0003-9969/90 $3.00 + 0.00 Copyright 0 1990 Pergamon Press plc
Archs oral Bid. Vol. 35, No. 6, pp. 425430, 1990 Printed in Great Britain. All rights reserved
EFF’ECTS OF MATERNAL CAFFEINE WITH ZINC INTAKE DURING GESTATION AND LACTATION ON BONE DEVELOPMENT IN NEWBORN RATS H.
SASAHARA,’
H. YAMANO~
and T.
NAKAMOTO’*
‘Laboratory of Perinatal Nutrition and Metabolism, Department of Oral Diagnosis, Nihon University School of Dentistry at Matsudo, Japan and *Department of Physiology, Louisiana State University Medical Center, New Orleans, LA 70119, U.S.A. (Accepted 4 January 1990)
day 9 of gestation, pregnant dams were randomly divided into 3 groups. Dams of group 1 were fed a 20% protein diet as a control. Dams of group 2 were fed a 20% protein diet supplemented with caffeine. Dams of group 3 were fed a 20% protein diet supplemented with caffeine and zinc. The amount of caffeine added to the maternal diet was 2 mg/lOO g body weight; the amount of zinc was 0.6 g/kg of diet. At birth, pups were mixed within each group, and 8 randomly selected pups from each group were assigned to each dam of the respective group and were continuously fed the same diet. On day 15, the pups were killed and cranial bones, mandibles and femurs removed. The bones were measured, and the mineral content of the mandibles and femurs was determined. Although there were no differences in the dimensions of the cranial bones among the groups, the measurements and mineral content of the mandibles and femurs were consistently affected by the caffeine in the diet. On the other hand, supplementation of the caffeine-added diets with zinc led to greatly improved bone development, reaching values up to 01: beyond control levels. Thus zinc supplementation of a caffeine diet given to the dams during gestation and lactation can favourably influence the otherwise impaired bone development of their offspring.
Summary--On
Key words: calfeine, zinc, newborn rats, bones.
INTRODUCTION
Caffeine (1,3,7_trimethylxanthine) is one of the most commonly consumed substances in daily life. Coffee, tea, cola, other carbonated soft drinks, and various over-the-counter medications contain caffeine. Because of its ubiquity, avoidance of caffeine consumption is extremely difficult. Caffeine taken during pregnancy is known to reach the fetus in humans (Goldstein and Warren, 1962) as well as in rats (Gilbert and Pistey, 1973), and a large dose of caffeine during pregnancy has been shown to induce various abnormalities in animal studies (Nishimura and Nakai, 1960). Furthermore, caffeine diffuses easily in breast milk (Aldridge, Aranda and Neims, 1975; Aeschbacher et al., 1980). Approximately 74% of pregnant women are reported to consume caffeine during pregnancy (Graham, 1978). According to National Cancer Health Statistics (1980), 52% of women with more than 12 years of schooling had chosen to breast-feed their infants. Supplementation with caffeine of the diet of the pregnant rat impairs fetal bone development (Nakamoto and Shaye, 1986) and results in reduced fetal mandibular weight and various decreases in mineral content (Na.kamoto, Grant and Yazdani, 1989). Of this decreased content, we have now specifi*Address correspondence to: Dr T. Nakamoto, Department of Physiology, Louisiana State University Medical Center, 1100 Florida Ave., New Orleans, LA 70119, U.S.A.
tally selected zinc because zinc is known to play an important role in growth and development (Dreosti, 1982) and may stimulate bone growth (Yamaguch and Yamaguchi, 1986). We have sought to determine whether zinc supplementation of the caffeine-added diet during gestation and lactation can alter and/or improve bone development in the offspring. MATERIALS
AND METHODS
Time-pregnant Sprague-Dawley rats were used (Holtzman Co., Madison, WI, U.S.A.). On the ninth day of gestation (the sperm-positive day counted as day l), dams were randomly divided into 3 groups. Dams of group 1 (n = 6) were fed a 20% protein diet as a control. Dams of group 2 (n = 6) were fed a 20% protein diet supplemented with caffeine. Dams of group 3 (n = 6) were fed a 20% protein diet supplemented with caffeine and zinc. Caffeine supplementation was 2 mg/lOO g body weight; zinc supplementation was 0.6 g zinc chloride/kg diet. The zinc chloride was dissolved in double distilled deionized water and added to the diet. The dietary composition of the 20% protein diet has been described by Nakamoto and Shaye (1986). Because maternal body weight gradually increases during pregnancy the amount of caffeine in the diet was adjusted at days 9, 14 and 18 to maintain a designated ratio. After birth, maternal weight is relatively constant, so thereafter the caffeine supplement was adjusted only once more. 425
H. SASAHARAet al.
426
Upon birth, litters delivered in each group within the span of 8 h were combined and designed as ‘day 1’. From these, each dam was assigned 8 randomly selected pups and was fed continuously with the same diet in each respective group. On day 15, pups from each group were killed between 09.00 and 11.OOh by an overdose of ether. The cranial bone, right mandible and right femur were removed and soft tissue and muscles were cleaned off after boiling in water for 2min. Then, a cranium, mandible and femur were randomly selected from each litter. Thus, 6 bones from the total number taken from each group were chosen. The bones were dried overnight at 110°C and weighed. In order to study the physical changes in each group, the linear distances between anatomically important points in the cranium, mandible and femur were measured. The dried bones were placed on plain white paper, the anatomical points marked on the paper with the pointed ends of a caliper and the
following distances measured: (a) from prosthion to basion; (b) from prosthion to sutua of basisphenoid and presphenoid; (c) from left orbita to right orbita; (d) from the buccal surface of the left first molar to that of the right first molar; (e) from the anterior aspect of the mandibular foramen to the farthest point on the head of mandibular condyle; (f) from the condylar process to the angular process; (g) from the posterior aspect of the mental foramen to the condylar process; (h) from the junction of the mesial surface of the first molar with the alveolar bone to the infradentale; and (i) between the fovea and the medial condyle of the femurs. The distances between the marked points were measured with a micrometer (Nakamoto and Wilson, 1969; Fig. 1). The developing teeth were removed from the mandibular body after soaking in water overnight. They were then dried again overnight at 110°C and weighed. A mandibular body or a femur was placed on a procelain crucible and put in a muffle furnace
Measuring points Cranial bones
Femur
fovaa
F-t%
A--: madal
condyls
Fig. 1. Diagrams of the anatomical measuring points in the cranial brones, mandible and femur.
Caffeine
with zinc
RESULTS
Maternal body weight during gestation did not show any difference among the groups (p > 0.05); neither did the pup’s body weight during lactation (p > 0.05; Table 1). Physical changes of the cranial bones showed no significant difference among the groups (p > 0.05; Table 2). On the other hand, in the mandible, the distance from first molar to infradentale in the caffeine-supplemented group (group 2) was significantly smaller than in the controls (group l), and in the caffeine plus zinc-supplemented group (group 3) this distance was larger (p < 0.05; Table 2). In the femurs, the distance from fovea to medial condyle in group 2 was less than in either group 1 or 3 (p < 0.05; Table 2). Although mandibular weight before removing the teeth showed no significant difference among the groups (p > 0.05) iafter removing the teeth, the mandibular bodies weighed significantly less in group 2 than either group I or 3 (p < 0.05; Table 3). The weight of the femur in group 2 was also significantly less than in either group 1 or 3 (p < 0.05). The mandibular body had an increased zinc content in group 3 compared to either group 1 or 2 (p < 0.05). The mag:nesium content in group 2 was less than in either group 1 or 3 (p < 0.05). Calcium and phosphorus content showed no difference among the groups (p > 0.05) and neither did the calcium/ phosphorus molar ratio (p > 0.05; Table 3). The zinc content c’f the femur in group 2 was less than in either group 1 or 3 (p < 0.05); furthermore, the zinc content of group 3 femurs was greater than that of group 1 (p 0.05), and neither did the calcium/phosphorus molar ratio (p > 0.05; Table 3). The mandibular zinc content in group 3 only showed a significant correlation with mandibular weight (r = 0.883; p < O.Ol), whereas for the femur, all 3 groups showed a significant correlation between that weight and zinc content (group 1, r = 0.893; group 2, r = 0.922; group 3, r = 0.998; p < 0.01 or p < 0.005). The magnesium content of the mandible (I = 0.975, 0.920 or 0.958, respectively) and femur (r = 0.915, 0.911 or 0.974, respectively) showed a highly significant correlation with respective bone weights in all 3 groups (p < 0.01 or p < 0.005). The mandibular calcium content showed a highly significant correlation with mandibular weight in all 3 groups (p < 0.005; Fig. 2). In contrast, femur calcium content in group 2 was not correlated with bone weight (p > 0.05), but such correlation was significant in group 1 and 3 (p < 0.01; Fig. 2). Mandibular phosphorus content became significantly correlated with weight in group 2 and 3 (p < O.Ol), but in group 1 showed no correlation (p > 0.05; Fig. 3). In contrast, in the femur, phosphorus content was significantly correlated with bone weight in group 1 and 3 (p < O.Ol), but not in group 2 (p > 0.05; Fig. 3).
overnight at 600°C. ‘The following day these bones were dissolved in 0.2-0.3 ml concentrated HCl, and the solution diluted with double-distilled deionized water in a 5 ml volumetric flask. Samples were taken to determine calcium, magnesium and zinc content by atomic absorption spectrophotometry (Model 280, Fisher Scientific Co., Fair Lawn, NJ, U.S.A.), and phosphorus by the method of Fiske and Subbarow (1925). The data were examined by analysis of variance and multiple cornparson (Student-Newman-Keuls). Correlation coefficients between bone weight and various mineral contents was calculated. The 5% level was considered statistically significant. Analyses were conducted on an Apple II microcomputer (Apple Corp., Cupertino, CA, U.S.A.).
Table
effects on bones
408+ 404+
6 10
399 * 9
of the dams is an average
(days)
1
8
15
6.43 k 0.09 6.43 + 0.08
16.7 + I.0 17.7 k 0.6
35.5 + 1.3 36.2 k 1.1
6.75 & 0.03
18.3 + 0.3
38.6 k 1.0
of 6; weight of the pups is an average
of
-_--_
-\
Cranial bones ‘.. %Y. \ “‘\.
-
14.36 _+0.23
23.01 -+ 0.26
b
14.29 + 0.08 14.3I k 0.14
a C
6.49 It 0.06
6.42 & 0.05 6.48 & 0.08
Mandibles
23.08 + 0.15 23.22 + 0.19
__~ e
-
3.41 + 0.07
3.46 + 0.05 3.38 + 0.05
f
6.14 + 0.07
6.25 f 0.09 6.14 + 0.04
R
13.59f0.17
13.43 + 0.10 13.56 & 0.06
Femurs
45.57 + 1.31 44.31 &I.14 50.03 k2.81
Mandible 28.65 kO.98 23.34 2 1.04 29.55 _t 1.90 f +
c
Femur
28.72 10.85 24.82 +0.70 30.03 _+I .76 -I-
Mandible*
Weight (mg)
~_~.~_~_,___. Mandible -~-__I______-. Zn Ocg) Ca (mg) P (mg) Mg (mg) 7.00 3.38 1.01 6.11 to.04 kO.25 kO.15 f 0.45 6.73 3.02 0.82 5.98 to.03 kO.14 kO.08 +0.31 7.30 3.38 0.98 7.73 +0.06 kO.37 + 0.23 kO.66 f + + +
_.I
.-..
_.
I
-
-
+0.01 I .67 f0.04
1.72
I .62 & 0.08
Ca/P**
c
+
-
+
---~&I k) Ca (mg) 4.22 5.89 +0.36 +0.27 3.20 4.75 +0.24 kO.33 5.67 5.99 1kO.29 20.37 + +
Mineral content
Table 3. Bone weight and mineral content of the mandibles and femurs of the newborn rats at day 15
Each value is mean F SEM. Weight of the bones is after drying at 110°C overnight. *Weight of mandibular body (after removal of the teeth). **Ca/P molar ratio. f: Statistically significant (p c; 0.05). -: Statistically not significant (p z 0.05).
20% protein + caffeine (G2) 20% protein + caffeine +zinc (G3) Gi vs G2 G1 vs G3 C,? vr cr?
20% protein (Gl)
Maternal diet
-
-
7.39 _+0.09
7.28 + 0.08 7.51 + 0.06
.d
Physical size (mm)
Table 2. Physical measurements of the cranial bones, mandibles and femurs of newborn rats at day 15
h
i
12.61 kO.17 -If
-t + +
12.58 F0.19 11.97 & 0.08
5.76kO.07
5.53 + 0.03 5.38 f 0.02
__
-
P tmg) 2.51 20.21 2.31 kO.15 2.70 +0.19
Femur
+
-
Mg (mg) 0.87 kO.04 0.72 +0.03 0.92 kO.07
-.___
-
& 0.02
1.72
---.Ca/P** 1.84 iO.11 1.60 +0.04
Each value is mean f SEM; each is an average of 6 measurements. a = Prosthion to basion, b = prosthion to sutua of basisphenoid and presphenoid, c = orbita to orbita, d = first molar to first molar, e = mandibular foramen to condylar process, f = condylar process to angular process, g = mental foramen to condylar process, h = first molar to infradentale, i = fovea to medial condyle. +: Statistically significant (p < 0.05). -: Statistically not significant (p > 0.05).
20% protein (Gt) 20% protein + caffeine (G2) 20% protein + caffeine -i-zinc (G3) Gl vs G2 Gl vs G3 G2 vs G3
Maternal diet
.L “\.._,.
/ cI/l.l.r Caffeine with zinc effects on bones
hblldlbb 8.0
7.2 r
0
7.0
7.5
0
6.6
6.6
7.Q
0
6.5 6.0
6.4
f - O.S?O
26
30
26
32
r-0,$92 pco.005
r-u947 p4l.005
p:o.w5
26
34
6.5
7.0 -
6.0
6.5 -
22
24
26
28
26
Fig. 2. Correlation between bone weight (s axis; mg) and calcium content mandible (p = 0.224 .r + 0.678), femur (y = 0.364 x _- 4.546); middle:
(y = 0.188 s + 2.&B),
femur (}I
(y = 0.148 .v + 1.259); right:
=0.209.x
+
l,Q17), femur
tended to be slighl.ly larger, suggesting that the amount of zinc ~up?lerncnt~tion did not exert any toxic effects on either dams or pups. The physical changes in the femur and mandible in the caffeine group were clearly improved by adding zinc to the caffeine diet, and this group grew to the same extent as the controls. However, the effects on the various bone were different, which could possibly be due to their different developmental origins:
cranial bones are a. mixture of membranous and endochondral bones. and the mandible and femur are
(y =
caffeine
28
28
30
30
32
32
34
34
36
36
3%
(y axis; mg). Left: control, caffeine group, mandibk
plus
zinc
group,
mandibte
0.189 x + 0.378).
membranous and endochrondral bone, respectively ~Weinmann and &her, 19553. Caffeine supplementation increases urinary calcium excretion (Whiting and Whitney, 1987). It is possible that maternal caffeine intake could unbalance the calcium homeostasis of the growing fetus, and of the neonate as a result of suckling milk containing caffeine. These processes may alter the mineralization of the bones in neonates. Urinary zinc excretion, on the other hand, is not affected by caffeine supplementation (Yeh et ul., 1986).
4.0 o 3Jl 0
3.6 0
3.4
0
3.2 3.0 2.6
r=wea p>ou5
00 ~
126
26
30
32
34
2.6'.
t “.
22
23
‘.
24
‘.“I
25
26
27
2.5--L-L-"*.'. 26 28
30
32
34
;6
3.4 3.2 3.0
-
r.0995 p
Archs oral Bid. Vol. 35, No. 6, pp. 425430, 1990 Printed in Great Britain. All rights reserved
EFF’ECTS OF MATERNAL CAFFEINE WITH ZINC INTAKE DURING GESTATION AND LACTATION ON BONE DEVELOPMENT IN NEWBORN RATS H.
SASAHARA,’
H. YAMANO~
and T.
NAKAMOTO’*
‘Laboratory of Perinatal Nutrition and Metabolism, Department of Oral Diagnosis, Nihon University School of Dentistry at Matsudo, Japan and *Department of Physiology, Louisiana State University Medical Center, New Orleans, LA 70119, U.S.A. (Accepted 4 January 1990)
day 9 of gestation, pregnant dams were randomly divided into 3 groups. Dams of group 1 were fed a 20% protein diet as a control. Dams of group 2 were fed a 20% protein diet supplemented with caffeine. Dams of group 3 were fed a 20% protein diet supplemented with caffeine and zinc. The amount of caffeine added to the maternal diet was 2 mg/lOO g body weight; the amount of zinc was 0.6 g/kg of diet. At birth, pups were mixed within each group, and 8 randomly selected pups from each group were assigned to each dam of the respective group and were continuously fed the same diet. On day 15, the pups were killed and cranial bones, mandibles and femurs removed. The bones were measured, and the mineral content of the mandibles and femurs was determined. Although there were no differences in the dimensions of the cranial bones among the groups, the measurements and mineral content of the mandibles and femurs were consistently affected by the caffeine in the diet. On the other hand, supplementation of the caffeine-added diets with zinc led to greatly improved bone development, reaching values up to 01: beyond control levels. Thus zinc supplementation of a caffeine diet given to the dams during gestation and lactation can favourably influence the otherwise impaired bone development of their offspring.
Summary--On
Key words: calfeine, zinc, newborn rats, bones.
INTRODUCTION
Caffeine (1,3,7_trimethylxanthine) is one of the most commonly consumed substances in daily life. Coffee, tea, cola, other carbonated soft drinks, and various over-the-counter medications contain caffeine. Because of its ubiquity, avoidance of caffeine consumption is extremely difficult. Caffeine taken during pregnancy is known to reach the fetus in humans (Goldstein and Warren, 1962) as well as in rats (Gilbert and Pistey, 1973), and a large dose of caffeine during pregnancy has been shown to induce various abnormalities in animal studies (Nishimura and Nakai, 1960). Furthermore, caffeine diffuses easily in breast milk (Aldridge, Aranda and Neims, 1975; Aeschbacher et al., 1980). Approximately 74% of pregnant women are reported to consume caffeine during pregnancy (Graham, 1978). According to National Cancer Health Statistics (1980), 52% of women with more than 12 years of schooling had chosen to breast-feed their infants. Supplementation with caffeine of the diet of the pregnant rat impairs fetal bone development (Nakamoto and Shaye, 1986) and results in reduced fetal mandibular weight and various decreases in mineral content (Na.kamoto, Grant and Yazdani, 1989). Of this decreased content, we have now specifi*Address correspondence to: Dr T. Nakamoto, Department of Physiology, Louisiana State University Medical Center, 1100 Florida Ave., New Orleans, LA 70119, U.S.A.
tally selected zinc because zinc is known to play an important role in growth and development (Dreosti, 1982) and may stimulate bone growth (Yamaguch and Yamaguchi, 1986). We have sought to determine whether zinc supplementation of the caffeine-added diet during gestation and lactation can alter and/or improve bone development in the offspring. MATERIALS
AND METHODS
Time-pregnant Sprague-Dawley rats were used (Holtzman Co., Madison, WI, U.S.A.). On the ninth day of gestation (the sperm-positive day counted as day l), dams were randomly divided into 3 groups. Dams of group 1 (n = 6) were fed a 20% protein diet as a control. Dams of group 2 (n = 6) were fed a 20% protein diet supplemented with caffeine. Dams of group 3 (n = 6) were fed a 20% protein diet supplemented with caffeine and zinc. Caffeine supplementation was 2 mg/lOO g body weight; zinc supplementation was 0.6 g zinc chloride/kg diet. The zinc chloride was dissolved in double distilled deionized water and added to the diet. The dietary composition of the 20% protein diet has been described by Nakamoto and Shaye (1986). Because maternal body weight gradually increases during pregnancy the amount of caffeine in the diet was adjusted at days 9, 14 and 18 to maintain a designated ratio. After birth, maternal weight is relatively constant, so thereafter the caffeine supplement was adjusted only once more. 425
H. SASAHARAet al.
426
Upon birth, litters delivered in each group within the span of 8 h were combined and designed as ‘day 1’. From these, each dam was assigned 8 randomly selected pups and was fed continuously with the same diet in each respective group. On day 15, pups from each group were killed between 09.00 and 11.OOh by an overdose of ether. The cranial bone, right mandible and right femur were removed and soft tissue and muscles were cleaned off after boiling in water for 2min. Then, a cranium, mandible and femur were randomly selected from each litter. Thus, 6 bones from the total number taken from each group were chosen. The bones were dried overnight at 110°C and weighed. In order to study the physical changes in each group, the linear distances between anatomically important points in the cranium, mandible and femur were measured. The dried bones were placed on plain white paper, the anatomical points marked on the paper with the pointed ends of a caliper and the
following distances measured: (a) from prosthion to basion; (b) from prosthion to sutua of basisphenoid and presphenoid; (c) from left orbita to right orbita; (d) from the buccal surface of the left first molar to that of the right first molar; (e) from the anterior aspect of the mandibular foramen to the farthest point on the head of mandibular condyle; (f) from the condylar process to the angular process; (g) from the posterior aspect of the mental foramen to the condylar process; (h) from the junction of the mesial surface of the first molar with the alveolar bone to the infradentale; and (i) between the fovea and the medial condyle of the femurs. The distances between the marked points were measured with a micrometer (Nakamoto and Wilson, 1969; Fig. 1). The developing teeth were removed from the mandibular body after soaking in water overnight. They were then dried again overnight at 110°C and weighed. A mandibular body or a femur was placed on a procelain crucible and put in a muffle furnace
Measuring points Cranial bones
Femur
fovaa
F-t%
A--: madal
condyls
Fig. 1. Diagrams of the anatomical measuring points in the cranial brones, mandible and femur.
Caffeine
with zinc
RESULTS
Maternal body weight during gestation did not show any difference among the groups (p > 0.05); neither did the pup’s body weight during lactation (p > 0.05; Table 1). Physical changes of the cranial bones showed no significant difference among the groups (p > 0.05; Table 2). On the other hand, in the mandible, the distance from first molar to infradentale in the caffeine-supplemented group (group 2) was significantly smaller than in the controls (group l), and in the caffeine plus zinc-supplemented group (group 3) this distance was larger (p < 0.05; Table 2). In the femurs, the distance from fovea to medial condyle in group 2 was less than in either group 1 or 3 (p < 0.05; Table 2). Although mandibular weight before removing the teeth showed no significant difference among the groups (p > 0.05) iafter removing the teeth, the mandibular bodies weighed significantly less in group 2 than either group I or 3 (p < 0.05; Table 3). The weight of the femur in group 2 was also significantly less than in either group 1 or 3 (p < 0.05). The mandibular body had an increased zinc content in group 3 compared to either group 1 or 2 (p < 0.05). The mag:nesium content in group 2 was less than in either group 1 or 3 (p < 0.05). Calcium and phosphorus content showed no difference among the groups (p > 0.05) and neither did the calcium/ phosphorus molar ratio (p > 0.05; Table 3). The zinc content c’f the femur in group 2 was less than in either group 1 or 3 (p < 0.05); furthermore, the zinc content of group 3 femurs was greater than that of group 1 (p 0.05), and neither did the calcium/phosphorus molar ratio (p > 0.05; Table 3). The mandibular zinc content in group 3 only showed a significant correlation with mandibular weight (r = 0.883; p < O.Ol), whereas for the femur, all 3 groups showed a significant correlation between that weight and zinc content (group 1, r = 0.893; group 2, r = 0.922; group 3, r = 0.998; p < 0.01 or p < 0.005). The magnesium content of the mandible (I = 0.975, 0.920 or 0.958, respectively) and femur (r = 0.915, 0.911 or 0.974, respectively) showed a highly significant correlation with respective bone weights in all 3 groups (p < 0.01 or p < 0.005). The mandibular calcium content showed a highly significant correlation with mandibular weight in all 3 groups (p < 0.005; Fig. 2). In contrast, femur calcium content in group 2 was not correlated with bone weight (p > 0.05), but such correlation was significant in group 1 and 3 (p < 0.01; Fig. 2). Mandibular phosphorus content became significantly correlated with weight in group 2 and 3 (p < O.Ol), but in group 1 showed no correlation (p > 0.05; Fig. 3). In contrast, in the femur, phosphorus content was significantly correlated with bone weight in group 1 and 3 (p < O.Ol), but not in group 2 (p > 0.05; Fig. 3).
overnight at 600°C. ‘The following day these bones were dissolved in 0.2-0.3 ml concentrated HCl, and the solution diluted with double-distilled deionized water in a 5 ml volumetric flask. Samples were taken to determine calcium, magnesium and zinc content by atomic absorption spectrophotometry (Model 280, Fisher Scientific Co., Fair Lawn, NJ, U.S.A.), and phosphorus by the method of Fiske and Subbarow (1925). The data were examined by analysis of variance and multiple cornparson (Student-Newman-Keuls). Correlation coefficients between bone weight and various mineral contents was calculated. The 5% level was considered statistically significant. Analyses were conducted on an Apple II microcomputer (Apple Corp., Cupertino, CA, U.S.A.).
Table
effects on bones
408+ 404+
6 10
399 * 9
of the dams is an average
(days)
1
8
15
6.43 k 0.09 6.43 + 0.08
16.7 + I.0 17.7 k 0.6
35.5 + 1.3 36.2 k 1.1
6.75 & 0.03
18.3 + 0.3
38.6 k 1.0
of 6; weight of the pups is an average
of
-_--_
-\
Cranial bones ‘.. %Y. \ “‘\.
-
14.36 _+0.23
23.01 -+ 0.26
b
14.29 + 0.08 14.3I k 0.14
a C
6.49 It 0.06
6.42 & 0.05 6.48 & 0.08
Mandibles
23.08 + 0.15 23.22 + 0.19
__~ e
-
3.41 + 0.07
3.46 + 0.05 3.38 + 0.05
f
6.14 + 0.07
6.25 f 0.09 6.14 + 0.04
R
13.59f0.17
13.43 + 0.10 13.56 & 0.06
Femurs
45.57 + 1.31 44.31 &I.14 50.03 k2.81
Mandible 28.65 kO.98 23.34 2 1.04 29.55 _t 1.90 f +
c
Femur
28.72 10.85 24.82 +0.70 30.03 _+I .76 -I-
Mandible*
Weight (mg)
~_~.~_~_,___. Mandible -~-__I______-. Zn Ocg) Ca (mg) P (mg) Mg (mg) 7.00 3.38 1.01 6.11 to.04 kO.25 kO.15 f 0.45 6.73 3.02 0.82 5.98 to.03 kO.14 kO.08 +0.31 7.30 3.38 0.98 7.73 +0.06 kO.37 + 0.23 kO.66 f + + +
_.I
.-..
_.
I
-
-
+0.01 I .67 f0.04
1.72
I .62 & 0.08
Ca/P**
c
+
-
+
---~&I k) Ca (mg) 4.22 5.89 +0.36 +0.27 3.20 4.75 +0.24 kO.33 5.67 5.99 1kO.29 20.37 + +
Mineral content
Table 3. Bone weight and mineral content of the mandibles and femurs of the newborn rats at day 15
Each value is mean F SEM. Weight of the bones is after drying at 110°C overnight. *Weight of mandibular body (after removal of the teeth). **Ca/P molar ratio. f: Statistically significant (p c; 0.05). -: Statistically not significant (p z 0.05).
20% protein + caffeine (G2) 20% protein + caffeine +zinc (G3) Gi vs G2 G1 vs G3 C,? vr cr?
20% protein (Gl)
Maternal diet
-
-
7.39 _+0.09
7.28 + 0.08 7.51 + 0.06
.d
Physical size (mm)
Table 2. Physical measurements of the cranial bones, mandibles and femurs of newborn rats at day 15
h
i
12.61 kO.17 -If
-t + +
12.58 F0.19 11.97 & 0.08
5.76kO.07
5.53 + 0.03 5.38 f 0.02
__
-
P tmg) 2.51 20.21 2.31 kO.15 2.70 +0.19
Femur
+
-
Mg (mg) 0.87 kO.04 0.72 +0.03 0.92 kO.07
-.___
-
& 0.02
1.72
---.Ca/P** 1.84 iO.11 1.60 +0.04
Each value is mean f SEM; each is an average of 6 measurements. a = Prosthion to basion, b = prosthion to sutua of basisphenoid and presphenoid, c = orbita to orbita, d = first molar to first molar, e = mandibular foramen to condylar process, f = condylar process to angular process, g = mental foramen to condylar process, h = first molar to infradentale, i = fovea to medial condyle. +: Statistically significant (p < 0.05). -: Statistically not significant (p > 0.05).
20% protein (Gt) 20% protein + caffeine (G2) 20% protein + caffeine -i-zinc (G3) Gl vs G2 Gl vs G3 G2 vs G3
Maternal diet
.L “\.._,.
/ cI/l.l.r Caffeine with zinc effects on bones
hblldlbb 8.0
7.2 r
0
7.0
7.5
0
6.6
6.6
7.Q
0
6.5 6.0
6.4
f - O.S?O
26
30
26
32
r-0,$92 pco.005
r-u947 p4l.005
p:o.w5
26
34
6.5
7.0 -
6.0
6.5 -
22
24
26
28
26
Fig. 2. Correlation between bone weight (s axis; mg) and calcium content mandible (p = 0.224 .r + 0.678), femur (y = 0.364 x _- 4.546); middle:
(y = 0.188 s + 2.&B),
femur (}I
(y = 0.148 .v + 1.259); right:
=0.209.x
+
l,Q17), femur
tended to be slighl.ly larger, suggesting that the amount of zinc ~up?lerncnt~tion did not exert any toxic effects on either dams or pups. The physical changes in the femur and mandible in the caffeine group were clearly improved by adding zinc to the caffeine diet, and this group grew to the same extent as the controls. However, the effects on the various bone were different, which could possibly be due to their different developmental origins:
cranial bones are a. mixture of membranous and endochondral bones. and the mandible and femur are
(y =
caffeine
28
28
30
30
32
32
34
34
36
36
3%
(y axis; mg). Left: control, caffeine group, mandibk
plus
zinc
group,
mandibte
0.189 x + 0.378).
membranous and endochrondral bone, respectively ~Weinmann and &her, 19553. Caffeine supplementation increases urinary calcium excretion (Whiting and Whitney, 1987). It is possible that maternal caffeine intake could unbalance the calcium homeostasis of the growing fetus, and of the neonate as a result of suckling milk containing caffeine. These processes may alter the mineralization of the bones in neonates. Urinary zinc excretion, on the other hand, is not affected by caffeine supplementation (Yeh et ul., 1986).
4.0 o 3Jl 0
3.6 0
3.4
0
3.2 3.0 2.6
r=wea p>ou5
00 ~
126
26
30
32
34
2.6'.
t “.
22
23
‘.
24
‘.“I
25
26
27
2.5--L-L-"*.'. 26 28
30
32
34
;6
3.4 3.2 3.0
-
r.0995 p
