This document discusses calcium, phosphorus, parathyroid hormone (PTH), and vitamin D. It provides information on: 1. The roles, regulation, and measurement of calcium, phosphorus, PTH, and vitamin D levels in the body. 2. How PTH and vitamin D work to regulate calcium and phosphorus absorption in the intestines and their reabsorption in the kidneys. 3. The clinical significance and reference ranges of measuring levels of calcium, phosphorus, PTH, and vitamin D through various assays.

1. CALCIUM,PHOSPHOROUS,PTH ,VITAMIN D
DR.M.MENAKA DEVI
M.D BIOCHEMISTRY-III YEAR PG
Stanley medical college , chennai
12.06.2019

2. CALCIUM
 Most abundant mineral in the body
 Bone-99%,soft tissue-1% ,ECF -<0.2%
 50%-free , 10% -complexed with small anions
(diffusible)40% -bound to plasma proteins (non
diffusible)
 Alteration in three physiological form
alteration in the concentration of protein
and small anions
change in pH

3. FORMS AND FUNCTIONS
 Free calcium –biologically active form
 Concentration is tightly regulated by PTH and
1,25 –dihydroxy vitamin D and calcitonin
 Intracellular calcium –muscle contraction ,
hormone secretion (insulin ,PTH ,calcitonin ,
vasopressin),glycogen metabolism ,cell
division , release of zymogens from pancreas
and histamine from mast cells
 Extracellular calcium –intracellular calcium
level ,bone mineralization ,blood coagulation
,stabilizes plasma membrane and influences
permeability and excitability

4. CALCIUM HOMEOSTASIS
 Principal organs –skin ,liver ,small intestine
,skeleton ,parathyroid gland and kidney
 Three hormones –PTH ,1,25(OH) 2 CC
,calcitonin
 Is taken through dietary sources as calcium
phosphate , carbonate ,tartrate and oxalate
 Absorption is from the first and second part of
duodenum

5.  Skeleton act as a mineral repository , releasing
calcium into blood on demand
 Excretion is through feces , urine and sweat
 Plasma Ca2+ is filtered in the kidneys
 98-99% of the filtered Ca2+ is reabsorbed
(proximal-60%) ,loop of henle and distal -40%

6. FACTORS INFLUENCING ITS ABSORPTION
 Vitamin D induce synthesis of the carrier
protein (calbindin) in the intestinal epithelial cells
–facilitate its absorption
 PTH increase calcium transport by enhancing
1α hydroxylase activity
 Acidity favors calcium absorption
 Amino acid lysine and arginine increase
calcium absorption

7.  Phytic acid : hexaphosphate of inositol in cereals
(fermentation and cooking reduces its content)
 Oxalate : present in leafy vegetable ((formation
of insoluble calcium oxalate)
 Malabsorption syndromes :FA is not absorbed ,
forms insoluble calcium complex of FA
 Phosphate :high phosphate content cause
precipitation as calcium phosphate

8. PTH AND VIT D ROLE
VIT D :1.Increases intestinal absorption of calcium
2.Reduces excretion of calcium (reabsorption in
distal renal tubules )
3.Mineralization of bone (coordinate the remodeling
of bone and increases bone mineral density)
PTH :1.increase absorption of calcium from intestine through
vit D
2.increase calcium reabsorption from distal convoluted
tubules of kidney
3.resorption of bone

9. TOTAL CALCIUM
 Serum or heparinized plasma
 Methods
Total calcium
 Photometric method
Arsenazo III method
 Atomic absorption spectrometry
 Adjusted calcium for albumin
=total calcium(mg/dL)+0.8[4-albumin(g/dL)]

10. IONIZED CALCIUM
Whole blood ,heparinized plasma or serum
Sample must be collected and handled anerobically
to minimize alteration in pH
Method :Ion selective electrode
Total calcium -8.5 – 10.1 mg/dL
Free calcium -4.6-5.3 mg/dL

11. URINARY CALCIUM
 The rate of urinary calcium excretion reflects
calcium intake , intestinal absorption , skeletal
resorption and renal tubular filtration and
reabsorption
 Healthy men and women -300mg / day on diet
with unrestricted calcium content ,200mg /day on
diet with restricted calcium content
 Uca(mg/100mL GF)
 =Uca(mg/dL) X serum creatinine
Urinary creatinine
Reference range is <0.16 mg/100ml for
spot fasting or timed collection after an
overnight fast

12.  Under fasting conditions ,the intestinal and
renal components are relatively fixed ,calcium
excretion in the fasting state is used to assess
skeletal component
 Value >0.16 mg/100 mL –osteoclastic
resorption
 Used in assessing renal stone disease and high
turnover osteoporosis
• Patient with hypercalciuria and normal sodium
excretion –renal leak hypercalciuria( result in
increased secretion of PTH)

13. PHOSPHOROUS
 Skeleton -85%, soft tissue -15%,extracellular-
<0.1%
 Free (ionized ) -55% , protein bound -10% ,
complexed -35%
 Inorganic phosphate –component of
hydroxyapatite in bone
 Exist in plasma as monovalent (H2PO4-) and
divalent form( HPO42-)
 Blood-organic phosphate esters are within cell-
nucleic acids , phospholipids ,phosphoproteins
, 2,3 BPG and “high energy compounds , and
and cyclic neucleotides (AMP) ,(NADP)

14.  Phosphate absorption from jejunum and ileum
 Phosphate level in blood is regulated by excretion
through kidney
 Phosphate excretion depends on muscle mass ,
renal function and age

15. FUNCTIONS OF PHOSPHATES
 Formation of bone and teeth
 Important constituent of cell
 Forms energy rich compounds
 Forms co-enzymes ,activation of enzymes
 Regulates blood and urine pH
 Forms organic molecules like DNA ,RNA

16. REGULATION OF PHOSPHATES
PTH - 1. increase absorption of phosphate from
intestine
2.decreases reabsorption of phosphate by the
proximal tubule
3.release from bone
Vit D –increase absorption from intestine
reabsorption from kidney

17. PHOSPHOROUS LEVEL TO BE CHECKED
 Renal tubular disease
 Hyperparathyroidism
 Hypoparathyroidism
 Bone disease
 Muscle weakness
 Renal failure

19. PHOSPHATE MEASUREMENT
 Serum or lithium heparin plasma
METHOD –ammonium phosphomolybdate method
REFERENCE RANGE
 serum-2.6 – 4.6 mg /dL
 Urine phosphate -0.4 to 1.3 g / day

20. VITAMIN D
 Steroid hormone ,synthesized in the skin
following exposure to UVB rays from the sun
7-dehydrocholesterol
liver
25hydroxy cholecalciferol (calcidiol) –
circulatory form
kidney 24,25(OH)2vit D
1,25dihydroxy cholecalciferol

21. TRANSPORT
 25(OH) Vitamin D,24,25- - dihydroxy vit D
and 1,25 dihydroxy vit D are bound to vit D
binding protein (DBP) –a specific high affinity
transport protein –group specific component
of serum or Gc – globulin
 <5% of vit D binding sites are occupied
 0.03% of 25(OH)D and 0.4% of 1,25(OH)2
are free in plasma

23. ABSORPTION OF CALCIUM FROM DIET
 1.Calcium entry into brush border cytoplasm ,
mediated by an epithelial Ca2+ tansporter or
channel(CaT1)-90% vit D dependent
 Diffusion of calcium within the cell by calbindin
–D9k (cytosolic calcium binding protein) -100%
vit D dependent
 3.exit of Ca from the cell across its basolateral
membrane by the action of CaATPase (eg a
Na+/Ca2+ exchanger)

24. ABSORPTION OF CALCIUM FROM
KIDNEY
increase secretion of klotho protein from
kidney
increase epithelial expression of epithelial
calcium channel TRPV5
stimulates reabsorption of calcium in distal
tubule

25. DIFFERENCE
 25 (OH)vit D
 Main circulating form
 Half life -2 -3 weeks
 Normal circulating
conc-10 -50 ng/mL
 Denotes nutritional
status
 1,25 (OH) 2vit D
 Biologically active
form
 Half life – 4-6 hours
 15 -60 pg/mL

26. 25 (OH) VIT D
1.Determination of nutritional status is assessed
by measurement of 25 (OH)D –main
circulating form (less day to day variation with
exposure to sunlight and with dietary intake )
2.Determination of 25(OH)D useful in
a. differential diagnosis of hypocalcemia –
b.assessment vit D status
c. bone disease and other disorders of mineral
metabolism
3. severe hepatocellular disease and
nephrotic syndrome

27. 1,25 (OH)2 D
measurement of 1,25 (OH)2 D is useful in inadequate or
excess production of hormone ,in the evaluation of
1.hypercalcemia 2.hypercalciuria 3.hypocalcemia
4.bone and mineral disorders
vit D dependent rickets type II
Primary hyperparathyroism
Renal failure
Vit D dependent rickets I
Hypoparathyroidism
Pseudohypoparathyroidism
Nephrotic syndrome

28. MEASUREMENT
 Serum
 Plasma is acceptable for assay using extraction and
chromatography
 Steps involved - deproteination or extraction ,
purification and quantification
25(OH) VIT D
 Competitive protein binding assay
 Competitive or noncompetitive
immunoassay(RIA ,EIA ,ICMA)
 UV absorption after separation by HPLC
 Mass spectrometry after separation by
chromatography

29. MEASUREMENT OF 1,25 (OH)2 VIT D
 Complication in its determination –extreme
hydrophobicity and instable
METHODS
1.LC-Tandem mass spectrometry after extraction
and purification
2.Ultra –performance liquid
chromatography(UPLC) electro spray tandem
MS -simultaneous quantification of vit D
metabolites [1,25(OH)2 D2 , 1,25(OH)2 D3
,25(OH)D2 , 25(OH)D3 ,24 ,25(OH)2VITD3]

30. MEASUREMENT OF 24,25(OH)2VIT D
METHODS
 Competitive protein binding assay
 LC – MS/MS
REFERENCE RANGE
 1,25(OH)2vit D -15-60 pg/mL
 25 (OH)vitD-10 -50 ng/mL
 24 ,25(OH)2D -2-7 nmol/L

31. PARATHYROID HORMONE
 Chief cells of parathyroid gland synthesize ,store
and secrete PTH
 Level in plasma determined by its synthesis and
secretion by parathyroid and its metabolism and
clearance by liver and kidney
Pre-pro-PTH intact PTH
enzymatic cleavage

33. BIOLOGICAL ACTION
 Interact with PTH/PTHrP receptors located in
plasma membrane  cyclic AMP cascade
of intracellular events
 Increases total and free plasma calcium
,decreases plasma phosphate and increases
urinary excretion of inorganic phosphate

34.  Circulate as intact and inactive carboxy(C)
terminal segments
 Active intact PTH(1-84 AA sequence) has a half
life in plasma<5 minutes
 C-terminal fragments have half life-<1 hour
 5%-25% of total PTH –intact hormone , 75%-
95% -C-terminal fragments
 Both increased in impaired renal function

35. CLINICAL SIGNIFICANCE
Determination of PTH is useful in
1.DD of hypercalcemia and hypocalcemia
2.for assessing parathyroid function in renal failure
3.to evaluate parathyroid function in bone and mineral
disease
4.Useful for monitoring therapy in dialysis pts
Free or total calcium is usually measured on the same
specimen as the PTH

36. HYPERCALCEMIA
1.PTH is important test for DD of hypercalcemia
A. Increased in pts with primary
hyperparathyroidism –solitary adenoma ,multiple
hyperplasic glands or parathyroid carcinoma
B.Below normal or in lower reference range in
nonparathyroid hypercalcemia

37. HYPOCALCEMIA
2.Useful in DD of hypocalcemia
A.hypoparathyroidism –low PTH
B.pseudohypoparathyroidism –high PTH
Secondary hyperparathyroidism

38. A. In ESRD –measurement of PTH is helpful in
1.assessing parathyroid function
2.estimating bone turnover
3.improving management
B.Patients with high turnover disease (advanced
osteitis fibrosa) have high concentration of PTH
 Low turnover or adynamic bone disease
(osteomalacia) have low concentration of PTH

39. MEASUREMENT OF PTH
 Serum or EDTA plasma
METHODS
 Two –site or sandwich immunoassay
 LC-Tandem Mass Spectrometry
REFERENCE INTERVAL
 first generation -radioimmunoassay
 Second generation assay –sandwich assay-
detected PTH fragments(eg-PTH[7-84]are
detected along with PTH(1-84)
 Third generation-true intact assay , whole PTH
assay , or cyclase activating PTH assays
 Intact PTH -10-65pg/ml ,PTH (1-84) -6 -40pg/ml