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Lipids ISSN 0024-4201 LipidsDOI 10.1007/s11745-013-3836-9

Aging Induces Tissue-Specific Changes inCholesterol Metabolism in Rat Brain andLiver

Kosara Smiljanic, Tim Vanmierlo,Aleksandra Mladenovic Djordjevic,Milka Perovic, Natasa Loncarevic-Vasiljkovic, Vesna Tesic, et al.

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ORIGINAL ARTICLE

Aging Induces Tissue-Specific Changes in Cholesterol Metabolismin Rat Brain and Liver

Kosara Smiljanic • Tim Vanmierlo • Aleksandra Mladenovic Djordjevic •

Milka Perovic • Natasa Loncarevic-Vasiljkovic • Vesna Tesic • Ljubisav Rakic •

Sabera Ruzdijic • Dieter Lutjohann • Selma Kanazir

Received: 21 February 2013 / Accepted: 22 August 2013

� AOCS 2013

Abstract Disturbance of cholesterol homeostasis in the

brain is coupled to age-related brain dysfunction. In the

present work, we studied the relationship between aging

and cholesterol metabolism in two brain regions, the cortex

and hippocampus, as well as in the sera and liver of 6-, 12-,

18- and 24-month-old male Wistar rats. Using gas chro-

matography-mass spectrometry, we undertook a compara-

tive analysis of the concentrations of cholesterol, its

precursors and metabolites, as well as dietary-derived

phytosterols. During aging, the concentrations of the three

cholesterol precursors examined (lanosterol, lathosterol

and desmosterol) were unchanged in the cortex, except for

desmosterol which decreased (44 %) in 18-month-old rats.

In the hippocampus, aging was associated with a significant

reduction in lanosterol and lathosterol concentrations at

24 months (28 and 25 %, respectively), as well as by a

significant decrease of desmosterol concentration at 18 and

24 months (36 and 51 %, respectively). In contrast, in the

liver we detected age-induced increases in lanosterol and

lathosterol concentrations, and no change in desmosterol

concentration. The amounts of these sterols were lower

than in the brain regions. In the cortex and hippocampus,

desmosterol was the predominant cholesterol precursor. In

the liver, lathosterol was the most abundant precursor. This

ratio remained stable during aging. The most striking effect

of aging observed in our study was a significant decrease in

desmosterol concentration in the hippocampus which could

reflect age-related reduced synaptic plasticity, thus repre-

senting one of the detrimental effects of advanced age.

Keywords Cholesterol metabolism �Hippocampus �Liver � Cholesterol precursors �24S-hydroxycholesterol � Aging

Abbreviations

24S-OHC 24S-hydroxycholesterol

27-OHC 27-Hydroxycholesterol

BBB Blood–brain barrier

CYP46A1 Cholesterol 24-hydroxylase

GC–MS Gas chromatography/mass spectrometry

GC-FID Gas chromatography/flame ionization

detection

TMS ethers Trimethylsilyl ethers

Introduction

As a multifaceted molecule, cholesterol fulfils a variety of

functions. It is an essential membrane component, a

cofactor for signaling molecules and a precursor for steroid

K. Smiljanic � A. M. Djordjevic � M. Perovic �N. Loncarevic-Vasiljkovic � V. Tesic � S. Ruzdijic �S. Kanazir (&)

Laboratory of Molecular Neurobiology, Department of

Neurobiology, Institute for Biological Research ‘‘Sinisa

Stankovic’’, University of Belgrade, Bulevar despota

Stefana 142, 11060 Belgrade, Serbia

e-mail: selkan@ibiss.bg.ac.rs

T. Vanmierlo � D. Lutjohann

Laboratory for Special Lipid Diagnostics/Centre Internal

Medicine/UG 68, Institute of Clinical Chemistry and Clinical

Pharmacology, University Clinics of Bonn, Bonn, Germany

Present Address:

T. Vanmierlo

Department of Immunology and Biochemistry, Biomedical

Research Institute, Hasselt University, Hasselt, Belgium

L. Rakic

Serbian Academy of Sciences and Arts, Belgrade, Serbia

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DOI 10.1007/s11745-013-3836-9

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hormones. Cholesterol plays crucial roles in major human

diseases. The brain contains 5–10 times more cholesterol

than any other organ, even though it represents only 2 % of

the body weight [1]. Experimental evidence has revealed

that the blood-brain barrier (BBB) renders homeostasis of

brain cholesterol independent of circulating cholesterol [1–

3]. As the peripheral and central cholesterol pools are not

readily interchangeable, the brain produces cholesterol by

de novo synthesis. Outside the brain, the requirement for

cholesterol is met by its de novo synthesis and cellular

uptake of dietary cholesterol in the form of lipoprotein-

cholesterol complexes. Cholesterol biosynthesis is a com-

plex process. The first steroid intermediate in cholesterol

synthesis, lanosterol, can be converted to cholesterol

through two alternative pathways: the Bloch pathway via

desmosterol, a direct cholesterol precursor, and the Kan-

dutsch-Russell pathway via lathosterol [4]. The liver is

responsible for bulk cholesterol synthesis. It also plays a

major role in the elimination of excess cholesterol from the

body. Cholesterol is oxidized by the liver into a variety of

bile acids [5]. A mixture of conjugated and nonconjugated

bile acids, along with cholesterol itself, is excreted from the

liver into the bile.

Cholesterol analogues, plant sterols, are solely derivable

from the diet. Unlike dietary cholesterol, they can pass the

BBB and accumulate in the brain, preferentially within the

lipid rafts of brain cells [6]. Moreover, plant sterols reduce

the molecular order in membranes by interacting less

efficiently with saturated phospholipids compared to cho-

lesterol as required in the formation of compact, liquid

ordered lipid rafts and therefore alter the membrane fluidity

[7]. However, accumulation of plant sterols in the brain

does not manifest any major effects on learning and

memory functions [8].

Aging represents a considerable risk for the develop-

ment of neurodegenerative diseases. While it has been

established that disturbance in cholesterol homeostasis

underlies many of these diseases, the influence of aging on

brain cholesterol metabolism has yet to be clarified since

data that link changes in cholesterol metabolism and the

aging brain are inconsistent (reviewed in [9]).

An increasing number of studies have highlighted the

relationship between cholesterol and synaptic plasticity

[10, 11]. We have previously shown that aging caused

reduction in the expression of synaptic proteins [12]. The

focus of this study was to explore the hitherto insufficiently

examined biochemistry of cholesterol metabolism during

aging. We present a detailed profiling of cholesterol, its

precursors and metabolites, as well as dietary-derived plant

sterols in rat brain regions, the cortex and hippocampus,

and in the serum and liver throughout the lifespan of the

rat, i.e., in 6-, 12-, 18- and 24-month-old animals. The

results obtained show that aging exerts a differential effect

on cholesterol synthesis in the brain and periphery. More-

over, in the brain, the most prominent changes were

observed in the hippocampus, exemplified by a significant,

age-related decrease in desmosterol levels.

Materials and Methods

Animals and Treatments

Experiments were performed on male Wistar rats main-

tained in the Institute for Biological Research, University

of Belgrade, Serbia. All animal handling was in accordance

with internal guidelines and the NIH Guide for the care and

use of laboratory animals (NIH publication No. 85-23).

Experimental protocols were approved by the Ethical

Committee of the Institute for Biological Research, Uni-

versity of Belgrade. The animals were kept in a 12 h light/

dark cycle, with free access to food and water. At 6, 12, 18,

and 24 months of age, the rats (n = 5 per experimental

group) were killed by decapitation. Their brains and livers

were quickly removed, dissected on ice and collected for

subsequent sterol analysis. Blood was collected from the

trunk and the serum was isolated and frozen until further

use.

Sterol Profile Determination

Prior to analysis, the tissue samples were spun in a speed

vacuum dryer (12 mbar; Savant AES 1000) for 24 h in

order to express the individual sterol concentrations rela-

tive to the dry weight. The sterols were extracted from the

dried tissue by placing in a 1.5 ml mixture of chloroform/

methanol (at a 2:1 ratio) for 24 h at 4 �C. Sterol levels were

determined by a gas chromatograph mass spectrometer as

described previously [13, 14]. A 100-ll sample of serum

was applied for sterol determination. Sterols were deter-

mined by gas chromatography/mass spectrometry (GC–

MS) as described previously. Briefly, 50 lg 5a-cholestane

and 1 lg epicoprostanol were added to 1 mL chloroform–

methanol reaction solution before evaporation. One mL of

1 M NaOH in 90 % ethanol was subsequently added to

perform saponification at 60 �C for 1 h in darkness and

under continuous agitation on a GFL-1086 shaker for

132 min. After saponification, 500 lL of distilled water

was added and neutral sterols were extracted with 3 mL

cyclohexane as follows: the samples were agitated for 30 s,

followed by centrifugation at 3,5009g for 10 min

(Heraeus multifuge 4 KR centrifuge). The cyclohexane

phase was transferred to a new glass tube. The extraction

procedure was repeated once more and the phases were

separated. The organic phase was evaporated under nitro-

gen at 65 �C. The residue was redissolved in 30 lL

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n-hexane and transferred to an injection vial for derivati-

zation. The sterols were derivatized to trimethylsilyl ethers

(TMS ethers) by the addition of 10 lL of silylation

reagent (pyridine:hexamethyldisilazane: trimethylchlorosi-

lane, 3:2:1; v/v/v, TMS reagent).

Gas chromatography-flame ionization detection (GC-

FID) analysis was performed for cholesterol detection on a

Hewlett Packard instrument (model HP 6890). High purity

hydrogen was used as the carrier gas at a constant flow rate

of 1.1 mL/min. Two microliters of the sample was injected

by an automated sampler injector at 270 �C in the splitless

mode. The oven program was as follows: 3 min at 150 �C,

followed by a temperature gradient of 20 �C/min, up to

290 �C; the samples were kept at 290 �C for 34 min. The

sterols were separated in a 30 m 9 0.25 mm J&W column

(122-1232 DB-XLB).

The trimethylsilyl ether derivatives of oxyphytosterols

were analyzed by GC–MS using an Agilent 6890 N Gas

Chromatograph coupled to a 5973 mass selective detector

(Agilent Technologies). High purity helium was used as the

carrier gas at a constant flow rate of 0.8 mL/min. The

sample was injected at 270 �C in a splitless mode with an

injection volume of 2 lL. The oven was programmed as

follows: 3 min at 150 �C, followed by a temperature gra-

dient of 20 �C/min up to 290 �C; the samples were kept at

290 �C for 30 min. A 30 m 9 0.25 mm J&W column

(122-1232 DB-XLB) was used. The electron impact ioni-

zation energy of the mass spectrometer was set to 70 eV.

To exclude false results due to the applied experimental

protocol, control experiment was performed. Brain

homogenates were incubated with d6-cholesterol and dried

by SpeedVac (n = 3) or extracted directly from the d6-

cholesterol-spiked homogenates (n = 3). No side-chain

auto-oxidation was detected upon spiking.

The sterol content in the food consumed by animals was

determined by the same procedure and is shown in Table 1.

Statistical Analysis

All values were expressed as the mean ± SEM and com-

pared with the corresponding values obtained for the

6-month-old control animals. Differences between the

experimental groups were tested using one-way ANOVA,

followed by the Fisher’s LSD test (Statistica v. 6.0, Stat-

Soft, Tulsa, OK). Significance was set at p \ 0.05.

Results

Age-Related Changes in Serum Sterol Concentrations

The serum sterol concentrations in different experimental

groups are shown in Table 2. While the cholesterol con-

centration remained unchanged, the concentrations of its

precursors, lanosterol and lathosterol, were elevated during

aging. An increase in the lanosterol concentration was

measured in 12-month-old rats (58 %). Lathosterol was

elevated in both 12- (66 %) and 24-month-old rats (58 %).

Unlike these precursors, serum desmosterol remained

unaltered throughout aging. Likewise, the concentrations

of the cholesterol metabolites, 24S-hydroxycholesterol

(24S-OHC), 27-hydroxycholesterol (27-OHC) and choles-

tanol, did not change during aging. The concentrations of

the phytosterols, sitosterol and campesterol, were

decreased (55 and 42 %, respectively) in 24-month-old

animals, whereas stigmasterol remained unaltered. Brassi-

casterol was elevated in 12-month-old rats (35 %) but

significantly reduced in the oldest group (28 %).

Aging Induces Opposite Changes in Concentrations

of Cholesterol Precursors in the Liver and Brain

Age-related changes in the concentrations of total choles-

terol and its precursors in the cortex, hippocampus and liver

are presented in Fig. 1. Apart from the changes in the con-

centration of precursors, the cholesterol concentration

remained unaffected by aging in both brain regions and in

the liver (Fig. 1a). In the cortex, the concentrations of all

three examined cholesterol precursors were unchanged

during aging, except for a decrease in desmosterol that was

observed in 18-month-old rats (44 %) (Fig. 1b–d, left col-

umns, respectively). In the hippocampus, aging was asso-

ciated with a significant reduction in lanosterol and

lathosterol concentrations at 24 months (28 and 25 %,

respectively) (middle columns in Fig. 1b, c, respectively). A

continual decrease in desmosterol concentration which was

statistically significant at 18 and 24 months (36 and 51 %,

respectively) (Fig. 1d, middle columns) was observed. It is

of interest to note that while desmosterol was predominant

in both brain regions, its concentrations were about three-

fold to fourfold higher in the hippocampus (700–1,200 ng/

mg) compared to the cortex (200–300 ng/mg).

In contrast to the hippocampus, in the liver, we detected

age-induced increases in lanosterol (47, 29 and 110 % in

12-, 18- and 24-month-old animals, respectively) and la-

thosterol (47 and 126 % in 12- and 24-month-old animals,

Table 1 The sterol content of the food

Food ng/mg %

Cholesterol 25.83 0.11

Sitosterol 405.48 1.67

Campesterol 111.00 0.46

Stigmasterol 31.19 0.13

Brassicasterol n.d. 0

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respectively), and no changes in desmosterol concentration

(right columns in Fig. 1b–d, respectively). The amounts of

these sterols were lower than in the brain regions. In both

brain regions, desmosterol was the predominant cholesterol

precursor. In the liver, lathosterol is the most abundant

precursor. This ratio remained stable during aging

(Fig. 1e).

Aging Elicits Different Changes of Cholesterol

Metabolites in the Liver and Brain

The age-related profile of cholesterol metabolites in the

cortex, hippocampus and liver is shown in Fig. 2. In the

cortex, aging induced a slight but significant decrease in

24S-OHC concentration in 18-month-old animals (16 %)

and reduced concentration of 27-OHC in 12-month-old rats

(31 %) (Fig. 2a, b, left columns, respectively). In contrast

to these metabolites, the concentration of cholestanol

increased in the cortex during aging. The change was sta-

tistically significant in 24-month-old animals (42 %)

(Fig. 2c, left columns). In the hippocampus, the amounts of

24S-OHC and 27-OHC remained unchanged during aging

(Fig. 2a, b, middle columns, respectively). As observed in

the cortex, the cholestanol concentration increased during

aging and was statistically significant at 24 months (39 %)

(Fig. 2c, middle columns). In the liver, aging had no

influence on any of the examined cholesterol metabolites

(Fig. 2a–c, right columns).

Profiling Liver and Brain Plant Sterols During Aging

The concentrations of the four most abundant plant sterols

(sitosterol, campesterol, stigmasterol and brassicasterol)

were assessed in the rat brain and liver during aging

(Fig. 3). In the rat cortex, sitosterol concentrations

remained unchanged during aging (Fig. 3a, left columns).

The concentration of campesterol was significantly

increased in 12-month-old animals (30 %) (Fig. 3b, left

columns). The increases in the amounts of stigmasterol and

brassicasterol were statistically significant in 18-month-old

rats (44 and 51 %, respectively) (Fig. 3c, d, left columns,

respectively). In the aging rat, the concentrations of sitos-

terol and brassicasterol remained stable in the hippocampus

(middle columns in Fig. 3a, d, respectively), while the

increase in campesterol and stigmasterol was statistically

significant in 18-month-old animals (50 and 31 %,

respectively) (middle columns in Fig. 3b, c, respectively).

It is of interest to note that the amount of phytosterol in the

oldest animal group was the same as in the control adult

group. The concentration of sitosterol in rat liver was

decreased in 24-month-old animals (36 %) (Fig. 3a, right

columns), whereas campesterol and stigmasterol remained

unaltered by aging (right columns in Fig. 3b, c, respec-

tively). Moreover, aging led to a reduction in brassicasterol

concentrations in the livers of 18- (20 %) and 24-month-

old rats (52 %) (Fig. 3d, right columns).

Discussion

In this study we present a detailed profile of sterols in the

course of aging in two brain regions highly vulnerable to

the aging process, the cortex and hippocampus, and

compare it with the sterol profiles in the liver and serum.

Cholesterol synthesis, as assessed by the concentrations of

its precursors, lanosterol, lathosterol and desmosterol, was

Table 2 Sterol concentrations in rat serum across the lifespan of the rat

Units Aging

6 m 12 m 18 m 24 m

Sterol

Cholesterol mg/dl 66.4 ± 4.0 75.7 ± 7.5 74.4 ± 10.0 67.9 ± 8.5

Lanosterol lg/dl 4.2 ± 0.5 6.7 ± 0.5* 5.2 ± 0.3 4.3 ± 0.1

Lathosterol lg/dl 38.1 ± 5.1 63.4 ± 5.9* 45.5 ± 3.8 60.2 ± 7.9*

Desmosterol lg/dl 59.5 ± 3.4 56.0 ± 3.9 81.8 ± 13.6 78.8 ± 11.5

24S-OHC ng/ml 21.1 ± 3.3 23.3 ± 0.3 24.1 ± 0.6 22.9 ± 0.8

27-OHC ng/ml 12.3 ± 0.7 12.2 ± 1.0 17.1 ± 3.9 13.1 ± 1.6

Cholestanol lg/dl 411.4 ± 32.7 541.3 ± 77.1 438.2 ± 60.7 371.2 ± 36.8

Phytosterols

Sitosterol mg/dl 1.8 ± 0.2 1.7 ± 0.2 1.6 ± 0.4 0.8 ± 0.1*

Campesterol mg/dl 1.1 ± 0.1 1.3 ± 0.2 1.2 ± 0.3 0.7 ± 0.1*

Stigmasterol lg/dl 21.2 ± 1.5 24.4 ± 1.7 22.6 ± 4.3 18.8 ± 1.5

Brassicasterol lg/dl 4.6 ± 0.5 6.3 ± 0.4* 6.0 ± 1.2 3.4 ± 0.3*

The data are presented as means ± SEM (five rats per group). * P \ 0.05 vs. control, 6-month-old animals

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increased with advanced age in extra cerebral tissues,

while in both brain regions the rates of cholesterol bio-

synthesis was decreased during aging. The major changes

were detected in the hippocampus. Aging did not influ-

ence the cholesterol content either in the periphery or in

the brain.

In the hippocampus, the most prominent effect of aging

was a decrease in the concentration of the direct precursor

of cholesterol, desmosterol. This is in agreement with the

data reported for the aging mouse brain [14–16]. Profiling

the cholesterol precursors revealed that in contrast to the

liver, in the cortex and hippocampus desmosterol

Fig. 1 Age-related changes of

cholesterol and related sterol

concentrations in the brain and

liver. Gas chromatography/mass

spectrometry analysis of the

concentration of cholesterol (a),

its precursors lanosterol (b),

lathosterol (c) and desmosterol

(d) in the rat cortex (left

columns), hippocampus (middle

columns) and liver (right

columns). The data are

presented as means ± SEM

(five rats per group). *P \ 0.05

vs. control, 6-month-old

animals. (e) Percentage

distribution of cholesterol

precursors in the cortex,

hippocampus and liver

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predominates, comprising 70–90 % of all sterols. This

indicates that desmosterol is a major cholesterol precursor

in the brain. Similar results were observed in adult [17] and

aged mouse brains [16]. Desmosterol remained the most

abundant precursor during the entire aging process. This

could point to an age-related adaptive mechanism in the

brain whereby the Bloch pathway which consumes less

energy is used for cholesterol synthesis. Although des-

mosterol predominates in both brain regions, its level in the

hippocampus is considerably higher than in the cortex

during aging. A substantially higher desmosterol concen-

tration in the hippocampus could be attributed to neuro-

genesis that takes place in the adult dentate gyrus [18]. It

has been shown that glial cells mainly use the Bloch

pathway for cholesterol synthesis in vitro, whereas differ-

entiated neurons use the Kandutsch-Russell pathway [19].

Yet, in the initial stage of neurogenesis/gliogenesis, there is

no clear distinction between young neurons and young glial

cells. The decreased desmosterol concentration in the aging

hippocampus could at least in part correlate with the

reduced number of progenitor cells differentiating into

neurons during aging [20]. Additionally, desmosterol has

an important role in the process of myelination [21]. Rel-

atively higher amounts of desmosterol are present in the

oligodendrocytes compared to stages later in life. Based on

the slow myelin turnover, the desmosterol will slowly

decrease over time as the myelin-sterols are replaced,

predominantly by cholesterol. This partially explains a

decrease in desmosterol concentration with aging, expected

to be more pronounced in areas rich in white matter, i.e. the

fimbriae fornix of the hippocampus.

The data that describe the fate of cholesterol in the aging

brain are conflicting. Moderate loss of cholesterol was

detected in vitro and in vivo in the membranes of old

hippocampal neurons [22, 23]. Also, there are reports that

in humans after the age of 20, the cholesterol content in the

frontal and temporal cortices is reduced [24], as well as in

the hippocampus and cerebellum [25]. Together, these

results suggest that aging is associated with a lower rate of

synthesis and increased catabolism of cholesterol and a

resulting decrease in its concentration. On the other hand,

there are data that provide evidence for increased amounts

of cholesterol in total brain extracts of old rats [26].

Reduced cholesterol synthesis in the aging human hippo-

campus has been reported, while the amount of cholesterol

was unchanged [27]. All these results should be interpreted

with caution given that despite the high cholesterol content,

cholesterol turnover in the brain was found to be much

slower, but far more stable than that in the rest of the body

[28]. Additionally, cholesterol in the brain resides in three

major compartments with different turnover rates. The

larger pool has the slowest turnover and is present in the

myelin membranes at half replacement times of *0.3 %/

day [29]. Consequently, the total amount of cholesterol is

Fig. 2 Age-related changes of

cholesterol metabolites content

in the brain and liver. Gas

chromatography/mass

spectrometry analysis of the

concentration of 24S-OHC (a),

27-OHC (b), and cholestanol

(c) in the rat cortex (left

columns), hippocampus (middle

columns) and liver (right

columns). The data are

presented as means ± SEM

(five rats per group). *P \ 0.05

vs. control, 6-month-old

animals

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not likely to change much during a rodent’s life. Therefore,

the precursors and metabolites are good predictors of its

turnover, which is very high in the neuronal fraction. A

continuous turnover of cholesterol in neurons may facili-

tate the cells’ ability to efficiently maintain cholesterol

homeostasis required for dynamic structural changes of

neurons, their extensions, and synapse functioning during

synaptic plasticity.

In order to maintain brain homeostasis, cholesterol is

converted by the neuron-specific enzyme cholesterol

24-hydroxylase (CYP46A1) into the more polar 24S-OHC,

and released from the brain into the circulation. The plasma

24S-OHC is almost completely of cerebral origin, and is

considered as an indicator of brain cholesterol homeostasis

[30]. Another oxidized derivative of cholesterol, 27-OHC

which mainly originates from extra-CNS sources, can cross

the BBB and be transformed into 7 alpha-hydroxy-3-oxo-4-

cholestenoic acid [31]. Our results show that both 24S-OHC

and 27-OHC were decreased in the aged rat cortex, while

remaining stable in the hippocampus. Together with the

unchanged amount of 24S-OHC in the circulation this result

indicates that aging did not significantly influence brain

cholesterol catabolism. In contrast to rats, in the hippocam-

pus of elderly people, a trend towards reduction of 24S-OHC

levels was observed when compared with young subjects

[27]. Available data demonstrated that the levels of 24S-

OHC are stable following adolescence while the variation of

its concentration is related to various age-related neurode-

generative diseases [32]. On the other hand, aging induced an

increase in cholestanol concentration in the cortex and hip-

pocampus. Cholestanol was observed to accumulate in the

brains of Cyp27a1 deficient mice [33, 34]. Thus, it can be

Fig. 3 Plant sterols in the brain

and liver of aging rats. Gas

chromatography/mass

spectrometry analysis of the

concentration of sitosterol (a),

campesterol (b), stigmasterol

(c) and brassicasterol (d) in the

rat cortex (left columns),

hippocampus (middle columns)

and liver (right columns) during

aging. The data are presented as

means ± SEM (five rats per

group). *P \ 0.05 vs. control,

6-month-old animals

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assumed that the elevated cholestanol concentrations mea-

sured herein resulted from decreased Cyp27a1 activity.

While the cholesterol content in the liver was stable

throughout the entire lifetime of rats, cholesterol synthesis,

as assessed by lathosterol and desmosterol concentrations,

was apparently elevated during aging. Aging had no

influence on cholesterol elimination since the concentration

of 27-OHC, a marker of cholesterol elimination from extra-

cerebral tissues [35], remained unchanged. Similar data

were reported for aging Sprague–Dawley rats [36]. In

contrast to these findings, Mulas and colleagues reported

that free cholesterol in rat liver was elevated in 24-month-

old animals [37]. Inconsistent literature data could be due

to the different methodologies used to determine the con-

centration of cholesterol.

A similar pattern was observed in rat serum during

aging. Even though Parini and colleagues [38] reported that

the concentration of plasma cholesterol increases with age

in rodents, our results demonstrate that the serum choles-

terol content remained stable during aging in Wistar rats.

Cholesterol synthesis was elevated throughout the aging

process, as judged by the measured concentrations of its

precursors, lanosterol and lathosterol. The unaltered serum

27-OHC concentration could indicate that advanced age

has no influence on the acidic pathway of bile acid

biosynthesis.

Our data revealed tissue-specific profiles of plant sterols

during aging. While in the serum and liver their concentra-

tions were decreased in aged rats, in the cortex and hippo-

campus they accumulated during aging, reaching a peak at

18 months of age. This could be indicative of a compromised

integrity of the BBB associated with aging [39]. After this

point, plant sterol concentrations exhibited a tendency to

decrease. This decrease, even more distinct in the liver and

serum, could reflect changes in the intestinal absorptive

capacity of plant sterols in aged rats. Our data also revealed

differences in the abundance ratio of plant sterols in the

periphery and brain. Although sitosterol was most abundant

in the serum and food provided to the animals, campesterol

prevailed over sitosterol in the hippocampus. A similar, but

less pronounced ratio was observed in the cortex, indicating

that campesterol more efficiently traverses the BBB, as also

shown to occur in the mouse brain [6, 13].

Our results show that aging influences cholesterol syn-

thesis in a different manner in the brain and periphery,

supporting the concept that brain cholesterol metabolism is

autonomous. Although overall cholesterol levels remained

relatively stable during aging, subtle changes in the cho-

lesterol content in membrane microdomains could not be

excluded. The decreased desmosterol content in the hip-

pocampus with advanced age reflects diminished synaptic

plasticity and could be attributed to the diverse effects of

aging.

Acknowledgments This work was supported by the Ministry of

Education, Science and Technological Development of the Republic

of Serbia, Grant ON173056., and by FWO Pegasus Marie Curie

Fellowship to T. Vanmierlo.

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