This document summarizes research on the relationship between calcium, vitamin D, and bone health. It discusses how calcium and vitamin D are important for bone health, and reviews epidemiological studies showing links between vitamin D and calcium intake and bone diseases. Animal and human studies are also summarized, providing evidence that calcium and vitamin D supplementation can help prevent bone loss and fractures. The document concludes that current nutrient guidelines for calcium and vitamin D intake are generally sufficient for bone health, based on the literature reviewed.

1. Intro (10%)
With the inevitable process of ageing; muscle loss and bone deterioration issues
become more relevant. The most prevalent bone related condition is Osteoporosis;
which is defined as an asymptomatic bone disease characterized by low bone
mineral density (BMD) and deterioration of microarchitecture of the skeleton, leading
to an increased fracture risk (Giusti 2015, p.105 ). In both men and women, the
fractures that are related to or resulting directly from Osteoporosis are recognized as
significant health problems. The latest data from the Australian NHMRC which has
recently been updated on the 20th of January 2015 estimates that 1 in 2 Australian
women and 1 in 3 Australian men over the age of 60 will have an Osteoporotic
fracture. It is one of the major causes of morbidity amongst ageing Australians
(NHMRC).
The complications of Osteoporosis and its associated fractures have been linked
with low intakes of Calcium, a mineral that is predominantly found in bone; 99% of
Calcium is stored in the skeleton as hydroxyapatite and is a major constituent of
bone mineral content. The remaining 1% of Calcium is located in the blood,
extracellular fluid and soft tissues (Stomberg 2014, p.47). Thus, Calcium is required
for the normal development and maintenance of the skeleton. However, recent
literature has concluded that calcium alone is unlikely to result in a clinically
significant decrease in fracture risk. Therefore an intervention consisting of both
calcium and vitamin D may be more effective in the treatment of Osteoporosis.
The major function of Vitamin D is to maintain appropriate serum calcium
concentrations by enhancing the ability of the small intestine to absorb calcium from
the diet. In adults, deficiency of vitamin D can lead to increased bone turnover and
eventually Osteoporosis (NHMRC). Recent observations suggest that the circulating
concentration of 25-hydroxy vitamin D (25-OHD), which is the biologically active form
of Vitamin D, is inversely associated with stress fractures and bone complications
(Stomberg, 2014).
Osteoporosis Australia (2005) defined mild deficiency of serum 25-OHD at levels
between 25-50 nmol/L, moderate deficiency at levels of 12.5 and 25 nmol/L and
severe deficiency at levels below 12.5 nmol/L.
General measures for the prevention of fractures related to Osteoporosis are
excellent nutrition, appropriate calcium intake, physical exercise, avoidance of

2. detrimental lifestyle factors and adequate serum 25-OHD levels through the use of
vitamin D supplementation (Giusti, 2015).
The aim of this review is to evaluate the current literature on the relationship
between calcium and vitamin D, and its associated bone diseases. The literature will
be compared with current NRV’s to determine if they are sufficient to treat and
prevent diseases relating to bone health.
Search Strategy
In order to critically evaluate the literature contributing to the evidence base for the
relationship between vitamin D, calcium and bone health, a literature search was
conducted from various sources on this particular topic area. The literature was
compiled using the following databases: Scopus, PubMed, ScienceDirect, Biomed
Central and PEN (Practise Based Evidence for Nutrition). The search criteria was
limited to the year 2000 to present with any articles published prior to the year 2000
being excluded to ensure the most current and valid results were used. The
predominant keywords used included, ‘vitamin d’, ‘calcium’, and ‘bone health’.
Boolean operators, such as ‘vitamin d AND calcium’, were also used in an attempt to
narrow the search and find more specific references. More relevant keywords for
each individual topic included ‘human’, ‘epidemiological’, ‘osteoporosis’, ‘rickets’,
‘animal’ and ‘in vitro’. The epidemiology studies had exclusion criteria; whereby
studies with less than 500 subjects lasting less than 3 years were excluded. Animal
and human evidence was mostly comprised of randomised controlled trials with
supporting evidence compiled from narrative reviews and cohort studies. A grey
search engine, such as Google, was used to collect official government documents
and social media data relating to the topic. All the articles included were from
reputable nutrition and health journals, with most retaining a significant impact factor.
Food Sources
As previously stated, Calcium and Vitamin D are required for maintenance of bone
structure and health; insufficient intakes of these nutrients are associated with
conditions of low bone mineral density such as osteoporosis, rickets, osteomalacia
and its related bone fractures. In order to maintain a sufficient intake and meet the

3. current NRV’s for calcium and vitamin D, individuals need to consume foods high in
calcium and vitamin D.
Calcium is found predominantly in dairy and milk-based foods, with smaller amounts
in bony fish, legumes, green leafy vegetables, nuts and fortified soy beverages. The
following five food sources contain the greatest amounts of calcium per 100 grams:
- Hard cheese, such as cheddar
o 660mg of calcium per 100g
o ~$1.60 per 100g
- Tofu (when fortified with calcium carbonate)
o 320mg per 100g
o ~$1.79 per 100g
- Almonds
o 230mg of calcium per 100g
o ~$2.40 per 100g
- Canned salmon (with bones)
o 200mg of calcium per 100g
o ~$1.90 per 100g
- Green leafy vegetables, such as broccoli
o 40mg of calcium per 100g
o ~$0.60 per 100g
Foods high calcium are widely available and relatively inexpensive. Calcium intake is
both feasible and sustainable as the nutrient is found across a wide range of food
groups. Specialised diets that exclude food groups may affect an individual’s
consumption of calcium. It is shown that lacto-ovo-vegetarians appear to have
similar calcium intakes to omnivores (Reed et al. 1994, p.214), however low levels of
calcium intake are experienced among the vegan population, as they do not
consume animal-derived products (Larsson & Johansson 2002, p.1414). People
suffering from lactose intolerance may also have difficulty meeting the current NRV’s
as they are unable to digest milk sugars from dairy products. However vegans and
those suffering from lactose intolerance can still meet their recommended daily
intake through the consumption of foods such as almonds, green leafy vegetables
and fortified foods.
Meeting the Vitamin D NRV’s can be more challenging. Vitamin D is synthesised by
the skin in the presence of sunlight, as vitamin D3 is converted to the biologically
active form of Vitamin D; 1,25 dihydroxy D3 (Figure.1). In most people, this may

4. meet 80-100% of the required amount. However, populations at higher risk of
deficiency may need to consume additional foods high in Vitamin D to reach the
recommended NRV. Food sources containing large amounts of Vitamin D are listed
below:
- Canned red salmon
- Cod liver oil
- Fortified margarine
For the most part, meeting the NRV for vitamin D is attainable through exposure to
sunlight. However, those who do not experience enough sunlight such as home-
bound elderly or people who live in a low sun exposure location, may need to
consume a diet high in Vitamin D, or supplement with vitamin D in order to meet their
recommended daily intake and prevent the onset on rickets, osteomalacia and
osteoporosis. The elderly may experience greater difficulty in meeting the NRV for
Vitamin D as the skin’s production of Vitamin D can decrease by 70% with age.
Epidemiology
Diseases, which impact bone health, are seen in different populations across the
world. Epidemiological studies show that people who have one or more factor/factors
that decrease their time in the sun and therefore UV absorption, will consequently be
more susceptible to these bone degenerative diseases and lack the NRV of Vitamin
D and Calcium. Vitamin D deficiency is extremely common around the world and an
estimated number of 1 billion people worldwide are thought to suffer from vitamin D
deficiency, this indicates that further study in greater detail needs to be performed to
gather a higher understanding of the associated diseases (Holick & Chen, 2008).
Individuals on a standard Australian diet are consuming roughly 60% of their dietary
Calcium from dairy products (Sanders et al., 2015). In order to reach an intake of
1000-1300mg calcium per day, which is the recommended amount for the majority of
the population, it is recommended that individuals consume three serves of dairy per
day with one being a fortified source. It is also highlighted that supplementation with
vitamin D and calcium may prevent fractures in the frail elderly population,
particularly women (Sanders et al., 2015). This is a large scale epidemiological study
with over 168,000 participants from 195 systematic reviews, recently being published

5. in 2015. This demonstrates a vital link between daily NRV values of vitamin D,
calcium, bone health and prevention of associated diseases.
A nationwide survey of the UK population showed that more than 50% of the adult
population have a deficiency in Vitamin D (Pearce & Cheetham, 2010). They also
determined that 16% of people suffer from a severe deficiency during winter and
spring. This concludes that deficiency is more prevalent in winter as there is a
greater amount of cloud cover and less UV penetration. It was stated that cloud
cover can block 99% of vitamin D production. The survey also established that
certain sub populations are at a higher risk of vitamin D deficiency, this includes
people with: pigmented skin, the elderly, obese, malabsorption issues, short bowel,
renal disease, liver disease and the intake of drugs which effects calcium and
Vitamin D.
A study carried out by (Garriguet D, 2011) show ranges of people who weren’t
meeting their daily NRV’s. Dietary and supplemental intake was recorded. The data
showed that 45% to 69% had inadequate intake of calcium and 54% to 66% had an
inadequate intake of vitamin D. The study included 136 participants over the age of
50. Despite showing relative evidence, the small subject size impacts the reliability of
the study and it may only be referred to as a guide rather than conclusive
epidemiology.
Furthermore, less time spent in direct UV light may cause a drop in serum 25-OHD
levels (Scragg & Camargo, 2008). This study shows a trend between different age
groups, leisure activity and serum 25-OHD levels. It hypothesised that with age; less
time is spent outdoors doing leisure activities in the exposure of sunlight and as a
consequence, lower serum 25-OHD levels are observed. However it is also stated
that the older age demographic of 60 years and over, who were spending the same
amount of time in the sun participating in leisure activities, had similar levels of
serum 25-OHD to the 20-39 year old age bracket (Scragg & Camargo, 2008). This
study included 5,148 participants above the age of 20 over a six-year period; this
demonstrates a significant subject number over a sufficient duration of time.
An interesting area of study is vitamin D absorption within Black and Hispanic
cultures, and its effects on bone health. It is believed that 90% of vitamin D

6. production results directly from UV radiation from the sun, therefore skin colour may
affect absorption. Darker skin, such as that present in Black and Hispanic
populations, contain higher levels of melanin, a pigment that gives skin its colour.
This interferes with UV absorption in the skin (Taksler, Cutler, Giovannucci &
Keating, 2014). This study presents the effects of variables such as clothing, direct
skin exposure, sunscreen, season, age, skin colour and their impacts on the ability to
absorb UV radiation. A total of 14,319 participants over the age of 18 participated.
The studied estimated that the Black population (84.2–100 %), Hispanic population
(56.3 –91.9 %) and even White population (34.8–78.2 %) showed insufficient vitamin
D levels in winter. The study presents measures for vitamin D consumption per day
and amount of time needed to reach 600UI. Figure 2 in the appendix provides
evidence of the amount of time needed to absorb enough sunlight to reach 600IU in
Black and Hispanic populations, the figure also takes into account sun penetration
dependent on location (Taksler, Cutler, Giovannucci & Keating, 2014).
Animal Evidence
In vitro and animal studies are not only an essential first step towards exposing the
effects of vitamin d and calcium on bone health, but also provide valuable insight that
allows one to carry on to human clinical trials and the subsequent development of
appropriate evidence-based nutrient reference values. The current nutrient reference
values (NRV’s) for the consumption of calcium and vitamin D are in place today due
to the underlying effects of calcium and vitamin D on bone health exposed in clinical
trials. Animal and In vitro studies have been conducted in recent years with the
intention of providing a significant body of evidence to support the relationship
between vitamin D, calcium and bone health and the consequent NRV’s.
A study conducted in 2004 examined the effect of vitamin D supplementation on
bone growth in young rats fed a normal or low calcium diet (Iwamoto et al. 2004, p.
293). 50 female rats, all 6 weeks of age, were randomised into five groups with 10
rats in each group: baseline control, adequate calcium intake, low calcium intake,
adequate calcium intake plus adequate vitamin D intake (via food) and low calcium

7. intake plus adequate vitamin D intake (via food). The study was carried out over 10
weeks and at conclusion it was shown that Vitamin D supplementation stimulated
intestinal calcium absorption in rats fed a low or normal calcium diet and that vitamin
D supplementation prevented the reduction in periosteal bone density that was
exposed in the groups not taking vitamin D. In order to provide stronger evidence to
support the current NRV’s for vitamin D and calcium intake, it may have been
beneficial to include both male and female rats in the study design. However, the
study did take into account differing weights between each of the 50 rats and studied
their progress over an extended period of time (2 and a half months). This study
conducted by Iwamoto et al. provides critical results to the evidence base that
supports the current NRV’s for vitamin D intake. The study did not show any
detrimental effects of vitamin D supplementation, and in fact highlighted the positive
effects of vitamin D supplementation on calcium absorption and bone growth in
young rats being fed a normal or low calcium diet.
A study conducted earlier this year (in February 2015) examined the effects of high
vitamin D and calcium intakes on bone mineral content in obese mice (Song &
Sergeev 2015, pg. 154). The study was undertaken as recent studies demonstrated
that an increased body mass was detrimental to bone health, however, whether an
increase in dietary vitamin D and calcium intakes in obesity is beneficial to bone
health was yet to be established. It was hypothesised that high vitamin D and
calcium intakes would promote mineralisation of the growing bone in obesity via the
calcium regulatory hormones; 1,25-dihydroxyvitamin D3 and parathyroid hormone
(PTH). Male mice were randomised into two groups. Group 1 was fed high levels of
vitamin D and calcium. Group 2 was fed high calcium diets of which 60% of the
energy came from fat. At the completion of the 10 week study, it was evident that
high vitamin D and calcium intakes significantly increased bone mineral content in
Diet-Induced-Obese mice. The results from this study are in parallel with the current
basis for calcium and vitamin D NRV’s as they provide evidence to support the
relationship between vitamin D and calcium intake and bone health.
Many clinical trials in recent years have examined the effects of increased calcium
and vitamin D on bone health. Two studies, both undertaken in 2014, observed the
effects of vitamin D and calcium on osteoporotic rats and rabbits (Lani et al. 2014, p.

8. 402), (Lee at al. 2014, p.159). These studies demonstrated that in animals with high
calcium consumption, an adequate serum level of Vitamin D (80nmol/L or more)
reduces osteoclastogenesis and inflammation- induced osteoporosis and maintains
optimal levels of bone volume measured in the femur, tibia, humerus and vertebral
rib of the animals. The results also showed that a relatively high dose of calcium or
calcium with vitamin D supplementation increases the bone mineralization index
significantly. On the other hand, vitamin D alone is not as effective in promoting
mineralization even with high intake.
Each of the aforementioned studies indicate a positive relationship between
adequate Vitamin D and calcium intake and bone health. However, as each of the
studies were conducted on animal specimens, the intervention amount for each
supplementation does not correlate to the amount of these supplements required in
human clinical trials to see the same results. Therefore it is not feasible to say that
these animal trials alone provide enough evidence to support the current NRV’s for
calcium and Vitamin D intake. Although the results from each of the aforementioned
clinical studies are not directly applicable to the current NRV’s for these nutrients,
they do indicate that the combination of vitamin D and dietary calcium
supplementation is effective in preventing bone diseases such as osteoporosis and
reducing fracture risk.
Human Evidence
The human evidence showed a consistent pattern of supplementing calcium with
vitamin D respectively to achieve optimal treatment for bone degenerative diseases.
Overall, the literature agreed consistently that “Vitamin D plays an important role in
calcium homeostasis and stimulates bone growth” (Schoor et al. 2008, p.260). The
literature agreed that vitamin D deficiency is associated with fractures, low bone
mineral density, muscular weakness and falls.
In the younger age groups (19-30 years), the Australian NRV’s recommend 1000mg
of calcium and 200IU of vitamin D respectively. Two randomized controlled trials
were selected from the literature to compare values that apply to this age group. The
first was a double blind placebo trial of 221 healthy, Danish caucasian girls aged
between the ages of 11-12 years old. The study hypothesised that “sufficient vitamin

9. D levels during puberty may be necessary for optimal bone accretion and obtaining a
high bone mass, consequently reducing the risk of osteoporosis later in
life” (Molgaard et al. 2010, p.432). The subjects were divided in 3 separate groups
(A) a placebo group (B) a group supplementing with 200IU/day of Vitamin D3 and
(C) a group supplementing with 400IU/ of Vitamin D3. At the completion of the study,
a significant increase in serum 25-OHD levels was seen in the intervention group,
specifically in the higher dose group. However the increase in serum 25-OHD had no
effect on bone accretion or bone turnover. A major shortcoming of the study is that
“to effectively examine whether increased vitamin D intake will be beneficial for the
attainment of optimal peak bone mass intervention studies lasting several years are
needed.” (Molgaard et al. 2010, p.438). Furthermore, a second study hypothesised
that “the consumption of vitamin D and calcium supplements may reduce stress
fracture risk in military personnel during initial military training” (Stomberg et al. 2014,
p.46). The current NRV for this group is 1000mg/day of calcium and 200IU/day of
vitamin D. The study included 85 subjects in a placebo group and 83 subjects in an
intervention group. The intervention group consumed fortified bars twice daily. The
bars contained 1034mg of calcium and 546IU vitamin D each, consumed twice daily
this provided a total intake of 2064mg/day of calcium, twice the value of the current
NRV, and 1092IU/day of vitamin D, five times the value of the current NRV. At
completion of the study, serum 25-OHD, the biologically active form of vitamin D
found as a biomarker in blood, increased in the intervention group compared to the
placebo group. Serum calcium levels increased in both groups but to a greater
extent in the intervention group. This study concluded that “supplemental Ca and
vitamin D intake during military training may reduce the risk of stress
fractures” (Stomberg et al. 2014, p.55).
Another study recruited vitamin D insufficient hip fracture patients, vitamin D
insufficiency was defined as serum 25-OHD under 50nm/L. The current NRV’s for
older age groups that may suffer from osteoporosis related hip fractures is
1000-1300mg/day of calcium and 600IU/day of vitamin D. The objective of this study
was to “determine if vitamin D2 and vitamin D3 are equipotent therapies in vitamin D
insufficient hip fracture patients” (Glendenning et al. 2009, p.870). The study was
conducted over 3 months, 48 subjects supplemented with 1000IU/day of vitamin D2

10. and 47 subjects supplemented with 1000IU/day of vitamin D3. At completion of the
study the Vitamin D3 group resulted in a 31% greater increase in serum 25-OHD
concentration. However the “treatment did not significantly increase ionised calcium
concentrations, this raises the a question of the biological importance of elevated 25-
OHD concentrations in the blood and whether it has significant affects on bone
mineral density and the prevention of fractures” (Glendenning et al. 2009, p.874).
Therefore, further studies are necessary to determine the most effective dosing
regime; long term prospective outcome trials in a larger population group.
Conclusively, a narrative review of 8 randomised controlled trials aimed to
“investigate the anti-fracture efficacy of pharmacological agents along with calcium
and vitamin D in the treatment of osteoporosis” (Giusti et atl. 2015, p.105). The
study states that general measures for fracture prevention in men and women are
similar, they include “excellent nutrition, calcium intake between 1000-1500mg per
day and a serum 25-OHD concentration of over 30ng/mL (~75 nmol/L)” (Giusti et al.
2015, p. 107). This evidence coincides with the current NRV recommendations for
the elderly population with calcium intake ranging between 1000-1300mg per day
and vitamin D intake of 600IU a day.

11. Figure 1
Fig. 1.
Schematic diagram of synthesis of vitamin D and regulation of calcium homeostasis.
Vitamin D is majorly synthesized in the skin during exposure to UV radiation and less
absorbed from the diet. 25-hydroxyvitamin D [25(OH)D], the major circulating and
storage form of vitamin D, is converted in the liver. The biologically active form of
vitamin D, 1,25-dihydroxyvitamin D [1,25(OH)2D], is generated in the kidney.
1,25(OH)2D increases the efficiency of intestinal calcium absorption and has been
shown to have antiproliferative effects on parathyroid cells to help maintain calcium
homeostasis between the blood and bones. PTH = parathyroid hormone.

12. Figure 2 (a)
Figure 2 (b)
Fig. 2 demonstrates the racial/ethnic differences in estimated number of minutes required to synthesise
15ug (600IU) of vitamin D from sunlight, in January. Results are assuming the maximum daily
temperature and sun exposure: face, neck, hands, arms exposed. The figure provides a measure of the
amount of time needed to reach 600UI of Vitamin D from sunlight in America during winter for the (a)
Black population (b) Hispanic population.

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