Erucic Acid is Differentially Taken up and Metabolized in Rat Liver and Heart

ORIGINAL ARTICLE

Erucic Acid is Differentially Taken up and Metabolizedin Rat Liver and Heart

Cameron C. Murphy Æ Eric J. Murphy ÆMikhail Y. Golovko

Received: 24 November 2007 / Accepted: 26 February 2008 / Published online: 19 March 2008

� AOCS 2008

Abstract Because X-linked adrenoleukodystrophy is

treated using erucic acid (22:1n-9), we assessed its

metabolism in rat liver and heart following infusion of

[14-14C]22:1n-9 (170 Ci/kg) under steady-state-like con-

ditions. In liver, 2.3-fold more tracer was taken up as

compared to heart, accounted entirely by increased incor-

poration into the organic fraction (4.2-fold). The amount of

tracer entering the aqueous fraction, which represents

b-oxidation, was not different between groups; however a

significantly elevated proportion of tracer was in the heart

aqueous fraction. In both tissues, 76% of the radioactivity

found in the organic fraction was esterified in neutral

lipids, while only about 10% was found esterified into

phospholipids. In liver, 56% of lipid radioactivity was

found in cholesteryl esters, whereas in heart 64% was

found in triacylglycerols. Because 22:1n-9 can be chain

shortened, we assessed tracer metabolism using phenacyl

fatty acid derivatives esterified from saponified esterified

neutral lipid (triacylglycerol/cholesteryl ester) and phos-

pholipid fractions. In heart esterified neutral lipids, 75% of

tracer was recovered as 22:1n-9 and only 10% as oleic acid

(18:1n-9), while in liver only 25% of the tracer was

recovered as 22:1n-9, while 50% was found as stearic acid

(18:0) and 10% as 18:1n-9. In liver and heart phospho-

lipids, the tracer was distributed amongst the n-9 fatty acid

family. Thus, 22:1n-9 under went tissue selective metab-

olism, with conversion to 18:0 the dominant pathway in the

liver presumably for export in the neutral lipids, while in

heart it was found primarily as 22:1n-9 in neutral lipids and

used for b-oxidation.

Keywords Erucic acid � Fatty acid uptake � Lipid �Phospholipid � Cholesteryl ester � Triacylglycerol �Fatty acid metabolism

Abbreviations

BBB Blood brain barrier

CE Cholesteryl esters

CerPCho Sphingomyelin

ChoGpl Choline glycerophospholipids

CNS Central nervous system

EtnGpl Ethanolamine glycerophospholipids

FFA Free fatty acids

PL Phospholipids

PtdIns Phosphatidylinositol

PtdSer Phosphatidylserine

SFA Saturated fatty acids

TAG Triacylglycerols

VLCFA Very-long-chain saturated fatty acids

X-ALD X-linked adrenoleukodystrophy

16:0 Palmitic acid

18:0 Stearic acid

18:1n-9 Oleic acid

20:4n-6 Arachidonic acid

22:1n-9 Erucic acid

C. C. Murphy � E. J. Murphy (&) � M. Y. Golovko

Department of Pharmacology,

Physiology, and Therapeutics,

School of Medicine and Health Sciences,

University of North Dakota,

501 N. Columbia Rd, Grand Forks,

ND 58202-9037, USA

e-mail: [email protected]

E. J. Murphy

Department of Chemistry, University of North Dakota,

Grand Forks, ND 58202-9037, USA

123

Lipids (2008) 43:391–400

DOI 10.1007/s11745-008-3168-3

Introduction

X-linked adrenoleukodystrophy (X-ALD) is characterized

by elevated very long chain saturated fatty acids (VLCFA)

in plasma [1, 2] and in tissue [2, 3]. The clinical manifes-

tations of X-ALD include adrenal insufficiency and rapid

demyelination in the central nervous system (CNS) [4].

Accompanying this demyelination is a rise in brain cho-

lesteryl ester mass [5, 6], which is extensively esterified

with 26:0 [2]. Lorenzo’s oil (LO) is a dietary therapy with

restricted saturated fatty acid ingestion in combination

with ingestion of a triacylglycerol (TAG) form of erucic

acid (22:1n-9), which effectively reduces plasma levels of

VLCFA found in X-ALD patients [7–9]. Although the use of

LO is controversial because early studies demonstrated the

absence of 22:1n-9 from the brain of treated patients [7, 10,

11] and because of its limited effectiveness in ameliorating

the progression of CNS demyelination in patients with

advanced X-ALD [7–9]. Recent evidence indicates that it

has a strong potential in limiting the onset of CNS demye-

lination following early intervention in X-ALD patients

[12–14]. The lack of elevated 22:1n-9 in brains from treated

X-ALD patients suggests limited uptake into the CNS,

perhaps as a result of poor movement of 22:1n-9 across the

blood brain barrier (BBB). Recent work from our laboratory

demonstrates that 22:1n-9 crosses the BBB and is found

esterified into brain esterified neutral lipid (NL) and phos-

pholipid (PL) pools as primarily 18:1n-9 due to the rapid

chain shortening of 22:1n-9 [15]. The entry and metabolism

of 22:1n-9 in the brain supports the results of recent clinical

trials demonstrating that LO reduces clinical symptoms of

X-ALD when administered early in life [12–14].

Although we have studied the metabolism of 22:1n-9 in

the brain [15], previous studies have focused on its

metabolism in the heart due to its reported impact on heart

physiology [16, 17]. In cultured cells, 22:1n-9 is rapidly

esterified into TAG and to a lesser extent into PL pools

[18–21]. Similar results are reported in isolated organs [22–

25], and in intact animals [26–28]. Although 22:1n-9 is

poorly oxidized to CO2 [29, 30], it is quickly converted

into oleic acid (18:1n-9) in vivo and in isolated tissues and

cultured cells [15, 18, 19, 24, 26–28, 31–35]. This con-

version is presumably through peroxisome localized

b-oxidation [19, 33, 36], although a possible mitochondrial

pathway for 22:1n-9 oxidation can not be excluded [26,

27]. Whole liver and liver cells are capable of converting

22:1n-9 into 18:1n-9 more efficiently than other organs and

cell types [18, 28–32], leading to the conclusion that liver

plays a central role in 22:1n-9 utilization with other organs

utilizing 18:1n-9 exported from liver after 22:1n-9 chain

shortening [33].

However, in most of these previous studies examining

22:1n-9 metabolism in vivo, the animals were fed high oil

diets rich in 22:1n-9. Under these experimental conditions,

peroxisomal b-oxidation is increased, thus increasing the

conversion of 22:1n-9 to 18:1n-9 in cultured heart and liver

cells [19, 33, 36, 37]. In contrast, constant exposure of the

brain to 22:1n-9 alters the pools in which it is found

esterified, with minimal impact on 22:1n-9 chain shorten-

ing to 18:1n-9 [15]. Pulse infusion studies using labeled

22:1n-9 in vivo also demonstrate an increase in chain

shortening even in animals fed with regular diets [26–28].

In these studies, the initial concentration of 22:1n-9 is

much higher than its normal physiological levels found in

plasma. These conditions would distort 22:1n-9 metabo-

lism because 22:1n-9 esterification into PL or TAG pools is

concentration dependent [22]. Similar concentration

dependence is observed for 20:4n-9 targeting to heart lipid

pools, where under non-physiologically high concentra-

tions it is incorporated into TAG pools [38–40], whereas

under conditions using a steady-state tracer infusion,

20:4n-6 is predominantly targeted to PL pools [41]. This

demonstrates the utility of the method used herein to study

fatty acid metabolism under conditions in which there is

minimal alteration of the plasma unesterified fatty acid

concentration during infusion of the radiotracer. Thus, high

superphysiological concentrations (mM range) of labeled

22:1n-9 used in experiments with perfused organs and

cultured cells would significantly alter 22:1n-9 metabolism.

Until now, no studies have been done to study 22:1n-9

metabolism in heart and liver under steady-state conditions

using low 22:1n-9 concentrations that do not perturb

plasma concentrations found in intact animals fed with a

regular diet.

Previously, we have demonstrated that under steady-

state-like conditions 7% of infused [14-14C]22:1n-9 is

recovered as [14C]18:0 from plasma [15], suggesting

possible contamination of the tracer. Although the

chemical purity of the tracer was [92% pure by GLC, no

18:0 was observed [15]. This observation led us to pro-

pose that 22:1n-9 may be rapidly converted into saturated

fatty acids (SFA) in liver and then exported into plasma.

Despite the fact the 22:1n-9 conversion into monounsatu-

rated fatty acids has been extensively studied, only one

study addressed the question of 22:1n-9 conversion into

SFA, but this study was done using cultured heart cells

[21], demonstrating a need to more fully understand the

dynamics of 22:1n-9 metabolism in liver and heart of

intact animals.

In the present study, we extend our previous work using

samples from our previous study [15] to examine the

uptake and incorporation of [14-14C]22:1n-9 into liver and

heart lipids as well as its metabolism under steady-state-

like conditions using a well established infusion protocol

where normal plasma fatty acid concentrations are unal-

tered by tracer infusion [42–48]. We demonstrate that 2.3-

392 Lipids (2008) 43:391–400

123

fold more [14-14C]22:1n-9 was taken up into liver as

compared to heart and that the bulk of [14-14C]22:1n-9

radioactivity was incorporated into the NL, mainly into

heart TAG and into liver cholesteryl esters (CE) pools,

while significantly lesser amounts were esterified into liver

and heart PL (\10%). In NL fraction, the bulk of the

radioactivity in heart was recovered as 22:1n-9, while in

liver NL the bulk of the radioactivity was recovered as

18:0. These data indicate that under steady-state-like con-

ditions, the predominant pathway for 22:1n-9 metabolism

in liver is conversion into 18:0 and subsequent incorpora-

tion into NL pool, mainly into CE, whereas in the heart, the

bulk of 22:1n-9 is incorporated into the NL pool as 22:1n-9

and then presumably used for b-oxidation.

Materials and Methods

Animals

Male Sprague-Dawley rats (150–200 g) were obtained

from Charles River Laboratories (St. Louis, MO) and

maintained on standard laboratory rat chow diet (Purina

rodent chow) and water ad libitum. This study was con-

ducted in accordance with the National Institutes of Health

Guidelines for the Care and Use of Laboratory Animals

(NIH Publication 80-23) under an animal protocol

approved by the IACUC at the University of North Dakota

(Protocol 0110-1).

Tracer Preparation

Tracer preparation was performed as previously described

[15]. Briefly, the custom synthesized [14-14C] 22:1n-9

(specific activity 53 mCi/mmol, Moravek Biochemical,

Brea, CA) was solubilized in 5 mM Hepes (pH 7.4) buffer

containing ‘‘essentially fatty acid free’’ bovine serum

albumin (50 mg/mL; Sigma Chemical Co, St. Louis, MO).

Solubilization was facilitated using a bath sonicator for

45 min at 45 �C. Tracer was infused at a dose of 170 lCi/

kg [15, 49]. Previously we determined tracer purity to be

[92% by GLC, based upon total peak area [15]. Because

we reported 6.5% of the tracer in plasma was found as

[14C]18:0, we assessed radiochemical purity of the

[14-14C]22:1n-9 using HPLC analysis and found that it the

radiochemical purity of the tracer was [99%.

Rat Surgery and Tracer Infusion

Rat surgery and tracer infusion was performed as previ-

ously described [15]. Briefly, rats were anesthetized with

halothane (1–3%) and PE-50 catheters inserted into the

femoral artery and vein. Using an infusion pump (BS-8000,

Braintree Scientific, Inc., Braintree, MA), awake rats were

infused 3–4 h following recovery from anesthesia with

170 lCi/kg of [14-14C]22:1n-9 via the femoral vein over

10 min at a constant rate of 0.4 mL/min to achieve steady-

state-like plasma radioactivity. Prior to and during the

infusion, arterial blood samples (200 lL) were taken to

determine plasma radioactivity (see Golovko et al. [15]).

Following infusion, the rats were killed using pentobarbital

(100 mg/kg, intravenous). Liver and heart were rapidly

removed and frozen in liquid nitrogen. The tissue was

stored at -80 �C until used.

Blood Extraction

To account for the contribution of residual blood to tissue

radioactivity, whole blood was extracted using a two-phase

extraction [50]. Residual blood in heart and liver was

estimated to be 24 and 17%, respectively based upon

literature values [51, 52]. Previously using this infusion

protocol, we demonstrated that 95% radioactivity in blood

is found as free fatty acid, whereas 2.5% is found in

phospholipids, and 2.5% is found in triacylglycerols [41,

53].

Tissue Lipid Extraction

To determine tracer incorporation into liver and heart

individual lipid compartments, lipids were extracted using

a two-phase extraction procedure [50]. Briefly, frozen tis-

sues were pulverized under liquid nitrogen temperatures

into a fine homogeneous powder and lipids from the tissue

powders were extracted using chloroform/methanol (2:1 by

vol) in a Tenbroeck tissue homogenizer. The tissue mass in

grams was multiplied by a correction factor of 1.28 to

convert it to an equivalent value in mL [54], which rep-

resents one volume. The tissue was homogenized in 17

volumes of chloroform-methanol (2:1 by vol), the solvent

removed and the homogenizer rinsed with 3 volumes of

chloroform/methanol (2:1 by vol). This rinse was added to

the original sample and 4 volumes of 0.9% KCl solution

were added to the combined lipid extract. The extract was

mixed by vortexing and the phases separated by centrifu-

gation. The upper aqueous phase and proteinaceous

interphase were transferred to a 20 mL scintillation vial for

counting. The lower organic phase was washed twice with

2 mL of theoretical upper phase (chloroform/methanol/

water, 3:48:47 by vol) and the phases again separated by

centrifugation. Each wash was removed and added to the

previously removed upper phase. The lower organic phase

was then dried under nitrogen and then dissolved in hex-

ane-2-propanol (3:2 by vol) containing 5.5% water.

To analyze tracer metabolism to other fatty acids and its

subsequent distribution within esterified fatty acids found

Lipids (2008) 43:391–400 393

123

in NL and PL fractions, lipids from liver and heart tissue

(200 mg, wet weight) were extracted using single phase

extraction procedure [55, 56]. Briefly, tissue powder was

homogenized in 1.8 mL hexane/2-propanol (3:2 by vol) per

0.1 g tissue and quantitatively transferred to a test tube.

The homogenizer was rinsed with an additional 3 mL of

hexane/2-propanol (3:2 by vol) and added to the original

extract. The protein-containing residue was pelleted by

centrifugation and the lipid bearing organic fraction was

removed and stored under a N2 atmosphere at -80 �C until

it was used to assess tracer elongation and chain

shortening.

Thin Layer Chromatography

Tissue PL were separated by thin layer chromatography

(TLC) on heat-activated Whatman silica gel-60 plates

(20 cm 9 20 cm, 250 lm) using two different solvent

systems. The first system used a chloroform/methanol/

acetic acid/water (60:30:3:1 by vol) solvent system that

resolves cardiolipin (Ptd2Gro), phosphatidic acid (PtdOH),

and ethanolamine glycerophospholipids (EtnGpl) [53].

Because this first system does not separate phosphatidyl-

serine (PtdSer) and phosphatidylinositol (PtdIns), these

two phospholipids were separated using a chloroform/

methanol/acetic acid/water (50:37.5:3.5:2 by vol) solvent

system [57]. Phospholipids were visualized using iodine

vapor.

Heart and liver NL were separated by TLC on heat-

activated Whatman silica gel-60 plates (20 cm 9 20 cm,

250 lm) and developed in petroleum ether/diethyl ether/

acetic acid (70:30:1.3 by vol) solvent system that resolves

cholesterol, cholesteryl esters, diacylglycerols, nonesteri-

fied fatty acids, and triacylglycerols [58].

All lipid fractions were identified using authentic stan-

dards (Doosan–Serdary, Englewood Cliffs, NJ, and

NuChek Prep, Elysian, MN). Bands corresponding to the

appropriate lipid fractions were removed from the TLC

plate by scrapping and transferred into 20 mL liquid

scintillation vials and 0.5 mL of water was added followed

by 10 mL of Scintiverse BD (Fisher Scientific, Pittsburgh,

PA). Radioactivity was quantified by liquid scintillation

counting using a Beckman LS 5000 CE liquid scintillation

counter.

Analysis of Tracer Conversion in Esterified NL and PL

Fractions

To determine the elongation and chain shortening of the

infused tracer by heart and liver, extracted heart and liver

lipids were separated into PL and NL fractions using silicic

acid column chromatography (Clarkson Chemical Co.,

Inc., Williamsport, PA) [59]. To remove nonesterified fatty

acids from the NL fraction, it was then separated by TLC

as described above using the petroleum ether/diethyl ether/

acetic acid (70:30:1.3 by vol) solvent system, except that

lipid fractions were visualized using 6-p-toluidino-2-

naphthalenesulfonic acid [60]. TAG and CE were collected

by scraping the bands off the TLC plate, and were then

extracted off the silica by adding 1.5 mL of water followed

by three successive washes of the aqueous phase with 3 mL

of hexane/2-propanol (3:2 by vol). For each wash, the

elution mixture was vigorously mixed for 1 min by vor-

texing and the two phases were separated by centrifugation.

The upper lipid-containing phase was aspirated and saved

for fatty acid analysis and subsequent washes were added

to this original wash.

Fatty acids from PL fraction and from the combined

TAG and CE fractions were separated and quantified after

conversion to their corresponding phenacyl esters [61,

62]. Briefly, solvent containing the PL and TAG/CE lipid

fractions was removed under a stream of nitrogen and the

lipids subjected to saponification at 100 �C for 30 min in

2% KOH in ethanol, which was then acidified with HCl.

The released fatty acids were extracted with hexane, the

hexane evaporated using nitrogen, and phenacyl esters

were then prepared by the addition of acetone containing

2-bromoacetophenone (10 mg/mL) (Sigma) and triethyl-

amine (10 mg/mL) (Sigma) followed by heating at

100 �C for 5 min followed by addition of acetic acid

(2 mg/mL) and then the samples were heated for an

additional 5 min.

Individual fatty acid phenacyl esters were separated

by high performance liquid chromatography (HPLC) on

a C-18(2) Luna column (Phenomenex, Torrance, CA) as

previously described [15] with a modified elution program

that allows resolution of VLCFA. Fatty acids were identi-

fied using fatty acid standards (NuChek Prep, Elysian, MN)

converted to phenacyl esters. The HPLC system was con-

trolled by a Beckman 127 solvent module (Fullerton, CA).

The eluent was monitored at 242 nm using a Beckman 166

ultraviolet/visible light detector. The gradient system used

was composed of water (solvent A) and acetonitrile (sol-

vent B). Column temperature was maintained at 37 �C. The

flow rate was 1 mL/min, and the initial percentage of B

was 80%. The percentage of B was increased to 90% over

1 min at 350 min, increased to 96% over 1 min. at

490 min, and returned to 80% over 1 min at 550 min.

Eluent from HPLC containing individual fatty acid

phenacyl esters was concentrated by reducing the volume

to 1 mL under a stream of nitrogen and 15 mL of Scinti-

verse BD was added and the samples were mixed by

vortexing. After mixing, all samples were allowed to sta-

bilize for at least 1 h before the radioactivity was quantified

by liquid scintillation counting using a Beckman LS 6500

liquid scintillation counter.

394 Lipids (2008) 43:391–400

123

Statistical Analysis

Statistical analysis was done using InStat (GraphPad, San

Diego, CA) and a two-tailed, unpaired Student’s t test.

Values were considered statistically significant when

p \ 0.05.

Results

Plasma Curve

The plasma curve for these awake, male rats infused with

[14-14C]22:1n-9 (i.v.) was previously published [15] and

indicates that the radiotracer was near steady-state-like

conditions and that the plasma concentration of unesterified

(free) 22:1n-9 is 6 lM.

Uptake and Distribution of 22:1n-9 in Liver and Heart

In liver and heart, the amount of tracer, expressed as nCi/g

ww, found in the total extract, in the organic fraction (lipid

containing), and in the aqueous fraction, which represents

products of b-oxidation [41, 63, 64] was determined

(Fig. 1). Under steady-state-like conditions, significantly

more tracer was taken up by the liver (2.3-fold) as com-

pared to the heart. When the unilateral incorporation

coefficient for liver and heart uptake was calculated [41–

45], which normalizes the amount of tracer taken up into a

given tissue by the amount of tracer infused into the rat, the

liver had taken up significantly more tracer (2.3-fold) as

compared to heart (data not shown). The percentage of the

infused tracer removed by the heart was 0.82 ± 0.33%,

while that removed by the liver was 1.63 ± 0.45%. Again,

using this calculation, the liver removed 2.0-fold more

tracer than did heart. The increase in liver total uptake was

accounted for by a significantly higher incorporation of

tracer (4.2-fold) into the liver organic fraction as compared

with heart. However, no differences were found between

liver and heart in amount of tracer found in the aqueous

fraction. However, in heart a significantly greater propor-

tion of radioactivity was found in the aqueous fraction

(63.2 ± 5.8%) as compared to liver (22.9 ± 2.9%), indi-

cating that in the heart 2.8-fold more tracer underwent

b-oxidation relative to the liver.

Distribution of Tracer in Liver and Heart Lipid Pools

To determine into which lipid compartments the tracer

entered in liver and heart, the incorporation of the tracer

into individual lipid fractions was determined (Fig. 2).

Significantly more tracer (nCi/g ww) went into liver total

PL, unesterified (free) fatty acids, and CE as compared to

heart (Fig. 2, top panel). However, the amount of tracer

entering into the TAG pool was not different between

groups. These values illustrate two important points. First,

that very little tracer entered into the phospholipid pools.

Total Organic Aqueous0

300

600

900

1200 Liver

Heart

*

*

nCi/g

ww

Fig. 1 Uptake of [14-14C]22:1n-9 by heart and liver values are

expressed as mean ± SD, where n = 5 heart and n = 6 for liver. The

* indicates statistical significance from liver, p \ 0.05

0

20

40

60

80

*

*

*

*

% O

rgan

ic F

ract

ion

Rad

ioac

tivity

PL FFA TAG CE

PL FFA TAG CE

0

100

200

300

400

500

***

Liver

Heart

nCi/g

ww

Fig. 2 Distribution of infused [14-14C]22: 1n-9 amongst different

heart and liver lipid fractions values are expressed as mean ± SD,

where n = 5 heart and n = 6 for liver. The * indicates statistical

significance from liver, p \ 0.05. In the top panel, values are

expressed as nCi/g ww, while in the bottom panel; these values are

expressed as percent of total organic fraction radioactivity. PL:

phospholipids; FFA: free fatty acids; TAG: triacylglycerols; CE:

cholesteryl esters

Lipids (2008) 43:391–400 395

123

Second, that the amount of tracer entering the CE pool was

tremendously different between groups.

However, it is also important to determine the propor-

tion of tracer targeted to each of the respective lipid pools

(Fig. 2, lower panel). Under steady-state-like conditions,

the bulk of the infused tracer in heart was found in TAG

pool (63.7 ± 4.4%), while the remainder was distributed

between the total PL, unesterified (free) fatty acids, and

CE. In stark contrast, in liver the bulk of the infused tracer

was found in CE (55.6 ± 5.1%), with the remaining tracer

distributed between the other lipid fractions. These data

indicate that under steady-state-like conditions the intact

liver targets 22:1n-9 for esterification into different lipid

metabolic pools than in the heart.

Distribution of Tracer in Individual Phospholipid

Classes

The distribution of the infused tracer in liver and heart

individual PL classes was also determined and values are

expressed as % of total phospholipid radioactivity (Fig. 3).

In both liver and heart, the bulk of the infused tracer was

incorporated into CholGpl, CerPCho, and EtnGpl. The rest

of the tracer was found distributed amongst the other

phospholipids.

Metabolism of [14-14C]22:1n-9 to Other Fatty Acids

in Liver and Heart

To determine whether the infused [14-14C]22:1n-9 was

chain elongated or shortened, we determined the distribu-

tion of radioactivity found in other monounsaturated and

saturated fatty acids in the total phospholipid fraction from

liver and heart (Fig. 4). We observed minimal of elonga-

tion of [14-14C]22:1n-9 to [16-14C]24:1n-9 in liver and

heart, however we did find that in liver and heart PL

fractions, 64.2 ± 10.9% and 41.1 ± 8.2% of radioactivity,

respectively, was found in products derived from chain

shortening of [14-14C]22:1n-9. The terminal pool was

found to be 18:1n-9, in which form 41.8 ± 5.4% and

30.8 ± 6.6% of the tracer was found in liver and heart PL,

respectively. Limited carbon recycling into SFA was found

in liver and heart, as only 6.9 ± 2.3% of the tracer was

found in liver PL SFA (mainly as 18:0, 3.0 ± 1.3%) and

only 7.0 ± 3.3% of the tracer was found in the heart PL

SFA fraction (mainly as 16:0, 4.2 ± 3.2%).

Similar to the PL pool in liver and heart, the combined

TAG/CE pool contained products of chain shortening,

mainly in the form of 18:1n-9 form. However, the distri-

bution into this pool was much lower (12.9 ± 3.1% and

10.0 ± 1.7% in liver and heart, respectively) than what

was observed in the PL pool. These data support previous

observations showing that 22:1n-9 is readily chain short-

ened and that it accumulates as 18:1n-9 as the terminal

pool. Limited carbon recycling into SFA was found in the

heart TAG/CE fraction (Fig. 5). In stark contrast, in the

liver TAG/CE fraction, 54.2 ± 9.0% of the tracer was

recovered in SFA form, mainly as 18:0 (48.4 ± 4.5%).

These data support our assumption that 22:1n-9 was rapidly

converted into 18:0 in liver, which can then be exported

into plasma, thereby accounting for our observation of

6.5% of the tracer in the plasma being in the form of

[14C]18:0.

0

15

30

45

60

75

*

*

*

CerPCho ChoGpl PtdSer PtdIns PtdGroEtnGpl

Liver

Heart

Ptd2Gro

% P

hosp

holip

id R

adio

activ

ity

Fig. 3 Distribution of infused [14-14C]22:1n-9 amongst individual

heart and liver phospholipid classes values are expressed as

mean ± SD, where n = 5 heart and n = 6 for liver. The * indicates

statistical significance from liver, p \ 0.05. CerPCho sphingomyelin,

ChoGpl choline glycerophospholipids, PtdSer phosphatidylserine,

PtdIns phosphatidylinositol, EtnGpl ethanolamine glycerophospho-

lipids, PtdOH phosphatidic acid, Ptd2Gro cardiolipin

16:1 18:1 20:1 22:1 24:1 16:0 18:0 20:0 22:0 24:00

20

40

60Liver PL

Heart PL

*

*

*

*

**

*

% P

L R

adio

activ

ity

*

Fig. 4 Distribution of radioactivity amongst different fatty acids

derived from the total tissue phospholipid fraction Values are

expressed as mean ± SD, where n = 5 heart and n = 6 for liver.

The * indicates statistical significance from liver, p \ 0.05. These

values are expressed as percent of total phospholipid fraction

radioactivity. The abbreviations are: 16:1: palmitoleic acid; 18:1:

oleic acid; 20:1: gondoic acid; 22:1: erucic acid; 24:1: nervonic acid;

16:0: palmitic acid; 18:0: stearic acid; 20:0: arachidic acid; 22:0:

behenic acid; 24:0: lignoceric acid

396 Lipids (2008) 43:391–400

123

Discussion

Although 22:1n-9 metabolism has been extensively studied

in different model systems, it is important to note that in

these other studies, the experimental conditions used may

dramatically impact normal 22:1n-9 metabolism, poten-

tially impacting interpretation. For example, feeding

animals high oil diets or diets rich in 22:1n-9 increases the

ability of the liver and heart to oxidize 22:1n-9 [19, 33, 36,

37], thus providing metabolic results that are difficult to

interpret and perhaps erroneous relative to more steady-

state conditions. Because the metabolism of 22:1n-9 is

concentration dependent [22], it is important to use studies

that avoid perturbing the normal plasma concentration of

the fatty acid of interest [41]. Recently, we demonstrated

the uptake into and metabolism of [14-14C]22:1n-9 in rat

brain [15]. Herein, we present data from hearts and livers

isolated from the rats used in our previous study [15].

Unlike the many other studies examining 22:1n-9 metab-

olism, this is the first study to examine liver and heart

22:1n-9 metabolism under steady-state-like conditions.

Under these steady-state-like conditions, 2.3-fold more

tracer was incorporated into liver as compared with heart

(Fig. 1). These data are consistent with results showing

increased liver uptake relative to that by heart under a

number of experimental conditions. Following pulse infu-

sion (i.v.) with [14-14C]22:1n-9, liver radioactivity is

10–15 fold higher than that found in heart, kidneys, or

spleen radioactivity after pulse i.v. infusion of [28]. Similar

results are observed following pulse infusion of tritiated

22:1n-9 [26, 27]. We did not see such a large difference

between heart and liver uptake, this is more than likely

because the concentration of labeled 22:1n-9 used in pulse

infusion experiments was about 10-fold higher than that

used in our experimental paradigm, certainly high enough

to alter the normal plasma concentration of 22:1n-9. This is

critical because liver fatty acid uptake is directly related to

fatty acid plasma concentration [65], which is consistent

with our lower difference between liver and heart uptake at

steady-state conditions.

Furthermore, targeting of tracer to lipid pools is also

highly dependent upon tracer concentration. We observed

that 7 and 16% of the tracer was in the unesterified free

fatty acid (FFA) fraction, in heart and liver, respectively

(Fig. 2), which is substantially lower than those obtained

under other experimental conditions when concentrations

of [14C]22:1n-9 are in the mM range. Under these supra-

physiological conditions, 80% of the tracer is recovered in

the unesterified FFA fraction in perfused liver [24], 36–

90% of the tracer in this fraction in hepatocytes [18, 34],

and 20–62% of the tracer in this fraction in perfused heart

[23, 25, 26, 66]. While we find only 23% of the infused

[14-14C]22:1n-9 was found in the aqueous fraction of the

liver, this value was two times higher than that observed in

vitro [24, 34, 35]. These data indicate that under steady-

state-like conditions, more 22:1n-9 that entered the liver

was subjected to b-oxidation rather than esterified into

lipids. Thus, the concentration of infused tracer is critical

for not only tissue uptake, but metabolism of the tracer by

the tissue as well as distribution of tracer into specific

metabolic pools.

In liver and heart, the bulk of the infused [14-14C]22:1n-

9 found in the organic fraction was incorporated into the

NL fractions, but the targeting to individual NL (TAG and

CE) was radically different between liver and heart

(Fig. 2). However, the distribution of radioactivity between

the total PL and NL fractions was similar for liver and

heart, which is consistent with tracer distribution between

these fractions following pulse-infusion [26, 27], in per-

fused heart [22, 23, 25, 66], in perfused liver [24, 35], and

in hepatocytes incubated with 22:1n-9 [34]. Interestingly,

22:1n-9 distribution between PL and NL fractions is

independent of fatty acid concentration used for heart

perfusion [22]. We also found that the tracer was mainly

found in CholGpl, CerPCho, and EtnGpl in liver and heart

phospholipids (Fig. 3), which appears to be independent of

experimental conditions, since a similar distribution was

shown in heart cell cultures [20], in liver cell cultures [67],

and in perfused heart [22].

We [15] and others have shown that 22:1n-9 is readily

converted into 18:1n-9 through chain shortening pathway

in various organs and cell types [18, 19, 24, 26–28, 31–35],

however none of these studies have addressed conversion

of 22:1n-9 to other fatty acids under steady-state-like

16:1 18:1 20:1 22:1 24:1 16:0 18:0 20:0 22:0 24:00

20

40

60

80Liver NL

Heart NL

*

* * * * *

% N

L R

adio

activ

ity

*

Fig. 5 Distribution of radioactivity amongst different fatty acids in

the combined tissue TAG/CE fraction Values are expressed as

mean ± SD, where n = 5 heart and n = 6 for liver. The * indicates

statistical significance from liver, p \ 0.05. These values are

expressed as percent of total phospholipid fraction radioactivity.

The abbreviations are: 16:1: palmitoleic acid; 18:1: oleic acid; 20:1:

gondoic acid; 22:1: erucic acid; 24:1: nervonic acid; 16:0: palmitic

acid; 18:0: stearic acid; 20:0: arachidic acid; 22:0: behenic acid; 24:0:

lignoceric acid

Lipids (2008) 43:391–400 397

123

conditions or its conversion into saturated fatty acids

(SFA). Previously we found that [91% of the infused

tracer was recovered as plasma 22:1n-9, with 6.5% of the

tracer found in plasma found as 18:0 under steady-state-

like conditions [15]. Because the radiochemical purity of

the custom-synthesized tracer was [99% for

[14-14C]22:1n-9 (as shown by radiochemical HPLC anal-

ysis), it is unlikely that the 18:0 was derived from the

tracer, but rather via conversion of 22:1n-9 to 18:0 in liver

through carbon recycling followed by export into plasma.

This is consistent with our observation reported herein that

the liver took up significantly more tracer and that by

others demonstrating that the liver has a central role in

22:1n-9 metabolism [33].

In brain, we demonstrated that [14-14C]22:1n-9 is chain

shortened predominantly to [10-14C]18:1n-9 [15]. In livers

and hearts isolated from these same rats, we demonstrate

that 31 and 42% of PL radioactivity was found as 18:1n-9,

respectively (Fig. 4). These results confirm previous

reports showing that 22:1n-9 is quickly converted into

18:1n-9 in all models studied [15, 18, 19, 24, 26, 27,

31–35]. Presumably, this conversion is through peroxisome

localized b-oxidation [33, 36, 68]. In contrast, 80% of heart

NL radioactivity was recovered as 22:1n-9 in which TAG

radioactivity represents 70% of total lipid radioactivity.

This is consistent with the observation that up to 80% of

heart TAG radioactivity remains in 22:1n-9 form after

pulse infusion of [14C]22:1n-9 [26, 28]. These data indicate

that the heart TAG pool may serve as a transient pool for

22:1n-9 before its b-oxidation, similar to the proposed role

for TAG pools comprised of other fatty acids in heart [41,

53] and this role may be independent of the 22:1n-9 con-

centrations used in the experiment.

Although 22:1n-9 conversion into monounsaturated FA

has been extensively studied, no studies have demon-

strated the conversion of 22:1n-9 into SFA in liver and

only one study addressed this conversion in cultured heart

cells [21]. In the present study, we demonstrate that half

of the tracer conversion was limited in the heart as it was

found in [14-14C]22:1n-9 (Figs. 4 and 5), which is con-

sistent with results from cultured heart cells [21]. The

high amount of radioactive 18:0 in the liver esterified NL

fraction explains our previous observation that 7% of

infused [14-14C]22: 1n-9 is found as radioactive 18:0 in

plasma [15].

There are two possible explanations for the high rate of

conversion of the tracer into SFA. First, the tracer could be

chain shortened to 18:1n-9 that is then saturated via the

reversal of the D-9 desaturase. However, there is no evi-

dence for a reversal of D-9 desaturase activity. Second, the

complete b-oxidation of 22:1n-9 and recycling of the

radioactive carbons for 18:0 biosynthesis. This is not

without precedence, as in brain a significant portion

([30%) of infused fatty acid radiotracer is found as

radioactive amino acids made via Krebs cycle intermedi-

ates within a 10 min time frame [63, 64]. Hence, the

rapidity of these processes has been observed in the past

and is an important indicator of how fast these processes

work in vivo.

In summary, under steady-state-like physiological con-

ditions more of the infused [14-14C]22:1n-9 (2.3-fold) was

taken up by the liver as compared to heart. It is important to

note that significantly more tracer was targeted for b-oxi-

dation in the heart as compared to liver, consistent with the

high distribution of radioactivity into heart TAG pools.

This is significant because these pools have a high rate of

incorporation in vivo [53], consistent with a high degree of

turnover to provide fatty acids for b-oxidation. In both

tissues, the bulk of tracer radioactivity was incorporated

into the esterified NL fraction, found mainly in TAG in

heart and CE in liver. In the heart NL fraction, the radio-

activity was found predominantly as 22:1n-9, whereas it

was mainly found as 18:0 in liver NL fraction, demon-

strating a rapid conversion of 22:1n-9 into saturated fatty

acids. The underlying importance of this finding is that

during the treatment of X-ALD using LO, which contains a

high level of 22:1n-9, its ingestion may lead to an increase

in saturated fatty acid burden. Because patients on LO have

a restricted saturated fatty acid diet our finding may have

clinical ramifications. The high levels of [14C]18:0 found in

liver TAG/CE is consistent with the presence of this fatty

acid (6.5%) in the plasma of rats, indicating that the liver

rapidly (\10 min) metabolized the [14-14C]22:1n-9 and

exported the TAG/CE containing the [14C]18:0 into the

plasma. Collectively, these data indicate that under steady

state-like conditions, the liver has a greater capacity to take

up 22:1n-9 as compared with heart, where significant

amounts of this fatty acid is metabolized to form saturated

fatty acids, while in the heart this fatty acid remains more

intact and it is targeted for pools destined for use in heart

for b-oxidation.

Acknowledgments The authors thank Dr. Carole Haselton for her

excellent surgical and technical assistance and editorial suggestions

and Mrs. Cindy Murphy for typing and preparation of the manuscript.

This work was supported by grant from The Myelin Project to EJM

and in part by a project (EJM) on a COBRE Grant from the National

Institute of Health P20 RR17699.

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