Journal of Perinatology

https://doi.org/10.1038/s41372-020-0693-2

ARTICLE

Effect on metabolic bone disease markers in the neonatal intensive

care unit with implementation of a practice guideline
Elizabeth Marie Sabroske1 Davis Harrison Payne2 Christina Nicole Stine3 Charmaine Marie Kathen4
● ● ● ●

Heather Marie Sollohub4 Katy Leanne Kohlleppel4 Pamela Louise Lorbieski4 Jennifer Elizabeth Carney4
● ● ● ●

Cheryl Leah Motta1,4 Maria Rodriguez Pierce1,4 Kaashif Aqeeb Ahmad 1,4,5
● ●

Received: 22 October 2019 / Revised: 22 April 2020 / Accepted: 11 May 2020

© The Author(s), under exclusive licence to Springer Nature America, Inc. 2020

Abstract
Objectives To determine the effect of implementing a 2015 policy for the screening, prevention, and management of
metabolic bone disease for very low birth weight (VLBW) infants in two Level IV NICUs.
Study design Retrospective cohort study of VLBW infants in the 2 years prior to (2013–2014) and after (2016–2017) policy
implementation.
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Results We identified 316 VLBW infants in 2013–2014 and 292 in 2016–2017 who met study criteria. After policy
implementation, vitamin D supplementation began earlier (20.1 ± 15.5 days vs 30.2 ± 20.1 days, p < 0.0005), the percentage
of infants with alkaline phosphatase obtained increased (89.7% vs 76.3%, p < 0.0005), while the percentage of infants with
alkaline phosphatase >800 IU/L (11.7 vs 4.5%, p = 0.0001) and phosphorous <4 mg/dL (14.2% vs 7.9%, p = 0.014) fell
significantly.
Conclusions After policy implementation, vitamin D supplementation began significantly earlier and the rate of detecting
abnormal biochemical markers of metabolic bone disease decreased significantly.

Background pregnancy. Despite current knowledge regarding MBD, an

Metabolic bone disease (MBD) is a common morbidity in
preterm infants and historically occurs in 16–40% of very
low birth weight (VLBW, <1500 g) and extremely low birth
weight (ELBW, <1000 g) infants [1–3]. The incidence of
MBD rises with increasing prematurity as the majority
of bone mineralization occurs in the third trimester of
Supplementary information The online version of this article (https://
doi.org/10.1038/s41372-020-0693-2) contains supplementary
material, which is available to authorized users.
* Kaashif Aqeeb Ahmad
kaashif.ahmad@bcm.edu
1
Baylor College of Medicine, San Antonio, TX, USA
2
University of Texas Health Sciences Center, San Antonio, TX,
USA
3
Central Ohio Newborn Medicine, Columbus, OH, USA
4
Pediatrix Medical Group of San Antonio, San Antonio, TX, USA
5
The Center for Research, Education, Quality, and Safety,
MEDNAX National Medical Group, Sunrise, FL, USA
exact incidence is unclear due to a lack of consensus
regarding diagnostic criteria [1].
Several screening recommendations for MBD exist
[1, 4, 5], and more recently the American Academy of
Pediatrics recommended routine evaluation of bone mineral
status for VLBW infants [6]. Unsurprisingly, survey data
has found significant variability in neonatologist practices
regarding MBD screening [7]. No single ideal biomarker for
the diagnosis of MBD exists [8, 9]. While alkaline phos-
phatase appears to be the most commonly utilized screening
tool among United States neonatologists [7], limitations
exist. Multiple studies have found an association between
elevated alkaline phosphatase and MBD [10–13] while
using cutoff values ranging from 500 to 900 IU/L due to a
lack of consensus threshold value. However, sensitivity and
specificity may be low if screening with alkaline phospha-
tase alone and may be improved with incorporation of
serum phosphorous level into algorithms [12]. Parathyroid
hormone expression has an important role in calcium
phosphorous metabolism and measurement levels may have
a role as well in screening for MBD according to recent
research [14].

In 2015, we implemented a policy regarding the pre-
vention, screening, diagnosis, and management of MBD at
two Level IV neonatal intensive care units (NICUs) in San
Antonio, TX. We hypothesized that policy implementation
would lead to more frequent screening for MBD and as a
result a reduction in risk for subsequent development of
MBD as determined by screening laboratory assessments in
VLBW infants.
Methods
Setting
All patients were admitted to the Level IV NICUs at The
Children’s Hospital of San Antonio and Methodist Children’s
Hospital, San Antonio, TX. Institutional Review Board (IRB)
approval was received from the CHRISTUS Health IRB and
the Methodist Healthcare IRB.
Intervention
In 2015, a new policy to standardize care practices for MBD
was implemented for all VLBW infants (Supplementary
Fig. 1). Policy recommendations included initiation of sup-
plemental vitamin D at 200 international units (IU) per day
when achieving full enteral feeds (typically 160 mL/kg/day if
not fluid restricted) and increasing vitamin D supplementa-
tion to 400 IU per day when weight is >1500 g. Prior to
policy implementation, vitamin D supplementation was
common, but timing was left to provider discretion.
The implemented protocol recommended that VLBW
infants receive enteral nutrition with fortified human milk
or premature formula to achieve 150–220 mg/kg/day
of calcium and 75–140 mg/kg/day of phosphorous.
The protocol recommended to follow persistent serum
phosphorous concentrations <5 mg/dL weekly and to
provide supplemental phosphorous if serum levels were
persistently <4 mg/dL (for 2 weeks or more) as phos-
phorous deficiency will enhance calcium excretion. When
supplementing phosphorous, the protocol recommended
starting at 10–20 mg/kg/day up to 40–50 mg/kg/day of
elemental phosphorous. When supplementing calcium, the
protocol recommended starting at 20 mg/kg/day up to
70–80 mg/kg/day of elemental calcium. Since calcium
products may bind phosphorous in feeds, calcium was
recommended to be given between feeds unless the infant
was on continuous feeds.
Laboratory screening recommendations included obtain-
ing alkaline phosphatase levels starting at 4 weeks of age and
every 2–4 weeks thereafter, through 36 weeks’ postmenstrual
age. Recommended actions based on alkaline phosphatase
levels are summarized in Supplementary Fig. 1. Guideline
E. M. Sabroske et al.
release to physicians and neonatal nurse practitioners was
conducted via electronic mail and presentation at a group
meeting. Independent to this retrospective data collection, we
conducted routine prospective audits for 14 months after
guidelines’ release and had results shared monthly with
clinicians.
Breast milk use and fortification
The Children’s Hospital of San Antonio had both Prolact+
H2MF® milk fortifier (Prolacta Bioscience) and donor
human milk available throughout the study period while
Methodist Children’s Hospital introduced these products in
February, 2014. The use of mother’s own milk or donor
human milk was considered standard of care for all VLBW
infants when available. All patients routinely received for-
tification of breast milk to optimize nutrient intake. For
infants with a birth weight <1250 g, the standard of care was
Prolact+ H2MF® and for infants ≥1250 g birth weight
(or prior to the introduction of Prolact+ H2MF) Similac®
Human Milk Fortifier (Abbott Nutrition). For infants <1250
g birth weight fortified with Prolact+ H2MF®, fortification
to 24 kcal/oz occurred in conjunction with feeding
advancement to 60 mL/kg/day and fortification to 26 kcal/
oz occurred in conjunction with feeding advancement to
100 mL/kg/day. For infants <1250 g birth weight fortified
with Similac® Human Milk Fortifier, fortification to 24 kcal/
oz occurred at 100 mL/kg/day and not in conjunction with
an advancement in volume. The overall goal for VLBW
infants in the protocol was to provide enteral nutrition with
fortified human milk or premature formula to achieve
150–220 mg/kg/day of calcium and 75–140 mg/kg/day of
phosphorous.
Patients
We retrospectively collected data for eligible infants
admitted in the 2 years prior to (2013, 2014) and after
(2016, 2017) policy implementation. We included all
VLBW infants admitted on day of life 0 or 1 who survived
at least 4 weeks after birth and remained through discharge
home. We excluded any infants with incomplete data (n =
25). No infants had major anomalies. We reviewed demo-
graphics, timing and results of MBD screening laboratory
tests, therapies, and outcomes.
Analysis
Data were hand collected into case report forms and entered
into SPSS v25. Tables were generated for patient demo-
graphics, medication exposures, laboratory results, and
patient outcomes. Chi-squared test, Fischer’s exact test, or
Mann–Whitney’s U test was used to compare demographic
Effect on metabolic bone disease markers in the neonatal intensive care unit with implementation of a. . .

data, outcomes variables, medication exposures, and labora-
tory trends between groups. We identified the highest risk
infants for MBD as those with an alkaline phosphatase level
>800 IU/L and phosphorous level <4 mg/dL. A binomial
regression analysis was performed to ascertain the effects of
epoch, small for gestational age, birth postmenstrual age,
birth weight, and furosemide exposure on developing an
alkaline phosphatase level >800 IU/L.
Results
Our cohort comprised of a total of 608 VLBW infants, 316
from prior to policy implementation and 292 after. We
found no differences in maternal characteristics between the
groups. Patients in the later cohort had a significantly
greater gestational age at birth but no significant difference
in birth weight. The rates of major neonatal outcomes were
not statistically different between groups (Table 1).
After policy implementation, enteral supplementation of
vitamin D by 14 days of age increased from 8.5% to 37.7%
(p < 0.0005) of infants and the mean age for vitamin D
initiation decreased from 30 to 20 days (p < 0.0005,
Table 2). These changes were consistent in subgroups of
ELBW infants as well as non-ELBW infants (1000–1499 g
at birth). After policy implementation, 28% of ELBW
infants had at least one alkaline phosphatase level >600 IU/
L and 9% had one alkaline phosphatase >800 IU/L, but for
infants ≥1000 g at birth we found these rates to be low at 6%
and 1%, respectively. Further, after policy implementation,
only 2% of non-ELBW infants experienced a phosphorous
level <4 mg/dL.
The percentage of infants with at least one alkaline
phosphatase level checked increased significantly (76.3% vs
89.7%, p < 0.0005); however, the overall total number of
alkaline phosphatase levels obtained declined by 12.7%
across epochs (1287–1124) for an overall decrease in mean
alkaline phosphatase levels obtained per patient from 4.1 to
3.8. Although a significant increase occurred in the per-
centage of infants screened, after policy implementation the
percentage of all infants with an alkaline phosphatase >800
IU/L fell by 62% (11.7% vs 4.5%, absolute difference
7.2%, p < 0.0005, Table 2). The majority of this difference
occurred in ELBW infants (20.9% vs 9.1%, absolute dif-
ference 11.8%, p = 0.008). We found a significantly overall
higher use of Prolact+ H2MF® in the later study period after
introduction of this product at one study site in early 2014.
With the change in screening guidelines, we found sig-
nificant increases in screening evaluations for phosphorous,
total calcium, and vitamin D after 21 days of age. Guideline

Table 1 Comparison of
2013–2014 2016–2017 p Value
maternal/infant demographics
and major outcomes across time n = 316 n = 292
periods.
Maternal characteristics
Maternal age (SD) 28.5 (6.8) 27.9 (6.5) 0.28
Primigravid (%) 91 (28.8) 96 (32.9) 0.92
Nulliparous (%) 115 (36.4) 106 (36.3) 1
Preeclampsia (%) 55 (17.4) 69 (23.6) 0.07
Gestational diabetes (%) 28 (8.9) 22 (7.5) 0.49
Chorioamnionitis (%) 13 (4.1) 22 (7.5) 0.082
Antenatal steroid exposure (%) 236 (74.7) 239 (81.8) 0.039
Infant characteristics
Gestational age (median, 10th/90th %ile) 27 (24, 31) 28 (24, 32) 0.037
Birth weight (median, 10th/90th %ile) 1005 (620, 1380) 1100 (663, 1436) 0.105
Sex (% female) 159 (50.3) 135 (46.2) 0.33
Apgar scores
1 min (median, 10th/90th %ile) 5 (1, 8) 6 (1, 8) 0.2
5 min (median, 10th/90th %ile) 8 (4, 9) 8 (4, 9) 0.375
Infant outcomes
Respiratory distress syndrome (%) 316 (94.3) 277 (94.9) 0.86
Grade 3/4 intraventricular hemorrhage (%) 37 (11.7) 32 (11) 0.8
Periventricular leukomalacia (%) 30 (9.5) 27 (9.2) 1
Oxygen requirement at 36 weeks PMA (%) 102 (32.3) 93 (31.8) 0.93
Retinopathy of prematurity stage 3+ (%) 24 (7.6) 20 (6.8) 0.76
Mortality (%) 4 (1.3) 1 (0.3) 0.38
 E. M. Sabroske et al.

Table 2 Changes in medication

All patients
exposures and laboratory values
after MBD guideline 2013–2014 2016–2017 p Value
implementation. n = 316 n = 292

Medication exposures
Vitamin D started on DOL 14 (%) 27 (8.5) 110 (37.7) <0.0005
If started, mean dose on Day 14 (SD) 277 (103) 266 (110) 0.2
Age at vitamin D initiation, mean days (SD) 30.2 (20.1) 20.1 (15.5) <0.0005
Enteral phosphorous exposure (%) 18 (5.7) 7 (2.4) 0.043
Enteral calcium exposure (%) 19 (6) 25 (8.6) 0.27
Furosemide exposure (%) 65 (20.6) 62 (21.2) 0.98
Caffeine citrate exposure (%) 296 (93.7) 269 (92.1) 0.75
Prolact+ H2MF® milk fortifier 132 (41.8) 292 (66.8) <0.0005
Laboratory trends
Alkaline phosphatase level checked (%) 241 (76.3) 262 (89.7) <0.0005
Mean number of alkaline phosphatase levels (SD) 4.1 (4.7) 3.9 (3.6) 0.51
Alkaline phosphatase >400 IU/L (%) 144 (45.6) 120 (41.1) 0.29
Alkaline phosphatase >600 IU/L (%) 79 (25) 44 (15.1) 0.002
Alkaline phosphatase >800 IU/L (%) 37 (11.7) 13 (4.5) 0.001
Phosphorous level obtained after 21 days of age (%) 282 (89.2) 285 (97.6) <0.0005
Minimum phosphorous level, mg/dL (SD) 4.7 (1.3) 5.3 (1) <0.0005
Phosphorous level <5 mg/dL (%) 85 (26.9) 63 (21.6) 0.131
Phosphorous level <4 mg/dL (%) 45 (14.2) 23 (7.9) 0.014
Alkaline phosphatase >800 IU/L and phosphorous level <4 mg/dL 20 (6.3) 7 (2.4) 0.028

Table 3 Univariate comparison

Max AP > 800 IU/L Max AP ≤ 800 IU/L p Value
of risk factors for one or more
alkaline phosphatase (AP) levels n = 50 n = 453
>800 IU/L in VLBW patients.
Post-intervention epoch (%) 13 (26) 249 (55) <0.001
Birth weight +/− SD 705 (452, 1053) 1000 (651, 1390) 0.011
Completed weeks GA +/− SD 25 (23, 30) 27 (24, 30) <0.001
SGA 14 (28) 53 (11.7) 0.003
Furosemide exposure (%) 23 (46) 96 (21.2) <0.001
Xanthine exposure (%) 50 (100) 437 (96.5) 0.39
Prolact+ H2MF® milk fortifier exposure (%) 30 (60) 261 (57.6) 0.77

implementation also had an association with improvement
in the lowest serum phosphorous levels. The percentage of
infants who experienced both an alkaline phosphatase ≥800
IU/L and phosphorous level <4 mg/dL decreased sig-
nificantly among ELBW infants (from 12.4% to 5%, p =
0.036. Overall, only 6 (1%) infants required wrist X-rays for
potential rickets and 1 (0.2%) received a formal diagnosis of
rickets.
For the entire study period, a total of 503 infants (82.7%)
had at least one alkaline phosphatase level obtained during
their hospital stay after 21 days of age—260 (94.9%) of
ELBW infants and 243 (72.8%) of non-ELBW infants. Of
these, ELBW infants had a significantly greater number
of alkaline phosphatase levels obtained (7 ± 5 vs 3 ± 3,
p < 0.005) and higher maximum alkaline phosphatase level
(604 ± 283 IU/L vs 413 ± 178 IU/L, p < 0.005). From the
fourth through tenth weeks after birth, ELBW infants con-
sistently had significantly higher alkaline phosphatase levels
than non-ELBW infants.
Among patients with at least one alkaline phosphatase
level checked, we examined risk factors for an alkaline
phosphatase level ≥800 IU/L. On univariate analysis,
patients with alkaline phosphatase levels ≥800 IU/L were
more likely to have lower birth weight, lower gestational
age at birth, to be small for gestational age, and have fur-
osemide exposure as well as less likely to be from the post-
intervention epoch (Table 3). We did not find a significant
difference in Prolact+ H2MF® exposure among infants with
Effect on metabolic bone disease markers in the neonatal intensive care unit with implementation of a. . .

Table 4 Binomial regression

Risk factors B SE Wald df p Value Odds ratio 95% CI for OR
analysis of risk factors for one or
more alkaline phosphatase levels Lower Upper
>800 IU/L in VLBW patients.
Later epoch −1.044 0.357 8.529 1 0.003 0.352 0.175 0.709
SGA 1.018 0.527 3.738 1 0.053 2.768 0.986 7.772
Birth PMA (completed weeks) −0.101 0.110 0.849 1 0.357 0.904 0.729 1.121
Birth weight (g) −0.002 0.001 5.184 1 0.023 0.998 0.996 1.000
Furosemide exposure 0.782 0.341 5.255 1 0.022 2.187 1.120 4.268
Constant 2.512 2.239 1.259 1 0.262 12.328

higher vs lower alkaline phosphatase levels. On regression
analysis, furosemide exposure had a higher odds of elevated
alkaline phosphatase while post-intervention patients had an
association with lower odds. Rising gestational age and
birth weight both had incrementally lower odds of elevated
alkaline phosphatase (Table 4). Adding exposure to Prolact
+ H2MF® to the regression model did not find a significant
association with elevated alkaline phosphatase level nor did
it change the significance of the other variables.
Discussion
We found implementation of a standardized approach to the
screening, prevention, diagnosis, and management of MBD
in VLBW infants was associated with significantly earlier
vitamin D supplementation (mean initiation from 30 to
20 days of age) and a significant decrease in biochemical
evidence of MBD as reflected by decreased alkaline phos-
phatase and increased phosphorous levels. While the per-
centage of infants who received laboratory screening for
MBD substantially increased with policy implementation,
the overall number of alkaline phosphatase levels obtained
declined. A formal diagnosis of rickets was very uncommon
in this cohort. In total, these data indicate that imple-
mentation of a standardized policy for prevention and
management of MBD in Level IV NICUs can be effective
in decreasing biochemical evidence of MBD in a high-risk
patient population without increasing the overall number of
alkaline phosphatase levels obtained. In regression analysis
including several risk factors for MBD, we found the post-
intervention time period to have a significantly decreased
odds of elevated alkaline phosphatase compared to the pre-
intervention period.
Consistent with previously reported data [5], we found
an inverse relationship between birth weight and sig-
nificantly abnormal laboratory values indicating MBD.
After policy implementation, 9% of ELBW infants had at
least one alkaline phosphatase level >800 IU/L, but for
infants ≥1000 g at birth we found this rates to be low at 1%
with only 2% of these infants having a phosphorous level
<4 mg/dL. In total, these data indicate that biochemical
abnormalities concerning for MBD are an uncommon
complication of prematurity among infants with ≥1000 g
birth weight when early vitamin D supplementation is
routinely implemented. The greatest risk appears to be
among those infants with <750 g birth weight.
We found lower birth weight and gestational age at birth to
be associated with elevated alkaline phosphatase levels. Fur-
ther we found furosemide exposure and small for gestational
age to be risk factors for abnormal screening labs.
There are several limitations that are important to
acknowledge. As with any investigation comparing two
epochs, underlying changes in clinical care or patient
population may have accounted for some of the changes
reported. We attempted to control for variations in the
underlying population in our analyses, including the intro-
duction of Prolact+ H2MF® milk fortifier at one facility.
While no other significant nutritional policy changes
regarding fortifier use were implemented during the study
period, we cannot rule out significant changes in parental
nutrition days. Further, we acknowledge that MBD is a
complex disease. While commonly utilized for this purpose,
alkaline phosphatase, with or without the addition of serum
phosphorous, is an inexact tool in screening for MBD and
this limits the generalizability of our findings.
In conclusion, we found implementing a standard guide-
line for the prevention and screening of MBD to be both
feasible and effective in a large population of VLBW infants.
These data indicate that routine screening and early vitamin D
supplementation can be successfully initiated in high-risk
ELBW infants. Despite an increase in numbers of patients
screened, the overall use of laboratory testing did not increase,
likely secondary to earlier supplementation and decreased
biochemical evidence of MBD of prematurity.
Acknowledgements We thank Dr. Reese Clark, Dr. Veeral Tolia, and
Dr. Blanca Molina for their critical review of this manuscript. We
thank our quality improvement team for support of this protocol after
initiation.
Compliance with ethical standards
Conflict of interest All authors have indicated that they have no
potential conflict of interest to disclose. The views expressed in this

publication represent those of the authors and do not necessarily
represent the official views of HCA or any of its affiliated entities.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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