Method for recovering purified 52,000 dalton fraction of human pancreatic cholesterol esterase using deae-cellulose, hydroxyapatite and heparin-sepharose

A method for recovering a purified 52,000 dalton fraction of human pancreatic cholesterol esterase is disclosed. The 52,000 dalton fraction of human pancreatic cholesterol esterase is purified by passing a solution having a pH of about 8.2 which contains two or more fractions of human pancreatic cholesterol esterase including the 52,000 dalton fraction over DEAE-cellulose to produce an effluent containing said 52,000 dalton fraction. Since the 52,000 dalton fraction of the esterase does not bind to the DEAE-cellulose whereas the other esterase fractions do bind to the DEAE-cellulose, the 52,000 dalton fraction is then collected in the effluent. The method can be further carried out by passing the purified 52,000 dalton fraction over both hydroxyapatite and heparin-Sepharose to obtain a homogeneous solution of the 52,000 dalton fraction.

BACKGROUND OF THE INVENTION 
This invention relates to the inhibition of intestinal cholesterol 
absorption in mammals and, more particularly, to inhibiting or decreasing 
intestinal cholesterol absorption by the oral administration of heparin or 
heparin subfractions. The invention is based upon our discovery of a novel 
molecular pathway for the absorption of ingested cholesterol/cholesteryl 
esters and the identification of specific sites in this pathway for drug 
intervention to decrease intestinal cholesterol absorption. 
Atherosclerosis is the leading killer in the United States, and yet 
detailed understanding of the absorption of cholesterol, an essential 
factor in the development of atherosclerosis, has remained rather sketchy, 
despite the fact that restrictions of dietary intake of cholesterol are 
the mainstay of therapeutic regimens according to current medical 
practice. It is known that the principal dietary form of cholesterol is a 
series of related fatty acid esters of cholesterol and that these must be 
hydrolyzed before intestinal absorption of cholesterol occurs (Treadwell 
et al., Handbook of Physiology, Alimentary Canal, Section 6, vol. III, 
1968). Pancreatic cholesterol esterase is known to catalyze the hydrolysis 
of cholesteryl esters but knowledge has remained rudimentary concerning 
these enzymes due to a lack of a general method for their preparation in 
homogeneous form, to the frequent use of artificial, colorimetric 
substrates in lieu of cholesteryl ester in their assay and to the failure 
to minimize or eliminate artifacts due to the presence of pancreatic 
proteases in their preparation (Srockerhoff et al., (1974) In: Lipolytic 
Enzymes, Chapter V, pp. 177-192, Academic Press, New York and Ruddet al., 
(1984) In: Lipases, pp. 185-204, Elsevier, New York). Accordingly, no 
unified conceptualization of the role of cholesterol esterase, buttressed 
by experimental documentation, has emerged. Thus, rat pancreatic 
cholesterol esterase, the most commonly studied one, is reported as a 
67,000 (67K) species that hexamerizes in the presence of taurocholate to 
form an active enzyme (Calame et al., (1975) Arch. Blochem. Biophys. 168: 
57-65). Bovine cholesterol esterase is also 67K, but the porcine and human 
enzymes are reported as 83K and 100K, respectively (Van den Bosch et al., 
(1973) Biochem. Biophys. Acta 296: 94-104; Momsen et al. (1977) Biochem. 
Biophys. Acta 486: 103-113; and Guy et al. (1981) Eur. J. Biochem. 117: 
457-460). These differences have not been satisfactorily explained nor the 
role of taurocholate rigorously examined, but some of these 
dissimilarities may reflect use of colorimetric substrates for assay 
instead of cholesterol esters. 
Because of these deficiencies, no generally accepted hypothesis has been 
advanced to explain in molecular details the nature of cholesterol 
absorption in the intestine, no selective inhibitors have been reported, 
and the mechanism of re-esterification of cholesterol in the intestinal 
cell before transport to the liver has not been elucidated. 
Despite these shortcomings in prior investigations, one of the primary 
forms of therapy for patients with elevated cholesterol in the blood has 
been modulation of intestinal cholesterol absorption, either by serious 
counseling to eat less cholesterol or through the use of bile salt 
(derived from cholesterol) binding resins such as cholestyramine which are 
poorly tolerated (Casdorph, H. R. (1976) In: Lipid Pharmacology, pp. 
222-256, Academic Press, New York). 
Also, because fatty acids, especially saturated ones, play an essential 
role in atherogenesis, inhibition of their intestinal absorption should 
diminish rates of atherogenesis. 
There has been a continuing need, therefore, for more fundamental knowledge 
of the mechanism of cholesterol absorption and improved and effective 
means for inhibiting intestinal cholesterol and fatty acid absorption in 
mammals. 
SUMMARY OF THE INVENTION 
Among the several objects of the present invention may be noted the 
provision of an effective method for inhibiting intestinal cell endogenous 
heparin mediated absorption of cholesterol or fatty acids in mammals; the 
provision of such a method which is conveniently effected through the oral 
administration of heparin, heparinase or an active heparin subfraction; 
and the provision of a method for inhibiting intestinal cholesterol or 
fatty acid absorption which minimizes or reduces toxic side effects. Other 
objects and features of the invention will be in part apparent and in part 
pointed out hereinafter. 
In brief, the present invention is directed to a method for inhibiting 
intestinal cell endogenous heparin mediated absorption of cholesterol or 
fatty acids in mammals by orally administering to a mammal an effective 
amount of heparin, heparinase or an active heparin subfraction.

EXAMPLE 1 
In a typical preparation, 30 gm of human pancreas received at autopsy were 
placed in 10 mM phosphate, pH 6.0, 50 mM benzamidine, 0.5% digitonin, and 
homogenized with a polytron. The mixture was centrifuged at 48,000 X g for 
30 minutes, the supernatant collected and then recentrifuged for one hour 
at 100,000 X g. The cytosol-containing supernatant was passed through 
glass wool to remove fat, and the clear solution was used as starting 
material for enzyme purification. 
Pancreatic cytosol was dialyzed overnight against 10 mM phosphate, pH 6.8, 
50 mM benzamidine and applied at 30 ml/hr. to hydroxylapatite 
(2.6.times.10 cm) equilibrated with the same buffer. The resin was washed 
with one column volume of equilibration buffer, one column volume of 50 mM 
phosphate, pH 6.8, 50 mM benzamidine and then developed with a linear 
gradient from 50 mM phosphate, pH 6.8, 50 mM benzamidine to 350 mM 
phosphate, pH 6.8, 50 mM benzamidine. Cholesterol esterase(s) was eluted 
as a single peak, pooled and concentrated ten-fold with an Amicon 
ultrafiltration cell, equipped with a YM-10 membrane. The concentrated 
protein was applied at 17 ml/hr. to Ultragel AcA 34 (2.6.times.90 cm) 
equilibrated with 0.50 M NaCl, 10 mM phosphate, pH 6.0 and the enzyme 
emerged as a single peak. Cholesterol esterase from the AcA 34 column was 
pooled, dialyzed against 10 mM phosphate, pH 6.0, and the sample was then 
chromatographed on heparin agarose, equilibrated with the same buffer (see 
FIG. 1a). All the activity was bound to the resin which was then washed 
with 50mM NaCl, 50 mM benzamidine, 10 mM Tris pH 7.2 to remove protein 
impurities. The resin was then washed with 30 mM NaCl, 20 mM taurocholate, 
50 mM bezamindine, 10 mM Tris pH 7.2, which removed a small amount of 
activity (&lt;1%). The bile salt was washed from the column with 50 mM NaCl, 
50 mM benzamidine, 10 mM Tris pH 7.2 and the resin was then washed with 
500 mM NaCl, 50 mM benzamidine, 10 mM Tris pH 7.2. For buffer wash 1 
indicated in FIG. 1a, 30 minute fractions were collected, while for buffer 
washes 2-4, 12 minute fractions were collected. The resulting high salt 
eluted 90% of the applied cholesterol esterase activity and produced two 
bands on SDS-PAGE; the major band had molecular weight 100,000 while the 
minor band had molecular weight 210,000. Both bands reacted positively in 
a Western blot to bovine anti-cholesterol esterase antibody. The overall 
yield of purification was approximately 40% with a 150-fold purification. 
EXAMPLE 2 
The following method allows isolation of a 67K species of human pancreatic 
cholesterol esterase. Human pancreatic cytosol was dialyzed against 5 mM 
benzamidine, 1 mM 2-mercaptoethanol, 3.5 mM Tris pH 8.0 and then applied 
to DEAE cellulose (2.5.times.15 cm), equilibrated with the same buffer. 
The resin was washed at 30 ml/hr with several column volumes of buffer and 
then developed with a linear gradient running from 5 mM benzamidine, 1 mM 
mercaptoethanol, 3.5 mM Tris pH 8.0 to 250 mM NaCl, 5 mM benzamidine, 1 mM 
mercaptoethanol, 3.5 mM Tris pH 8.0. Cholesterol esterase activity was 
eluted at approximately 0.10M NaCl, pooled, concentrated to 5 ml and 
applied to a gel filtration column. The column was developed as described 
in Example 1. Activity was pooled and dialyzed against 10 mM phosphate pH 
6.0 The pH of the sample was raised to 7.2 by adding 1M Tris and it was 
then applied to heparin agarose equilibrated with 10 mM Tris pH 7.2. The 
column was washed with 100 mM NaCl, 10 mM Tris pH 7.2 and finally, with 
500 mM NaCl, 10 mM Tris pH 7.2 to remove active enzyme. Examination on 
SDS-PAGE showed several bands centered at 67,000. 
EXAMPLE 3 
Human pancreatic cholesterol esterase (52K form) can be isolated as 
follows. Cytosol prepared as above from 30 g pancreas is dialyzed versus 
10 mM Tris-C1, 50 mM benzamidine, pH 8.2 and then chromatographed over 
DEAE cellulose (2.6.times.25 cm). Unlike the higher molecular weight forms 
of cholesterol esterase which bind under these conditions, the 52K species 
rapidly passes through DEAE cellulose at pH 8.2, with a 40-fold 
purification and 80% yield. Subsequent chromatography over hydroxylapatite 
and heparin-Sepharose yields a homogeneous protein (SDS-PAGE, M.W.=52K) 
with enzymatic activity expressed principally in the synthetic direction 
for cholesterol esters (rate of synthesis/rate of hydrolysis=10/1). This 
species also cross-reacts in Western blotting to anti-cholesterol esterase 
antibodies. 
EXAMPLE 4 
The procedures described above have been extended to other species with the 
following results (See FIG. 1b and FIG. 2). 
For the results shown in FIG. 1b, thirty milligrams of commercially 
available enzyme were dissolved in 10 mM Tris, pH 7.2 and pumped at 15 
ml/hr onto a heparin-Sepharose column equilibrated with the same buffer. 
The column was then washed with buffers 1 and 2 as indicated in FIG. 1b. 
For buffer 1, 30 minute fractions were collected; for buffer 2, 10 minute 
fractions were collected. 
FIG. 2 is an immunoblot of cholesterol esterase from cow, pig and rat 
pancreas. Fresh tissue was homogenized in 50 mM benzamidine, 10 mM 
phosphate, pH,6.8, plus digitonin and then dialyzed against 50 mM 
benzamidine, 10 mM phosphate, pH 6.8, followed by dialysis against 10 mM 
phosphate, pH 6.0. The sample was then pumped onto a heparin-Sepharose 
column and washed with several column volumes of 50 mM NaCl, 50 mM 
benzamidine, 10 mM Tris, pH 7.2. All the cholesterol esterase activity was 
removed with 500 mM NaCl, 50 mM benzamidine, 10 mM Tris, pH 7.2, and then 
lyophilized. Samples were then run on SDS-PAGE using 7.5% acrylamide as 
the separating gel and 4.5% acrylamide as the stacking gel. Immediately 
following electrophoresis, the protein was transferred to nitrocellulose 
and then incubated with rabbit anti-bovine cholesterol esterase antibody. 
After washing, the nitrocellulose sheet was incubated with .sup.125 
I-Protein A, dried and exposed to x-ray film. 
Using fresh pig pancreas, cholesterol esterase forms are found at .sup.180 
K, 83K and 52K. The purification summary for pig cholesterol esterase 
(83K) is set forth in the following table: 
______________________________________ 
PURIFICATION SUMARY FOR PIG CHOLESTEROL 
ESTERASE (83K) 
Purifi- 
Protein Total Act..sup.a 
Sp. Act. 
Recovery 
cation 
Step (A.sub.280) 
.times. 10.sup.-3 
.times. 10.sup.-3 
(%) (x-fold) 
______________________________________ 
Cytosol 2,034 5,715 2.8 100 1.0 
Hydroxyl- 
498 3,445 6.9 60 2.5 
apatite 
AcA 34 58 2,110 36.2 37 12.9 
Heparin 9.8 1,309 134.2 23 50.0 
______________________________________ 
.sup.a = nmoles/hr 
Using rat pancreas, cholesterol esterase forms are found at 180K, 67K and 
52K. Using bovine pancreas, cholesterol esterase forms are found at 180K, 
72K and 52K. Human pancreatic cholesterol esterase forms are 210K, 100K, 
67K and 52K. 
EXAMPLE 5a 
Exogenous heparin inhibits the interaction and binding of pancreatic 
cholesterol esterase to small intestine vesicles prepared from proximal 
small intestine segments homogenized in 10 mM Tris-C1, pH 7.2, and 
centrifuged at 48,000 X g for 45 minutes. Vesicles contained 1.0 mg 
protein/ml, 30 ug total cholesterol/mg protein and 50 ug endogenous 
heparin/mg protein. .sup.125 I-cholesterol esterase (100K or 67K form) was 
added (0 to 400 pmol; 2,000 dpm/pmol) to 100 ug of protein of small 
intestine vesicles in 10 mM Tris-Cl, pH 7.2 for 15 min. at 37.degree.. The 
concentration of offered .sup.125 I-cholesterol esterase varied between 10 
nM and 1 uM. Experiments were performed utilizing 1% blotto to decrease 
nonspecific protein binding to the vesicles. After the 15 minute 
incubation at 37.degree., the reaction was quenched and the assay mixtures 
were filtered through wells of a Millipore system containing 0.2 micron 
pore filters presoaked in buffer. The filtrate was-collected to assess 
free (unbound) enzyme, and the filters were then immediately washed with 
15 ml of cold buffer and counted for .sup.125 I. Total binding was defined 
as .sup.125 I-dpm bound to filter paper minus .sup.125 I-dpm detected in 
the same reaction without vesicles (background). Nonspecific binding 
(radioactivity bound to the vesicles in the presence of excess cold 
ligand) was quantitated as the amount of .sup.125 I-cholesterol esterase 
binding in the presence of a 1000-fold molar excess of unlabeled 
cholesterol esterase as described above. Specific binding is defined as 
the difference between total bound dpm and nonspecific-bound dpm 
(converted to moles bound by dividing by the SRA). 
Graphical analysis indicated saturation binding. Binding in the presence of 
a 1000-fold molar excess of cold cholesterol esterase established a 
nonspecific binding curve. A difference curve between the two represents 
specific binding of .sup.125 I-cholesterol esterase to small intestine 
vesicles with a K.sub.D of 100 nM (FIGS. 3A and 3B). When included in the 
binding assay, exogenous heparin decreases binding of .sup.125 
I-cholesterol esterase to vesicles in a concentration-dependent manner 
with increasing concentrations of heparin from a 0.5 to 100-fold molar 
excess compared to surface-bound vesicular heparin: specific binding in 
the presence of heparin decreased by 5% to 72% in the presence of 0.5 to 
100-fold molar excesses of heparin, respectively. Specificity of binding 
was demonstrated since chondroitin sulfate at 0.5 to 100-fold molar 
excesses decreased specific binding less than 10%, isotonic saline had no 
effect and pretreatment of vesicles with bacterial heparinase diminished 
specific binding by 75%. Lastly, binding of the 100K and 67K form of 
cholesterol esterase in the presence of 5 mM taurocholate showed that the 
100K form retained 72% specific binding while the 67 form retained only 20 
%. These results indicate that pancreatic cholesterol esterase binds to 
small intestine through a receptor-like interaction with endogenous 
heparin and that exogenous heparin can significantly reduce this 
interaction. 
EXAMPLE 5b 
Cellular uptake of .sup.3 H-cholesterol from .sup.3 H-cholesterol oleate is 
dependent on the presence of cholesterol esterase and is markedly 
inhibited by the addition of soluble heparin. For example, intestinal 
cells (colonic adenocarcinoma cells CaCo-2, American Type Cell Culture) 
were grown in T150 flasks in medium consisting of DME, 10% v/v fetal 
bovine serum, 50 ug/ml gentamicin and 2 mM sodium pyruvate. Uptake 
experiments were conducted using 10.sup.5 intestinal cells in PBS buffer 
containing 5 mM CaCl.sub.2, pH 7.4 in the presence of 5 uM .sup.3 
H-cholesterol oleate (SRA=50 dpm/pmol) embedded in phosphatidylcholine 
vesicles along with 10% v/v lipoprotein-depleted serum and 1.5 mM 
taurocholate. At time zero, 5 nM human pancreatic cholesterol esterase 
(100K, homogeneous) was added to one set of flasks, an equivalent volume 
of buffer to another set, and 1 mg/ml final concentration bulk heparin 
together with 5 nM enzyme to a third set. At selected times, i.e., 0, 15, 
30, 60, 120 and 480 min, medium was removed and the cells were washed with 
medium to allow quantitation of .sup.3 H-cholesterol uptake. In the 
absence of enzyme, cellular uptake of .sup.3 H-cholesterol was linear and 
occurred at a rate of 400 dpm/hr/10.sup.5 cells. In the presence of 
cholesterol esterase, the rate of .sup.3 H-cholesterol uptake was 1,800 
dpm/hr/10.sup.5 cells. Importantly, in the presence of heparin, uptake was 
significantly suppressed and occurred at a rate of 500 dpm/hr/10.sup.5 
cells. Thus, exogenous soluble bulk heparin can significantly inhibit the 
uptake of cholesterol into intestinal cells commonly used to study 
transport phenomena. 
EXAMPLE 6 
Subfractionation of heparin produces a high reactive form that is 100-fold 
more effective in displacing bound cholesterol esterase from small 
intestine vesicles. First, low density lipoprotein (LDL) was isolated by 
density gradient centrifugation from a rabbit which had been fed 
cholesterol for three weeks. After immobilization of LDL to Sepharose, 
bulk commercial heparin (from pig intestine) was applied to the LDL 
conjugate in 10 mM Tris-C1, pH 7.2, the resin washed with five column 
volumes of buffer and a fraction eluted by addition of 0.5M NaCl. 
Approximately 5% of applied heparin was recovered in the NaCl wash. After 
removal of salt, this high reactive form of heparin was evaluated in the 
vesicle assay. On a weight basis, this subfraction was 100-fold more 
potent in displacing bound .sup.125 I-cholesterol esterase when compared 
to bulk heparin. The unreactive heparin that passed through the LDL 
Sepharose resin was completely ineffective. 
Importantly, this high reactive heparin is also a very potent inhibitor of 
human pancreatic cholesterol esterase (100K). Whereas bulk heparin has a 
K.sub.i of 200 nM for this enzyme, unreactive heparin is non-inhibitory 
and high-reactive heparin has a K.sub.i of 2 nM. 
EXAMPLE 7 
Male New Zealand white rabbits (1.sup.+.sub.- 0.1 kg) were fed (n=4 in each 
group) 2% cholesterol Purina Rabbit Chow for two weeks plus either tap 
water or oral heparin (2 mg/ml) for 48 hours prior to the experiment. On 
day 15, the heparin pretreated rabbits received 100 mg heparin in 10 ml 
water followed by 10 ml of radiolabeled liquid rabbit Chow containing 50 
uCi of [1,2,6,7-.sup.3 H] cholesteryl oleate and the control group 
received 10 ml H.sub.2 O followed by 10 ml of the same radiolabeled Chow. 
After four hours without food and water, the rabbits were sacrificed and 
the amount of .sup.3 H-cholesterol and .sup.3 H-cholesteryl esters 
quantitated in liver and blood in both groups. Compared to the control 
group, the heparin-treated group had 30% less .sup.3 
H-cholesterol/cholesterol esters per ml of blood and 50% less per gram of 
liver. 
EXAMPLE 8 
Small intestine vesicles containing approximately 50 ug of endogenous 
heparin were treated with 1.25 units of bacterial heparinase, the vesicles 
centrifuged and the amount of uronic acid residues in the supernatant and 
pellet quantified as a function of the length of exposure to heparinase. A 
time-dependent release of uronic acid fragments from the pellet into the 
supernatant was noted, with approximately 50% of total vesicular heparin 
appearing in the supernatant after four hours. Aliquots of these vesicles 
were then incubated with .sup.125 I-labeled cholesterol esterase to assess 
the binding of cholesterol esterase to these heparin-depleted vesicles. In 
contrast to control experiments in which cholesterol esterase was found to 
bind avidly to small intestine vesicles, it was observed that specific 
binding of .sup.125 I-cholesterol esterase to the heparin-depleted 
vesicles was decreased by 95%.+-.5%. Therefore, removal of surface-bound 
vesicular heparin by treatment with bacterial heparinase eliminated 
specific binding of cholesterol esterase which in turn should eliminate 
absorption of free cholesterol produced at the membrane. 
EXAMPLE 9 
Bovine pancreatic amylase, phospholipase A.sub.2, deoxyribonuclease, 
cholesterol esterase and triglyceride lipase were radiolabeled with 
Na.sup.125 I and utilized in binding assays as described in Example 5. 
Blotto (1%) was utilized to block nonspecific protein binding sites and a 
50-fold molar excess of unlabeled pure enzyme was added to assess 
nonspecific binding. As noted above for cholesterol esterase, triglyceride 
lipase binds to small intestine membrane vesicles and is displaced by 
exogenous heparin (see FIG. 4). In contrast, pancreatic enzymes (amylase, 
deoxyribonuclease, and phospholipase A.sub.2) which hydrolyze hydrophilic 
substrates such as sugars do not bind to membrane-bound heparin. These 
results indicate that interfering with the interaction between heparin and 
neutral lipolyric enzymes secreted by the pancreas should markedly 
diminish fatty acid absorption, but have little or no effect on the 
metabolism of other nutrients. 
EXAMPLE 10 
Human pancreatic cholesterol esterase (100 kDa) and human pancreatic 
triglyceride lipase (52kDa) were purified to homogeneity (Examples 1 and 
3), and their abilities to cleave triglycerides and cholesteryl esters 
were compared. The homogeneous 52 kDa enzyme hydrolyzed triolein at a rate 
of 27 nmol/hr/g pancreas and this activity was inhibited by 0.15M NaCl 
(68%), but it was stimulated by a 10-fold excess of colipase (375%). On 
the other hand, the purified 100 kDa esterase protein hydrolyzed triolein 
at a rate of 1,970 nmol/hr/g pancreas in the presence of 5 mM 
taurocholate, and this activity was unaffected either by 0.15M NaCl or by 
a 50-fold molar excess of colipase. Since pancreatic cytosol cleaves 
triolein at a rate of 2,200 nmol/hr/g, these results indicate that the 
vast majority of triglyceride hydrolytic activity resides with the 100 kDa 
protein, and not with the 52 kDa species. Virtually the same results were 
found with cholesteryl esters; the 100 kDa enzyme cleaved cholesterol 
oleate at a rate at least 490-fold greater than that found for the 52 kDa 
triglyceride lipase. Taken together, these results suggest that in man the 
100 kDa enzyme is responsible for hydrolyzing both triglycerides and 
cholesteryl esters. 
EXAMPLE 11 
The importance of heparin for the uptake of fatty acid from triolein was 
demonstrated in a similar way. Confluent monolayers of 2.times.10.sup.6 
CaCo cells (Example 5b) were incubated with 2 mM taurocholate and with 
[.sup.14 C]triolein (5 uM) embedded in phosphatidylcholine vesicles. In 
the absence of triglyceride lipase, cellular uptake of [.sup.14 C] oleate 
was linear and at eight hours was 204 pmol. In the presence of human 
triglyceride lipase (25 nM) and colipase (400 nM), uptake was brisk, 
reaching 706 pmol at eight hours. Moreover, enzyme-mediated uptake was 
also dependent on the presence of colipase in the medium, since uptake was 
only 229 pmol at eight hours in its absence. Heparin (400 uM) added to 
medium containing triglyceride lipase and colipase reduced uptake from 706 
pmol to 329 pmol at eight hours. Thus, heparin decreased cellular uptake 
of oleate (oleic acid) from cleaved triglyceride by 75%. 
EXAMPLE 12 
The 500 MHz high resolution proton NMR spectra of LDL-bound heparin and 
bulk heparin were gathered at 24.degree. C. in D.sub.2 O. Proton 
assignments were determined from a comparison of published values for 
heparin and its chemically modified derivatives (Ayotte, L. and Perlin, 
A.S., Carbohydrate Research 145:267-277, 1986). The spectrum for LDL-bound 
heparin has two differences when compared to that for bulk heparin. Thus, 
for LDL-bound heparin, there is a new signal at 4.85 ppm (FIG. 5), and 
moreover, the signal at 5.1 ppm for bulk heparin (FIG. 6) is shifted 
downfield to 5.3 ppm. Both these changes are consistent with the 
replacement of an --OSO.sub.3 group in bulk heparin by an --OH group in 
LDL-bound heparin at the C-2 position of .alpha.-1-idopyranosyluronic acid 
residue. 
As can be seen from a comparison of FIGS. 5 and 6, there is a clear 
difference between LDL-bound heparin (heparin subfraction) and bulk 
heparin. The heparin subfraction is characterized by greater inhibitory 
potency, LDL binding and a downfield proton on NMR spectra. 
In view of the above, it will be seen that the several objects of the 
invention are achieved and other advantageous results attained. 
As various changes could be made in the above methods and compositions 
without departing from the scope of the invention, it is intended that all 
matter contained in the above description shall be interpreted as 
illustrative and not in a limiting sense.