Long-term effects of lactational zinc deficiency on bone mineral composition in rats fed a commercially modified Luecke diet

The purpose of this study was threefold: 1. to determine the long-term effects of interactions between lactational zinc deficiency and gender on bone mineral composition in repleted rat offspring, 2. to determine the nutritional efficacy of the second of two commercially designed, modified Luecke diets (ML2) during the gestational and lactational stress, and 3. determine the ultratrace element contents of Ralston Rodent Laboratory Chow #5001. The ML2 basal diet, based on dextrose, sprayed egg white, and corn oil contained 0.420 μg Zn/g, was supplemented with Zn (as zinc acetate) at 0 (diet 0ML2) or 30 (diet 30ML2) μg/g, and was mixed and pelleted commercially. all rat dams were fed the 30ML2 diet ad libitum during gestation. Beginning at parturition, the dams were fed either the 1. 0ML2, 2. 30ML2 (food restricted), or 3. 30ML2 (ad libitum) diets. All pups were fed the 30ML2 diet ad libitum from 23 to 40 d of age. From d 40 to 150, all pups were fed Ralston Rodent Laboratory Chow. The 30ML2 diet was found to be nutritionally efficacious; litter size and pup growth were normal and pup mortality was only 1.2%. Pups (ZD) with access to the 0ML2 diet until 23 d of age and nursed by dams fed the 0ML2 diet, when compared to pups (PF) fed restricted amounts of the 30ML2 diet, exhibited increased mortality and decreased concentrations of tibial zinc but no change in growth. Inadequate zinc nutriture during infancy, despite postlactational zinc repletion, induced imbalances in adult bone mineral metabolism. Thus, at 150 d of age, the ZD pups exhibited increased levels of bone P and Mg and decreased concentrations of K as compared to the PF pups.     

Teratogenicity of zinc deficiency in the rat: study of the fetal skeleton

Zinc deficiency (ZD) is teratogenic in rats, and fetal skeletal defects are prominent. This study identifies fetal skeletal malformations that affect calcified and non-calcified bone tissue as a result of gestational zinc deficiency in rats, and it assesses the effect of maternal ZD in fetal bone calcification. Pregnant Sprague-Dawley rats (180-250 g) were fed 1) a control diet (76.4 micrograms Zn/g diet) ad libitum (group C), 2) a zinc-deficient diet (0 microgram/g) ad libitum (group ZD), or 3) the control diet pair-fed to the ZD rats (group PF). On day 21 of gestation, laparotomies were performed. Fetuses were weighed, examined for external malformations, and stained in toto with a double-staining technique for the study of skeletal malformations. Maternal and fetal tissues were used for Zn, Mg, Ca, and P determinations. Gross external malformations were present in 97% of the ZD fetuses. No external malformations were found in fetuses from groups C and PF. Ninety-one percent of cleared ZD fetuses had multiple skeletal malformations, whereas only 3% of the fetuses of group PF had skeletal defects; no skeletal malformations were found in fetuses from group C. Some of the skeletal malformations described in the ZD fetuses, mainly affecting non-calcified bone, were not mentioned in previous reports, thus stressing the importance of using double-staining techniques. Examination of stained fetuses and counting of ossification centers revealed important calcification defects in ZD fetuses. These effects were confirmed by lower Ca and P concentrations in fetal bone with alteration of the Ca:P ratio.     

Effects of dietary zinc deficiency on homocysteine and folate metabolism in rats

In rats, zinc deficiency has been reported to result in elevated hepatic methionine synthase activity and alterations in folate metabolism. We investigated the effect of zinc deficiency on plasma homocysteine concentrations and the distribution of hepatic folates. Weanling male rats were fed ad libitum a zinc-sufficient control diet (382.0 nmol zinc/g diet), a low-zinc diet (7.5 nmol zinc/g diet), or a control diet pair-fed to the intake of the zinc-deficient rats. After 6 weeks, the body weights of the zinc-deficient and pair-fed control groups were lower than those of controls, and plasma zinc concentrations were lowest in the zinc-deficient group. Plasma homocysteine concentrations in the zinc-deficient group (2.3 +/- 0.2 micromol/L) were significantly lower than those in the ad libitum-fed and pair-fed control groups (6.7 +/- 0.5 and 3.2 +/- 0.4 micromol/L, respectively). Hepatic methionine synthase activity in the zinc-deficient group was higher than in the other two groups. Low mean percentage of 5-methyltetrahydrofolate in total hepatic folates and low plasma folate concentration were observed in the zinc-deficient group compared with the ad libitum-fed and pair-fed control groups. The reduced plasma homocysteine and folate concentrations and reduced percentage of hepatic 5-methyltetrahydrofolate are probably secondary to the increased activity of hepatic methionine synthase in zinc deficiency.     

Renal metallothionein responds rapidly and site specifically to zinc repletion in growing rats

Metallothionein (MT) is important for heavy metals and free radical protection in the kidney. MT is responsive to zinc and primarily localized within the renal cortex. However, site-specific renal responses to dietary zinc repletion are understudied. The objective of this study was to examine the effects of dietary zinc deficiency and repletion on renal MT concentration and immunolocalization in rats. Weanling male Sprague Dawley rats were randomly assigned to either a zinc-deficient, zinc control, or pair-fed to zinc-deficient group. Half of the zinc-deficient and pair-fed rats were repleted with the control diet ad libitum for an additional 24 h. Renal tissue samples were assessed for total zinc, MT concentrations and MT immunostaining. Dietary zinc deficiency reduced renal zinc and MT concentrations, and attenuated intensity and localization of MT. Dietary zinc repletion for 24 h restored renal zinc and MT concentrations, the latter primarily in the proximal convoluted tubules of the cortex. Concentrations of renal MT, but not zinc, were elevated by diet restriction and MT (μg/mg protein) and partially normalized by 24 h diet repletion. In conclusion, renal MT modification due to zinc deficiency or diet restriction can be rapidly normalized in a site-specific manner with normal dietary zinc intake. The results support a role for MT in kidney homeostasis, in particular at the level of the proximal tubules in the cortex. The speed of MT repletion may have clinical implications for dietary zinc in the treatment of acute and chronic renal pathology due to toxins and free radicals.     

Dietary zinc deficiency decreases plasma concentrations of vitamin E

Experiments were conducted to examine the effects of dietary zinc (Zn) upon plasma vitamin E (E) concentrations to test the hypothesis that there may be a significant dietary interaction between these two nutrients. Weanling female Sprague-Dawley rats were fed diets that were (i) Zn-deficient (less than 0.9 micrograms Zn/g diet) ad libitum; (ii) Zn-adequate (50.9 micrograms Zn/g diet), pair-fed to the Zn-deficient group; and (iii) Zn-adequate (50.9 micrograms Zn/g diet) ad libitum. Plasma E in Zn-deficient animals (4.02 +/- 1.20 micrograms/ml) was significantly reduced (P less than or equal to 0.05) compared with results in both Zn-adequate pair-fed (9.21 +/- 0.70 micrograms/ml) and Zn-adequate ad libitum-fed (9.47 +/- 0.90 micrograms/ml) animals. Zn deficiency in this model system also resulted in significant (P less than or equal to 0.05) reductions in femur and plasma Zn concentrations as well as in plasma retinol, plasma triglyceride, and plasma cholesterol concentrations. Plasma albumin and total plasma protein concentrations were normal in Zn-deficient animals. With dietary Zn deficiency, the decrease in plasma E appeared to be out of proportion to associated decreases in plasma triglyceride and plasma cholesterol concentrations. Since E is associated with plasma lipoproteins, these data suggest that lipid and/or E malabsorption may be a consequence of Zn deficiency. In response to increased dietary intake of E, increments of plasma E were lower in Zn-depleted than in Zn-adequate, pair-fed animals. These findings suggest that dietary Zn deficiency possibly may increase the nutritional requirement for E necessary to maintain adequate plasma concentrations.     

Effect of an acute zinc depletion on rat lipoprotein distribution and peroxidation

The aim of this study was to determine the extent to which zinc depletion leads to lipoprotein modifications by measuring both lipoprotein-fraction distribution and peroxidation in zinc-depleted rats. The animals were divided into three groups and fed for 8 wk a zinc-adequate diet (100 ppm) ad libitum (AL), a zinc-deficient diet (0.2 ppm) ad libitum (ZD), or a zinc-adequate diet according to the pair feeding method (PF). Trace-element status, tissular lipids, and lipoprotein-fraction study were performed. The MDA production by the lipoprotein fraction was measured before and after induced peroxidation. Cholesterol and phospholipids were increased in ZD rats. An important increase of VLDL and IDL was observed and a significant enhanced production of MDA by the LDL was related to zinc deficiency. From this observation, we may conclude that LDL fractions of ZD rats are more susceptible to induced oxidative damage. These results suggest that in zinc deficiency, the lipoprotein fragility is an aggravating factor of peroxidation and the dyslipoproteinemia may lead to an atherogenic risk.     

Iron and zinc status in rats with diet-induced marginal deficiency of vitamin A and/or copper

The hypothesis was tested that there are interactions of marginal copper and vitamin A deficiency regarding iron and zinc status. Copper restriction (1 vs 5 mg Cu/kg diet) significantly lowered copper concentrations in plasma and tissues of rats and reduced blood hemoglobin, hematocrit, and iron concentrations in tibia and femur, but raised iron concentrations in liver. Vitamin A restriction (0 vs 4000 IU vitamin A/kg diet) reduced plasma retinol concentrations and induced a fall of blood hemoglobin and hematocrit. Neither copper nor vitamin A restriction for up to 42 d affected feed intake and body wt gain. There were no interrelated effects of vitamin A and copper deficiency on iron status. Copper deficiency slightly depressed liver, spleen, and kidney zinc concentrations. Vitamin A deficiency lowered zinc concentrations in heart, but only when the diets were deficient in copper.     

Decreased food intake rather than zinc deficiency is associated with changes in plasma leptin, metabolic rate, and activity levels in zinc deficient rats( small star, filled)

This study investigated the hypothesis that the reduced food intake and poor weight gain in zinc deficient rats is due to: increased plasma leptin concentration, increased physical activity and/or increased metabolic rate. Weanling rats were assigned to three groups: controls fed ad libitum (C), zinc deficient (ZD), and pair-fed controls (PF), and tested in a metabolic chamber and activity monitor at baseline and weekly for four weeks. At the end of the study, all groups were compared for differences in plasma leptin concentrations. ZD and PF animals had markedly reduced food intake and weight gain. ZD had reduced stereotypic and locomotor activity compared to PF animals and both groups demonstrated an abolished peri-nocturnal activity spike and were much less active than controls. This was associated with a reduced total metabolic rate by day 30: ZD (0.73 +/- 0.07 kcal/hr, p = 0.0001) and PF (0.83 +/- 0.06 kcal/hr, p = 0.0001) groups vs. controls (1.82 +/- 0.09 kcal/hr). Plasma leptin concentrations in ZD (1.55 +/- 0.06 &mgr;g/L) were lower than controls (2.01 +/- 0.18 &mgr;g/L, p < 0.03), but neither ZD nor controls were statistically different from PF (1.68 +/- 0.05 &mgr;g/L). Both low leptin concentrations and low metabolic rates in the ZD and PF rats were associated with decreased food intake rather than zinc deficiency. The reduced food intake and poor weight gain observed in zinc deficient rats could not be explained by elevated leptin concentrations, hypermetabolism, or increased activity. Low serum leptin concentrations, hypometabolism, and decreased activity are more likely the result of the anorexia of zinc deficiency.     

Glucose tolerance, plasma and pancreatic insulin levels in zinc deficient rats.

Two experiments were conducted to examine the effect of zinc deficiency on glucose tolerance, and on blood and pancreatic insulin concentrations. In the first study, no significant differences in blood glucose or plasma insulin levels were noted between pair-weighted zinc deficient and zinc sufficient rats after an oral glucose load. In the second experiment, the concentration of pancreatic insulin in pair-fed zinc sufficient rats did not differ significantly from that of zinc deficient rats. However, a zinc deficient group fed ad libitum had significantly lower pancreatic insulin levels, suggesting that food restriction may cause increased pancreatic insulin. Thus, zinc deficiency per se had no apparent effect on oral glucose tolerance or pancreatic insulin concentrations.     

Relationship between plasma zinc, angiotensin-converting enzyme, alkaline phosphatase and onset of symptoms of zinc deficiency in the rat

Recent evidence suggests that changes in plasma zinc concentration may play a central role in the development of early lesions of zinc deficiency. The aim of the following work was to better understand events occurring in plasma during the onset of zinc deficiency, and to investigate biochemical mechanisms by which plasma zinc may exert its effects. Fifty male weanling rats of 90 g weight were allocated to five treatment groups of ten rats each. Treatments were: 1, zinc deficient, mixed diet (1-2 mg Zn per kg): 2, zinc deficient, self-select diet; 3, zinc repleted; 4, control, pair fed; 5, control, ad libitum fed. With the exception of treatment 1, which consisted of a 25% casein diet, all rats were offered protein as a separate component of the diet. Control rats received zinc in the drinking water (100 mg l-1). The sequence of events following initiation of zinc deficiency were: reduced plasma zinc concentration (2 days), reduced plasma angiotensin-converting enzyme and alkaline phosphatase activities (3-4 days), reduced feed intake and growth (5-6 days) and reduced percentage protein intake (12 days). Plasma zinc concentration in the deficient rats was inversely correlated with the growth rate of the rat over the previous 24 h. Zinc repletion resulted in marked overshoot in plasma zinc concentration (300%) and converting-enzyme activity (150%) within 24 h, but a return to normal within 72 h. Alkaline phosphatase activity responded likewise, albeit more slowly. Protein self selection had no effect on the manifestations of zinc deficiency, although reduced protein intake was associated with lower plasma zinc concentration. The results provide evidence of a role for plasma zinc in the development of early clinical signs of zinc deficiency, possibly acting biochemically through reduced activity of zinc-dependent peptidases such as angiotensin-converting enzyme.     

Maternal zinc deficiency reduces NMDA receptor expression in neonatal rat brain, which persists into early adulthood

Prenatal and early postnatal zinc deficiency impairs learning and memory and these deficits persist into adulthood. A key modulator in this process may be the NMDA receptor; however, effects of zinc deficiency on the regulation of NMDA receptor activity are not well understood. Female Sprague-Dawley rats were fed diets containing 7 (zinc deficient, ZD), 10 (marginally zinc deficient, MZD) or 25 (control) mg Zn/g diet preconception through postnatal day (PN) 20, at which time pups were weaned onto their maternal or control diet. Regulation of NMDA receptor expression was examined at PN2, PN11, and PN65. At PN2, expression of whole brain NMDA receptor subunits NR1, NR2A, and NR2B was lower in pups from dams fed ZD and MZD compared to controls, as analyzed using relative RT-PCR and immunoblotting. At PN11, whole brain and hippocampi NR1, NR2A, NR2B and PSA-NCAM (polysialic acid-neural cell adhesion molecule) expression and the number of PSA-NCAM immunoreactive cells were lower in pups from dams fed ZD compared to controls. Whole brain brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) concentrations were lower in pups from dams fed ZD or both low zinc diets, respectively. Whole brain NR1 expression remained lower in previously zinc-deficient rats at PN65. These data indicate potential mechanisms through which developmental zinc deficiency can impair learning and memory later in life.     

Changes in the fetal tibial growth plate secondary to maternal zinc deficiency in the rat: a histological and histochemical study

Zinc deficiency (ZD) is teratogenic in rats, and fetal skeletal defects are prominent. To elucidate further the effects of maternal ZD in the fetal skeleton, we performed a morphological and histochemical study of tibial growth plate (GP) in ZD rat fetuses. The histochemical study included the identification of calcium, of hydrolytic enzymes associated with the process of calcification, and of oxidative enzymes related to energy production and to the synthesis of proteoglycans. Pregnant Sprague-Dawley rats were fed (1) a control diet (76.4 micrograms Zn/g diet) ad libitum (group C), (2) a zinc-deficient diet (0 micrograms/g) ad libitum (group ZD), or (3) the control diet pair-fed to the ZD rats (group PF). On day 21 of gestation, laparotomies were performed, the fetuses were removed, and fetal tibiae obtained. Specimens were stained with hematoxylin-eosin (H&E) and Masson Trichrome and were processed for identification of alkaline phosphatase, adenosine triphosphatase, succinic dehydrogenase, NADH dehydrogenase, and calcium. The morphologic patterns found in ZD fetal tibiae indicated defects in various cell types implicated in bone metabolism. Staining for hydrolytic enzymes revealed alterations in the size and distribution of matrix vesicles and a weaker staining for ATPase in ZD fetuses. Staining for oxidative enzymes was overall more intense in ZD fetal tibiae. ZD fetuses also presented irregular and defective calcification. These findings indicate that severe maternal ZD in the rat results in structural and functional alterations in the GP of fetal bone, leading to a defective endochondral ossification.     

The effect of dietary n-3 long-chain polyunsaturated fatty acids on femur mineral density and biomarkers of bone metabolism in healthy, diabetic and dietary-restricted growing rats

INTRODUCTION: Dietary fish oil promotes bone formation in healthy states, but its effect during insulin deficiency or nutrient restriction is unclear. METHODS: Eighty weanling male rats were randomized to receive an injection of streptozotocin to induce insulin deficiency (diabetes) or saline (control) and a diet containing soy oil or corn + fish oil for 35 days. Half of the saline-injected rats were randomized to 20% dietary restriction. Measurements were growth, biomarkers of bone metabolism and femur bone mass. RESULTS: Density of femur was elevated in the corn + fish group and reduced in the diabetes group. Plasma osteocalcin and bone prostaglandin E2 (PGE2) were reduced by the corn + fish diet. N-telopeptide, IGF-1, bone PGE2 and urinary Ca were highest and calcitriol lowest in the diabetes group. CONCLUSIONS: These data suggest that the benefit of a diet high in n-3 long-chain polyunsaturated fatty acid is most advantageous to long bone density in healthy states.     

Effects of Zinc Supplementation and Deficiency on Bone Metabolism and Related Gene Expression in Rat

One hundred male rats were randomly divided into four groups (n = 25) and fed a Zn-adequate diet (ZA, 46.39 mg/kg), Zn-deficient diet (ZD, 3.20 mg/kg), Zn-overdose diet (ZO, 234.39 mg/kg), or were pair-fed a Zn-adequate diet (PF) for 5 weeks, respectively. The body weight, femur weight, and activity of alkaline phosphatase (ALP) were reduced in the ZD group but were increased in the ZO group. Zn concentrations in both liver and femur were elevated in the ZO group, whereas femur Zn was decreased in the ZD group. The concentrations of calcium and phosphorus were lower in the ZD than those in other groups. Serum calcium concentration was decreased in the ZD. The relative expression level of ALP was decreased in both ZD and PF, and no significant differences were observed between ZO and ZA. Insulin-like growth factor-I (IGF-I) mRNA level was reduced in the ZD but unchanged in the ZO and PF group. Zn deficiency also decreased ALP mRNA level as compared with that of PF group. Carbonic anhydrase II mRNA level was not affected by Zn. Nevertheless, dietary Zn influenced the growth, bone metabolism, and expression of IGF-I and ALP in male growing rats.     

Comparative study of zinc, copper, manganese, and iron concentrations in organs of zinc-deficient rats and rats treated neonatally with l-monosodium glutamate

The effects of a zinc-deficient (ZD) diet on the growth and trace element concentrations of various organs (body hair, liver, kidney, gastrocnemius muscle, and femur) of male rats were studied. Furthermore, these trace element concentrations of the above-mentioned organs in male rats neonatally treated with l-monosodium glutamate (MSG) are compared with those of the ZD rats. The ZD rats showed growth retardation compared to rats fed a zincadequate diet (controls). The feed efficiency of the ZD rats was only one-fifth of the controls. This is one reason why the ZD rats showed retarded growth. Body hair concentration of zinc (Zn) in the ZD rats was significantly lower than in the controls. On the other hand, copper (Cu), manganese (Mn), and iron (Fe) concentrations in the body hair were significantly higher in the ZD rats than in the controls. Moreover, the apparent absorption rate of these trace elements was significantly higher in the ZD rats than in the controls. The reason for the decrease in Zn contents of the body hair in the ZD rats is probably the reduced dietary Zn intake. Liver and kidney concentrations of Zn in the ZD rats were significantly lower than in the controls. Femur Zn concentrations in the control rats showed higher values than in the ZD rats. Cu and Mn concentrations in the femur in the ZD rats showed higher values than in the controls. Ninh et al. suggested that growth retardation in ZD rats is the result of a decrease in protein biosynthesis. The results of this study support their theory. The reasons for the use of MSG-treated rats in this study are as follows. (1) We reported on the head hair concentration of the above-mentioned elements from pituitary dwarfism (human growth hormone deficient) patients. In that study, the sample was restricted to head hair from pituitary dwarfism patients. More detailed physiological data may be obtained by the used of MSG-treated rats. (2) We took notice of many resemblances between the pituitary dwarfism patients and the MSG-treated rats in morbidity. The MSG-treated rats showed a severe growth retardation compared to NaCl-treated controls. Zn concentration in the body hair was significantly higher in the MSG-treated rats than in the NaCl-treated controls. For the other trace element concentrations, there were no significant differences between the MSG-treated rats and the NaCl-treated controls. The concentrations of these trace elements in the liver of the MSG-treated rats were lower than in the NaCl-treated controls. In the MSG-treated rats, the concentrations of Zn and Cu in the femur were higher than in the NaCl-treated controls. However, the Fe concentration in the femur of the MSG-treated rats showed lower values compared with NaCl-treated controls. The results of this study suggest that the reduction of rat growth hormone (rGH) secretion and/or its synthesis are a consequence of the impairment of rGH anabolic effects. Furthermore it indicates that MSG-treated rats are useful as an in vivo model for the study of the effects of GH.     

Increased methionine synthetase activity in zinc-deficient rat liver

To study the effect of zinc deficiency on folate metabolism, three groups of male Sprague-Dawley rats (zinc deficient (ZD), restricted-fed (RF + Zn), and ad libitum-fed control (control] were given a semipurified 25% egg white protein diet. The ZD group received less than 10.3 nmol zinc/g of diet, while the RF + Zn and control groups were given 1620 nmol zinc/g of diet. After 6-7 weeks of feeding, severe zinc deficiency developed in ZD rats. Hepatic methionine synthetase activity was increased in the ZD group compared to both the RF + Zn and control groups, but hepatic 5,10-CH2-H4folate reductase activity was similar in all groups. This increased methionine synthetase activity found in zinc-deficient rats might induce secondary alterations in folate metabolism. These changes include significantly lowered plasma folate levels, decreased 5-CH3-H4folate in liver, and increased rates of histidine and formate oxidation. The latter two findings suggest that the available non-5-CH3-H4folate is increased in zinc deficiency.     

Skeletal effects of zinc deficiency in growing rats.

There is ample evidence that zinc plays an important role in bone metabolism and zinc deficiency has been implicated as a risk factor in the development of osteoporosis. It was the aim of the present study to investigate the skeletal effects of alimentary zinc deficiency in growing rats using quantitative bone histomorphometry. Twenty-four male Sprague Dawley rats with a mean initial body weight of 101 +/- 2 g were allocated in two groups of 12 rats each and had free access to a semi-synthetic, casein-based, zinc-deficient diet (0.76 mg zinc/kg) or to the same diet supplemented with 60 mg zinc per kg. All rats were sacrificed 42 days after the start of the experiment and the right distal femur was removed for bone histomorphometry. Relative to controls (+Zn), the zinc-deficient rats (-Zn) had a significantly lower body weight and about an 80% reduction in plasma and femur zinc concentration. The histomorphometric evaluation of the distal femoral metaphysis showed that zinc deficiency led to a 45% reduction (p < 0.01) in cancellous bone mass and to a deterioration of trabecular bone architecture, with fewer and thinner trabeculae. The osteopenia in -Zn rats was accompanied by significant reductions in osteoid perimeter (-31%, p < 0.05), osteoblast perimeter (-30%, p < 0.05), and osteoclast number (-38%, p < 0.01) relative to +Zn controls. We conclude that zinc deficiency induced low turnover osteopenia in femoral cancellous bone of growing rats. These results support the hypothesis that zinc deficiency during growth may impair the accumulation of maximal bone mass in humans; additionally, they suggest that zinc deficiency may play a role as a risk factor in the pathogenesis of osteoporosis.