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Milan - Bone Calcium
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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
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
Bone
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 bone resorption
Increased net bone resorption Elevated PTH
Malignancy
Increased bone turnover Paget’s disease
Hyperthyroidism
Decreased bone mineralisation
Elevated PTH
Aluminium toxicity
Decreased urinary excretion
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
Decreased GI absorption
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 /
Increased bone mineralisation
Hypoparathyroidism
PTH resistance (pseudohypoparathyroidism)
Vitamin D deficiency
Hungry bone syndrome
Osteoblastic metastases
Increased urinary excretion
Low PTH
Thyroidectomy
I131 treatment
Autoimmune hypoparathyroidism
PTH resistance
Vitamin D deficiency
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 deficiency
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
Vitamin D deficiency
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
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
Increased bone resorption
Decreased bone mineralisation
Decreased urinary excretion
Hypophosphataemia
Decreased GI absorption
Decreased bone resorption
Increased bone mineralisation
Increased urinary excretion
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