimal hyperplasia is a persistent problem after implantation of prosthetic grafts. Although the mechanisms underlying this hyperplastic response are unknown, it has been proposed that such responses may be due to chronic vascular injury similar to that of atherogenesis. Thus, the role of oxidation was explored using the potent antioxidant drug probucol. Adult New Zealand White rabbits fed a modestly (0.25%) cholesterol-enriched diet had a polytetrafluoroethylene prosthetic graft placed into the lower aorta. After the grafting procedure, a group of 11 rabbits was placed on the cholesterol-enriched diet supplemented with 1% wt/wt probucol while a control group of 10 rabbits was placed on the cholesterol-enriched diet alone. The rabbits were maintained for a further 10 weeks before histological examination of the area surrounding the graft. Although administration of probucol did not significantly alter the dimensions of lesions at the anastomotic sites, the drug promoted striking histological changes in the surrounding tissue. Both groups of rabbits had a similar intimal hyperplastic response of the aortic tissue surrounding the graft. The vascular lesions present in the perigraft region of the control group consisted of a normal-appearing media but a thickened intima. The thickened intima contained numerous smooth muscle cells in a network of extracellular matrix. Regions in the neointima that were rich in smooth muscle cells exhibited modest staining for proliferating cell nuclear antigen. A few macrophages were present in the control group as determined by immunostaining with the monoclonal antibody RAM-11. In contrast, administration of probucol led to a marked reduction in the presence of RAM-11-staining macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)
Anastomosis, Surgical↗
Presence of LDL receptor-related protein/alpha 2-macroglobulin receptors in macrophages of atherosclerotic lesions from cholesterol-fed New Zealand and heterozygous Watanabe heritable hyperlipidemic rabbits.
Atherosclerotic lesions are composed of a complex mixture of cell types that are engorged with lipid and enveloped in extracellular matrix elements. This manifestation probably results from imbalances in the cellular processing of cholesterol-delivering lipoproteins, changes in extracellular matrix deposition, and growth factor elaboration. One receptor class that could modulate these processes is LDL receptor-related protein/alpha 2-macroglobulin receptors (LRP/alpha 2-MR). Consequently, the presence of LRP/alpha 2-MR was determined on a temporal basis in lesions of distinct morphologies that were developed in cholesterol-fed New Zealand and heterozygous Watanabe heritable hyperlipidemic (WHHL) rabbits. The two strains of rabbits developed similar degrees of hypercholesterolemia in response to 0.5% wt/wt cholesterol in their diet. Lipoprotein-cholesterol distribution was also similar in the two strains. Aortic intimal areas covered by grossly discernible atherosclerotic lesions were extensive and not statistically different between the strains. Despite the similarities in the extent of hypercholesterolemia, lipoprotein distribution, and extent of atherosclerosis, the cellularity of the lesions formed was different in the two groups. Atherosclerotic lesions in cholesterol-fed New Zealand rabbits were uniformly rich in macrophages and deficient in smooth muscle cells, as determined by immunocytochemical staining with the cell-specific monoclonal antibodies RAM-11 and HHF-35. In contrast, atherosclerotic lesions formed in cholesterol-fed heterozygous WHHL rabbits covered a spectrum ranging from macrophage-rich lesions to those predominantly composed of disaggregated smooth muscle cells that were embedded in dense layers of extracellular matrix.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions.
Oxidatively modified lipoproteins have been implicated in atherogenesis, but the mechanisms that promote oxidation in vivo have not been identified. Myeloperoxidase, a heme protein secreted by activated macrophages, generates reactive intermediates that oxidize lipoproteins in vitro. To explore the potential role of myeloperoxidase in the development of atherosclerosis, we determined whether the enzyme was present in surgically excised human vascular tissue. In detergent extracts of atherosclerotic arteries subjected to Western blotting, a rabbit polyclonal antibody monospecific for myeloperoxidase detected a 56-kD protein, the predicted molecular mass of the heavy subunit. Both the immunoreactive protein and authentic myeloperoxidase bound to a lectin-affinity column; after elution with methyl mannoside their apparent molecular masses were indistinguishable by nondenaturing size-exclusion chromatography. Peroxidase activity in detergent extracts of atherosclerotic lesions likewise bound to a lectin column and eluted with methyl mannoside. Moreover, eluted peroxidase generated the cytotoxic oxidant hypochlorous acid (HOCl), indicating that enzymatically active myeloperoxidase was present in lesions. Patterns of immunostaining of arterial tissue with antihuman myeloperoxidase antibodies were similar to those produced by an antimacrophage antibody, and were especially prominent in the shoulder region of transitional lesions. Intense foci of myeloperoxidase immunostaining also appeared adjacent to cholesterol clefts in lipid-rich regions of advanced atherosclerotic lesions. These findings identify myeloperoxidase as a component of human vascular lesions. Because this heme protein can generate reactive species that damage lipids and proteins, myeloperoxidase may contribute to atherogenesis by catalyzing oxidative reactions in the vascular wall.
Arteriosclerosis↗
Lipoprotein receptors in arterial tissue: relation to the pathology of atherosclerosis.
The molecular and cellular physiology of several classes of lipoprotein receptors has been extensively characterized in vitro. However, the evidence that these lipoprotein receptors mediate the morphological events characteristic of atherosclerotic development in vivo is limited. The increasing availability of reagents to characterize receptor mRNA and protein will enable the definition of receptors involved in atherosclerotic lesion progression. These studies should set the stage for the more difficult task of modulating lipoprotein receptors to determine whether their activity is a critical component of atherogenesis.
Animals↗
Quantitative ultrasonic characterization of lesion composition and remodeling in atherosclerotic rabbit aorta.
We have previously shown that high-frequency, high-resolution ultrasound can characterize the acoustic properties and composition of fatty plaques in cholesterol-fed rabbits. To determine whether quantitative ultrasound can delineate the regression of atherosclerotic lesions by detecting a change in their composition from fatty to fibrous types induced by alterations in dietary regimen, we fed six New Zealand White rabbits a 2% cholesterol diet for 3 months, followed by a standard diet for 3 additional months to promote the development of fibrous intimal lesions. Segments of aortas were excised, and backscattered radiofrequency data were acquired from 400 to 600 independent sites in each specimen with an acoustic microscope operated at 50 MHz. Control data were provided by measuring backscatter from adjacent portions of the aortas devoid of lesions. Histological and immunocytochemical analyses of the fibrous intimal lesions confirmed the presence of smooth muscle cells and abundant connective tissue with little appreciable lipid. Backscatter from normal aortic segments (-30.7 +/- 1.0 dB) was approximately 10-fold greater than that from fibrous lesions (-42.4 +/- 1.0 dB; P < .05). We previously reported that integrated backscatter from fatty lesions was -50.6 +/- 0.7 dB, or approximately 10-fold less than that from fibrous lesions (P < .05). Values for integrated backscatter from the media of each tissue type were approximately equal (-30.0 +/- 1.7 versus -30.7 +/- 1.6 versus -33.4 +/- 0.8 dB for normal versus fibrous versus fatty tissues, respectively; P = not significant).(ABSTRACT TRUNCATED AT 250 WORDS)
Actins↗
Quantification of the accumulation and degradation of beta-very-low-density lipoproteins in vivo using a 19F-containing residualizing label and n.m.r. spectroscopy.
beta-Very-low-density lipoproteins (beta-VLDL)O were conjugated to the 19F-containing residualizing label, NN-dilactitol-3,5-bis(trifluoromethyl)benzylamine (DLBA), to determine whether the metabolism of this lipoprotein fraction could be characterized in vivo with n.m.r. spectroscopy. Solution state 19F high-resolution n.m.r. spectroscopy of DLBA-beta-VLDL, containing either intact apoproteins or selectively enzymically digested products, demonstrated that the extent of degradation could be distinguished by differences in spin-spin relaxation times (T2 times). DLBA-beta-VLDL was injected intravenously into rabbits, and accumulation of 19F in hepatic tissue was quantified non-invasively by n.m.r. spectroscopy 5 and 30 h after injection. In addition to quantifying the accumulation of DLBA-beta-VLDL in hepatic tissue, a marked decrease (approx. 100 Hz) in the linewidth of 19F resonance from labelled lipoproteins was observed at 30 h compared with the 5 h interval in continuously monitored animals. The change in linewidth was consistent with a decrease in molecular size that occurred during protein degradation, resulting in increased T2 times. To demonstrate that T2 times can be used as an index to quantify apoprotein degradation in vivo, relaxation measurements were performed on livers excised 20 h after injection of DLBA-beta-VLDL into rabbits. Two molecular motional fractions were revealed by relaxation profiles representing either an intact or an extensively degraded form of apoprotein. The amplitudes of each component were compared with results from trichloroacetic acid precipitation of liver homogenates acquired from rabbits 20 h after injection of beta-VLDL labelled with the radioiodinated analogue of DLBA, dilactitol-125I-tyramine. The amount of degraded apoprotein determined by n.m.r. spectroscopy and acid precipitation was 68.6 +/- 7.0% and 58.7 +/- 7.5% (n = 4) respectively. The results of this study demonstrate that 19F n.m.r. spectroscopy can be used to define the temporal characteristics of the hepatic metabolism of lipoproteins in vivo by quantifying both the tissue-specific accumulation and extent of apoprotein degradation. The methodology developed offers promise for the non-invasive, sequential and longitudinal evaluation of lipoprotein metabolism in vivo.
Animals↗
Short-term interruption of training affects both fasting and post-prandial lipoproteins.
Exercise training alters plasma lipoprotein profiles in a manner compatible with decreased coronary artery disease risk. The aim of this study was to ascertain whether interruption of training (detraining) was associated with potentially undesirable changes in the metabolism of post-prandial lipoproteins and plasma levels of Lp(a). Eight normolipidemic, male runners who ran 30-40 miles/week were studied in the trained state and after 14-22 days of detraining. Two of the subjects were studied in the reverse order to control for any confounding effects of exercise sequence. Detraining resulted in (1) a 12% (P = 0.002) reduction in the subjects' aerobic capacity, (2) a 7.7% (P = 0.007) reduction in fasting concentrations of high density lipoprotein cholesterol (HDL-C), (3) a 21% (P = 0.01) reduction in post-heparin lipoprotein lipase activity. Lp(a) concentrations did not change significantly (mean increase 15%, P = 0.076). Fasting plasma concentrations of total cholesterol (TC), triglycerides (TG) and low density lipoprotein-cholesterol (LDL-C) did not change in the detrained state. There was little fluctuation over 24 h in plasma concentrations of TC, LDL-C and HDL-C in either the trained or detrained states. TG concentrations fluctuated over the 24 h in accord with food intake, but there were no exercise-related changes. Exercise had a dramatic effect on chylomicron and chylomicron remnant metabolism as measured by retinyl palmitate measurements. The mean areas under the concentration vs. time curves (AUC) for chylomicron-retinyl esters increased by 41% (P = 0.013) and for chylomicron remnant-retinyl ester by 37% (P = 0.058) following detraining.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
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.
Animals↗
Imaging of thrombi with tissue-type plasminogen activator rendered enzymatically inactive and conjugated to a residualizing label.
BACKGROUND: Contemporary cardiovascular practice relies increasingly on thrombolysis as a therapeutic modality. Its optimal use requires prompt, noninvasive delineation of thrombotic occlusion in arterial beds and rapid detection of reocclusion after initially successful thrombolysis. METHODS AND RESULTS: We have been developing an approach to noninvasively image thrombi in which plasminogen-activating properties of tissue-type plasminogen activator (t-PA) are attenuated by treatment with D-Phe-L-Pro-L-Arg-chloromethyl ketone (PPACK) and have shown that the inactive t-PA avidly and promptly binds to clots in vitro. In the present study, we conjugated this material to a residualizing label, radioiodinated dilactitol tyramine (*I-DLT), and characterized the potential use of the inactivated, conjugated t-PA as a radiopharmaceutical for imaging thrombi in vivo. The approach developed requires not only avid binding of the tracer to thrombi but also rapid clearance from plasma and a lack of prompt release of radiolabeled degradation products from the liver. The rapid clearance of unaltered or PPACK-treated t-PA was not influenced by conjugation to *I-DLT, but the release of radioiodinated degradation products into plasma after injection of *I-DLT-conjugated t-PA was markedly less than release of degradation products of directly radioiodinated t-PA. When 131I-DLT-PPACK-t-PA was infused for 15 minutes intravenously after a bolus injection of 20% in dogs with coronary, pulmonary, or carotid artery thrombi, clearance was rapid. Mean +/- SEM thrombus-to-blood ratios of radioactivity were high, ranging from 37 +/- 9:1 and 2.8 +/- 0.6:1 with carotid thrombi formed concomitantly or approximately 30 minutes before infusion of tracer, respectively, to 35:1 for concomitantly formed coronary thrombi, 42 +/- 7:1 and 8.1 +/- 0.8:1 for concomitantly formed and preformed pulmonary thrombi, respectively, and 18:1 for a preformed femoral artery thrombus. Thrombi were detectable by planar gamma scintigraphy even though image quality was affected adversely by low concentrations of radioactivity that in aggregate composed a relatively large amount of radioactivity in underlying and overlying tissues. This limitation was overcome by tomographic imaging, which was used to detect both femoral and pulmonary thrombi. CONCLUSIONS: Use of enzymatically inactivated t-PA coupled to a residualizing label permits rapid detection and localization of thrombi in vivo.
Amino Acid Chloromethyl Ketones↗
The volume of gas emitted from five metered dose inhalers at three levels of fullness.
UNLABELLED: We conducted this study to determine the volume of gas emitted from five commonly used metered dose inhalers (MDIs). MATERIALS & METHODS: We used the following MDIs: Alupent (Boehringer Ingelheim), Atrovent (Boehringer Ingelheim), Beclovent (Allen & Hanburys), Intal (Fisons), and Ventolin (Allen & Hanburys). The test system consisted of plastic bag, MDI adapter, 4-way stopcock, and 60-mL calibrated syringe. This system was glued together, and absence of leaks was confirmed by underwater testing. Each evaluation consisted of 10 puffs from the MDI into the bag, after which the volume in the bag was determined using the syringe, and the volume/puff was calculated by dividing by 10. Each MDI was evaluated at 3 levels of fullness: nearly full, partially empty, and nearly empty. Five measurements were made with each MDI brand, using a new MDI for each set of measurements. RESULTS: Although there was a significant difference in the volumes emitted between MDI (p less than 0.001) and the levels of fullness (p = 0.001), the volume of gas emitted from these MDIs was small (approximately 15-20 mL). A significant interaction existed between MDI brand and level of fullness (p = 0.001). CONCLUSIONS: Based on prior studies of propellant toxicity coupled with our findings on the volumes of gas emitted from MDIs, we conclude that the volumes of gas emitted are too small to be clinically important in the care of adult patients. The volume of gas emitted from an MDI is only potentially important if MDIs are used with very small tidal volumes in a closed system (eg. infants).
Chlorofluorocarbons, Methane↗
Failure of the intracellular itinerary of beta very low density lipoproteins to augment cholesterol esterification in macrophages from Watanabe heritable hyperlipidemic rabbits.
beta very low density lipoproteins (beta-VLDL) interact with mouse peritoneal macrophages via specific receptors leading to pronounced stimulation of cholesterol esterification. The present study has defined an alternative pathway for the processing of beta-VLDL in alveolar macrophages from Watanabe heritable hyperlipidemic (WHHL) rabbits. Macrophages from either New Zealand (NZ) or WHHL rabbits degraded 125I-beta-VLDL to an equivalent extent. Degradation was competed to a similar extent in both cell types by either excess unlabeled beta-VLDL or low density lipoprotein, indicative of a specific receptor involvement. Accumulation of intracellular degradation products of beta-VLDL labeled with the residualizing label, dilactitol-125I-tyramine, was similar in both cell types demonstrating that degradation was not due to secreted proteolytic enzymes. beta-VLDL promoted the incorporation of [3H]oleate into cholesteryl-[3H]oleate and increased the cellular mass of cholesterol in NZ macrophages. In contrast, beta-VLDL did not augment cholesteryl-[3H]oleate deposition in WHHL macrophages. This lack of cholesterol esterification occurred despite equivalent acyl-CoA:cholesterol acyltransferase activity in microsomal fractions of both cell types, and similar augmentations in cholesteryl-[3H]oleate during incubation with phospholipase C-treated LDL. Incubation of WHHL macrophages with beta-VLDL increased cellular cholesterol mass, although the response was attenuated compared to NZ cells. To determine whether these disparities in cholesterol esterification were related to the catabolic fate of beta-VLDL-derived cholesterol esters, [3H]cholesteryl oleate was exchanged into the core of beta-VLDL and incubated with macrophages in medium containing [14C]oleate. NZ macrophages accumulated both [3H]cholesterol and [3H]cholesteryl-[14C]oleate after 5 h, indicating hydrolysis and re-esterification of cholesterol esters. In contrast, WHHL macrophages only accumulated [3H]cholesterol esters, suggesting uptake of cholesterol esters without subsequent hydrolysis. These data demonstrate that WHHL macrophages possess a pathway for the intracellular processing of beta-VLDL that permits internalization of the particle without stimulation of cholesterol esterification.
Animals↗
The effects of probucol on the progression of atherosclerosis in mature Watanabe heritable hyperlipidaemic rabbits.
1. Probucol was administered to mature Watanabe heritable hyperlipidaemic (WHHL) rabbits (approximately 9 months old). Groups of WHHL rabbits were randomly selected and treated as follows: Group 1 killed at 9 months (n = 9); Group II placed on sham-treated diet at 9 months and followed for 6 months (n = 8); Group III placed on probucol at 9 months and followed for 6 months (n = 8). Probucol was administered by mixing 1% wt/wt drug with standard laboratory diet. 2. Plasma concentrations of probucol increased to 93 +/- 11 micrograms ml-1 in Group III during the initial 2 weeks and increased further to 149 +/- 24 micrograms ml-1 at the end of the treatment period. 3. Plasma concentrations of total cholesterol, unesterified cholesterol and phospholipids were significantly reduced overall by probucol, while triglycerides were not affected. 4. No statistically significant differences were observed in the presence of oxidized products in low density lipoproteins (LDL) isolated from plasma of controls compared to probucol-treated rabbits. However, LDL from probucol-treated animals was resistant to oxidation in the presence of Cu2+ (3 microM). 5. Group I had aortic atherosclerosis covering 70 +/- 5% of intimal area of thoracic aortae, that increased to 91 +/- 3% in Group II. This was associated with cholesterol contents of aortae increasing from 1.4 +/- 0.2 microgram mg-1 in Group I to 2.7 +/- 0.3 microgram mg-1 in Group II. Probucol administration did not produce a statistically significant reduction of atherosclerotic lesion area (78 +/- 7%). However, probucol treatment reduced cholesterol content to 1.9 + 0.3,ugmg-' (P < 0.01). Collagen content of aortae was not affected by probucol treatment. 6. Thus, while probucol did not promote regression, the drug did retard the continued deposition of cholesterol esters into atherosclerotic lesions of mature WHHL rabbits.
Animals↗
Metabolic imaging with gallium-68- and indium-111-labeled low-density lipoprotein.
Low-density lipoprotein (LDL) labeled with either gallium-68 (68Ga) or indium-111 (111In) was evaluated as a potential PET or SPECT radiopharmaceutical for determination of hepatic lipoprotein metabolism in rabbits. Gallium-68 or 111In was linked to LDL via diethylenetriaminepentaacetic acid (DTPA) with a 25-70% radiochemical yield. Studies in vivo that compared 68Ga- or 111In-DTPA-LDL with dilactitol-[125I]-tyramine LDL and 131I-LDL showed that both 68Ga- and 111In-labeled LDL behaved as residualizing radiotracers. Localization of radioactivity within the liver of normal rabbits was visualized clearly with [68Ga]DTPA-LDL by PET and with [111In]DTPA-LDL by gamma scintigraphy. Significant differences were observed in hepatic uptake of normal compared with hypercholesterolemic rabbits in which low-capacity LDL receptor-mediated catabolism was saturated. Gallium-68 and 111In-DTPA-LDL are attractive radiopharmaceuticals for noninvasive delineation of tissue LDL metabolism under normal and pathophysiologic conditions.
Animals↗
Enhanced clearance from plasma of low density lipoproteins containing a truncated apolipoprotein, apoB-89.
Previously we have reported on a kindred with hypobetalipoproteinemia in which three sisters were found to be compound heterozygotes for two newly described truncated forms of apoB. apoB-40 and apoB-89. ApoB-89-containing low density lipoproteins (LDL) bound with increased affinity to cultured normal human fibroblasts and were internalized and degraded at increased rates, suggesting that the low plasma concentrations of apoB-89-LDL of the patients could be due to enhanced rates of clearance through LDL-receptors. To examine this hypothesis, apoB-89-LDL was isolated from the three study subjects and apoB-100-LDL from two control subjects. LDL was conjugated to the radioiodinated residualizing label, dilactitol tyramine (*I-DLT, containing either 125I or 131I). *I-DLT-apoB-89-LDL and *I-DLT-apoB-100-LDL were simultaneously injected into ear veins of rabbits. The clearance from plasma and hepatic accumulations of both radiolabeled LDL fractions were followed over 24 h. Fractional catabolic rates (FCR) of apoB-89-LDL were 0.105 +/- 0.012 h-1 compared to 0.054 +/- 0.007 h-1 for apoB-100-LDL. In agreement with the enhanced clearance from plasma, 1.72 to 1.87 times more *I-DLT-apoB-89-LDL than *I-DLT-apoB-100-LDL accumulated in the livers 24 h after injection. There was no significant difference in splenic accumulation, suggesting that LDL-receptors rather than scavenger receptors mediated the enhanced clearance of apoB-89-LDL. To assess further the importance of LDL-receptors, *I-DLT-apoB-89-LDL and *I-DLT-apoB-100-LDL were reductively methylated to inhibit their interactions with LDL-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Non-invasive detection of protein metabolism in vivo by n.m.r. spectroscopy. Application of a novel 19F-containing residualizing label.
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.
Animals↗