What is Osteoporosis?
Osteoporosis is a systemic skeletal disease characterised by low bone mass and deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture.

Diseases of the bone, particularly osteoporosis, represent a major health problem. Affecting approximately one-half of all women over age 50 and one-fourth of all men over age 50. As the median age of the population increases, diagnosis, treatment, and monitoring of skeletal diseases will become an even more prominent health care issue, and the medical community will be compelled to focus even more resources toward patients with bone disorders.

Biochemical markers of bone metabolism may be the answer to cost-effective monitoring of the aging population. A relatively recent development in the clinical arena, bone markers provide a more timely assessment of bone resorption, formation, and turnover than does the current gold standard 'dual- energy X-ray absorptiometry (DEXA). While DEXA provides an accurate (³ 95%) and precise (~1%) means for diagnosing decreased bone mass and predicting fracture, use of this technology is limited because the efficacy of any intervention can be measured reliably by DEXA only after two to three years of treatment. Biochemical bone markers, on the other hand, can help physicians make clinical decisions on therapy and monitoring within three months of initiating treatment. Biochemical markers are effective because they quickly measure by-products of osteoclast and osteoblast activity-the biological machinery that carries out bone turnover.

Cells Involved in Bone Metabolism
Osteoclasts act early in the bone remodelling cycle to resorb existing bone and make way for new bone matrix. They are usually multinucleated with apical and basolateral poles that differ both morphologically and functionally. The osteoclasts apical pole has a fenestrated membrane that attaches to the bone's surface, isolating a microenvironment or "sealing zone" in which pH is lowered and where potent enzymes such as acid phosphatase are released to erode bone. The osteoclast's basolateral pole has receptors for hormones and other substances active in physiological, regulatory, and other cellular functions. Osteoblasts carry out bone formation. After osteoclast-mediated erosion, osteoblasts deposit replacement bone matrix by filling the eroded area with osteoid material, and the bone formation process then stops. During the course of bone formation, some osteoblasts become trapped in the bone matrix where they become osteocytes. In the past, physicians and researchers considered osteocytes metabolically inactive, but new studies indicate that osteocytes may play an important role in activating osteoclasts by detecting microfractures or other flaws in bone structure.

The Bone Cycle
All adult human bone is derived from previously existing bone via the bone emodelling cycle-a dynamic and continuous process that is normally maintained in a tightly coupled balance between resorption of old or injured bone and formation of new bone. Bone metabolism occurs on the surface of bone at focused sites, each of which is termed a bone metabolism unit (BMU).

The bone cycle proceeds in one direction only, beginning with the initial stage of bone remodelling-the quiescent phase. After osteoclasts receive the biochemical signal to become active, they are attracted to the new BMU site, where they erode bone and form a lacunae approximately 100 mm in diameter and 50 mm deep; this process takes about 10 days. At this point, resorption comes to a halt and osteoblasts are then recruited to the site, where they begin to deposit osteoid (collagen) matrix at the bottom of the lacunae, thereby forming the base for new bone material. This formation process constitutes the majority of the 90- to 120-day bone cycle, which then passes into the quiescent phase. Over the next three to four months, the newly formed bone matrix mineralizes with hydroxyapatite, giving tensile strength to the BMU. In general, the proteins and other substances produced, modified, released, or degraded by active osteoclasts and osteoblasts during the different phases of the bone cycle represent the biochemical markers used for monitoring. Most individuals experience a net increase in bone mass until the third or fourth decade of life, followed by a balanced period where total mass remains constant for a relatively few years. Thereafter, a net decrease in bone mass occurs at the level of the BMU. In men, this decrease is "osteoblast-mediated" because even though osteoclasts continue to tunnel lacunae to the usual 50-mm depth, osteoblasts do not completely fill the eroded areas to their original dimension, leaving with each bone cycle a small deficit at each BMU.

Because about 10% of the total body bone mass is normally replaced each year, approximately 1 million BMUs are in various phases of the bone cycle at any given time. Therefore, even a slight deficit in remodeling can lead to substantial cumulative bone loss over time.

Defects in the organisation of bone formation or imbalances on the side of bone resorption can result in substantial changes in functional integrity over time. And while these usually build up slowly over time, changes can occur rapidly when the rate of bone turnover increases. For example, most women experience a decrease in bone density at approximately 45-60 years of age. This loss is termed "osteoclast-mediated" because the osteoclasts of postmenopausal women erode lacunae deeper than the standard 50-mm depth. As with men and their osteoblast-mediated bone loss, the matrix of this deeper lacunae in middle-aged women is incompletely restored during the bone formation phase. In addition, the characteristic estrogen deficit of menopause causes an increased frequency of BMU activation, thereby accelerating bone loss.

Basic Bone Structure and Composition
The skeleton consists of two basic types of bone. The first type, comprising 80% of the skeleton, is cortical bone. This bone type is well suited for mechanical, structural, and protective functions because it is 80%-90% calcified and is quite dense. Cortical bone comprises the outside protective surfaces of all bones and is the major component of long bones. The second type of bone, comprising 20% of the skeleton, is called ancellous or trabecular bone. Because only 5%-20% of cancellous bone mass is calcified, it is much less dense than cortical bone. Under the microscope, cancellous bone has a honeycombed appearance because it is laced with trabeculae that greatly increase surface area. Since bone metabolism occurs only at surface sites, this honeycomb structure confers high metabolic activity to cancellous bone. Even though cortical bone comprises four-fold more of the skeleton than cancellous bone, their metabolic activity is approximately equal.

The organic matrix of cortical and cancellous bone is mostly comprised of type I collagen. Although connective and some other tissues also contain type I collagen, bone has a far higher proportion of this protein and a much higher rate of collagen turnover.

Type I collagen has a high proportion of the amino acids proline and hydroxyproline, and its precursor protein has relatively large extension peptides on both the carboxyterminal and aminoterminal ends. These extension peptides are cleaved during the secretion and fibril-formation process. Figure 3 shows the basic crosslinking of collagen fibers, each of which contains aminoterminal and carboxyterminal ends. In the case of type 1 collagen, these ends are each linked to a helical portion of a nearby molecule by a pyridinium crosslink. The amino- and carboxy- non-helical ends are termed the N-telopeptides and C-telopeptides, respectively.

Bone degradation by osteoclasts during the resorption process releases fragments of various sizes-some still attached to helical portions of a nearby molecule by a pyridinium crosslink-for metabolism or excretion in urine. With further degradation in the liver and kidneys, the fragments are finally broken down to their constituent modified and unmodified amino acids and the pyridiniums pyridinoline (Pyr) and deoxypyridinoline (D-Pyr). Assays for peptides of the N-terminal and C- terminal region, as well as for the Pyr and D-Pyr molecules, have been developed for use in monitoring bone resorption.

Regulation of Bone Metabolism
Bone metabolism is regulated by a complex interaction of many hormones and factors that affect progenitor cells, osteoblasts, and/or osteoclasts. Regulators of bone metabolism include parathyroid hormone (PTH), vitamin D, estrogen, and calcitonin, among others.

Increased PTH stimulates bone metabolism dependant on circulating PTH concentrations, as well as the length of exposure to excess PTH. Glucocorticoid therapy may magnify osteoclastic sensitivity to the resorbing effects of circulating PTH. Vitamin D is comprised of two substances, calcifediol (25-dihydroxyvitamin D) and calcitriol (1,25-trihydroxyvitamin D). It affects osteoblast maturation as well as normal growth and inhibition of bone. Decreased concentrations of vitamin D are associated with increased BMU activation and, as a result, a greater rate of bone turnover.

Estrogen is a critical regulator of bone metabolism because it decreases production of osteoid matrix and, researchers hypothesize, promotes formation of trabecular bone. The estrogen deficit that occurs with the onset of menopause promotes bone resorption and increases bone turnover. In contrast to estrogen, calcitonin is a calcitropic hormone that is an effective inhibitor of bone resorption. Although scientists have not yet pinpointed the mechanism of calcitonin's action, the hormone has been used to treat patients with high bone turnover, osteoporosis, Paget's disease, and hypercalcemia of malignancy. Other hormones that play a role in bone metabolism include thyroid hormone and prolactin. Hyperthyroidism or therapeutic administration of thyroid hormone may cause increased bone turnover, and prolactin secondarily accelerates bone loss by suppressing synthesis of estrogen and testosterone. Cortisol and related steroids increase bone turnover by directly stimulating both bone resorption and formation. Also, in patients receiving long-term glucocorticoid treatment, osteoporosis can be a common and serious secondary side effect that may go undetected until substantial bone loss has occurred.

Specific Markers of Bone Metabolism
Based on the phases of the bone cycle, biochemical markers are conveniently classified as indicators of bone formation, bone resorption, or overall bone turnover. Markers of bone formation assess either osteoblastic synthetic activity or metabolism of procollagen. Resorption markers reflect osteoclast activity and/or collagen degradation. Laboratorians can assess bone turnover by comparing concentrations of marker released during resorption and formation. Alternatively, it may be possible to measure a marker that is both "leaked" into circulation during bone formation as well as incorporated into the bone matrix for release during the resorption phase.

Markers of Bone Formation
Alkaline phosphatase (ALP)
The exact function of ALP remains unknown. Associated with the cell plasma membrane, this enzyme appears to play a role in the transport of substances from the intracellular compartment across the cell membrane to the extracellular region. Four ALP isoenzymes may be found in blood, each of which is relatively specific for the respective liver, bone, placental, and intestinal tissues with which it is associated. The liver-, renal-, and bone-specific isoenzymes are coded by the same gene; therefore, differences between these isoenzymes are the result of post-translational modification.Bone alkaline phosphatase (B-ALP) is produced by the osteoblast, and researchers have identified the enzyme in matrix vesicles called "buds," which are derived from the cell membrane. These deposits appear to play an essential role in the bone formation process. B-ALP is produced in extremely high amounts during the bone cycle formation phase and is therefore a good indicator of overall bone formation activity.

For therapeutic monitoring of patients, B-ALP measurements are a good indicator of the metabolic activity of bone. Rising B-ALP concentrations may also indicate estrogen deficiency, and scientists have shown that postmenopausal women have significantly higher B-ALP levels than premenopausal women.

As a result of the relatively large molecular size of ALP, all assays for this enzyme are serum-based. Laboratories measure B-ALP by numerous methods including electrophoresis, wheat germ lecithin precipitation, heat stability, and immunoassay. However, many laboratorians consider immunoassay the method of choice because these assays have better analytical sensitivity and reported imprecision in the range of 5%-8%.

Because of the similar epitopes, however, tissue specificity of B-ALP immunoassays may be confounded by potential crossreactivity with ALP liver and bone fractions. Zero crossreactivity is therefore difficult to attain, but clinical performance of these assays appears to be acceptable.

Osteocalcin (Ocn)
Ocn is a relatively small protein (Mr = 5800 daltons) produced by osteoblasts during the matrix mineralization phase. It is released into blood and incorporated into the bone matrix where it is the most abundant non-collagenous protein. Ocn synthesis is dependent on vitamin K, which post-translationally modifies the gene product with g-carboxyglutamate (Gla) residues. As a result of this modification, Ocn is also known as bone Gla protein. Ocn is secreted into blood and, in individuals having normal renal function, it is excreted in urine. The body degrades Ocn during bone resorption, with up to 70% entering the circulatory system. Because circulating Ocn may be either newly synthesized during bone formation or released during resorption, there is some question whether Ocn should be considered a marker of osteoblast activity or an indicator of bone matrix metabolism and turnover. If one considers Ocn a marker of bone turnover, it may hold substantial clinical utility for monitoring tightly coupled formation/resorption processes. When formation and resorption are uncoupled, most experts consider Ocn a marker of osteoblast activity. Markers of Bone Resorption

Acid phosphatase
Five isoenzymes of the lysosomal enzyme acid phosphatase circulate in blood, the major sources of which are bone, prostate, platelets, erythrocytes, and spleen. Bone acid phosphatase is presumably released into circulation by "leakage" during resorption as well as after detachment of the osteoclast's sealing zone. Due to its relatively large molecular size, assays for acid phosphatase are serum or plasma based. Data for the use of acid phosphatase as a marker of bone metabolism is incomplete. This may be the result of numerous analytical problems and the fact that the enzyme is released into the sealed osteoclast microenvironment rather than directly into the blood stream.

N-telopeptide
Fragments from the N-terminus are released into circulation as a result of osteoclast degradation of type I collagen during the resorption process. Because the vast majority of these fragments are relatively small, they readily pass through the glomerulus into urine. Researchers report that N-telopeptides are very specific for bone tissue breakdown because other tissues comprised of type I collagen (e.g., skin) are not actively metabolized by osteoclasts. A urine assay for N-telopeptides that monitors bone resorption has been commercially developed. This assay is based on a monoclonal antibody that specifically recognizes the a -2 chain N-telopeptide fragment, which contains pyridinium crosslinks. However, this assay does not cross-react with Pyr or D-Pyr. Recommended specimens are a second morning spot urine or a 24-hour collection.

C-telopeptide
Fragments derived from the C-terminus are also released into circulation as a result of the osteoclast-mediated degradation of type I collagen during the bone resorption process. Again, these fragments are highly bone specific because osteoclasts are not active in degrading other type I collagen-containing tissues. Diagnostic companies have developed both serum and urine assays for these peptides.

Pyridinoline and deoxypyridinoline crosslinks
Pyr and D-Pyr crosslinks, resulting from post-translational processing of lysine and hydroxylysine residues, are essential for stabilizing the mature forms of collagen fibers and elastin. Although both Pyr and D-Pyr are found in bone, D-Pyr is more bone specific. Fluorimetric detection of both the free and protein-bound forms of Pyr and D-Pyr by high performance liquid chromatography (HPLC) provides a highly specific marker of bone resorption, but can be technically demanding and is not routinely offered in all clinical laboratories. Immunoassays for free Pyr and free D-Pyr are available. These assays provide a quantitative measure of free crosslinks that reflect bone resorption. Scientists have evaluated free Pyr and D-Pyr assays in menopause and hormone replacement therapy, as well as in a number of other diseases including osteoporosis, hyperthyroidism, hyperparathyroidism, and Paget's disease.

Why Are People Talking about Bone Markers?
Clearly, biochemical markers of bone metabolism provide a potentially important clinical tool for assessing and monitoring skeletal diseases. They complement DEXA and respond more quickly, often showing clinical changes within three months. The EPIDOS study, which included 7,598 patients, illustrated the potential "added value" of bone markers to conventional tools and clinical acumen for evaluating fracture risk in patients.