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Mild to moderate zinc deficiency in short children: Effect of zinc supplementation on linear growth velocity
Mild to moderate zinc deficiency in short children: Effect of zinc supplementation on linear growth velocity Toshiro Nakamura, MD, PhD, Soroku Nishiyama, MD, Yoshiko Futagoishi-Suginohara, MD, Ichiro Matsuda, MD, PhD, a n d Akimasa Higashi, MD, PhD From the Departments of Pediatrics, Kumamoto City Hospital and Kumamoto University Medical School, Kumamoto, Japan Twenty-One prepubertal, short Japanese children ( t t boys) without endocrine abnormalities were identified as having mild-to-moderate zinc deficiency by zinc kinetics studies (zinc b o d y clearance _>20 ml/kg per hour). Only one child had a serum zinc level <65/~g/dl (cutoff level). A total of 10 children (5 boys) received 5 mg/kg per day of zinc sulfate for 6 months; 11 untreated children (6 boys) served as control subjects. During treatment, calorie intake (p <0.01), growth velocity (p <0.01), serum zinc, calcium, and phosphorus concentrations, alkaline phosphatase activity (p <0.001), percentage of tubular reabsorption of phosphorus (p <0.05), ratio of maximal tubular reabsorption rate for phosphorus to the glomerular filtration rate (p <0.05), serum osteocalcin level (p <0.01), and plasma insulin-like growth factor I (p <0.05) were significantly increased, but urinary excretion of growth hormone was unchanged in the zinc-supplemented group. All these values were unchanged in the untreated children. We conclude that zinc supplementation is effective for inducing growth in short children with zinc deficiency, and that b o d y zinc clearance tests facilitate detection of marginal zinc deficiency. (J PEDIATR1993;123:65-9)
Zinc is required for metabolic activity of about 200 enzymes and is considered essential for cell division, DNA synthesis, and protein synthesis.l3 Growth and development, testicular function, taste and appetite, wound healing, behavior, resistance to infection, and memory are impaired as a result of zinc deficiency in human beings. 1, 4-6 In most studies the levels of zinc in plasma and in hair were measured, 7' 8 but the former measurement is not useful for estimating the marginal status of zinc nutrition, and a drawback of the latter is the seasonal variation in the rate of hair growth) Accordingly, how to diagnose zinc deficiency remains controversial, especially in its mild or moderate stages. ComSubmitted for publication Aug. 10, 1992; accepted Feb. 19, 1993. Reprint requests: Toshiro Nakamura, MD, PhD, Department of Pediatrics, Kumamoto City Hospital, Koto 1-1-60, Kumamoto 862, Japan. Copyright | 1993 by Mosby-Year Book, Inc. 0022-3476/93/$1.00 + .10 9/20/46548
pared with zinc values in plasma and hair, measurement of leukocyte zinc content2,9 or of body zinc clearance 1~ 11 seems more reliable. We report here the findings in short children with mild-to-moderate zinc deficiency, determined Clzn GFR GH IGF- 1 Kel TmP Vd
Total body zinc clearance Glomerular filtration rate Growth hormone Insulin-like growth factor 1 Elimination constant Maximal tubular reabsorption rate for phosphorus Volume of distribution
by using body zinc clearance methods, and the effect of supplemental zinc on growth. METHODS A total of 21 Japanese children (11 boys) with short stature were studied. They were selected by the following tests: a Tanner evaluation, growth hormone provocation test, and
65
66
N a k a m u r a et al.
The Journal of Pediatrics July 1993
group l, and 6.1 _+ 2.5 years and 3.8 +_ 1.0 years in group 2. All anthropometric measurements were made by the same pediatrician in the early morning at the initial and the 2-, 4-, and 6-month clinical visits. Height was measured with the children standing barefoot; the mean was calculated from three serial measurements. Linear growth velocity and z score for height for age were calculated according to reference data from the Ministry of Health and Welfare of Japan. 12
= 15I~ -o
Cp~.
r
'.*" -~~'q
100~
c
I
~Co
ro o rE
Co
50
~
0
30
i
I
60 90 Tim e (minutes)
I
120
Fig. I. Serum decay curve after intravenous zinc administration. AC0 = Cp - Co, Estimated initial increase of serum zinc concentration after zinc infusion;Cp, theoretical serum zinc concentration; Co, initial serum zinc concentration. Volumeof distribution, Kel of serum zinc, and Clzn were calculated as described in Methods section.
body zinc clearance test. The tests were performed on 220 patients with short stature hospitalized in our clinic. Informed consent for the study was obtained from the parents. Three provocation tests for GH secretion, including intravenous administration of insulin (0.1 U/kg), oral administration of clonidine (0.1 mg/m2), and intravenous administration of growth hormone releasing factor (1 #g/kg), were used to evaluate GH secretion. Zinc kinetics were studied as described below. The final criteria for inclusion in the study were (1) height for age < - 2 . 0 SD, (2) apparent good health with no evidence of endocrinologic disorder, peak serum GH level >10 ng/ml in insulin and clonidine stimulation tests, and >20 ng/ml in the growth hormone releasing factor loading test, (3) zinc clearance >_20 ml/kg per hour, and (4) prepubertal status (Tanner breast and genitalia growth stage) throughout the study period. The children were divided randomly into two groups: group 1 (five boys and five girls) received zinc sulfate, 5 mg/kg per day, orally for 6 months, and group 2 (six boys and five girls) were control subjects. The chronologic age and bone age were 5.6 +_ 1.2 years and 3.8 _+ 1.0 years, respectively, in
The body zinc clearance test was performed as followslO, H: At 10 A M ( 3 hours after breakfast), zinc sulfate solution, 1 #mol/kg, was injected into a mid-cubital vein. Blood specimens were obtained from a mid-cubital vein of the contralateral arm at 0, 30, 60, 90, and 120 minutes after the inj ection. The samples were immediately transferred to an acid-washed tube and allowed to clot. The serum was then transferred to a second acid-washed tube and kept frozen until analysis. Serum zinc levels were measured by atomic absorption spectrophotometry (model 403, PerkinElmer/Cetus, Norwalk, Conn.). The serum zinc level declined exponentially for up to 120 minutes after the intravenous injection of zinc. These levels can therefore be used to predict the turnover rate of zinc. The volume of distribution was calculated by using the following equations: Vd-
Dose s
AC0 = Cp - Co where the dose is the amount of intravenously administered zinc, Cp is the theoretical serum zinc concentration immediately after injection as calculated from the serum concentration versus time profile, and Co is the initial serum zinc concentration (Fig. l). The elimination constant of serum zinc was calculated by using the following equation: Kel =
0.693
where tl/2 is the biologic half-life of serum zinc calculated directly from the serum concentration versus time profile. Total body zinc clearance was calculated as follows: Clzn = Kel X Vd. Serum calcium, phosphate, creatinine, and alkaline phosphatase levels were measured by using an AutoAnalyzer (Technicon, Tarrytown, N.Y.). The ratio of maximal tubular reabsorption rate for phosphorus to the glomerular filtration rate was calculated by the method of Walton and Bijvoet} 3 Serum insulin-like growth factor 1 levels were measured by using radioimmunoassay kits (Nichols Institute Diagnostics, San Juan Capistrano, Calif.). The intraassay and interassay coefficients of variation were 5.7% and 9.2%, respectively. Serum GH levels were measured by
The Journal o f Pediatrics Volume 123, Number 1
N a k a m u r a et al.
67
Table I. Effect of zinc supplementation on growth Patients with zinc s u p p l e m e n t a t i o n
Height (SDS) Height velocity (SDS) Calorie intake (cal/day)
Patients without zinc supplementation
Before
After
p
Before
After
p
-2.54 • 0.20 -3.14 • 0.91 1312 _+ 32
-2.17 • 0.16 2.26 _+ 1.06 1456 • 38
<0.05 <0.01 <0.01
-2.34 _+ 0.23 -2.29 + 0.25 1278 • 34
-2.57 • 0.53 -2.42 _+ 0.62 1351 _+ 41
NS NS NS
NS, Not significant; SDS, SD score. Values are mean _+ SEM.
T a b l e II. Effect of zinc supplementation on serum, plasma, a n d urinary biochemical values Normal range
Clzn (ml/kg/hr) -Serum zinc (#mol/L) 10.0 _+ 19.9 Serum zinc (/zg/dl) 65-130 Serum calcium (mmol/L) 2.15-2.70 Serum calcium (mg/dl) 8.6-10.8 Serum phosphorus (retool/L) 1.36-1.58 Serum phosphorus (mg/dl) 4.2-4.9 Serum ALP (U/L) 210-320 TRP (%) 85-95 TmP/GFR ratio 4.9-5.3 Serum osteocalcin 0zg/L) 15-20 Plasma IGF-1 (U/m1) 0.6-1.2 Urinary GH (ng/gm of Cr) 15-20
Patients with zinc supplementation Before
29.3 11.9 78 2.29 9.16 1.60 4.97 219 91.3 5.31 15.3 0.84 17.3
• 2.9 • 0.9 _+ 6 • 0.04 • 0.16 _+ 0.05 • 0.14 • 9 • 1.3 • 0.21 _+ 2.2 • 0.27 _+ 5.6
After
p
-• • • • • • • + • • + •
-<0.01 <0.01 <0.05 <0.05 <0.05 <0.05 <0.001 <0.05 <0.05 <0.01 <0.01 NS
15.9 104 2.40 9.61 1.68 5.19 275 93.6 5.91 24.2 1.23 16.2
0.9 6 0.03 0.12 0.04 0.11 11 0.7 0.26 2.3 0.28 2.2
Patients without zinc supplementation Before
30.7 13.0 85 2.31 9.24 1.49 4.62 235 92.4 5.13 16.5 0.49 18.3
• 3.7 • 0.8 • 5 • 0.03 _+ 0.11 • 0.06 • 0.20 _+ 14 • 2.0 _+ 0.41 • 2.4 _+ 0.14 + 4.3
After _
12.1 79 2.28 9.13 1.46 4.53 251 92.7 4.95 17.1 0.50 17.9
_
_+ 0.3 _+ 2 _+ 0.04 • 0.15 _+ 0.06 _+ 0.19 • 14 • 1.5 • 0.45 _+ 2.0 _+ 0.17 • 3.5
p b
NS NS NS NS NS NS NS NS NS NS NS NS
ALP, Alkaline phosphatase; Cr, creatinine; NS, not significant; TRP, tubular reabsorption of phosphorus. Values are mean _+ SEM.
r a d i o i m m u n o a s s a y with a n t i h u m a n G H a n t i s e r u m obtained by i m m u n i z a t i o n of a r a b b i t with synthetic h u m a n G H (Eiken Kizai Co., Ltd., Tokyo, Japan). A n t i r a b b i t 3,-globulin goat serum, the same synthetic G H , and synthetic G H labeled with iodine 125 were used as a second antibody, a standard, a n d a tracer, respectively. This assay detected G H levels as little as 1 ~ g / L ; the intraassay and interassay coefficients of variation were 6.2% a n d 8.5%, respectively. S e r u m osteocalcin levels were measured by using r a d i o i m m u n o a s s a y kits (Incstar Corp., Stillwater, Minn.), as described elsewhere. 14 U r i n a r y G H concentrations were m e a s u r e d in duplicate by the highly sensitive enzyme i m m u n o a s s a y described by H a s h i d a et al. ~5 a n d Sukegawa et al. 16 Dialyzed urine samples or G H s t a n d a r d were incubated with monoclonal a n t i h u m a n G H I g G coated polystyrene balls at 37 ~ C for 6 hours. T h e polystyrene balls were then i n c u b a t e d with affinity-purified r a b b i t a n t i h u m a n G H F a b ' - h o r s e r a d i s h peroxidase conjugated at 4 ~ C for 16 hours a n d at 20 ~ C for 6 hours. Peroxidase activity bound to the balls was assayed by fluorimetry, with 3-(4-hydroxyphenyl)propionic acid used as the substrate. T h e intraassay and interassay coefficients of variation were 5.8% and 8.9%, respectively. T h e detection limits of h u m a n
G H in serum and urine were 1.5 p g / m l with 20 #1 serum and 0.2 p g / m l with 0.15 ml urine, respectively. U r i n a r y h u m a n G H was expressed in terms of n a n o g r a m s per day and n a n o g r a m s per g r a m of creatinine. (To convert urinary hum a n G H values to n a n o g r a m s per millimole of creatinine, divide by 8.84.) T h e S t u d e n t paired t test was used for statistical assessment.
RESULTS As shown in Tables I and II, the biologic and biochemical values at the time of initiation of our study were similar in groups 1 and 2. All biochemical m e a s u r e m e n t s were within the normal range. S e r u m zinc levels were within the normal r a n g e in all subjects, except for one in group 1, whose serum zinc level was 9.9 # m o l / L ( < 6 5 # g / d l ) , the cutoff point. 17 The Clzn values were 29.3 _+ 2.9 m l / k g per hour (range, 20.0 to 48.8 m l / k g per hour) in group 1, and 30.7 _+ 3.7 m l / k g per hour (range, 20.2 to 40.7 m l / k g per hour) in group 2. C o m p a r e d with a g e - m a t c h e d control values in our clinic population (15.1 + 0.6 m l / k g per hour),1~ these values were significantly elevated (p <0.001), thereby indicating a mild-to-moderate zinc deficiency. A m o n g these children, only the s u b j e c t with the lowest serum zinc level
68
Nakamura et al.
The Journal of Pediatrics July 1993
zinc-supplemented group
tO
control group
.=_
-5 I o
I
I O
.Q
Fig. 2. Effects of 6 months of zincsupplementation on linear growth velocity. In the zinc-supplementedgroup, SD score (SDS) was improved(p <0.01), and there was only one significantchange in the control group. 7.7 ~mol/L (50 /~g/dl) and with the highest Clzn (48.8 ml/kg per hour) could have a diagnosis of zinc deficiency in the classic sense/7 After oral administration of zinc sulfate for 6 months, all biologic and biochemical measurements, including linear growth velocity (p <0.01), calorie intake (p <0.01), the serum levels of zinc (p <0.01), calcium (p <0.05), phosphorus (p <0.05), and alkaline phosphatase (p <0.001), the percentage of tubular reabsorption of phosphorus (p <0.05), T m P / G F R ratio (p <0.05), the serum level of osteocalcin (p <0.01), and the plasma level of IGF-1 (p <0.01), were improved, whereas the urinary calcium/creatinine ratio and GH excretion rates were unchanged (Tables I and lI; Fig. 2). All mean values in the untreated subjects in group 2 remained unchanged during the same period of observation. DISCUSSION Zinc supplementation can improve rates of growth in children with zinc deficiency,8, is, 19but related mechanisms are poorly understood. Zinc clearance has also been evaluated in prepubertal diabetic children, in whom a significant
negative correlation was found between linear growth velocity and the Clzn value; zinc supplementation resulted in a considerable increase in height. 1~Other investigators reported an association with improvements in serum levels of GH, 2~ thyrotropin-releasing hormone, 23 IGF-1, 24-26 and vitamin D, as well as bone protein metabolism.262s In our study, urinary GH excretion and other pituitary hormone levels in the plasma were unchanged with zinc supplementation (data not shown), possibly because of the mild or moderate deficiency of zinc in these patients. An important observation was the significant elevation in the IGF-1 level after zinc supplementation. We found only one short communication that the plasma IGF-1 level increased after zinc supplementation in malnourished children aged 6 to 24 months. 29 Elevated reabsorption of phosphorus in the renal tubule, as shown by increases of the T m P / G F R ratio and percentage of tubular reabsorption of phosphorus, resulted in a significant increase in serum phosphorus levels in the zincsupplemented children, a condition that seems linked with the serum IGF-I level. 3~Caverzasio and Bonjour3t reported that IGF- 1 can stimulate renal phosphate transport, both in vivo and in vitro. Receptors for IGF-1 have been isolated from the proximal tubular cell plasma membrane. 32 Increased plasma levels of IGF-1, despite unchanged GH production (urinary excretion), can be explained if zinc has a role in making a strong combination between GH and the GH receptor. 29 Yamaguchi et al. 27, 28 found that zinc stimulates bone protein synthesis at the translational level in rat bone cells and synergistically enhances 1,25-hydroxyvitarain D3-stimulated bone metabolism. A similar phenomenon may have occurred in our zinc-supplemented children because there was evidence of enhanced serum levels of calcium, phosphorus, and osteocalcin. The cause of mild-to-moderate zinc deficiency in our patients is uncertain; none seemed to have a dietary intake of zinc any less than that of the normal children. We conclude that body zinc kinetics studies will reveal some cases of marginal zinc deficiency. We propose that children with nonendocrinologicshort stature be treated for at least 6 months with zinc, even if the serum zinc concentration is within normal ranges. The pediatrician can thus determine whether there is a response to zinc even when Clzn studies are not feasible. We are indebted to Ms. Ohara for critical comments. REFERENCES
1. Prasad AS. Discovery and importance of human nutrition. Federal Proceedings 1984;43:2829-34. 2. Prasad AS, Oberleas D. Thymidine kinase activity and incorporation of thymidine into DNA in zinc deficient tissue. J Lab Clin Med 1974;83:634-9.
The Journal of Pediatrics Volume 123, Number 1
3. Vallee BL, Williams RJ. Metalloenzyme. The entatic nature of their active status. Proc Natl Acad Sci USA 1968;59:498505. 4. Hambidge KM, Silverman A. Pica with rapid improvement after dietary zinc supplementation. Arch Dis Child 1973;48: 567-8. 5. Prasad AS. Clinical manifestations of zinc deficiency. Aanu Rev Nutr 1985;5:341-63. 6. Laitinen R, Vouri E, Dahlstrom S, Akerblom HK. Zinc, copper, and growth status in children and adolescents. Pediatr Res 1988;25:323-6. 7. American Academy of Pediatrics Committee on Nutrition. Zinc. Pediatrics 1978;62:408-12. 8. Gibson RS, Smit Vanderkooy PD, MacDonald AC, Goldman A, Ryan BA, Berry M. A growth-limiting, mild zinc deficiency syndrome in some southern Ontario boys with low height percentiles. Am J Clin Nutr 1989;49:1266-73. 9. Pai LH, Prasad AS. Cellular zinc in patients with diabetes mellitus. Nutr Res 1988;8:889-97. 10. Nakamura T, Higashi A, Nishiyama S, Fujimoto S, Matsuda I. Kinetics of zinc status in children with insulin dependent diabetes mellitus (IDDM). Diabetes Care 1991;14:553-7. 11. Nakamura T, Higashi A, Takano S, Akagi M, Matsuda 1. Zinc clearance correlates with clinical severity of Crohn's disease: a kinetic study. Dig Dis Sci 1988;33:1520-4. 12. Committee for Child Growth. The data on child growth and a report of statistical investigations in Japanese schools in Japan. Tokyo: Ministry of Health and Welfare of Japan, 1988. 13. Walton R J, Bijvoet OLM. Nomogram for deviation of renal threshold phosphate concentration. Lancet 1975;2:309-10. 14. Nishiyama S, Tomoeda S, Ohta T, Higuchi A, Matsuda I. Difference in basal and postexercise osteocalcin levels in athletic and nonathletic humans. Calcif Tissue Int 1988;43:150-4. 15. Hashida S, Ishikawa E, Kato U, Imura H, Mohri Z, Murakami Y. Human growth hormone (hGH) in urine and its correlation to serum hGH examined by a highly sensitive sandwich enzyme immunoassay. Clin Chim Acta 1987;162:229-35. 16. Sukegawa I, Hizuka N, Takano K, et al. Urinary growth hormone (GH) measurements are useful for evaluating endogenous GH secretion. J Clin Endocrinol Metab 1988;66:1119-23. 17. Higashi A, lkeda T, Uehara I, Matsuda I. Effect of low content zinc and copper formula on infant nutrition. Eur J Pediatr 1982;138:237-40. 18. Castillo-Duran, Heresi G, Fisberg M, Uauy R. Controlled trial of zinc supplementation during recovery from malnutrition:
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19.
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24. 25.
26.
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29. 30.
31.
32.
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effects on growth and immune function. Am J Clin Nutr 1987;45:602-8. Walravens PA, Hambidge KM, Koepfer DM. Zinc supplementation in infants with a nutritional pattern of failure to thrive: a double-blind, controlled study. Pediatrics 1989;83: 532-8. Collip P J, Castro-Magana M, Petrovic M, et al. Zinc deficiency: improvement in growth and growth hormone levels with oral zinc therapy. Ann Nutr Metab 1982;26:287-90. Root AW, Duckett G, Sweetland M, Reiter EO. Effects of zinc deficiency upon pituitary function in sexually mature and immature male rats. J Nutr 1979;109:958-64. Nishi Y, Hatano S, Aihara K, Fujie A, Kihara M. Transient partial growth hormone deficiency due to zinc deficiency. J Am Coll Nutr 1989;8:93-7. Jordan D, Suck C, Veisseire M, Chazot G. Zinc may play a role in the regulation of thyrotropin function. Horm Res 1986;24:263=8. Cossack ZT. Somatomedin-C in zinc deficiency. Experientia 1984;40:498-500. Oner G, Bhaumick B, Bala RM. Effect of zinc deficiency on serum somatomedin levels and skeletal growth in young rats. Endocrinology 1984;14:1860-5. Bolze MS, Reeves RD, Lindbeck FE, Elders MJ. Influence of zinc on growth, somatomedin, and glycosaminoglycan metabolism in rats. Am J Physiol 1987;252:E21-6. Yamaguchi M, Inamoto K. Differential effect of calcium regulating hormones on bone metabolism in weanling rats orally administered zinc sulfate. Metabolism 1986;35:1044-7. Yamaguchi M, Oishi H, Suketa Y. Zinc stimulation of bone protein synthesis in tissue culture. Biochem Pharmaeol 1988; 37:4075-80. Payne-Robinson HM, Golden MHN, Golden BE, Simeon OT. The zinc sandwich and growth. Lancet t99I;337:925-6. Nishiyama S, Ikuta M, Nakamura T, Tomoeda S, Matsuda I. Renal handling of phosphate can predict height velocity during growth hormone therapy for short children. J Clin Endocrinol Metab 1992;74:906-9. Caverzasio J, Bonjour J-P. Stimulatory effect of recombinant IGF-1 (somatomedin C) on renal phosphate (Pi) transport in vivo and in epithelial cell culture [Abstract]. Proceedings of the 10th International Congress of Nephrology, London, 1987:418. Hammerman M, Rogers S. Distribution of IGF receptors in the plasma membrane of proximal tubular cells. Am J Physiol 1987;253:F841-7.
Zinc is required for metabolic activity of about 200 enzymes and is considered essential for cell division, DNA synthesis, and protein synthesis.l3 Growth and development, testicular function, taste and appetite, wound healing, behavior, resistance to infection, and memory are impaired as a result of zinc deficiency in human beings. 1, 4-6 In most studies the levels of zinc in plasma and in hair were measured, 7' 8 but the former measurement is not useful for estimating the marginal status of zinc nutrition, and a drawback of the latter is the seasonal variation in the rate of hair growth) Accordingly, how to diagnose zinc deficiency remains controversial, especially in its mild or moderate stages. ComSubmitted for publication Aug. 10, 1992; accepted Feb. 19, 1993. Reprint requests: Toshiro Nakamura, MD, PhD, Department of Pediatrics, Kumamoto City Hospital, Koto 1-1-60, Kumamoto 862, Japan. Copyright | 1993 by Mosby-Year Book, Inc. 0022-3476/93/$1.00 + .10 9/20/46548
pared with zinc values in plasma and hair, measurement of leukocyte zinc content2,9 or of body zinc clearance 1~ 11 seems more reliable. We report here the findings in short children with mild-to-moderate zinc deficiency, determined Clzn GFR GH IGF- 1 Kel TmP Vd
Total body zinc clearance Glomerular filtration rate Growth hormone Insulin-like growth factor 1 Elimination constant Maximal tubular reabsorption rate for phosphorus Volume of distribution
by using body zinc clearance methods, and the effect of supplemental zinc on growth. METHODS A total of 21 Japanese children (11 boys) with short stature were studied. They were selected by the following tests: a Tanner evaluation, growth hormone provocation test, and
65
66
N a k a m u r a et al.
The Journal of Pediatrics July 1993
group l, and 6.1 _+ 2.5 years and 3.8 +_ 1.0 years in group 2. All anthropometric measurements were made by the same pediatrician in the early morning at the initial and the 2-, 4-, and 6-month clinical visits. Height was measured with the children standing barefoot; the mean was calculated from three serial measurements. Linear growth velocity and z score for height for age were calculated according to reference data from the Ministry of Health and Welfare of Japan. 12
= 15I~ -o
Cp~.
r
'.*" -~~'q
100~
c
I
~Co
ro o rE
Co
50
~
0
30
i
I
60 90 Tim e (minutes)
I
120
Fig. I. Serum decay curve after intravenous zinc administration. AC0 = Cp - Co, Estimated initial increase of serum zinc concentration after zinc infusion;Cp, theoretical serum zinc concentration; Co, initial serum zinc concentration. Volumeof distribution, Kel of serum zinc, and Clzn were calculated as described in Methods section.
body zinc clearance test. The tests were performed on 220 patients with short stature hospitalized in our clinic. Informed consent for the study was obtained from the parents. Three provocation tests for GH secretion, including intravenous administration of insulin (0.1 U/kg), oral administration of clonidine (0.1 mg/m2), and intravenous administration of growth hormone releasing factor (1 #g/kg), were used to evaluate GH secretion. Zinc kinetics were studied as described below. The final criteria for inclusion in the study were (1) height for age < - 2 . 0 SD, (2) apparent good health with no evidence of endocrinologic disorder, peak serum GH level >10 ng/ml in insulin and clonidine stimulation tests, and >20 ng/ml in the growth hormone releasing factor loading test, (3) zinc clearance >_20 ml/kg per hour, and (4) prepubertal status (Tanner breast and genitalia growth stage) throughout the study period. The children were divided randomly into two groups: group 1 (five boys and five girls) received zinc sulfate, 5 mg/kg per day, orally for 6 months, and group 2 (six boys and five girls) were control subjects. The chronologic age and bone age were 5.6 +_ 1.2 years and 3.8 _+ 1.0 years, respectively, in
The body zinc clearance test was performed as followslO, H: At 10 A M ( 3 hours after breakfast), zinc sulfate solution, 1 #mol/kg, was injected into a mid-cubital vein. Blood specimens were obtained from a mid-cubital vein of the contralateral arm at 0, 30, 60, 90, and 120 minutes after the inj ection. The samples were immediately transferred to an acid-washed tube and allowed to clot. The serum was then transferred to a second acid-washed tube and kept frozen until analysis. Serum zinc levels were measured by atomic absorption spectrophotometry (model 403, PerkinElmer/Cetus, Norwalk, Conn.). The serum zinc level declined exponentially for up to 120 minutes after the intravenous injection of zinc. These levels can therefore be used to predict the turnover rate of zinc. The volume of distribution was calculated by using the following equations: Vd-
Dose s
AC0 = Cp - Co where the dose is the amount of intravenously administered zinc, Cp is the theoretical serum zinc concentration immediately after injection as calculated from the serum concentration versus time profile, and Co is the initial serum zinc concentration (Fig. l). The elimination constant of serum zinc was calculated by using the following equation: Kel =
0.693
where tl/2 is the biologic half-life of serum zinc calculated directly from the serum concentration versus time profile. Total body zinc clearance was calculated as follows: Clzn = Kel X Vd. Serum calcium, phosphate, creatinine, and alkaline phosphatase levels were measured by using an AutoAnalyzer (Technicon, Tarrytown, N.Y.). The ratio of maximal tubular reabsorption rate for phosphorus to the glomerular filtration rate was calculated by the method of Walton and Bijvoet} 3 Serum insulin-like growth factor 1 levels were measured by using radioimmunoassay kits (Nichols Institute Diagnostics, San Juan Capistrano, Calif.). The intraassay and interassay coefficients of variation were 5.7% and 9.2%, respectively. Serum GH levels were measured by
The Journal o f Pediatrics Volume 123, Number 1
N a k a m u r a et al.
67
Table I. Effect of zinc supplementation on growth Patients with zinc s u p p l e m e n t a t i o n
Height (SDS) Height velocity (SDS) Calorie intake (cal/day)
Patients without zinc supplementation
Before
After
p
Before
After
p
-2.54 • 0.20 -3.14 • 0.91 1312 _+ 32
-2.17 • 0.16 2.26 _+ 1.06 1456 • 38
<0.05 <0.01 <0.01
-2.34 _+ 0.23 -2.29 + 0.25 1278 • 34
-2.57 • 0.53 -2.42 _+ 0.62 1351 _+ 41
NS NS NS
NS, Not significant; SDS, SD score. Values are mean _+ SEM.
T a b l e II. Effect of zinc supplementation on serum, plasma, a n d urinary biochemical values Normal range
Clzn (ml/kg/hr) -Serum zinc (#mol/L) 10.0 _+ 19.9 Serum zinc (/zg/dl) 65-130 Serum calcium (mmol/L) 2.15-2.70 Serum calcium (mg/dl) 8.6-10.8 Serum phosphorus (retool/L) 1.36-1.58 Serum phosphorus (mg/dl) 4.2-4.9 Serum ALP (U/L) 210-320 TRP (%) 85-95 TmP/GFR ratio 4.9-5.3 Serum osteocalcin 0zg/L) 15-20 Plasma IGF-1 (U/m1) 0.6-1.2 Urinary GH (ng/gm of Cr) 15-20
Patients with zinc supplementation Before
29.3 11.9 78 2.29 9.16 1.60 4.97 219 91.3 5.31 15.3 0.84 17.3
• 2.9 • 0.9 _+ 6 • 0.04 • 0.16 _+ 0.05 • 0.14 • 9 • 1.3 • 0.21 _+ 2.2 • 0.27 _+ 5.6
After
p
-• • • • • • • + • • + •
-<0.01 <0.01 <0.05 <0.05 <0.05 <0.05 <0.001 <0.05 <0.05 <0.01 <0.01 NS
15.9 104 2.40 9.61 1.68 5.19 275 93.6 5.91 24.2 1.23 16.2
0.9 6 0.03 0.12 0.04 0.11 11 0.7 0.26 2.3 0.28 2.2
Patients without zinc supplementation Before
30.7 13.0 85 2.31 9.24 1.49 4.62 235 92.4 5.13 16.5 0.49 18.3
• 3.7 • 0.8 • 5 • 0.03 _+ 0.11 • 0.06 • 0.20 _+ 14 • 2.0 _+ 0.41 • 2.4 _+ 0.14 + 4.3
After _
12.1 79 2.28 9.13 1.46 4.53 251 92.7 4.95 17.1 0.50 17.9
_
_+ 0.3 _+ 2 _+ 0.04 • 0.15 _+ 0.06 _+ 0.19 • 14 • 1.5 • 0.45 _+ 2.0 _+ 0.17 • 3.5
p b
NS NS NS NS NS NS NS NS NS NS NS NS
ALP, Alkaline phosphatase; Cr, creatinine; NS, not significant; TRP, tubular reabsorption of phosphorus. Values are mean _+ SEM.
r a d i o i m m u n o a s s a y with a n t i h u m a n G H a n t i s e r u m obtained by i m m u n i z a t i o n of a r a b b i t with synthetic h u m a n G H (Eiken Kizai Co., Ltd., Tokyo, Japan). A n t i r a b b i t 3,-globulin goat serum, the same synthetic G H , and synthetic G H labeled with iodine 125 were used as a second antibody, a standard, a n d a tracer, respectively. This assay detected G H levels as little as 1 ~ g / L ; the intraassay and interassay coefficients of variation were 6.2% a n d 8.5%, respectively. S e r u m osteocalcin levels were measured by using r a d i o i m m u n o a s s a y kits (Incstar Corp., Stillwater, Minn.), as described elsewhere. 14 U r i n a r y G H concentrations were m e a s u r e d in duplicate by the highly sensitive enzyme i m m u n o a s s a y described by H a s h i d a et al. ~5 a n d Sukegawa et al. 16 Dialyzed urine samples or G H s t a n d a r d were incubated with monoclonal a n t i h u m a n G H I g G coated polystyrene balls at 37 ~ C for 6 hours. T h e polystyrene balls were then i n c u b a t e d with affinity-purified r a b b i t a n t i h u m a n G H F a b ' - h o r s e r a d i s h peroxidase conjugated at 4 ~ C for 16 hours a n d at 20 ~ C for 6 hours. Peroxidase activity bound to the balls was assayed by fluorimetry, with 3-(4-hydroxyphenyl)propionic acid used as the substrate. T h e intraassay and interassay coefficients of variation were 5.8% and 8.9%, respectively. T h e detection limits of h u m a n
G H in serum and urine were 1.5 p g / m l with 20 #1 serum and 0.2 p g / m l with 0.15 ml urine, respectively. U r i n a r y h u m a n G H was expressed in terms of n a n o g r a m s per day and n a n o g r a m s per g r a m of creatinine. (To convert urinary hum a n G H values to n a n o g r a m s per millimole of creatinine, divide by 8.84.) T h e S t u d e n t paired t test was used for statistical assessment.
RESULTS As shown in Tables I and II, the biologic and biochemical values at the time of initiation of our study were similar in groups 1 and 2. All biochemical m e a s u r e m e n t s were within the normal range. S e r u m zinc levels were within the normal r a n g e in all subjects, except for one in group 1, whose serum zinc level was 9.9 # m o l / L ( < 6 5 # g / d l ) , the cutoff point. 17 The Clzn values were 29.3 _+ 2.9 m l / k g per hour (range, 20.0 to 48.8 m l / k g per hour) in group 1, and 30.7 _+ 3.7 m l / k g per hour (range, 20.2 to 40.7 m l / k g per hour) in group 2. C o m p a r e d with a g e - m a t c h e d control values in our clinic population (15.1 + 0.6 m l / k g per hour),1~ these values were significantly elevated (p <0.001), thereby indicating a mild-to-moderate zinc deficiency. A m o n g these children, only the s u b j e c t with the lowest serum zinc level
68
Nakamura et al.
The Journal of Pediatrics July 1993
zinc-supplemented group
tO
control group
.=_
-5 I o
I
I O
.Q
Fig. 2. Effects of 6 months of zincsupplementation on linear growth velocity. In the zinc-supplementedgroup, SD score (SDS) was improved(p <0.01), and there was only one significantchange in the control group. 7.7 ~mol/L (50 /~g/dl) and with the highest Clzn (48.8 ml/kg per hour) could have a diagnosis of zinc deficiency in the classic sense/7 After oral administration of zinc sulfate for 6 months, all biologic and biochemical measurements, including linear growth velocity (p <0.01), calorie intake (p <0.01), the serum levels of zinc (p <0.01), calcium (p <0.05), phosphorus (p <0.05), and alkaline phosphatase (p <0.001), the percentage of tubular reabsorption of phosphorus (p <0.05), T m P / G F R ratio (p <0.05), the serum level of osteocalcin (p <0.01), and the plasma level of IGF-1 (p <0.01), were improved, whereas the urinary calcium/creatinine ratio and GH excretion rates were unchanged (Tables I and lI; Fig. 2). All mean values in the untreated subjects in group 2 remained unchanged during the same period of observation. DISCUSSION Zinc supplementation can improve rates of growth in children with zinc deficiency,8, is, 19but related mechanisms are poorly understood. Zinc clearance has also been evaluated in prepubertal diabetic children, in whom a significant
negative correlation was found between linear growth velocity and the Clzn value; zinc supplementation resulted in a considerable increase in height. 1~Other investigators reported an association with improvements in serum levels of GH, 2~ thyrotropin-releasing hormone, 23 IGF-1, 24-26 and vitamin D, as well as bone protein metabolism.262s In our study, urinary GH excretion and other pituitary hormone levels in the plasma were unchanged with zinc supplementation (data not shown), possibly because of the mild or moderate deficiency of zinc in these patients. An important observation was the significant elevation in the IGF-1 level after zinc supplementation. We found only one short communication that the plasma IGF-1 level increased after zinc supplementation in malnourished children aged 6 to 24 months. 29 Elevated reabsorption of phosphorus in the renal tubule, as shown by increases of the T m P / G F R ratio and percentage of tubular reabsorption of phosphorus, resulted in a significant increase in serum phosphorus levels in the zincsupplemented children, a condition that seems linked with the serum IGF-I level. 3~Caverzasio and Bonjour3t reported that IGF- 1 can stimulate renal phosphate transport, both in vivo and in vitro. Receptors for IGF-1 have been isolated from the proximal tubular cell plasma membrane. 32 Increased plasma levels of IGF-1, despite unchanged GH production (urinary excretion), can be explained if zinc has a role in making a strong combination between GH and the GH receptor. 29 Yamaguchi et al. 27, 28 found that zinc stimulates bone protein synthesis at the translational level in rat bone cells and synergistically enhances 1,25-hydroxyvitarain D3-stimulated bone metabolism. A similar phenomenon may have occurred in our zinc-supplemented children because there was evidence of enhanced serum levels of calcium, phosphorus, and osteocalcin. The cause of mild-to-moderate zinc deficiency in our patients is uncertain; none seemed to have a dietary intake of zinc any less than that of the normal children. We conclude that body zinc kinetics studies will reveal some cases of marginal zinc deficiency. We propose that children with nonendocrinologicshort stature be treated for at least 6 months with zinc, even if the serum zinc concentration is within normal ranges. The pediatrician can thus determine whether there is a response to zinc even when Clzn studies are not feasible. We are indebted to Ms. Ohara for critical comments. REFERENCES
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