99mTc-low density lipoprotein: Intracellularly trapped radiotracer for noninvasive imaging of low density lipoprotein metabolism in vivo

99mTc-Low Density Lipoprotein: Intracellularly Trapped Radiotracer for Noninvasive Imaging of Low Density Lipoprotein Metabolism In Vivo Shankar Vallabhajosula and Stanley J. Goldsmith L o w density lipoprotein (LDL) is the major transport protein for endogenous cholesterol in human plasma. LDL can be radiolabeled w i t h SS=Tc using sodium dithionite as a reducing agent. Biodistribution studies of 9SmTc-LDL in normal rabbits confirm that sgmTc-LDL acts as an intracellularly "trapped ligand" similar to radioiodinated tyramine callobiose-LDL (the previously validated trapped radioligand). In addition, studies p e r f o r m e d in hypercholesterolemic rabbit models demonstrated the feasibility of imaging hepatic LDL-receptor concentration noninvasively. 9SmTc-LDL imaging studies in a number of hypercholesterolemic and hypocholesterolemic patients have proven useful in understanding the abnormal uptake and metabolism of LDL. In patients w i t h

hypercholesterolemia (HC), sgmTC-LDL appears to be taken up well by the actively evolving atherosclerotic lesions and x a n t h o m a t a t h a t contained foam cells and macrophages. In patients with myeloproliferative disease and chronic hypocholesterolemia, 99mTcLDL images showed intense accumulation of radioactivity in the spleen and bone marrow; t h i s demonstrated extensive proliferation of the macrophage population suggesting t h a t hypocholesterolemia in these patients may be due to increased uptake of LDL uptake by the macrophages. 9gmTc-LDL is a powerful tool for the noninvasive exploration of a variety of disorders of lipoprotein metabolism in patients.

I P O P R O T E I N S are water-soluble complexes composed of lipids (cholesterol, triglycerides, phospholipids) and one or more specific proteins, called apolipoproteins. Plasma lipoproteins transport water-insoluble lipids in the blood. The lipoproteins are divided into various categories according to density as determined by ultracentrifugation. 1 The principal categories are chylomicrons, very low-density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). While the large chylomicrons have a lipid-protein ratio of -99:1,the small HDL consist o f - 5 0 % lipid and 50% protein. VLDL is secreted into the bloodstream by the liver, and undergoes degradation in the plasma to IDL and further to LDL. The major transport protein for endogenous cholesterol in human plasma is LDL. The role and significance of plasma lipoprotein levels in health and disease will be discussed later.

LDL: CHEMISTRY AND METABOLISM

From the Department of Physics-Nuclear Medicine, The Mount Sinai Medical Center, New York, NY. Address reprint requests to Shankar Vallabhajosula, PhD, Department of Physics-Nuclear Medicine, The Mount Sinai Medical Center, One Gustave L. Levy PI, New York, NY, 10029. 9 1990 by W.B. Saunders Company. 0001-2998/90/2001-0006505.00/0 68

9 1990 by W.B. Saunders Company.

LDL is a large spherical particle with a mass of 3 million d and a diameter of 22 nm. It is composed of some 1,500 molecules of cholesteryl esters. The oily core is shielded from the aqueous plasma by a detergent coat composed of 800 molecules of phospholipid, 500 molecules of unesterified cholesterol, and one large protein molecule, apoprotein B-100. 2 The tissue uptake and degradation of LDL occurs in part by way of a high-affinity saturable mechanism involving the specific LDL receptor first reported by Goldstein and Brown. 3 After binding to the receptor, LDL is internalized and degraded, presumably by lysosomes.3 The receptors recognize the apoprotein B-100 component of LDL. In addition, LDL can also be taken up and degraded by scavenger cells (macrophages of reticuloendothelial system) by low-affinity adsorptive endocytosis4 or, like other solutes, can be taken up by fluid or bulk endocytosis. 5 Two thirds of LDL particles in plasma are metabolized after binding to specific receptors that are located on the surfaces of liver cells6 and other body cells 7'8 while the remaining one third of plasma LDL is metabolized by receptor-independent mechanisms. 9 RADIOIODINATED LDL PREPARATIONS

Studies designed to understand the pharmacokinetics and metabolism of LDL have traditionSeminars in Nuclear Medicine, Vol XX, No 1 (January), 1990: pp 68-79

99MTc: I M A G I N G

STUDIES

IN V I V O

69

ally employed radioiodinated native-LDL as a tracer of LDL. Radioiodination of LDL was performed using the iodine monochloride method.l~ A number of animal and human studies 1116 have used 125I-LDL to study the plasma clearance of LDL. Proteins labeled by direct radioiodination, however, yield radiolabeled catabolic products that rapidly leave cells as radioiodide or radiotyrosine. 13'14'17 Therefore, to study the metabolism and determine the sites of LDL degradation in vivo, Pittman et a118 developed 14C-sucroseLDL by covalently coupling 14C-sucrose to LDL. Following cellular uptake the lysosomal degradation of 14C-sucrose-LDL, the ligand 14C-sucrose was trapped intracellularly within lysosomes since sucrose cannot be degraded by lysosomal enzymes. The 14C-sucrose labeling method, however, is limited by the low specific activities (< 10 #Ci/mg of apoprotein) achievable. 17 Subsequently, Pittman et a117developed another intracellularly trapped radiotracer, 125I-tyramine cellobiose-LDL (125I-Tc-LDL), which can be prepared in high specific activities (2 to 5 mCi/ mg of apoprotein). The preparation of 125I-TcLDL involves two steps: electrophilic radioiodination of tyramine-cellobiose (Tc) followed by conjugation of 125I-Tc to LDL using cyanuric chloride. 17 A number of animal studies demonstrated that radioiodinated tyramine cellobiose LDL is a trapped radioligand for quantitative determination of LDL metabolism in vivo.13,14,17,19 The development of intracellularly trapped ligands has permitted investigators to identify the sites of in vivo degradation of LDL in animals. These methods, however, require killing the animals, thus excluding repeat studies in the same animal. Noninvasive imaging of LDL distribution would be of considerable value in animals and patients with abnormal LDL levels to understand metabolism and to follow the effects of therapy. For this purpose, neither 125Inor 1311are satisfactory for noninvasive imaging studies. 123I on the other hand is suitable for imaging studies, 14'2~but is quite expensive. In addition, LDL labeled with radioiodinated Tc may not be approved for studies in human subjects.

99mTCLOW-DENSITY

LIPOPROTEIN

99mTc-LDL: Preparation Lees et a121 first reported the technique of labeling LDL with 99mTC,a radionuclide ideal for

imaging studies. LDL was labeled with 99mTcby the reduction of 99mTc-pertechnetate with sodium dithionite in the presence of native LDL. 21'22 Subsequently, 99mTc-LDL was separated from unbound 99mTcby gel filtration. The reduction of pertechnetate by dithionite at mildly alkaline pH does not aggregate or denature LDL, and is one of the most efficient methods for attaching 99mTc to proteins. 21'23The purity and stability of 99mTCLDL was also demonstrated based on agarose electrophoresis, ultracentrifugation, and immunoelectrophoresis.13'21

99mTc-LDL Versus Radioiodinated LDL Preparations Plasma clearance. In normal rabbits, the disappearance of 99mTc-LDL, 125I-native-LDL, and 131I-Tc-LDL from the circulation reflects a biexponential decay mode (Fig 1). The plasma disappearance curves of 99mTc-LDL and 1251native-LDL are quite similar and are more rapid than that of 131I-Tc-LDL.13 In a study comparing radioiodinated native LDL with Tc-LDL in monkeys, Portman and Alexander 19 also observed that 131I-Tc-LDL was removed from circulation at a slightly slower rate than 125I-native-LDL. However, the plasma disappearance of 131I-TcLDL and 125I-native-LDL were reported by other investigators to be similar. 14'17 While there is some discrepancy between the reports about the plasma disappearance of 125I-native-LDL and 131I-Tc-LDL, our results suggest that the stability and behavior of 99mTc-LDL in the circulation is similar to that of radioiodinated native LDL. 13 >I--

x 13LlTC LDL o I25i-lOL

I.O

I-
. 9"Tc LOL

~

0.5 nm Z

Z 0 r~

"0.10

i

i

I

2 5 4

J

II

I

I

I

I

I

12 16 TIME(H0urs)

8

I

I

20

I

I

24

i

28

Fig 1. Plasma decay curves of 99"Tc-LDL, 1251-native LDL, and 1311-Tc-LDL in a normal rabbit. The plasma disappearances of SS'Tc-LDL and 12SI-LDL were quite similar and more rapid than the disappearance of l s l I - T c - L D L ,

70

VALLABHAJOSULA A N D GOLDSMITH

Biodistribution. The accumulation of radioactivity in several selected tissues in normal rabbits 24 hours after a simultaneous injection of the three radiolabeled LDL preparations is summarized in Table 1. The uptake of all three tracers (expressed as a percent of injected dose per gram of tissue) was highest in the adrenal glands followed by spleen, liver, and kidney. 13 The accumulation of 99mTc in these organs was somewhat similar to that of 1311 (from Tc-LDL), but - 1 0 times higher than 1251 (from nativeLDL). Similarly, imaging studies performed in monkeys 13 showed equivalent accumulation of radioactivity in the liver with each of the three tracers in the early images. Over the next several hours, however, only radioiodine counts (from native-LDL) diminished drastically from the liver and were seen over the area of lower abdomen. These results demonstrate that the usefulness of radioiodinated native-LDL as a tracer of L D L for biodistribution studies is severely limited by the rapid and significant deiodination (dehalogenation) within the tissues.13'14'17 99mTc-LDL as a Trapped Ligand The tissue distribution results (Table 1) comparing 99mTc-LDL with 131I-Tc-LDL clear show that these two tracers behave quite similarly. In contrast to radioiodinated native-LDL, both 99mTc and 131I were taken up and retained by various tissues and organs, although the accumulation of J31I in adrenals, spleen, and liver was less than that of 99mTc.These differences are probably due to slower removal of 131I-Tc-LDL from circulation (Fig 1) and leakage of 131I-Tc from tissuesJ 7 The pattern of biodistribution of 99mTc-LDL in normal rabbits shown in Table 1 is similar to those observed with the well characterized Table 1. Comparison of the Accumulation of Three LDL Radiotracers in Selected Tissues of Normal Rabbits Percent Injected Dose Per Gram of Tissue at 24 h ~m

Organ Blood Adrenal glands

Tc-LDL 0.05 • 0.03*

1311-Tc-LDL

1251-native-LDL

0 . 1 5 _+ 0 . 0 2

0 . 0 5 -+ 0 . 0 5

2 . 0 2 _+ 1.03

1.23 _+ 0 . 5 6

0.23 • 0.23

Spleen

0 . 7 4 _+ 0 . 5 9

0 . 6 2 +_ 0 . 2 0

0.08 • 0.03

Liver Kidneys Gallbladder Lungs

0.46 + 0.08

0 . 2 2 _+ 0 . 0 3

0.06 • 0.02

0.17 • 0.05

0 . 2 8 _+ 0 . 0 9

0.03 • 0.02

0 . 1 3 -+ 0 . 1 0

0.10 • 0.08

0.07 • 0.04

0 . 0 7 _+ 0 . 0 4

0.07 + 0.03

0.05 • 0.03

* M e a n _+ SD; (n = 4).

trapped ligands, 14C-sucrose-LDL,18 and 125I-TcLDL. 17'23In addition, 99mTc-LDL biodistribution studies in normal rabbits, reported by Lees et al, 21 also showed that adrenal glands accumulated maximum 99mTcactivity per gram of tissue followed by spleen, liver, and kidney, although the absolute amounts varied. Intracellular trapping of 99mTc-LDL should be associated with minimal urinary excretion of 99mTc activity. Both in animals and human subjects, only 5% to 12% of injected 99mTc activity was excreted in the urine within a 24-hour period following an injection of 99mTc-LDL.13 These results strongly support our hypothesis that 99mTcLDL acts as an intracellularly trapped ligand similar to that of 14C-sucrose-LDL and radioiodinated-Tc-LDL. 25 Recently, Lees et a126 demonstrated in an in vitro model that 99mTc-LDL is preferentially taken up by cultured fibroblasts and that the 99mTc activity is retained within the cells. Since LDL receptors are known to be present on fibroblasts, 3 this observation further confirms that 99mTc-LDL acts as an intracellularly trapped ligand. 99,,Tc-LDL: Imaging Studies in Hypercholesterolemic Rabbits Goldstein and Brown 3 showed that the tissue uptake of LDL is partially mediated by LDL receptors present in various tissues. The liver is the primary organ for LDL catabolism 6'1~ and the vast bulk of LDL receptors in animals are in the liver. 6'24 These hepatic LDL receptors are subject to regulation. LDL receptor activity can be suppressed by high cholesterol diets 6'27 or even by prolonged fasting. 28 The number of LDL receptors is also diminished as a result of defects in the gene encoding the receptor. 3'8'29 When the number of LDL receptors are diminished, IDL is not cleared normally by the liver and is converted to L D L in increased amounts. 3~ Catabolism of LDL is also diminished. Finally, as a result of overproduction and undercatabolism, the level of LDL in plasma rises resulting in hypercholesterolemia (HC). 3~ In order to evaluate the LDL catabolism by hepatic L D L receptors noninvasively, we studied the biodistribution of 99mTc-LDL in normal and hypercholesterolemic rabbits. 31Two animal models with H C were used: normal rabbits fed on a

99"Tc: IMAGING STUDIES IN VlVO

high cholesterol diet (HCR) and rabbits with a genetic defect designated as Watanabe heritable hyperlipidemic (WHHL).29 The defect in WHHL rabbits resides in the gene for the LDL receptor-the same gene that is defective in patients with familial HC. 3~ The W H H L homozygous (WHHL-HO) rabbits exhibit a total lack of LDL receptors while the WHHL heterozygotus rabbits (WHHL-HE) exhibit half the number of functional receptors. Following an intravenous (IV) injection of 99mTc-LDL into different groups of rabbits (normal, HCR, WHHL-HO, and WHHL-HE), gamma camera images of the whole body were obtained at 0.5, 1.0, 4.0 and 24.0 hours. The 99mTc-LDL distribution in normal, HCR, and WHHL-HE at 4 hours is shown in Fig 2. Regions of interest (ROI) were drawn around the liver and heart and the ratio of liver to heart (L:H) 99mTcactivity was calculated. The L:H ratio as a function of time for different animal models is shown in Table 2. The images in Fig 2 clearly show intense 99mTcactivity in the liver of normal rabbit compared with that of HCR and W H H L

71

rabbits. The L:H ratio increases as a function of time in all rabbits. The L:H ratio in normal rabbits at 4 and 24 hours, however, is significantly higher than in HCR and W H H L rabbits. The normal rabbits with normal functional hepatic LDL receptors have a higher L:H ratio compared with WHHL-HO rabbits with lower L:H ratio while the HCR and WHHL-HE rabbits have L:H ratios between the two. This inverse relationship between the hepatic LDL receptor concentration and L:H ratios of 99mTC activity clearly demonstrates the feasibility of imaging hepatic LDL receptor activity in vivo, and in addition confirm the validity of 99mTcLDL as a tracer of LDL distribution in vivo. Similarly, Williams et al 3z demonstrated, based on imaging studies, that the hepatic trapping of 99mTc-LDL was significatly greater in normal rabbits than WHHL rabbits. As control experiments, biodistribution studies of 99mTc-HDL, the other principal lipoprotein in plasma that has been implicated as a major link in the transport of cholesterol from peripheral tissues to the liver, were performed. 33 Imaging

Fig 2. 9g'Tc-LDL biodistribution in normal (A), HCR (B), and WHHL-HE rabbits (C) 24 hours after injection of the tracer. The images show intense accumulation of 99mTc activity in the liver and adrenals (arrows) of normal rabbit and reduced uptake by liver with nonvisualization of adrenals in HCR and WHHL rabbits. In addition, kidney uptake in these HC rabbits is much greater than in normal rabbits.

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VALLABHAJOSULA A N D GOLDSMITH

Table 2. 99mTc-LDL: Liver to Heart Ratios as a Function of Time Rabbit Normal

0.5 h

1h

4h

24 h

3.6 • 0 . 3

5.2 • 1.1

11.3_+ 1.5

2 9 . 3 - + 6.7

HCR

3.5•

4.1•

5.9 • 1.4

11.5 • 1.7

WHHL-HE

2.7 • 0 . 5

3.2-+ 0.4

5.6 • 0 . 9

11.9 • 1.3

WHHL-HO

2.9-+ 0.4

3.2 • 0.3

4.4 •

6 . 8 • 1.5

studies with99mTc-HDL were performed in normal and hypercholesterolemic rabbits; L:H ratios are shown in Table 3 (previously unpublished). The L:H ratios of 99mTc-HDL in normal rabbits at 4 and 24 hours are lower than the L:H ratios of 99mTc-LDL, suggesting that the plasma clearance of HDL is slower than LDL in normal rabbits. In addition, the L:H ratios of 99mTcHDL are similar in both normal and hypercholesterolemic rabbits suggesting that the hepatic clearance of HDL is not affected by hepatic LDL receptors. These results are in agreement with the in vivo behavior of HDL. The apoprotein component of HDL consists of several apoproteins: apo A-I, apo A-IL, and apo E. The classic LDL receptor also recognizes the apo E component of HDLfl4 It has been shown that one LDL particle binds to one receptor while one apo E of HDL binds to four LDL receptors) 5 As a result, hepatic uptake of HDL via LDL receptors is very limited. These results demonstrate that 99mTcLDL is a true tracer of LDL biodistribution in vivo, since another lipoprotein, HDL labeled with 99mTc, under similar conditions behaved distinctly different from 99mTc-LDL, ie, no differences were observed with 99mTc-HDL clearance between normal and hypercholesterolemic rabbits.

Adrenal Imaging." Effect of Diet and Drugs The 99mTc-LDLdistribution in normal rabbits (Fig 2) clearly shows intense accumulation of 99mTcactivity in the adrenal glands. The biodistribution studies (Table 1) also showed that the adrenal glands accumulate a greater percentage of 99mTc activity per gram of tissue than the Table 3. 99mTc-HDL: Liver to Heart Ratios as a Function of Time Rabbit

0,5 h

1h

4h

24 h

Normal

3.3 -+ 0 . 4

3.8 -+ 0 . 5

5.3 • 0 . 7

8 . 9 • 1.2

HCR

3.5 • 0 . 8

3.8 • 0.9

4.4 •

1.2

7.9 • 2 . 0

WHHL-HE

4.0 • 0.4

4.5 • 0.4

5.2 • 0 . 4

7.6 + 0 . 9

spleen, liver, or kidney) 3'21 It has been previously shown that the adrenal glands have the highest LDL receptor activity per gram of tissue24'36and are expected to be most affected by dietaryinduced HC with subsequent down-regulation of LDL receptors. 6'27As expected, there is a significant decrease in the amount of 99mTc-LDLactivity in rabbits fed on a high cholesterol diet. 13'25 The 99mTc-LDL distribution in HCR rabbits (Fig 2) shows nonvisualization of adrenal glands. Similarly, adrenal glands are not visualized in the WHHL rabbit (Fig 2). Dexamethasone, a synthetic glucocorticosteroid, is known to suppress adrenal cortical function by decreasing adrenocorticotrophic hormone (ACTH) secretion, which in turn leads to a decrease in cortisol production. 37 Isaacsohn et a137 recently demonstrated that the uptake of 99mTc-LDL by adrenals in rabbits was significantly decreased by prior treatment with dexamethasone and the gamma camera images showed nonvisualization of adrenal glands. 99mTc-LDL: IMAGING STUDIES IN PATIENTS

Patients With Abnormal Plasma LDL Levels Serum LDL concentrations are altered in many disease states. Ideally, plasma cholesterol levels for adults range from 130 to 190 mg/dL or 3.36 to 4.91 mmol/L. 38Approximately two thirds of total cholesterol in plasma is transported in LDL. Serum LDL concentrations are altered in such primary (genetically determined) metabolic defects as familial HC (FH) and hyperapobetalipoproteinemia.8'38 Abnormally high levels are also acquired secondary to heart disease, diabetes, nephrotic syndrome, hepatoma, and diet or drug induced. 38'39 The most dramatic form of primary HC, known as (FH), occurs because of abnormalities in the gene encoding for the LDL receptor. Heterozygotes for FH have one normal gene and one mutant gene for the LDL receptor, while homozygotes for FH have inherited two mutant genes. These patients develope severe, premature atherosclerosis.30 Hypocholesterolemia is commonly observed in patients with myeloproliferative diseases and Gaucher's disease in whom hepatosplenomegaly and increased numbers of macrophages occurs. 40'41There appears to be increased receptorindependent catabolism of LDL in these states.

99MTc: IMAGING STUDIES IN VIVO

73

Hypocholesterolemia is also a frequent finding in patients with acute myelocytic leukemia. 42 99mTc-LDL Imaging in Hypercholesterolemic and Hypocholesterolemic Patients 99mTc-LDL imaging studies in a number of HC and hypocholesterolemic patients 43'44'45 have proven beneficial in understanding the biochemical basis of some of the findings in these diseases. The plasma lipid and lipoprotein levels in four patients with these disorders are shown in Table 4. In order to image LDL in vivo biodistribution, 10 mCi of 99rnTc-LDL is injected IV and gamma camera images are obtained over the next 24 hours. In normal human subjects, 99mTc activity was seen in the cardiac blood pool, in major blood vessels, and in the liver immediately after the injection. At 4 hours postinjection (Fig 3), 99mTc activity in the major blood vessels had decreased while the activity over the liver had increased. There was minimal activity in the spleen (in most normal subjects the spleen is not visualized), kidney, and central bone marrow, but no uptake in the peripheral marrow. Over the next 24 hours of the study, activity in the major blood vessels declined continuously while that over the organs (liver, kidney, and intestine) increased. 45 The biodistribution of 99mTc-LDL in normal, HC, and hypocholesterolemic patients 4 hours after the injection of the tracer is shown in Fig 4 (previously unpublished). Compared with the normal subject, liver uptake of 99mTcactivity in the hypercholesterolemic patient (patient no. 1) is lower while the 99mTcactivity in the blood pool is significantly higher and the spleen is not visualized. In contrast, the biodistribution in the hypocholesterolemic patient with myeloproliferative disease (patient no. 4) shows intense uptake in the spleen. In this patient, as a result of splenomegaly, the liver uptake does not appear to

Fig 3. gSmTc-LDL distribution in a normal human subject. The four anterior images of abdomen, pelvis, upper, and lower legs w e r e taken 4 hours after the injection of the tracer. SgmTc activity is mostly seen in the liver and major blood vessels. Minimal uptake is seen in central marrow and spleen while no activity is present in peripheral marrow (femur or tibia).

be higher than in the normal subject, even though the blood pool activity is lower. These images of 99mTc-LDL biodistribution in patients with altered LDL metabolism are consistent with the expected LDL behavior and LDL receptor density. 6,4~ Imaging Studies in Patients With Atherosclerosis Atherosclerosis is a chronic, progressive disease of the blood vessels with metabolic changes in the arterial wall characterized by cellular proliferation and accumulation of LDL. The ubiquitous fatty streak is the earliest lesion in atherosclerosis (commonly found in children), and is a grossly fiat, lipid-rich lesion consisting of both macrophages and some smooth muscle while the fibrous plaque is made up of increased smooth muscle cells surrounded by connective

Table 4. Lipoprotein and Lipid Levels (mg/dL) in Patients

Patient No. 1 2 3 4

Diagnosis

Age (yr)/sex

Tg

T-C

LDLC

HDLC

Betasitosterolemia Homo-FH Hetero-FH Post-polycythemia Myeloid metaplasia

57/F 16/F 45/F 72/F

125 80 120 75

280 675 350 101

215 627 251 50

40 32 75 36

Abbreviations: Tg, triglycerides; T-C, total cholesterol; LDLC, low density lipoprotein cholesterol; HDLC, high density lipoprotein cholesterol.

74

VALLABHAJOSULA AND GOLDSMITH

Fig 4. The distribution of Sg"Tc-LOL in human subjects with normal and abnormal levels of plasma lipoproteins. The anterior images were obtained 4 hours after the injection of the tracer. In the normal subject (B), liver uptake is relatively greater than the blood pool activity. In one hypercholesterolemic patient (A), SSmTcactivity in the blood pool (heart) is greater than the liver. In a hypocholesterolemic patient with myeloproliferative disease (C), the enlarged spleen shows intense accumulation of "mTC while the liver uptake appears to be similar to the uptake seen in the normal subject. There is also some reduction of the cardiac blood pool activity.

tissue matrix and containing variable amounts of intracellular and extracellular lipid. 46 External imaging of human atherosclerosis (carotid lesions) was first demonstrated by Lees et a147 using 125I-LDL and by Sinzinger et a148 using 123I-LDL. Subsequently, we have shown that 99mTc-LDL is useful to image the atherosclerotic lesions in rabbits fed high cholesterol diets. 49 These rabbits develop fatty streaks (in the ascending aortas), which demonstrated selective accumulation of 99mTc-LDL. We also studied the biodistribution of 99rnTcLDL in many patients with HC. One of the patients is a 57-year-old woman with a long history of HC and beta-sitosterolemia (patient no. 1), a disorder in which significant quantities of normally nonabsorbable plant sterols are absorbed from the Small intestine. 5~ 99mTc-LDL imaging studies (Fig 5A, previously unpublished) in this patient showed an intense uptake of 99mTcactivity in the right pelvic area, corresponding to the iliofemoral artery. This abnormal focus of 99mTcactivity was seen both in early (4 hours) and delayed (24 hours) imaging studies. This abnormal focus, of 99mTc-LDL accumulation is interpreted as an atherosclerotic lesion. Angiographic evidence of atherosclerosis in this area of abnormal 99mTcaccumulation, however, is not available. For comparison,99mTc-LDL im-

ages in another hypercholesterolemic patient (patient no. 3) are shown in Fig 5B. In this patient, no significant abnormal focus of 99rnTcaccumulation was seen in the pelvic area either in the early or in the delayed image. Lees et a144 recently studied the diagnostic usefulness of 99mTc-LDL imaging in 17 patients with atherosclerosis. Asymmetrical accumulation of 99mTc activity was considered as an abnormal focus representing an atherosclerotic lesion. In four patients, arteries with focal accumulation of 99rnTcincluded iliac, femoral, and carotid vessels. Based on the data obtained from carotid endarterectomy specimens, Lees et al identified that the carotid plaque that did image well was made up of tissue that contained abundant foam cells and macrophages. 44 From these studies, they hypothesized that if a lesion is visualized with 99mTc-LDL, it is likely to be an actively evolving lesion. Imaging Studies in Patients with Xanthomas Xanthomas are accumulations of lipids that may be present in the skin and tendons of patients with various forms of hyperlipidemia. 5~ Pathological studies have demonstrated that foam cells are the major cell type found in most xanthomata, although lipid-filled fibroblasts and dermal cells have also been described: 2 Scott

99MTc: IMAGING STUDIES IN VIVO

75

vasive approach to assess the metabolic state of atheromatous lesions.

Imaging Studies in Patients with Myeloproliferative Disease

Fig 5. (A) 99mTc-LDL distribution in the pelvic area of a patient (patient no. 1, Table 4) w i t h a long history of HC and betasitosterolemia. The abnormal uptake of SS~Tc is seen in the right distal iliac (arrow) at 4 and 24 hours post-injection and is interpreted as an atherosclerotic lesion. (S) For comparison, similar images taken in another hypercholesterolemic patient w h o is heterozygous for FH (patient no. 3, Table 4) show no significant abnormal gSmTc uptake.

and Winterbourn 53 demonstrated that radioiodinated LDL accumulates in xanthomata. We have recently observed that 99mTc-LDL is actively taken up by xanthomata.43 99mTc-LDL imaging studies were performed in four hypercholesterolemic patients (2 FH homozygotes, 1 FH heterozygote, and 1 with beta-sitosterolemia). One of the FH homozygotes (patient no. 2) is a 1 6-year-old girl who had large tendinous, tuberous, and planar xanthomata over her knees and both Achilles tendons. In this patient, intense accumulation of 99mTcactivity (Fig 6A) was seen over the knees and Achilles tendons corresponding to the area of xanthomata 4 hours after the injection of 99mTc-LDL. A scan (Fig 6B) of the heterozygous FH subject (patient no. 3), who had small, hard elevations over both pretibial tuberosities and some minor thickening over the Achilles tendons, is shown for comparison. This patient's scan, however, did not demonstrate any significant focal uptake of 99mTcactivity. The mechanism whereby LDL is taken up by macrophages in xanthomata is not clearly known. These studies, however, suggest that external imaging of xanthomata might be a useful, nonin-

Myeloproliferative disorders (MPD) are a group of chronic syndromes that includes polycythemia vera, myeloid metaplasia, essential thrombocythemia, and chronic myelogenous leukemia.54 MPD are low-grade malignancies characterized by ineffective proliferation of bone marrow elements, extramedullary blood production, and in some stages of disease, reactive fibrosis of bone marrow. Our colleagues at the Mount Sinai Medical Center, Gilbert et al, 40 described a state of chronic hypocholesterolemia in MPD, in which total cholesterol, LDL cholesterol, and HDL cholesterol concentrations were decreased in proportion to proliferative disease activity and to the degree of splenomegaly. To elucidate the mechanism responsible for the hypocholesterolemia and to determine the sites of LDL catabolism in MPD patients, imaging studies were performed with 99mTc-LDLin a group of MPD patients. 45 Five patients with MPD were injected with 99mTc-LDL and total body images were obtained at several times over the next 24 hours. One of the patients was a

Fig 6. ggmTc-LDL images of hypercholesterolemic patients obtained 4 hours after the injection of the tracer. (A) Images show increased uptake of gSmTc activity over the knees and Achilles tendons (arrows) of a homozygous FH patient (patient no. 2, Table 4) who has xanthomata in these areas. Similar images (B) obtained in a heterozygous FH patient (patient no. 3, Table 4) do not show abnormal accumulation over the knees and Achilles tendons.

76

VALLABHAJOSULA A N D GOLDSMITH

Fig 7. The abnormal distribution of eemTc-LDL in a patient with post-polycythemia myeloid metaplasia (patient no. 4}. The four anterior images of abdomen, pelvis, upper, and lower legs were taken 4 hours after the injection of the tracer, The images show normal liver uptake, but abnormally high uptake of ~mTc by the enlarged spleen and peripheral bone marrow.

72-year-old woman (patient no. 4) with a 15-year history of polycythemia vera who had marked hypocholesterolemia. 99mTc-LDL images (Fig 7) showed intense accumulation of 99mTcactivity in the spleen and peripheral bone marrow. By contrast, 99mTc-LDL images (Fig 3) in a normal subject showed no uptake of 99raTeby the spleen and peripheral bone marrow. In addition, there was marked uptake of 99roTe activity by the enlarged spleen in patient no. 4 in whom the bone marrow expansion and splenomegaly was also demonstrated by 99mTc-sulfur colloid imaging,45 which showed the proliferation of a macrophage (RES) population in these tissues. Recently, Nimer et a155showed that treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF) altered cholesterol homeostasis in patients with aplastic anemia and suggested that GM-CSF treatment may be potentially useful in the treatment of HC. 55 Our studies with 99mTcLDL indicate that the uptake of LDL by macrophages in spleen and bone marrow may be the mechanism for the increased catabolism of LDL in patients with MPD 4~ and suggest that increased numbers of activated monocyte-macrophages may play a significant role in the overall removal of LDL from plasma. UPTAKE OF LDL BY MACROPHAGES

In patients with atherosclerosis, Lees et al observed that the carotid plaque that did image

well with 99mTc-LDL was made up of tissue that contained abundant foam cells and macrophages. But the fundamental questions of just how lipid accumulation occurs in the arterial wall and what is the exact role of LDL receptors with regard to macrophage accumulation of cholesterol are not clearly understood. 56Our own observation that 99mTc-LDL is taken up by xanthomas (rich in foam cells) in the hypercholesterolemic subjects, and by the spleen in bone marrow (rich in activated monocyte-macrophages) in patients with myeloproliferative disease, also implicates macrophages in the uptake of LDL. Since macrophages have very few LDL receptors for native-LDL,57the exact mechanisms involved in the uptake of LDL by the macrophages is not very clear. The acetyl-LDL receptor or scavenger cell receptor on the macrophages,57'5~ however, appears to play a major role in the accumulation of LDL. Modification of LDL structure is now considered a prerequisite for generation of foam cells from macrophages. LDL, when modified chemically by acetylation, loses the ability to bind to the classic LDL receptor, but is recognized by another highaffinity receptor on macrophages called the acetylLDL receptor or scavenger cell receptor. 57'58This acetyl-LDL receptor seems to be present only on macrophages and endothelial cells. 57The scavenger cell receptor also recognizes LDL treated with agents such as glutaraldehyde and malondialdehyde (MDA) that alter lysine residues on LDL apo-B. 59 Modification of LDL in vivo may result from an interaction with MDA, which is released from blood platelets or produced by lipid peroxidation. 59 Based on the above discussion, it is expected that the distribution of 99mTc-MDA-LDL will be different from that of 99mTc-LDL. In order to test this hypothesis, the biodistribution of 99mTc-LDL in normal rabbits was compared with that of 99mTc-MDA-LDL. Normal rabbit LDL was modified by treatment with MDA and MDA-LDL was labeled with 99mTcusing the same technique as previously described for LDL. 13'2199mTc-MDALDL cleared rapidly from circulation with accumulation in liver and bone marrow. No adrenal glands were visualized. The gamma camera images of 99mTc-MDA-LDL distribution in normal rabbits 24 hours post-injection is shown in Fig 8B (previously unpublished). Compared with the images of 99mTc-LDL(Fig 8A), the 99mTc-MDA-

99MTc: IMAGING STUDIES IN VIVO

77

SUMMARY

Fig 8. Distribution of 99"Tc-LDL (A) and sSmTc-MDA-LDL (B) in a normal rabbit 24 hours after the injection of the tracer. The images show intense accumulation of 99rnTc in the bone marrow with MDA-LDL (B) than with native-LDL

(A).

L D L i m a g e s show intense a c c u m u l a t i o n o f activity in bone m a r r o w suggesting t h a t the m a c r o phages in m a r r o w a r e involved in the p r e f e r e n t i a l u p t a k e of 99mTc-MDA-LDL. Based on these results, we would s p e c u l a t e t h a t c h e m i c a l l y modified L D L p r e p a r a t i o n s such as a c e t y l - L D L or M D A - L D L labeled with 99mTc m i g h t be more suitable t h a n 99mTc-native-LDL for noninvasive i m a g i n g of atherosclerotic lesions.

P l a s m a lipoproteins, L D L , and H D L can be l a b e l e d efficiently with 99mTc using sodium dithionite as a reducing agent. The labeling technique is also a p p l i c a b l e to chemically modified L D L p r e p a r a t i o n s such as M D A - L D L . 99mTc-LDL is an ideal t r a c e r for studying L D L biodistribution and m e t a b o l i s m since it behaves in vivo as an intracellularly trapped radioligand. Hepatic L D L - r e c e p t o r concentration in a n i m a l models and in patients with H C - and hypocholestero l e m i a can be i m a g e d noninvasively. In addition, atherosclerotic lesions, x a n t h o m a t a , and organs rich in m a c r o p h a g e population a p p e a r to accumulate significant a m o u n t s of 99mTc-LDL. C h e m i cally modified L D L p r e p a r a t i o n s such as M D A L D L , when labeled with 99mTc, show even greater affinity for m a c r o p h a g e s . I n conclusion, lipoproteins labeled with 99mTc provide powerful rad i o t r a c e r s for noninvasive exploration of a variety o f disorders of lipoprotein metabolism. ACKNOWLEDGMENT

The authors thank Helena Lipszyc for her expert and devoted technical assistance in this research project.

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a~"Tc: IMAGING STUDIES IN VlVO

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