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Clinical Research Center Studies |
Veterans Affairs Medical Center and the Department of Internal Medicine, Division of Endocrinology, Metabolism, and Diabetes and the Division of Hematology and Oncology, Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, Utah 84132; and University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4
Address all correspondence and requests for reprints to: Dana E. Wilson, M.D., Veterans Administration Hospital (111E), 500 Foothill Drive, Salt Lake City, Utah 84148. E-mail: minerals{at}xmission.com
Abstract
Recombinant human interleukin-2 (rIL-2) is used to treat refractory cancers. During such treatment, patients develop severe hypocholesterolemia along with striking alterations in the concentration and composition of the circulating lipoproteins. The present study was undertaken to gather information about the pathogenesis of these abnormalities. Patients were studied before-, during- and after a 5-day course of high dose iv rIL-2.
Whole plasma cholesterol was markedly reduced by rIL-2 administration (52%; P < 0.001), whereas the triglyceride concentration did not change. Thus, the lipoproteins became triglyceride enriched (P = 0.004). Low density lipoprotein cholesterol, apolipoprotein B (apoB), high density lipoprotein cholesterol, and apoA-I concentrations all decreased. Esterified cholesterol levels were markedly reduced. Total plasma apoE increased markedly, and two kinds of abnormal particles appeared: 1) ß-migrating, very low density lipoproteins; and 2) discoidal, apoE- and phospholipid-containing particles with abnormal density and electrophoretic mobility. The activities of two lipoprotein triglyceride hydrolases, lipoprotein lipase and hepatic lipase, fell significantly during treatment and returned promptly to pretreatment levels after rIL-2 was discontinued. Lecithin:cholesteryl acyltransferase (LCAT) activity also decreased significantly (64%) during treatment, but in contrast to the lipases, remained low for at least 5 days after the last dose of rIL-2 (P < 0.001).
High dose iv rIL-2 induces severe dyslipidemia with deficiencies of both postheparin lipases and acute LCAT deficiency. Most, if not all, of the lipoprotein changes observed are explained by the LCAT deficiency that follows IL-2-induced hepatocellular injury and cholestasis.
HUMAN recombinant interleukin-2 (rIL-2) is used to treat patients with metastatic melanoma and renal cell cancer (1, 2). It has recently been used to increase CD4 T lymphocyte counts in patients infected with human immunodeficiency virus (3). High dose rIL-2 has serious toxicities in humans that involve virtually every organ system. A generalized vascular leak results in decreased intravascular volume, prerenal azotemia, and increased cardiac work. Acute cholestatic jaundice is nearly universal (4). Striking hypocholesterolemia, reported by several investigators, is accompanied by the near disappearance of high density lipoprotein (HDL) cholesterol and a marked reduction in the concentration of low density lipoprotein (LDL) cholesterol (5, 6, 7). Hypocholesterolemia has also been reported in subjects receiving two other cytokines, granulocyte-macrophage colony-stimulating factor (8) and macrophage colony-stimulating factor (9). The abrupt onset and prompt reversal of these changes suggested that either IL-2 itself or secondarily released cytokines (10) was responsible.
In this study we sought clues to the pathogenesis of these abnormalities by characterizing further the changes in lipoprotein composition and distribution during and after rIL-2 treatment and by measuring the activities of three key enzymes in lipoprotein metabolism: lipoprotein lipase (LPL), hepatic lipase (HL), and lecithin:cholesteryl acyltransferase (LCAT). As features of the complex dyslipidemia accompanying rIL-2 treatment are also seen in cholestasis with a variety of causes, we examined the temporal relationship between dyslipidemia and abnormal hepatic function.
Subjects and Methods
Patients
Patients with metastatic malignant melanoma or renal carcinoma were studied during a multiinstitutional trial of high dose, bolus rIL-2 therapy, given by itself or together with lymphokine-activated killer cells (1). The protocol was approved by the university institutional review board. After giving their signed informed consent, patients received two 5-day courses of iv rIL-2 (100,000 U/kg every 8 h) on days 1–5 and days 11–15, separated by a 5-day recovery period. Patients randomized to receive lymphokine-activated killer cells underwent leukapheresis daily for 4 days (days 7–10). The harvested activated lymphocytes were readministered together with rIL-2 on days 11, 12, and 14. rIL-2 was provided by Cetus/Chiron (Emeryville, CA).
Study conditions
To be eligible, patients had to be older than 16 yr, have Karnofsky scores of 70% or higher, and demonstrate adequate lung, liver, kidney, and bone marrow function. No concurrent chemotherapy or radiotherapy was permitted. Acetominophen, indomethacin, and ranitidine were given routinely to prevent fever, chills, and gastrointestinal bleeding. Hydroxyzine, diphenhydramine, meperidine, antiemetics, and antidiarrheal agents were given as required. Glucocorticoids were not used.
Pretreatment blood samples (day 0 samples) were drawn, and patients
were started on continuous total parenteral nutrition (TPN) via a
central venous catheter to maintain adequate nutritional intake during
periods of anorexia and mucositis. The TPN solution consisted of equal
volumes of 50% dextrose and a standard amino acid solution
supplemented with electrolytes, multivitamins (ascorbic acid, retinol,
ergocalciferol, thiamine, riboflavin, pyridoxine, niacinamide,
dexpantethol, d,l-
-tocopheryl acetate, folate,
biotin, and cyanocobalamin), and trace minerals (Aminosyn, Abbott
Laboratories, North Chicago, IL). TPN was delivered at a fixed rate of
80–100 mL/h in a total amount calculated from the estimated daily
caloric needs of each patient (1920–2400 Cal). Twice a week, patients
received 250 mL 10% iv fat emulsion (Lyposyn II, Abbott) to provide
essential fatty acids (275 Cal/250 mL). The infusion of the fat
emulsion was timed so that it preceded serum lipid or lipoprotein
measurements by at least 24 h.
Lipid and lipoprotein analyses
Blood for lipoprotein analysis was collected in Vacutainer tubes containing disodium ethylenediamine tetraacetate (EDTA; 1 mg/mL final concentration) and chilled on crushed ice. Plasma was separated by centrifugation (2000 x g for 20 min) and kept refrigerated at 4 C. HDL cholesterol was routinely measured in the plasma supernatant after heparin-Mn2+ precipitation of the apoB-containing lipoproteins (11). LDL cholesterol (milligrams per dL) was calculated as: total cholesterol - [(triglyceride (TG)/5) + HDL cholesterol]. TG and cholesterol analyses of whole plasma and fractionated lipoproteins were carried out with coupled enzymatic assays using microbial lipase, or cholesterol esterase and cholesterol oxidase, respectively (Beckman Instruments, Carlsbad, CA). Unesterified cholesterol in isolated lipoprotein fractions was determined enzymatically (Boehringer Mannheim Corp., Indianapolis, IN). Esterified cholesterol in the lipoprotein fractions was calculated as the difference between total and unesterified cholesterol. Phospholipid was measured as inorganic phosphate equivalents (12), and protein was quantified by a modification of the Lowry method (13). Analytical electrophoresis of whole plasma and isolated lipoproteins was performed using a Lipoprotein Kit (Ciba Corning, Palo Alto, CA).
Lipoproteins were isolated by density gradient ultracentrifugation in a VTi50 vertical rotor (Beckman Instruments, Palo Alto, CA) (14) with pre- and post-IL-2 treatment samples carried through the procedure in parallel. Cholesterol and TG concentrations were determined in each fraction. Preparative molecular exclusion chromatography (15) was carried out by applying 5 mL fresh whole plasma containing 100 mg sucrose to a 2.5 x 100-cm column of 4% agarose (Bio-Gel A-15 M, 200–400 mesh, Bio-Rad, Richmond, CA) and eluting with 0.2 mol/L NaCl, 1 mmol/L EDTA, 0.02% NaN3, aprotinin (50 kallikrein inhibitor units/mL), and 10 mmol/L sodium phosphate, pH 7.0, at room temperature under a hydrostatic pressure of 50 cm, resulting in a flow rate of approximately 15 mL/h over 30 h. The void volume of the column was approximately 150 mL (fraction 30), and the included volume was 425–450 mL (fractions 85–90). Cholesterol, TG, and apolipoprotein E (apoE) (16) concentrations were determined in each fraction.
To display the relative proportions of cholesterol and TG on a continuous proportional scale, molar ratios were calculated using formula weights of 387 and 885 g/mol for unesterified cholesterol and triolein, respectively. Enrichment ratios were calculated for fractions in which cholesterol and TG concentrations allowed quantitatively reliable assay: [(cholesterol/TG) -1] with a cholesterol/TG molar ratio greater than 1, [(TG/cholesterol) - 1] with a cholesterol/TG molar ratio less than 1, and [(cholesterol/TG) - 1 = 0] with a cholesterol/TG molar ratio of 1. A cholesterol/TG molar ratio of 10:1 gives a molar excess of 9, a cholesterol/TG molar ratio of 1:10 gives a molar excess of -9, and equimolar proportions are signified by a molar enrichment of 0. Thus, positive values indicate cholesterol enrichment, and negative values indicate TG enrichment.
Apo
Apo in the lipoprotein fractions were separated by electrophoresis in 15% polyacrylamide gels according to the method of Laemmli (17), stained with Fast Stain (Zoion Research, Allston, MA), and quantitated with the 512 x 512-pixel Visage 100 system (BioImage/Kodak, Ann Arbor, MI) in the transmission mode using whole band analysis. ApoB and apoA-I were quantitated by nephelometry using a Beckman Astra system (18).
ApoE was quantitated with a monoclonal sandwich enzyme-linked immunosorbent assay. The enzyme-linked immunosorbent assay was developed using human apoE isolated from very low density lipoprotein (VLDL) by SDS-PAGE to immunize female BALB/c mice. Mouse spleen cells were separated and fused with P3X myeloma cells in polyethylene glycol (19), and the resulting hybridomas that produced antibody against human apoE were cloned and injected peritoneally into Pristane-primed mice. Monoclonal antibodies (II42A5 and II4C4) were isolated from the ascites fluid by affinity chromatography on Immunopure protein G (Pierce Chemical Co., Rockford, IL). The II4C4 was conjugated with horseradish peroxidase (Sigma Chemical Co., St. Louis, MO) (20). Immunolon 4 (Dynatech, Chantilly, VA) 96-well microtiter plates were coated with 100 µL II42A5 (15 µg/mL in phosphate buffered saline) for 3 h at room temperature, followed by 16-h incubation at 4 C. The plates were then blocked for 1 h at room temperature with 1% BSA in phosphate-buffered saline. ApoE was then "captured" in a 2-h incubation at 37 C. Peroxidase-conjugated II4C4 was added, and the plates were incubated at 37 C for 2 h. Peroxidase activity was quantified by the addition of o-phenylenediamine (1 mg/mL) in citrate phosphate buffer, pH 5.3, and H2O2 (0.012%). Reactions were terminated with 4 N H2SO4, and optical density was measured at 405 nm in a MicroTek plate reader (Flow Laboratories, McLean, VA). The assay was standardized with authentic apoE, generously provided by Dr. Gustav Shonfeld, Washington University (St. Louis, MO). Pre- and post-IL-2 treatment samples from each subject were assayed together. The significance of differences in mean values on days 1, 4, 7, and 10 was determined by one-factor ANOVA.
HDL characterization
Fractionated lipoproteins from density gradient ultracentrifugation were pooled according to their density and cholesterol concentrations. The pooled fractions were further purified from plasma proteins by ultracentrifugation at densities of 1.019 g/mL [VLDL and intermediate density lipoprotein (IDL)], 1.062 g/mL (LDL), or 1.21 g/mL (HDL) and dialyzed against 0.125 mol/L ammonium acetate, 2.6 mmol/L ammonium carbonate, and 0.26 mmol/L tetrasodium EDTA, pH 7.4. Lipoproteins were then negatively stained by mixing with an equal volume of 2% sodium phosphotungstate, pH 7.4, placed on Formvar-carbon-coated grids using the droplet method (21), and examined on a Geol transmission electron microscope (JEM-100 CXII, Joel Ltd., Peabody, MA) at 80 kV under magnifications of x10,000–50,000.
HDL size was estimated by electrophoresis at 125 V for 24 h at 4 C in 90 mmol/L Tris base, 80 mmol/L boric acid, 3 mmol/L EDTA, and 3 mmol/L sodium azide, pH 8.35, using a 4–30% gradient gel in a Hoeffer MiniGel apparatus (Hoeffer, San Francisco, CA). Samples were mixed (4:1; vol/vol) with a solution consisting of sucrose (40%) and bromophenol blue (0.05%) immediately before electrophoresis. The HDL particles were visualized by staining the gels with Fast Stain.
LPL and HL
After patients had fasted for 12 h, and 10 min after the iv injection of 60 U/kg heparin, venous blood was collected in chilled EDTA tubes. Plasma lipase activities were determined as described in detail previously (22). Briefly, the substrate for lipase activity was radioactive triolein emulsified with lecithin, with the addition of pooled serum as a source of apoCII activator. LPL and HL activities were calculated as the fractions of total lipolytic activity that were and were not inhibited (23), respectively, by a specific polyclonal antiserum prepared against pure bovine milk LPL.
LCAT
The endogenous rate of cholesterol esterification in plasma was determined as previously described (24, 25). An exogenous substrate assay using artificial proteoliposomes containing human apoA-I, phosphatidylcholine, and unesterified [3H]cholesterol was used to measure plasma LCAT activity in vitro (25).
Data analysis
The data are shown as the mean ± SEM. Between-group comparisons were made by two-tailed Student’s t test for paired or unpaired data as appropriate. In cases where the normal distribution of data was in doubt, nonparametric analyses were used.
Results
Whole plasma constituents
Plasma lipids and apo levels before rIL-2 treatment (day 0), after
4 days of treatment (day 4), and after 2 or 5 days of recovery (days 7
and 10, respectively) are shown in Table 1
. Total
cholesterol fell in all subjects, from 3.96 ± 0.37 mmol/L
(153 ± 15 mg/dL) on day 0 to 2.07 ± 0.25 mmol/L (82.6
± 10.6 mg/dL) on day 4 (mean ± SE). In contrast,
plasma TG did not change significantly. The mean plasma cholesterol
concentration decreased 52% (P < 0.001), and the
molar ratio of cholesterol to TG decreased 53% (3.47 to 1.64;
P = 0.004, by Wilcoxon’s signed rank). These data
confirmed the pronounced hypocholesterolemic effect of rIL-2 and the
relative TG enrichment of the circulating lipoproteins (4, 6). Plasma
total cholesterol levels remained low during recovery.
|
). By day 10, 5 days after the
last dose of rIL-2, plasma apoB had returned to the pretreatment level,
whereas apoA-I remained approximately 50% of the pretreatment value.
In contrast, there was a pronounced increase in total apoE during
treatment, which persisted into the recovery period; apoE was 51%
higher on day 4 and 159% higher on day 7 than that on day 0 (Table 1). Plasma lipoproteins
During treatment, HDL cholesterol fell from 0.97 ± 0.07
mmol/L on day 0 to 0.34 ± 0.07 mmol/L on day 4 and remained
depressed on posttreatment day 7 (0.21 ± 0.05 mmol/L). LDL
cholesterol fell concomitantly. To better understand the effect of
rIL-2 on lipid metabolism, the compositions of the various lipoprotein
fractions pre- and posttreatment were compared. Plasma lipoproteins
obtained on days 0 and 7 were separated by density gradient
ultracentrifugation. Representative profiles from a single study
subject are shown in Fig. 1. Consistent with our
previous findings (6), HDL cholesterol was reduced, and the HDL peak
was shifted to a lower density. Similar changes were observed in LDL,
although the density shift was not as pronounced. In addition, there
was an increase in TG in the IDL/LDL density region. Isolated HDL from
day 7 was heterogeneous and had abnormal electrophoretic mobility (Fig. 1). Although the pretreatment HDL (fraction 27, day 0) showed a single
-migrating species, the posttreatment HDL (fraction 21, day 7)
exhibited two bands with ß and pre-ß migrating mobilities,
respectively.
|
). Ultracentrifuged fractions, pooled according to
cholesterol content and density (Table 2), were analyzed first. The HDL
were phospholipid enriched after rIL-2 treatment and were protein poor
in comparison to pretreatment HDL, but failed to show significant
changes in the relative proportions of TG or total cholesterol. Changes
were also observed in LDL composition, with TG and phospholipid
enrichment and proportional reductions in cholesterol and protein. The
IDL also became TG enriched and cholesterol depleted, but failed to
show changes in either relative phospholipid or protein content. The
VLDL were phospholipid enriched 
(40%) and displayed reciprocal
reduction in cholesterol content.
|
). Cholesterol in the LDL and HDL decreased more than
two thirds by day 4, whereas VLDL TG and cholesterol were no different
from pretreatment values. TG increased in the LDL and HDL, particularly
among the smaller LDL (Fig. 2). These effects were most evident on day
7, when there was a distinct shift in LDL TG toward larger particles
(Fig. 2).
|
; see Materials and
Methods). The cholesterol to TG molar ratio in LDL fell from
10–20:1 before treatment to 2:1 after rIL-2, and in HDL from a
pretreatment value of 10:1 to 2:1 posttreatment. Changes in LDL
and HDL evident on day 4 of treatment persisted at least until day 7, 2
days after rIL-2 had been discontinued. VLDL composition was more
variable, with some, but not all, of the subjects demonstrating VLDL
cholesterol enrichment on day 7.
|
)
We previously noted remnant-like lipoprotein particles in
rIL-2-treated subjects, raising the possibility that abnormal TG
hydrolase activity might account for TG enrichment in the IDL and LDL
(6). Therefore, we measured postheparin LPL and HL activities in eight
subjects before rIL-2 administration, on the fourth day of rIL-2
administration (day 4), and 5 days after the last dose of rIL-2 (day
10). As shown in Fig. 4, LPL and HL activities both fell significantly
by day 4 of rIL-2 administration (186 ± 37 to 68 ± 10, and
274 ± 52 to 82 ± 14 nmol/mL·min, respectively; both
P < 0.01). The reduction in lipase activities was
reversible, returning to pretreatment values by day 10, 5 days after
the last dose of rIL-2 (P > 0.5 for the differences
between days 0 and 10).
|
)
Total and nonesterified cholesterol were measured in pooled
density fractions before (day 0) and after (day 7) rIL-2 treatment.
Esterified cholesterol was reduced in all four lipoprotein density
classes after rIL-2 administration, whether it was expressed on an
absolute basis or as a proportion of total cholesterol (Table 3). There
was no statistically significant difference in nonesterified
cholesterol content. Thus, the reduction in whole plasma total
cholesterol reflected the decrease in esterified cholesterol.
|
)
Because of the striking reduction in cholesterol esterification,
LCAT activity was measured (18) (11 subjects, 12 treatment courses).
LCAT activity against proteoliposomes (Fig. 5A) fell significantly by
day 4 of rIL-2 treatment (64%), as did the in vitro rate of
cholesterol esterification (43%; Fig. 5B). In contrast to the
abnormalities in LPL and HL (Fig. 4), the defect in cholesterol
esterification persisted for at least 5 days after the last dose of
rIL-2 (P < 0.001 for either, by repeated measures
ANOVA).
|
Plasma lipoproteins from days 0 and 7 were separated by agarose
gel permeation chromatography, and the apoE content of each fraction
was determined. The sum of apoE recovered in the eluted fractions (data
not shown) confirmed a markedly increased plasma concentration of apoE.
The distribution of apoE on day 7 was unusual; the majority was
associated with particles with an elution volume intermediate between
and distinct from those of normal HDL and LDL (Fig. 6B).
Phospholipid distribution was also strikingly abnormal; phospholipid in
the IDL-LDL and VLDL size regions was markedly increased, to a lesser
extent in the inter-LDL-IDL region (fraction 70). Particles isolated
from the latter region had cholesterol/TG ratios of 3:1 (Fig. 3) and
contained phospholipid and apoE (Fig. 6, B and C).
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Electron microscopy of 1.063–1.21 g/mL HDL from day 7 revealed a
heterogeneous population of particles ranging in size from about 6 nm
to nearly 50 nm (Fig. 7). Two distinct classes of
spherical particles were seen with mean diameters of 6–10 and 10–20
nm. Disk-shaped particles were also abundant in aggregates, resembling
"stacked coins" or crinoid stems (26). The stacks ranged in width
from 10–25 nm, and total length ranged from 50–180 nm, with an
individual disc thickness of approximately 3–5 nm. In addition, there
were variably sized larger particles that appeared to be associated
with and adherent to the ends of many of the stacks (Fig. 7). These
ultrastructural abnormalities are typically associated with familial
LCAT deficiency (27, 28) and acquired LCAT deficiency accompanying
severe liver disease (29). Ultrastructural abnormalities in
lipoproteins of densities less than 1.063 g/mL were not found (data not
shown).
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Clinical chemistry profiles obtained during rIL-2 treatment reflected widespread organ dysfunction with hypoalbuminemia and hyponatremia, as described by others (30). Abnormalities in renal function were relatively mild. Fasting hyperglycemia (6.22 ± 0.38, 11.49 ± 1.55, and 7.22 ± 0.72 mmol/L on days 0, 4, and 7, respectively; P < 0.0002) developed in this series of patients, none of whom had diabetes mellitus previously (W. E. Samlowski, G. Wiebke, M. McMurry, M. Mori, and J. H. Ward, manuscript submitted). Hepatocellular enzymes (aspartate aminotransferase/alanine aminotransferase) were increased, peaking on days 4–5 of treatment. In contrast, alkaline phosphatase rose progressively, further increasing in the 2- to 5-day interval after rIL-2 was discontinued (112 ± 6.7, 158 ± 15, and 252 ± 24 U/L for days 0, 4, and 7, respectively; P < 0.0001). Direct (conjugated) hyperbilirubinemia also reached a peak on day 7, 2 days after the 5-day course of rIL-2. Visualization of the biliary tract and liver by ultrasound at the time of maximal hyperbilirubinemia excluded physical obstruction. Thus, hepatocellular injury was most prominent during rIL-2 administration, whereas cholestasis, like the compositional and ultrastructural abnormalities in HDL, was most severe during recovery.
Discussion
Patients treated with rIL-2 for metastatic cancer develop profound hypocholesterolemia, with dyslipidemia manifest by the presence of ß-migrating VLDL (5, 6). The present work extends previous studies (5, 6, 7) of rIL-2-induced dyslipidemia and provides new information about these abnormalities. The reduction in total serum cholesterol observed in this study (46%) was comparable to that reported previously by ourselves and others (5, 6, 7). LDL cholesterol fell 34%, and HDL cholesterol fell 66%, changes paralleled by reductions in apoB and apoA-I (5, 7). Plasma TG levels were unchanged or rose slightly, so the circulating lipoproteins became enriched in TG relative to cholesterol.
We used two complementary methods to separate the plasma lipoproteins (density gradient ultracentrifugation and gel filtration chromatography) to avoid arbitrary distinctions between density classes and to minimize apo dissociation. ApoB-containing lipoproteins from the IDL and LDL density regions were cholesterol depleted, and TG and phospholipid enriched. The VLDL had anomalous ß-mobility on agarose gel electrophoresis. Plasma lipoproteins were preferentially depleted in cholesteryl ester. As the concentrations of normal LDL and HDL decreased, a new lipoprotein species with unusual composition and physical properties appeared; normal HDL (judging from the cholesterol content in its typical density range) virtually disappeared, whereas abnormal apoE-bearing, phospholipid-enriched particles accumulated in the inter-LDL-HDL region. These particles were most abundant on day 7, after the course of rIL-2 had ended. Electron microscopy revealed abnormally small discoidal HDL in "stacked coin" aggregates.
The HDL from rIL-2-treated patients resembled HDL in LCAT deficiency, either inherited deficiency or that accompanying severe liver disease (31). LCAT is synthesized by the liver and secreted into the plasma, where it circulates together with HDL (31). This enzyme catalyzes the formation of HDL cholesteryl ester by transesterifying a fatty acid residue from phosphatidylcholine to free cholesterol. HDL cholesteryl esters then transfer to apoB-containing lipoproteins (VLDL and LDL) through heteroexchange with TG in the presence of cholesteryl ester transfer protein. LCAT deficiency results in decreased cholesteryl ester in all lipoprotein classes and in phospholipid (phosphatidylcholine) enrichment. LCAT activity decreased during rIL-2 administration and remained low after rIL-2 was discontinued. The compositional abnormalities in HDL after IL-2 treatment are entirely consistent with LCAT deficiency (32, 33). The ultrastructure of the HDL from rIL-2-treated patients (discoidal "stacked coins" and abnormally large particles) also strongly suggested LCAT deficiency (27, 28).
The role of HL deficiency was of interest because the lipoprotein abnormalities present during rIL-2 administration had features seen in inherited (34, 35) or acquired (36) HL deficiency in humans. Moreover, similar changes have been induced in experimental animals by in vivo inhibition of HL with specific anti-HL antibodies (37). Lastly, both acquired LCAT deficiency and HL deficiency have been reported in patients with advanced primary biliary cirrhosis (38). In the latter condition, similarities in the lipoprotein profile include the presence of VLDL with ß-electrophoretic mobility, an increase in IDL (Sf = 12–20) mass, the accumulation of large, buoyant, TG-enriched LDL2 (39), and reduced LDL mass (37, 40). In contrast to rIL-2-treated subjects, HDL2 concentrations are usually increased in patients with early primary biliary cirrhosis and in those with inherited HL deficiency (38, 41). Thus, although HL deficiency may contribute to the accumulation of TG-enriched, ß-migrating, remnant-like VLDL-IDL, it cannot account for reduced HDL concentrations.
Reversible cholestatic jaundice is seen regularly in patients receiving rIL-2 (4); the most pronounced abnormalities in hepatic function are observed in the days following rIL-2 treatment. The abnormalities in HDL composition and ultrastructure were also most pronounced after the cessation of rIL-2 administration, coinciding with peak cholestasis.
Some, but not all, of the changes in rIL-2-treated subjects are seen in
cholestasis from a variety of causes (29, 42). Reduced cholesterol
esterification, hyperphospholipidemia, ß-migrating VLDL, TG-enriched
LDL, reduced -migrating HDL concentrations, the appearance of
discoidal apoE-enriched HDL, and reduced LCAT and hepatic lipase
activities are common to both situations. The most striking difference
between the two conditions is the magnitude of the observed changes. In
typical cholestasis (e.g. that which accompanies primary
biliary cirrhosis), there is development of hypercholesterolemia and
the accumulation of Lp(X), a discoidal LDL particle enriched in free
cholesterol and phospholipid. In contrast, we found that rIL-2-treated
subjects become hypocholesterolemic, exhibit very low concentrations of
lipid in the LDL density region, and lack discoidal LDL [Lp(X)].
Thus, rIL-2 dyslipidemia may reflect cholestatic injury together with
more specific effects of IL-2; the present study provides no
information about the direct effects of this cytokine on the synthesis
or catabolism of key enzymes or apo or whether rIL-2 affects LCAT
expression directly.
The activities of the two heparin-released TG hydrolase activities, LPL and HL, were also of interest because LPL is inhibited in vivo by other cytokines (tumor necrosis factor) (43, 44), and lipoproteins during the acute phase response (45) and in subjects with inherited HL deficiency bore similarities to the changes induced by IL-2. Both LPL and HL activities were depressed, but in contrast to LCAT, they returned promptly to pretreatment values by day 10, 5 days after the last rIL-2 treatment. It is difficult to ascribe any major role to LPL deficiency in the pathogenesis of rIL-2 dyslipidemia in the absence of overt hypertriglyceridemia, without the accumulation of chylomicrons or large VLDL. It remains possible that LPL deficiency contributed to TG enrichment of the apoB-containing lipoproteins.
The effects of TPN on lipid and carbohydrate metabolism were considered. Hyperglycemia is a recognized complication of TPN in the seriously ill, particularly in neonates (46, 47). TPN is likely to have contributed to fasting hyperglycemia in the present series of patients (W. E. Samlowski, G. Wiebke, M. McMurry, M. Mori, and J. H. Ward, manuscript submitted). TPN is associated with the development of dyslipidemia. However, this is characterized by hypercholesterolemia (48) and the accumulation of Lp(X) (29), features absent in our subjects. To minimize the effect of TPN on plasma lipoproteins, blood samples for lipoprotein analysis were obtained at least 24 h after iv fat administration. Furthermore, TPN alone could not explain the present findings, as IL-2 dyslipidemia has been seen in patients who had never received TPN (6). Nevertheless, although an independent contribution of TPN (particularly with preparations containing excess phospholipid) to the observed lipoprotein abnormalities seems unlikely, it cannot be ruled out completely.
In summary, patients with refractory metastatic cancer who are treated with rIL-2 develop severe hypocholesterolemia that mirrors striking alterations in the concentration and composition of all normal lipoprotein classes. Likely explanations include a marked inhibition of LCAT activity along with reversible suppression of LPL and HL activities; it is not surprising that the consequent lipoprotein abnormalities are widespread and complex. The striking changes in HDL are best explained by LCAT deficiency accompanied by rIL-2-induced cholestasis and hepatocellular injury. Still other mechanisms may be involved: the extravasation of small molecules via a capillary leak (30), defective cholesteryl ester transfer, and the utilization of lipoprotein cholesterol by rapidly proliferating cells (49) [as documented in monocyte-macrophage (50, 51) and myelocytic cell lines (52)]. It is tempting to speculate that the pathological production of cytokines in vivo may contribute to other dyslipidemic clinical states, such as the hypocholesterolemia of terminal illness.
Acknowledgments
The authors are grateful for the expert technical assistance of Kristie Allen and Bruce Dugan, the editing assistance of Steve Sugden, and the dedicated nursing care provided by Carol Bowcutt, R.N., and her staff at the University of Utah General Clinical Research Center.
Footnotes
1 This work was supported by the Department of Veterans Affairs and
grants from the NIH to the General Clinical Research Center (RR-00064)
and the Utah Regional Cancer Center (CA-42014). Nonclinical portions of
the work were assisted by a grant-in-aid from the Nora Eccles Treadwell
Foundation. 
Received May 22, 1996.
Revised January 8, 1997.
Accepted January 15, 1997.
References
) interferon induces hypertriglyceridemia and inhibits
post-heparin lipase activity in cancer patients. J Exp Med. 164:1093–1101.This article has been cited by other articles:
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  S. Barlage, D. Fröhlich, A. Böttcher, M. Jauhiainen, H. P. Müller, F. Noetzel, G. Rothe, C. Schütt, R. P. Linke, K. J. Lackner, et al. ApoE-containing high density lipoproteins and phospholipid transfer protein activity increase in patients with a systemic inflammatory response J. Lipid Res., February 1, 2001; 42(2): 281 - 290. [Abstract] [Full Text]
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 A. Stachon, A. Boning, H. Weisser, A. Laczkovics, G. Skipka, and M. Krieg Prognostic Significance of Low Serum Cholesterol after Cardiothoracic Surgery Clin. Chem., August 1, 2000; 46(8): 1114 - 1120. [Abstract] [Full Text] [PDF]
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