milan - bone calcium

Bone Homeostasis Calcium, Phosphate & Magnesium

Anna Milan

Principal Clinical Scientist

Department of Clinical Biochemistry & Metabolic Medicine

Royal Liverpool & Broadgreen University Hospital NHS Trust

Liverpool

Outline

Regulation pathway

Bone structure and function

Basic bone physiology

Common disorders of bone

metabolism

Calcium and Phosphate metabolism

Magnesium Metabolism

Introduction

Bone growth and turnover is influenced by Calcium, phosphate, and magnesium

metabolism

PTH and 1,25(OH)2D

Other hormones and factors such as thyroid hormones, oestrogens, androgens, cortisol, insulin, GH, IGFs, TGF, FGF, PDGF

Significant research has increased understanding of bone and mineral metabolism and the pathogenesis of associated disorders

Improvement in lab methods/technology have enabled these hormones/factors to be measured

Ageing population will drive this further with the need for bone markers of resorption and formation, expressed at differing disease stages

Introduction (2)

Homeostatic systems work to ensure that the extracellular

[Ca2+] is tightly controlled

Achieved through interaction between calciotropic

hormones and their effector tissues in the kidney, intestine

and bone

Key to this is the calcium-PTH axis

Vitamin D and vitamin D receptors expressed within nucleus

of parathyroid cells, play important role in calcium

homeostasis

Calcium regulating hormones

Calcium in the ECF

is tightly controlled

Largely regulated by

two hormones

PTH

1,25(OH)2D

(calcitrol)

Also regulate

phosphate

concentration

Calcitonin proposed

minor role in Ca

homeostasis

Parathyroid Hormone

Secreted by parathyroid glands

Chief and oxyphil cells

PTH synthesised, stored and secreted by chief cells

Concentration PTH in plasma determined by its synthesis and secretion by parathyroid glands

Metabolism and clearance determined by liver and kidneys

PTH acts directly on bone and kidney

Indirectly on intestine to regulate [Ca] and [PO4]

Parathyroid Hormone

PTH exerts its influence by interacting with PTH/PTHrP

receptors on plasma membrane of target cells

This initiates a cascade of intracellular events

Generation of cAMP

Activation of kinases

Phosphorylation of proteins

Increased entry of calcium and intracellular calcium

Stimulated phospholipase C activity

• Generation of DAG and PI activate enzyme transport

systems

Secretion of lysosomal enzymes

Vitamin D ??

Several forms of Vitamin D occur (vitamers) D1 – D5

Two major forms parent molecules, known collectively as calciferol Vitamin D2 – Ergocalciferol

Vitamin D3 – Cholecalciferol

25(OH) Vitamin D Calcidiol, Calcifediol, 25-hydroxycholecalciferol, 25-

hydroxyvitamin D

1,25(OH)2D 1,25-dihydroxycholecalciferol, 1,25-dihydroxyvitamin D,

Calcitriol

Alphacalcidol 1-hydroxycholecalciferol

Vitamin D analogue with less of an effect on calcium than calcitriol

Calcichew D3 Forte Vitamin D3 with calcium

http://en.wikipedia.org/wiki/File:Protein_GC_PDB_1j78.png

Vitamin D ? Non-hydroxylated parent compounds,

short t1/2= 24hrs, conc transient based on recent sun exposure and diet

Very liphophilic and difficult to measure

25(OH) Vitamin D, D2 and D3 Effectively a pre-cursor of active form

of Vitamin D

t1/2= 3 weeks

Direct indicator of available Vitamin D

1,25(OH) Vitamin D Active form, very short t1/2= 4hrs

Limited clinical utility

Vitamin D endocrine system

Senses low serum Ca

and PTH secretion

1,25(OH) D formation

Ca excretion absorption of dietary Ca

Releases

Ca & PO4

In the kidneys

PTH

Induces 25-OH Vit D-1-hydroxylase which increases production 1,25(OH)2D which stimulates intestinal absorption of calcium and phosphate

Increases calcium reabsorption in the DCT

Decreases reabsorption of phosphate by PT

Inhibits Na+-H+ antiporter activity which favours a mild hyperchloremic metabolic acidosis in hyperparathyroid states

In Bone

Effects of PTH are complex as can stimulate bone resorption or bone formation depending on [PTH] and duration of exposure

Chronic exposure to high [PTH] leads to increased bone resorption

PTH acts directly by altering the activity or number of osteoblasts and indirectly on osteoclasts

Bone resorption, a quick response is important for maintenance of calcium homeostasis

Delayed effects are important for extreme systemic needs and skeletal homeostasis

Renal Failure

Fall in calcium conversion 25(OH)D to 1,25(OH)D

Increase in phosphate Kidneys not excreting excess

FGF23 role

Increase in PTH Stimulated by low Ca

Continual stimulation of parathyroid glands leads to 2° hyperparathyroidism

Patients with end stage renal failure become hypercalcaemiac Probably due to development of autonomous PTH secretion from prolonged

hypocalcaemic stimulus

Such hypercalcaemia may manifest for the first time in a renal transplant patient who becomes able to metabolise vitamin D normally 3° hyperparathyroidism

PTH-Calcium

Integration of direct and indirect effects of PTH lead to alterations in calcium and phosphate in serum and urine

PTH mobilisation of calcium is biphasic A rapid phase involving existing cells

Long term response dependent on proliferation of osteoclasts

In serum total and free calcium are increased, phosphate decreased

In urine, inorganic phosphate and cAMP are increased

Urinary calcium is usually increased Larger filtered load of calcium from bone

resorption and intestinal reabsorption overrides increased tubular reabsorption of calcium

In absence of disease the increase in serum calcium reduces PTH secretion through negative feedback loop maintaining homeostasis.

PTH-PO4/Mg

Despite PTH being important in control of phosphate secretion

Changes in phosphate do not directly affect secretion of PTH

Mild hypomagnasaemia stimulates PTH secretion

More severe hypomagnasaemia reduces PTH secretion as it is a Mg dependent process

Functions of Bone

Support Framework of body supporting softer connective tissues and muscles

Protection Mechanical protection for internal organs

Assisting in movement Muscles attached to bones so when they contract bones will move

Mineral storage Calcium and phosphate reservoirs

Production of blood cells Bone marrow inside some long bones

Storage of energy With age, bone marrow changes from ‘red’ to ‘yellow’ and is

predominantly adipose cells providing a chemical energy reserve

Types of Bone

Long bones Greater length than width, shaft (diaphysis) with variable number of endings, curved for strength

Predominantly compact bone with lesser amounts of marrow and spongy bone • e.g. femur, tibia, ulna and radius

Short bones Roughly cube shaped with approximately equal length and width

Thin layer of compact bone surrounding spongy interior • e.g. ankle and wrist bones

Flat bones Thin structure providing mechanical protection and extensive surface area for muscle attachment

Two parallel layers of compact bone surrounding spongy interior • e.g. cranial bones, sternum, shoulder blades

Irregular bones Complicated shapes due to function they fulfil within body

Thin layers of compact bone surrounding spongy interior • e.g. vertebrae and some facial bones

Sesamoid bones Develop in some tendons where there is considerable friction, tension and physical stresses;

quantity varies considerably person to person • e.g. common to all are patellae (kneecaps)

Structure of Bone

Long bones grow from the ends and under normal circumstances stop growing in late teens or early 20’s

Two main types of (lamellar) bone tissue

Compact • Forms outer shell of bones consisting of very hard

bones arranged in concentric layers (Haversian systems)

• Accounts for 80% of total bone mass of adult

Cancellous (trabecular, spongy bone) • Located beneath the compact bone

• Consists of a meshwork of bony trabeculae with many interconnecting spaces containing bone marrow

• Accounts for remaining 20% of total bone mass but nearly 10x surface area of compact bone

Bone Cells

Osteoblasts Produce matrix which mineralises to form ‘osteoid’

Become quiescent and flatten to become lining cells

Respond to hormonal control to activate osteoclasts

Osteocyctes Cells inside the bone which sense mechanical stress to initiate remodelling

Transports mineral into and out of bone

Osteoclasts Dissolve bone by solubilising mineral - resorption

Effects change in bone structure

Bone Remodelling

Process of resorption followed by replacement

Lifelong process

In 1st year of life almost 100% of bone is replaced

In adults approx 10% per year

Little change in shape and occurs throughout life Regulates calcium homeostasis

Repairs micro-damaged bones (everyday stress)

Shapes and sculptures skeleton during growth

Imbalance leads to metabolic bone disorders

Osteoblasts produce RANKL which activates RANK on osteoclast precursor cells Stimulates cell to differentiate

in mature osteoclast

Activated RANK induces expression of c-Fos which binds to DNA and activates genes required for osteoclast function

c-FOS also activates Interferon- which prevents further osteoclast differentiation

Osteoprotegerin is soluble protein released from osteoblasts that binds to RANKL preventing RANK activation

Molecular structure

Matrix

40% organic • Type 1 collagen (tensile strength)

• Proteoglycans (compressive strength)

• Osteocalcin / osteonectin

• Growth factors / cytokines

Inorganic

60% inorganic - hydroxyapatite

Organic

Osteoblasts / osteocytes / osteoclasts

Formation (ossification) of Bone

Begins 3rd month foetal life and completed late adolescence

Two processes occur

Intramembranous ossification

Occurs during flat bone formation

Formed from mineralisation of connective tissue rather than cartilage

Endochondral ossification

Occurs in long bones

Involves initial hyaline cartilage model which continues to grow (growth plate) and mineralise at the metaphysis

Once skeletal maturity is reached, bones stop growing in length and the plate is replaced with an epiphyseal line

Defects in the continued division of these plates can lead to growth disorders e.g. achondroplasia where there is a defect in cartilage formation leading to dwarfism

Disorders of Bone

206 bones which can be affected by various diseases and disorders!!! Osteomalacia – inadequate mineralisation of bone

• Rickets in children

• Insufficient Ca absorption due to lack Ca or Vit D def

• Phosphate deficiency caused by increased renal loss

Osteoporosis – Reduced bone mineral density

Pagets disease – excessive resorption and formation leading to weak and misshapen bones

Renal osteodystrophy – kidneys fail to maintain Ca and PO4

Rheumatoid osteoarthritis – systemic inflammatory disease

Malignancy

Many others….

Calcium

Functions of Calcium

Functions of calcium

Bone growth and remodeling

Secretion (exocytosis)

Excitation-contraction coupling

Stabilization of membrane potentials

Enzyme co-factor (e.g. in blood coagulation)

Second messenger – intracellular signalling

Different forms of Calcium

Majority of calcium is in the skeleton (reservoir) Serum Calcium 2.20 – 2.60

mmol/L

Ionised calcium 1.1-1.3 mmol/L • 45% exists in ionised form

(physiologically active form)

• 45% bound to proteins (predominantly albumin)

• 10% complexed with anions (citrate, sulphate, phosphate)

Report adjusted calcium and calcium Ionised calcium difficult to

measure – ABG machine, calcium electrode, not readily available, dependent on pH

Adj Ca accounts for changes in albumin Useful when a decrease in albumin may

mask hypercalcaemia

Conversely not useful in very low albumin states <20g/L

Interpret with caution in extremes of pH • Acidosis decreases binding

• Alkalosis increases binding

Standard ACa formula ACa = Total Ca + 0.02 x (40-[albumin])

More appropriate to develop in-house adjustment formula

Remember it is the unbound calcium which the body regulates and in low protein states ACa may be inaccurate

Biochemical Homeostasis

Blood Input Output

Internal

Reservoir

(Not directly

measurable)

Lungs

GI Tract

Skin

Lungs

GI Tract

Skin

Kidneys

Calcium Homeostasis

Blood Input Output

Bone

GI Absorption

of Ca

Urinary

excretion of Ca Mineralisation Resorption

Hypercalcaemia

Increased GI absorption

Increased bone resorption

Decreased bone mineralisation

Decreased urinary excretion

Hypocalcaemia

Decreased GI absorption

Decreased bone resorption

Increased bone mineralisation

Increased urinary excretion

Aetiologies of Hypercalcaemia

Increased GI Absorption

Elevated Vitamin D Excess exogenous (therapeutic)

Excess endogenous (e.g. sarcoidosis)

Elevated PTH

Hypophosphataemia

Milk-alkali syndrome


Increased net bone resorption Elevated PTH

Malignancy

Increased bone turnover Paget’s disease

Hyperthyroidism


Elevated PTH

Aluminium toxicity


Thiazide diuretics

Elevated Vitamin D

Elevated PTH

Common Causes

Primary hyperparathyroidism (99% ambulant patients) Single adenoma (80%)

Hyperplasia (15%)

Double adenoma (2%)

Carcinoma (<1%)

Malignant disease (99% of ill patients) Metastases and myeloma

PTHrp secreting

Lymphoma

PTH secreting (v. rare)

Uncommon Causes

Vitamin D excess

Tertiary hyperparathyroidism

Hyperthyroidism

Rare Causes

Familial hypocaliuric hypercalcaemia

PTHrP

Discovered in 1987 when studying the mechanism by which certain cancers produce humoural hypercalcaemia of malignancy

The N-terminal shows homology with PTH with 8 of first 13 aa matching

Remainder of molecule shows little homology

The common N-terminal explains how PTHrP can interact with PTH/PTHrP receptors, mimicking biological actions of PTH in target tissues such as bone and kidney

Like PTH, PTHrP causes hypercalcaemia and hypophosphataemia and increases urinary cAMP.

Signs & Symptoms Hypercalcaemia

Aetiologies of Hypocalcaemia


Poor dietary intake

Impaired absorption of Ca Vitamin D deficiency

• Poor dietary intake of Vit D

• Malabsorption

Decreased conversion of Vitamin D • Liver failure

• Renal failure

• Low PTH

• Hyperphosphataemia

Decreased bone resorption /


Hypoparathyroidism

PTH resistance (pseudohypoparathyroidism)

Vitamin D deficiency

Hungry bone syndrome

Osteoblastic metastases


Low PTH

Thyroidectomy

I131 treatment

Autoimmune hypoparathyroidism

PTH resistance


Causes

Parathyroid Causes

Parathyroid agenesis Isolated

Part of complex developmental anomaly eg DiGeorge Syndrome

Parathyroid destruction Surgery

Radiation

Infiltration: eg haemochromatosis, Wilson’s

Autoimmune Isolated

Polyglandular

Reduced parathyroid function PTH gene defects

Hypomagnesaemia

Neonatal hypocalcaemia

Hungry bone disease

Non-parathyroid Causes


Vitamin D resistance

Altered vitamin D metabolism eg phenytoin, ketoconazole

PTH resistance Pseudohypoparathyroidism

Magnesium deficiency

Bisphosphonates

Acute pancreatitis

Acute rhabdomyolysis

Most Common Causes

Acute or chronic renal failure

Hypoparathyroidism

Hypomagnesaemia


Always exclude

EDTA contamination

Multiple transfusions with citrated blood

products

Signs & Symptoms of Hypocalcaemia

Neuromuscular irritability Tetany

Carpopedal spasm

Muscles cramps

Seizures – all types

Prolonged QT interval on ECG

Bronchospasm

Laryngospasm

Longterm hypocalcaemia ectopic calcification eg in basal ganglia causing

extrapyramidal neurological symptoms

Cataract, papilloedema

Abnormal dentition

http://drweather.files.wordpress.com/2009/04/spasm-carpopedal.jpg

Phosphate

Functions of Phosphate

Functions of phosphate

Formation of: High energy compounds e.g. ATP, creatinine

phosphate

Second messengers e.g. cAMP, inositol phosphates

Component of: DNA/RNA

Phospholipid membranes

Bone

Phosphorylation (activation/inactivation) of enzymes

Intracellular anion

Distribution of Phosphorous

85% is within the skeleton and teeth

14% is located within the cells

Only 1% is present in the extracellular fluids

Present as organic (phosphoproteins, phospholipids) and inorganic (phosphate)

Inorganic phosphate component is what we measure

RR 0.70-1.40 mmol/L

• Mild deficiency 0.35 – 0.70 mmol/L

• Severe deficiency <0.35 mmol/L

Phosphate Flux (mmol/24hr)

Phosphate

Pool

Kidneys

Sweat

1.0

Bone

Intestine

52

Food

45

Faeces

19

Intestinal

absorption

33

Digestive Juice

7

Formation

7 Resorption

7

Filtered

160

Urine

25

Reabsorbed

135

PTH Action 1,25(OH)

Vit D

Action

Phosphate Homeostasis

Blood Input Output

Bone

GI Absorption

of PO4

Urinary

excretion of

PO4 Mineralisation Resorption

Hyperphosphatemia

Increased GI absorption




Hypophosphataemia


Decreased bone resorption



Intracellular

Redistribution

Refeeding, recovery

from DKA, Alkalosis etc

Delayed separation,

Rhabdomyolysis, Renal Failure etc

Causes of Hyperphosphataemia

Pseudohyperphosphataemia

Haemolysed specimen

Myeloma

Delayed separation / Old sample

Increased Phosphate Input

IV PO4

Rectal PO4

Cell death Tumour lysis syndrome

Rhadbomyolysis

Malignant hyperpyrexia

Heat stroke

Reduced phosphate excretion

Reduced eGFR Acute renal failure

Chronic renal failure

Increased renal tubule reabsorption Physiological

• Recovery from Vit D def

• Lactation

Pathological • Reduced PTH or PTH resistance

• Vitamin D toxicity

• Thyrotoxicosis

• Acromegaly

Causes of Hypophosphataemia

Inadequate phosphate absorption

Low dietary intake v rare

Phosphate binders (dialysis patients)

Phosphate binding antacids (rare due to new therapies for peptic ulcers)

Abnormal urinary phosphate loss

Primary and secondary hyperparathyroidism

Osmotic diuresis e.g. hyperosmolar hyperglycameic state

Diuretics

Fanconi syndrome

Genetic conditions e.g.X-linked hypophosphataemia

Shifts of phosphate from extracellular fluid into cells

<1% in extracellular space

Recovery from DKA Treatment with insulin causes phosphate to

move back into cells

Refeeding syndrome Starving or chronically malnourished are

refed or given IV glucose

Carbohydrates stimulate insulin which drives phosphate and glucose intracellularly

Cells swithc to anabolic state resulting infurther depletion

Respiratory alkalosis Activating phsophofructokinase which

stimulates intracellular glycolysis

Increased muscle intake

Hepatic encephalopathy

Salicylate toxicity

Acute leukaemia Rapid growing malignancies may consume

phosphate preferentially

FGF23

Most important regulatory of serum phosphate and 1,25 (OH) Vitamin D

Secreted by osteocytes and osteoblasts in response to oral phosphate loading or increased 1,25(OH)D

In CKD, FGF23 sensitive biomarker of abnormal renal phosphate handling increasing during early stages

Raised FGF23 increases fractional phosphate excretion, reducing phosphate levels and 1,25(OH)D formation, thereby increasing PTH

Responsiveness to FGF23 declines as number of intact nephrons reduces FGF23 therefore cannot reduce PO4 as effectively and exerts other off-

target effects including premature mortaility

Lowering PO4 through binding agents reduces FGF23 and may improve patient outcomes

Magnesium

Magnesium Flux (mmol/24hr)

Magnesium

Pool

Kidneys

Sweat

0.2

Bone

Intestine

13.5

Food

12

Faeces

7.3

Intestinal

absorption

6.2

Digestive Juice

1.5

Formation

0.1 Resorption

0.1

Filtered

100

Urine

4.5

Reabsorbed

96

Functions of Magnesium

Cofactor for 300+ enzymes

Mg-ATP complex is substrate for many ATP requiring enzymes

Critical role for DNA replication, transcription and translation

Maintenance of structure of ribosomes, nucleic acids and some proteins

Interacts with calcium

Affects permeability of excitable membranes and their electrical properties

ECF depletion of Mg causes hyperexcitability

Magnesium

Hypomagnesaemia symptoms include Loss of appetite

Nausea and vomitting

Fatigue

Weakness & numbness

Tingling

Muscle cramps

Siezures

Personality changes

Hypokalaemia

Hypocalcaemia

Hypermagnesaemia Sympoms usually not apparent

unless > 2mmol/L

Concomitant HypoCa, HyperK or uraemia exaggerate symptoms of hyperMg

Non-specific symptoms include nausea, vomiting and flushing

Neuromuscular symptoms Blockage of neuromuscular

transmission

Conduction system symptoms Mild decrease in blood pressue

Higher concentrations lead to symptomatic hypotension

Heart block >7mmol/L

Hypocalcaemia

Predominantly intracellular cation

Serum Mg inaccurate way to

assess total body Mg stores and

can be misleading

Causes of

hypomagnesaemia

Decreased intake +/- absorption Starvation (protein calorie

malnutrition)

Malabsorption syndrome

Prolonged gastric suction

Inadequate parenteral nutrition

Loss from body Extra renal

Diarrhoea

Laxative abuse

Gut fistula

Excessive lactation (rare)

Misc Acute pancreatitis

Multiple transfusions

Insulin therapy

Hungry bone syndrome

Renal Alcoholism

Interstitial nephropathy

Diuresis e.g. DKA, post ATN

Drugs e.g. loop diuretics, cis-platinum (65-75% reabsorbed in Loop of Henle)

Hypercalcaemia

RTA

Bartter’s syndrome, Gitelman’s

Endocrine e.g. hypoparathyroidism, primary hyperaldosteronism, hyperthyroidism

K depletion

PO4 depletion

Post renal Tx

Primary renal Mg wasting

Causes of hypermagnesaemia

Significant hypermagnesaemia is uncommon as readily excreted in urine

Cardiac conduction is affected at concentration >2.5-5.0 mmol/L

Very high concentrations >7.5 mmol/L cause respiratory paralysis and cardiac arrest

Generally either Impaired renal function

Large Mg load • IV Contamination

• Post cardiac surgery or in pre-eclampsia where it is used to decrease neuromuscular excitability

• Enema / laxitive abuse

Rare causes include Excessive tissue breakdown

Lithium therapy ( renal excretion)

Hypothyroidism

Addisons disease

Familial hypocalciuric hypercalcaemia

Summary Points

Ca and PO4 homeostasis is controlled by Vitamin D and PTH

Commonest causes of HyperCa are primary hyperparathyroidism, malignancy and medications

Comment causes of HypoCa are Vitamin D def, hypoPTH, malabsorption, hypoMg

HyperPO4 – associated with renal failure

HypoPO4 – redistribution

Mg measurement important in inadequate PTH response to low Ca

HypoCa and / or hypoK may not require supplementation or may not response to replacement if the Mg is low

Measure Mg in patients on TPN, with chronic diarrhoea and alcoholics

milan - bone calcium

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