Zinc Deficiency Causes to Decrease Plasma Calcium and Ionized Calcium Concentrations Prior to an Increase in the Plasma Parathyroid Hormone Concentration in Rats

Goto et al., Nano Biomedicine 8(2), 67-73, 2016

ORIGINAL ARTICLE Zinc Deficiency Causes to Decrease Plasma Calcium and Ionized Calcium Concentrations Prior to an Increase in the Plasma Parathyroid Hormone Concentration in Rats

Tomoko GOTO, Hitoshi SHIRAKAWA, and Michio KOMAI

Laboratory of Nutrition, Department of Science of Food Function and Health, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan

Synopsis The effects of zinc deficiency on plasma calcium and ionized calcium concentrations were exam- ined in rats. We monitored plasma concentrations of calcium, ionized calcium (Ca2+), parathyroid hormone (PTH) and ionized magnesium (Mg2+) in male 4 week-old Sprague-Dawley rats, that were divided into three groups: zinc-deficient (Zn-Def), low-zinc (Low-Zn), and control pair-fed to Zn-Def (Pair-fed) groups, for 28 days. Comparing to Pair-fed rats, plasma calcium and Ca2+ concentrations of Zn-Def rats were found to be significantly lower after 4, 14, 21, 28 days of feeding. There was no significant change of the plasma Mg2+ concentration among three groups. Comparing to Pair-fed rats, the plasma PTH concentration in Zn-Def rats tended to be higher after 21 days of feeding, and significantly higher after 28 days of feeding. The results suggest that zinc deficiency causes to decrease plasma calcium and Ca2+ concentrations prior to an increase of the plasma PTH concentration.

Key words: zinc, calcium, parathyroid hormone, rats

Introduction Calcium in blood occurs in 3 forms: the pro- Zinc is one of the essential trace elements. It is tein-bound form, the chelated form that is com- well known that zinc deficiency leads to ano- plexed with phosphate and citrate, and the ion- rexia, epilation, failure of immunological com- ized form that is believed to be physiologically petence, and growth retardation in animals [1]. It active. Extracellular ionized Ca2+ concentration has been reported that zinc deficiency impairs is maintained in a narrow concentration range bone growth and development, suggesting a controlled by PTH [8,9]. Zinc compounds added physiologically important role of zinc in the in tissue culture cells derived from calvaria in- regulation of bone metabolism [2,3]. Skeleton hibited the PTH-induced increase of glucose contains a large proportion of the total body consumption or accumulation of lactic acid in burden of zinc [4]. Zinc is secondly abundant in the culture medium [10], suggesting zinc has an bone tissue following the muscle, which con- inhibitory role for PTH-induced bone resorption tains approximately 30% of total zinc in the in vitro. However, there is little information on body [5]. Parathyroid hormone (PTH) is a well the interaction between zinc deficiency and known calciotropic hormone controlling calcium plasma calcium concentration in vivo. Suzuki et homeostasis, promoting bone resorption and in- al. reported that rats fed a zinc-deficient diet for creasing the serum calcium concentration [6,7]. 4 wk decreases the serum concentration of cal-

67 Goto et al., Zinc Deficiency Affects Plasma Calcium, Nano Biomed 8(2), 67-73, 2016 cium along with an increase in the serum PTH by zinc-deficiency, such as bone growth retarda- concentration [11]. They suggested that zinc de- tion and increased NaCl preference, may be as- ficiency is suspected to cause an increase in the sociated with impaired calcium balance in blood. serum PTH concentration owing to an inability It is a reasonable assumption that PTH may be to maintain calcium homeostasis, resulting in involved in the process as it is an important fac- bone fragility. Although their report suggests tor to control both plasma Ca2+ level and bone involvement of zinc for bone formation/ resorption. Based on these findings, we specu- resorption through calcium/PTH, it remains un- lated that zinc-deficiency may alter calcium clear how concentrations of calcium, ionized balance in vivo. In order to determine the effect calcium (Ca2+), ionized magnesium (Mg2+) and of zinc deficiency on calcium balance in rats, we PTH in plasma may change chronologically af- investigated the plasma concentrations of cal- ter feeding a zinc-deficient diet. Particularly, it cium, Ca2+, Mg2+, and PTH during the feeding of should be important to measure plasma concen- the experimental diet. tration of Ca2+, since it is more relevant to clinical abnormalities than total plasma calcium Materials and Methods [12,13]. 1. Animals and diets Zinc deficiency also leads to taste disorder Male Sprague-Dawley rats (age 4 weeks; weight (decreased taste acuity) in both humans and rats, 80-90 g at the time of delivery) (Japan SLC, a symptom that can be reversed by zinc admini- Hamamatsu, Japan) were used in this experi- stration [14-16]. Because of habitual problems ment: all rats were housed individually in of an unbalanced diet, or the zinc-chelating ef- stainless steel cages. The rats were fed a fect of certain kinds of drugs taken by patients, it zinc-sufficient diet for 3 days before commenc- has been suggested that a potentially large num- ing the experimental diets, and they were then ber of individuals could be suffering marginal divided into three groups: zinc-deficient zinc deficiency. McConnell and Henkin demon- (Zn-Def), low-zinc (Low-Zn), and Pair-fed strated that within 3 days of the start of feeding, groups. The Zn-Def and Low-Zn rats were given the intake of 0.30 mol/L NaCl, which is a nor- free access to the appropriate diet, but the mally aversive concentration, was greater in rats Pair-fed rats, which were fed the zinc-sufficient fed a zinc-deficient diet than in zinc-replete ones diet, were pair-fed 1 day later with respect to the [17]. In previous study, we also confirmed that Zn-Def rats. Food consumption and body weight NaCl preference suddenly increased on day 4 in changes in the various experimental groups were the zinc-deficient (Zn-Def) and low-zinc as shown in our previous study [21]. All rats (Low-Zn) rats [18]. Furthermore, we demon- were maintained on a 12 h light/dark cycle at 22 strated that long-term zinc deficiency decreased ± 2 °C with a constant humidity of 50 ± 10%. taste sensitivity at the level of the chorda tym- The experimental plan of the present study was pani (CT) nerve in rats [18]. In order to deter- approved by the Animal Research-Animal Care mine whether such a rapid change of NaCl pref- Committee of the Graduate School of Agricul- erence at the early stage of zinc deficiency is tural Science, Tohoku University. The entire ex- triggered by decreased taste sensitivity at the periment closely followed the guidelines issued level of peripheral nerves, we measured the by that committee, which strictly follows gov- changes in the CT nerve responses against sev- ernment legislation in Japan (1980). The care eral kinds of taste stimulations. As a result, we and use of the rats involved in the present study found that the CT nerve responses to NaCl solu- were under the surveillance of the tions are normal at the early stages of zinc defi- above-mentioned committee. ciency, although long-term zinc deficiency de- Three types of diets with different zinc lev- creases taste sensitivity at the level of the CT els were used as previously reported [18,21,22]. nerve [18]. The added amounts of zinc in each diet of the It has also been reported that rats fed a Zn-Def, Low-Zn and zinc-sufficient diet were 0, low-calcium diet showed increased NaCl pref- 3.8 and 33.8 mg Zn/kg diet, respectively, while erence [19,20]. It suggests that symptoms caused the amounts of zinc in each diet analyzed by

68 Goto et al., Zinc Deficiency Affects Plasma Calcium, Nano Biomed 8(2), 67-73, 2016 atomic absorption spectrophotometry (SAS-727; -80 °C until assayed for PTH with an ELISA kit SEIKO Denshikogyo, Tokyo, Japan) were 2.1, (Rat Intact PTH ELISA Kit, Immutopics, Inc.). 4.1 and 33.7 mg Zn/kg diet, respectively. We confirmed previously that we can create a sub- 4. Statistical analysis clinical zinc deficiency (although overt signs The results are expressed as mean values with and symptoms of zinc deficiency do not appear, their standard errors. The data were analyzed plasma zinc levels are reduced) by feeding statistically using one-way analysis of variance Low-Zn diet, and a severe zinc deficiency (ano- (ANOVA). Post hoc multiple comparisons were rexia and growth retardation, as well as a deple- made using the Scheffé’s test. The StatView tion of the plasma zinc concentration are ob- program (StatView J-5.0; Abacus Concepts, served) by feeding Zn-Def diet [21]. Berkeley, CA, USA) was used for the analysis in each case. 2. Plasma concentrations of calcium, Ca2+ and Mg2+ Results We measured plasma concentrations of calcium, We monitored plasma calcium and Ca2+ concen- Ca2+ and Mg2+ in experimental rats on day 0, 4, trations after 0, 4, 7, 14, 21, and 28 days of 7, 14, 21, 28 of the experimental diet (n = 3~5). feeding. Comparing to Pair-fed rats, plasma cal- Each rat was sacrificed by decapitation and cium and Ca2+ concentrations of Zn-Def rats blood was collected in a heparinized tube. The were found to be significantly lower after 4, 14, blood was centrifuged (1000 × g, 15 min) to 21, 28 days of feeding (-). Comparing to collect plasma. The concentration of calcium Low-Zn rats, plasma calcium and Ca2+ concen- was measured with atomic absorption spectro- trations of Zn-Def rats were also found to be photometric analysis (SAS-727; SEIKO Den- significantly lower after 14, 21, 28 days of feed- shikogyo, Tokyo, Japan). Concentrations of Ca2+ ing (Figure 1, 2). In Low-Zn rats, plasma cal- and Mg2+ were measured with Nova CRT8 cium concentration was significantly lower than ion-selective electrode Analyzer (Nova Bio- that in Pair-fed rats after 4 days of feeding (Fig- medical; MA, USA). ure 1). Moreover, plasma Ca2+ concentration in Low-Zn rats was significantly lower than that in 3. Plasma concentrations of PTH Pair-fed rats after 28 days of feeding (Figure 2). We measured the plasma concentration of PTH On the other hand, there was no change of the on day 0, 4, 7, 14, 21, 28 of the experimental plasma Mg2+ concentration among three groups, diet. Each rat (n = 3~5) was sacrificed by de- although plasma Mg2+ concentration in Zn-Def capitation and blood was collected. The blood rats tended to be higher than other groups (Fig- was centrifuged (1000 × g, 15 min) at 4 °C and ure 3). Comparing to Pair-fed rats, the plasma plasma was collected. The plasma was stored at PTH concentration in Zn-Def rats tended to be

Figure 1 Plasma calcium concentrations during the experimental period. Values are means + SEM, n = 3-5; those at a time with different superscripts are significantly different, P < 0.05. 69 Goto et al., Zinc Deficiency Affects Plasma Calcium, Nano Biomed 8(2), 67-73, 2016 higher after 21 days of feeding, and significantly significantly higher than that in Pair-fed rats af- higher after 28 days of feeding (Figure 4). In ter 28 days of feeding (Figure 4). Low-Zn rats, the plasma PTH concentration was

Figure 2 Plasma Ca2+ concentrations during the experimental period. Values are means + SEM, n = 3-5; those at a time with different superscripts are significantly different, P < 0.05.

Figure 3 Plasma Mg2+ concentrations during the experimental period. Values are means + SEM, n = 3-5.

Figure 4 Plasma intact parathyroid hormone (PTH) concentrations during the experimental period. Values are means + SEM, n = 3-5; those at a time with different superscripts are significantly different, P < 0.05.

70 Goto et al., Zinc Deficiency Affects Plasma Calcium, Nano Biomed 8(2), 67-73, 2016

Discussion rats was significantly lower than that in Pair-fed Plasma calcium and Ca2+ concentrations of rats after 28 days of feeding (Figure 2), while Zn-Def rats were found to be significantly lower the plasma PTH concentration in Low-Zn rats than that in Pair-fed rats after 4, 14, 21, 28 days found to be significantly higher than that in of feeding (Figure 1, 2), while the plasma PTH Pair-fed rats after 28 days of feeding (Figure 4). concentration in Zn-Def rats was significantly The results suggest that marginally low zinc higher than that in Pair-fed rats after 28 days of status can also trigger an decrease of the plasma feeding (Figure 4). The results demonstrate that Ca2+concentration and may lead to elevate zinc deficiency has an effect to decrease plasma plasma PTH level, although the timing of an de- calcium and Ca2+ concentrations, prior to an increase of the Ca2+ concentration was much later, crease in the plasma PTH concentration. comparing to Zn-Def rats. Nielsen showed that Suzuki et al. investigated the effect of zinc although marginal zinc deficiency significantly deficiency on bone remodeling, measuring the decreased the urinary excretion of magnesium serum concentration of mineral component of and calcium, the changes did not alter the mag- bones such as calcium, magnesium and phos- nesium and calcium concentrations in tissues phorus, as well as hormones related to bone re- [25]. However, his experimental condition for modeling in rats fed a zinc-deficient diet after 4 inducing marginal zinc deficiency in rats are wk [11]. Based on their findings, they reported milder (our condition for inducing low-zinc that a zinc-deficient diet causes an impairment in condition was 4.1 mg Zn/kg diet, while he used calcium utilization that is not regulated by an the diet containing 8 mg Zn/kg diet) than that of increase in the serum PTH or the l,25 (OH)2 vi- our present experiments, which may be the rea- tamin D3. In addition, they stated that the im- son why plasma zinc concentrations in his mar- pairment in calcium utilization was suggested to ginally zinc deficient rats did not show measur- lead to a decrease in the serum calcium concen- able decrease of the plasma calcium concentra- tration, resulting in an increase in PTH secretion. tion. Our present study supports their findings, since There was no change of the plasma Mg2+ it showed that a decrease of the plasma Ca2+ concentration among three groups, although concentration occurred well ahead of an increase plasma Mg2+ concentrations in Zn-Def rats tend of the plasma PTH concentration. Although the to be higher than other groups (Figure 3). Al- exact mechanism of the phenomena is still un- though physiological meanings of the data are clear, as Suzuki et al. reported, it is noteworthy not fully understood, there is a possibility that that the rate of apparent calcium absorption was maintaining plasma Mg2+ concentrations at significantly reduced despite increases in PTH higher level may be involved in altered calcium and l,25 (OH)2 vitamin D3 concentrations [11]. utilization in Zn-Def rats. In this respect, there There are several reports showing that are interesting reports suggesting that marginal zinc-deficiency causes to reduce calcium uptake zinc deficiency affects calcium utilization and by cells [23]. It is probable that zinc deficiency magnesium retention in other experimental sys- may cause impaired intestinal calcium uptake, tems. Browning and O’Dell showed that low which may lead to decrease plasma calcium zinc status in guinea pigs impairs calcium uptake level. by glutamate-stimulated brain synaptosomes Although it has not been denied that zinc [26]. In addition, O’Dell has described that in- may also be involved in homeostatic regulations creased retention of cellular magnesium may be of plasma Ca2+ concentrations controlled by involved in the impaired calcium uptake by ex- hormones like PTH, there are supportive evi- citable (e.g., platelets and neurons) and nonex- dence indicating that zinc is directly associated citable cells (i.e., fibroblasts) [27]. with the process of bone formation and resorption provided by in vitro observations, as re- Conclusion viewed by Yamaguchi [24]. Our data should add Zinc deficiency has an effect to decrease plasma in vivo evidence supporting the idea. calcium and Ca2+ concentrations prior to an in- The plasma Ca2+ concentration in Low-Zn crease in the plasma PTH concentration. It sug-

71 Goto et al., Zinc Deficiency Affects Plasma Calcium, Nano Biomed 8(2), 67-73, 2016 gests that zinc should be directly involved in the calcium, serum magnesium, and N-terminal process of calcium utilization in rats. pro-B-type natriuretic peptide in intensive care unit inpatients. Clinical Chemistry 2016; 62: 824-830. Financial Support 13) Huang CY, Zheng CM, Wu CC, Lo L, Lu KC, This work was supported by The Salt Science Chu P. Effects of pamidronate and calcitriol on Research Foundation Grant No.16D4. the set point of the parathyroid gland in postmenopausal hemodialysis patients with sec- References ondary hyperparathyroidism. Nephron Clin 1) Mills CF, Quarterman J, Chesters JK, Williams Pract 2012; 122: 93-101. 14) Henkin RI, Bradley DF. Hypogeusia cor- RB, Dalgarno AC. Metabolic role of zinc. Am ++ ++. J Clin Nutr 1969; 22: 1240-1249. rected by Ni and Zn Life Sci 1970; 9: 2) Calhoun NR, Smith JC Jr, Becker KL. The 701-709. effects of zinc on ectopic bone formation. Oral 15) Hambidge KM, Hambidge C, Jacobs M, Baum Surg Oral Med Oral Pathol 1975; 39: 698-706. JD. Low levels of zinc in hair, anorexia, poor 3) Hsieh HS, Navia JM. Zinc deficiency and growth, and hypogeusia in children. Pediatr bone formation in guinea pig alveolar implants. Res 1972; 6: 868-874. J Nutr 1980; 110: 1581-1588. 16) Yoshida S, Endo S, Tomita H. A double-blind 4) Haumont S. Distribution of zinc in bone tis- study of the therapeutic efficacy of zinc glu- sues. J Histochem Cytochem1961; 9, 141-145. conate on taste disorder. Auris Nasus Larynx 5) Jackson MJ. Physiology of zinc, general as- 1991; 18: 153-161. pects. In Mills CF (eds); Zinc in Human Biol- 17) McConnell SD, Henkin RI. Altered preference ogy 1989; Germany, Springer-Verlag: pp 1-14. for sodium chloride, anorexia, and changes in 6) Aurbach GD, Marx SJ, Spiegel AM. Para- plasma and urinary zinc in rats fed a thyroid hormone, calcitonin, and the calcif- zinc-deficient diet. J Nutr 1974; 104: erols. In Wilson JD, Foster DW (eds): Text- 1108-1114. book of Endocrinology (ed 7) 1985; USA, 18) Goto T, Komai M, Suzuki H, Furukawa Y. Saunders: pp 1137-1217. Long-term zinc deficiency decreases taste sen- 7) Neer RM, Arnaud CD, Zanchetta JR, Prince R, sitivity in rats. J Nutr 2001; 131: 305-310. Gaich GA, Reginster JY, Hodsman AB, Erik- 19) Tordoff MG. The importance of calcium in the sen EF, Ish-Shalom S, Genant HK, Wang O, control of salt intake. Neuroscience and Mitlak BH. Effect of parathyroid hormone Biobehavioral Reviews 1996; 20: 89-99. (1-34) on fractures and bone mineral density in 20) Tordoff MG, Okiyama A. Daily rhythm of postmenopausal women with osteoporosis. NaCl intake in rats fed low-Ca2+ diet: relation N Engl J Med 2001; 344: 1434-1441. to plasma and urinary minerals and hormones. 8) Brown EM. Extracellular Ca2+ sensing, regula- Am J Physiol 1996; 270: (Regulatory Integra- tion of parathyroid cell function, and role of tive Comp Physiol 39), R505-R517. Ca2+ and other ions as extracellular (first) 21) Goto T, Komai M, Bryant BP, Furukawa Y. messengers. Physiol Rev 1991; 71: 371-411. Reduction in carbonic anhydrase activity in 9) Brown EM, Pollak M, Hebert SC. Sensing of the tongue epithelium and submandibular extracellular Ca2+ by parathyroid and kidney gland in zinc-deficient rats. Int J Vit Nutr Res cells: cloning and characterization of an ex- 2000; 70: 110-118. tracellular Ca2+-sensing receptor. Am J Kid- 22) Goto T, Shirakawa H, Furukawa Y, Komai M. ney Dis 1995; 25, 506-513. Decreased expression of carbonic anhydrase 10) Yamaguchi M, Segawa Y, Shimokawa isozime II, rather than of isozyme VI, in sub- N,Tsuzuike N, Tagashira E. Inhibitory effect of mandibular glands in long-term zinc-deficient b-alanyl-L-histidinato zinc on bone resorption rats. Brit J Nutr 2008; 99: 248-253. in tissue culture. Pharmacology 1992; 45: 23) O’Dell BL, Browning JD. Impaired calcium 292-300. entry into cells is associated with pathological 11) Suzuki T, Kajita Y, Katsumata S, Matsuzaki H, signs of zinc deficiency. Adv Nutr 2013; 4: Suzuki K. Zinc deficiency increase serum 287–293. concentration of parathyroid hormone through 24) Yamaguchi M. Role of zinc in bone metabo- a decrease in serum calcium and induces bone lism and preventive effect on bone disorder. fragility in rats. J Nutr Sci Vitaminol 2015; Biomed Res Trace Elements 2007; 18: 61: 382-390. 346-366. 12) Moyer AM, Saenger AK, Willrich M, Donato 25) Nielsen FH. Marginal zinc deficiency in- LJ, Baumann NA, Block DR, Botz CM, Khan creases magnesium retention and impairs cal- MA, Jaffe AS, Hanson CA, Karon BS. Imple- cium utilization in rats. Biol Trace Elem Res mentation of Clinical decision support rules to 2009; 128: 220-231. reduce repeat measurement of serum ionized

72 Goto et al., Zinc Deficiency Affects Plasma Calcium, Nano Biomed 8(2), 67-73, 2016

26) Browning JD, O’Dell BL. Low zinc status in guinea pigs impairs calcium uptake by brain synaptosomes. J Nutr 1994; 124: 436-443. 27) O’Dell BL. Role of zinc in plasma membrane function. J Nutr 2000; 124, 436-443.

(Received: July 23, 2016/ Accepted: August 22, 2016)

Corresponding author: Tomoko GOTO, Ph.D Laboratory of Nutrition, Department of Science of Food Function and Health, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-Ku, Sendai, 981-8555, Japan Fax: +81-22-717-8813 E-mail: [email protected]