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ORIGINAL ARTICLE
Milk ribonuclease-enriched lactoferrin induces positive
effects on bone turnover markers in postmenopausal women
S. Bharadwaj & A. G. T. Naidu & G. V. Betageri &
N. V. Prasadarao & A. S. Naidu
Received: 17 October 2008 / Accepted: 12 December 2008
# International Osteoporosis Foundation and National Osteoporosis Foundation 2009

Osteoporos Int
DOI 10.1007/s00198-009-0839-8
S. Bharadwaj : A. G. T. Naidu : A. S. Naidu (*)
N-terminus Research Laboratory,
981 Corporate Center Dr., # 110,
Pomona, CA 91768, USA
e-mail: asnaidu@nterminus.com
G. V. Betageri
Department of Pharmaceutical Sciences, College of Pharmacy,
Western University of Health Sciences,
309 E Second Street,
Pomona, CA 91766, USA
N. V. Prasadarao
Division of Infectious Diseases, Childrens Hospital Los Angeles
and Keck School of Medicine, University of Southern California,
4650 Sunset Blvd.,
Los Angeles, CA 90027, USA

Abstract
Summary Current treatments for postmenopausal osteoporosis
suffer from side effects. Safe and natural milk
proteins, ribonuclease, and lactoferrin promote formation
of new capillaries and bone formation. A ribonucleaseenriched
lactoferrin supplement studied here, demonstrates
significant reduction in resorption and increase in formation,
towards restoring the balance of bone turnover within
6 months.

Introduction Osteoporosis, a major health issue among
postmenopausal women, causes increased bone resorption
and reduced bone formation. A reduction in angiogenesis
could also contribute to this imbalance. Current treatments
such as hormone replacement therapy and bisphosphonates
have drawbacks of severe side effects. Milk ribonuclease
(RNase) is known to promote angiogenesis and lactoferrin
(LF) to stimulate bone formation by osteoblasts. We
examine the effect of ribonuclease-enriched lactoferrin
supplement on the bone health of postmenopausal women.
Methods A total of 38 healthy, postmenopausal women,
aged 45 to 60 years were randomized into placebo or
RNAse-enriched-LF (R-ELF) supplement groups. The bone
health status was monitored by assessing bone resorption
markers, serum N-telopeptides (NTx), and urine deoxypyridinoline
(Dpd) crosslinks and serum bone formation
markers, bone-specific alkaline phosphatase (BAP), and
osteocalcin (OC).

Results R-ELF supplementation demonstrated a decrease in
urine Dpd levels by 14% (19% increase for placebo) and
serum NTx maintained at 24% of the baseline (41% for
placebo), while serum BAP and OC levels showed a 45%
and 16% elevation (25% and 5% for placebo).

Conclusions R-ELF supplementation demonstrated a statistically
significant reduction in bone resorption and
increase in osteoblastic bone formation, to restore the
balance of bone turnover within a short period.

Keywords Lactoferrin . Osteoporosis . Postmenopause .
Ribonuclease

Introduction
Osteoporosis is a bone and joint disorder that causes a
significant reduction in bone density and alteration of the
bone microstructure that primarily affects elderly women.
These chronic changes in the interior of bone tissue lead to
fractures that could have adverse influence on the quality of
life of the elderly. The US National Osteoporosis Foundation
has estimated that by 2010, about 12 million people over
the age of 50 are expected to have osteoporosis and
another 40 million to have low bone mass (osteopenia). In
addition, osteoporosis also affects one in eight men
worldwide [1, 2]. Bones undergo a continuous remodeling
process through repeated cycles of destruction and
rebuilding. In healthy young adults, the amount of new
bone formation approximately balances the amount of
bone resorption. As the age increases, however, the
balance shifts to favor bone resorption. Among the
numerous factors that contribute to the development of
osteoporosis in women, postmenopausal estrogen deficiency
accelerates bone loss of 1–5% per year during the
first decade after menopause. Current efforts to treat bone
diseases have primarily concentrated on the development
of drugs to block bone resorption, which decrease the
formation or activity of osteoclasts. Present treatment
options for postmenopausal osteoporosis include hormone
replacement therapy (HRT) and bisphosphonates. HRT is
known to significantly reduce osteoclast activity but is
prone to adverse effects, such as increased risk of breast
cancer [3, 4], and bisphosphonates have a risk of
development of esophageal ulcers [5]. Therefore, it is
imperative to explore and develop strategies for enhancing
the bone formation and simultaneously prevent the bone
loss without side effects.
Milk, a superior source of bioavailable calcium, has
pronounced effects on bone metabolism. The basic protein
fraction of milk contains several active components that
have been shown to suppress osteoclast-mediated bone
resorption and osteoblast differentiation. Studies on milk
basic protein (MBP) intake have demonstrated bonestrengthening
effects in cell culture studies and animal
experiments [6]. A limited number of studies report the
influence of MBP supplementation on bone metabolism
and radial bone mineral density in healthy adult women [7,
8]. Recently, the component of MBP responsible for the
inhibition of osteoclastic resorption has been shown to be
identical to milk angiogenin, a 14-kDa milk basic protein
that exhibits both angiogenic and RNAse properties [9].
Angiogenesis or formation of new capillaries within bone
tissue could facilitate supply of nutrients and removal of
waste products and may be responsible for healing of
fractures, remodeling and regeneration [10, 11].
Lactoferrin (LF), a multi-functional protein, present in
neutrophils and exocrine secretions like milk and tears, is
also present in synovial fluid of bone joints. LF is a major
constituent of colostrum responsible for the rapid growth
and development of skeletal and immune system of the
newborn. Recent studies have established LF as a bone
growth factor as it stimulates osteoblast differentiation and
new bone formation; and reduces bone resorption in vitro
and in animal models [12, 13]. LF is also a transport protein
that could facilitate absorption of many essential minerals
and nutrients that bind to it; however, the effect of LF on
bone health in humans is not known. The objective of the
present study is to investigate the benefits of milk RNAseenriched
LF (R-ELF) supplement along with calcium on
bone health of postmenopausal women in comparison to an
appropriate age-matched control that received only calcium
supplement. The bone health status was assessed by wellestablished
assays for markers of bone resorption and bone
formation. Our studies have, for the first time, demonstrated
that R-ELF significantly reduced the bone resorption
markers and simultaneously increased the bone formation
markers; and suggest possible utilization of R-ELF in
natural supplements for osteoporosis without significant
side effects.

Materials and methods
Subjects
More than 50 women responded to the announcement about
this study made through notification in a local community
organization. All of the prospects completed a questionnaire
on general bone health status, previous injury/disease,
current or previous treatment, and consumption of calciumrich
foods. General health was determined by routine
standard medical assessment of physical and mental health.
The exclusion criterion for the study included potential risk
factors for osteoporosis, dietary calcium intake, and
medical history. Women who had history of any illness
(active Paget’s disease, hyperthyroidism, hypothyroidism,
type I diabetes mellitus, calcium intolerance, kidney
problems; cancer diagnosis for solid malignancies, and
inflammatory bowel disease) that affect bone mineral
metabolism were excluded. Women that received treatment
with estrogens, progesterone, raloxifene, or tamoxifen and
antiresorptive agents like calcitonin or bisphosphonates
were also excluded. We also excluded women with life
expectancy of less than 2 years; current and ongoing use of
methotrexate, phenytoin, phenobarbital, or inhaled corticosteroids
at a dose greater than 800 μg/day; and a body mass
index above 32 or below 20. On evaluation, 38 healthy,
ambulatory postmenopausal women, 45–60 years old, with
no menses for at least 12 months were registered for the
study. Every effort was made to recruit only those women
who fulfilled the inclusion and exclusion criteria. Nonetheless,
there were three exclusions, one with a history of
treatment for bone health and other two with hypothyroidism.
The study was approved by the appropriate Institutional
Review Board of the Western University of Health
Sciences in Pomona, CA, USA. Prospective participants
were advised of the nature of the study and provided
written informed consent before participation.

Study design
Thirty-five women included into the study were randomly
assigned to one of the two groups: placebo group or R-ELF
Osteoporos Int
group. R-ELF is a ribonuclease (angiogenin)-enriched lactoferrin
either co-isolated from bovine milk (50:50 ratio wt/wt)
or both proteins admixed to obtain required ratios, as
previously described [14, 15]. There were 15 subjects in
the placebo group and were supplemented with 100%
recommended daily allowance (RDA) of calcium, in tablet
form, whereas 20 subjects of the R-ELF group were
administered with two R-ELF capsules of 125 mg each,
along with 100% RDA of calcium administered orally from
day 1 to day 180. Venous blood (by standard venipuncture
technique) and urine samples were collected from each
subject on day 0 (baseline before the commencement of
supplementation), day 15, day 30, day 60, day 90, and
day 180 of the study. Standard systolic/diastolic blood
pressures as well as the body weight were also monitored at
baseline and the aforementioned days. All the participants
were interviewed at each visit for adherence to the regimen
and occurrence of adverse events. A flow diagram of the
study is shown in Fig. 1.
Biochemical markers of bone turnover
Venous blood was drawn into a vacutainer (BD Biosciences,
NJ, USA) by standard venipuncture technique. The
blood was allowed to clot and the serum was separated by
centrifugation. The serum specimens were stored at −80°C
until analysis. Urine was collected after an overnight fast
and frozen at −20°C until analysis. On the day of analysis,
serum and urine samples were thawed, mixed, centrifuged,
and the clear supernatant was used for the assay. Bone
formation rates were assessed by measuring two wellestablished
markers of osteoblast activity: (1) change in
levels of bone-specific alkaline phosphatase in the serum
(sBAP) [16] and (2) change in serum levels of intact bone gla
protein or osteocalcin (sOC) [17]. Bone resorption was also
investigated by identifying the change in levels of two
resorption markers, N-telopeptides in the serum (sNTx) [18]
and free deoxypyridinoline crosslinks in urine (uDPD) [19].
These four bone turnover markers were monitored in the
urine and blood samples collected from each subject, before
R-ELF supplementation (baseline) and with R-ELF supplementation
at the end of 15, 30, 60, 90, and 180 days. Metra-
BAP, Metra-OC, and Metra-DPD antibody immunoassay
kits used for the study were obtained from Quidel, San
Diego, CA, USA. Osteomark NTx enzyme immunoassay
kit was purchased from Inverness, Princeton, NJ, USA.
Urinary creatinine was determined using standard colorimetric
assay (Oxford Biomedical Research, Oxford, MI,
USA) based on well-known Jaffe reaction [20].

Statistical analysis
Biochemical bone turnover marker data for each observational
day was analyzed for measures of central tendency,
deviation, and distribution of data. Data were considered
outliers, if they were >1.5 times the inter-quartile range
above the third quartile or below the first quartile. Outliers
were discarded from datasets for statistical tests of
significance. In view of the small size of placebo and RELF
groups, median and standard error of the mean were
used as the preferred measures of central tendency
throughout this study, as it is least influenced by the
extremes of data. The Kolmogorov–Smirnov test (KS test)
was used for normal distribution of data within each set
[21]. Non-linear least-squares regression of the marker data
to a logistics curve was performed to evaluate the peak
levels of bone turnover markers at the end of the study.
Student’s unpaired two-sample t test was used for comparison
of the mean observed change in markers for placebo
and R-ELF datasets in order to establish the effect of RELF
supplementation. OriginPro Ver. 8 (OriginLab, MA,
USA) software was used for data analysis.

Results
Baseline characteristics, compliance, and adverse events
The baseline characteristics of placebo and R-ELF groups
are shown in Table 1. The data shows a comparable match
between the placebo and R-ELF groups, i.e. generally good
bone health status and distribution of the bone turnover
marker levels. The median and range of markers are within
the accepted levels for generally healthy, postmenopausal
women [22–32]. Three participants from the control group
Registration
(n = 38)
EXCLUSIONS
Past treatment for bone health (n = 1)
Hypothyroidism (n = 2)
Randomization
(n = 35)
CONTROL Group
(n = 15)
Adverse Events (n = 0)
Withdrawal (n = 3)
Analyses
Completed (n = 12)
R-ELF Group
(n = 20)
Adverse Events (n = 0)
Non-compliance (n = 1)
Analyses
Completed (n = 19)
Fig. 1 Flow-chart of the study
Osteoporos Int
dropped out of the study before day 15 and one from the
R-ELF group was dropped out from the study due to noncompliance.
There was >95% compliance to the supplement
regimen among the subjects. The body weight and
blood pressures of all the subjects essentially maintained
within ±3% of their baseline values. No adverse events
were reported during the 6-month study or 3-month poststudy
follow-up.
Bone resorption markers
The median sNTx and uDpd changed gradually to a peak
level for both placebo as well as R-ELF groups, as shown
in Fig. 2. The resorption markers for each observational day
were also normally distributed (p=0.34–0.99). sNTx for the
placebo group increased from 15.8±2.6 nM bone collagen
equivalents (BCE) on day 0 to 22.1±1.7 nM BCE on
Fig. 2 Variation of bone resorption
markers—sNTx (a) and
uDpd (b) and bone formation
markers—sBAP (c) and sOC (d)
with the progress of the study.
Median±SEM data are shown
for placebo (unfilled square) and
R-ELF (filled square) groups
Table 1 Baseline characteristics
of the study population
a Reference [20–30]
b Bone collagen equivalents
c Median±SEM given for all
markers. Range shown in
parenthesis
d Creatinine
Characteristic Typical values for
PM womena
Control R-ELF
Number of participants (n) 15 20
Age (years)
Mean±SD 51.0±4.4 53.5±5.4
Range 45–59 45–60
Weight (lbs)
Mean±SD 134±20 141±24
Range 104–174 107–208
Blood pressure (mm Hg)
Mean systolic 121 127
Mean diastolic 78 80
Bone resorption status
Serum NTx (nM BCEb) 15.9 (8–28) 15.8±2.6c (5.3–29.1) 12.9±3.3 (7.8–41.6)
Urine Dpd (nM/mM CRd) 7.5 (4–12) 6.6±0.7 (5.2–11.7) 8.2±0.6 (6.0–13.2)
Bone formation status
Serum BAP (U/L) 25 (14–43) 25.0±1.3 (18.9–30.6) 19.7±2.4 (13.1–44.8)
Serum OC (ng/mL) −3–10 4.1±0.5 (2.1–7.0) 4.2±0.4 (2.6–9.8)
Osteoporos Int
day 180, while R-ELF group displayed a relatively smaller
rise (12.9±3.3 to 16.1±2.0 nM BCE). The change in bone
turnover markers, calculated as a percent of the
corresponding baseline levels are represented in Fig. 3.
Median sNTx for the placebo group showed an increase of
40.1% in 180 days, reflecting significant bone resorption
while the R-ELF group showed a relatively smaller rise of
24.5% during the same period (pduration to achieve 80% of the peak change in sNTx is
about 25 days for R-ELF group compared to 45 days for the
placebo group, indicating that R-ELF supplementation
induces its effects within a short time. The calculated
parameters for the bone markers and the results of statistical
tests are summarized in Table 2.
An interesting reversal of trend was observed with
median uDpd levels although the placebo group showed an
increase from 6.6±0.8 to 7.7±0.7 by day 180, the R-ELF
group decreased from 8.2±0.6 to 6.9±0.6 nM Dpd/mM
Creatinine. The overall rise of 17.8% indicated a significant
level of bone resorption in the placebo group. In contrast,
Fig. 3 Change from the baseline
of median bone turnover
markers with the progress of the
study—sNTx (a), uDpd (b),
sBAP (c), and sOC (d). Open
bars denote the placebo group
and filled bars denote R-ELF
group in all the panels. In the
case of sOC, change in mean
from baseline level is shown
Table 2 Progress of biochemical
bone turnover markers and
statistical analysis
a Median±SEM
b Peak change in the marker
obtained from a non-linear least
squares fit to logistics curve
cValue of the t statistic. Significance
p (95% CI) is given in
parenthesis
d Not determined as there was
no significant change in serum
OC for the control group
Marker Change from baseline (%) Time to reach 80% of peak
level in days
t testc
Mediana Peakb
Bone resorption markers
Serum NTx
Placebo 32.0±7.6 41.6 45 15.18
R-ELF Group 20.2±4.0 24.1 25 Urine Dpd
Placebo 5.6±3.3 19.2 95 4.11
R-ELF Group −10.7±2.2 −14.0 31 Bone formation markers
Serum BAP
Placebo 9.1±3.9 25.4 115 4.10
R-ELF Group 33.2±7.2 44.9 45 Placebo 0.4±1.0 5.1 –d −20.3
R-ELF Group 7.2±2.8 16.4 – Osteoporos Int
Dpd levels of the R-ELF group showed a reduction in resorption
by ∼10% within 30 days and continued to fall to 14.6%
by the end of the study (p=0.0021, 95% CI). The R-ELF
group achieved 80% of peak change in Dpd levels in about
31 days, compared to 95 days taken by the placebo group.
Bone formation markers
The two markers of bone formation, sBAP and sOC, were
determined at the same pre-defined intervals during the study.
Figure 2 shows the measured variation of median sBAP and
sOC levels for both groups. The range of observed sBAP and
sOC values are similar to that reported in other recent studies
[22–32]. As with the bone resorption markers, the sBAP and
sOC datasets for each observational day were normally
distributed (p=0.39–0.98).
The variation of sBAP and sOC from their respective
baseline levels is depicted in Fig. 3. Median sBAP
gradually increased to a peak level for both the groups
(25.0±1.3 to 31.4±3.0 U/L for placebo and 19.7±2.4 to
28.1±3.1 U/L for the R-ELF group). Although median
levels for R-ELF group are lower than those for the placebo
group, the percentage change from baseline was better for
the R-ELF group. The 42.7% elevation for R-ELF group
compared to 25.7% for the placebo was also statistically
significant (pabout twice that of the placebo group and 80%of peak change
was achieved within ∼45 days of R-ELF supplementation. In
the case of sOC, mean marker levels maintained within ±3%
of baseline for placebo (4.1±0.5 to 4.2±0.4 ng/mL), while
those for the R-ELF group increased linearly by 18.8% from
4.3±0.4 to 5.2±0.5 ng/mL. The results summarized in Table 2
indicate that change in sOC with supplementation was
statistically significant (pBone formation markers were observed to be highly
correlated with bone resorption markers for both groups,
with Pearson correlation coefficient (r) values in the range
0.58–0.93 for the placebo and 0.66–0.90 for the R-ELF
group, respectively. The correlation plots along with the
statistical parameters are shown in Fig. 4. The positive
correlation observed for placebo group is generally improved
with R-ELF supplementation, as seen from the higher r and
smaller p values for sNTx with sBAP as well as sOC. In
case of uDpd, the positive correlation seen with the placebo
group is changed to negative for the R-ELF group—r
changing from 0.93 to −0.87 for sBAP and from 0.83 to
−0.66 for sOC, respectively. This change reflects the effect
of R-ELF supplementation to reduce resorption markers
while increasing the formation markers to restore balance of
bone turnover.
Discussion
Clinical studies of large populations of postmenopausal
women have established that biochemical markers of bone
metabolism such as serum osteocalcin, bone-specific alkaline
phosphatase, and the urinary excretion of cross-linked
collagen N-telopeptides are indicators of loss of bone
mineral density, which in turn is a risk for osteoporosis and
Fig. 4 Correlation plots of bone formation markers vs. bone resorption markers. Top row corresponds to the placebo group and the bottom row to
the R-ELF group. The Pearson correlation coefficient (r) and significance (p) are shown for each plot
Osteoporos Int
fractures. In the present study, R-ELF supplementation has
been shown to improve bone formation markers, while
reducing the bone resorption in postmenopausal women. In
addition, R-ELF supplementation is able to achieve this
significant change in bone turnover markers within a short
duration. The ability of R-ELF supplementation to improve
bone metabolism is attributed to the characteristics of its key
components, RNAse and LF. Firstly, LF is a transport
protein and present in all exocrine secretions in the body
including synovial fluid in the joints. Specific LF receptors
have been described in mammalian cell types and tissues
including monocytes, lymphocytes, platelets, liver, mammary
epithelial cells, and intestine [33]. LF also interacts with
low-density lipoprotein receptor-related protein 1 in osteoblasts
and increases the mitogenic activity, indicating that LF
enhances the bone growth. [34]. It can also form complexes
with many nutrients like the glucosaminoglycans, due to its
highly positive charge. Therefore, LF is able to interact with
cells and may deliver the nutrients to key locations in bones
and joints. Furthermore, LF is also known to stimulate
osteoblastic activity [12, 13]. Secondly, formation of new
capillaries and supply of nutrients is essential for the
rejuvenation of the aging bone tissue. Milk RNAse present
in the R-ELF may accomplish this function, as it is known to
stimulate angiogenesis [35].
Several strategies for the restoration of postmenopausal
bone turnover have been developed and established with
the support of bone mineral density data over the last
decade, and have been approved for therapy. Bisphosphonates,
like alendronate and its variants, have been used for
treatment of age-related bone loss in postmenopausal
women and have consistently shown substantial reduction
in bone resorption. However, this reduction in resorption is
also accompanied by up to 40% decrease in bone formation
as reflected by sBAP and sOC markers in general, and as
high as 52% reduction in one trial [29, 30, 36]. In HRT or
estrogen therapy trials, a reduction by 30–50% of resorption
has been observed along with 20–45% reduction in
bone formation makers [26, 36]. Selective estrogen receptor
modulator such as raloxifene hydrochloride has shown a
gradual 25–30% decrease of resorption markers in 3 years
along with 10–20% decrease in formation markers [37, 38].
In contrast to above treatment options, R-ELF not only
reduced the resorption markers by about 20% but also
affected bone formation rate positively (up to 40%
increase), within 3 months of supplementation.
Strontium renelate, isosorbide mononitrate, and teriparatide
(synthetic parathyroid hormone (PTH)) are among the
few other therapeutic agents that show a positive effect on
bone formation as well as a reduction in bone resorption
activity [39–41, 32]. Organic nitrates stimulate osteoclasts
and osteoblasts via the production of NO and have been
observed to elevate bone formation levels. In a recent study,
isosorbide mononitrate (ISMO) showed a 45% decrease in
resorption markers and a 23% increase in formation
markers compared to the placebo group [40]. However,
ISMO induced mild to moderately severe headaches in
some participants in that study. In cardiovascular health
studies, nitrates have been known to cause this side effect in
as many as 36–52% of participants [42]. In a recent clinical
study of PTH [32, 41], there was a 92% enhancement of
sBAP, but was accompanied by 72% increase in bone
resorption as measured by uDpd. Although teriparatide has
been approved, PTH treatment is known to have adverse
effects, at least in animal model studies [43, 44] and
requires further long-term evaluation [41]. Of note, similar
elevation of bone formation marker was observed by daily
oral supplementation of R-ELF in the present study,
compared to subcutaneous injection of PTH. Therefore, RELF
supplementation would provide alternative treatment
without significant adverse effects.
Correlations between bone turnover markers for some
studies of interventions with HRT, prednisone and teriparatide
[26, 28, 32] were compared with our results. Similar to
the present study, a generally improved correlation between
resorption and formation markers with the intervention was
observed. However, the correlation did not change from
positive (for placebo) to negative for the treatment group.
This is so because reduction in resorption by HRT and
bisphosphonate treatments is accompanied by significant
decrease in bone formation marker like BAP [45].
Although teriparatide treatment resulted in favorable increase
in BAP, it was associated with increased resorption
levels [41]. In both these cases, the correlation between the
formation and resorption markers remained positively
correlated even after intervention, leading away from a
balance of bone turnover. A negative correlation between
the markers is a condition of increased bone formation and
reduced resorption leading to attainment of the balanced
bone turnover and is the preferred state of outcome. In the
present study, uDpd is negatively correlated with formation
markers, sBAP and sOC, after R-ELF supplementation; and
therefore, appears to lead towards restoring the balance.
In summary, R-ELF supplementation in preliminary
human clinical trials, for the first time, has shown
promising and favorable effect on biomarkers of bone
turnover in postmenopausal women. Despite the small
number of subjects, short duration and lack of bone density
data in the present study; and in view of the several
shortcomings of drug therapy for postmenopausal bone
loss, the results of R-ELF supplementation are very
significant, as it is based on safe and natural milk proteins.
Acknowledgements We thank Tiffani Davis (phlebotomist), Natver
Patel, and Sreus Naidu for coordinating with the clinical study.
Osteoporos Int
Funding This project was funded by N-terminus Research Laboratories,
Pomona, CA, USA.
Conflicts of interest S. Bharadwaj, A. G. Tezus Naidu, and A. S.
Naidu declares conflict of interest – all are employed by the N-terminus
Research Laboratory. All other authors have no conflict of interest.
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