CHRISTINE VOJNIK ANDLUCILLE S. HURLEY Department of Nutrition, University of California, Davis, California 95616 ABSTRACT The effect of maternal zinc deficiency during gestation on fetal lung development was studied. Sprague-Dawley rats were fed from the day of mating (day zero) a zinc deficient diet (0.4 ±0.1 ppm zinc) ad libitum, or a zinc supplemented control diet (100 ppm zinc) either ad libitum or with restricted intake. Fetuses were removed by cesarean section on days 17 to 21 of gestation. Fetuses of zinc deficient dams had smaller lungs both in absolute weight and relative to body weight on all days than did either ad libitum-fed or restricted-intake controls. On days 20 and 21 of gestation, concentration of fetal lung lecithin and phosphatidylethanolamine was lower in zinc deficient fetuses than in control groups, indicating a reduced production of pulmonary surfactant. The lecithin to sphingomyelin ratio of amniotic fluid was lower in zinc deficient rats than in controls on days 19, 20, and 21 of gestation. On days 18 through 21 of gestation, fetal lung DNA concentration in zinc deficient fetuses was lower than in con trols, but there were no differences in fetal lung zinc concentration. Histological examination of lungs from zinc deficient fetuses at term showed air spaces that were slightly collapsed with smaller lumina of the alveolar ducts than in controls. J. Nutr. 107: 862-872, 1977. INDEXING KEY WORDS lung development •zinc deficiency • phospholipids •pregnancy Hurley and Swenerton ( 1 ) demonstrated that maternal dietary zinc deficiency in rats results in the development of young with a high incidence of congenital malformations. Ninety percent of the full term young of rats fed a zinc deficient diet from the beginning of pregnancy had one or more gross congenital malformations. Lung abnormalities consisting of small or missing lobes occurred in about 40% of the fetuses, When short-term and transitory periods of zinc deficiency were evaluated, the incidence of lung malformations varied (2), indicating that there is a critical period in the development of the lung during which it is adversely affected by absence of zinc, In the present study, the effect of prenatal zinc deficiency on pulmonary devel-
a lipoprotein complex lining the aveoli of mammalian lungs, functions in lowering the surface tension of the lung, thus preventing alveolar collapse. The major surface activity of surfactant resides in the phospholipid fraction, especially phosphatidylcholine (lecithin) (3-7). Pulmonary surfactant is not present in early fetal life, but appears in association with the development of highly differentiated alveolar cells. The ratio of lecithin to sphingomyelin ( L :S) in amniotic fluid has been used clinically as an index of fetal maturity (8-11). It was therefore of interest to determine the concentration of the classes of lipids as well as of zinc in lungs
and
ana Human Development.
Opment was J u- u
Studied both -11 TÕi
biochemically.
morphologically _r i. i
Pulmonary
surfactant,
Receivedfor publicationAugust 12, 1976.
' Supported In part by NIB research grant HD01743 from the National Institute of Child Health
862
Downloaded from https://academic.oup.com/jn/article-abstract/107/5/862/4769086 by University of Otago user on 18 December 2018
Abnormal Prenatal Lung Development Resulting from Maternal Zinc Deficiency in Rats1
ZINC DEFICIENCY AND PRENATAL LUNG DEVELOPMENT
of developing rats, from 17 days of gesta tion to term, and to correlate these with lung morphology and analyses of amniotic fluid.
Animals. Virgin female Sprague-Dawley rats weighing 190 to 200 g were purchased from a commercial source2 and were fed a purified complete diet for at least 3 days prior to breeding. They were caged over night with normal males fed stock diet,3 and matings were confirmed by the pres ence of sperm in the vaginal smear. The day of finding sperm was considered day 0 of gestation. All female rats were individually housed in a temperature and light controlled room (13 hours light, 11 hours dark) in stainless steel cages and racks, except for the brief periods when a male was introduced into the cage for breeding purposes. Feed was supplied in food cups made of aluminum with stainless steel covers. Distilled deionized water was freely available to all rats from Pyrex bottles with stainless steel mouthpieces and vinyl plastic stoppers. Careful precautions were taken to mini mize zinc contamination from the envi ronment (12). Diets. The composition of the zinc defi cient diet was as follows, in percent: iso lated soybean protein, 30.0 *; sucrose, 57.3; corn oil, 8.0; salt mix, 4.0s; and DL-methionine, 0.7. The zinc content of the soybean protein was reduced by treatment with the tetrasodium salt of ethylenediaminetraacetic acid (Na4EDTA). This diet con tained 0.4 ±0.1 ppm zinc as determined by atomic absorption spectroscopy6 ( 12 ). The zinc supplemented control diet was the same as the zinc deficient diet except that zinc was added in the salt mixture to provide 100 ppm zinc in the diet. Crystal line vitamins 7 were given three times per week in small glass dishes. Experimental procedure. On day 0 of gestation, the female rats were weighed and were assigned to one of three groups: zinc deficient, zinc supplemented controlad libitum fed, or zinc supplemented con trol-restricted intake. The rats were fed the deficient or control diets, and the restricted intake controls were fed the control diet in
amounts restricted to those eaten by the zinc-deficient group.8 On days 17 to 21 of gestation, the dams were weighed, and following ether anesthesia, terminal blood samples were collected by cardiac punc ture in heparinized plastic zinc-free syringes for zinc analysis. Amniotic fluid was collected in a syringe for analysis of the lecithin :sphingomyelin (L:S) ratio and then centrifuged to remove cell debris. Both amniotic fluid and plasma were trans ferred to zinc-free polyethylene snap-cap vials and stored frozen until analyzed. Fetuses were delivered by cesarean sec tion, weighed, and their lungs were re moved. Some were stored frozen and some were fixed in Bouin's solution for 48 hours, then stored in 70% alcohol. At 21 days of gestation, five litters from each group were allowed to breathe for at least 10 minutes after removal the uterus perfused with from Bouin's solutionbefore and being were stored in 70% alcohol. Gross examination of the lungs was made with the aid of a dissecting microscope. For histological ex amination, paraffin sections were stained with hematoxylin and eosin. Analytical methods. Plasma samples were diluted with 0.1 N HC1 and analyzed for zinc content by atomic absorption spec troscopy (13). Diet and tissue samples were weighed, dried, and ashed in por celain crucibles at 450°and dissolved in zinc-free 2 N HC1 for zinc analysis by atomic absorption spectroscopy (12). Each 2Horton Laboratories, Oakland. California. 3 Purina Rat Chow, Ralston Purina Co., St. Louis, Missouri ad libitum and crystalline vitamins as de scribed below. 4Purina Assay Protein RP-100, Ralston Purina Company, St. Louis, Missouri. 6Composition of the salt mix : (in g) CaCOj, 600 ; Ca(H.PO4)a-H2O, 200; KjHPO,, 650; NaCl, 336; MgSO4-7H«0, 250; FeSO.-THjO,50; MnSO4-H2O,4.6; KI, 1.6 ; CuSO4-5H2O,0.6. 6Unicam SP-90 atomic absorption spectrophotometer with a detection limit of 0.02 ppm zinc. 7A mixture of crystalline vitamins in glucose was given providing the following intake In mlcrograms per day ; Ca pantothenate, 500 ; p-aminobenzoic acid and riboflavin, each 100 ; thiamin HC1, pyridoxine HC1 and nicotlnlc acid, each 300 ; menadlone, 250 ; folie acid, 6 ; biotin, 2.5 ; vitamin Bu, 0.3 ; and chollne chloride, 10 mg ; inosltol, 5 mg ; ascorbic acid, 1 mg ; ot-tocopheryl acetate, 1.2 international units (IU) ; retinyl palmitate, 150 IU, and cholecalciferol, 15 IU. 8The mean daily food consumption of females fed the zinc deficient diet calculated from a previous experiment was as follows : day 0, 16 g ; day 1, 15 g ; day 2, 12 g ; day 3, 7 g; day 4, 11 g; day 5, 12 g ; day 6, 9 g; day 7. 11 g; day 8, 13 g ; day 9, 11 g; day 10, 6 g; day 11, 9 g; day 12, 10 g; day 13, 7 g; day 14, 7 g ; day 15, 12 g ; day 16, 8 g ; day 17, 6 g ; day 18, 8 g; day 19, 5 g; day 20, 5 g.
Downloaded from https://academic.oup.com/jn/article-abstract/107/5/862/4769086 by University of Otago user on 18 December 2018
MATERIALS AND METHODS
863
864
CHRISTINE VOJNIK AND LUCILLE S. HURLEY TABLE 1 Effects of zinc deficiency on maternal body weight and reproduction weightHats,Vo.9617130201161713U1513519Terminal body gain'a87
young per litterNo.11
17Day
libitumControl, ad restrictedZinc-deficientad
±12.8248 4.0'228 ±
±10.234 3.2'7± ±
±0.911 ±0.912 ±0.99±0.8I-i11±0.012
libitumControl,
3.6«.'31 ±
2.4'.'95
18Day
libitumControl, ad restrictedZinc-deficientad
8.0252 1± 5.3'217 ±
5.933 ± 7.0«1± ±
libitumControl,
3.4«.'328 ±
19Day
libitumControl, ad restrictedZinc-deficientad
8.8205 ± 4.1«212± ±
libitumControl, 20Day
libitumControl, ad restrictedZinc-deficientControl,
21GroupMaternal libitumControl, ad restrictedZinc-deficientad libitumMaternal
2.2'.'108
tion sitesNo.12
±0.012
tions per litterfÃ-o.1±O.S1±0.33 tiong%2
±0.97 ±0.3'20 ±0.0'0±0.20±0.34 ±0.0'.'1±0.22
±0.49±.128 ±0.410±1.211
±0.727 ±0.7«10
±3.311
±0.7«.'2 ±0.0'.'0±0.22±1.22
8.049 ± 7.2'3± ±
±0.59±1.48 ±0.511 ±0.911
3.8'.'345
2.2«.'120
±0.5»12±4.412 ±0.312
±0.0'.'0±0.00±0.033 ±0.0»0±0.00±0.04
5.5200 ± 0.7'22a± ± 4.1«.'343
5.054 ± 3.2«2± ± 3.0«.'132
±0.012 ±0.97*1.1«.'11±0.910 ±0.511
±1.3'.'0±0.00±0.020 ±0.8'.'0±0.00±0.03
±10.7203 7.9'209 ±
8.051 ± ±10.4'-9±
±0.511 ±0.511 ±0.58±0.7>.«Implanta ±0.511
± 4.4«.'Wt.
2.9«.'Live
±0.310 ±2.9'17
±0.7'.'Hesorp-±0.9-.' ±0.4Résorp
1 Wt. gain = (Terminal wt. —initial wt.). ' Significantly different from control ad libitum P < 0.05. ' Significantly differ ent from control ad libitum P < 0.01. « Significantly different from control ad libitum P < 0.001. >Significantly different from control restricted P < 0.05. ' .Significantly different from control restricted P < 0.01. ' Significantly different from control restricted P < 0.001.
sample of lung tissue consisted of 0.3 to 0.6 g pooled from a single litter. Sample blanks were carried through the entire ashing procedure. Amniotic fluid and fetal lungs were ana lyzed for individual classes of phospholipids. The fetal lungs from each litter were pooled and homogenized. Lipids were ex tracted with absolute methanol and chloro form, and surface active phospholipids were precipitated with acetone (6) and dissolved in chloroform:methanol. Separa tion of the compounds was achieved by two dimensional thin layer chromatography, using chloroform :methanol :ammo nium hydroxide as the vertical solvent, and chloroform :acetone :methanol :acetic acid : water as the horizontal solvent (14). Individual phospholipids were visualized on air dried plates with iodine vapor and total phosphorous was determined for each phospholipid sample after collection from the plates (15). The DNA content of fetal lung homogenates was analyzed using the methods of
Burton (16) and Munro and Flee (17). determination of statistical aFor one-tailed Student's T-test wassignificance, used (8). RESULTS Reproduction. The effect of zinc defi ciency on maternal body weight is reported in table 1. Only dams with living young were included. Rats fed the zinc deficient diet had body weights lower than those of either control group, but restriction of food intake also caused depression of body weight gain. The weight gain of the re stricted-intake group was less than half that of the ad libitum-fed control group on all days. The effects of zinc deficiency on repro duction are summarized in table 1. The number of live young per litter was lower in the dams fed the zinc deficient diet than in control groups, but there were no dif ferences among the groups in the number of implantation sites per litter. The num ber of résorptionsper litter and the per centage of résorptions were significantly
Downloaded from https://academic.oup.com/jn/article-abstract/107/5/862/4769086 by University of Otago user on 18 December 2018
wt.a301
GestationDay dietControl,
ZINC DEFICIENCY
AND PRENATAL
865
LUNG DEVELOPMENT
in the control-ad libitum fed group, while in the zinc deficient group there was no change. On days 20 and 21 of gestation the L:S ratio of the zinc deficient rats con tinued to be lower than that of the control 1.00 group. U) Fetus. The effect of zinc deficiency on ", 0.75 weight of the fetal body and lung is shown in table 3. The fetal body weights of the 0.50 restricted-intake controls were Tower than those from ad libitum-fed controls on days 025 20 and 21 of gestation, but the fetal weights of the zinc deficient group were lower than those of both control groups on 18 19 20 17 DAY OF GESTATION all days. Weight of fetal lung, both abso Fig. 1 Lecithin :sphingomyelin ratio in amnilute and on a body weight basis, was lower otic fluid from rats on days 17 to 21 of gestation. for the zinc deficient group on all days of •Control ad libitum; Q zinc deficient. Means ± gestation than in both control groups. SE. Number of rats: control ad libitum, 3, 6, 3, Weight of fetal lung in restricted-intake 8, 3 and zinc deficient: 7, 5, 5, 8, 3 for days 17, controls was lower than in ad libitum-fed 18, 19, 20 and 21, respectively. controls on all days of gestation, but their higher on all days for the females fed the lung weight relative to body weight was zinc deficient diet than in the control smaller only on days 17, 18, and 19 of ges groups. Dams with 100% of implantation tation. On days 20 and 21 of gestation, sites resorbed were not included. there were no differences in fetal lung Maternal plasma zinc levels on days 17 weight per body weight between the re to 21 of gestation are shown in table 2. The stricted-intake and ad libitum-fed controls. plasma zinc level of dams fed the zinc de The zinc level of fetal lungs from the ficient diet was severely reduced compared zinc deficient group was lower than that of to both ad libitum-fed and restricted-intake ad libitum-fed controls on days 18 and 19 controls on all days measured. of gestation when lung zinc was expressed Amniotic fluid. The L :S ratio in amniotic on a wet weight basis (table 3), but no fluid was determined on days 17 to 21 of other differences in fetal lung zinc were gestation (fig. 1). There was no difference observed. There was no difference in the phosin the ratio on days 17 and 18 of gestation between control and zinc deficient groups. phatidylethanolamine ( PE ) concentration of fetal lung (table 4) between the reOn day 19 of gestation, the ratio increased 1.50
Plasma zinc levels Day 18
Day 17 Group
Day 19
Day 21
Day 20
Rata
Zinc
Rats
Zinc
Rats
Zinc
Rats
Zinc
Rats
Zinc ¡it/100ml
No.
iie/100 ml
No.
w/100 ml
No.
ita/100 ml
No.
M/100 ml
No.
Control, ad libitum
4
101.1±4.72"
4
86.8±3.71
4
76.4±8.89
4
69.6±9.98
4
67.5dt 7.14
Control, restricted
6
90.8 ±5.79
6
79.8 ±2.86
6
72.8 ±4.14
5
57.2 ±3.99
4
61.0±12.43
Zinc-deficient ad libitum
4
19.9±1.32».«6
25.9±2.68'.»
5
32.1±5.95>.'
7
27.8± 8.88M
34.5±4.46«.> 7
'Mean±sE. *Significantly lower than control ad libitum P < 0.01. ' Significantly lower than control ad libitum P < 0.001. 4Significantly lower than control restricted P < 0.05. ' Significantly lower than control restricted P < 0.001.
Downloaded from https://academic.oup.com/jn/article-abstract/107/5/862/4769086 by University of Otago user on 18 December 2018
TABLE 2 Maternal plasma zinc levels on days 17 to HI of gestation
CHRISTINE VOJNIK AND LUCILLE S. HURLEY
866
Sd sö-H8co00N•tiIco8o-H*— d-H -HSII
1J1«oo
S
M
5
cot*B
O)CN
S
-Hd
o•" 3CN 1~ s¥j
io
1s
d1 -Hs
C-H-t-;9o^
-HS
>oi
rs?s!CO
CN05rtH«
idylethanolamin•t&câJj1P.M1_^^a3QwOHeaj3oO CÌ¡2
CÌ
1"2« iCOO «O
|g
iO
CO 10Ã-
33Q«2s^»— gestation is reported in table 7. On days 17, 18, and 20, DNA concentration was lower in the restricted-intake controls than in the ad libitum-fed controls, but by days 19 and 21 of gestation there were no dif ^Hsodò| o o ferences in lung DNA. The zinc deficient £§4j -H group had a lower concentration of DNA in lung tissue on all days of gestation than o.j® ö did either control group. At 18 days of gestation, there appeared cO *ß?i 'ß to be no morphological differences in fetal lungs among the groups as seen by light microscopy (not shown). The lung tissue was composed principally of mesenchymal Q^ OS tissue with epithelium many alveolar lined with S2 S § columnar givingducts a "gland-like" 04o.
t~
-H";> o öJ o -HS -H i-H¿
t~
q ö -H ^i
O
I-H
O5•^ t** *Oö
O O5
ö"S ö
"HO ~H
ö n
appearance. Formations of branched al veolar ducts could also be observed at this age in ah1groups. Ö| Ö At 20 days of gestation, the lungs of the « >0l =0 control group appeared to have lost the glandular appearance seen earlier and showed numerous and branched alveolar ducts. Zinc deficient fetuses, on the other ÕÕg 'S hand, showed compact lungs composed 0¿£'•3 % mainly of mesenchyme in which oblong ^ -g "*?~ ducts were embedded. Sections of fetal lung tissue of 21 day old *o o * o h .9 ö SoöV^
a lipoprotein complex lining the aveoli of mammalian lungs, functions in lowering the surface tension of the lung, thus preventing alveolar collapse. The major surface activity of surfactant resides in the phospholipid fraction, especially phosphatidylcholine (lecithin) (3-7). Pulmonary surfactant is not present in early fetal life, but appears in association with the development of highly differentiated alveolar cells. The ratio of lecithin to sphingomyelin ( L :S) in amniotic fluid has been used clinically as an index of fetal maturity (8-11). It was therefore of interest to determine the concentration of the classes of lipids as well as of zinc in lungs
and
ana Human Development.
Opment was J u- u
Studied both -11 TÕi
biochemically.
morphologically _r i. i
Pulmonary
surfactant,
Receivedfor publicationAugust 12, 1976.
' Supported In part by NIB research grant HD01743 from the National Institute of Child Health
862
Downloaded from https://academic.oup.com/jn/article-abstract/107/5/862/4769086 by University of Otago user on 18 December 2018
Abnormal Prenatal Lung Development Resulting from Maternal Zinc Deficiency in Rats1
ZINC DEFICIENCY AND PRENATAL LUNG DEVELOPMENT
of developing rats, from 17 days of gesta tion to term, and to correlate these with lung morphology and analyses of amniotic fluid.
Animals. Virgin female Sprague-Dawley rats weighing 190 to 200 g were purchased from a commercial source2 and were fed a purified complete diet for at least 3 days prior to breeding. They were caged over night with normal males fed stock diet,3 and matings were confirmed by the pres ence of sperm in the vaginal smear. The day of finding sperm was considered day 0 of gestation. All female rats were individually housed in a temperature and light controlled room (13 hours light, 11 hours dark) in stainless steel cages and racks, except for the brief periods when a male was introduced into the cage for breeding purposes. Feed was supplied in food cups made of aluminum with stainless steel covers. Distilled deionized water was freely available to all rats from Pyrex bottles with stainless steel mouthpieces and vinyl plastic stoppers. Careful precautions were taken to mini mize zinc contamination from the envi ronment (12). Diets. The composition of the zinc defi cient diet was as follows, in percent: iso lated soybean protein, 30.0 *; sucrose, 57.3; corn oil, 8.0; salt mix, 4.0s; and DL-methionine, 0.7. The zinc content of the soybean protein was reduced by treatment with the tetrasodium salt of ethylenediaminetraacetic acid (Na4EDTA). This diet con tained 0.4 ±0.1 ppm zinc as determined by atomic absorption spectroscopy6 ( 12 ). The zinc supplemented control diet was the same as the zinc deficient diet except that zinc was added in the salt mixture to provide 100 ppm zinc in the diet. Crystal line vitamins 7 were given three times per week in small glass dishes. Experimental procedure. On day 0 of gestation, the female rats were weighed and were assigned to one of three groups: zinc deficient, zinc supplemented controlad libitum fed, or zinc supplemented con trol-restricted intake. The rats were fed the deficient or control diets, and the restricted intake controls were fed the control diet in
amounts restricted to those eaten by the zinc-deficient group.8 On days 17 to 21 of gestation, the dams were weighed, and following ether anesthesia, terminal blood samples were collected by cardiac punc ture in heparinized plastic zinc-free syringes for zinc analysis. Amniotic fluid was collected in a syringe for analysis of the lecithin :sphingomyelin (L:S) ratio and then centrifuged to remove cell debris. Both amniotic fluid and plasma were trans ferred to zinc-free polyethylene snap-cap vials and stored frozen until analyzed. Fetuses were delivered by cesarean sec tion, weighed, and their lungs were re moved. Some were stored frozen and some were fixed in Bouin's solution for 48 hours, then stored in 70% alcohol. At 21 days of gestation, five litters from each group were allowed to breathe for at least 10 minutes after removal the uterus perfused with from Bouin's solutionbefore and being were stored in 70% alcohol. Gross examination of the lungs was made with the aid of a dissecting microscope. For histological ex amination, paraffin sections were stained with hematoxylin and eosin. Analytical methods. Plasma samples were diluted with 0.1 N HC1 and analyzed for zinc content by atomic absorption spec troscopy (13). Diet and tissue samples were weighed, dried, and ashed in por celain crucibles at 450°and dissolved in zinc-free 2 N HC1 for zinc analysis by atomic absorption spectroscopy (12). Each 2Horton Laboratories, Oakland. California. 3 Purina Rat Chow, Ralston Purina Co., St. Louis, Missouri ad libitum and crystalline vitamins as de scribed below. 4Purina Assay Protein RP-100, Ralston Purina Company, St. Louis, Missouri. 6Composition of the salt mix : (in g) CaCOj, 600 ; Ca(H.PO4)a-H2O, 200; KjHPO,, 650; NaCl, 336; MgSO4-7H«0, 250; FeSO.-THjO,50; MnSO4-H2O,4.6; KI, 1.6 ; CuSO4-5H2O,0.6. 6Unicam SP-90 atomic absorption spectrophotometer with a detection limit of 0.02 ppm zinc. 7A mixture of crystalline vitamins in glucose was given providing the following intake In mlcrograms per day ; Ca pantothenate, 500 ; p-aminobenzoic acid and riboflavin, each 100 ; thiamin HC1, pyridoxine HC1 and nicotlnlc acid, each 300 ; menadlone, 250 ; folie acid, 6 ; biotin, 2.5 ; vitamin Bu, 0.3 ; and chollne chloride, 10 mg ; inosltol, 5 mg ; ascorbic acid, 1 mg ; ot-tocopheryl acetate, 1.2 international units (IU) ; retinyl palmitate, 150 IU, and cholecalciferol, 15 IU. 8The mean daily food consumption of females fed the zinc deficient diet calculated from a previous experiment was as follows : day 0, 16 g ; day 1, 15 g ; day 2, 12 g ; day 3, 7 g; day 4, 11 g; day 5, 12 g ; day 6, 9 g; day 7. 11 g; day 8, 13 g ; day 9, 11 g; day 10, 6 g; day 11, 9 g; day 12, 10 g; day 13, 7 g; day 14, 7 g ; day 15, 12 g ; day 16, 8 g ; day 17, 6 g ; day 18, 8 g; day 19, 5 g; day 20, 5 g.
Downloaded from https://academic.oup.com/jn/article-abstract/107/5/862/4769086 by University of Otago user on 18 December 2018
MATERIALS AND METHODS
863
864
CHRISTINE VOJNIK AND LUCILLE S. HURLEY TABLE 1 Effects of zinc deficiency on maternal body weight and reproduction weightHats,Vo.9617130201161713U1513519Terminal body gain'a87
young per litterNo.11
17Day
libitumControl, ad restrictedZinc-deficientad
±12.8248 4.0'228 ±
±10.234 3.2'7± ±
±0.911 ±0.912 ±0.99±0.8I-i11±0.012
libitumControl,
3.6«.'31 ±
2.4'.'95
18Day
libitumControl, ad restrictedZinc-deficientad
8.0252 1± 5.3'217 ±
5.933 ± 7.0«1± ±
libitumControl,
3.4«.'328 ±
19Day
libitumControl, ad restrictedZinc-deficientad
8.8205 ± 4.1«212± ±
libitumControl, 20Day
libitumControl, ad restrictedZinc-deficientControl,
21GroupMaternal libitumControl, ad restrictedZinc-deficientad libitumMaternal
2.2'.'108
tion sitesNo.12
±0.012
tions per litterfÃ-o.1±O.S1±0.33 tiong%2
±0.97 ±0.3'20 ±0.0'0±0.20±0.34 ±0.0'.'1±0.22
±0.49±.128 ±0.410±1.211
±0.727 ±0.7«10
±3.311
±0.7«.'2 ±0.0'.'0±0.22±1.22
8.049 ± 7.2'3± ±
±0.59±1.48 ±0.511 ±0.911
3.8'.'345
2.2«.'120
±0.5»12±4.412 ±0.312
±0.0'.'0±0.00±0.033 ±0.0»0±0.00±0.04
5.5200 ± 0.7'22a± ± 4.1«.'343
5.054 ± 3.2«2± ± 3.0«.'132
±0.012 ±0.97*1.1«.'11±0.910 ±0.511
±1.3'.'0±0.00±0.020 ±0.8'.'0±0.00±0.03
±10.7203 7.9'209 ±
8.051 ± ±10.4'-9±
±0.511 ±0.511 ±0.58±0.7>.«Implanta ±0.511
± 4.4«.'Wt.
2.9«.'Live
±0.310 ±2.9'17
±0.7'.'Hesorp-±0.9-.' ±0.4Résorp
1 Wt. gain = (Terminal wt. —initial wt.). ' Significantly different from control ad libitum P < 0.05. ' Significantly differ ent from control ad libitum P < 0.01. « Significantly different from control ad libitum P < 0.001. >Significantly different from control restricted P < 0.05. ' .Significantly different from control restricted P < 0.01. ' Significantly different from control restricted P < 0.001.
sample of lung tissue consisted of 0.3 to 0.6 g pooled from a single litter. Sample blanks were carried through the entire ashing procedure. Amniotic fluid and fetal lungs were ana lyzed for individual classes of phospholipids. The fetal lungs from each litter were pooled and homogenized. Lipids were ex tracted with absolute methanol and chloro form, and surface active phospholipids were precipitated with acetone (6) and dissolved in chloroform:methanol. Separa tion of the compounds was achieved by two dimensional thin layer chromatography, using chloroform :methanol :ammo nium hydroxide as the vertical solvent, and chloroform :acetone :methanol :acetic acid : water as the horizontal solvent (14). Individual phospholipids were visualized on air dried plates with iodine vapor and total phosphorous was determined for each phospholipid sample after collection from the plates (15). The DNA content of fetal lung homogenates was analyzed using the methods of
Burton (16) and Munro and Flee (17). determination of statistical aFor one-tailed Student's T-test wassignificance, used (8). RESULTS Reproduction. The effect of zinc defi ciency on maternal body weight is reported in table 1. Only dams with living young were included. Rats fed the zinc deficient diet had body weights lower than those of either control group, but restriction of food intake also caused depression of body weight gain. The weight gain of the re stricted-intake group was less than half that of the ad libitum-fed control group on all days. The effects of zinc deficiency on repro duction are summarized in table 1. The number of live young per litter was lower in the dams fed the zinc deficient diet than in control groups, but there were no dif ferences among the groups in the number of implantation sites per litter. The num ber of résorptionsper litter and the per centage of résorptions were significantly
Downloaded from https://academic.oup.com/jn/article-abstract/107/5/862/4769086 by University of Otago user on 18 December 2018
wt.a301
GestationDay dietControl,
ZINC DEFICIENCY
AND PRENATAL
865
LUNG DEVELOPMENT
in the control-ad libitum fed group, while in the zinc deficient group there was no change. On days 20 and 21 of gestation the L:S ratio of the zinc deficient rats con tinued to be lower than that of the control 1.00 group. U) Fetus. The effect of zinc deficiency on ", 0.75 weight of the fetal body and lung is shown in table 3. The fetal body weights of the 0.50 restricted-intake controls were Tower than those from ad libitum-fed controls on days 025 20 and 21 of gestation, but the fetal weights of the zinc deficient group were lower than those of both control groups on 18 19 20 17 DAY OF GESTATION all days. Weight of fetal lung, both abso Fig. 1 Lecithin :sphingomyelin ratio in amnilute and on a body weight basis, was lower otic fluid from rats on days 17 to 21 of gestation. for the zinc deficient group on all days of •Control ad libitum; Q zinc deficient. Means ± gestation than in both control groups. SE. Number of rats: control ad libitum, 3, 6, 3, Weight of fetal lung in restricted-intake 8, 3 and zinc deficient: 7, 5, 5, 8, 3 for days 17, controls was lower than in ad libitum-fed 18, 19, 20 and 21, respectively. controls on all days of gestation, but their higher on all days for the females fed the lung weight relative to body weight was zinc deficient diet than in the control smaller only on days 17, 18, and 19 of ges groups. Dams with 100% of implantation tation. On days 20 and 21 of gestation, sites resorbed were not included. there were no differences in fetal lung Maternal plasma zinc levels on days 17 weight per body weight between the re to 21 of gestation are shown in table 2. The stricted-intake and ad libitum-fed controls. plasma zinc level of dams fed the zinc de The zinc level of fetal lungs from the ficient diet was severely reduced compared zinc deficient group was lower than that of to both ad libitum-fed and restricted-intake ad libitum-fed controls on days 18 and 19 controls on all days measured. of gestation when lung zinc was expressed Amniotic fluid. The L :S ratio in amniotic on a wet weight basis (table 3), but no fluid was determined on days 17 to 21 of other differences in fetal lung zinc were gestation (fig. 1). There was no difference observed. There was no difference in the phosin the ratio on days 17 and 18 of gestation between control and zinc deficient groups. phatidylethanolamine ( PE ) concentration of fetal lung (table 4) between the reOn day 19 of gestation, the ratio increased 1.50
Plasma zinc levels Day 18
Day 17 Group
Day 19
Day 21
Day 20
Rata
Zinc
Rats
Zinc
Rats
Zinc
Rats
Zinc
Rats
Zinc ¡it/100ml
No.
iie/100 ml
No.
w/100 ml
No.
ita/100 ml
No.
M/100 ml
No.
Control, ad libitum
4
101.1±4.72"
4
86.8±3.71
4
76.4±8.89
4
69.6±9.98
4
67.5dt 7.14
Control, restricted
6
90.8 ±5.79
6
79.8 ±2.86
6
72.8 ±4.14
5
57.2 ±3.99
4
61.0±12.43
Zinc-deficient ad libitum
4
19.9±1.32».«6
25.9±2.68'.»
5
32.1±5.95>.'
7
27.8± 8.88M
34.5±4.46«.> 7
'Mean±sE. *Significantly lower than control ad libitum P < 0.01. ' Significantly lower than control ad libitum P < 0.001. 4Significantly lower than control restricted P < 0.05. ' Significantly lower than control restricted P < 0.001.
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TABLE 2 Maternal plasma zinc levels on days 17 to HI of gestation
CHRISTINE VOJNIK AND LUCILLE S. HURLEY
866
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S
M
5
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S
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o•" 3CN 1~ s¥j
io
1s
d1 -Hs
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rs?s!CO
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idylethanolamin•t&câJj1P.M1_^^a3QwOHeaj3oO CÌ¡2
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1"2« iCOO «O
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CO 10Ã-
33Q«2s^»— gestation is reported in table 7. On days 17, 18, and 20, DNA concentration was lower in the restricted-intake controls than in the ad libitum-fed controls, but by days 19 and 21 of gestation there were no dif ^Hsodò| o o ferences in lung DNA. The zinc deficient £§4j -H group had a lower concentration of DNA in lung tissue on all days of gestation than o.j® ö did either control group. At 18 days of gestation, there appeared cO *ß?i 'ß to be no morphological differences in fetal lungs among the groups as seen by light microscopy (not shown). The lung tissue was composed principally of mesenchymal Q^ OS tissue with epithelium many alveolar lined with S2 S § columnar givingducts a "gland-like" 04o.
t~
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t~
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O
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appearance. Formations of branched al veolar ducts could also be observed at this age in ah1groups. Ö| Ö At 20 days of gestation, the lungs of the « >0l =0 control group appeared to have lost the glandular appearance seen earlier and showed numerous and branched alveolar ducts. Zinc deficient fetuses, on the other ÕÕg 'S hand, showed compact lungs composed 0¿£'•3 % mainly of mesenchyme in which oblong ^ -g "*?~ ducts were embedded. Sections of fetal lung tissue of 21 day old *o o * o h .9 ö SoöV^
