Biochemical markers of bone remodeling and diseases

Bone resorption markers

Marker	Tissue specificity	Metabolism
Pyridinoline (PYD) and deoxypyridinoline (DPD)	Formed between the end region of one collagen molecule and the helical region of another. Contribute collagen stabilization. PYD is mainly present in the type II collagen in cartilage and to a lesser extent in bone tissue. DPD is present primarily in bone, and released only when bone resorption	Excreted with urine; not metabolized in the liver
Carboxy- and aminotermal telopeptides of type I collagens (CTX, NTX)	Present in all tissues containing type I collagen. Throw out the bones only in the process of resorption	Excreted with urine
Hidroxyproline (OHP)	Present in the molecules of collagen and elastin. Appears in the extracellular fluid during the bone formation or resorption	Metabolized in the liver; 10-15% excreted by the kidneys
Tartarate-resistant acid phosphatase (TR-ACP)	Present in osteoclasts and other macrophages	Do not metabolized in the liver; excreted with urine

 

Bone alkaline phosphatase (B-ALP)

Bone alkaline phosphatase (B-ALP) is secreted by osteoblasts and it is involved in matrix maturation and mineralization. Synthesis of bone alkaline phosphatase (B-ALP) is increased in differentiation of osteoblasts in accelerated bone formation.

A significant increase in the activity of bone alkaline phosphatase (B-ALP) in the serum observed in the increased activity of osteoblasts:

• bone growth (in children activity is higher than in adults)
• the last trimester of pregnancy
• the renewal movements after prolonged bed rest
• fractures
• osteitis deformans
• Paget's disease
• rickets
• hyperparathyroidism
• osteomalacia (malignant bone tumors, myeloma)
• bone tuberculosis
• leukemias

 

Osteocalcin (OC)

Osteocalcin (OC) — collagenous vitamin-K-dependent calcium-binding protein of the bone matrix with a molecular mass of 5.7 kDa, synthesized by osteoblasts and odontoblasts. More than 90% of the synthesized osteocalcin is included in the bone matrix, a small portion immediately diffuses into the blood.

The function of osteocalcin in the blood is unknown, assume that it is necessary for the implementation of the effects of 1,25(OH)2D3. Osteocalcin is excreted from the bloodstream by the kidneys: filtered in the glomeruli and degraded in renal tubules. In marked decrease in glomerular filtration rate, particularly in chronic renal failure, the level of osteocalcin in the blood can be too high.

Osteocalcin in the blood is considered as one of the most informative markers of bone formation and rate of remodelling.

The concentration of osteocalcin in serum is increased in most cases, accompanied by the mineralization of bones, but the concentration changes do not always parallel changes of bone alkaline phosphatase (B-ALP), mainly due to renal excretion and methodical features of determination of fragments of osteocalcin.

The most adequate methods of investigation of osteocalcin are considered to radioimmunoassay and enzyme-linked immunosorbent assay using antibodies.

Clinical-diagnostic value of osteocalcin changes in the serum:

Increase	Decrease
- Age (women 50-60 years) - Puberty - Menstrual cycle (luteal phase) - Lactation - Nighttime - Paget's disease - Hyperthyroidism - Chronic renal failure (chronic renal insufficiency) - Osteomalacia - Bone with metastases - Anticonvulsants - Elevated levels of growth hormone	- Age (0-40 years) - Pregnancy - Early morning - Summertime - Growth hormone deficiency - Hypothyroidism - Estrogens - Corticosteroids - Hyperparathyroidism (acute) - Calcitonin

 

Carboxy- and aminotermal propeptides of type I procollagens (PINP, PICP)

The type I collagen is the main protein that constitutes 90% of the organic matrix of bone. It is synthesized by osteoblasts in the form of a precursor of procollagen type I, which is a large molecule, containing partially globular fragments of carboxy- and aminoterminal propeptides of procollagen type I (PINP and PICP) from the C- and N-ends. PINP and PICP are separated from the main molecule by specific peptidases after the release of procollagen from the cell.

Purified collagen type I molecule involved in the construction of the fibrils of bone matrix, and PINP and PICP released into the extracellular fluid. The ratio between the amount of collagen is deposited in the bone matrix, and the number of PINP and PICP, enters the bloodstream, it is theoretically equal to 1, so by the level of PINP and PICP is possible to judge the ability of osteoblasts to synthesize type I collagen. Neither PINP, nor PICP can not pass through the kidney glomerular filter, so the level of propeptide in the blood is not dependent on renal filtration. They are metabolized in the liver.

 

Pyridinoline (PYD) and deoxypyridinoline (DPD)

In bone collagen cross-links are present between the individual collagen molecules, which play a major role in its stabilization and presented in the form of pyridinoline and deoxypyridinoline. Cross-links are formed extracellularly after the deposition of collagen molecules in the matrix. As a result of resorption carried out by osteoclasts, the destruction of collagen possible their exit from the bone into the bloodstream.

Bones most specific to DPD, because it contains predominantly type I collagen of bone tissue and a small amount in the dentin, aorta, and ligaments. PYD is mainly present in the type II collagen in cartilage and to a lesser extent in bone tissue. PYD and DPD are not metabolised in the body and excreted with urine.

The level of DPD in the urine in women slightly higher than in men and increases with age. In women during menopause the excretion of DPD in the urine is 2-3 times higher than in women of childbearing age, and excretion does not depend on the diet and physical activity. Both women and men, the excretion of PYD and DPD increased in primary hyperparathyroidism (about 3 times), hyperthyroidism (about 5 times), Paget disease (10-12 times). Less important, but nevertheless significantly increased excretion of DPD in osteoporosis, osteoarthritis and rheumatoid arthritis. Excretion in all cases decreases with successful treatment.

For analysis use daily or (for ease of collection) – morning urine.

 

Carboxy- and aminotermal telopeptides of type I collagens (CTX, NTX)

Carboxy- and aminotermal telopeptides of type I collagens (CTX, NTX) formed in tissues that contain type I collagen. The molecular weight of telopeptides is from 9 to 20 kDa, they are effectively excreted with urine. Deterioration of renal function leads to elevated levels of telopeptides in the serum. The CTX and NTX concentration in serum correlate closely with the rate of bone resorption. NTX (commercial name of the test – Cross Laps) can be used to determine telopeptide collagen in serum and in urine. During menopause the Cross Laps marker is increased in serumalmost in 2 times.

Dynamically determine the level of telopeptide is important for predicting recovery of bone mineral density in conducting anti-resorption therapy in women in postmenopausal period and patients with osteopenia and Paget's disease. The advantage of using Cross Laps is that this marker of bone resorption allows you to quickly evaluate the efficiency of all types of osteoporosis therapy within 3 months after the start of treatment. The increase in Cross Laps from the mean value at 2SD associated with a 2-fold increased risk of hip fracture.

 

Hidroxyproline (OHP)

Hidroxyproline (OHP) is around 14% of the amino acid composition of collagen produced by osteoblasts. 85-90% of hydroxyproline released from bone as a result of destruction of collagen, is metabolized in the liver and only 10-15% appears in urine. However, about 10% of hydroxyproline present in the urine, hydroxyproline is formed not as a result of resorption, as a result of degradation of newly synthesized peptides or procollagenase of new collagen molecules to be used to build bone matrix.

Thus, appearing in the urine reflects total hydroxyproline and function of osteoblasts (formation process), and the function of osteoclasts (resorption process), however, prevails the proportion of hydroxyproline produced as a result of resorption.

Using the study of hydroxyproline in urine to estimate the rate of remodeling, it should also be borne in mind that it is not specific for bone, because it contains (though in smaller numbers) in all types of collagen molecules. In addition, it can appear in the urine resulting from the ingestion of food containing collagen, so for laboratory analysis requires that the patient at least for 3 days not consuming collagen containing food.

 

Tartarate-resistant acid phosphatase (TR-ACP)

Acid phosphatase is a heterogeneous group of enzymes that hydrolyze monoesters of orthophosphoric acid in an acidic environment. In humans allocated at least 6 structural isoforms. In osteoclasts showed the presence of 2 forms of acid phosphatase: the "big" isoenzyme with a molecular mass of 100 kDa, which is sensitive to the inhibitory tartrate action, and the "small" isozyme with molecular weight 34 kDa, which are resistant to tartrate (tartarate-resistant acid phosphatase TR-ACP).

TR-ACP is localized in the rough endoplasmic reticulum of osteoclasts and is released from them in bone lacunae in the process of bone resorption. The level of TR-ACP in osteoclasts is increased by the action of parathyroid hormone and decreased by calcitonin.

Since the activity of TR-ACP in the blood is increased in conditions, characterized by increased bone resorption process, and due to correlation between its activity and the data of histomorphometry, TR-ACP are used to determine the severity of the resorptive processes in the skeleton.

 

The clinical conditions associated with changes in serum activity of TR-ACP:

Conditions	Rate of change
Increase   - tumor metastasis in bone   - osteomalacia   - Paget's disease   - primary hyperparathyroidism   - osteoporosis   - hyperthyroidism   - multiple myeloma   - Cushing's disease   - "hairy cell" leukemia Decrease   - hypothyroidism	+++ +++ ++ ++ + + + ++ ++ -

 

Prognostic significance of bone remodeling markers

Prognostic significance of bone remodeling markers suggests that:

1. High levels of bone resorption markers (premenopausal levels exceeded 2 standard deviations) is associated with a twofold increase in the risk of fractures
2. Resorption markers can be used further in deciding the appointment of therapy when densitometry and clinical risk factors are not unique for a decision
3. Patients with osteoporosis, in which bone turnover markers level exceeds the norm by more than 3-fold, presumably have a different metabolic bone pathology, including malignant
4. The normal reference values are determined for healthy premenopausal women aged 30-45 years

Thus, the ratio of the change in markers of resorption and formation, it is possible to judge the speed of bone loss, predict the risk of bone fracture and to choose the most appropriate therapy.