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Bioelectrical Impedance Analysis of the Body Composition of Nigerian Children with Calcium-deficiency Rickets by Dorothy J. VanderJagt,a Marti Morales,a Tom D. Thacher,b Marco Diaz,a and Robert H. Glewa of Biochemistry and Molecular Biology, University of New Mexico, School of Medicine, Albuquerque, NM, 87131, USA bDepartment of General Medical Practice, Jos University Teaching Hospital, Jos, Nigeria aDepartment

Summary Children with calcium-deficiency rickets experience increased urinary excretion of both essential and non-essential amino acids compared to non-rachitic children of the same age. Chronic wastage of amino acids into the urine could lead to a deficit in fat-free mass (FFM) in rachitic children. We therefore determined the FFM of children with calcium-deficiency rickets in northern Nigeria using bioelectrical impedance analysis. Because of the leg deformities present in the rachitic subjects, we calculated a ‘corrected’ height for each rachitic subject using the relationship between arm length and height in non-rachitic subjects over the same age range (r = 0.94). A total of 28 children (4 male and 24 female), 2–9 years of age with calcium-deficiency rickets and an equal number of age- and gender-matched controls were recruited into the study. Resistance and reactance measurements were obtained for all subjects and the total body water was calculated using age-specific equations. FFM was then calculated using age- and gender-specific hydration constants. No significant differences were found in the weights or FFM between the rachitic subjects and the controls (8.41 kg ± 2.19 vs. 8.85 kg ± 1.90, respectively). We conclude that chronic urinary wastage of amino acids by rachitic children does not result in a deficit in FFM.

Introduction Vitamin D deficiency, which results in impaired bone mineralization in children, is associated with generalized aminoaciduria, as well as increased urinary excretion of phosphate and bicarbonate.1–3 The mechanism underlying the aminoaciduria in vitamin D deficiency is obscure; however, it is thought to be related to the secondary hyperparathyroidism that is a consequence of the hypocalcemia4 and low levels of 1,25-dihydroxyvitamin D.5 Muscle abnormalities also occur in humans with vitamin D deficiency rickets,6–8 and rats made deficient in vitamin D exhibit increased myofibrillar protein degradation.9 In some parts of the world, Africa in particular, inadequate dietary intake of calcium rather than vitamin D deficiency is the main cause of rickets in children over the age of 2 years.10–15 We recently demonstrated that children in northern Nigeria who

Acknowledgements This work was supported by a Minority International Research Training (MIRT) grant from the Fogarty International Center of the National Institutes of Health. Correspondence: Robert H. Glew, Ph.D, Department of Biochemistry and Molecular Biology, University of New Mexico, School of Medicine, Room 249, BMSB, Albuquerque, NM 87131, USA. Tel. 505 272-2362; Fax 505 272-6587. E-mail .

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have active calcium-deficiency rickets excrete significantly greater amounts of both essential and nonessential amino acids in their urine than age-matched non-rachitic children.16 Although the concentrations of the majority of the 20 common amino acids were elevated in the serum of rachitic children relative to age-and sex-matched non-rachitic controls, the mean serum glutamine concentration was significantly lower for the rachitic children (260 ± 201 µmol/l vs. 469 ± 216 mol/l, p = 0.04). Muscle is the main source of circulating glutamine, and low plasma and muscle glutamine levels are seen in patients with conditions associated with muscle wasting, such as sepsis and trauma. In addition, the concentration of 3-methylhistidine, an amino acid derived solely from muscle protein breakdown, was elevated four-fold in the sera of the rachitic children compared to controls. However, after 12 weeks of calcium supplementation, the mean serum glutamine concentration in the rachitic subjects increased to 638 ± 53 µmol/l and the 3-methylhistidine concentration decreased from 12.9 ± 11.9 to 3.1 ± 2.6 µmoles/l. The serum prealbumin concentration, an indicator of protein status, was significantly lower in the rachitic subjects at baseline compared to control subjects, indicating that the rachitic children were less well nourished. The central hypothesis of the present study is that chronic urinary wastage of amino acids leads to a deficit in fat-free mass. In a recent study of children Vol. 47

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D. J. VANDERJAGT ET AL.

in Nigeria with sickle cell disease (SCD), a hematological disorder which is characterized by growth deficits, we demonstrated that urinary excretion of amino acids was significantly increased in SCD subjects compared to age- and sex-matched controls.17 The children with SCD had a lower mean weight-for-age than the controls. Furthermore, using bioelectrical impedance analysis (BIA) to assess body composition of the SCD children, we determined that the weight deficit was due to the decreased fat-free mass.18 The aim of the present study was to use BIA to appraise the body composition of children with active rickets in order to determine if the elevated urinary excretion of amino acids in this disorder leads to an imbalance in muscle protein degradation and synthesis, ultimately presenting as a reduction in fat-free mass (FFM). BIA was used to measure FFM because the analyser is portable and the method is rapid and non-invasive. Although 3 months of calcium supplementation can normalize urinary amino acid excretion and serum amino acid concentrations in children with rickets, it may be beneficial for these children to receive long-term supplemental dietary protein, in addition to calcium supplements, as part of their treatment. Methods Subjects Children aged of 2–9 years with clinical and radiographic evidence of rickets were recruited from the area around Jos, Nigeria. Each child had the bone deformities that are characteristic of rickets (e.g. genu varum, genu valgum, widened wrists). Active rickets was confirmed by radiography of the wrist or knee. Radiographic features used to define active rickets were metaphyseal cupping, fraying and widening of the epiphysus. An equal number of age- and sex-matched controls were also recruited from the General Outpatient Department of the Jos University Teaching Hospital. The control group consisted of healthy children who had returned to clinic for follow-up of an acute illness (e.g. pneumonia, malaria) from which they had fully recovered. Measurements of weight, height, mid-arm circumference, triceps skin-fold thickness and arm length were also obtained for each subject. The arm length was determined by measuring the distance between the lateral projection of the acromial process and the inferior border of the olecranon process of the ulna while the elbow was flexed to 90º. Body mass index (BMI) was calculated as kg/m2. This study was approved by the Human Research Review Committee of the University of New Mexico School of Medicine and the Ethics Review Committee of the Jos University Teaching Hospital, Nigeria. Journal of Tropical Pediatrics

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Bioelectrical impedance analysis Resistance (R) and reactance (Xc) were obtained using a portable bioelectrical impedance analyser (BIA-Quantum, RJL Inc., Clinton Township, MI). The system delivers an alternating current of 800 mA at a frequency of 50 kHz. Subjects were instructed to void urine prior to the impedance measurement. Impedance measurements were made while the subjects lay on a non-conducting examination table with limbs abducted approximately 30º from the body. Signal introducing electrodes were placed on the first joint of the middle finger and just below the middle toe. The detecting electrodes were placed with the upper edge of the electrode bisecting the ulnar head of the wrist and the median malleolus of the foot. Care was taken to ensure that the signal and detecting electrodes were at least 5 cm apart. Duplicate measurements were made on all subjects. Calculation of FFM Total body water (TBW) was calculated using agespecific equations that have been validated by 18O dilution19,20 in healthy and malnourished children (Table 1). Because the pronounced leg deformities that were present in the rachitic subjects prevented us from obtaining accurate measurement of their heights, we derived an equation for estimating the heights of the rachitic children which was based on the relationship between the arm length (AL) and height of the control children (Fig. 1): estimated height (cm) = 3.69 AL (cm) + 25.8. The FFM was then calculated from the amount of TBW using the appropriate age- and gender-specific hydration constants (Table 1).21 Results Subjects A total of 28 rachitic subjects (22 females and 6 males) between the ages of 2 and 9 years were enrolled in the study (Table 2), along with an equal number of age- and gender-matched controls. Since the number of male rachitic subjects was small (n = 6) and because we did not find significant differences in weight and height for male and female subjects, the

TABLE 1 Calculation of total body water Age (years) 0.4–4 5–18

Equation 0.67 (S2/Z) + 0.48a 0.60 (S2/R) –0.50b

S, height; R, resistance; Z, impedance (√R2 + Xc2); Xc2) reactance. a Reference 19; b Reference 20.

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FIG. 1. The relationship between arm length and height in control subjects. Estimated height = 3.69 (arm length) + 25.8; r = 0.94.

data for male and female subjects were combined for all comparisons and statistical analyses. Although the rachitic children tended to weigh less than the controls, the weight difference was not statistically significant (Table 2). As expected, because of their skeletal deformities, the measured heights of the rachitic children were significantly lower than those of the controls. However, there was no significant difference between the ‘corrected’ heights of the rachitic subjects and controls when the heights of the rachitic children were estimated using the equation based on the relationship of arm length and height of the control children. On average, the ‘corrected’ height of the rachitic children was 5.3 per cent greater than the height determined using a stadiometer. The triceps skinfolds and mid-arm circumference measurements were also similar for the two groups, indicating that the overall nutritional status of the experimental and control groups was equivalent.

TABLE 2 Summary of the characteristics of the rachitic subjects and controls Rachitic subjects (n = 28) Age (years) Gender (M/F) Height (cm) Measured Calculateda Weight (kg) Body mass index (kg/m2) Arm length (cm) Triceps skin fold (mm) Mid-arm circumference (cm) Rickets scoreb 1–3 4–7 8–10

Controls (n = 28)

3.6 ± 1.5 6/22

3.7 ± 1.7 6/22

85.5 ± 8.7 90.4 ± 8.3 11.2 ± 2.3 15.1 ± 1.9 17.4 ± 2.2 7.8 ± 2.2 13.5 ± 1.6

94.2 ± 12.1 — 15.3 ± 1.5 13.9 ± 1.6 18.5 ± 3.1 8.0 ± 2.6 13.9 ± 1.4

14 10 4

N/A

a

Bioelectrical impedance parameters As shown in Table 3, there was no significant difference in the mean resistance (R) of the rachitic compared to control children; however, the mean 94

Height calculated from arm length (AL): height (cm) = 25.77 AL + 3.69. b Radiographic analysis score based on the number of bone deformities on wrists and knees. Score: 1 (mild) to 10 (severe). Values given are mean ± SD.


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FIG. 2. The relationship between weight and age in calcium-deficient rachitic children () and non-rachitic controls ().

reactance (Xc) values for the two groups were significantly different (p = 0.04). Although both mean TBW and mean FFM of the rachitic subjects were slightly lower than the means for the control group, the differences were not statistically significant. Discussion The main result of this study was the finding that children with calcium-deficiency rickets have a FFM that is similar to that of non-rachitic controls of the same age. This result was unexpected based on our previous observation of decreased FFM in children with SCD, another disease that is associated with increased urinary loss of amino acids. The preservation of lean body mass in rachitic children, despite the increased urinary loss of amino acids they experience, may be due to other compensatory factors. Growth is controlled by complex interactions between hormonal and nutritional factors, including growth hormone.22 Growth hormone promotes the growth of skeletal and soft tissues indirectly through the actions of insulin-like growth factors (IGF-I and IGF-II) and six different binding proteins (IGHBF Journal of Tropical Pediatrics

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TABLE 3 Bioelectrical impedance parameters of rachitic subjects and controls

Resistance Reactance Reactance/Resistance Total body water (l) Fat-free mass (kg)

Rachitic subjects (n = 28)

Controls (n = 28)

838 ± 143 57.6 ± 12.4 0.070 ± 0.014 6.53 ± 1.69 8.41 ± 2.19

849 ± 93 63.6 ± 8.5 0.075 ± 0.009 7.04± 1.67 9.06 ± 2.10

1–6). IGFBP-3 is the most abundant of the IGF binding proteins in circulation. The circulating levels of IGFBP-3 correlate positively with birthweight23 and serum IGFBP-3 increases during growth and puberty.24 Although the function of IGFBP-I has not been clearly established, it is thought to oppose the actions of IGFBP-3, and may inhibit IGF binding to cell surface receptors, thereby affecting its activity. IGFBP-I is increased by fasting, malnutrition and diabetes.25 The regulation of IGFBP-I expression by 95


FIG. 3. The relationship between fat-free mass and age in calcium-deficient rachitic children () and nonrachitic controls ().

amino acid concentrations in vitro has been demonstrated.26 Levels of amino acids comparable to those found in protein–energy malnutrition increase the expression of IGFBP-I and may therefore block the effects of IGF. In our previous study of rachitic children,16 we observed that the serum concentrations of 20 of the 22 serum amino acids that we measured were significantly higher in the rachitic children than in the agematched controls. The expression of IGFBP-I in these subjects may have been suppressed by these high amino acid levels, thereby allowing the action of IGF to be modulated by IGFBP-3. In this study, we have also demonstrated the utility of using an estimated height from arm length in the rachitic subjects. The formulas that are used to calculate TBW from bioelectrical impedance measurements require an exact measure of a subject’s height. Because of the severe skeletal deformities associated with rickets, that is, the bending of the legs in either a bow-leg or k-leg orientation, accurate determination of the true body length of rachitic subjects is not possible. Since we determined that there was no difference in the arm length of the rachitic vs. the non-rachitic control subjects, we hypothesized that a relationship exists between their arm length and stature. The correlation coefficient for the 96

relationship between arm length and height in the control children was 0.94. The relationship between arm length and height was also highly correlated for the rachitic subjects (r = 0.91), but the intercept differed from that observed for the non-rachitic controls. Whereas the measured height of the rachitic children was significantly different from the height of the control children, when we estimated the heights for the rachitic children using the regression equation for the relationship between arm length and height for non-rachitic children, no difference in height was observed between the rachitic children and controls. We concluded therefore, that the overall growth of the rachitic children is not diminished and that the empirically estimated height of the rachitic children reflects their true height. We conclude that the urinary wastage of amino acids into the urine by the calcium-deficient rachitic children does not compromise their ability to maintain a FFM comparable to that of age-matched controls. References 1. Chisholm JJ, Harison HE. Aminoaciduria in vitamin D deficiency states in premature infants and older children with rickets. J Pediatr 1962; 60: 206.


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2. Jonxis JHP, Huisman THJ. Aminoaciduria in rachitic children. Lancet 1953; 2: 428–31. 3. Cusworth DC, Dent CE. Renal clearances of amino acids in normal adults and in patients with aminoaciduria. Biochem J 1960; 74: 550–60. 4. Phillips ME, Havard J, Otterud B. Aminoaciduria in chronic renal failure—its relationship to vitamin D and parathyroid status. Am J Clin Nutr 1980; 33: 1541–45. 5. Fraser D, Kooh SW, Scriver CR. Hyperparathyroidism as the cause of hyperaminoaciduria and phosphaturia in human vitamin D deficiency. Pediatr Res 1967; 1: 425–35. 6. Ritz E, Boland R, Kreusseer W. Effects of vitamin D and parathyroid hormone on muscle: potential role in uremic myopathy. Am J Clin Nutr 1980; 33: 1522–29. 7. Schott GD, Wills MR. Muscle weakness in osteomalcia. Lancet 1976; 1: 626–29. 8. Floyd M, Ayyat DR, Barwick DD, Hudgson P, Weightman D. Myopathy in chronic renal failure. Quart J Med 1974; 43: 509–24. 9. Wassner SJ, Li JB, Sperduto A, Norman ME. Vitamin D deficiency, hypocalcemia, an increased skeletal muscle degradation in rats. J Clin Invest 1983; 72: 102–12. 10. Walter EA, Scariano JK, Easington CR, et al. Rickets and protein malnutrition in northern Nigeria. J Trop Pediatr 1997; 43: 98–102. 11. Thacher T, Glew RH, Isichei C, et al. Rickets in Nigerian children: response to calcium supplementation. J Trop Pediatr 1999; 45: 202–7. 12. Oginni LM, Worsfold M, Oyelami A, Sharp CA, Powell DE, Davie MWJ. Etiology of rickets in Nigerian children. J Pediatr 1996; 128: 692–94. 13. Okonofua F, Gill DS, Alabi ZO, Thomas M, Bell JL, Dandona P. Rickets in Nigerian children: a consequence of calcium malnutrition. Metabolism 1991; 40: 209–13. 14. Pettifor JM, Ross FP, Travers R, Glorieux FH, DeLuca HF. Dietary calcium deficiency: a syndrome associated with bone deformities and elevated 1,25-dihydroxyvitamin D concentrations. Metab Bone Dis Rel Res 1981; 2: 301–5. 15. Thacher TD, Fischer PR, Pettifor JM, et al. A comparison of calcium, vitamin D, or both for nutritional rickets in Nigerian children. N Engl J Med 1999; 341: 563–68.


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16. VanderJagt DJ, Peery B, Thacher T, Pastuszyn A, Hollis BW, Glew RH. Aminoaciduria in calcium-deficiency rickets in northern Nigeria. J Trop Pediatr 1999; 45: 258–64. 17. VanderJagt DJ, Kanellis GJ, Isichei C, Pastuszyn A, Glew RH. Serum and urinary amino acid levels in sickle cell disease. J Trop Pediatr 1997; 43: 220–25. 18. VanderJagt DJ, Okolo SN, Rabasa A, Glew RH. Bioelectrical impedance analysis of the body composition of Nigerian children with sickle cell disease. J Trop Pediatr 2000, 46; 67–72. 19. Fjeld CR, Freundt-Thurne J, Schoeller DA. Total body water measured by 18O dilution and bioelectrical impedance in well and malnourished children. Pediatr Res 1990; 27: 98–102. 20. Davies PSW, Preece MA, Hicks CJ. The prediction of total body water using bioelectrical impedance in children and adolescents. Ann Hum Biol 1988; 15: 237–40. 21. Fomon SJ, Haschke F, Ziegler EE, Nelson SE. Body composition of reference children from birth to age 10 years. Am J Clin Nutr 1982; 35: 1169–75. 22. Baker J, Liu JP, Robertson EJ, Efstratiadis A. Role of insulinlike growth factors in embryonic and postnatal growth. Cell 1993; 75: 73–82. 23. Cance-Rouzaud A, Laborie S, Bieth E, et al. Growth hormone, insulin-like growth factor-1 and insulin-like growth factor binding protein-3 are regulated differently in small-forgestational-age and appropriate-for-gestational-age neonates. Biol Neonate 1998; 73: 347–55. 24. Blum WF, Ranke MB, Kietzman K, Gauggel E, Zeisel HJ, Bierich J. A specific radiommunoassay for the growth hormone (GH)-dependent somatomedin binding protein: its use for diagnosis of GH deficiency. J Clin Endocrinol Metab 1990; 70: 1292–98. 25. Lee PDK, Conover CA, Powell DR. Regulation and function of insulin-like growth factor-binding protein-1. Proc Soc Biol Med 1993; 2204: 4–29. 26. Jousse C, Bruhat A, Ferrara M, Fafournoux P. Physiological concentration of amino acids regulate insulin-like-growthfactor-binding protein 1 expression. Biochem J 1998; 334: 147–53.

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