D irc e u S a le , B rig itte R ie c k m a n n , R a q u e l M a tto s C o s ta L ip p e tt, R o n a ld a T a d e u T u c c i L ip p e lt, O lg a M a ria S ilv e rio A m a n c io , S u z a n a d e S o u z a Q u e iro z , C h a rle s K iro v N a s p itz
Z inc deficient diet consequences for pregnancy and
offsprings of w istar rats
Department of Pediatry, Escola Paulista de Medicina - Silo Paulo, Brazil
A d u lt fe m a le W is ta r ra ts (9 0 d a y s o ld ; w e ig h t 1 8 0 to 2 2 0 g ra m s ) w e re s u b m itte d to d iffe re n t z in c d e fic ie n t d ie ts (Z n ; s e v e re = 2 .6 p p m ; m ild
=
9 .0 p p m a n d n o rm a l d ie t=
8 1 .6 p p m ), d u rin g 6 w e e k s . A fte r th is tim e th e y w e re c o u p le d w ith n o rm a l m a le W is ta r ra ts .N o d iffe re n c e s re g a rd in g fe c u n d ity a n d s te rility w e re o b s e rv e d b e tw e e n th e g ro u p s . D u rin g p re g n a n c y , p a rt o f th e a n im a ls fro m s e v e re a n d m ild Z n d e fic ie n t g ro u p s re c e iv e d th e s a m e d ie t a n d th e o th e rs re c e iv e d n o rm a l d ie t. T h e a n im a ls fro m th e g ro u p re c e iv in 9 ..n o rm a l d ie t, w e re d iv id e d in to o th e r 3 : th e firs t re c e iv e d s e v e re , th e s e c o n d m ild Z n d e fic ie n t d ie t a n d th e th ird n o rm a l d ie t.
D u rin g th e s tu d y w e o b s e rv e d th a t a n im a ls s u b m itte d to a Z n d e fic ie n t d ie t (a c u te o r c h ro n ic ) h a d te n d e n c y to lo w e r w e ig h t g a in , lo w e r w e ig h t o f th e o ffs p rin g , lo w e r s e ru m le v e ls o f Z n in m a te rn a l a n d n e w b o rn (p o o l) b lo o d . A s ig n ific a n t re d u c tio n in th e n u m b e r o f a liv e n e w b o rn s w a s o b s e rv e d in th e g ro u p o f a n im a ls s u b m itte d to s e v e re Z n d e fic ie n c y . T h e s e d a ta re in fo rc e s th a t Z n is a v e ry
im p o rta n t tra c e e le m e n t o v e ra ll d u rin g p re g n a n c y .
U N IT E R M S : Z in c . P re g n a n c y . O ffs p rin g . D e fic ie n c y . M a lfo rm a tio n s .
IN T R O D U C T IO N
B
a se did e n tify in go n th e stu d ie sth e z in c (Z n ) a s in d isp e n sa b leo f K E lL IN & M A N N c a rb o n ic(1 5 ,1 6 ), a n h y d ro u s c o m p o n e n t o f th e b lo o d re d c o rp u sc le s,a ro u n d 2 0 0 m e ta llo e n z y m e s o r m e ta llo p ro te in s w e re
id e n tifie d a s Z n d e p e n d e n ts in a ll P h y la (1 ,2 9 ). It is
b e lie v e d th a t th is n u m b e r d o e s n o t su rp a ss 3 0 in th e h u m a n
A d d re s s fo r c o rre s p o n d e n c e :
D irc e u S a le
A v . P a e s d e B a rro s , 8 4 4 -a p to . 6 1 S a o P a u lo - S P - B ra s il- C E P 0 3 1 1 4 -0 0 0
b e in g s, a lso b e in g p re se n t in so m e a ld o se s,
d e sh y d ro g e n a se s, p e p tid a se s a n d p h o sp h a ta se s (1 7 ), w ith
p a rtic ip a tio n in th e m e ta b o lism o f th e c a rb o h y d ra te s,
lip id e s a n d p ro te in s m a in ly in sy n th e sis a n d d e g ra d a tio n
o f n u c le ic a c id s. .
D e p e n d in g o n th e p e rio d th a t th e Z n d e fic ie n t d ie t
in sta ls, th e c lin ic a l e v a lu a tio n c a n b e v a ria b le . D u rin g th e
p re g n a n c y , e ffe c ts o c c u r in a se v e re w a y a n d in te rfe rin g
w ith th e a c tio n o f th e e n z y m e s, d e p e n d e n t a n d n e c e ssa ry
fo r th e c e llu la r d iv isio n a n d re p ro d u c tio n (D N A a n d R N A
p o ly m e ra se s, th y m id in o c k in a se ), m a y le a d to fo e tu s d e a th .
In th is stu d y w e h a v e e v a lu a te d th e re fle c tio n o f th e
d iffe re n t ty p e s o fZ n d e fic ie n t d ie ts c o n c e rn in g W ista r ra ts
~IDmmm,'i13' ;m; iil11;~~.m 1:l'm . ~;m;~;.,:m.~t:~~.:w.;&;,.;mnmf~iil11;',,:,':iil11;.re::sm : m :-.o:,.;.~:.;...:~, ..~~~~ilSl$~~~ilSl$mml~~~~~~mm'iil11;' ,W W.',W ",,'rm :::m ':'m'ii;',.:fm'ii ~m'ii~.~.(;m;.. (..:i13(,W ;m;:.:.~::w :m m A A .:~.m'.,.m .,..'.ml:",ml:.. y.;:m.. '.,..Im '(.~~W ( Wm :w :., .
M A T E R IA L A N D M E T H O D S
Female W istar rats, adults, 180 - 220 grams weight,
were submitted, after 97 days born, to three diets during 6
weeks. These animals were kept in individual polypropilein
cages, glassed floor and covered by steel, fixed to the cage
by rubber tape. Received disionized water and special diet
"ad libitum". They were classified in the following groups,
according to the ration received: D - Ration with severe
deficiency of Zn (2.6 ppm); M '- Ration with moderate Zn
deficiency (9.0ppm) and C - ration without deficiency of
Zn (81.6ppm). After these 6 weeks period, they were
coupled with normal males during 12 hours/night, being
kept only the disionized water. Female W istar rats
presenting spermatozoids in the vaginal secretion were
considerd "pregnant"(G) and continued under study
forming the final seven groups, according to the ration
received during the 21 days pregnancy: DGD - severe Zn
deficiency before and during pregnancy; DGC - severe
Zn deficiency before and ration control during pregnancy;
M GM - M oderate deficiency before and no deficiency
during pregnancy; CGD - severe deficiency during
pregnancy only;: CGM - moderate deficiency during
pregnancy only and CGC - no deficiency before and during
pregnancy. The female that did not become pregnant in
the one week deadline period, even being coupled daily,
were sacrified.
After 21 days pregnancy, rats were submitted .to
cesarea, under ethylic ether anesthesy, and their newborn
constituted the RN groups according denomination of their
mothers.' group. At this moment, were observed the
number of concepts (RN alive, natimorti, foetus
reabsorptions) and the weight of the RN (offspring
average). Samples oftotal heparinized blood (Liquimine@ )
were collected from mothers through cardiac puncture and
from the RN after decapitation ("pool") for the Zn levels
determination, by spectrumphotometry of atomic
absorption (Perkin-Elmer; wave length 213.8nm).
Placentas were colfected as well and, after dried .in the
glasshouse, 250 mg of dried tissues were submitted to
liquid digestion, with a nitric-perchloric acid (2) mixture
and to the determination of the Zn levels through
spectrumphotometry of atomic absorption.
During the research, ingestion of ration and the
ponderal evolution of the animals were followed. The
deficient rations were produced in the Re~earch Laboratory
of the Pediatric Department of the Faculdade de M edicina
in Botucatu, presenting a basic composition of 21 % of
proteins (28). Zn levels were controlled by the addition of
a mineral salt mixture with a known Zn concentration.
682
Statistical analysis was performed through: Student
"T" test, Kruskal- W allis,W i1coxon, and participation of
the qui-square establishing alpha-5% .
R E S U L T S
All the animals presented ponderal gain during
pregnancy. However, animals submitted to the deficient
diet presented moderate results (Tale 1). No difference
was observed in the quantity of animals fecunded
considering the initial type of ration received, D or M or
C. Number of concepts (RN alive, natimorti or foetus
reabsorption) was not different for all the other groups
studied (Table' 2). However, considering the number of
RN alive, smaller values were observed between the
animals of the DGB group (Table 2). The offsprings
average weight in the different groups was almost the same
(Table 1). About 65% of the animals whose mothers were
submitted to deficient diet during pregnancy, presented
paw malformations (fusion and ankles absence).
The Zn plasmic levels of the mothers pertaining to
M GM group were meaningly smaller than the level of
mothers pertaining to the M GC group. In the RN groups,
differences were observed between the M GM < CGM
groups (Table 1). No difference in the Zn placentary levels
were observed (Table 1). No correspondence was observed
between the maternal Zn levels, the "pool" of the RN and
the placentary "pool"of the different groups studied.
D IS C U S S IO N
The Zn nutritional deficiency has been described as
associated to anorexia in a premature installation,
dermatitis, alopecia and growth discontinuing. Generally,
they will be more intensive according to the degree,
duration and the period of life the deficiency was imposed.
During pregnancy, lactation and wean, nutritional Zn needs
are the maximum (18,20,24, 25,28).
W e have observed in this research that, considering
same weights in the begining of pregnancy, the ponderal
gain of animals submitted to Zn acute deficiency (CGM .
andCGD) or chronic (DGD,M GM ), was smaller than the
informed by others (4,7,8,11,18,22-25). It is agreed that
the lower ponderal gain is consequence of smaller
ingestion. Our animals consumed the same ration volume,
fact that did not obligate us to criate "pair fed" groups.
••
D uring the experience, w e observed fur fall, diarreic
occurrence and ulcerous lesions in tails, in the anim als
pertaining to several groups. In spite of descriptions on
higher index of sterility and infecundity in fem ales
subm itted to Zn diet deficiency (3,12,22,25), this has not
been observed in our anim als. A t the end of pregnancy,
the num ber of concepts w as not different from the several
other groups. H ow ever, the num ber of RN alive in the
D G D group w as significantly sm aller than the verified in the others (Table 2).
H U RLEY & TA O (11), studying W istar rats during
pregnancy, obse'rve~ that only 68% of the anim als
subm itted to an acute Zn diet deficiency (0.4 ppm ) had
RN alive and 56% of the foetus im plantation in the w om b
of the m others w ere absorbed or death and 83% of the RN
presented crude m alform ations. These results are
confirm ed by others (8,18,23,24). Pregnancy itself, m ainly
w hen associated to Zn nutritional deficiency, is able to
decrease the Zn serum levels and cause serious problem s
to foetus, since m alform ation till death (3,5,10,14,26,32).
RECO RD et col. (22) studied the Zn deficiency im posed to "Sprange-Faw ley" rats during the first 11 days
pregnancy. They concluded that Zn. deficiency affects the
cellules in large part in w hat refers to cellular division,
m ainly the lateral portion of the cranioneural tube and the
tail region, justifying a higher incidence of m alform ations in the SN C.
In order to avoid any eventual m isunderstanding in
the evaluation of the RN anim als, w e m ade use of the
offspring average w eight. Poor diet anim als show ed
tendency to low er w eights. G enerally, diets w ith m ore
deficiency are usually follow ed by a w eight reduction of
the RN . H ow ever this relation is not very clear w hen rations
w ith lim ited deficiencies are used and that could probably
be partially corrected by using m aternal reserves (7,11,23-25).
D eterm ination of the Zn plasm atic levels'in the
pregnant rats, at the end of this period, show ed sm aller
values for the M G M group anim als in com parison the the
M O C ones (Table 1).
Zn levels in "pool" of the RN plasm a (Table 1),
w hose m others w ere subm itted to any kind of deficiency
(acute or chronic, intensive or slight), show ed tendency
to low er value~ w hen com pared to the control group
(CO C). H ow ever, difference w as only m eaning in the
M O M and CO C groups. These values w ere significantly
higher than the ones of their m others (Table 1). Sim ilar
occurrence has been verified in the hum an beings (13,31).
, A ccording to PRA SA D (21), m aternal Zn w ould be
transfered to foetus by active transportation through
placenta. SIM M ER et col. (27) by m eans of guinea-pigs,
studied the 65Zn distribution furnished to m other, through
a foetusplacentary unit and observed the m aintenance, in
. the RN , of a higher ,gradient betw een the plasm atic and
the m uscular Zn of the RN in com parison to the m aternal
values. They also got sim ilar 65Zn concentrations in the
m aternal liver and foetus. Capacity of the Zn concentration
by the placenta, observe one hour after the unique
intravenous ,dose of 65Zn w as sim ilar to' the liver
concentration. Through controled perfusion of the
Table 1
Main Characteristics Evaluated in Animals Pertaining to the Different Groups Studied (Average Data)
V a r ia b le G r o u p s
O G O O G C M G M M G C C G O C G M C G C
N u m b e r 1 1 1 0 1 3 8 1 1 8 8
I n it ia l w e ig h t ( g ) 2 1 4 , 3 0 2 2 1 , 6 0 2 2 5 , 1 0 ' 2 2 7 , 8 0 2 2 3 , 9 0 2 1 8 , 6 0
F in a l w e ig h t ( g ) 2 8 1 , 4 0 3 2 7 , 5 0 3 0 0 , 4 0 3 1 8 , 6 0 2 9 7 , 7 0 3 1 0 , 8 0 3 2 1 , 3 0
N e s t f u l a v e r a g e w e ig h t ( g ) 4 , 2 7 4 , 7 3 4 , 3 7 4 , 5 7 4 , 3 0 4 , 3 3 4 , 5 0
P la s m a t ic Z n ( m g / d l) 1 2 3 , 8 0 1 4 2 , 4 0 8 4 , 7 0 * 1 5 1 , 0 0 1 1 3 , 6 0 1 3 9 , 7 0 1 3 7 , 6 0
Z n " p o o l" p la s m a R N ( u g / d l) b 2 7 4 , 2 0 - 2 6 8 , 0 0 1 9 9 , 5 0 * 2 5 3 , 3 0 2 2 2 , 7 0 2 6 3 , 2 0 3 4 7 , 0 0
Z n " p o o l" p la c e n t a ( u g / g d r y t is s u e ) 5 1 , 8 0 5 0 , 8 0 4 1 , 6 0 5 2 , 7 0 4 4 , 4 0 4 4 , 0 0 4 5 , 1 0 .
K r u s k a l W a llis a , b - P la s m a t ic Z n M G M < M G C
Z n " p o o l" p la s m a R N - M G C < e G C
Table2
Quantity of concepts, after birth, alive and not, in the different groups studied
G r o u p A liv e N o t a liv e T o ta l % A liv e
D G D 1 0 7 2 0 1 2 7 8 4 .2 5 D G C 1 1 7 5 1 1 6 9 5 .6 5 M G M 1 3 0 8 1 3 8 9 4 .2 0 M G C 7 7 8 8 5 9 0 .5 8 C G D 1 0 7 6 1 1 3 9 4 .6 9 C G M 8 4 1 8 5 9 8 .8 2 C G C 8 4 3 8 7 9 6 .5 5 T O T A L 7 0 0 5 0 7 5 0 9 3 .3 3
Q u i- s q u a r e
x c a lc u la te d
=
2 2 .4 6 * x c r itic a l ( 6 g l;5 % )=
1 2 .5 9 Q u i- s q u a r e p a r titio nD G D x ( D G C + M G M + M G C + C G D + C G M + C G C ) x c a lc u la te d = 1 7 .2 2 * x c r itic a l ( 1 g l;5 % ) = 3 .8 4 D G D < D G C < M G M < M G C < C G D < C G M < C G C
placentary tissue, they observed that the Z n m ass transfered through the placenta w as closely related w ith the plasm atic concentrations, that vary betw een 0.7 and 24.1 ug/m l. U nder norm al conditions, daily transference of the Z n serum is of 0.12m g/day, quantity sufficient for foetus suplim ent and sim ilar to the one incorporated to it in the last third part of the pregnancy. T he authors conclude that the m aternal foetus transfer suggests an active transport at m aternal surface level, com bined w ith slow liberation to the foetus, under a concentration gradient. U nder norm al conditions, 32% of plasm atic Z n circulates linked to alpha-2 m acroglobuline, 66% to album ine and in less quantity to am inoacids, m ainly histidine and cysteine (9). Z IM M E R M A N N et col. (33) observed at the end of pregnancy in hum an beings, sim ilar levels of album ine and inferior to alpha-2 m acroglobuline in sam ples of m aternal serum , w hen com pared to the obtained through
the um bilical vein. H ow ever, the Z n quantity per album ine/ gram w as higher in the R N serum w hile in the one, linked to alpha-2, m acroglobuline w as higher in the m aternal serum . T his w ay, authors discus~the fact that the album ine should be the m ain protein transporter ofZ n to the placenta. T hey consider that album ine is im portant for the Z n transport to tissues of rapid transference (for exam ple intestine, liver and. placenta) and that alpha-2 m acroglobuline interacts w ith the cellules for the transport of Z n for com plex m etabolic requirem ents such as synthesis of D N A through foetus (33).
FA IR W E A T H E R - T A IT and col (8) in the serial of experim ents w ith W istar pregnant rats, subm itted to a discrete Z n deficiency, observed a significant decrease in the Z n levels of placentary tissues of the deficient diet anim als in com parison to those of the control group, on the 19th day of pregnancy. T his low er placentary concentration of Z n resulted in a low er Z n total content in the foetus of these anim als, although the placentary w eight has show ed higher than the one of the control group.
T E R R Y et col. (30) studying Z n placentary transport in pregnant fem ale rabbits, in the third quarter of pregnancy, observed bigger 65Z n passage through the placenta and a better foetus capture w ith the evolution of the pregnancy. T he absence of65Z n in the placentary tissue m ade these authors define placenta as a transport organ and not a Z n reservatory. JO A Q U IM (13) observed, in hum an beings, significantly higher levels of Z n in the placentary tissue in com parison to the m aternal and R N serum , and does not believe, therefore, that placenta is a deposit organ, considering the higher Z n level found as a consequence, probably, of the grow ing that occurs in the placentary tissue until the end. A ccording to M E T H FE SSE L & SPE N C E R (19), the hom eostasis of Z n is regulated, m ainly, through its secretion. A lthough Z n levels in foetus and in the respective placentas of the pregnant anim als subm itted to deficiency are low er than the one of the control group, FA IR W E A T H E R - T A IT observed a bigger Z n transference to foetus. T his w ay, they affirm that under inadequate nutrition conditions of Z n the pregnant anim al is able to m obilize the endogen Z n from m etabolically active deposits such as the liver, and transfer it to foetus.
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1 9 7 4 .
2 2 . R E C O R D , I.R .; T U L S I, R S .; D R E O S T I, I.E . &F R A S E R , F .J . - C e llu la r n e c ro s is in z in c o -d e fic ie n t ra t e m b ry o s . T e ra to lo g y 3 2 : 3 9 7 -4 0 5 , 1 9 8 5 .
2 3 . R E IN S T E IN , N .H .; L O N N E R D A L , B .; K E E N C , L . & H U R L E Y , L .S . - Z in c c o p p e r in te ra c tio n s in th e p re g n a n t ra t: fe ta l o u tc o m e a n d m a te rn a l a n d fe ta l z in c , c g p p e r a n d iro n .
J
N u tr 1 1 4 : 1 2 6 6 -7 9 , 1 9 8 4 .2 4 . R O G E R S , J .M .; K E E N , C .L . & H U R L E Y , L .S . - Z in c d e fic ie n c y in p re g n a n t L o n g -E v a n s h o o d e d ra ts : te ra to g e n ic ity a n d tis s u e tra c e e le m e n ts . T e ra to lo g y 3 1 : 8 9
-1 0 0 , -1 9 8 5 .
2 5 . S A N D S T E A D , H .H . - Z in c d e fic ie n c y a p u b lic h e a lth p ro b le m ? A m
J
D is C h ild 1 4 5 : 8 5 3 -9 ,1 9 9 1 .2 6 . S A T O , F .; W A T A N A B E , T .; H O S H I, E . & E N D O , A . -T e ra to g e n ic e ffe c t o f m a te rn a l z in c d e fic ie n c y a n d its c o te ra to g e n ic e ffe c t w ith c a d m iu m . T e ra to lo g y 3 1 : 1 3 -8 ,
1 9 8 5 .
2 7 . S IM M E R , K .&T H O M P S O N , R P .H . - Z in c in th e fe tu s a n d n e w b o rn . A c ta P a e d ia tr S c a n d 3 1 9 : 1 5 8 -6 3 , 1 9 8 5 .
2 8 . S O L , D . - E s tu d o d a c a re n c ia n u tric io n a l d e z in c o e m ra ta s a d u lta s n a o g e s ta n te s , g e s ta n te s e s e u s re c e m -n a s c id o s . S a o P a u lo , 1 9 8 7 (T e s e d e D o u to ra d o - E s c o la P a u lis ta d e M e d ic in a ) S a o P a u lo .
2 9 . S Z E R D A H E L I, P .&K A S A , P . - H is to c h e m is try o f z in c a n d c o p p e r. In t R e v C y to l8 9 : 1 -3 3 ,1 9 8 4 .
3 0 . T E R R Y . C .W .; T E R R Y , B .E . & D A V IS , J . - T ra n s fe r o f 6 5 z in c a c ro s s th e p Ia c e n te a n d fe ta l m e m b ra n e s o f a ra b b it. A m J P h y s io l1 9 8 : 3 0 3 -8 , 1 9 6 0 .
3 1 . T R IN D A D E , C .E .P .; S A R T O R , M .E .A .; T O N E T E , S .S .Q .; Z U L IA N I, A .; N a B R E G A , F J .; R U D G E , M .V .C .&C U R l, P .R - Z in c o n o s o ro d e m a e s e d e c o rd a o d e re c e m n a s c id o s d e te rm o c o m p e s o s a d e q u a d o s , te rm o c o m p e s o s b a ix o s e d e p re -te rm o c o m p e s o s a d e q u a d o s p a ra a id a d e g e s ta c io n a l. R e v P a u l P e d ia tr 1 : 1 0 -3 , 1 9 8 3 .
3 2 . V IL L A -E L IZ A G A , I. & C U N H A F E R R E IR A , R .M .C . -Z in c , p re g n a n c y a n d p a rtu ritio n . A c ta P a e d ia tr S c a n d 3 1 9 : 1 5 0 -7 , 1 9 8 5 .
3 3 . Z IM M E R M A N , A .W .; D U N H A M , B .S .; N O C H IM S O N , D J .; K A P L A N , B .M .; C L IV E , J .M . &K U N K E L , S .L . - Z in c tra n s p o rt in p re g n a n c y . A m
J
O b s te t G y n e c o l 1 4 9 : 5 2 3 -9 ,1 9 8 4 . .
