original

Effect of maternal
malnutrition
lactation
on hepatic
protein
infant
rat: biochemical
and
0.

Enwonwu,3

D.Sc.,

M.D.S.,

and

Vicki

The prenatal
and suckling
periods
of life
in mammals
are characterized
by marked
anabolic
changes
(1-3),
and
the
rate
of
growth
and development
of various
organs
during
these
stages
shows
a critical
dependency on the supply
of adequate
nutrients
as
well as the ability
of the young
to utilize
the
nutrients
(2, 3). In underpriviledged
societies,
poor
lactation
performance
resulting
from
maternal
malnutrition
is a common
finding,
and this is believed
to play
a major
role in
the genesis
of protein-calorie
malnutrition,
which
is widespread
in such communities
(4,
5). Available
data from experimental
animal
studies
(1, 2, 6, 7) do confirm
the importance
of adequate
maternal
nutrition
during
pregnancy
and lactation
in the subsequent
growth
of the progeny.
Some
of the latter
studies
(1,
8) have attributed
a dominant
role to the protein value
of the maternal
diet.
Developmental
changes
in livers
of growing rats have
been
the subject
of extensive
biochemical
studies
(1, 2), and some of these
investigations
(1, 6, 7) have
examined
the
consequences
of maternal
malnutrition.
The
emphasis
on the liver in many
of these
nutritional
studies
derives
partly
from
the fact
that
it is one of the organs
most
severely
affected
in prolonged
protein-calorie
malnutrition
in the human
(9) and in experimental
animals
(10, 11). The purpose
of the present
study was to make further
contribution
to the
subject
of the influence
of early malnutrition
on hepatic
protein
metabolism
by correlating
the biochemical
alterations
with morphologic
changes.
The

American

Journal

of clinical

Nutrition

26:

JANUARY

Glover,4

pregnancy
in the
studies2

and

B.S.

Materials
A izima/s

and

methods

and diets

Timed
pregnant
rats of the Sprague-Dawley
strain
(Simonsen
Laboratory,
Gilroy,
California).
obtained
at day
14 of the
first
pregnancy
were
randomized
group.
The

into
large

two large
groups
groups
were
fed

and
ad

a third
libitum

small
diets,

supplying
either
18 or 3.5%
protein.
The
third
group
was fed a 6% protein
diet. The compositions
of the synthetic
diets are shown
in Table
1. Food
intake
by the rats was monitored
throughout
the
duration
of the experiment.
Animals
fed the 18%
protein
diet had average
daily food intakes
of 18
to 23 g, compared
with
16 to 21 g in the group
fed a 6% protein
diet.
Rats
fed
a diet
containing
19

3.5%
g/day.

continuously.
litter sizes

protein
Water

were

consumed
was
made

on the
available

Immediately
reorganized

tion

on

days

2, 5, 12, and

Results.

Most

following
as indicated
the

12
the

to
rats

parturition,
in the sec-

were
killed
on
and in order to
eliminate
possible
effects of diurnal
rhythm
(12),
the rats were
killed at essentially
the same
time
of the day at each stage of the study. The suckling

21,

of

average
to

pups

postnatally,

rats were removed

from
their mothers
just before
being
killed
as other
studies
(2, 13) have
shown
that early
in life, relatively
short
fasting
periods
produce
considerable
disaggregation
of
hepatic
polyribosomes
with an associated
decrease
in protein synthesis
in the rat. Each
rat was decapitated
without
anesthesia.
1

From

the

Center

for

Research

in Oral

Biology

and Department
of Oral
Biology,
University
of
Washington,
Seattle,
Washington
98195.
Supported
in part by Grant
FR05346-08
made
available
by the University
of Washington,
and by
Public
Health
Service
Research
Grant
DE-0260004 from
the National
Institute
of Dental
Research,
Bethesda,
Associate

1973,

pp.

Maryland.
Professor.

3-16.

Printed

Research
in U.S.A.

Technician.

Downloaded from ajcn.nutrition.org at Ghana: ASNA Sponsored on July 29, 2014

Cyril

during
metabolism
ultrastructural

communications

ENWONWU

TABLE

Composition

sodium
deoxycholate
treatment
was
omitted in
order
to obtain
profiles
of monomeric
and dimeric
ribosomes
sedimenting
free
of the endoplasmic
membrane
(18, 19). In evaluating
the polyribosomal
tracings,
any
260
m
absorbing
material
larger than a dimer
(n > 2) was considered
to be
polysome.

of synthetic diets
6%
protein

18%
protein

3.5%

protein

g/kg

Salt

mixture

Vitamin
mixture
OiI, ml
Choline
chloride,c
ml (20%)

10.0

Rogers

Wesson

and

Harper
percent

solution

amino

fractionation
hepatic ribosomes
The
several
0.375

and isolation

livers
were quickly
volumes
of ice-cold
M

Laboratory,

ribonuclease-free
New York),

sucrose
0.005
pH

liver

ribonuclease

and chilled
in
1 containing

(Mann

Research

MgCl2,
0.025
M
7.6. Subsequent
opM

enzyme

inhibitor

(nine

parts of medium
I to one part of inhibitor
fraction),
using a Potter-Elvehjem
homogenizer
with a closefitting
Teflon
pestle.
The
crude
ribonuclease
enzyme inhibitor
from rat liver was prepared
as previously
reported
(14) and stored
at -20
C for no
longer
than
1 week
before
use. In some
of the
experiments
aimed
at evaluating
the effectiveness
of liver postmicrosomal
supernatant
as a source
of RNase
inhibitor, the test liver
samples
were
homogenized
in either
medium
I alone
or in undiluted
liver
ribonuclease
inhibitor fraction. Following
treatment
of the homogenate
with
antihorse

ferritin

serum

(Nutritional

Biochemicals,

Cleveland, Ohio) to remove liver ferritin (15), cell

debris,
nuclei, and mitochondria
were sedimented
by spinning in a refrigerated Sorvall-SS1 rotor for
15 mm at 1,230 X g average.
The relatively
low
speed
of centrifugation
was undertaken
to minimize
loss of bound
polysomes
into the nuclear
pellet, as the popular conventional method
of preparing
postmitochondrial
supernatant
by spinning
the homogenate
at 10,000
to 15,000
X g average
yields
a fraction
representing
a mixed
population
of

polyribosomes

but

skewed

more

the

liver

for

analysis

of

free

microscopy

Immediately

KC1, and 0.05 M tris-HCI,

erations were carried
out at a temperature
near
0 C. The minced
liver sample was homogenized
in
two volumes (w/v)
of a mixture
of medium
I and

crude

of

(w/v).

of

removed
medium

pooi

amino
acid
concentration
was as previously
reported
(10). Chromatography
was carried
out with
a Beckman
Model
120B AutoAnalyzer
using DC-i
and DC-2
resins
(Durrum
Instrument,
Palo Alto,
California)
in the
long and short columns,
respectively.
The
reproducibility
of this analytical
system
was 6% in four separate
runs using
aliquots
of the same sample.
Electron

Tissue

acid

Fractionation

b Wesson
Fullerton,

(25).

Oil Sales Company,

Twenty

Free

in

favor

of

the

free polysomes
(16). Analysis
of the liver ribosomal
profiles in continuous
sucrose
gradients
(10
to
50%)
and identification
of the major peaks were
as previously
described
(10, 17). The
profiles
of
total
ribosomes
(riembrane-bound
and free ribosomes)
were
resolved
from
postmitochondrial
supernatant
treated
with sodium
deoxycholate
(DOC)
to a final concentration
of 0.8%.
In some
cases,

following

decapitation

of

the

ani-

mals,
portions
of the liver were fixed for 2 hr in
6.25%
glutaraldehyde
and 0.5%
acrolein
in Daltons buffer
(20), washed
overnight
in 0.14 M sucrose in Daltons
buffer,
and postfixed
for 1.5 hr at
room
temperature
in 1.0% osmium
in Caulfields
buffer
(0.057
M veronal
acetate
and 0.13 M sucrose,
pH 7.4). Following dehydration and embedding
in
Araldite, sections approximately
500 A were cut on
a Huxley
ultramicrotome,
mounted
on carboncoated
acetate

copper
grids,
double-stained
and
lead
hydroxide
(21),

an RCA
Oilier

EMU-3G

uranyl

examined

in

analyses

Liver
were

electron

with

and
microscope.

contents

estimated

of
as

RNA,

previously

DNA,
reported

and

protein
(10,

11).

For assay of the nucleic acids, liver samples were

homogenized
in 0.2 N perchioric
acid.
Liver
ribonuclease
enzyme-inhibitor
activity
was assayed
according
to Shortman
(22). Partially
purified
liver RNase
inhibitor
was prepared
as described
by Sirakov
and
Kochakian
(23).
Bovine
pancreatic
ribonuclease
(EC
2.7
7.16)
(Sigma
Chemical
Company,
St. Louis,
Mo.)
was
used.
The
substrate
RNA
was processed
according
to
Wojnar
and Roth
(24) to eliminate
metallic
ions
which inactivate
ribonuclease
inhibitor
activity.

Results
Liver

composition

2 summarizes
the effects
of maternal
diet, age, and litter
size on body
weight
and
liver
composition
of the young
rat. Among
the offspring
of rats fed the 18% protein
diet
during
pregnancy
and lactation,
changes
in
liver
weight
and composition
were
minimal
during
the 1St week
of postnatal
life. In the
young
rats suckled
in litter
sizes
of 9 or 10
Table

Downloaded from ajcn.nutrition.org at Ghana: ASNA Sponsored on July 29, 2014

After

#{176}

Oil from
California.

35.0
544.6
263.4
50.0
5.0
100.0
10.0

60.0
519.6
263.4
50.0
5.0
100.0
10.0

180.0
442.0
221.0
50.0
5.0
100.0

Lactalbumin
Dextrin
Sucrose

GLOVER

AND

PERINATAL

TABLE
Effect

AND

Maternal
diet,
protein

diet,

Litter
Size

age,

No. of
litters

and

litter

Body

size

weight,
g

011

lIVer

compOsit

18.0
18.0

9
13

1(4)
2(8)

3(12)

3.5

9
10

1(7)

18.0

15

2(6)

3.5
18.0

12

18.0
3.5

9
10
9
5-9

9
15
4
7

3(8)
2(8)

3(11)
4(15)
2(6)
1(9)
2(4)
1(7)
1(14)
3(12)

6.71
6.48
5.30
9.13
9.57
5.61
11 .40
21 .69
8.84
45.38
28.03
36.23
21 .67
10.81
10. 15

0.23
0.17
0.04
0.38
0.29
0.48
0.60
0.69
0.33
1.20
0.49
2.62
0.27
0.25
0.54

of the

young

rat

Liver
Weight,

mg

261.5
254.2
206.4
272.3
336.0
165.4
377.2
593.5
232.1
1,875.0
920.0
1,483.3
783.4
326.3
361.8

4.7

9.9

8.0

10.0

13.6

10.8
19.2
26.3

10.0

137.6

40.6

92.8
20.6
18.0
21.6

mg/liver

2.45
2.26
1.68
2.06
3.22
1.56
3.27
4.99
1.96
13.56
6.94
10.61

0.01
0.12
0.04
0.16
0.22
0.09
0.17
0.35
0.18
1.12
0.28
0.86

5.64
2.47
2.87

0.15
0.18
0.16

DNA,

mg/liver

Protein,

mg/liver

2.33
2.67
1.53
2.02

48.64
39.32
35.91
41.37
52.06
29.00
55.57
93.35
41.10
372.18
153.19
219.00
33.55

1.78
1.59
0.71
2.36
1.99
1.53
2.53
:1::8.38
1.94
19.29
2.60
8.15

3.07
1.67

59.06

2.15

1.36
2.46
3.21
1.55
8.70
4.73
7.09
4.49
2.24

2.18

0.08
0.12
0.06
0.07
0.07
0.06
0.16
0.15
0.09
0.46
0.18
0.54
0.16
0.13
0.11

103.63

4.07

#{176}Data
based
on duplicate
analysis
for RNA
and triplicate
assays
for DNA
and protein.
Each
rat
assayed separately. Values are expressed as mean SE. Numbers
in parentheses refer to number
of rats
studied.
b Pregnant
rats fed respective
diets from day 14 of pregnancy all through lactation.

by adequately
fed
mothers,
alterations
in
body
weight,
liver weight,
and liver contents
of RNA,
DNA,
and protein
between
days
2
and
6 were
+70,
+44,
+33,
+6,
and
+14%,
respectively.
By approximately
the
end of the 1st week
of life, more
marked
elevations
in liver weight
and liver composition occurred
in the well-fed
young
rats, and
at day 12, the net increases
in liver weight,
RNA,
DNA,
and
total
protein
compared
with
corresponding
values
at day
2 were
127,
104,
38, and
92%,
respectively.
Between
days
2 and
21,
body
weight,
liver
weight,
and liver
contents
of RNA,
DNA,
and protein
increased
6.8-,
7.2-,
5.5-,
3.7-,
and
7.6-fold,
respectively,
in
young
rats
suckled
in groups
of 9 or 10 by mothers
fed
the
18%
protein
diet.
Comparative
values
in rats raised
in litter
sizes of 13 to 15 by
adequately
fed mothers
were
4.3,
3.6,
3.1,
1.8, and 3.9, respectively.
By day 2, the offspring
of mothers
fed the 3.5%
protein
diet
showed
significant
reduction
in liver
weight
(P < 0.005)
and liver
nucleic
acids
(P <
0.001)
compared
with rats suckled
in similar
litter sizes by well-fed
mothers.
Between
days
2 and 21, body weight,
liver weight,
and liver
contents
of RNA,
DNA,
and protein
in the
young
of malnourished
mothers
fed the 3.5%
protein
diet increased
1.9-,
1.8-,
1.7-,
1.4-,
1.6-fold,
respectively,
so that at weaning
on

day
21, these
malnourished
weanlings
resembled
6-day-old
rats raised
by adequately
fed mothers.
Table
2 shows
the combined
effects
of maternal
protein-calorie
malnutrition and large
litter size on the development
of the rat liver.
By day 21, postnatally,
rats
raised
in a litter
size of 14 by a mother
fed
6% protein
diet during
pregnancy
and lactation were indistinguishable
from the offspring
(litter
size ranging
from
five
to eight)
of
mothers
fed 3.5% protein.
In contrast,
rats
that were suckled
in groups
of four by dams
fed
6%
protein
had
body
weights,
liver
weights,
and liver
contents
of nucleic
acids
and protein
which
were comparable
to findings in weanling
rats raised
in a litter size of
nine by mothers
fed the 18% protein
diet.
Free

amino

acid

pooi

Table
3 shows
concentrations
of 17 free
amino
acids in the livers of young
rats raised
by well-fed
and protein-deficient
mothers.
In
the progeny
of mothers
fed 18% protein,
no
prominent
differences
were
observed
b#{128}tween
liver aminograms
of rats killed
at 10 or 21
days
of age except
for a higher
concentration of tyrosine
and lower
methionine
in the
younger
animals
compared
with the 21-dayold rats.
The
10-day-old
offspring
of adequately
fed mothers
were
included
ii this
aspect
of the study to serve as a body-weight

Downloaded from ajcn.nutrition.org at Ghana: ASNA Sponsored on July 29, 2014

18.0
18.0
6.0
6.0

Loll

RNA,

18.0

21

LIVER

- -

%h

RAT

2
of maternal

Age,
days

MALNUTRITION

ENWONWU

TABLE
Effect

AND

GLOVER

3
of maternal

diet on

concentrations

of free amino

acids

in the liver of the young

Maternal

Amino

acid

18%

I)ay

2.4
13.6
28.0

Valine
Lysine

16.8
20.0

Phenylalanine
H istidine
Tyrosine

86.4
24.8

Aspartic

acid

Glutamic

acid

381.6
330.4
97.6

Serine
Asparagine-glutarnine

552.8

Glycine
Alanine
Citrulline

107.2
107.2
6.4
40.0
21.6

Ornithine

Proline
Total essential (E)
Total

nonessential

Cummulative

(E,+

0.8
0.8
1.4
0.8
1.6
1.4
1.6
16.9

N)

6.9

0.6

13.2
25.1

0.5
0.7
0.5
4.2
0.4
5.4
0.6
9.4

2.1

2.2

24.2

2.3

18.5

18.4
29.3
8.8

79.9
9.9
259.4
330.4
84.5
649.2
100.5

15.3

4.5
24.4
5.6
5.6

0.8

153.0
13.6

0.8

32.9

2.4

22.0

180.0
,669.6

(N)

total

0.0

1,849.6

Day

21c

40.0
9.2

19.1
5.4
5.6

181.6

1,655.4
1,837.0

2l

Signiiicance

17.0
11.8
29.5
8.0
58.4
5.1

193.9

0.4
0.8
1.2
1.1
2.4
0.4

285.8

93.6

30.7

54.5

5.3
9.4

367.3
166.6

163.0
8.5

protein

3.4
0.3

P
P
P

<0.005
< 0.001
< 0.001

P
P

<

0.01

<

0.001

P
P

<
<

0.001
0.05

17.0
12.9
26.1
21.9
1.3
2.3
0.4

139.4
1,326.5
1,465.9

Students
i test
(comparison
of values
of day 21).
Concentrations
are expressed
as micromoles
per 100 g (wet weight)
of liver tissue.
Data
based
on
six separate assays for each group
with each assay performed
on pooled
sample
from several
rats. Values
are expressed
as mean SE.
b 10 rats.
c 30 rats.
36 rats.
Includes threonine.

control
for the malnourished
weanlings
aged
21 days. Comparison
of the offspring
of wellfed and malnourished
mothers
at 21 days
of age revealed
a significant
reduction
in concentrations
of many
amino
acids
and a net
depression
(-20%)
in total
pooi
size
in
the latter
group.
Among
the
amino
acids
most
affected
were
leucine
(-32%),
valine
(36%),
tyrosine
(-48%),
isoleucine
(-29%),
histidine
(-27%),
and asparagineglutamine
(-43%).
Concentration
of glycine
(+ 66%)
was
higher
in malnourished
than in adequately
fed weanling
rats.
Sedimentation

profiles

of liver

ribosomes

The use of polyribosomal

profiles
to estimate
the potential
for protein
synthesis
in a
mammalian
tissue
requires
that the isolated
profile
reflect
as accurately
as possible
the
size-distribution
of active
ribosomes
in the
intact
cells. The method
of choice
must therefore
insure
quantitative
recovery
of poly-

somes
without
degradation
of the
heavy
mRNA-ribosomal
complexes
that can readily
be produced
by release
of endogenous
ribonucleases
(RNase)
during
tissue
fractionation.
Because
the liver
of a newborn
rat is
characterized
by the presence
of high RNase
activity
which
decreases
with increasing
age
of the animal
(26-29),
preliminary
evaluation
of various
media
for isolating
hepatic
polysomes
was
undertaken
in the present
study.
Figure
1 shows
the
polyribosomal
profiles
isolated
from
deoxycholate-treated
postmitochondrial
supernatants
of livers
from
the 2- (A, B, C) and 5-day-old
(D, E, F)
offspring
of rats fed the 18%
protein
diet
during
pregnancy
and lactation.
At each age,
pooled
livers
from
several
rats were
divided
into three
fractions
and homogenized
in sucrose-TKM
medium
(A, D), 10% postmicrosomal
supernatant
from rat liver (B, E), and
undiluted
liver
postmicrosomal
supernatant
(C, F). Details
of the composition
and prep-

Downloaded from ajcn.nutrition.org at Ghana: ASNA Sponsored on July 29, 2014

Methionine
lsoleucine
Leucine

12.8

35(

Day

rat

diet

protein

lOt

suckling

PERINATAL

M,1.NUTR11lON

AND

aration
of the various
homogenizing
media
were
described
in Materials
and
methods.
Findings
in Fig. 1 indicate
that fractionation
of liver
samples
from
newborn
rats in diluted
postmicrosomal
supernatant
or in sucroseTKM
medium
increased
the yield
of slow
sedimenting
fast

ribosomes

sedimenting

observations
studies
livers

were

(pH

the

7.8)

expense

of

LIVFR

045.f 4

the

aggregates.
These
by biochemical

ribosomal

which
showed
were
endowed

activity

at

RAT

supported
that
with

the
high,

resulting

newborn
rat
free
RNase

from

0
00

presence

activity
Until

030

8ttom

Top

Top

BoTom

to

5Q/

Bottom

0--

Bttom

Ttop

-----500.

-50%

i---

-5O

2. Sedimentation
profiles
of liver
ribosomes
from
2-day-old
rats. The rats were the offspring
of well-fed
(A, C) and malnourished
mothers (B, D). Profiles
were
isolated
from
sodium
deoxycholate
treated
(C, D) and untreated
(A, B)
postmitochondrial
supernatants.
The
tissues
were
homogenized
in undiluted liver postmicrosomal
suFIG.

pernatant,
taining

FIG.
1. Sedimentation
profiles
of rat liver ribosomes
in continuous
sucrose
gradients
(10 to 50%).
Pooled
livers
from
four
rats aged
2 days
(A, B,
C) or 5 days
(D, E, F) were
divided
into three
portions
and homogenized
in either
0.375
M
sucrose-TKM
medium
(A, D) or 10% liver postmicrosomal
supernatant
(B, E) or undiluted liver
postmicrosomal
supernatant
(C, F). Profiles
were
isolated
from the offspring
of mothers
fed an 18%
protein
diet. The liver postmicrosomal
supernatant,
used
as a crude
source
of RNase
inhibitor,
was
prepared
by homogenizing
livers
from
rats fasted
overnight
(150
to 200 g) in one volume
(w/v)
of
0.375
trifuging

sucrose-TKM
the

medium,

postmitochondrial

pH

7.6,
and
supernatant

cenat

50,000
rpm for 3 hr. Each
polysome
profile
was
isolated
from
deoxycholate-treated
(0.8%)
postmitochondrial
supernatant.
Other
details
are as described
in Materials
and methods.

and
the
approximately

postmitochondrial
8 mg liver

samples

con-

tissue
were layered
on 4.6 ml sucrose
gradient
(10 to 50%).
Centrifugation
in a Beckman
L2 preparative
ultracentrifuge using
rotor
SW 50.1 was for 1 hr,
at 45,000
rpm.
Extinction
at 260 mz was monitored
automatically
in a Gilford
Model
2400
recording
spectrometer.
Each profile was from pooled
tissues of two rats.

the
of

end

of the

undiluted

tant

sential

for

1st week

life, use
supernathe
samples
was esinhibition
of the free

postmicrosomal
homogenizing

for

adequate

RNase
activity.
Figure
2 shows

the

of postnatal
liver

typical

polyribosomal

profiles
isolated
from liver samples
of 2-dayold offspring
of adequately
fed mothers
(A,
C) and mothers
fed 3.5%
protein
(B, D).
The profiles
were isolated
from deoxycholate
treated
(C, D) and untreated
(A, B) postmitochondrial
supernatants.
Isolation
of the poly-

Downloaded from ajcn.nutrition.org at Ghana: ASNA Sponsored on July 29, 2014

of low endogenous
RNase-inhibitor
(Enwonwu,
unpublished
observations).

ENWONWU

AND

GLOVER

to
to

00

030

os

TC5
-

Bottom

3. Sedimentation
from
12-day-old
(A, C) and malnourished
were from
detergent-treated
FIG.

somes

(A,
same

B) supernatants.
as for Fig.
2.

Tp

-50%

Bottom
-

-.50%

profiles
of liver
nborats suckled
by well-fed
(B, D) mothers.
Profiles
(C, D) and untreated
Other

conditions

were

the

to

somal
profiles
in the absence
of detergent
treatment
(A, B) revealed,
starting
from
top
of the gradient,
peaks
for monomeric
and
dimeric
ribosomes
(80-120
S), and towards
the bottom
of the gradient
is a conspicuous
ultraviolet
absorbing
area,
corresponding
to
the fast sedimenting
ribosomal
aggregates
as
well as to the membranes
of the rough
endoplasmic
reticulum
to which
some of the latter
are attached.
Treatment
of aliquots
of the
same
postmitochondrial
supernatants
with
sodium
deoxycholate
before
isolating
the
profiles
(C, D) produced
a marked
loss of
the large
ultraviolet
absorbing
peak
at the
bottom
of the gradient,
thus indicating
that
a good proportion
of this peak was attributable to the endoplasmic
membrane.
The profiles from
livers
of 2-day-old
offspring
of
malnourished
mothers
(B, D) are character-

ized by prominent
monomer
and dimer peaks
compared
with findings
in the young of ade-

00
0

lop
0

FIG.

4.

Liver

old
and

Bottom
-50%

ribosomal

profiles

from

21-day--

offspring
of mothers
fed 18% protein
(A, C)
3.5%
protein
(B, D) diets.
Profiles
were
isolated in absence
(A, B) and presence
(C, D) of sodium
deoxycholate.
Other conditions
were the same
as for Fig. 2.

Downloaded from ajcn.nutrition.org at Ghana: ASNA Sponsored on July 29, 2014

quately
fed mothers
(A, C). With increase
in
age,
the well-fed
young
rats
demonstrated
increased
prominence
of the polysomes
(n >
2) with concomitant
decrease
in the proportion of liver ribosomes
sedimenting
as monomers and dimers
as revealed
by profiles
isolated
from
12- (Fig.
3A, C) and 21-day-old
(Fig. 4A, C) rats. This was in marked
contrast
to findings
in the progeny
of malnourished
mothers
(Figs.
3 and 4B, D).
Values
for polysomes
(n > 2) and polysome-membrane
complex
obtained
by planimetric
quantitation
of the
relative
proportions
of the
major
peaks
in the
total
ultraviolet
tracing
are summarized
in Table
4. In the profiles
of total
ribosomes
(membrane-bound
and
free)
isolated
from
the
young
of well-fed
mothers
at day
2, fast
sedimenting
polysomes
(n > 2) accounted

MALNUTRITION

PERINATAL

TABLE
4
Yield of polyribosomes
and

protein-calorie

(n

2) from

>

livers

AND

of offspring

RAT

LIVER

of well-fed

deficient mothers
Yield

of polyribosomes

Malnourished1

Well-fed
Age,

days

+ 1)0Cc

Percent

57.5

21

69.2

rat

61.9
72.5

liver

was

and methods.

1.8
1.2
1.0
0.9

homogenized

Percent

(6)
(10)
(17)
(8)

72.5
73.3
84.1
83.1

in the presence

contribution

of 260 m,a absorbing

1.6
1.1
1.9
1.7

55.4
48.3
56.3
55.6

(7)
(10)
(10)
(8)

of excess

of polysomes

exogenous
(ii

>

2)

DOCd

profile

0.6 (6)

70.3

1.1 (9)

1.6
1.6

(6)
(13)

69.0
70.1

2.0

(10)

72.8

0.7
1.9
0.6

RNase

inhibitor

as described

to the total
ultraviolet
tracing
SE. Figures
in parentheses

(8)
(9)
(8)

in
was

calculated
using a compensating
planlmeter.
Data expressed
as mean
refer
to number
of animals.
All rats used were raised
in litter
sizes of 5 to 8 for the malnourished
group and
7 to 9 for the well-fed
group.
Young
of mothers
fed 18% protein.
b Young
of mothers
fed 3.5% protein.
c Sodium
deoxycholate
added
to final concentration
of 0.8%.
d Values
for polysomes
(ii > 2) include
absorption
of
membranes
and
attached
polysomes
as well
as free
polysomes.
Significantly
different
(P < 0.001 or
<0.005)
from
corresponding
values
for the well-fed
age-counterparts.

for 57.5%
of the ultraviolet
tracing,
and
this value
did not change
significantly
until
approximately
the age
of 12 days
(P <
0.00 1). In this group
of well-fed
growing
rats,
the yields
of hepatic
polyribosomes
at
days
12 and 21 after birth were comparable.
At all ages
examined,
except
day
2, the
offspring
of mothers
fed the 3.5%
protein
diet
had
low liver
concentrations
of polysomes
and
polysome-membrane
complex
compared
with the well-fed
age controls.
In
marked
contrast
to findings
in the control
rats, the malnourished
animals
demonstrated
no increase
in liver
polysome
content
with
increasing
age (Table
4).
Electron

,nicroscopy

of

liver

The
morphological
cytodifferentiation
of
the liver in rodents
during
the early postnatal
period
of development
has been described
by
numerous
investigators
(30-32)
and will not
be detailed
here.
The present
report
will focus mainly
on the cytoplasmic
organdies
in
which
the main
bulk
of protein
metabolism
occurs.
At birth,
the rat liver shows
a sudden
decrease
in the number
of cells of the erythropoietic
series
with concomitant
increase
in
hepatocytes.
At approximately
3 days
after
birth,
the normal
rat liver
presents
an ul-

trastructure
virtually
similar
to that
of the
mature
liver
and is characterized
by abundant
smooth
and rough
endoplasmic
reticulum, decreased
population
of free ribosomes,
reduction
in volume
density
of glycogen
areas,
and
more
mitochondria
and
iysosomes
than
were
present
at birth
(31-33).
Figure
5 is an electron
micrograph
of a
liver
section
from
a 10-day-old
offspring
(litter
size 10) of rats fed an 18%
protein
diet. The membranes
of the granular
endoplasmic
reticulum
are arranged
in parallel
stacks,
and
most
of the cytopiasmic
ribosomes
are attached
to the membranes.
In
the cytoplasmic
matrix
are few ribonucleoprotein
particles
with no discernible
attachment
to membranes.
The
cisternae
of the
granular
endopiasmic
reticulum
are narrow
except
for some
small
dilation
at their
ends
on which
attached
ribosomes
are often
conspicuously
absent.
Vesicles
of agranular
endoplasmic
reticulum
are numerous,
and in
most
of the sections
from
well-fed
rats examined
at this
age,
the Golgi
complex
is
well developed.
Marked
variation
was
observed
in the
liver
ultrastructure
of rats suckled
by malnourished
mothers.
The
features
showed
good correlation
with the litter size in which

Downloaded from ajcn.nutrition.org at Ghana: ASNA Sponsored on July 29, 2014

Each

Materials

2
5
12

+ DOC

DOCd

10

ENWONWU

AND

GLOVER

6
E
0

-*

4.
.

-:

C)

4:

p.,:.

.9
ce.9
U-.

_i

..c

:-

.uil

.,-

V0

00.0
-C)
-

C)-.

C)-Cu

0
00
-C)

.-.-,>

0
>0

COO
.

0.

000
0

E
U-

C)

r.

;.

-:

01}

-.-.i
I-

Downloaded from ajcn.nutrition.org at Ghana: ASNA Sponsored on July 29, 2014

Cu,,

PERINATAL

MALNUTRITION

the young
suckling
rats
were
raised.
The
present
report
shows
the ultrastructural
findings in livers
of rats suckled
in a litter
size
of eight
by malnourished
mothers.
By day
10 (Fig.
6), many
of the malnourished
rats
examined
revealed
little abnormal
alterations
in cellular
architecture.
Parallel
stacks
of
membranes

studded

usually

with

prominent

ribosomes

although

sections,

evidence

of

granular

endoplasmic

in

were

some

disorganization
reticulum

was

of

the

of

the

present,

parallel
These

arrangement
abnormal
features

nounced

at

weaning

of

the
became

on

day

membranes.
more
pro21

(Fig.

In some of the livers, abnormal

lipid
lation
was
quite
prominent,
and
invariably
associated
with
profound
ganization
and loss of the endoplasmic
ulum
(Fig.
7B).

7A).

accumuthis
was
disorretic-

Discussion
Protein
biosynthesis
is probably
the most
fundamental
of all the processes
concerned
with
the
rapid
growth
and
development
that
characterize
the prenatal
and suckling
periods

of

life

metabolism
phases

of

(1,

as

evaluated

2).

with

In

the

rat

age

34).

in hepatic

several

reviewed

synthesis

of

radio-

microsomal

at

birth

pro-

but

Associated
protein

Mil-

by

protein

into
low

these

by

incorporation

acids

(1,

Protein

during

studied

liver,

the

is relatively

crease

been
recently

by

amino

(1-3).

organs

has
and

ler

teins,

mammals

various

life

investigators

active

in

in

increases

with

synthesis

acids (I), a decrease

(37)

as well

during

and

total

monomers
sedimenting

in free

liver

Biochemical
in the young

tein
study

that
were
were
in

and
of

aggregates
morphologic
mothers
fed

investigated
good
accord

this

aged

2 to

pool

of free

Milk
ing

the

(38,
observa-

18%
proin the present
with
these
re-

increased

to

12

suckling

70%

at

weaning

thereby
able
to

be achieved
suckling
rats
been

attributed

to

2).
of mammals

period,

and

by increasing
raised
by

claimed

such

that

neonatal
to
(1,

low
dur-

studies

in

In

many

ex-

the

of

the

the

sequelae
to

study,

of the

suckling

was

of
It

can

than

present

mothers

the
severity
varied
which
the
rats
were
weaning
on day
21,
composition

of

size
46).

restriction
rather

in growth

malnourished

the
litter
dam
(1,

the

food

protein

2).

retardation
of

and

animals
(44,
45)
have
demonthat
maternal
malnutrition
has
a
effect
on lactation
performance,
reducing
the amount
of milk availthe offspring.
The
latter
can
also

quantitative

has

days

amino
acids
(1,
is the
natural
food

perimental
strated

deficit

ribosomes

value

(Table
4). Although
Wittman
and
Miller
(40)
interpreted
the
functional
nature
of
polyribosomes
as being
a reservoir
of active
ribosomes
that can be but are not necessarily
used
for
protein
synthesis,
many
reports
(11, 41-43)
indicate
a close
correlation
between
tissue
polysome
content
and the magnitude
of protein
synthesis.
The present
study
also showed
that
quantitative
recovery
of
intact
liver polysomes
during
the early
postnatal
period
requires
the presence
of excess
RNase
inhibitor
during
cell
fractionation
(Fig.
1) in view
of the imbalance
between
RNase
enzyme
and inhibitor
activities
at this
stage
of development.
This
finding
calls
for
reassessment
of some
of the previous
reports
regarding
protein
synthesis
in neonatal
rat liver.
The
presence
of high
RNase
activity
due to low endogenous
RNase
inhibitor may account
for the marked
polysomal
instability
that was observed
in livers
of rats

the

concentrations
of free
ribosomes
sedimenting
as
concomitant
increase
in fast

polyrihosomal

39).
tions

well-fed
suckling
rats,
polyribo> 2) accounted
for 57%
of the
ribosomal
profiles
at 2 days
of age,
(ii

attributed

as in the
with

and

11

LIVER

In

in-

early
postnatal
period
are increased
organ
contents
of nucleic
acids
and proteins
(35,
36), an increase
in liver
concentration
of free
amino

ports.
somes
total

of

the

RAT

evident,

be

caloric
marked

young
and

with
the
litter
size
in
raised
(Table
2).
At
body
weight
and
liver
offspring

of

mothers

fed

the 6% protein
diet were similar
to findings
in the young
of well-fed
mothers
if the former were
raised
in a litter
size of four rats.
This
and
in

suggested
not
such

just

that
proteins

developmental

adequacy
was
and

of
the

food

crucial
growth

intake
factor
studies.

Downloaded from ajcn.nutrition.org at Ghana: ASNA Sponsored on July 29, 2014

and this was often

in the form
of focal
cisternal
dilations,
fragmentation
into short
segments
and vesicles,
increase
in monomeric
free ribosomes,
and some
loss of the usual

AND

12

ENWONWU

AND

GLOVER

Cu
0

Cu

Cu

0
.

C).

.9
CO

U
CO
.

nCu
Cu

>0

C)
0.

00

c.
0...

to

(5

Cu
.

C,,
-0

-1:
0

Cu

0
0.

>0Cu
Con

CO
C.)

0
U....
C.)

.
.-

C.)

C)

Downloaded from ajcn.nutrition.org at Ghana: ASNA Sponsored on July 29, 2014

C,.)

PERINATAL

MALNUTRITION

AND

RAT

13

LiVER

,i

Downloaded from ajcn.nutrition.org at Ghana: ASNA Sponsored on July 29, 2014

Li
7. A) Liver secticn form 21-day-old offspring of rats fed 3.5% protein diet. N, nucleus; E, fragments of
granular
endoplasmic
reticulum:
MI, mitochondria;
(solid arrow),
lysosome;
(open arrow),
free ribosomes
and
vesicles of granular endoplasmic reticulum. Magnification x9,220.
B) Electron
micrograph
of liver section
from 21-day-old offspring of rats fed a 3.5% protein diet. LI, lipid; MI, mitochondria; E, fragments of rough
endoplasnie
reticulum;
(solid arrow),
lysosomes.
Magnification
x 11,760.
FIG.

14

ENWONWU

liver
is minimal
if protein
deficiency
is accompanied
by a gross caloric
deficit
(47).
In underpriviledged
societies
with
a high
prevalence
of the
clinical
conditions
of
kwashiorkor
and marasmus,
severe
maternal
malnutrition
during
pregnancy
and lactation
is a common
feature
(4, 5). In such women,
the yield of breast
milk is markedly
reduced,
especially
after the first few months
of lactation (4, 5). Supplementary
feeding
of suckling infants
is virtually
nonexistent
in such
impoverished
societies.
Although
it may not
be completely
valid to extrapolate
findings
in
suckling
rats
to human
infants,
especially
in view of the faster
rate of growth
in the
former
relative
to the latter
(3), the present
study
suggests
possible
widespread
morphological
as well as functional
defects
in livers
of infants
fed solely
breast
milk by severely
malnourished
mothers.
It is against
this
background
of possible
structural
and functional
liver defects,
albeit
subclinical,
which

GLOVER

are already
present
at the end of the suckling period,
that one must
view
the consequences
of the grossly
inadequate
diets
to
which such infants
are usually
weaned.
Summary
Diets
containing
18, 6, and 3.5%
protein
were fed ad libitum
to pregnant
rats during
the last
week
of gestation
and
continued
throughout
the period
of lactation.
Evaluation of hepatic
protein
metabolism
in the
progeny
of these
well-fed
and malnourished
mothers
showed
that
both
the dietary
protein level of maternal
diet and the litter
size
in which
the young
rats were suckled
were of
crucial
importance.
Between
days 2 and 21,
body weight,
liver weight,
and liver contents
of RNA,
DNA,
and protein
increased
6.8-,
7.2-,
5.5-, 3.7-,
and
7.6-fold,
respectively,
in young
rats suckled
in groups
of nine
by
mothers
fed 18%
protein.
Comparative
values in rats raised
in litter
sizes of 13 to 15
by adequately
fed mothers
were 4.3, 3.6, 3.1,
1.8, and 3.9, respectively.
During
the same
time interval,
liver weight
and liver contents
of

nucleic

acids

and

proteins

increased

one-

to twofold
in the young
(litter
size 5 to 8)
of malnourished
mothers
fed 3.5%
protein.
At weaning,
the livers
of the malnourished
suckling
rats
showed
marked
disorganization of the cytoplasmic
organelles
as exemplified by the breakdown
of the granular
endoplasmic
reticulum
into
short,
often
dilated
fragments
and vesicles,
with
loss of the usual
parallel
stacking
of the granular
membranes.
Such
morphologic
equivalent
of impaired
protein
synthesis
correlated
well
with
the
biochemical
findings
of reduced
liver
contents of nucleic
acids
and proteins,
disaggregation
of polyribosomes
(n > 2) into slow
sedimenting
monomeric
and
dimeric
ribosomes,

hepatic

and
free

reduced
amino
acid

as
well
pool.

as

distorted

We wish to thank
Professor
Leo Sreebny
who
made
facilities
available
for
this
work.
Special
thanks
are due to Patricia
Lewis,
Joan Askew, and
Richard
Granberg
for their
technical
assistance.
The excellent secretarial assistance of Barbara
Carlsson is highly appreciated.

References
1.

S.

MILLER,

growth

and

A.
Protein
development.

metabolism
In: Mammalian

during
Pro-

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The liver,
morphologically
and functionally,
was responsive
to nutritional
status.
Among
the morphological
changes
in livers
of protein-calorie
deficient
suckling
rats at weaning
were
the disorganization
of the cytoplasmic
architecture
as exemplified
by the profound
breakdown
of the granular
endoplasmic
reticulum
into
short,
often
dilated
fragments
and vesicles,
and loss of the usual
parallel
stacking
of the granular
membranes.
Such
alterations
of
the
hepatocellular
granular
endoplasmic
reticulum
constitute
a good
morphologic
index
of impairment
in protein
biosynthesis
although
they are by no means
specific
for malnutrition
(47-49).
The morphologic
changes
were
supported
by biochemical
observations
of reduced
liver contents
of nucleic
acids
and proteins,
marked
disaggregation
of polyribosomes,
and
reduced
as well as distorted
hepatic
free amino
acid
pool.
Abnormal
accumulation
of fat,
although
present
in some
of the livers,
was
not as prominent
as was observed
in previous
studies
(10,
11) in which
rats
aged
4 to 5
weeks
were fed ad libitum
for several
weeks
a diet containing
0.5%
lactalbumin
as the
sole source
of protein.
The latter
finding
indicates
that
the
limiting
nutrients
in the
young
of rats fed protein-deficient
diets during pregnancy
and lactation
are both calories
rand proteins
as accumulation
of fat in the

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