Calcium, Phosphate and Magnesium

Mineral Metabolism

Calcium, Phosphate and Magnesium
Lecture Outline • Calcium – Intake and excretion – Distribution – Function – Control – Abnormality – Assessment

Calcium, Phosphate and Magnesium
• Calcium
– Mostly in bones – Prolonged deficiency causes bone disease – Total body calcium depends on Ca2+ absorbed from diet and Ca2+ lost from body – 1,25-dihydroxyvitamin D (1,25dihydroxycholecalciferol) – the active metabolite of Vitamin D needed for calcium absorption

Calcium
~ 25 mmol (1g) ingested per day Net absorption = 6 – 12 mmol

Calcium
• Loss: in faeces and urine • Faecal calcium – Derived from diet and large amount intestinal secretions that has not been reabsorbed – Intestinal Ca2+ + phosphate or fatty acids  insoluble, poorly absorbed – Steatorrhoea: excess fatty acids in intestinal lumen → Ca2+ malabsorption

Calcium
• Loss: in faeces and urine • Urinary calcium – Amount of Ca2+ reaching glomeruli, glomerular filtration rate, renal tubular function  amount of urinary excretion – Parathyroid hormone and 1,25dihydroxy-vitamin D increase calcium reabsorption

Calcium Distribution
• 5th most common element in the body • 99%  crystalline hydroxyapatite in bones and teeth • 1%  extracellular fluid; contains ~25 mmol of calcium • Blood contains 7.5 mmol = virtually all in the plasma • ~125 mmol of calcium is in the exchangeable calcium pool, located mainly on the bone forming surfaces and in soft tissue where Ca2+ is readily available for physiological functions

Calcium: Function
• • • • Prime inorganic messenger for the regulation of cell function Activator of blood coagulation Activator of enzymes Control secretion of endocrine glands: – Parathyroid glands – Thyroid cells – Pancreatic beta cells Function at the plasma membrane: – Regulating membrane permeability – Regulating transmitter release – Regulating neuromuscular excitability Involved in cell to cell adhesion and possibly communication In striated muscles  activates the contraction of myosin fibril in combination with troponin Contribute to structure of bone and teeth

• • •

Plasma Calcium
• Normal plasma concentration = 2.5 mmol/L ( 10 mg/dL) • Calcium circulates in three physiochemical states a. Protein-bound (45%) b. Complexed with small, diffusible ligands: citrate, lactate, phosphate, carboxylate and bicarbonate (10%) c. Ionized states (ionized calcium) • Circulate freely in bloodstream • [ ] is affected by pH and plasma protein concentration • Acidosis favors dissociation • Alkalosis has the opposite effect

Plasma Calcium
• Ionized calcium is the physiologically important fraction • Form (b) and (c) can pass through porous membrane, leaving bound form (a) behind

• Which form can be found in urine?

Control of plasma calcium levels
• Control of plasma level is very important • Level must be maintained • Low levels  tetanic contraction of muscles, seizures and death • High levels  depression, coma, death • 3 hormones control maintenance of calcium homeostasis 1)Parathyroid hormone 2)Calcitonin 3)Vitamin D

Hormones controlling calcium
Hormones Target Actions  Plasma Ca  Plasma PO Properties
84 amino acids Polypeptide Stimulated by  Ca Inhibited by  Ca

Second Messengers cAMP

Parathyroid Kidney hormone (PTH) Bones

Calcitonin Vitamin D

Bone Osteoclasts GI tract Bone

 Plasma Ca  Plasma Ca  Plasma PO

32 aa plopeptide cAMP Nuclear receptor

Steroid-like

Control of plasma calcium levels

• Parathyroid hormone (PTH)
– Most important controlling factor – Secreted by parathyroid glands – Increases circulating free-ionised calcium

Control of plasma calcium levels
• Calcitonin
– Produced in C-cells of thyroid glands – Slows calcium release from bones by decreasing osteoclastic activity – Decreases circulating calcium

Control of plasma calcium levels

• Vitamin D
– Increases calcium absorption from the intestine – Necessary for PTH activity

Parathyroid hormone
• Biological actions
– Stimulate osteoclastic bone resorption → release free-ionised calcium and phosphate into extracellular fluid üPlasma [calcium and phosphate ] 

Parathyroid hormone
• Biological actions
– Decrease renal tubular reabsorption of phosphate → phosphaturia and increased reabsorption of calcium üPlasma [calcium ]  ; üPlasma [ phosphate ] 

Parathyroid hormone
• Control of PTH secretion depends on concentration of free-ionised calcium circulating through parathyroid glands • Calcium   PTH secretion  • Affected by extracellular [ magnesium ] • Decreased by severe, chronic hypomagnesaemia

Parathyroid hormone
• Control of PTH secretion depends on concentration of free-ionised calcium circulating through parathyroid glands • Calcium   PTH secretion  • Affected by extracellular [ magnesium ] • Decreased by severe, chronic hypomagnesaemia

Low blood calcium Normal Range

High blood calcium

Vitamin D
• Derived from: – Cholecalciferol (Vitamin D3) – formed in skin by action of UV light on 7dehydrocholestrol – Ergocalciferol (Vitamin D2) – Diet: important when requirements are high, e.g. during growth, pregnancy, individuals confined indoors (no exposure to sun) • Transport: bound to specific carrier proteins • Inactive until metabolised

Control of plasma calcium levels
• General rule: control of extracellular [calcium] rather than total body content • Effective control depends on: – Normal functioning of: üParathyroid glands üKidneys üIntestine – Adequate supply of üCalcium üVitamin D

Control of plasma calcium levels

• Impairment of control →→ loss of calcium from bone: plasma [calcium] maintained at the expense of bone calcification

Control of plasma calcium levels

Other factors
• Serum Protein Concentration
– Amount of protein in circulation affects calcium concentration by altering the bound to free calcium ratio – Low serum albumin,  unbound fraction – Parathyroid hormone detects this and increases the unbound calcium • By reducing PTH production, lowers the calcium concentration – This may result in reduction of total calcium levels but keeps the unbound level normal

Other factors
• Other Hormones – Thyroxine • Stimulates bone turnover – Oestrogens and androgens • Affect production of bone matrix • With menopause,  oestrogens,  matrix production – Glucocorticoids • Influence bone metabolism by their action on protein catabolism affecting production of bone matrix

Abnormal plasma calcium

• Hypercalcaemia
– Raised protein-bound Ca; normal freeionised Ca – Raised free-ionised Ca  high  PTH – Raised free-ionised Ca  due to other causes + low [ PTH ]

Abnormal plasma calcium

• Hypocalcaemia
– Reduced protein-bound Ca; normal free-ionised Ca – Reduced free-ionised Ca  PTH deficiency – Reduced free-ionised Ca  due to other causes + high [ PTH ]

Assays for calcium
• Total Calcium
– Colorimetric methods – Atomic absorption spectroscopy • Reference method for total calcium assay • Very sensitive and specific
• http://www.chemsoc.org/pdf/LearnNet/rsc/AA_t xt.pdf

Atomic absorbance spectrometry (AAS)

•Analytical technique that measures the concentrations of elements •So sensitive, it can measure down to parts per billion of a gram (μg/dm3) •Makes use of the wavelengths of light specifically absorbed by an element •They correspond to the energies needed to promote electrons from one energy level to another, higher, energy level.

Assays for calcium
• Total Calcium
– Photometric: Simple spectrophotometry of the colored reaction products formed by various indicators/dyes • Indicators/dyes change colour when calcium bound • easy for automation on chemistry analyzers • Allow direct measurement of calcium in serum and other biological fluid • 2 dyes commonly used: Ocresolphthalein complexone and arsenazo III •

Assays for calcium
• Total Calcium
– Photometric: O-cresolphthalein complexone (CPC) – Form red chromophore with calcium in alkaline solution – Measured at 570 – 580 nm – Release bound / complexed calcium by diluting with acid and buffer with organic base – Interference by magnesium reduced by: • Adding 8-hydroxyquinoline • Buffering near pH 12 • Measuring near 580 nm

Assays for calcium
• Total Calcium
– Photometric: arsenazo III – High and specific affinity for calcium – At pH 6 has higher affinity for calcium than magnesium – Must be buffered since spectral properties very dependent on pH – Use is increasing

Assays for calcium
• Total Calcium
– Titrimetric method • Complex calcium with calcein • Form flourescent complex that is maximally stimulated at 490 nm and emits at 520 nm • Titrate with EDTA (ethylenediamine tetraacetic acid) or EGTA (ethyleneglycol tetraacetic acid) • EDTA / EGTA binds calcium with higher affinity and therefore replaces calcein • End point of titration: when flourescence return to baseline • Amount of EDTA / EGTA is directly proportional to amount of calcium • Can be automated

Assays for calcium
• Total Calcium
– Serum: preferred specimen – Certain anti-coagulants can interfere with analysis – Total calcium stable in serum for: • Days at 4°C • Months if frozen – Plastic ware / glass ware adsorb calcium upon long storage • wash well if re-using ware –

Assays for calcium
• Ionised Calcium
– i.e. ‘free’ calcium available to cells; Ca2+ bound to proteins are not accessible to cells – Ion selective electrode: instrument for quantitating ionised calcium in serum – Ultrafiltration + colorimetric methods: calcium not bound to proteins is filtered and separated

Assays for hormones
• Parathyroid Hormone
– Radioimmunoassay

• Calcitonin
– Radioimmunoassay – Serum samples yield higher calcitonin levels than plasma samples – Haemolysis: false elevated values

Assays for hormones
• Vitamin D
– Complex process: low concentration of vitamin D and presence of a variety of materials with very similar structures – Assays involve extraction of vitamin from its transport protein → purification and separation of vitamin D components → immunoassay procedure

Diagnosis of disorders of calcium metabolism
• Plasma parathyroid hormone assay • Urinary calcium and phosphate estimation • Plasma Vitamin D assay

Abnormal plasma calcium
• There are bone disorders that do not affect plasma [ Ca ] – Osteoporosis – Paget’s disease of bone – Osteomalacia caused by renal tubular disorders of phosphate reabsorption – Rickets

http://en.wikipedia.org/wiki/Beer-Lambert_law

A = absorbance  = absorption coefficient l = distance c = concentration

Diagram of Beer-Lambert absorption of a beam of light as it travels through a cuvette of size l.

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