Calcium and Bone Health

what is the recommended dietary allowance of calcium per day for adults 19-50?

1000 mg

what is the calcium consumption in the general population? in the lactose intolerant population?

800-830 mg (below the RDA)

200-560 mg

more recently, what have been the average intakes for Canadian males and females?

males - 1038 mg

females - 904 mg

what are the best foods with high calcium contents? not as good foods?

good: yogurt (non fat, low-fat), swiss cheese, cheddar cheese

not as good: whole wheat bread, cooked broccoli, okra, kale

how many cups of cooked broccoli is about equal to the calcium content of a cup of milk?

~ 9-10 cups of cooked broccoli

what might be indicated/prescribed for patients with LI?

calcium (and vit D?) supplements

lactose intolerance is associated with …

associated with low milk consumption and low bone mineral density (BMD)

what are the differences in average milk consumption according to lactose intolerance symptoms?

lactose intolerant people drink 55% less milk per day compared to lactose tolerant people

what is osteoporosis?

a systemic skeletal disorder characterized by low bone mineral density (BMD) and microarchitectural deterioration of the bone tissue/structure that compromises bone strength and predisposes a person to an increased risk of fracture (can often lead to permanent and severe disability - e.g. in elderly populations, there is strong correlation between hip fractures and congestive heart failure)

• reduction in bone mineral density (decreases bone quantity)

• changes in bone structure (decreases bone quality)

what are the difference between spongy bone and compact bone? in cases of osteoporosis, which is usually lost first?

spongy bone: inner bone, porous, found in lumbar vertebrae (usually lost first)

compact bone: outer bone, rigid, found in femur

what is the most important determinant of bone strength and BMD?

dual X-ray absorptiometry (DXA/DEXA) - used clinically as a surrogate measure for the diagnosis of osteopenia and later osteoporosis

what is osteopenia?

reduced bone mass and mineral density, less severe compared to osteoporosis - precedes osteoporosis and is a silent condition, one doesn’t know there’s a problem until they take a fall

what are the risk factors for osteoporosis beyond calcium status?

traditional

• female gender

• increasing age

• decreased physical activity

• decreased bone acquisition

• amenorrhoea/premature menopause

secondary

• renal dysfunction

• malnutrition/low BMI

• vit D deficiency

• immunologic effects (e.g. cytokines: TNF, IL-6)

all of these result in decreased bone mineral density

how does an individual obtain calcium?

all calcium is provided through the diet

what is calcium needed for in the body?

needed for skeletal and cardiac muscle contractions

where is calcium found in the body?

99% of calcium stores are in bone and teeth

1% in extracellular fluid and soft tissues

how is calcium concentration in the extracellular fluid regulated?

very tightly regulated through hormones

what must blood calcium be maintained at?

between 9-10.5 mg/dL (homeostatic set point/range)

describe absorption of calcium

occurs in the small intestine and acts to increase dietary calcium uptake

describe reabsorption of calcium

occurs in the kidney

calcium can be excreted in the urine when blood calcium levels are high or reabsorbed into the blood when levels are low

describe resorption of calcium

occurs in the bone

involves dissolving bone structure to release calcium stored in bone into the blood stream

describe the hormonal control in maintaining calcium homeostasis

1. PTH is first response to low blood calcium secreted by the parathyroid gland

• stimulates production of calcitriol (active form vit D) in kidney by activating 1a-hydroxylase enzyme

• stimulates resorption of bone calcium → blood

• maximizes tubular resorption of calcium in kidney → blood

2. calcitriol is second response to low blood calcium

• active metabolite of vit D made in the kidney

• stimulates resorption of bone (immediate response)

• facilitates absorption of calcium from SI (short term response)

• maximizes tubular resorption of calcium in kidney (short term response)

3. calcitonin - response to HIGH blood calcium

• secreted by thyroid parafollicular cells

• suppresses tubular reabsorption of calcium in kidney

• inhibits bone resorption and facilitates demineralization

• longer term response which improves bone density

describe how inactive vit D becomes activated

vit D3 (cholecalciferol) → 25-hydroxycholecalciferol (via 25-hydroxylase in liver) → 1,25-dihydroxycholecalciferol (via 1a-hydroxylase in kidney) - this is the active form (calcitriol) which functions to increase blood calcium levels

calcitriol functions as a ____

transcription factor

how does calcitriol influence many disease processes?

• decreases adaptive immune system activation by:

  • decreasing B and T cell proliferation

  • decrease antigen presenting cell maturation (cannot present antigen)

  • decrease inflammatory cytokine production (IL-6, TNFa)

  • inhibit Th17 cell activation and IL-17 secretion

  • stimulates antimicrobial protein secretion (e.g. cathelocidin, beta-defensins)

describe the two ways of intestinal absorption of calcium

1. passive diffusion (paracellular transport) active when consuming a high Ca diet to allow for maximal uptake of dietary Ca (as lumen Ca decreases, the mechanism also decreases

2. calcium channel (TRPV6, apical border)

• Ca forms a complex with calbindin which transports Ca to and from intracellular stores in the mitochondria and ER to help maintain intestinal Ca levels and contribute to blood Ca levels; can also shuttle Ca to the basolateral membrane where it can be transported in 1 of 2 ways:

1. sodium calcium exchanger (3 Na in / 1 Ca out)- energy efficient

2. calcium pump (Ca out, H in) - requires ATP

describe the role of calcitriol in the intestinal absorption of calcium

calcitriol stimulates gene expression and drives mRNA expression for protein synthesis of ALL calcium apical and basolateral transporters AND calbindin

what does PTH inhibit?

inhibits many bone forming reactions:

• type 1 collagen formation

• osteocalcin production in osteoblasts

what does PTH stimulate? what does it decrease the activity of?

stimulates the activity of 1-a-hydroxylase in the kidney

decreases renal excretion of Ca

why is a rapid PTH response needed to low blood calcium? how is this executed?

if Ca is low, the heart may not have enough Ca to continue beating - a rapid response is needed (don’t have time to wait for transcription/translation of PTH)

pre-formed active PTH is stored in vesicles so the release of active PTH can happen quickly

describe the cleavage activity to form active PTH

in the ribosome, there is pre-pro PTH → the “Pre” sequence is cleaved

in the ER, the “pro” sequence is cleaved

the active part of PTH is left to be secreted into vesicles and stored for when needed

when does PTH release from vesicles stop?

when blood [Ca] is high - Ca binds to the CaSR (calcium sensing receptor) which activates a signalling pathway to stop PTH release from vesicles

describe how FHH mutations in the CaSR can impact Ca levels in the blood

FHH mutation in CaSR - not responsive to changes in blood Ca levels (body perceives a lack of Ca levels and sustains secretion of PTH past the homeostatic range (higher blood Ca levels needs to signal a stop to the release of PTH)

describe the phenotype of FHH

hypercalcemia:

• high or increased blood Ca levels → sustained by bone resorption (more aggressive bone loss)

• over time, this increases the risk of osteoporosis and fractures

hypocalciuric:

• low levels of Ca in the urine (excess Ca not being excreted) → instead develop kidney stones because Ca stays trapped in the kidney and is neither lost in the urine OR reabsorbed back into the blood

explain the differences between the 2 types of bone

cortical bone ~ 80% of total bone mass

• outer layer of all bones and forms the bulk of the interior of the long bones of the body

• dense tissue composed of bone minerals and extracellular matrix proteins

• provides much of the strength for weight bearing by the long bones

trabecular bone ~ 20% of total bone mass

• found in interior of bones and is prominent within vertebral bodies

• composed of thin spicules of bone that extend from the cortex into medullary cavity

• lacework of bone spicules lined by areas of osteoblasts and osteoclasts

• undergoes constant turnover at a higher rate compared to cortical bone (b/c we don’t want a constant breakdown of the outside of our bones, trabecular bone is not as functionally important for weight-bearing)

describe and differentiate the different types of mature bone cells

osteoblasts - bone forming cells through the process of ossification (bone deposition)

• secrete organic matrix (largely composed of collagen) and other proteins

osteoclasts - bone resorbing cells (breakdown), found on the growth surfaces of bone

osteocytes - old osteoblasts that are embedded in bone matrix (after they’ve secreted it in the process of forming new bone)

• sense mechanical stress on bone and secrete growth factors that stimulate new osteoblasts and bone formation

describe how osteogenic cells become mature osteoblasts and osteoclasts

osteogenic cells are stem cells

osteogenic cell → mesenchymal stem cells → pre-osteoblasts → mature osteoblasts (via Runx2 - a key transcription factor for osteoblast maturation)

osteogenic cell → hematopoietic stem cell → pre-osteoclasts (via expression of M-CSF - stimulates RANK expression) → mature osteoclast (via RANK-L binding to RANK) → mature osteoclast

what would happen if an individual was deficient in mesenchymal stem cells?

they would not be able to mature into an osteoblast - cannot form new bone matrix

what would happen if an individual was deficient in hematopoietic stem cells?

these stem cells will not be able to express M-CSF which would eventually stimulate RANK expression, RANK-L would not have a receptor to bind to and would not form mature osteoclast - cannot break down bone (if deficient in Ca, can lead to heart failure)

what would happen if an individual had no expression of M-CSF?

RANK would not be able to be expressed and form pre-osteoclast, RANK-L would not have a receptor to bind to and there would be no new osteoclasts formed - no bone resorption

describe the bone remodeling cycle

1. becomes activated in response to low blood Ca, osteoclast resorption

2. osteoblast activity, matrix formation (osteoid) - no minerals added yet

3. mineralization - hydroxyapatite, add structure to bones to resist compression (mineralization hardens and strengthens new bone)

4. resting phase

describe the composition of bone

70% tough organic matrix that is strengthened by deposits of calcium salts (30%)

organic matrix (70%) aka osteoid

• majority is type 1 collagen fibers (gives bone its tensile strength and stability)

• ground substance = chondroitin sulfate and hyaluronic acid (provide sites for nucleation of hydroxyapatite crystals)

• organized matrix of proteins produced by osteoblasts (osteocalcin and osteonectin)

bone salts (30%)

• crystalline salts deposited within organic matrix composed of calcium and phosphate (hydroxyapatite)

a small amount of osteoblastic activity occurs continually in all living bones, what percentage of all bone surfaces does it account for?

~4%

how is type 1 collagen hardened?

hardened by deposits of hydroxyapatite

describe and differentiate osteocalcin vs osteonectin

osteocalcin - strongly binds Ca within hydroxyapatite

osteonectin - binds to hydroxyapatite AND collagen fibers → forms a lattice work holding the organic matrix and bone salts together; facilitates mineralization of collagen fibers

give examples of some proteins secreted by osteoblasts that regulate the activation of osteoclasts. what is the secretion of each stimulated by?

RANK-L

• binds to RANK on surface of osteoclast progenitor cells (pre-osteoclast) which stimulates the cell to mature into a functional osteoclast capable of breaking down bone

• can be expressed on osteoblast surface (cell-contact dependent) OR secreted as a free protein (cell-contact independent)

• secretion/expression stimulated by:

  • calcitriol

  • PTH

  • inflammatory cytokines (TNFa, IL-6)

  • PGE2

Osteoprotegerin (OPG)

• RANK-L antagonist secreted and binds to RANK-L forming a complex that can’t bind to RANK receptor and inhibits osteoclast maturation

• secretion is stimulated by estrogen and IL-4

what are the stimulants of RANK-L

• PTH

• calcitriol

• inflammatory cytokines (TNFa, IL-6)

• prostaglandin E2 (PGE2)

after expression on the osteoblast surface or secretion, what happens to RANK-L?

RANK-L will bind to RANK on the surface of the osteoclast precursor (pre-osteoclast)

what does RANK-RANKL signalling result in?

results in osteoclast maturation → bone resorption

what happens when OPG binds to RANK-L?

it prevents RANK-L from binding to RANK and prevents the activation/maturation of the pre-osteoclast and thus prevents bone resorption

what are stimulants of OPG secretion?

estrogen and IL-4

on how much bone surface are osteoclasts active?

less than 1% (higher in growing children/adolescents)

osteoclast expression of RANK is stimulated by…?

M-CSF (if there is no M-CSF expression, no RANK expression and no osteoclast maturation and activity)

how do osteoclasts carry out bone resorption?

1. send out villus-like projections toward the bone, forming a “ruffled border” = cell membrane adjacent to the bone (increases SA of the cell membrane in contact with the bone surface)

2. secrete proteolytic enzymes (e.g. Cathespin K) stores in lysosomes - digests the organic matrix of the bone (e.g. osteonectin, osteocalcin, type 1 collagen proteins)

3. secrete citric acid (pH 4.5) - dissolves hydroxyapatite and digest bone matrix → releases free Ca and P

what are the osteoclast surface receptors?

• RANK

• cytokine receptors (e.g. IL-6, TNF receptors - stimulate activation of osteoclast)

• calcitonin receptor → signals to reduce osteoclast activity and reduce bone resorption)

describe the process that occurs during osteoclast’s activity in bone resorption

1. edges of the osteoclast are sealed against the surface of bone through INTEGRIN and VITRONECTIN protein interactions (integrin expressed by osteoclast, vitronectin expressed on bone surface)

2. release of citric acid and acidification of bone matrix

3. proteolytic enzymes degrade bone matrix (e.g. type 1 collagen, osteocalcin, osteonectin)

4. results in bone salt (hydroxyapatite) dissolution, bone matrix protein degradation, collagen and protein fragments, free Ca and phosphate released from bone into blood

5. secretion of TRAP proteins allow osteoclasts to move along the bone surface and continue to break down bone

what happens to the Ca, phosphate and collagen and protein fragments from bone resorption?

calcium and phosphate go to blood

collagen and protein fragments degraded in bone microenvironment

what are the stimulators of bone resorption?

• inflammatory cytokines (TNFa, IL-6)

• calcitriol

• lipid mediators (e.g. PGE2)

• growth factors (e.g. epidermal growth factor - EGF)

• MAIN 2: PTH and RANK-L bound to RANK

what are the inhibitors of bone resorption?

• cytokines - IL-4, interferon-gamma

• calcitonin

what are stimulators of bone formation?

• insulin-like growth factor (IGFs)

• bone morphogenic proteins (BMP)

• estrogen (MAIN)

  • increases OPG → decreases osteoclast activity

  • increases osteoblast activity

  • increases osteoclast apoptosis

  • decreases osteoblast apoptosis

  • decreases NFkB activity (transcription factor that drives the expression of inflammatory cytokines - decreases activity and decreases the signal for osteoclast maturation) results in decreased TNFa production

are vertebral or hip fractures more commonly seen? why?

vertebral fractures because it contains more spongy bone (trabecular bone) which is broken down more often in bone resorption compared to cortical bone

osteoporotic fractures has similar prevalance to which other diseases?

vertebral ~ heart attack

hip ~ breast cancer

describe the net gain/loss in the following groups: pubertal girls, premenopausal women, postmenopausal women

pubertal girls: net gain of bone (more bone formation than breakdown)

premenopausal women: small net loss (not growing anymore, bone being replenished)

postmenopausal women: larger net loss (loss of estrogen which is a potent stimulator of bone formation)

why is there higher levels of bone formation in postmenopausal women versus premenopausal?

in postmenopausal women:

• start BMD scans using DEXA

• recommendations and use of Ca and vit D supplements is higher

• drug interventions are available that stimulate bone formation are commonly prescribed (e.g. Prolia injections) - RANK-L inhibitor

during a DEXA scan, what is the reference mean?

T score compared BMD to a reference mean for a 30 year old man/woman

what is the T score critical cut-off for osteoporosis?

-2.5

peak BMD occurs when?

sometime between 25 and 30

when is approximate age of menopause? what happens to BMD during this time and beyond?

~ 55-60

BMD deficiency occurs which may lead to/past the critical cutoff and lead to osteoporosis

what happens to BMD during early menopause?

they eventually reach the same point as other post-menopausal women around 55-60 but reach osteopenia earlier (more susceptible to fractures)

describe the influence of activity level on BMD over the life cycle

during growth, active children can be seen to have a greater BMD compared to their inactive counterparts

during the rest of the life span, active men/women have a greater BMD compared to their sedentary counterparts

what are the mechanisms connecting exercise to bone health?

exercise has been shown to decrease pre-osteoclast activation/maturation by:

1. increasing OPG secretion by the osteoblast (preventing osteoclast activation and maturation)

2. decreasing RANKL expression and secretion by the osteoblast (less RANKL finding RANK receptor = fewer active osteoclasts)

what are influences on peak bone mass?

• genetics (e.g. how much receptors/transporters are transcribed)

• hormones (e.g. PTH level)

• physical activity

• nutrition

flaxseed and fish oil are high in which PUFAs?

flaxseed → high in alpha-linoleic acid (ALA) - plant-derived n3 PUFA

fish oil → high in EPA and DHA (long chain n3 PUFA, primarily from marine sources)

how does the consumption of n3 PUFAs found in foods such as flaxseed and fish oil impact fracture threshold?

can delay the fracture threshold by 10+ years (reached later in life)

what is the ideal dietary ratio of n6 : n3 PUFA? what is the typical north american diet?

ideal - 4:1

average - 12:1

describe the mechanisms used by long chain n-6 PUFA (e.g. arachidonic acid) with regards to bone resorption

arachidonic acid is a precursor for inflammatory eicosanoid (PGE2) synthesis which disrupts the balance between osteoclast and osteoblast activity

• decreases OPG secretion and increases RANKL secretion from osteoblasts

• stimulates RANK expression on osteoclasts

• inhibits osteoclast apoptosis (continued activity of mature osteoclasts)

describe the mechanisms used by long chain n-3 PUFA (e.g. DHA and EPA) with regards to bone resorption

• increase Ca pump expression on the intestinal epithelial cell basolateral membrane (facilitate dietary Ca uptake into the blood)

• decrease NFkB activation and TNFa (as well as some IL-6) secretion

• decrease STAT3 activation and IL-6 secretion

• decrease RANKL secretion from osteoblasts and subsequent osteoclast maturation

• decrease M-CSF secretion and osteoclast differentiation from precursor cells

• increase growth factor secretion to stimulate osteoblast activity (e.g. IGF)

• precursor for non-inflammatory eicosanoids (e.g. PGE3) produced in favour over PGE2 → more dietary n3 PUFA instead of n6 PUFA is better for bone health (PGE3 is bone neutral)

  • COX2 enzyme has steric preference for metabolizing DHA and EPA to make PGE3 over PGE2

what is the role of plant polyphenols (e.g. resveratrol in red grapes, catechin in tea leaves and berries) in bone resorption?

• attenuate inflammatory signalling via suppression of NFkB activation, the TF that drives TNFa and IL-6 expression and secretion

• inhibit RANK-L secretion from osteoblasts

• inhibit osteoclastogenesis (stem cell to pre-osteoclast)

• inhibit activation of eicosanoid synthesizing enzymes such as COX-2, decreasing PGE2 synthesis and its effects

• stimulate osteoblast secretion of PG

• stimulate osteoblast differentiation (increasing expression of RUNX2 → stimulates pre-osteoblasts to differentiate into mature osteoblasts)

• upregulate growth factors that stimulate osteoblast activity (e.g. BMP)

what are the two major types of soy isoflavones typically studied?

genistein and diadzein

describe the mechanisms used by soy isoflavones with regards to bone resorption

• function as phytoestrogens, bind to estrogen receptor

• synergize with vit D (calcitriol) to increase estrogen receptor expression on osteoblasts

• stimulate expression of 1-a-hydroxylase in the kidney

• increase OPG and osteocalcin secretion and reduce RANK-L secretion from osteoblasts

• induce osteoclast apoptosis

• decrease inflammatory cytokine production (TNFa, IL-6)

• stimulate osteoblast maturation via upregulation of RUNX2

with regards to soy isoflavones, what was witnessed during human intervention trials?

• increased BMD in the lumbar spine and femur

• higher intake is associated with lower risk of fractures

describe the stats associated with osteoporosis risk in females with age

• 40% of postmenopausal women will suffer at least one osteoporotic fracture (age-related BMD loss)

• 10 times more common in women vs. men

how do obese post-menopausal women have a higher BMD than women with a healthy BMI?

they can still produce some estrogen because the enzyme aromatase is expressed in obese adipose tissue, allowing for some estrogen production and release into the blood

with regard to osteoporosis risk, what happens to individuals with very low BMI (underweight)?

increased risk for fragility-related fractures (no cushion, independent of site)

risk increases by 40% for every std dev decrease in fat mass

with regard to osteoporosis risk, what happens to individuals with high BMI (obese)?

site-specific (ankles and leg fractures) increased risk of fractures + inflammation driven increases in bone resorption (TNFa and IL-6)

why may obese individuals be more frature-prone?

increased fat mass imposes a greater mechanical stress on bones; in response, bone mass increases to accommodate the greater load but eventually increases in bone mass can’t match increases in fat mass

studies find ~20% increased risk of ankle and upper leg fractures

describe the fracture site risk in obese women

tend to see increased leg and ankle fractures (at risk)

fewer hip/spine and upper body fractures (some protection)

why might % body fat be a better assessment of obesity than BMI?

muscle weighs more than adipose tissue, so BMI may not be an accurate representation of obesity

what are the adverse effects of obesity on bone health?

• inflammatory cytokines (TNFa, IL-6) are produced in obese adipose tissue and can stimulate osteoclast activity → bone resorption

• 25-hydroxyvitamin D levels are commonly reduced in obese individuals

  • impacts on immune system, more inflammatory cytokine production

  • increased bone resorption due to RANK-L secretion from osteoblasts

  • stimulate the expression of transporters (Ca2+ to bloodstream) - calbindin

  • transcription factor - other effects

• serum PTH levels are commonly high in obesity - adverse effect on bone (constant bone breakdown signal, excess blood Ca2+ wasted at the kidney)

what are the beneficial effects of obesity on bone health?

• increased body mass increases the mechanical load on bones - stimulate bone formation (to a point)

• estrogen stimulates osteoclast apoptosis → estrogen synthesized in obese adipose tissue via increased aromatase expression (may lead to ectopic production of estrogen)

what is metabolic syndrome?

a cluster of medical disorders that when present together, increase the risk for cardiovascular disease and diabetes (many may not know that they have metabolic syndrome)

what is the diagnosis of metabolic syndrome based on?

the WHO diagnosis is based on the presence of ONE of:

• type 2 diabetes

• impaired glucose tolerance

• impaired fasting glucose

• insulin resistance

• (last 3 may be problems with glucose homeostasis, pre-diabetes)

AND 2 of:

• high blood pressure (>140/90 mmHg)

• dyslipidemia (high triglycerides)

• high waist circumference (measure of obesity)

• microalbuminuria (kidney function)

metabolic syndrome increases the risk of developing:

• atherosclerosis

• heart attack

• stroke

• type 2 diabetes

describe the trend of metabolic syndrome prevalence in Canada

risk increases over the life span (by over 20% from 18 to 79)

describe the relationship between obesity prevalence and T2D prevalence

1 in 3 people will be obese by 2034

1 in 10 people will develop T2D

how does the combined high intake of calcium and vit D impact the risk of developing T2D?

combined high intake if both decreases risk of T2D by 33%

(1 - 0.67 = 0.33 = 33% less risk)

describe how dairy product intake (primarily as milk) influences metabolic syndrome risk factors

over the trends of obesity, abnormal glucose homeostasis and hypertension, the % incidence dropped between low consumers and high consumers

*there was no significance in change in dyslipidemia

describe the changes in body parameters in very low calcium obese consumers before and after a weight loss intervention, with or without the supplementation of calcium and vit D

body weight, waist circumference and fat mass all decreased more relative to the placebo group

• no statistical significance in BMI changes between the two groups

calcium intake can be correlated with weight loss, risk of diabetes and risk of metabolic syndrome in only specific populations. which populations does it impact?

only overweight and obese and/or very low calcium consumers (<600 mg daily)

how could calcium combined with vit D work?

• increases fecal fat excretion (decreasing lipid absorption in the small intestine and not storing it in adipose tissue)

• favours or promotes fat oxidation in adipose tissue (using fat for energy)

• calcium specific effects on appetite