Intravascular metabolism of an artificial transporter of triacylglycerols. Alterations of serum lipoproteins resulting from total parenteral nutrition with intralipid

176

BBA 5 1068

INTRAVASCULAR METABOLISM OF AN ARTIFICIAL TRANSPORTER OF TRIACYLGLYCEROLS

ALTERATIONS OF SERUM LIPOPROTEINS RESULTING FROM TOTAL PARENTERAL NUTRITION WITH INTRALIPID *

STEVEN H. UNTRACHT **

(Received November 18th. 1981)

As a model for transport in the bloodstream of exogenous triacylglycerols, we have studied the intravascular metabolism of Intralipid, an artificial, cholesterol- and protein-free ‘chylomicron’. Two patients with Crohn’s disease and one patient with intermittent abdominal pain received total parenteral nutrition including between 50 and 100 g of Intralipid per day. Samples of serum were analyzed chemically for lipids, assayed for lecithin:cholesterol acyltransferase (EC 2.3.1.43), and analyzed by isopycnic density-gradient ultracentrifugation and electron microscopy. After 3 weeks of infusion, single-bilayer vesicles, 300-600 A in diameter, were present in the serum of each patient. Comprising equimolar amounts of phospholipids and unesterified cholesterol, and approximately 5% by weight of protein, these vesicles accumulated to levels of approximately 100,300 and 50 mg/dl for the three patients indicated above, causing hyperlipidemia in the first two cases. In conjunction, the levels of HDL and lecithin:cholesterol acyltransferase had decreased by 40% in each patient. Upon stopping the infusions, vesicles disappeared with monophasic kinetics with a half-life of 2 days, and were not converted to HDL or other lipoproteins; HDL and lecithin: cholesterol acyltransferase returned to normal levels after a lag period of at least 4 days. Two normal subjects were studied during and after a single 4-h infusion of 50 g of Intralipid. Triacylglycerols were cleared with a half-life of 1 h, but at least 40% of the phospholipids remained in the serum as vesicles. Although Intralipid contains no steroids, unesterified cholesterol progressively entered the serum after the vesicles appeared. During these short-term studies, the levels of HDL and lecithin:cholesterol acyltransferase did not change. The observations indicate that vesicles, derived from a variable fraction of the Intralipid phospholipids, extract unesterified cholesterol into plasma. Inert and slowly cleared, the resulting mixed vesicles accumulate in the blood. The levels of HDL and 1ecithin:cholesterol acyltransferase decrease only when the intestine is bypassed for long periods of time, probably because the lipoprotein and enzyme are synthesized more slowly than usual. Our results suggest that in chylomicrons other mechanisms prevent phospholipids from being released into the bloodstream as vesicles.

* Accounts of this work have appeared in abstract form

(( 1979) Clin. Res. 27, 444A; Clin. Res. 27, 504A).

** Present address: Department of Surgery; Massachusetts General Hospital; Boston, MA 021 14. U.S.A

OOOS-2760/82/OOOC-OooO/$O2.75 Q I982 Elsevier Biomedical Press

Abbreviations: HDL, high-density lipoproteins, i.c.. serum

lipoproteins with density between 1.063 and 1.21 g/ml: LDL,

low-density lipoproteins, i.e.. serum lipoproteins with density

between I.019 and I.063 g/ml; VLDL. very low density lipo-

proteins, i.e., serum lipoproteins with density less than 1.006

g/ml; LpX. lipoprotein X.

Introduction

As they transport lipids through the bloodstream, the four major classes of serum lipo-

proteins - chylomicrons, VLDL, LDL, and

HDL-are interconverted through a variety of

pathways [l-3]. Chylomicrons and VLDL prim-

arily serve to deliver absorbed and endogenously

synthesized triacylglycerols, respectively, to

adipose tissue, skeletal muscle and lactating

mammary glands [2,4]. In the case of chylomicrons,

it has long been known [5-71 that when the tri-

acylglycerols are removed from the circulation,

new phospholipids appear in the HDL fraction-

presumably through the action of lecithin : cholesterol acyltransferase [ 1,8- 111. Recent ra- dioisotope tracer studies in rats [ 12,131 have dem-

onstrated that these HDL-bound phospholipids are derived directly from chylomicrons. Radioac-

tive-labeling studies in humans [14] and in rats

[ 12,131 have also shown that apolipoproteins are

transferred simultaneously with phospholipids

from the chylomicrons to HDL.

Such interconversions seem to be important in

maintaining homeostasis of serum lipids and lipo-

proteins, and may be governed in part by the

complicated chemical make-up of these particles.

We were, therefore, prompted to study the in-

travascular metabolism of Intralipid, and artificial

transporter of triacylglycerols whose chemical

composition is much simpler than that of

chylomicrons. Intralipid is an aqueous dispersion

of egg-yolk lecithin (12% by weight), soybean-oil

triacylglycerols (88% by weight), and trace amounts of cholesterol. The lipids are emulsified into a

heterogeneous group of particles that range in diameter from 200 to 1000 A [ 151. This phos-

pholipid-stabilized triacylglycerol emulsion is infused intravenously as a source of calories in pa-

tients who suffer from inflammatory bowel dis-

ease, malnutrition, gastro-intestinal pain or ob-

struction, or any other disoder in which the gastro-

intestinal tract is not functional [15]. In many

cases, patients receive balanced diets in which all of the fat is supplied as Intralipid, carbohydrates are infused as glucose, and amino acids, vitamins and minerals are administered as such [ 15- 171. In these ‘total parenteral nutrition’ regimens, all of the calorie sources enter the circulation directly

and are not processed by the small intestine.

Therefore, during total parenteral nutrition

chylomicrons do not enter the bloodstream; under

these conditions the Intralipid particle carries exo-

geous triacylglycerols and functions as a

chylomicron.

A variety of studies suggest that Intralipid and

chylomicrons are metabolized similarly. For exam-

ple, Hallberg [ 181 has shown that normal adults

clear Intralipid triacylglycerols from the blood at a

rapid, dose-dependent, and saturable rate. Subse-

quently, Have1 et al. [7] demonstrated that In-

tralipid acquires C-peptides from HDL, and

thereby becomes a suitable substrate for lipopro-

tein lipase. More recently, Robinson and Quar-

fordt [19] have found that Intralipid particles can also obtain apolipoprotein A-I, the arginine-rich

peptide, and other proteins from HDL in vitro.

Moreover, ultra-histochemical studies have shown

that, as is the case with chylomicrons, the tri-

acylglycerols of Intralipid are hydrolyzed at the

surface of endothelial cells in capillaries [20,21].

For the above-mentioned reasons, total

parenteral nutrition with Intralipid is a good model

system to study the intravascular transport of ex-

ogenous fats. With this purpose in mind, we now

present our studies of five human subjects who

have been infused with the triacylglycerol emulsion.

Materials and Methods

Subjects. Three of the five subjects were pa-

tients from the Gastroenterology Service at the

University of Chicago.

Subject 1 (case l), a lCyear-old white male with a 7-year history of Crohn’s disease, was admitted

to the hospital for correction of secondary malnutrition and to be evaluated for arrested

growth and development. Initially, he was in-

travenously hyperalimented with 50% glucose.

However, this therapy affected his liver adversely:

serum glutamic-oxaloacetic transaminase, glutamic -pyruvic transaminase, lactate dehydrogenase and alkaline phosphatase all rose to abnormal levels, and he developed hepatomegaly. The treatment, therefore, was changed to a 5-week total parenteral nutrition regimen comprising 1000 ml/day of 10% Intralipid (Cutter Laboratories: Berkeley, CA),

17x

Fig I. Levels of total cholesterol (unesterified and esterified) in

the serum of subject 1 during total parenteral nutrition with

Intralipid. This patient received 1000 ml of Intralipid per week

prior to day 0. On day 0. he began to receive 1000 ml of

Intralipid per day for approximately 6 weeks. These data were

obtained from the patient’s clinical records.

2000 ml/day of 20% glucose in water, and 1000

ml/day 10% glucose in water containing 3% Freamine II (total calories: 3130/day). Under the

second treatment, his hepatic function returned to

normal. However, serum-cholesterol levels began

to rise immediately, and they reached a total value

of nearly 500 mg/dl by the end of 2 weeks, as

shown in Fig. 1. The cholesterol level remained

elevated for the duration of the infusion period *.

Subject 2 (case2), a 35-year-old white female,

required total parenteral nutrition because of in-

termittent abdominal pain. She was given 1000

ml/day of 10% Intralipid, and 3000 ml/day 5%

glucose in aqueous 0.45% sodium chloride contain-

ing 3% Frearnine II (total calories: 2350/day). In

this patient, total parenteral nutrition lasted for 3 weeks. Her serum-cholesterol level was 168 ml/d1

on the day that parenteral nutrition was started,

but this value rose to 276 mg/dl by the end of the

infusion period. Subject 3 (case 3) was a 16-year-old black female

with a 3-year history of Crohn’s disease. She was hospitalized for treatment of an anal fistula and for ‘bowel rest’. The latter was achieved with a

* In three patients receiving chronic total parenteral nutrition

in which 60% of the non-protein calories were supplied as

hypertonic glucose and the remaining 40% were supplied as

Intralipid. Broviac et al. [ 161 found that the steady-state

concentration of total serum cholesterol was 315-’ 100

mg/dl. When the same patients chronically received all of their non-protein calories as glucose, their serum cholesterol

levels averaged I56 i 15 mg/dl.

5-week course of total parenteral nutrition with

10% Intralipid (500 ml/day) and 3000 ml/day of

20% glucose in water containing a 4.5% Freamine

II (total calories: 3058/day).

During their hospitalizations, none of the three

patients had icterus, elevated levels of serum bi-

lirubin, or other clinical signs of cholestasis. No

other known causes of hyperlipidemia were pre-

sent in any of these cases.

Two healthy, male, student volunteers served as

subjects 4 and 5 (ages 23 and 22 years, respec-

tively). Neither subject thad received Intralipid

before this study or had a history of liver disease

or serum-lipid abnormalities.

The clinical details of the five subjects are listed

in Table I. In no case did the dose of tri-

acylglycerols exceed that in a normal diet. For

example, after total parenteral nutrition was dis-

continued, subjects 1 and 2 continued to consume

approximately the same amount of fat and total

calories. Therefore, in these two cases, the dose of

Intralipid was equivalent to the normal daily flux

of chylomicrons. Each of the other three subjects

received much smaller relative doses (Table I).

Thus, none of the results can be attributed to ‘fat

overloading’.

Sampling schedule. Approximately 8 ml of blood

was obtained from subjects 1 and 2 at 48-h or, in

some cases, 24-h intervals, for a least 2-weeks after

the last infusion. Subject 3 was studied 4 weeks after the first infusion, as well as 6 and 11 weeks

after parenteral nutrition was discontinued. The

blood was allowed to clot for 1 h at room tempera-

ture. The formed elements were then sedimented

by low-speed centrifugation for 10 min, and the

serum was analyzed by the procedures outlined

below.

The two normal subjects were studied after an

overnight fast and, except for the intravenous fat,

both subjects fasted during the 8-h experiment. An Angiocath, inserted into a forearm vein for pur- poses of obtaining blood, was maintained patent by infusing sterile physiological saline at a rate of 250 ml/h. After a control blood sample was obtained, 500 ml of 10% Intralipid was infused intravenously through the opposite arm at a constant rate of 125 ml/h, using an Imed (San Diego, CA) Model 922 Volumetric Infusion Pump. Fur- ther samples of blood were obtained at 0.5, 1, 2, 3,

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and 4 h-during the infusion, and at various inter-

vals after the infusion was completed. Each of the

resulting sera were analyzed as outlined below. In

addition, parameters of hepatic and renal func-

tion, which remained normal in both subjects,

were measured periodically by the Clinical

Laboratories of the University of Chicago.

Informed consent was obtained from all par-

ticipants before studies were undertaken. The

single-infusion protocol (i.e., the studies on sub-

jects 4 and 5) was approved by the Clinical Inves-

tigation Committee of the University of Chicago (Protocol No. 2779).

Analytical methods. Density-gradient ultracentrifugation was performed on l-ml aliquots of

whole serum, according to the method of Foreman

et al. [22]. The resulting gradient was fractionated

and monitored at 280 nm, using an Isco density-

gradient flow cell. A typical analysis is shown in

Fig. 2.

Triacylglycerols and total cholesterol were mea-

sured with a Technicon AutoAnalyzer II [23]; un-

esterified cholesterol was measured as the dig-

itonide [24,25]. Phospholipids were extracted from serum into chloroform/methanol (2: 1 by vol.),

and then measured in the extract, as described by

Bartlett [26].

Free fatty acids were determined by the method

of Mikac-Devic et al. [27]; sodium palmitate was used as the standard. HDL was quantitated chemi-

cally through its cholesterol after precipitating

other lipoproteins with dextran sulfate [28]. Cholesterol weight was multiplied by 8.4 (based on

chemical composition), to obtain the weight of

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Fig. 2. Representative isopycnic density-gradient pattern of I ml

of normal human serum (total cholesterol. 180 mg/dl; tri-

acylglycerols, SO mg/dl). The gradients were prepared and

analyzed as described in Materials and Methods.

Lecithin: cholesterol acyltransferase activity was

assayed as previously reported [29]. With this

method, normal activity of lecithin : cholesterol

acyltransferase ranges from 700 to 1400 dpm/h.

For electron microscopy, specimens of nega-

tively stained lipoproteins were prepared as de-

scribed by Ohtsuki et al. [30], and viewed with a

Philips EM 300 electron microscope operated at a

beam-acceleration voltage of 80 kV and a primary

magnification of 70000 X Gel-filtration chromatography was performed

at 23’C, using a column (1.5 X 90 cm; Pharmacia;

Uppsala, Sweden) packed with 4% agarose (Biogel

A 15 m; Biorad; Richmond, CA). Samples were

eluted with 0.15 M NaCl/O.OS% EDTA/O.OS%

NaN,, pH 7.4, at a downward flow rate of 15

ml/h. Absorbance of the effluent was monitored

at 280 nm.

Protein was measured by the procedure of

Lowry et al. [31], with bovine serum albumin as

the standard. Radioimmunoassays for

apolipoproteins A-I, A-II and B were performed

as previously described [32-341.

Results

Long-term infusions of Intrulipid in patients.

After the last infusion of Intralipid, the serum of

subjects I and 2 contained abnormally large con-

centrations of unesterified cholesterol (2 13 and

180 mg/dl, respectively) and phospholipids (550 and 450 mg/dl, respectively), whereas cholesteryl

esters and triacylglycerols were present in normal

amounts. Subject 3, who received a relatively small

dose of Intralipid, was not hyperlipidemic. After

each patient was infused for at least 3 weeks,

lecithin : cholesterol acyltransferase activity and

HDL concentrations were reduced by approxi-

mately 40%. Upon stopping the treatment in subjects 1 and

2, the concentrations of phospholipids and unesterified cholesterol decreased immediately and reached normal levels approximately 2 weeks later (Figs. 3A and 4A) *. After terminating total

-~ * Between days 5 and 8. the concentration of cholesteryl

esters increased sharply in subject 2 (Fig. 4A). This second

hypercholesterolemia resolved slowly over the ensuing

3 weeks. Density-gradient ultracentrifugation revealed that

this second hyperlipidemia was caused by increased lcvcls of LDL.

TABLE II

SERUM CHEMISTRIES IN CASE 3

During total parenteral refers to day 26 of the 39.day infusion

period. After total parenteral refers to I I weeks after the last

infusion. Values are mg/dl, except for lecithin : cholesterol

acyltransferase activity where results are given as meanirange

of duplicate determinations. Values for HDL were determined

from dextran-sulfate-soluble cholesteroi.

During After

total total

parenteral parenteral

nutrition nutrition

Cholesterol

Total 120 141

Unesterified 51 39

Esterified 69 102

Phospholipids 185 186

Triacylglycerols 59 62

HDL 134 294

Lecithin : cholesterol

acyltransferase activity 464% 32 631263

parenteral nutrition in subject 3, levels of

cholesteryl esters increased by SO%, whereas the level of unesterified cholesterol decreased by 25%

(Table 11). Lecithin: cholesterol acyltransferase ac-

tivity and HDL concentrations simultaneously

returned to normal values, following a lag period

ranging from 4 days in subject 1 (Fig. 3B) to 6 weeks in subject 2 (Fig. 4B). In all cases, as

illustrated in Fig. 5, HDL concentrations corre-

lated with lecithin: cholesterol acyltransferase activity.

To obtain complementary information of the

manner in which the lipids were distributed among

the serum lipoproteins, the post-infusion sera were

analyzed by isopycnic density-gradient ultracentrifugation. The results are shown in Figs. 6 and 7. Initially, the profiles were very unusual, but after approximately 2 weeks, they were similar to

those of most healthy donors (cf., Fig. 2, lower

tracings in Fig. 6, and thin-line tracings in Fig. 7). In conjunction with the elevated levels of serum phospholipids, a large, asymmetric peak was present over the density range between 1.020 and 1.034 g/ml (3-5 ml, arrow A in the top panel of Figs. 6 and 7). This component, which is absent from normal sera (e.g., Fig. 2), disappeared rapidly

in subjects 1 and 2 and was not detectable 10 or 11

days after the last infusion (Fig. 6 and upper panel

of Fig. 7). Moreover, although subject 3 was not hyperlipidemic, the abnormal peak was present

during the infusion period in this case as well

(Fig. 7, lower panel, arrow A).

The density-gradient profiles, in addition, con-

firmed the reduced levels of HDL (Figs. 6 and 7;

8-12 ml). The areas of the HDL peaks increased in parallel with the serum levels of dextran-sulfate-

soluble cholesterol (e.g., Fig. 3B) *. Despite the

reduced levels of HDL, however, the contours of the HDL peaks were normal in all cases. There-

fore, it is likely that the HDL remained normal

qualitatively.

The lipoproteins corresponding to the two major

peaks (Fig. 6 and 7, arrows A and B) were

examined by electron microscopy. Representative

micrographs are displayed in Fig. 8. The denser,

minor component (arrow B, Figs. 6 and ‘7) con-

sisted of homogeneous spherical particles with a

mean diameter of 249 k 1.6 A (mean t S.E. of 100

measurements for Subject 1) and appeared identical to normal LDL (Figs. 8B, D, and F). The

major component, which banded over the density range between 1.020 and 1.034 g/ml (arrow A,

Figs 6 and 7), contained a few particles that may

be LDL, but consisted mostly of vesicular complexes 300-600 A in diameter (Figs. 8A, C, and E).

These latter complexes tended to stack; when

viewed in cross-section they each had a hollow interior and a 50-A-thick limiting membrane (e.g.,

Fig. SA, arrow).

From 4% agarose columns the vesicular lipo-

protein eluted near the void volume, although it

penetrated the gel considerably. These characteris-

tics are consistent with particle sizes that range

from 300 to 6OOA. Only a small amount of

material, approximately 2% of the total mass, eluted in the position that corresponds to normal LDL.

* Case 3 was an exception to this general rule. Although

chemical analyses demonstrated the rise of serum cholestetyl

ester and HDL levels (Table II), the density-gradient peaks corresponding to HDL did not increase in size after total

parenteral nutrition was stopped. The reasons for this dis-

crepancy are uncertain; however, the ultracentrifugal

method measures protein, whereas the chemical method

measures cholesterol, and the latter is probably more rele-

vant in this case.

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Fig. 3. Concentrations of lipids and HDL. and activity of

lecithin: cholesterol acyltransferase (LCAT), in the serum of

subject I after Intralipid was infused for 6 weeks. A: Cholesterol

(esterified, 0 ; unesterified, 0; total, n ) and phospholipid (0)

levels at various times after the last infusion. B: Corresponding

lecithin : cholesterol acyltransferase activity (A) (mean and

range of duplicate determinations) and HDL levels. The solid

hexagons show the HDL levels determined from the dextran-

sulfate-soluble cholesterol. The open hexagons show the HDL

levels determined by integrating the peaks corresponding to

HDL in Fig. 6.

Chemically, the vesicles comprised 66% phospholipids, 28% cholesterol, all unesterified, and 5%

protein, as listed for subject 1 in Table III. Be-

cause of the relatively small quantities of protein,

we could not investigate thoroughly the poly-

peptide composition. However, as assessed by ra- dioimmunoassay, 74% of the protein mass was contributed by apolipoprotein B-probably due to minor contamination by LDL. Trace amounts (0.1%) of apolipoprotein A-II and no apolipoprotein A-I were detected. The remaining 26% of the protein was uncharacterized.

The chemical composition, hydrated density, and electron-microscopic appearance of these vesicles are similar to those reported for the

Fig. 4. Concentrations of lipids and HDL, and activity of

lecithin: cholesterol acyltransferase (LCAT; A) in the serum of

subject 2 after Intralipid was infused for 3 weeks. See text and

Fig. 3 for further explanation.

abnormal lipoprotein, known as lipoprotein X, or LpX, originally described in patients with cholestasis [35-371. Because of this similarity, we will

refer to the abnormal lipoprotein as ‘LpX’ or

LpX-like particle.

300 I 6 . ’

250 - n 8

y.O.IW~0.024)~ +75.6(‘12) . . PO.72

m . 1 400 500 800 700 800

LCAT ACTIVITY (dpmlhr)

Fig. 5. Scatter diagram which shows that the levels of HDL

correlate with the activity of lecithin: cholesterol acyltransferase

(LCAT) in the serum of subjects I (m), 2 (A) and 3 (0). The

points represent all of the unaveraged data in each of the

patients. Therefore, the correlation coefficient and standard

errors reflect the uncertainty in the analytical techniques and

assays as well as the inter- and intra-patient variability.

183

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Fig. 6. Isopycnic density-gradient patterns of the serum of

subject I. The samples were obtained on the indicated days

after the last infusion of Intralipid. Intralipid was also studied

by this method; it floated completely to the top of the gradient.

For further explanation, see text and Fig. 2.

Fig. 9 illustrates the kinetics by which the hyperlipidemia resolved in subjects 1 and 2. ‘Lipo- protein X’ was quantitated by integrating peaks A in the density-gradient profiles. As shown by the squares in Fig. 9, it was cleared from the circula-

a6

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a4

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Fig. 7. Isopycnic density-gradient profiles of serum from sub-

jects 2 and 3. In each case, the bold curves are the profiles of

serum obtained near the end of the infusion period. The

thin-line curves are the profiles of serum obtained 34 and 38

days (subjects 2 and 3, respectively) after the last infusion. Peak

C (case 2) corresponds to lipoproteins which, in electron micro-

graphs, are spherical, with a mean diameter of 239.7*4.3 A.

Similar studies of large numbers of normal sera (unpublished

data) have shown that such minor lipoproteins occur in at least

40% of the population. For further explanation, see text

tion in a monoexponential fashion, at the same

rate in both cases. Similarly, the excesses of phos-

pholipids and unesterified cholesterol in the serum

of subject 1, calculated from the data shown in

Fig. 3A, were both cleared with simple first-order

kinetics (Fig. 9; closed and open circles, respec-

tively) *. Within experimental error, the rate con-

stants for the clearance of ‘LpX’, phospholipids,

and unesterified cholesterol in subject l-0.331 *

0.024, 0.359 * 0.052, and 0.306 -C 0.028 days-‘, re-

spectively-are identical (Table III). Moreover,

from the ordinate-intercepts of these semilogarith-

mic plots, the serum concentrations in subject 1 of excess phospholipids and excess cholesterol were

642.3 i 1.4 and 322.1 + 1.2 mg/dl, respectively,

immediately after the last infusion (Table III).

Thus, the serum levels of these two lipids were

elevated in the same proportion as found in ‘LpX’

* It is not possible to analyze kinetically the excess serum

lipids in subject 2, since this patient developed a second.

independent hyperlipidemia while the ‘LpX’ was being

cleared (see footnote, p. 180).

Fig. 8. Electron micrographs (negative staining with 1% sodium phosphotungstate, pH 7) of lipoproteins from subJccta I (A, B), 2

(C, D). and 3 (E, F). A: Abnormal lipoprotein obtained from the density gradient shown in the top panel of Fig. 6 (arrow A). B:

Lipoprotein obtained from the same gradient in Fig. 6, at the position corresponding to arrow B. These are normal LDL particles. C

and E show the abnormal lipoproteins obtained from subjects 2 and 3. respectively; the corresponding density-gradient peaks arc

indicated by arrows A in Fig. 7. D and F show the normal LDL for subjects 2 and 3. respectively: these were also obtained from the

density gradients shown in Fig. 7 (arrows B). All micrographs arc at the same magnification. The bar in pane1 F reprcscnts 1000 A.

185

TABLE III

ABNORMALITIES OF SERUM LIPIDS IN CASE I

Values for abnormal lipoprotein were obtained from the pooled fractions of the density gradients (Fig. 6) that correspond to peak A.

Excess serum level is the concentration in serum, in excess of the normal values for this patient, at the end of the last infusion. The

values were determined by analyzing the data according to the equation: In X(t)= -X(+lnX(O), where X(f) is the excess

concentration in the serum at time t after the last infusion, and k is the first-order rate constant for clearance of X (k is expressed in

units of I/time). X(0) is the value given in the table. Results are expressed as meancS.E. r,,2 =0.693/k. where k is as defined above.

Abnormal lipoprotein Excess serum level Clearance half-life

percent composition (mg/dl) (t,,r) (days)

(by wt.)

Density-gradient peak A

Phospholipids

Unesterified cholesterol

Cholesteryl esters

Protein

Weight ratio

Phospholipids/unesterified cholesterol

_ 2.09”0.15 a

66.2 642.3 * I .4 1.93-co.33

28.1 322.1 * 1.2 2.21-0:23

0 0 _

5.7

2.36 to.24 1.99-cO.012 _

a In case 2, ‘LpX’ was cleared with a half-life of 2. I8 to.06 days.

(Table III). These results confirm that the hyper-

lipidemia resulted exclusively because ‘LpX’ accu-

mulated. This abnormal lipoprotein is removed

directly from the circulation as intact units, with

3-

0 2 4 6 6 IO 12 OI\ys

Fig. 9. Semi-logarithmic-plots showing monoexponential first-

order clearance of ‘LpX’ and excess lipids from the serum of

subjects 1 and 2. w, 0: Serum level of ‘LpX’ in subjects I and 2, respectively, determined by integrating peak A in the ap-

propriate density-gradient profiles (i.e., Fig. 6 in the case of

subject 1, upper panel of Fig. 7 and other data (not shown) in the case of subject 2. The peak areas were normalized so that

for both subjects the semi-logarithmic plots would have the

same ordinate-intercept); 0: excess phospholipids in subject 1:

0 : excess unesterified cholesterol in subject I.

monoexponential first-order kinetics and a half-life

of 2 days.

Single infusions of Intralipid in normal subjects.

Two normal subjects were studied during and immediately following a 4-h infusion of Intralipid.

Results of chemical analyses are displayed in Fig.

10. Serum lecithin : cholesterol acyltransferase ac-

tivity, HDL concentrations, and cholesteryl levels

remained constant, except for some minor dilution

effects during the 1st h. Venous blood pH was

measured in subject 5; it remained constant at 7.34

despite the large elevation of serum free-fatty-acid

levels (see below). Wright-stained smears of pe-

ripheral blood were normal at the beginning and

end of the experiment.

In both cases, levels of serum triacylglycerols

rose to their maximum values by the end of the

infusion and then rapidly declined thereafter (Fig. 10, top panels). In subject 5, triacylglycerol con-

centrations quickly reached a steady-state level of

300 mg/dl. After the infusion was terminated, this excess lipid was completely eliminated within 4 h,

with first-order kinetics and a half-life of 1 h. In subject 4, triacylglycerol concentrations rose at -a nearly constant rate to 600 mg/dl by the end of infusion, with no indication that a steady-state level had been attained. In this case, approximately 50% of the excess lipid was eliminated within 2 h after the end of the infusion, whereas

0 2 4 6 6 0 2 4 6 a

Fig. 10. Concentrations of lipids and HDL, and activity of lecithin : cholesterol acyltransferase (LCAT) in the serum of subjects 4 and

5. These subjects were each studied during and immediately after the intravenous infusion of 500 ml of Intralipid. Results for subject 4

are shown on the left; those for subject 5 are on the right, The upper panels show the levels of triacylglycerola, free fatty acids, and

cholesteryl esters. The lower panels show the corresponding lecithin: cholesterol acyltransferase activity (mean and range of duplicate

determinations) and the concentrations of HDL, phospholipids, and unesterified cholesterol. The infusion period is indicated by the

solid black bar along the abscissa.

the remainder cleared slowly, with a half-life greater than 18 h.

Serum levels of free fatty acids also increased to their maximum values by the end of the infusion, and subsequently became normal within 3 h (Fig. 10, upper panels). Because Intralipid does not contain free fatty acids, they were presumably produced by hydrolysis of the triacylglycerols.

The clearance kinetics of the triacylglycerols and free fatty acids are not directly relevant to this

article and will be described elsewhere (Ref. 38 and unpublished data).

As shown in the lower portion of Fig. 10, the serum concentration of phospholipids increased by

100 mg/dl in each subject. Approximately 50% of this excess lipid was eliminated during the following 4 h. Furthermore, despite the fact that Intrali- pid contains no sterols, in both subjects the level of serum cholesterol rose significantly during the infusion, and this elevation was sustained during

187

HOURS

Fig. I I. Graph which demonstrates that some of the phos-

pholipids of Intralipid remain in the circulation after the tri-

acylglycerols are cleared, and that Intralipid extracts unesteri-

fied cholesterol into serum. The thin curves show the ratio

[TG-TG,]/[PL-PL,], where TG and TG, are the concentra-

tions of triacylglycerols in the serum at time t and time 0.

respectively, and PL and PL, are the corresponding values for

phospholipids. If the triacylglycerols and phospholipids are

cleared together, then this ratio should remain constant at the

corresponding value for Intralipid (i.e., the value indicated by

the open hexagon). Because this ratio decreases during and

after the infusion, phospholipids must remain in the serum as

the triacylglycerols are cleared. The bold curves show the ratio

of [FC-FC,]/[PL-PL,], where FC and FC, are the concentra-

tions of unesterified cholesterol in the serum at time t and time

0, respectively. If Intralipid does not extact cholesterol from

tissues-if cholesterol were merely injected with the emul-

sion-then this ratio should remain constant at the negligible

ratio corresponding to Intralipid (i.e., the value indicated by the

solid hexagon). Because this ratio increases continuously during

and after the 4-h infusion, the Intralipid. or phospholipids

therefrom, must mobilize rapidly unesterified cholesterol into

the bloodstream. (The solid black bar indicates the infusion

period.)

the ensuing 4 h. Based on the calculated volumes of distribution [38], the total quantity of cholesterol transferred into the circulation was at least 700 mg in each case.

With the information described above and pre- sented in Fig. 10, two parameters were calculated. One of these, the ratio excess serum tri-

acylglycerols: excess serum phospholipids ([TG-

TG,]/[PL-PL,]), decreased continuously during

the experiment, to values below the tri-

acylglycerols: phospholipid ratio of Intralipid (Fig.

11). This result indicates that phospholipids re-

mained in the circulation as the triacylglycerols

were removed. The second parameter, the ratio

excess serum cholesterol : excess serum phospholi-

pids ([FC-FC,]/[PL-PL,]), increased progressively

from the negligible cholesterol: phospholipid ratio

of Intralipid to one approaching the corresponding

ratio in ‘LpX’ (0.42 by weight) (Fig. 11). Thus,

unesterified cholesterol, which is not present in

Intralipid, was mobilized rapidly into the circula-

tion from the surrounding tissues.

During the course of the experiment, the serum-

lipoprotein distributions exhibited only two sig- nificant changes, as illustrated in Fig. 12. First,

during the infusion, the HDL fraction shifted to

lower density. This effect paralleled the increase of

the serum free-fatty-acid levels, possibly indicating

that free fatty acids bound to HDL, and was

totally reversed within 4 h after the infusion was

ended. Second, a shoulder appeared on the low-

density side of the LDL peak (Fig. 12, arrow). In

both cases, this additional component, most likely

corresponding to ‘LpX’, became apparent 2 h after

the experiment was started, reached its maximum

level by the end of the infusion period, and was

still present 4 h later.

Studies in vitro. When HDL was extracted with

organic solvents, redispersed in aqueous media,

sonicated, and subjected to isopycnic density- gradient ultracentrifugation, phospholipids and

cholesterol were isolated in the region of the den-

sity gradient corresponding to peak A of Fig. 6.

However, with 300 mg of Intralipid, containing

approximately 30 mg of phospholipid-a quantity

IO-fold greater than the 3 mg of phospholipid that

gave rise to peak A in the top panel of Fig. 6 (the

corresponding serum sample contained 300

mg/lOO ml of excess phospholipids, as shown in

Fig. 3, and 1 ml of this serum was placed on the density gradient)-all of the lipid floated to the top of the gradient, and we did not observe a peak corresponding to phospholipid vesicles. These results prove that vesicles are not contained as such in the Intralipid preparation, but are generated as the triacylglycerol emulsion is metabolized.

188

0.6 -

3 I I I I 1 I I

!$ 0.6- S&ct 5 Q

0.6 -

‘Q w

VOLUME (ml)

Fig. 12. Representative isopycnic density-gradient patterns of

serum from subjects 4 (upper panel) and 5 (lower panel). The

thin curves are patterns for serum obtained before Intralipid

was infused. The bold curves are patterns for serum obtained at

the end of the infusion period (i.e.. at 4 h). The shoulder due to

‘LpX’, which first appeared after 2 h. is shown by the arrows.

In the lower panel, the dotted curve is the pattern of serum

obtained at R h (i.e., 4 h after the infusion was stopped); it

shows that the HDL peak shifted back to its pre-infusion

position. (Before the sample was applied to the density gradi-

ent, any unmetabolized Intralipid was centrifuged out of the

serum by spinning at 10000 rev./min for 30 min.)

Discussion

Parenteral nutrition with Intralipid, as we have

demonstrated in the present work, results in un-

usual metabolic effects. During long-term infu-

sions, a vesicular, cholesterol-phospholipid lipo-

protein (‘LpX’) is produced, and serum lecithin : cholesterol acyltransferase activity and

HDL concentrations decrease jointly. With physiological doses of the emulsion (i.e., 100 g/day), large quantities of ‘LpX’ accumulate, causing hyperlipidemia.

The same type of hyperlipidemia occurs most characteristically in individuals with cholestasis [35-37,39,40], in which case it is believed [41] that bile, after overflowing into the bloodstream, forms LpX. But none of the subjects of this study had cholestasis; therefore, a different mechanism must explain why the vesicular lipoprotein accumulated.

In this context, the following observations are

relevant: (a) Lipid levels increased immediately

after long-term infusions were begun (Fig. 1) and

decreased immediately after they were discon-

tinued (Fig. 9), and (b) ‘LpX’ was generated dur-

ing single infusions (Fig. 12). These facts demon-

strate that ‘LpX’ is produced directly from In- tralipid.

Some of the steps that effect this precursor-

product relationship were elucidated by examining

the manner in which serum-lipid levels vary during

single infusions. In each of the two normal sub-

jects, the injected phospholipids remained in the

circulation while the triacylglycerols were cleared

(Fig. 11). Moreover, even though Intralipid con-

tains no sterols, the amount of cholesterol in the

serum increased during the 8-h experiments. The

lipid levels varied in such a manner that the ratio

of excess cholesterol : excess phospholipids rose

progressively (Fig. 11). These observations suggest that when the core of the Intralipid particle is

depleted, the phospholipids form vesicles, and these

vesicles rapidly extract cholesterol from peripheral

tissues until they attain an equimolar ratio of

cholesterol : lecithin. Elevation of the serum-

cholesterol level is consistent with the numerous

studies which have demonstrated that cholesterol is transferred readily in vitro from cell membranes

to dispersions of pure phospholipids [42,43].

The results from subjects 1 and 2 have also shown that the ‘LpX’ particle is removed from the

circulation as a unit, with first-order kinetics and a

half-life of approximately 2 days. The monoex-

ponential decay suggests that a single tissue is

responsible for eliminating ‘LpX’ from the blood.

Although that tissue has not been identified di-

rectly, lipofuschin, a pigment associated with the

degradation of excess phospholipids, has been re-

ported to accumulate in the hepatic parenchyma after infusions of Intralipid and other fat emulsions [44-471. Thus, the liver may play an important role in this process.

In addition to the rate of infusion of Intralipid and the slow clearance of ‘LpX’, other factors determine the extent to which lipid accumulate. If ‘LpX’ is distributed in the same volume as In- tralipid (8.17 * 0.7% of body weight, Ref. 38), and if all of the infused phospholipids remain in the circulation as ‘LpX’, then the expected steady-state

Fig. 13. Suggested scheme for the metabolism of Intralipid. Shortly after Intralipid enters the circulation, the triacylglycerols are

hydrolyzed to free fatty acids and glycerol, which are in turn cleared rapidly (cf., Fig. I I). Between 60 and 90% of the phospholipids

are also removed from the plasma within several hours; the pathway for this process is at unknown present. The remainder of the

phospholipids form single-bilayer vesicles, which rapidly extract unesterified cholesterol from tissues until the molar ratio of

cholesterol : phospholipids reaches I : I These mixed vesicles, ‘LpX’ are cleared slowly from the circulation with monoexponential

first-order kinetics-the half-life is approximately 2 days. In particular, these mixed vesicles are not converted to other lipoproteins.

Evidence from the literature suggests that the hepatocyte may clear the ‘LpX’ from the blood, although there is no direct evidence to

support this possibility. For further explanation, see text. LCAT, lecithin : cholesterol acyltransferase: triglycerides, triacylglycerols.

level of excess phospholipids, PL,,, can be calcu-

lated with the formula [38,48]:

PL,, =gg, d

where Q is the rate of infusion of phospholipids

(in mg/day), [l/z is the clearance half-life of ‘LpX’

(in days), Vd is the volume of distribution (in dl), and PL,, is expressed in mg/dl. Although the ‘LpX’ was cleared at the same rate in subjects 1 and 2, the steady-state phospholipid levels were not proportional to the rate at which Intralipid was administered (Table1 and Figs. 3A and 4A), even after correcting for differences in V,. Morever, the serum-lipid levels were, in all cases, elevated by

less than 50% of the amount that would be ex-

pected on the basis of Eqn. 1. These observations,

together with the simple first-order clearance of all

of the excess lipids (Fig. 9), indicate that a signifi-

cant fraction of the phospholipids are cleared from

the circulation with a half-life of approximately

5 h. Using Eqn. 1 and the data given in Tables I, II

and III, the fraction of rapidly cleared phospholi-

pids was estimated to be 52, 80 and 85% in sub-

jects 1, 2 and 3, respectively. During the single infusions as well, between 40 and 60% of the excess phospholipids was cleared at a rate com- parable to, although slower than, that of the triacylglycerols (Fig. 10, lower panels). Thus, the degree of hyperlipidemia depends on the rate at which Intralipid is administered, on the slow rate

190

at which ‘LpX’ is cleared, and also on the fraction

of infused phospholipids that is transformed into

‘LpX’.

At present, we cannot draw firm conclusions

about the factors that determine the fraction of

slowly cleared phospholipids. Future studies must

elucidate whether that fraction as well as the half-

life of ‘LpX’ vary-for example, increase-during

the course of long-term parenteral nutrition, or

whether they are predetermined and constant in

each individual; these are the only alternatives

with which the current data are consistent. In the

first case, hyperlipidemia should develop during

the later stages of total parenteral nutrition; in the

latter case, hyperlipidemia, if it occurs, should

begin to develop immediately. The various possi-

bilities can only be resolved by studying serum-

lipid levels during the course of long-term infusion

periods. The data shown in Fig. 1 favor the latter

alternative, although more precise measurements

should be made in a larger number of subjects.

On the basis of the above-mentioned evidence,

the metabolism of Intralipid can be represented

schematically, as shown in Fig. 13. A principal

feature of this scheme is that ‘LpX’ does not serve

as a precursor for other lipoproteins. In subject 1,

for example, the excess phospholipids and

cholesterol were removed from the serum at the

same rate as the ‘LpX’ (Fig. 9) thereby suggesting

that net transfer of lipids, from ‘LpX’ to other

lipoproteins, did not occur. Moreover, as ‘LpX’ (peak A, Fig. 6) was cleared, additional peaks did

not appear in the density-gradient profiles, further

demonstrating that new complexes were not formed. Although, in subject 1, the concentrations

of HDL increased as ‘LpX’ was removed (Fig. 6)

these two processes did not occur in parallel

(Fig. 3). And in subject 2, the ‘LpX’ was also

cleared with a half-life of 2 days, but the HDL

levels remained low for at least 3 weeks after all of the abnormal lipoprotein was eliminated from the serum (Fig. 4). Thus, the HDL is resynthesized independently of the clearance of ‘LpX’.

The levels of HDL and the activity of

lecithin : cholesterol acyltransferase decline only during long-term infusions. Moreover, these quantities are not affected by incubating Intralipid with serum under various conditions in vitro (Ref. 38 and unpublished data). These findings demon-

strate that the enzyme is not directly inhibited,

and the lipoprotein particles are not disrupted

(e.g., by losing apolipoprotein A-I), by either In-

tralipid or its metabolites. Most likely, HDL and

lecithin : cholesterol acyltransferase are synthe-

sized, during total parenteral nutrition, more slowly

than usual. This explanation is consistent with the

notion that whereas chylomicrons donate lipids

and proteins to HDL, Intralipid does not. How-

ever, we cannot presently rule out other long-term effects on the small intestine and liver-organs

which are also involved in synthesizing HDL and

lecithin : cholesterol acyltransferase [ 12,13,49,50].

It is important to emphasize that the failure of

‘LpX’ to be converted to other lipoproteins, the slow rate at which ‘LpX’ was cleared in subjects 1

and 2, and the consequent hyperlipidemia in subjects 1 and 2, cannot reasonably be ascribed to the

modest reduction of lecithin : cholesterol

acyltransferase activity. In each of the three pa-

tients the activity was not sufficiently low to cause

any of the classical disturbances-such as virtual

absence of cholesteryl esters from serum, or accu-

mulation of large amounts of grossly abnormal,

discoidal HDL-that invariably ensue when the

enzyme activity falls below acceptable physiologi-

cal levels [51]. In subject 3, moreover,

lecithin : cholesterol acyltransferase activity was

moderately reduced during total parenteral nutrition (Table II), and hyperlipidemia did not occur.

Also, as demonstrated by Wengeler and Seidel [52] and Patsch [53], LpX (from patients with cholesta-

sis) is a poor substrate for lecithin: cholesterol

acyltransferase. It therefore seems reasonable to

conclude that the slow clearance of ‘LpX’ reflects

the properties of the vesicles themselves and is not

related to extrinsic factors such as the reduced

levels of HDL or lowered activity of

lecithin: cholesterol acyltransferase.

Because ‘LpX’ accumulates as an abnormal product when Intralipid is metabolized (Figs. 6 and 7), vesicles could not be similarly released from chylomicrons under physiological conditions. On the basis of other evidence, it has been suggested [54,55] that single-bilayer vesicles, such as ‘LpX’, may be one intermediate through which phospholipids are transferred post-prandially from chylomicrons to HDL: In vitro, single-bilayer vesicles can serve directly as substrates for

lecithin: cholesterol acyltransferase [56-581, or they

can first be converted to small protein-

phospholipid discs after acquiring apolipoprotein

A-l from HDL [59]. These processes can occur,

however, only if the molar ratio of choles-

terol : phospholipid is not larger than 1 : 2 [57,60].

‘LpX’ cannot undergo such physico-chemical

transformations and yield other lipoproteins,

probably because the initial phospholipid vesicles

rapidly acquire, in vivo, equimolar amounts of

unesterified cholesterol from a virtually unlimited

tissue pool (Figs. 11 and 13). This explanation is

supported by recent studies [61,62] on the fate in

vivo of cholesterol-containing vesicles. Future research efforts in this clinically and

theoretically important field must be directed to-

ward four general goals: establishing the precise

physical form in which surface components are

transferred from chylomicrons to HDL; finding

the long-term cause(s) for the joint reduction of

HDL levels and lecithin: cholesterol acyltransferase

activity; determining the kinetic parameters of the

phospholipids during the course of long-term total

parenteral nutrition with Intralipid; and preparing

other triacylglycerol emulsions in which the

surfactant is metabolizable rapidly and which could

therefore be administered without causing hyper-

lipidemia. The studies described in this article

have illustrated that excess surface is produced during the intravascular metabolism of at least

some lipid-laden particles. And the disposition of

those moieties, which solubilize and transport neu- tral lipids, is one important function fulfilled by

the intricate system of serum lipoproteins.

Acknowledgments

I wish to express my sincere appreciation to the

many colleagues who made this work possible.

Drs. Patricia McElroy and Joseph B. Kirsner sup-

plied most of the blood samples from the patients.

Mr. Roger Franz, Mrs. Rita Grammas and Mr.

Lance Lusk generously provided expert technical assistance in determining the serum lipid levels. Dr. Jiwhey Chung and Mrs. Darla Abano analyzed numerous samples for lecithin : cholesterol acyltransferase activity. Mrs. Judy Swanson, R.N., as- sisted with the single infusions. I thank Drs. Ron Goldberg and Arthur Rubenstein for their interest

191

in this work and for carrying out radioimmunoas-

says on ‘LpX’. Drs. Ferenc J. KCzdy, Jayme Borensztajn, Godfrey S. Getz, Robert Josephs,

Marvin W. Makinen, Irwin H. Rosenberg, Gunther

M. Fless and Mrs. Celina Edelstein offered many

thoughtful comments and suggestions on various

versions of the manuscript. In addition, Mrs. Rose

Scott, Miss Sharron Riley, Mrs. Delores Walker

and Mrs. Julia Crawford provided excellent secre-

tarial assistance. Finally, I would like to thank the

patients as well as the normal subjects for their

genuine enthusiasm, limitless cooperation, and

profound interest in this work. These studies were

supported by Grants HL 18577 and HL 15026

from the United States Public Health Service, and

partially by Grant 78-09815 from the National

Science Foundation. The author is recipient of Medical Scientist Training Program Grant 5 T32

GM07281, from the National Institute of General

Medical Sciences, United States Public Health

Service.

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