Mechanisms of Ageing and Development
105 (1998) 1 – 18

Age-related changes in the inner zone of the
adrenal cortex of the rat—a morphologic and
biochemical study
Henrique de Almeida, Maria C. Magalhaes,
˜
Manuel M. Magalhaes *
˜
Institute of Histology and Embryology of the Faculty of Medicine,
and Institute of Molecular and Cellular Biology of the Uni6ersity of Porto, 4200 Porto, Portugal
Received 17 March 1998; received in revised form 29 May 1998; accepted 8 June 1998

Abstract
In this work, a correlative morphologic and biochemical study on the effects of ageing on
the rat adrenal Inner Zone (IZ) was made. Male Wistar rats were studied at 2, 6, 12, 18 and
24 months. Structural data of Zona Fasciculata (ZF) showed age-related increase in cell
volume (P B0.05), decrease in mitochondria (PB 0.01) and smooth endoplasmic reticulum
(SER) volumes, and increase in lipid droplets (P B0.01) and lipofuscin granules (P B 0.01)
volumes. In Zona Reticularis, the main change observed was the increase in lipofuscin
granules (P B0.001). Serum corticosterone from unstimulated rats increased until 12 months
but decreased thereafter (PB0.01), to levels below those from 2-month-old rats. Similarly,
plasma adrenocorticotrophic hormone (ACTH) presented a maximum at 12 months, followed by a decrease to levels higher than at 2 months (PB 0.05). In rats injected either with
only ACTH or dexamethasone, before ACTH stimulation, corticosterone level had a
maximum at 12 months. In aged rats, serum high density lipoprotein (HDL) and adrenal
cholesterol ester increased signiﬁcantly (PB 0.05 and PB 0.01, respectively), whereas adrenal
corticosterone decreased. Products of lipid peroxidation, assayed with the thiobarbituric acid
reaction and ﬂuorimetry showed an age-related increase (PB 0.05). The age-related decrease
in mitochondria and SER volumes is consistent with the decrease of serum corticosterone.
The increase in lipid droplet and HDL and the reduction of adrenal corticosterone level
correlate with the increase of adrenal cholesterol ester content. These suggest a continued
* Corresponding author. Present address: Institute of Histology and Embryology, Faculty of
Medicine, 4200 Porto, Portugal. Tel.: + 351 2 591468; Fax: +351 2 5505728.
0047-6374/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved.
PII S0047-6374(98)00099-2

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H. de Almeida et al. / Mechanisms of Ageing and De6elopment 105 (1998) 1–18

uptake of steroid precursor but a reduced steroid synthesis. On the whole, the data provide
evidence for an age-related reduced functional ability of IZ and particularly of ZF. © 1998
Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Adrenal cortex; Ageing; Age-related; Lipofuscin; Steroids; ACTH

1. Introduction
The rat adrenal cortex is a steroidogenic gland structured in three different layers.
The Zona Glomerulosa (ZG), the outermost, is the site of synthesis of the
mineralocorticoid aldosterone. The Zona Fasciculata (ZF), the middle layer, and
the Zona Reticularis (ZR), close to the adrenal medulla, are distinct from ZG, and
considered together as the Inner Zone (IZ) (Long, 1967). In response to adrenocorticotrophic hormone (ACTH), IZ accounts for the synthesis and secretion of
corticosterone, the main glucocorticoid of the rat.
The IZ of the adrenal cortex of the aged rat has been a subject for morphological
and biochemical studies. Early structural reports in the male rat emphasised an
age-related increase of lipid droplets in ZF (Jayne, 1957) and ﬂuorescent lipofuscin
pigment in ZR (Jayne, 1957; Reichel, 1968). The increase in lipofuscin pigment in
ZR was conﬁrmed by ultrastructural studies in male (Shaposhnikov, 1985; Rebuffat
et al., 1992; Cheng and Kowal, 1994) and female rats (Szabo et al., 1970; Nickerson
´
et al., 1979) and mice (Samorajski and Ordy, 1967). Other studies in ZF showed a
quantitative increase in mitochondria and smooth endoplasmic reticulum (Rebuffat
et al., 1992), but there was controversy in relation to the increase of lipid droplets
(Shaposhnikov, 1985; Rebuffat et al., 1992).
In contrast to the small number of structural studies, biochemical data concerning IZ functioning was reported more frequently; however, the results are conﬂicting. Corticosterone levels in old unstimulated animals, when compared to young
animals, were found either increased (Sapolsky et al., 1983; Scaccianoce et al., 1990;
Rebuffat et al., 1992), unchanged (Hess and Riegle, 1970; Tang and Phillips, 1978)
or decreased after a maximum at an intermediate age (Cizza et al., 1994). Similarly,
controversy exists regarding ACTH level in aged rats (Tang and Phillips, 1978;
Sonntag et al., 1987; Rebuffat et al., 1992; Cizza et al., 1994).
The mechanism underlying such changes is still a matter of debate. Functional
studies in the hypothalamus – pituitary–adrenal (HPA) axis suggested either an
increase (Scaccianoce et al., 1990) or a decrease (Cizza et al., 1994) of hypothalamic
activity, whereas others emphasised an age-related lesser functional ability of the
adrenal cortex, as evidenced by decreased response to ACTH administration in vivo
(Hess and Riegle, 1970; Reaven et al., 1988; Cizza et al., 1994) and in vitro
(Malamed and Carsia, 1983).
Part of this controversy in rat studies is consequent to variations in the criteria
used to consider the animal as young or old and also to the number of time points
used in the study. In fact, some studies only used a few time points, which contrasts

H. de Almeida et al. / Mechanisms of Ageing and De6elopment 105 (1998) 1–18

3

with recommendations to use several time points (Coleman et al., 1990) in order to
disclose variations during the ageing period.
In attendance to these considerations and also due to the scarcity of morphological studies, particularly those correlating structural and biochemical changes in the
IZ of the rat adrenal cortex during ageing, it seemed worthwhile to re-examine the
subject. Therefore, we performed a quantitative ultrastructural, cytochemical and
biochemical study, and extended it to ﬁve different time points, starting at 2 months
and ending at 24 months.

2. Materials and methods
Wistar male rats from the colony of the Gulbenkian Institute of Science (Oeiras,
Portugal) with free access to water and laboratory diet, were kept at room
temperature and natural day – night light cycles. At 2, 6, 12, 18 and 24 months,
randomly selected and apparently healthy animals were sacriﬁced after anaesthesia
with 50 mg/kg of pentobarbital intraperitoneally (i.p.). A laparotomy was performed, the adrenals were dissected from surrounding fat, weighed and processed
for morphological or biochemical study. Blood was obtained by decapitation and
prepared for assay of ACTH and corticosterone.
ACTH, 0.2 mg/kg of i 1 – 24-Corticotropin Zinc (Synacthen Deposito, Ciba-Geigy
´
SA) was injected i.p. 30 min before sacriﬁce. This concentration was used taking
into account a previous report (Magalhaes et al., 1979) and a recent evaluation of
˜
the effects of three different concentrations of ACTH in 2-month-old rats (personal
data). Similarly, 30 min was the time required to obtain a maximum effect, after
comparing with 5, 10, 20, 45, 60 and 120 min. To evaluate adrenal response to
exogenous ACTH after chemical blockade of the pituitary, rats were injected with
4 mg/kg of dexamethasone phosphate (Decadron®, Merck Sharp and Dhome), i.m.,
12– 14 h before ACTH administration. This time resulted in undetectable levels of
corticosterone and also dexamethasone, necessary for its use as internal standard in
high-performance liquid chromatography (HPLC).
After sacriﬁce, all the animals were subjected to autopsy. When there was
evidence of tumours or pulmonary changes, the animals were taken off the study.

2.1. Morphologic studies
The right adrenal was cut into two pieces. One, used for ultrastructural morphological studies, was ﬁxed in 2% glutaraldehyde, post-ﬁxed in 1% osmium tetroxide
and Epon embedded; the other was processed for acid phosphatase (AP) ultrastructural cytochemistry (Barka and Andersen, 1962; Magalhaes et al., 1985).
˜

2.2. Morphometric study
2.2.1. Light microscopy
In 1 vm thick Epon sections obtained from ﬁve different blocks per animal

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(n=6) and stained with methylene blue-azur II (Richardson et al., 1960), ZF and
ZR were analysed with aid of the program IMA, version 1.4, Biocom 200 (Biocom,
Les Ulis, France). A square lattice having a test line of 10 vm was used, a total of
1750 points per animal/zone were counted and volume density (Vv), nuclear and
cellular volumes and numerical density (Nv) were determined (Weibel and Bolender,
1973).

2.2.2. Electron microscopy
The Vv of cytoplasmic structures was obtained according to a previously described method (Almeida et al., 1986).
2.3. Biochemical study
2.3.1. Blood samples
Serum corticosterone was extracted with dichloromethane, washed with sodium
hydroxide, 0.1 N, and dried under nitrogen ﬂow. The residue was redissolved in
acetonitrile:water (40:60 v/v) and analysed by reversed-phase HPLC (Waters) with
similar mobile phase ﬂowing at 1 ml/min, using dexamethasone (Sigma) as internal
standard. The separation was carried out by a Nova-Pak® C18 column (Waters)
and the detection was made by UV with the wavelength set at 254 nm.
Dichloromethane and acetonitrile were of chromatographic grade (E. Merck) and
water was tridistilled. Total circulating corticosterone (Total B) was estimated from
serum corticosterone considering the plasma volume per weight as 3.88 ml/100 g
(Ringler and Dabich, 1978). The average amount of circulating corticosterone per
mg of adrenal weight, B/mg, was obtained as previously (Reaven et al., 1988).
ACTH assay in plasma was made with the kit Elsa-ACTH (CIS Bio International, Gif-sur-Yvette, France), following the manufacturer’s instructions.
Serum triglycerides (TG), total cholesterol (TC) content and HDL-cholesterol
(HDL-C) were determined using clinical chemistry kits from Boehringer-Mannheim
in a Hitachi 917 automatic analyser.
2.3.2. Adrenal homogenates
After removal of capsules, the adrenals were homogenised in 0.1 M phosphate
buffer, pH 7.2, for 90 s and aliquots stored at − 20°C until use.
For ﬂuorescence studies, extraction was carried out with a 2:1 mixture of
chloroform – methanol (E. Merck) and 0.05% of butylated hydroxytoluene
(Aldrich), processed according to a previous method (Dillard and Tappel, 1984)
and analysed with a Jasco FP 770 spectroﬂuorometer (Japanese Spectroscopic)
equipped with a Xenon arc lamp 150 W and a photomultiplier R376. The
sensitivity was set at 100 and the standard used was quinine sulphate, 1 vg/ml of
0.1 N H2SO4, which had a relative ﬂuorescence intensity of 22.6. The ﬂuorescence
intensity was measured with excitation and emission wave lengths at 365 and 455
nm, respectively, and calculated in arbitrary units of ﬂuorescence per gram of tissue
(AUF/g) (Dillard and Tappel, 1984).

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5

The assay of thiobarbituric acid reactive substances (TBArs) was made by
spectrophotometry (Ohkawa et al., 1979). As standard, malonaldehyde bis(dimethyl
acetal) (MDA) (Sigma) was used and the ﬁnal values were expressed as nmol of
MDA/g of tissue. The same technique was employed in the assay of total and
esteriﬁed cholesterol (Sperry and Webb, 1950) and protein content (Lowry et al.,
1951). HPLC was also used for adrenal corticosterone determination.

2.4. Statistical analysis
The means of the measurements at the different times were compared using
analysis of variance (ANOVA). A multiple range analysis was used to contrast the
means at the different times studied and the statistical signiﬁcance of differences
between individual pairs of means was assessed using the least signiﬁcant difference
procedure of Fisher (LSD test). In some cases, the Student t-test was used.

3. Results

3.1. General data pertaining to animals
The weights of the rats and pairs of adrenals are depicted in Table 1. For both,
the age-related increase was signiﬁcant (PB 0.001).

3.2. Morphologic studies
3.2.1. Light microscopy
ZF and ZR parenchyma cells from young rats displayed structural features
similar to previous descriptions (Deane, 1962). In ZF cells from rats aged 6 months
and older, there was an increase in lipid droplets, but the usual ovoid or polyhedral
shape of the cells was present. In older rats, there were occasional areas of intense
lipid accumulation suggesting an increase in the dimensions of the droplets.
Table 1
Rat and adrenal weight (mean 9S.E., n=22–29)
Age

Rat (g)

Adrenals (mg)

2 months
6 months
12 months
18 months
24 months
P

291.349 11.09
546.969 17.82A
662.239 16.38AB
703.419 30.17AB
667.879 24.15AB
B0.001

51.00 91.98
54.40 91.56
60.60 91.85Ab
64.77 92.24AB
73.57 93.06ABCD
B0.001

a,b,c,d

Refer to comparisons for PB0.05, in the LSD procedure of Fisher, at 2, 6, 12 and 18 months,
respectively; A, B, C and D report comparisons for PB0.01.
S.E., standard error.

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Table 2
Nuclear volume, cellular volume and Nv of Zona Fasciculata and Zona Reticularis at different ages
(mean9 S.E., n=6)
Nuclear volume (mm3)
Zona Fasciculata
2 months
6 months
12 months
18 months
24 months
P
Zona Reticularis
2 months
6 months
12 months
18 months
24 months
P

Cell volume (mm3)

Nv (×103/mm3)

235.119 7.37
231.089 13.70
232.3997.41
238.359 7.81
262.279 13.76b
n.s.

2369.22 979.65
3012.16 9 196.09
2714.16 962.48
3270.87 9 212.72a
3356.44 9426.15A
B0.05

399.28912.94
322.31 920.87a
351.48 9 7.00
297.96 9 18.75A
300.99 935.27A
B0.05

203.329 7.10
186.099 2.57
184.0595.59
191.379 14.27
201.499 15.91
n.s.

1463.67 943.63
1371.73948.09
1448.56 9 81.05
1498.76 9 105.81
1568.42 9 121.06
n.s.

534.88913.21
579.59 921.46
538.34 920.18
531.27 943.42
514.53 947.93
n.s.

a,b,c,d

Refer to comparisons for PB0.05, in the LSD procedure of Fisher, at 2, 6, 12 and 18 months,
respectively; A, B, C and D report comparisons for PB0.01.
n.s., not signiﬁcant.

Changes such as congestion, ﬁbrosis or distortions of the tissue were not observed.
Macrophages were sometimes seen. The cells in ZR from either young or old
animals were almost devoid of lipids. However, in older animals, an increase in
macrophages occupying the whole ZR was observed (Almeida et al., 1995). In some
animals, the capillaries were ﬁlled with diverse material which stained similarly to
the surrounding cells.
The quantitative studies are presented in Table 2. In ZF, nuclear volume
increased only slightly whereas cell volume had a signiﬁcant increase (PB 0.05) and
Nv a signiﬁcant decrease (P B0.05). In ZR, there were no signiﬁcant changes in all
three parameters.

3.2.2. Electron microscopy
The ultrastructure of ZF cells at 2 months was similar to previous descriptions
(Nussdorfer, 1986). In short, most of the cells exhibited a large, round nucleus with
a prominent nucleolus. The mitochondria had the usual vesicular cristae and dense
matrix. The lipid droplets were abundant, scattered, and displayed low, homogeneous, electron density; frequent vesicular proﬁles of the smooth endoplasmic
reticulum (SER), lysosomes and free ribosomes were observed. The Golgi complex
was frequently a prominent structure (Fig. 1). In contrast, the cells from ZR were
smaller, their nuclei contained more condensed chromatin and the nucleolus was
less prominent. In the cytoplasm, only a few lipid droplets were present, but
abundant proﬁles of SER and mitochondria with tubulo-vesicular cristae could be
seen (Fig. 2).

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7

In ZF from older rats (especially at 18 and 24 months), mitochondria, SER and
Golgi complex did not present remarkable ultrastructural changes. However, the
lipid droplets were more numerous, larger, and frequently displayed a slight
increase in the density of their content. The lysosomes seemed more numerous. The
most remarkable ultrastructural change was the appearance of lipofuscin granules.
They were round or ovoid in shape, presented a surrounding membrane and
contained variable amounts of structures with heterogeneous shape and electronic
density: some were dense, round or irregular in outline, others resembled small lipid
droplets and still others appeared as thin, dense, membrane-like. In ZR cells, the
most conspicuous change were the numerous lipofuscin granules displaying similar
features to those observed in ZF (Fig. 3). Increased numbers of macrophages and
features of phagocytosis of debris of parenchyma cells were common in this zone.
Electron microscopy quantitative studies (Table 3), showed an increase of lipid
droplet Vv (P B 0.01) and a decrease of mitochondria (PB 0.01) and SER Vv
(P=0.050) in ZF; lipofuscin granules increased signiﬁcantly (PB0.001). In ZR,
only lipofuscin had a signiﬁcant age-related quantitative change (PB 0.001).

3.2.3. Ultrastructural cytochemistry
Although the preservation of the structures was poorer, the organelles could still
be identiﬁed. AP activity was evidenced by a granular electron-dense precipitate in
lysosomes and at the trans side of Golgi cisternae at all ages. The precipitate was
present in some lipofuscin granules and absent in others (Fig. 4).

Fig. 1. Zona Fasciculata at 2 months, showing common ultrastructural cytoplasmic features: mitochondria with vesicular cristae (m), lipid droplets (l), smooth endoplasmic reticulum (r), lysosomes (arrow)
and Golgi complex (G). Bar: 1 vm.

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H. de Almeida et al. / Mechanisms of Ageing and De6elopment 105 (1998) 1–18

Fig. 2. Zona Reticularis at 2 months with structures similar to Fig. 1. The mitochondria, however,
display tubulovesicular cristae and lipid droplets present enhanced electron density. Bar: 1 vm.

3.3. Biochemical studies
3.3.1. Blood samples
The assay of serum corticosterone (B), total circulating corticosterone (Total B),
total B/mg of adrenal (B/mg) and plasma ACTH are shown in Table 4. All
parameters increase until 12 months and decrease signiﬁcantly thereafter.
The effects of ACTH on serum corticosterone at the different ages studied are
depicted in Fig. 5. In animals only injected with ACTH, corticosterone increased
from 241.0 9 33.6 ng/ml at 2 months to 319.8 9 42.1 ng/ml at 12 months, and
decreased to 201.0 9 17.4 ng/ml at 24 months. In rats previously injected with
dexamethasone, the most relevant values were: 239.69 38.4 ng/ml at 2 months,
258.09 34.8 ng/ml at 12 months and 143.09 27.9 ng/ml at 24 months. Serum
corticosterone from ACTH injected rats was signiﬁcantly higher than corticosterone
from unstimulated animals (P B 0.05 at 6, 12 and 24 months; PB 0.01 at 2 and 18
months).
In Fig. 6, the results of the assays of serum TG, TC and HDL-C are displayed.
For all, ANOVA revealed a statistically signiﬁcant age-related increase (PB 0.05
for TG and HDL-C; P B 0.01 for TC). TG values (g/l 9 S.E.) were: 1.209 0.12 at
2 months, 1.390.08 at 12 months and 1.939 0.24 at 24 months; the LSD test
revealed signiﬁcant differences (PB 0.05) when 18- and 24-month-old rats were
compared with 2-month-old animals. Concerning TC, the results were: 0.69 g/l 9
0.09 at 2 months, 21 9 0.12 at 12 months and 1.40 9 0.13 at 24 months; in the LSD
test, statistical signiﬁcance was found comparing 2-month-old rats with 12-, 18- and

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9

24-month-old rats (P B0.01). Serum HDL-C increased from 0.38 g/l 9 0.05 at 2
months to 0.8 9 0.08 at 12 months and 0.76 9 0.13 at 24 months; when 2-month-old
rats were compared with 12-, 18- and 24-month-old rats, statistically signiﬁcant
differences (P B 0.01) were found.

3.3.2. Adrenal homogenates
The results, depicted in Fig. 7, showed non-signiﬁcant changes in free cholesterol (FC): 2.98 vg/mg at 2 months, 3.32 vg/mg at 24 months. Esteriﬁed cholesterol (EC) increased from 15.1292.08 vg/mg at 2 months to 28.969 4.08 at 12
months and 32.34 9 5.99 vg/mg at 24 months. ANOVA showed a statistically
signiﬁcant difference (P B 0.05). Adrenal corticosterone decreased from 25.99 ng/
mg at 2 months to 19.8 at 12 months and 19.36 ng/mg at 24 months. Protein
content (vg of protein/mg of adrenal) did not differ signiﬁcantly and did not
show any age-related trend (253.2 at 2 months, 244.0 at 12 months and 253.1 at
24 months).
Either TBArs (Fig. 8, left), or ﬂuorescence intensity (Fig. 8, right), had an
age-related increase, signiﬁcant for TBArs (pB 0.05). The assay of TBArs were
the following (in mmol of MDA/g tissue9 S.E.): 191.19 18.2 at 2 months,
231.09 52.3 at 12 months and 455.49 54.8 at 24 months. In the LSD test,

Fig. 3. Zona Reticularis at 24 months. Mitochondria and lipid droplets are present as in young rats.
However, in the cytoplasm, membrane-bound lipofuscin granules with heterogeneous shape and content
are observed. Note membranous structures (arrow) and areas with electron density similar to lipids
(double arrow). Bar: 1 vm.

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Table 3
Volume density (%) of cytoplasmic structures from Zona Fasciculata and Zona Reticularis at different
ages (mean 9S.E., n= 6)
Mitochondria SER
Zona Fasciculata
2 months 41.229 1.59
6 months 38.6991.42
12 months 34.7391.48a
18 months 30.729 2.50AB
24 months 32.189 1.45Ab
P
B0.01
Zona Reticularis
2 months 35.4291.47
6 months 34.849 1.43
12 months 33.919 1.73
18 months 32.3192.05
24 months 32.7890.66
P
n.s.

Lipid droplet Golgi

Lysosomes

Lipofuscin

42.0591.42
38.21 91.83
35.459 1.49A
36.519 1.35a
35.439 2.12A
0.050

13.72 91.04
19.35 91.47
27.16 92.38Ab
29.68 93.85Ab
28.6293.39Ab
B0.01

1.4790.15
1.449 0.16
1.30 9 0.14
1.71 9 0.17
1.89 9 0.17
n.s.

1.35 9 0.26
1.06 9 0.22
0.68 90.14a
0.51 90.18Ab
0.57 90.09A
B0.05

0.2090.06
0.5390.11
0.68 9 0.10a
0.86 9 0.10A
1.30 9 0.30ABc
B0.001

50.5491.25
50.979 2.38
48.829 1.96
49.399 0.65
49.7091.13
n.s.

9.98 90.88
10.33 9 1.34
11.34 91.70
12.61 92.18
10.75 91.73
n.s.

1.169 0.34
1.2190.24
1.6690.14
1.259 0.11
1.719 0.37
n.s.

2.37 9 0.38
1.85 90.28
2.07 90.22
1.97 90.13
2.16 9 0.38
n.s.

0.529 0.17
0.80 90.13
2.209 0.34AB
2.469 0.25AB
2.90 9 0.43AB
B0.001

a,b,c,d

Refer to comparisons for PB0.05, in the LSD procedure of Fisher, at 2, 6, 12 and 18 months,
respectively; A, B, C and D report comparisons for PB0.01.
S.E., standard error.

signiﬁcant diﬀerences were found when comparing 24- and 2-month-old animals
(PB0.01) and comparing 24- and 6-month-old, and 12- and 18-month-old rats
(PB0.05). Fluorescence intensity in AUF/g tissue was: 13.05× 103 at 2 months,
21.03 × 103 at 12 months and 29.36×103 at 24 months. A signiﬁcant difference
was found between 2- and 24-month-old rats (PB 0.05). TBArs assay and
ﬂuorescence intensity in the homogenates from ACTH-injected rats presented an
age-related increase (P B0.05 for TBArs). In general, the values obtained were
lower than those from unstimulated rats with similar age. However, when individual pairs were assessed there were no statistically signiﬁcant differences at all
ages studied.

4. Discussion
The study of the effects of ageing on the rat adrenal cortex was made at ﬁve
different ages, ranging from 2 to 24 months old. For the purpose of comparing such
effects, 2-month-old animals representing early adulthood, were used as the starting
point, taking into account that by that age they had become sexually mature
(Farris, 1949; Baker, 1978). The use of 24 months as the end-point of this study was
due to an apparent increase in the incidence of morbid situations, such as tumours
and respiratory conditions, among the rats; on the other hand, the 50% survival

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11

Fig. 4. Ultrastructural acid phosphatase cytochemistry of Zona Fasciculata cells at 12 months. A dense,
ﬁne granular precipitate is present on some lipofuscin granules (arrow) but absent in others (double
arrow), supporting the lysosomal origin of lipofuscin. The precipitate is absent from the nucleus (N).
Bar: 1 vm.

age, an index used for ageing studies (Burek and Hollander, 1980), was found to be
24 months in Wistar male rats (Schlettwein-Gsell, 1970), the strain used in this
study.
The growth of the adrenal cortex is mainly regulated by ACTH (Hornsby, 1985;
Estivariz et al., 1992). In the rat, failure of ACTH secretion, either by hypophysecTable 4
Unstimulated rats: serum corticosterone (B), Total B, Total B/mg of adrenal and plasma ACTH
(mean9 S.E.)
Age

B (ng/ml)*

Total B (ng)*

Total B/mg*

ACTH (pg/ml)**

2 months
6 months
12 months
18 months
24 months
P

163.37911.08
179.489 12.82
214.029 17.21A
150.809 13.23C
146.099 9.98C
B0.01

1894.94 9125.93
3748.18 9302.67A
5618.22 9 466.68AB
4102.57 9402.64AC
3904.90 9303.35AC
B0.01

36.78 9 2.60
70.15 9 5.63A
93.24 98.88AB
62.20 95.52AC
54.32 94.34aC
B0.01

723.009 93.29
1085.27 9 112.35
1593.17 9 163.94Ab
1280.43 9 192.56a
1198.07 9 189.34a
B0.05

a,b,c,d

Refer to comparisons for PB0.05, in the LSD procedure of Fisher, at 2, 6, 12 and 18 months,
respectively; A, B, C and D report comparisons for PB0.01.
*n = 18.
**n=14.

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Fig. 5. Serum corticosterone (ng/ml9S.E.). In unstimulated rats (n = 18), a maximum is reached at 12
months (m); the level at 18 and 24 months is lower than in 2- and 6-month-old rats. In animals
stimulated with ACTH alone (n= 4–5) or previously injected with dexamethasone (n = 4 – 5), corticosterone values are also higher in younger than in aged animals.

tomy or chronic glucocorticoid administration, is associated with marked atrophy
of the cortex and decreased steroid secretion; whereas stimulation in vivo originates
an increase in adrenal weight, cell and nuclear volume, corticosterone output and,
at the ultrastructural level, an increase in volume density of mitochondria and
smooth endoplasmic reticulum (Nussdorfer, 1986).
Although the effects of ACTH may extend to the entire cortex, its main
stimulatory action is exerted on the cells of the IZ (Estivariz et al., 1992). The IZ
of the adrenal cortex is considered together as ZF and ZR, but ZF is by far the
largest, accounting for over 70% of the IZ volume in young rats (Deane, 1962;
Rohr et al., 1976; Reaven et al., 1988), a relation maintained in older rats (Reaven
et al., 1988). In addition, ZF cells’ corticosterone secretory capacity in response to

Fig. 6. Serum triglycerides (TG), total and HDL-cholesterol (TC and HDL-C) in g/l9 S.E. from 2 to 24
months (m). For the three items assayed, an age-related increase occurs (n = 9 – 13).

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13

Fig. 7. Adrenals assays of free and esteriﬁed cholesterol (FC and EC) in vg/mg of tissue9 S.E. and
corticosterone (B) in ng/mg of tissue 9 S.E. Cholesterol ester increases steadily from 2 to 24 months (m),
whereas a decline in corticosterone is observed (n =10 – 14).

ACTH is much greater when compared to ZR cells (Hyatt et al., 1980). This
indicates that ACTH effects in the IZ are expected to be more pronounced in the
ZF as well as circulating levels of corticosterone are more likely to result from ZF
cells’ capacity to synthesize steroids.
Steroidogenesis is a multi-step pathway involving the utilization of cholesterol
and the activity of enzymes present in the mitochondria and SER (Malamed, 1975).
Cholesterol may be obtained by de-novo synthesis from acetate in which 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA red) is the rate-limiting
enzyme. A second source of cholesterol is the hydrolysis of cholesteryl esters present
in lipid droplets of steroid-producing cells, in a reaction catalyzed by a neutral
cholesterol ester hydrolase (CEH) (Vahouny et al., 1984). However, in basal
conditions, most of the cholesterol is derived from the uptake of circulating
lipoproteins, of which HDL is preferentially utilised in the rat (Gwynne and

Fig. 8. Adrenal assay of thiobarbituric acid reactive substances (left chart, nmol MDA/g of tissue9 S.E.,
n = 10) and ﬂuorescence intensity of extracts (right chart, arbitrary units of ﬂuorescence × 103/g of
tissue 9 S.E., n=4–5). For either item assayed, an age-related increase occurs (PB 0.05 for TBArs),
which is maintained after ACTH stimulation.

14

H. de Almeida et al. / Mechanisms of Ageing and De6elopment 105 (1998) 1–18

Strauss, 1982); unlike the endocytic pathway of LDL, HDL-C appears to be taken
up and used for steroidogenesis or storage without prior lysosomal degradation of
its apoprotein (Gwynne et al., 1984).
In the present study, there was an age-related increase in adrenal weight and an
increase of ZF cell volume, but not of ZR. Apparently, these ﬁndings suggested a
persistent trophic action on the ZF cells of the adrenal cortex, which is supported
by the increase in plasma ACTH observed in the older animals.
However, the ultrastructural and other biochemical data failed to conﬁrm that
correlation. As a matter of fact, in ZF, the volume density of mitochondria and
SER decreased in aged animals compared to young ones, a variation usually
correlated with decreased, rather than increased, steroid synthesis (Nussdorfer,
1986). Moreover, serum corticosterone presents an additional support. In fact, until
12 months, possibly due to a maturational effect, an increase in corticosterone
followed by a decrease at 18 and 24 months to levels lower than those from younger
ages was observed. Similar ﬁndings were obtained in respect of total circulating
corticosterone and its ratio to adrenal weight.
The increase in lipid droplet volume density from 13.7% at 2 months to 28.6% at
24 months is the most likely reason for the increase in ZF cell volume and provides
an additional ﬁnding in favour of a decrease in steroid synthesis by these cells. In
fact, the increase of lipid droplets, which store esteriﬁed cholesterol as a result of
the activity of acyl-CoA-cholesterol acyltransferase (ACAT), has been commonly
associated to steroid output depression (Nussdorfer, 1986) as occurs in the aged
animals of the present study.
Thus, there is an apparent discrepancy between the increase in ACTH and the
lack of proportional corticosterone output in aged rats. This disagreement is more
evident after ACTH administration, which shows a smaller corticosterone increase
in older rats when compared to younger ones, indicating a lesser ability of ZF cells
from aged rats to respond to ACTH. To explain this by means of a reduction in the
cholesterol available for steroidogenesis or a decrease in corticosterone secretion by
the cells, serum HDL-C and adrenal corticosterone were assayed. However, the
results showed an increase in HDL-C and a decrease in corticosterone adrenal
content.
Hence, a decrease in the sensitivity of aged ZF cells to ACTH or in the activity
of the enzymes that mobilise cholesterol from the ester stores in lipid droplets, or
both, seem to be the more likely mechanisms. In fact, previous studies indicated a
decrease in ACTH receptors of adrenocortical cells from aged rats (Malamed and
Carsia, 1983) or bovine adrenocortical cells aged in culture (Hornsby et al., 1980).
Moreover, a decrease of neutral CEH and ACAT activities, 60 and 33%, respectively, was found in aged rats as compared to younger ones (Popplewell and Azhar,
1987). As a result, the effect of ACTH is diminished, lesser cholesterol is transferred
to the droplets, and much less is mobilised for steroidogenesis, which is consistent
with the increase of cholesterol ester content of the adrenals, Vv lipid droplet
increase and serum corticosterone decrease observed.
Lipofuscin granules, a common structure in aged IZ cells, deserve additional
comments. They are frequently observed in post-mitotic cells and their age-related

H. de Almeida et al. / Mechanisms of Ageing and De6elopment 105 (1998) 1–18

15

accumulation has made of them a classic marker of ageing. They exhibit cellular
remnants and AP activity in a manner similar to those observed in hepatocytes
(Essner and Novikoff, 1961), Leydig cells (Frank and Christensen, 1968), cardiac
muscle cells (Topping and Travis, 1974), neurons (Brunk and Ericsson, 1972) and
corpus luteum cells (Paavola, 1978), among others, which supports their origin in
autolysosomes. The progressive accumulation of remnants is associated with a
decrease in AP activity (Daems et al., 1969) and results in an enzymatically inactive
structure — the residual body or post-lysosome (de Duve and Wattiaux, 1966). Our
ultrastructural and cytochemical ﬁndings are consistent with this view. The spectral
similarities between extracts of lipofuscin rich tissues from aged subjects and
N,N%-disubstituted 1-amino-3-iminopropenes, conjugated Schiff bases, led to the
assumption that these products are probably responsible for the ﬂuorescent properties of lipofuscin granules (Chio and Tappel, 1969; Dillard and Tappel, 1984), whose
maximum intensity for excitation and emission wave lengths is 360–395 and 430–460
nm, respectively (Sohal, 1984). Schiff bases are thought to result from cross-linking
between peroxidised lipids, or their carbonyls derived from auto-oxidation, and
amino groups from incompletely degraded proteins, nucleic acids and other
molecules (Gershon, 1988; Brunk et al., 1992). Additional ﬂuorescent compounds
may be present as occurs with rethynil stearate, recognised in adrenal extracts (Cheng
et al., 1996). The ﬂuorescence analysis of extracts and the TBA reaction are methods
to study lipid peroxidation (Slater, 1984). Our results showed an age-related increase
in ﬂuorescent and TBArs substances in the extracts of the adrenals, which is
consistent with their presence in lipofuscin granules. Lipofuscin does not exert any
obvious damaging effect on cells (Gershon, 1988) but it is possible that, at a certain
moment, this accumulation may interfere with cellular functions (Brunk et al., 1992).
Such effects may occur in the IZ cells, particularly in ZF, and therefore contribute
to a lesser functioning.
Beyond an age-related decline in the function of ZF cells, the pattern of age-related
decline in ACTH levels observed after a maximum functioning at around 12 months
is possibly related to a post-maturational impairment in higher levels of the HPA
axis. In the hypothalamus, there is an age-related decline in corticotropin-releasing
factor (CRF) and pro-CRF mARN content (Cizza et al., 1994) and a similar decrease
in anterior pituitary CRF receptors in animals older than 12 months (Heroux et al.,
1991). However, POMC mRNA content was found unchanged (Heroux et al., 1991)
or minimally decreased (Cizza et al., 1994) in aged animals. In fact, both pituitary
ACTH content and pituitary cells ability to secrete ACTH in response to CRF
stimulation were found to be higher in aged animals (Cizza et al., 1994). It is thus
possible that, beyond the cells of the IZ of the adrenal cortex, age-related functional
impairment may affect other areas of the HPA axis, higher than the pituitary.

Acknowledgements
We thank Prof. Henrique Barros, Department of Hygiene and Epidemiology,
Faculty of Medicine of Porto, for valuable comments and help in the statistical study.

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References
Almeida, H., Magalhaes, M.M., Serra, T.A.M., Hipolito-Reis, C., Magalhaes, M.C., 1986. Ultrastruc˜
´
˜
tural and biochemical alterations produced in rat adrenal cortex by 4-aminopyrazolopyrimidine. J.
Ultrastruct. Mol. Struct. Res. 97, 1–9.
Almeida, H., Magalhaes, M.C., Magalhaes, M.M., 1995. Adrenal zonation and age-related changes in
˜
˜
macrophage number. Age 18, 194–195.
Baker, D.E.J., 1978. Reproduction and breeding. In: Baker, H.J., Russell-Lindsey, J., Weisbroth, S.H.
(Eds.), The Laboratory Rat. Biology and Disease, Vol. 1. Academic Press, Orlando, FL, pp.
105–121.
Barka, T., Andersen, P.J., 1962. Histochemical methods for acid phosphatases using hexazonium
pararosanilin as coupler. J. Histochem. Cytochem. 10, 741 – 753.
Brunk, U., Ericsson, J.L.E., 1972. Electron microscopical studies on rat brain neurons. Localization of
acid phosphatase and mode of formation of lipofuscin bodies. J. Ultrastruct. Res. 38, 1 – 15.
Brunk, T.U., Jones, C.B., Sohal, R.S., 1992. A novel hypothesis of lipofuscinogenesis and cellular aging
based on interactions between oxidative stress and autophagocytosis. Mutation Res. 275, 395 – 403.
Burek, J.D., Hollander, C.F., 1980. Experimental gerontology. In: Baker, H.J., Russell-Lindsey, J.,
Weisbroth, S.H. (Eds.), The Laboratory Rat. Research applications, Vol. 2. Academic Press,
Orlando, FL, pp. 149–159.
Cheng, B., Kowal, J.C., 1994. Adrenal aging: accumulation of cholesteryl esters and lipofuscin granules.
Endocrine 2, 1097–1106.
Cheng, B., Tserng, K., Kowal, J., 1996. Characterization and identiﬁcation of an adrenal age-related
nonpolar ﬂuorescent substance. Endocrinology 137, 2447 – 2456.
Chio, K.S., Tappel, A.L., 1969. Synthesis and characterization of the ﬂuorescent products derived from
malonaldehyde and amino acids. Biochemistry 8, 2821 – 2827.
Cizza, G., Calogero, A.E., Brady, L.S., Bagdy, G., Bergamini, E., Blackman, M.R., Chrousos, G.P.,
Gold, P.W., 1994. Male Fischer 344/N rats show a progressive central impairment of the hypothalamic–Pituitary–Adrenal axis with advancing age. Endocrinology 134, 1611 – 1620.
Coleman, P., Finch, C., Joseph, J., 1990. The need for multiple time points in aging studies (Editorial).
Neurobiol. Aging 11, 1–2.
Daems, W.T., Wisse, E., Bredero, P., 1969. Electron microscopy of the vacuolar apparatus. In: Dingle,
J.T., Fell, H.B. (Eds.), Lysosomes in Biology and Pathology, Vol. 1. North Holland, Amsterdam, pp.
64–114.
de Duve, C., Wattiaux, R., 1966. Functions of the lysosomes. Ann. Rev. Physiol. 28, 435 – 492.
Deane, G., 1962. The anatomy, chemistry and physiology of adrenocortical tissue. In: Eichler, O.,
Farrah, A. (Eds.), Handbuch der Experimentellen Pharmakologie. Springer, Berlin, pp. 1 – 185.
¨
Dillard, C.J., Tappel, A.L., 1984. Fluorescent damage products of lipid peroxidation. Methods Enzymol.
105, 337–341.
Essner, E., Novikoff, A.B., 1961. Localization of acid phosphatase activity in hepatic lysosomes by
means of electron microscopy. J. Biophys. Biochem. Cytol. 9, 773 – 784.
Estivariz, F.E., Lowry, P.J., Jackson, S., 1992. Control of adrenal growth. In: James, V.H.T. (Ed.), The
Adrenal Gland, 2nd edn. Raven Press, New York, pp. 1 – 42.
Farris, E., 1949. Breeding of the rat. In: Farris, E.J., Grifﬁth, J.Q., Jr. (Eds.), The Rat in Laboratory
Investigation, 2nd ed. Lippincott, Philadelphia, PA, pp. 1 – 18.
Frank, A.L., Christensen, A.K., 1968. Localization of acid phosphatase in lipofuscin granules and
possible autophagic vacuoles in interstitial cells of the guinea pig testis. J. Cell Biol. 36, 1 – 13.
Gershon, D., 1988. Oxidative and peroxidative damage and its role in the aging process. In: Quintanilha,
A. (Ed.), Reactive Oxygen Species in Chemistry, Biology and Medicine. NATO ASI Series — Series
A: Life Sciences, Vol. 146. Plenum, New York, pp. 211 – 219.
Gwynne, J.T., Strauss, J.F. III, 1982. The role of lipoprotein in steroidogenesis and cholesterol
metabolism in steroidogenic glands. Endocr. Rev. 3, 299 – 329.
Gwynne, J.T., Hess, B., Hughes, T., Rountree, R., Mahaffee, D., 1984. The role of serum high density
lipoproteins in adrenal steroidogenesis. Endocr. Res. 10, 411 – 430.

H. de Almeida et al. / Mechanisms of Ageing and De6elopment 105 (1998) 1–18

17

Heroux, J.A., Grigoriadis, D.E., De Souza, E.B., 1991. Age-related decreases in corticotropin-releasing
factor (CRF) receptors in rat brain and anterior pituitary gland. Brain Res. 542, 155 – 158.
Hess, G.D., Riegle, G.D., 1970. Adrenocortical responsiveness to stress and ACTH in aging rats. J.
Gerontol. 25, 354–358.
Hornsby, P.J., Simonian, M.H., Gill, G.N., 1980. Aging of adrenocortical cells in culture. Int. Rev.
Cytol. Suppl. 10, 131–162.
Hornsby, P.J., 1985. The regulation of adrenocortical function by control of growth and structure. In:
Anderson, D.C., Winter, J.S.D. (Eds.), Adrenal Cortex. Butterworth, London, pp. 1 – 31.
Hyatt, P.J., Wale, L.W., Bell, J.B.G., Tait, J.F., Tait, S.A.S., 1980. Cyclic AMP levels in puriﬁed rat
adrenal Zonae Fasciculata and Reticularis cells and the effect of adrenocorticotrophic hormone. J.
Endocr. 85, 435–442.
Jayne, E.P., 1957. Histochemical and degenerative changes in the adrenal cortex of the rat with age. J.
Gerontol. 12, 2–8.
Long, J.A., 1967. Zonation of the mammalian adrenal cortex. In: Greep, R.O., Astwood, E.B. (Eds.),
Handbook of Physiology, Section 7 (Endocrinology), Vol. 6. American Physiological Society,
Washington, DC, pp. 25–39.
Lowry, O.H., Rosebrough, N.Y., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin
Phenol reagent. J. Biol. Chem. 193, 265 – 275.
Magalhaes, M.C., Resende, C., Hipolito-Reis, C., Magalhaes, M.M., Coimbra, A., 1979. Ultrastructural
˜
´
˜
changes in rat adrenal cortical cells after chronic stimulation with i 1 – 24-Corticotropin. Cell Tissue
Res. 204, 267–277.
Magalhaes, M.C., Vitor, A.B., Magalhaes, M.M., 1985. Effects of vinblastine and colchicine on the rat
˜
˜
adrenal cortex. Morphometric and cytochemical study. J. Ultrastruct. Res. 91, 149 – 158.
Malamed, S., 1975. Ultrastructure of the mammalian adrenal cortex in relation to secretory function. In:
Greep, R.O., Astwood, E.B. (Eds.), Handbook of Physiology, Section 7 (Endocrinology), Vol. 6.
American Physiological Society, Washington, DC, pp. 25 – 39.
Malamed, S., Carsia, R.V., 1983. Aging of the rat adrenocortical cell: response to ACTH and cyclic
AMP in vitro. J. Gerontol. 38, 130–136.
Nickerson, P.A., Feld, L.G., VanLiew, J.B., 1979. Zona reticularis in aging spontaneously hypertensive
rats. Am. J. Pathol. 93, 433–448.
Nussdorfer, G.G., 1986. Cytophysiology of the adrenal cortex. Int. Rev. Cytol. 98, 1 – 405.
Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric
acid reaction. Anal. Biochem. 95, 351 – 358.
Paavola, L.G., 1978. The corpus luteum of guinea pig: III. Cytochemical studies on the Golgi complex
and GERL during normal postpartum regression of luteal cells, emphasizing the origin of lysosomes
and autophagic vacuoles. J. Cell Biol. 79, 59 – 73.
Popplewell, P.Y., Azhar, S., 1987. Effects of aging on cholesterol content and cholesterol-metabolizing
enzymes in the rat adrenal gland. Endocrinology 121, 64 – 73.
Reaven, E., Kostrna, M., Ramachandran, J., Azhar, S., 1988. Structure and function changes in rat
adrenal glands during aging. Am. J. Physiol. 255, E903 – E911.
Rebuffat, P., Belloni, A.S., Rocco, S., Andreis, P.G., Neri, G., Malendowicz, L.K., Gottardo, G.,
Mazzocchi, G., Nussdorfer, G., 1992. The effects of aging on the morphology and function of the
zonae fasciculata and reticularis of the rat adrenal cortex. Cell Tissue Res. 270, 265 – 272.
Reichel, W., 1968. Lipofuscin pigment accumulation and distribution in ﬁve rat organs as a function of
age. J. Gerontol. 23, 145–153.
Richardson, K.C., Jarret, L., Finke, E.H., 1960. Embedding in epoxy resins for ultrathin sectioning in
electron microscopy. Stain Technol. 35, 313 – 325.
Ringler, D.H., Dabich, L., 1978. Hematology and clinical biochemistry. In: Baker, H.J., Russell-Lindsey,
J., Weisbroth, S.H. (Eds.), The Laboratory Rat. Biology and Disease, Vol. 1. Academic Press,
Orlando, FL, pp. 105–121.
Rohr, H.P., Bartsch, G., Eichenberger, P., Rasser, Y., Kaiser, Ch., Keller, M., 1976. Ultrastructural
morphometric analysis of the unstimulated adrenal cortex of rats. J. Ultrastruct. Res. 54, 11 – 21.
Samorajski, T., Ordy, J.M., 1967. The histochemistry and ultrastructure of lipid pigments in the adrenal
glands of aging mice. J. Gerontol. 22, 253 – 267.

18

H. de Almeida et al. / Mechanisms of Ageing and De6elopment 105 (1998) 1–18

Sapolsky, R.M., Krey, L.C., McEwen, B.S., 1983. The adrenocortical stress-response in the aged male
rat: impairment of recovery from stress. Exp. Gerontol. 18, 55 – 64.
Scaccianoce, S., Sciullo, A.D., Angelucci, L., 1990. Age-related changes in Hypothalamo – Pituitary –
Adrenocortical axis activity in the rat. In vitro studies. Neuroendocrinology 52, 150 – 155.
Schlettwein-Gsell, D., 1970. Survival curves of an old age rat colony. Gerontologia 16, 111 – 115.
Shaposhnikov, V.M., 1985. The ultrastructural features of secretory cells of some endocrine glands in
aging. Mech. Ageing Dev. 30, 123–142.
Slater, T.F., 1984. Overview of methods used for detecting lipid peroxidation. Methods Enzymol. 105,
283–293.
Sohal, R.S., 1984. Assay of lipofuscin/ceroid pigment in vivo during aging. Methods Enzymol. 105,
484–487.
Sonntag, W.E., Goliszek, A.G., Brodish, A., Eldridge, J.C., 1987. Diminished diurnal secretion of
Adrenocorticotropin (ACTH), but not corticosterone, in old male rats: possible relation to increased
adrenal sensitivity to ACTH in vivo. Endocrinology 120, 2308 – 2315.
Sperry, W.M., Webb, M., 1950. A revision of the Schoenheimer – Sperry method for cholesterol
determination. J. Biol. Chem. 187, 97 – 110.
Szabo, D., Dzsinich, C., Okros, I., Stark, E., 1970. The ultrastructure of the aged rat zona fasciculata
´
¨
under various stressing procedures. Exp. Gerontol. 5, 335 – 337.
Tang, F., Phillips, J.G., 1978. Some age-related changes in Pituitary – Adrenal function in the male
laboratory rat. J. Gerontol. 33, 377–382.
Topping, T.M., Travis, D.F., 1974. An electron cytochemical study of mechanisms of lysosomal activity
in the rat left ventricular mural myocardium. J. Ultrastruc. Res. 46, 1 – 22.
Vahouny, G.V., Chanderbhan, R., Noland, B.J., Scallen, T.J., 1984. Cholesterol ester hydrolase and
sterol carrier proteins. Endocr. Res. 10, 473 – 505.
Weibel, E.R., Bolender, R.P., 1973. Stereological techniques for electron microscopic morphometry. In:
Hayat, M.A. (Ed.), Principles and Techniques of Electron Microscopy, Vol. 3. Van Nostrand-Reinhold, New York, pp. 237–296.

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