LABORATORY ASPECT OF

BONE METABOLISM

Windarwati
RSUP Dr. Sardjito/FK UGM
Mineral and Bone Metabolism
 The skeleton is a metabolically active organ that
undergoes continuous remodeling throughout life.
 This remodeling is necessary both to maintain the
structural integrity of the skeleton and to fulfill its
metabolic functions as a storehouse of calcium and
phosphorus
 The skeleton also serves as the second line of
defense against acidosis, and it is able to liberate
buffers in the form of inorganic phosphates
A, Compact bone, long bone, cross-section. B, Cancellous bone, longitudinal section

B
 STRUKTUR TULANG

Tulang Kortikal:
- Bagian yg tersusun rapat  penyusun
utama tulang panjang

Tulang Trabekular/spongy:
- Lapisan-lapisan tipis yang menyusun
bagian terdalam tulang belakang,
tulang panggul, kosta, skapula, dan
ujung tulang panjang.
 Struktur Tulang
Matriks Komponen
Organik Sel Tulang
(Osteoid) anorganik
Magnesium
Kolagen
(10%)
Osteoklast
Kalsium

Phospat
Non Kolagen
Kristal Osteoblast
(90%)
Hidroksiapatit
Calsium
A healthy adult contains approximately 1–1.3 kg of
calcium
99% : hydroxyapatite in the skeleton
1% : extracellular fluid (ECF) and soft tissues.
Additionally, less than 1% of the skeletal content of
calcium is in bone fluid and exchanges freely with
the ECF
Serum (plasma) calcium exists in three
distinct forms:

1 • free or ionized calcium

• ± 50%

• complexed calcium (bicarbonate, lactate,

2 phosphate, and citrate)
• ± 10%

• Plasma protein-bound calcium albumin

3 80%
• ± 40%
 Function
 skeletal mineralization,
 blood coagulation,
 neural transmission,
 plasma buffering capacity and enzyme activity
 maintenance of normal muscle tone and
 excitability of skeletal and cardiac muscle
 activator of intracellular signal transduction processes
 DNA and RNA biosynthesis
 glandular synthesis and in regulation of exocrine and
endocrine glands,
Calcium homeostasis

Solid arrows and block arrows

 that increase serum calcium
levels;
dashed arrows i  negative
effects that decrease serum
calcium
. Respon hormonal

Lewandrowski, 2002
Analytic Techniques
 Total Calcium
(1) colorimetric analysis with metallochromic
indicators;
(2) atomic absorption spectrometry (AAS); and
(3) indirect potentiometry.
 Ionized Calcium

Calcium ion-selective electrodes (ISEs)

Reference Interval
 The reference interval for total calcium in normal
adults ranges between 8.8 and 10.3 mg/dL (2.20–
2.58 mmol/L).
 Specimen for total calcium determination
: serum
heparinized plasma
Calsium : 6,6 mg/dL
Albumin ; 2,4 mg/dL
Total calsium corrected : 6,6 + (4,4 -2,5) x 0,8
: 8,2 mg/dL
Reference Interval
 The reference interval for ionized (free) calcium in
normal adults is 4.6–5.3 mg/dL (1.16–1.32 mmol/L).
 Specimen for ionized calcium determination :
- whole blood
- heparinized plasma
- serum
 Proper collection technique is important to ensure
accurate ionized calcium results; a tourniquet left on too
long can lower pH at the site of collection and falsely
elevate levels.
Lewandrowski, 2002.
Reference Interval
 urinary calcium up to 300 mg/day (7.49 mmol/day).
 spesimen urin tampung maupun urin puasa setelah
puasa semalam adalah <0,16 mg (<0,04 mmol)
/100mL glomerular filtrate (GF)
[UCa (mg/dL) x SCr (mg/dL)] / UCr (mg/dL).
UCa adalah kadar kalsium urin,
SCr adalah kadar kreatinin serum dan
UCr adalah kadar kreatinin urin (Burtis et al., 2006).
PHOSPHORUS
 The total body phosphorus content in normal adults is
around 700–800 g
 ± 80%–85% : skeleton  inorganic phosphate
(hydroxyapatite and calcium phosphate)

± 15% : - ECF  inorganic phosphate,

- Intracellularly in the soft tissues 
organic phosphates (phospholipids,
nucleic acids, and ATP)
 Function
 skeletal mineralization ,
 important constituent of nucleic acids in that both RNA and
DNA
 Cofactors (nicotinamide adenine dinucleotide phosphate
[NADPH])
 muscle contractility,
 Neurologic function,
 electrolyte transport, and
 oxygen carrying by hemoglobin (2,3-diphosphoglycerate).
Analytic Techniques
 enzymatic method
 Specimen for phosphat determination :
- Serum
- heparinized plasma
 Serum phosphate is best measured in fasting morning
specimens (higher levels in the afternoon and evening)
as well as a reduction in serum phosphate after meals.
 Levels are influenced by dietary intake, meals, and
exercise.
Reference Interval
 normal adults : 2.8 and 4.5 mg/dL or
0.89–1.44 mmol/L
 growing children : 4.0–7.0 mg/dL or
1.29–2.26 mmol/L
MAGNESIUM
 The normal body magnesium content in an adult is
approximately 1000 mmol, or 22.66 g,
- 50%–60% is in bone,
- 40%–50% is in the soft tissues
- Only 1% (TBMg) is in extracellular fluid
 Only 1% of the total body magnesium (TBMg) is in
extracellular fluid.
 In serum,
- 55% : ionized or free magnesium (Mg++)
- 30% : associated with proteins (albumin)
- 15% : complexed with phosphate, citrate
Function
 Magnesium is essential for the function of more than 300
cellular enzymes (transfer of phosphate groups, all reactions
that require ATP, and replication and transcription of DNA
and the translation of mRNA).
 This cation is also required for cellular energy metabolism
and membrane stabilization, nerve conduction, ion
transport, and calcium channel activity.
 critical role in the maintenance of intracellular potassium
concentration by regulating potassium movement through the
membranes of the myocardial cells.
 magnesium deficiency  refractory plasma electrolyte
abnormalities (especially depressed potassium) and cardiac
arrhythmias most often observed after stress such as cardiac
surgery
Analytic Techniques

 Total Magnesium : photometric method

 Ionized (Free) Magnesium : ion-selective electrodes

Reference Interval
 The reference interval for serum total magnesium in
normal adults ranges between 0.75 and 0.95
mmol/L (1.7–2.2 mg/dL or 1.5–1.9 mEq/L).
Parathyroid Hormone
 PTH is synthesized and secreted by the chief cells of
the parathyroid gland.
 PTH secretion is regulated on a time scale of
seconds by extracellular ionized calcium and
represents a simple negative-feedback loop
Parathyroid Hormone
 The primary physiologic function of PTH is to maintain
the concentration of ionized calcium in the ECF, which is
achieved by the following mechanisms:
(1) stimulation of osteoclastic bone resorption and
release of calcium and phosphate from bone
(2) stimulation of calcium reabsorption and inhibition
of phosphate reabsorption from the renal tubules
(3) stimulation of renal production of 1,25-(OH)2D3,
which increases intestinal absorption of calcium and
phosphate.
Analytic Techniques
 immunoradiometric assay (IRMA)
 immunochemiluminometric assay

 These immunometric assays have several advantages :

(1) increased sensitivity and specificity through the use
of sequence-specific and affinity-purified antibodies,
(2) extended assay concentration range, and
(3) decreased incubation time, and
(4) do not utilize radioactive compounds.
Reference Interval
 intact PTH in normal adults is 10–65 pg/mL (ng/L)
when a two-site immunometric method is used
 Serum is the preferred specimen for measurement
of PTH
Pathways of vitamin D synthesis and
their end-organ effects
 Analytic Techniques
 25(OH)D is a better marker than vitamin D for
evaluation of vitamin D status because of its longer
half-life (2–3 weeks vs. 5–8 hours)
 Most other assays for vitamin D metabolites are
measured by RIA or chemiluminescent immunoassay.

Reference Interval
 25(OH)D serum : 10–50 ng/ mL (25–125 nmol/L),
1,25(OH)2D : 15–60 pg/mL (36–144 pmol/L)
Disorders of Mineral Metabolism
HYPOCALCEMIA
 HYPOCALCEMIA

Relationships leading to renal

osteodystrophy in chronic renal failure.
HYPOCALCEMIA
Graph correlating alterations in serum calcium levels and parathyroid
hormone levels with the diseases most frequently causing these alterations
 Biochemical Markers of
Bone Remodeling

 Bone remodeling is a coupled process that begins

with resorption of old bone by osteoclasts, a process
that takes approximately 50 days, followed by
formation of new bone by osteoblasts, which takes
another 150 days, for a total turnover cycle lasting
approximately 200 days (Erikson, 1994).
Bone Remodeling
 Three major diagnostic procedures are available to
monitor bone turnover and evaluate metabolic bone
disease:
1. bone imaging techniques,
2. bone biopsy,
3. biochemical markers of bone turnover.
BONE RESORPTION MARKERS
Bone tissue has three components:
 organic matrix (called osteoid),

 bone mineral,

 bone cells.
BONE RESORPTION MARKERS
 calcium
 collagen degradation products hydroxyproline,
pyridinium crosslinks, and telopeptides,
 cellular products involved with degradation of the
mineralized matrix  tartrate-resistant acid
phosphatase (TRAP).
 Urinary calcium is affected by diet and renal
function; thus, it is not sensitive or specific for
assessment of bone remodeling
BONE FORMATION MARKERS
 Alkaline Phosphatase
 Osteocalcin
 Procollagen Type I N-Terminal and C-Terminal
Peptides
√

√
√

(Sumber: Seibel, 2005)

biochemical markers of bone turnover.

Penanda biokimiawi metabolisme tulang
Metabolic Bone Disease
 OSTEOPOROSIS
 OSTEOMALACIA AND RICKETS
OSTEOPOROSIS
 It is a systemic skeletal disorder characterized by
decreased organic bone matrix and
microarchitectural deterioration of bone tissue, with
a subsequent increase in bone fragility and
susceptibility to fracture
 Resorption and Resorption Exceeds
Formation are Equal Formation

Normal Trabecular Bone Osteoporosis Bone

Schematic comparison of femoral cortex in a
30-year-old male (left) and a 75-year-old
male (right).

Osteoporosis of lumbar vertebra

 KRITERIA WHO
Severe
Normal Osteopenia Osteoporosis
Osteoporosis
• BMD > - 1 • BMD antara - • BMD kurang • BMD kurang
SD rata-rata 1 – -2,5 SD dari -2,5 SD dari -2,5 SD
masa tulang rerata masa rerata masa rerata masa
wanita tulang wanita tulang wanita tulang wanita
normal normal. normal.
normal
• Satu atau
lebih fraktur

Mauck & Clarke ; Mayo Clin Proc : 81(5), 2006

 Bone turnover markers

International Osteoporosis Foundation (IOF) dan International

Federation of Clinical Chemistry and Laboratory Medicine (IFCC)
merekomendasikan:
 Marker resorpsi : CTX serum

 Marker formasi : P1NP serum

 Sampel : serum atau plasma EDTA

 Pengukuran : automated immunoassay analysers, ELISA.

(Vasikaran et al, 2010)

 Procollagen type I propeptide (PINP)

Molekul kolagen tipe I

 disintesis osteoblast
 berasal kolagen tipe 1
 elongated protein BM 35 kDa
 Banyak proline dan hydroxyproline
Stabilitas Variabilitas

23-25⁰C : 2 hari,
2-8⁰C : ±7 hari, Diet sedikit
mempengaruhi
-20⁰C ± 6 bulan

Freezing dan thawing

berulang  stabil
C-terminal cross-linking telopeptide of type I collagen (CTX)
/β Cross Labs

CTX: fragmen proteolitik dari kolagen tipe I yang dihasilkan selama

proses resorpsi tulang (Wasnich, 2004).
C-terminal cross-linking telopeptide of type I collagen (CTX)

40C  24 jam Variasi diurnal

-300C  12 minggu a. Tinggi 8 – 11
b. Rendah: sore hari
Freezing dan thawing 9 kali 
tdk mempengaruhi kadar CTX
Serum Diet sedikit mempengaruhi
kadar CTX serum  sampel
puasa
- Stabil thd efek penyinaran
UV
 Clinical performance s-CTX and s-P1NP
Commercial Automate Assay Reference interval Analytical Intra-individual
assay d analytical range Premenopausal CV (%) CV (%)
(μg/L)
S CTX Β-CrossLaps Yes 0,01-6 0,11-0,63 1,3-4,3 9,4
ECLIA
0,10-0,62 3,7-5,7 < 4,1
S-CTX Β-CrossLaps No 0,02-3,38 0,20-0,90 2,2-5,5 10%
ELISA
S-P1NP Total P1NP Yes 5-1,200 16,3-78,2 2,7-4,4 7,2
ECLIA
16,2-60,9
S-P1NP Intact P1NP Yes 2-230 < 6%
CLIA
S-P1NP Intact P1NP No 5-250 16-83 4,1-7,6 3,7-5
RIA

(Vasikaran et al, 2010)

Recent national guidelines on utility of BTMs in
the management of patients with osteoporosis
Country, year Recomendation

Predictor Monitor Patient

fracture risk TX Compliance
Belgium, 2009 Additional risk √ √
factor in decicion
Canada,2009 Bone loss + √ √ √

Europe, 2008 √

Latin America,2009 √ √

Singapura,2008 √ √

UK,2008 √ √

USA,2008 √ √

Vasikaran et al, Osteoporosis Int (2011) 22:391-420

Garnero et al : penelitian OFELY, wanita osteoporosis, follow up 5
thn. Kombinasi prediktor independen utk identifikasi risiko fraktur.
BMD : ≤ 2,5 rerata wanita dewasa, CTX urin : kuartil tertinggi.
(Journal of Bone and Mineral Reseaarch 15 : 8, 2000)
Changes (% ± SEM) in markers of bone resorption (NTX) and bone
formation (P1NP) following treatment with an anti resorptive
therapi and an anabolic therapy.
Vasikaran et al, Osteoporosis Int (2011) 22:391-420
 50 tahun 25 tahun
kemudian

the National Osteoporosis Foundation,

Osteoporosis: The Silent Disease, National Osteoporosis Foundation,
Partners in Prevention Slide Presentation, 1993
OSTEOMALACIA AND RICKETS
 Osteomalacia is a failure to mineralize newly
formed organic matrix (osteoid) in the mature
skeleton.
 Rickets, a disease of children, is the designation for
osteomalacia that occurs before cessation of
growth, that is, before closure of the epiphyseal
plates of long bones.
 Optimal mineralization requires
(1) an adequate supply of calcium and phosphate
ions from the extracellular fluid,
(2) an appropriate pH (≈7.6),
(3) bone matrix of normal chemical composition
and rate of synthesis, and
(4) control of inhibitors of mineralization.
 The major categories of diseases that produce
osteomalacia or rickets are
1. vitamin D deficiency states,
2. Phosphate depletion,
3. systemic acidosis,
4. inhibitors of mineralization

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