Calcif Tissue Int (2007) 80:3943 DOI: 10.

1007/s00223-006-0149-5

Assisted Exercise and Bone Strength in Preterm Infants

I. Litmanovitz,1 T. Doln,1 S. Arnon,1 R. H. Regev,1 D. Nemet,2 A. Eliakim2
Department of Neonatology1 and Child Health and Sports Center2, Pediatrics, Meir Medical Center, Kfar-Saba, Sackler School of Medicine, Tel-Aviv University, 59 Tchernichovsky Street, Kfar-Saba 44281, Israel

Received: 28 May 2006 / Accepted: 12 August 2006 / Online publication: 8 December 2006

Abstract. Studies have previously demonstrated that brief (4 weeks) passive range-of-motion exercise is benecial for bone development in very low birth weight (VLBW) preterm infants. However, the optimal duration of exercise for bone development in preterm infants is yet unknown. The aim of the present study was to examine the eect of 8 weeks of assisted exercise on bone strength and metabolism in VLBW premature infants. Sixteen infants (mean standard error of the mean birth weight 1,009 55 g and gestational age 27.3 0.3 weeks) were randomly assigned into exercise (n = 8) and control (n = 8) groups. The intervention started at the rst week of life and involved 8 weeks of daily passive extension and exion range-of-motion exercise of the upper and lower extremities. Biochemical markers of bone turnover were measured at enrollment and after 8 weeks. Bone strength was measured weekly by quantitative ultrasound measurement of tibial bone speed of sound (SOS). Bone SOS decreased signicantly in the control group ()108.1 33.7 m/second, P < 0.0001) during the study period, while remaining stable in the exercise group (11.3 22.8 m/second). The main benecial eect of exercise occurred in the rst 4 weeks of the intervention. There were no signicant dierences in the bone turnover marker changes between the groups. There is a signicant postnatal decrease in bone SOS in VLBW preterm infants. Eight weeks of assisted range-of-motion exercise attenuates the decrease in bone strength and may decrease the risk of osteopenia in premature infants. Key words: Preterm Exercise Speed of sound Bone strength

The period of greatest bone mineral accretion in utero occurs during the last trimester of pregnancy, and it is very dicult to match bone requirements in the postnatal extrauterine environment [1, 2]. Therefore, osteopenia is relatively common in very low birth weight (VLBW) preterm infants. In addition, severe morbidity during the neonatal period (e.g., bronchopulmonary dysplasia) and chronic drug therapy further increase the risk of bone demineralization [3]. Consistent with that, most therapeutic eorts to prevent osteopenia of prematurity have
Correspondence to: A. Eliakim; E-mail: eliakim.alon@clalit. org.il

focused on nutritional changes. However, despite the use of mineral-enriched special preterm formulas, major improvements in postnatal intensive care, and the reduction in systemic steroids and calcium-wasting diuretics use, these eorts have been only partially successful in improving preterm infant bone mineralization [4]. Recent studies using physical activity interventions, after the initial period of stabilization, have demonstrated promising protective eects for bone metabolism in preterm infants. These studies indicated that passive range-of-motion assisted exercise of the large joints resulted in increases of the circulating bone turnover markers, leptin and insulin-like growth factor I level [5, 6] and improvements of bone mineralization [7, 8]. In addition, when assisted exercise was applied in VLBW preterm infants from the rst week of life, it attenuated the natural postnatal decrease in bone strength, determined by qualitative ultrasound measurements of bone speed of sound (SOS) [9]. All these studies used the same 4-week exercise protocol. However, it is not yet known whether this exercise regimen is the best for premature infants, and eorts should be made to nd the optimal exercise duration for bone development in this unique population. Therefore, the aim of the present study was to determine the eects of a longer (8 weeks) passive range-of-motion assisted exercise intervention on bone strength assessed by measurements of tibial bone SOS and bone turnover markers. We hypothesized that exercise will lead to increased bone formation and improved bone strength in VLBW preterm infants. In addition, we speculated that extending the assisted exercise period to 8 weeks would lead to additional benecial eects in bone strength compared to an exercise regimen of 4 weeks.
Materials and Methods Experimental Subjects The sample size calculation for this study was based on the previously reported intervention changes in SOS following assisted exercise [9]. With a two-sided 0.05 signicance level (a = 0.05), with SOS change as the primary variable, six

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I. Litmanovitz et al.: Exercise, Bone, and Preterm Infants

subjects in each group would allow us to detect a signicant dierence at 95% power. To account for possible dropout, two infants were added to each group. Sixteen VLBW infants from the neonatal intensive care unit at the Meir Medical center, Kfar Saba, Israel, participated in the study. Infants were included in the study if they met the following criteria: birth weight of <1,500 g, body size appropriate for gestational age, postnatal age of <1 week. The study was approved by the institutional human research committee, and informed parental consent was obtained. Premature infants with intrauterine growth retardation, severe central nervous system disorder, or other major congenital anomalies were excluded. Preterm infants were randomly assigned to exercise (n = 8) and control (n = 8) groups. Randomization was made by birth order: the rst born preterm infant was recruited to the exercise group, the second to the control group, etc. Nutritional Management All preterm infants received intravenous glucose 510% supplemented with calcium gluconate 10% (300 mg/100 mL) in the rst 24 hours. Total parenteral nutrition (TPN) was initiated at 24 hours of age (Primine 10% Baxter, protein 2 g/100 cc, calcium 200400 mg/100 cc, phosphor 0.76 mmol/100 cc). Enteral feeding was introduced gradually according to the attending physicians decision (day 19). Subjects were fed either fortied human milk (Similac Human Milk Fortier from Ross, Columbus, OH; calcium 117 mg/100 cc, phosphor 67 mg/100 cc) or preterm special formula (Similac Special Care from Ross; calcium 146 mg/100 mL, phosphor 73 mg/100 mL). TPN was stopped when enteral feeding reached 100 mL/kg daily. Enteral feeding was targeted to 120 kcal/kg. Oral vitamin D 200 IU/day was added for all premature infants (control and exercise) receiving enteral feeding at 2 weeks of age. Exercise Protocol The physical activity program was based on the Moyer-Mileur et al. [8] protocol and started after initial cardiorespiratory stabilization (day 47). Briey, this protocol involves extension and exion range-of-motion exercise against passive resistance of both the upper and lower extremities. Both extension and exion were performed ve times at the wrist, elbow, shoulder, ankle, knee, and hip joints (about 10 minutes for each session). This activity was performed ve times per week for 8 weeks by the same person. Since it is possible that tactile stimulation might inuence bone growth and development, control group subjects had a similar time (10 minutes/ day) of daily interactive periods of holding and stroking without range-of-motion activity. Other activities, such as bathing (every day) and kangaroo care (30 minutes/day), were done by both the control and exercise group subjects according to our neonatal intensive care unit (NICU) recommendations. Quantitative Ultrasound Measurements of Bone SOS The left tibial SOS was measured by quantitative ultrasound (Omnisense, PremierTM; Sunlight, Tel Aviv, Israel), a method designed to measure SOS at multiple skeletal sites by axial transmission. Briey, the SOS measurement is based on the fact that ultrasound waves propagate faster through bone than through soft tissue. The device consists of a desktop main unit and a number of small probes, designed to measure SOS at dierent sites. The probe was moved across the mid-tibial plane, searching for the site with maximal reading. The measurement site was dened as the midpoint between the apex of the medial malleolus and the distal patellar apex. The mean of three measurements of tibial SOS was selected for data analysis. All measurements were performed by the same technician, who was blinded to the group assignment. The instrumental accuracy is 0.250.5%, and the precision is 0.40.8%. The

precision for the present study population (based on two separate measurements of 35 preterm infants) was 0.32% (9.1 m/second). Measurements were done once a week throughout the intervention follow-up (8 weeks). Blood Sampling Protocol Early-morning venous blood samples for the evaluation of bone turnover markers were collected before and at the end of the program in both the control and exercise groups as a part of the routine follow-up blood tests (i.e., routine chemistry panel and complete blood count that are performed weekly in our NICU). Bone osteoblastic activity was assessed by measurements of circulating bone-specic alkaline phosphatase (BSAP) [10]. Bone resorption was assessed by measurements of serum levels of the carboxy-terminal cross-links telopeptide of type I collagen (ICTP), which reect osteoclastic activity [11]. All serum samples were kept frozen at )70C until analyzed. All specimens from each individual were analyzed in the same batch by the same laboratory worker, who was blinded to the subjects group and to the order of the samples. BSAP. Circulating BSAP levels were measured by enzyme immunoassay, utilizing a monoclonal anti-BSAP (Alkaphas-B kit; Metra Biosystems, Mountain View, CA). The interassay coecient of variation (CV) was 5.07.6%, and the intra-assay CV was 3.95.8%. Assay sensitivity was 0.7 U/L. ICTP. ICTP levels were determined by equilibrium radioimmunoassay, using the Diasorin (Stillwater, MN) ICTP kit. The interassay CV was 4.17.9%, and the intra-assay CV was 2.86.2%. Assay sensitivity was 0.5 ng/mL. Statistical Analysis The unpaired t-test was used to determine dierences in birth weight, gestational age, age at enrollment, initial and full day of enteral feeding, bone SOS, and bone turnover markers between the exercise and control subjects prior to the training intervention. A two-way repeated measure analysis of variance was used to compare the eect of the intervention on body weight, bone SOS, and bone turnover markers, using time as the within-group and exercise as the between-group factors. To adjust for possible confounding factors (e.g., birth weight, weight at enrollment, daily Ca intake, and full enteral feeding), we used a multivariate regression model analysis. Sample size calculation was performed using Systat version 10 software (SPSS, Richmond, CA). Statistical signicance was set at P < 0.05. Data are presented as mean standard error of the mean (SEM). Results

Baseline measurements of birth weight, gestational age, and age at enrollment as well as information on feeding and medical history of the participants are summarized in Table 1. There were no signicant dierences in birth weight, gestational age, gender, ethnicity, age or weight at enrollment, feeding history, daily Ca intake, or medical history between the control and exercise group participants. Measurements of bone SOS are shown in Figures 1 and 2. No signicant baseline dierences in bone SOS were found between the groups. There was a signicant postnatal decrease in bone SOS in the control group

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60 40

Table 1. Characteristics of the study participants Control group Exercise group (n = 8) (n = 8) Gestational age (weeks) Birth weight (g) Gender (female/male) Ethnicity (Jewish/Arab) Respiratory distress syndrome Oxygen at 28 days Sepsis Feeding (fortied human milk/preterm formula) Age at enrollment (day) Weight at enrollment (g) Initial enteral feeding (day) Full enteral feeding (day) Daily calcium intake (mg/kg) 27.5 0.5 1,017.6 73.8 4/4 5/3 4 4 1 3/5 5.4 0.4 919 87.3 3.2 0.5 13.2 1.8 197.5 8.1 27.0 0.5 998.1 87.8 4/4 4/4 3 3 1 4/4 5.5 0.4 917 83.4 3.8 0.6 12.6 2.3 193.8 10.2
Bone SOS (m/sec)

Weeks 1-4

Weeks 4-8

20 0 -20 -40 -60 -80 -100

Control Exercise

Fig. 2. Changes in bone SOS in preterm infants during the rst postnatal 8 weeks. The major attenuating eect of exercise on the decline of bone SOS occurred in the rst 4 weeks of life (*P < 0.05). Data are presented as mean SEM.

There were no signicant dierences in any of the parameters between the groups. Data are presented as mean SEM.

Bone Speed of Sound (m/sec)

100

control

exercise

(P < 0.05) in both the control and exercise group participants. The changes in bone turnover markers were not accompanied by between-group dierences (Fig. 3).
Discussion

50

* * *

-50

-100

-150

Time (weeks)
Fig. 1. Weekly changes in bone SOS in study participants. While there was a signicant decrease in bone SOS in the control group (black circles), no change was found in the exercise group (white circles, *P = 0.01 for between-group dierence). Data are presented as mean SEM.

participants (P < 0.0001), mainly due to a decrease in the rst 4 weeks of observation. In contrast, there was no change in bone SOS in the exercise group participants. The major attenuating eect of exercise on the decline of bone SOS occurred in the rst 4 weeks of life (P < 0.05, Fig. 2). Using a multivariate regression model analysis adjusting for confounders such as birth weight, weight at enrollment, daily Ca intake, and full enteral feeding, between-group SOS changes were signicantly aected only by exercise. The eect of the intervention on body weight and bone turnover markers is shown in Table 2. Body weight increased signicantly in both the control and exercise group participants, without a between-group dierence. There was a signicant increase in BSAP (P < 0.01) and a signicant decrease in ICTP

We examined the eect of passive range-of-motion exercise on bone metabolism in VLBW preterm infants. The study demonstrated a signicant decrease in bone SOS during the rst 8 postnatal weeks in VLBW premature infants. This is consistent with previous reports from our own and other laboratories showing a postnatal decrease in bone strength (determined by quantitative ultrasound measurements of bone SOS) [9, 12] and a progressive decrease in bone mineral density (measured by single- and dual-photon X-ray absorptiometry) in VLBW infants [7, 8]. The decrease in bone strength occurred despite overall growth, reected by a remarkable weight gain and a biochemical indication of bone growth suggested by a signicant increase in bone formation markers (i.e., BSAP) and a decrease in bone resorption markers (i.e., ICTP). The mechanism for the postnatal decrease in bone SOS in the control VLBW preterm infants is not clear. Tomlinson et al. [12] recently suggested that this decrease may be attributed to higher morbidity or to nutritional factors. The major nding of this study was that 8 weeks of assisted, daily physical activity prevented the decrease in bone SOS in this unique population. These results are consistent with recent reports emphasizing the importance of intrauterine movements on fetal bone development [13]; with previous reports demonstrating that a shorter, 4-week, passive range-of-motion exercise intervention, applied to VLBW preterm infants after an initial period of stabilization, increases bone mineral content and density [7, 8]; and with our own report indicating that use of the same 4-week protocol starting at the rst

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I. Litmanovitz et al.: Exercise, Bone, and Preterm Infants

Table 2. Eect of assisted exercise intervention on body weight and circulating bone turnover markers Control (n = 8) Pre Weight (g) BSAP (u/L) ICTP (ng/mL) 1,017.6 73.8 119.7 23.9 122.3 12.6 Post 1,803.1 155.9 179.3 21.9 92.3 7.7 P <0.0001 <0.01 <0.05 Exercise (n = 8) Pre 998.1 87.8 152.4 24.2 121.4 14.7 Post 1,739.3 161.8 235.6 11.6 100.8 8.2 P <0.0001 <0.01 <0.05

P value indicates signicance for within-group dierences. Data are presented as mean SEM

140 120 100 80

BSAP

ICTP

60 40 20 0 -20 -40

Control

Exercise

Fig. 3. Eect of the exercise intervention on changes in bone turnover markers. BSAP increased signicantly and ICTP decreased signicantly in both groups (*P < 0.05). These changes were not accompanied by between-group dierences. Data are presented as mean SEM.

week of life prevented the decrease in bone SOS in VLBW preterm infants [9]. In the present study, the decrease in bone SOS in control subjects was greater during the rst 4 weeks of life (Fig. 2). Therefore, the major protective eect of exercise occurred during this period. Moreover, exercise prevented the decline in bone SOS from the rst week of life in the VLBW preterm infants (Fig. 1). However, the eects of exercise on bone strength during the second month (weeks 48) of postnatal life should not be ignored since bone SOS continued to decline in the control group but remained stable in the exercising preterm infants. These results suggest that mechanical stimulation is benecial for bone strength and metabolism in preterm infants and that exercise has an important role in bone development during the neonatal period. The exact mechanisms that lead to the exercise-induced bone changes in premature infants are yet unknown. The exercise protocol includes passive range-of-motion exercise with gentle compression at the end of exion/extension in the exercised joints. This exercise regimen may cause an increase in joint pressure and muscle tension, leading to increased mechanical load on the bone-muscle unit, increased lean/muscle mass, and change in bone metabolism and mineralization. The assisted exercise regimen may also lead to increased overall daily movement in preterm infants.

In addition, there is now substantial evidence to demonstrate the utility of quantitative ultrasound measurement of bone SOS as an inexpensive, portable, noninvasive, and ionizing radiation-free method for the assessment of bone strength in infants [14, 15]. This method is based on the concept that the propagation of sound waves through a medium depends upon its physical properties. The denser the medium, the faster the sound waves propagate through it. In addition to bone density, bone SOS is inuenced by other qualitative bone properties, such as cortical thickness, elasticity, and microarchitecture, providing a more complete picture of bone strength [16, 17]. Therefore, the postnatal decrease in bone SOS in VLBW preterm infants may reect a combined contribution of reduced qualitative and quantitative bone properties. Our results suggest that exercise was able to prevent the decline in these two elements for bone strength. Despite the favorable eects of exercise on bone SOS, physical activity was not accompanied by signicant changes in both bone turnover markers (BSAP and ICTP). An exercise-induced increase in bone formation markers (BSAP and terminal procollagen peptide (PICP)) and a decrease in bone resorption markers (ICTP) were previously reported in VLBW premature infants who started their exercise intervention at a postnatal age of 45 weeks [6, 18]. However, consistent with the ndings of the present study, exercise-related changes in bone turnover were not found when exercise was applied to premature infants early after birth (rst or second week of life) [9, 7]. The rst 4 weeks of life are characterized by a remarkable increase in bone formation markers [19]. Therefore, it is possible that the marked early postnatal increase in bone turnover markers masked more subtle physical activity-associated eects. Previous reports indicated that when assisted exercise interventions were applied to preterm infants at the age of 46 weeks they resulted also in a signicantly greater weight gain [6, 8] but that when introduced in the rst week of life or before the premature infants reached body weight of 1.82 kg weight gain was not enhanced [7, 9]. In the present study, exercise was not associated with a greater increase in body weight in VLBW preterm infants. These observations indicate that the exerciseassociated eects on body weight in VLBW preterm

ng/ml

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infants probably depend on the timing of the assisted exercise intervention. The results also suggest that the positive bone eects were not related to increased weight gain. It is possible, however, that changes in body composition and increased lean mass, rather than changes in body weight, contribute to the exerciseassociated eect on bone strength in preterm infants. In conclusion, using quantitative ultrasound, we have successfully shown that early onset of a daily 8-week range-of-motion exercise program (of only about 10 minutes/day) prevented the postnatal decline of bone SOS in VLBW infants. The results suggest that exercise plays an important role in bone development and metabolism in this unique population. While the major benecial eect of exercise occurred during the rst 4 weeks of the program, continuing the exercise program also proved to be benecial. It is still unclear if the positive bone eects are related just to the short assisted exercise session or to metabolic changes related to greater overall daily physical activity in the exercising infants. Moreover, the optimal duration, frequency, and type of exercise for bone development in premature infants still need to be determined.
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