October 2007 Clinical Laboratory News: Bone Markers

 
October 2007: Volume 33, Number 10

Bone Markers
A Valuable Tool for Detection and Management of Osteoporosis
By Catherine Hammett-Stabler, PHD, DABCC, FACB

 

 

 

An active 6-year-old, Allen fractures his right femur after falling from a slide on the playground. His records reveal that he’s had two previous fractures over the past two years. Mary Jane, a 59-year-old female, learns that her height is 5’9”. This is a full 1.5” shorter than previous measurements. While playing in a department softball game, 61-year-old professor Richard breaks his wrist sliding into home base. The individuals in these brief scenarios are very different, and at first glance, one might wonder how they are related. But upon closer examination, they may share a common disease—osteoporosis.

Defined as a skeletal disorder in which the density and quality of the bone is decreased, osteoporosis com-promises the strength of affected bones. No longer able to withstand the stresses of daily life, the bone is now susceptible to fracture, as perhaps evidenced in each of the above cases. Osteoporosis has long been thought of as a disease of older women, but for many patients it begins much earlier as an insidious attack on bone, progressing quietly and often with few visible signs or symptoms.

Not Just Grandma’s Problem Anymore

By most estimates, more than 10 million individuals in the U.S. currently have a diagnosis of osteoporosis, while another 34–44 million are on their way to developing the disease. This later group of patients has osteopenia, which is defined as decreased bone density, and is likely to progress on to osteoporosis if no interventions are undertaken. In the intervening years, their risk of experiencing a fracture is about double that of comparable individuals whose bone density is considered adequate. While it is true that Caucasian women over the age of 50 have the greatest risk of osteoporosis, men and non-Caucasian women are being diagnosed with increasing frequency, as are children.

Not long ago osteoporosis was considered primarily a “woman’s disease” and a natural course of aging that had few treatment options. The diagnosis was made when the woman lost height, developed a dowager’s hump, or experienced a more obvious fracture. Fortunately for baby boomers, much has been learned about this disease in recent years: key details of its pathogenesis have been uncovered, tools for patient identification have been developed, and a number of drugs used to treat the disease have been released.

However, the fact remains that without intervention, osteoporosis and osteopenia predispose individuals to increased risk of fracture. For older patients, this represents a serious, potentially life-altering event as these individuals are faced with a loss of mobility and independence, a decreased quality of life, and even death. About one-third of patients who experience a hip fracture die within a year, and the risk is greatest for men. The direct healthcare costs resulting from osteoporosis-related fractures currently exceed $17 billion per year and are expected to continue increasing. While unknown, the indirect costs far exceed this.

Setting the Stage

Bone is a dynamic tissue. In a year’s time, 10–20% of the skeleton undergoes a process known as remodeling in which old and damaged bone is removed and replaced. This highly regulated and continuous process involves the combined efforts of the three bone cells: osteoblasts, osteocytes, and osteoclasts. From birth into late adolescence and early adulthood, growth and remodeling favors the formation of bone so that there is a net increase in bone density. Depending on the bone type and its location, maximum or peak bone density is achieved between the late teens to the mid-30s. For a brief period, bone density is somewhat stable before beginning to decline. For many women, this point comes soon after menopause when estrogen, a known regulator of bone remodeling, becomes deficient. In this environment, the osteoclasts are no longer restrained. For most women, the rate of loss accelerates dramatically after menopause with osteoclasts resorbing bone at a rate much faster than the osteoblasts can form anew. There is evidence that estrogen plays a role in regulating remodeling for men as well.

If estrogen were the only contributor to these events, life would be simple, but a number of other factors are involved. Vitamin D and mineral metabolism, genetics, local growth factors and cytokines, receptors, not to mention a number of diseases and drugs, all come into play (Table 1, below). These factors respond to daily stresses and events as needed. In addition, remodeling follows a diurnal pattern with most activity occurring during the early morning hours. In some patients, the osteoclasts are not overactive and go about their business of resorbing bone at an appropriate rate. Instead, the osteoblasts fail to make enough new bone to replace the bone resorbed. One factor to pay attention to, however, is the actual level of an individual’s bone mass at peak. Everyone loses bone density with age, and a number of factors come into play to determine the amount and rate of loss. But where an individual starts that loss can make a difference in whether that person goes on to develop osteoporosis. This fact has lead to a number of initiatives to encourage teenagers and young adults to develop strong bones early.

 

Table 1
Factors Known to Influence Bone Remodeling

Electrical and mechanical forces

Estrogen

Androgens

Parathyroid hormone

Calcitonin

Thyroid hormones

Cortisol

Growth hormone

Osteoprotegerin

Receptor Activator of Nuclear Factor-κβ (RANK) and its ligand, RANKL

Vitamin D

Vitamin K

Vitamin B12

Macrophage colony-stimulating factor

Growth factors (IGF-1)

Cytokines (IL-1, IL-6, TGF-b)

Leptin

Indian Hedgehog gene

Vitamin D receptor gene

Collagen genes

Protein Dickkopf , LRP-5

Wnt, bone morphogenic protein

Core-binding factors

 

Bone density is measured using any of several techniques. For now, dual energy X-ray absorptiometry (DXA) is not only the most commonly used method, but also defines the diagnosis. The instruments used to perform this testing range from small, portable screening units to those designed to scan the entire skeleton. Bone density, expressed as g/cm2, is interpreted by comparing the patient’s result to control populations, as T- and Z-scores. The T-score compares the patient’s bone density to that of a young Caucasian of the same gender, while the Z-score compares the patient to a matched population in terms of age, sex, and ethnicity. The more negative the score, the greater the severity of the disease and the greater one’s risk of fracture. Using criteria adopted by several organizations, patients whose T-scores are more than 2.5 SD below the mean have osteoporosis. Those whose T-scores fall between –1.0 and –2.5 have low bone density, or osteopenia. T-scores are used to assess post-menopausal women and men over age 50. Children and younger adults are assessed using Z-scores.

Bone density has received much of the focus in osteoporosis, but other factors come into play. The overall quality of the bone is perhaps even more important in determining who will sustain a fracture. Unfortunately, bone quality is difficult to both define and quantify. It involves assessment of the microarchitecture, mineralization, and all aspects of bone remodeling. In the past, biopsies of bone were taken in order to assess quality by studying the bone at the cellular level. Before the procedure, patients were often given several doses of fluorescing drugs that bind to bone in order to determine rates of bone formation and mineralization. Because of the invasive nature of the procedure and the focus on DXA, it is rarely conducted today. There are several promising imaging techniques on the horizon that are likely to change this, notably quantitative computed tomography, quantitative ultrasound, and magnetic resonance imaging. As illustrated in the cases at the beginning of this article, osteoporosis is not a “one-size fits all” disease in terms of pathogenesis. Therefore, it makes sense that there are different needs for diagnosis and treatment.

Biochemical Markers of Bone Metabolism

Bone markers include a variety of compounds either expressed by bone cells or released during bone formation or degradation, and as such they provide information about the health of the bone. For example, markers released during resorption provide information about the activity of osteoclasts, and whether resorption is accelerated. The key to understanding the relevance of bone markers, however, is to understand that they originate at different points in bone turnover or even in different points within the life-cycle of a given cell. Following is a brief description of the most widely used bone markers and their role in bone metabolism.

The primary job of the osteoclasts is to demineralize and degrade a very localized portion of bone. To do this, the cells attach to the bone surface, form a tight seal to avoid damaging nearby bone, and secrete HCl and proteolytic enzymes. Tartrate-resistant acid phosphatase (TRAP) was one of the first enzymes associated with this activity, and the relationship between increased enzymatic activity in the serum and increased osteoclast activity has been well documented; however, the exact role of osteoclast TRAP remains unclear. Its intracellular co-localization with bone degradation products and studies of TRAP-deficient mice suggest TRAP is involved in processing some of the bone proteins taken up by the osteoclasts. Investigators have successfully used electrophoretic techniques to distinguish osteoclast-derived TRAP, known as TRAP 5b, from that originating in other cells, designated 5a. Several methods for the measurement of TRAP 5b in serum have been developed, including a kinetic assay based on 5a inhibition, chromatography methods, and immunoassays.

The cross-linking that takes place between neighboring collagen molecules in bone confers strength and flexibility. During bone resorption, fragments containing these cross-links are released into the circulation and eventually excreted in the urine. The fragments include free and peptide-bound cross-linking amino acids, pyridinioline and deoxypyridinoline, and larger fragments containing specific sequences of the telopeptide or non-helical portions near the amino- and carboxy-terminals. NTx immunoassays recognize a specific peptide sequence of the cross-linked α2(I)NH2-terminal, while CTx immunoassays recognize a region of the α1(I) chain near the carboxy-terminal.

One of the first steps of bone collagen formation involves the cleavage of the extension peptides at the amino- and carboxy-terminals of the procollagen precursors. The resulting fragments, procollagen amino-terminal propeptide (PINP) and procollagen carboxy-terminal propeptide (PICP), are specific for bone collagen activity. There is some controversy that PINP may be recycled and re-used to make additional procollagen, but PICP appears to be released as an intact subunit with one PICP fragment released for each collagen molecule incorporated into a collagen fibril. Both may be measured using immunoassay and chromatographic methods.

Bone alkaline phosphatase (BALP) is a member of a family of membrane-associated glycoproteins widely distributed throughout the body. Expressed with the liver and kidney isoenzymes by the tissue non-specific alkaline phosphatase gene, it acquires specificity through post-translational glycosylation and sialylation. The exact mechanisms involved in transitioning an area of bone from resorption to formation have eluded investigators. However, there is evidence that the BALP originating from osteoblast precursors plays a role. Studies involving cell cultures and animal models show an increase in BALP activity immediately preceding cessation of osteoclast activity and deposition of osteoid, another early step as osteoblasts begin making new bone.

The majority of osteocalcin found in the circulation originates from osteoblast activity. The protein originates from a pro-peptide that is carboxylated before additional post-translational processing and release. The degree of carboxylation, which enhances the peptide’s affinity for calcium and calcium-containing proteins, depends on the individual’s vitamin K status. After carboxylation, a 25-amino acid sequence is removed to yield biologically active osteocalcin. Most of the osteocalcin produced is incorporated into bone matrix where it is thought to limit the extent of mineralization. There is evidence that osteocalcin is also involved in osteoblast maturation and osteoclast recruitment. Circulating osteocalcin is considered a marker of bone formation, but a small amount of the protein is released during resorption. After release, intact osteocalcin is rapidly degraded to produce a number of fragments that can be measured. Fragments may also originate from degradation of bone matrix during resorption.

Samples and Assays

Various methods are used for laboratory measurement of bone markers in a variety of sample matrices (Table 2, below). Commercial assays that use serum, plasma, and urine are available. Regardless of the analyte or specimen type, concentrations tend to be highest early in the day before declining steadily after approximately 10 a.m., reflecting the diurnal pattern of bone turnover. Ideally, sample collection should be arranged to take place before the concentrations decline and samples for follow-up repeat testing should be similarly collected. The randomly collected urine specimen for NTx favored by many physicians is far from an ideal sample. If this test is desired, the best specimen comes from a 24-hour urine collection. Since this collection is not the easiest to perform as an outpatient, an alternative is to have the patient collect a first or second morning void and bring the specimen to the laboratory or their doctor’s appointment. While consistent sample collection will not eliminate the intra-individual variability observed, it will minimize it. Serum- or plasma-based specimens should be processed quickly, as several of the analytes are prone to degradation.

Table 2
Current Bone Markers and Methods
Analyte Abbreviation Sample Analysis
Bone alkaline phosphatase BALP

Serum

Heparinized plasma

Immunoassay

Electrophoresis

C-telopeptide CTx

Serum

EDTA plasma

Immunoassay
Deoxypyridinoline DPD

Serum, fasting

Urine

Immunoassay

Chromatography

N-telopeptide NTx

Serum

Urine

Immunoassay
Osteocalcin OC

Serum

Heparinized or EDTA plasma (not suitable for all assays)

Immunoassay

Chromatography

Procollagen amino terminal extension peptide PINP

Serum

Heparinized or EDTA plasma

Immunoassay
Procollagen carboxy terminal extension peptide PICP

Serum

Plasma

Immunoassay
Pyridinoline PYD

Fasting serum

Urine

Immunoassay

Chromatography

Tartrate resistant acid phosphatase TRAP 5b

Serum

Plasma

Kinetic assay

Immunoassay

Chromatography

Electrophoresis

For most clinical labs, immunoassays are the preferred methods for analysis. Immunoassays are available for NTx, CTx, PINP, deoxypyridinoline, pyridinoline, osteocalcin, and BALP. Since few automated platforms have multiple bone markers available, however, labs generally must refer some of their bone marker testing to another lab.

Of the assays mentioned above, the NTx and CTx assays are standardized in terms of what is measured, but differences are seen between assays from different vendors. Assays for deoxypyridinoline and pyridinoline vary with respect to the percentage of cross-reactivity that is observed between the assays and with respect to whether the antibody detects free versus peptide-bound cross-linked material, or a combination of the two. Similarly, the antibodies employed in the osteocalcin immunoassays vary both in the measurement of intact molecule versus fragments and with the degree of carboxylation.

Clinical Utility

While none have sufficient specificity to be diagnostic for osteoporosis, the existing group of bone markers has several uses. Physicians use them to monitor patients after initiation of drug therapy, to determine if turnover is accelerated, to assess fracture risk independent of DXA, and to manage patients who have a malignancy.

In concert with the progress made in discovering aspects of the pathophysiology of osteoporosis, pharmaceutical manufacturers have developed a number of drugs. Bisphosphanates, selective estrogen receptor modulators (SERMs), and a parathyroid hormone (PTH) analog are now available, along with calcitonin, calcium, vitamin D, and estrogen. Of the prescribed medications, only the PTH analog stimulates bone formation; the others reduce osteoclast activity or resorption.

Although DXA is used to diagnose osteopenia and osteoporosis, it typically takes 1–2 years of drug therapy before significant changes in bone density are measured, which is a long time for patients to wait to find out if their drug therapy is working. Bone markers, on the other hand, provide a more rapid means of assessment. If antiresorptive therapy is successful, markers of resorption decline from their initial starting point within 1–3 months, while the formation markers decline in approximately 3–6 months. With pro-formation therapy using the PTH analogue, patients typically show an increase in formation markers within 1–2 months of starting drug therapy with an initial decline in resorption bone markers, followed by an increase. Measurement of one of the bone markers often provides positive feedback to the patient and encourages compliance.

Several studies have demonstrated that the rate of bone turnover independently predicts the risk of fracture for osteoporosis patients. In these studies, researchers saw a significant reduction in the risk of non-spine fractures in conjunction with decreases in bone marker concentrations following initiation of antiresorptive drug therapies. Using bone markers to determine if turnover is increased is also useful in following patients at risk of secondary osteoporosis due to drug therapy with antiepileptic drugs, glucocorticoids, methotrexate, and immunosuppressives.

Some have also suggested that a woman’s marker baseline at menopause is a good indicator of her risk of developing osteoporosis. Women whose DXA T-scores were considered “normal,” but whose baseline concentrations were increased at menopause, lost more bone compared to those whose markers were within the premenopausal ranges.

An additional role for bone markers lies in the management of patients with multiple myeloma, monoclonal gammopathy of undetermined significance, and other malignant bone diseases. Establishing a baseline and monitoring these patients periodically throughout their treatment appears to provide early indications of bone metastases.

An Adjunct to Patient Management

In summary, much has been learned about the pathogenesis of osteoporosis and the markers that result from bone metabolism. But the area continues to evolve and there is much work remaining. Although none of the analytes we call bone markers are diagnostic of osteoporosis, when collected properly they are useful tools in the assessment and monitoring of patients who have or who are at risk of developing osteoporosis.

Suggested Reading

    1. Hamano T, Fujii N, Nagasawa Y, Isaka Y, Moriyama T, Okada N, Imai E, Horio M, Ito T. Serum NTX is a practical marker for assessing antiresorptive therapy for glucocorticoid treated patients with chronic kidney disease. Bone 2006;39:1067–72.
    2. Henriksen K, Tanko LB, Qvist P, Delmas PD et al. Assessment of osteoclast number and function: applications in the development of new and improved treatment modalities for bone diseases. Osteoporos Int 2007;18:681–85.
    3. Keene GS et al. Mortality and morbidity after hip fractures. BMJ 1993;307:1248–50.
    4. Knott L, Bailey AJ. Collagen cross-links in mineralizing tissues: a review of their chemistry, function, and clinical relevance. Bone 1998; 22:181–87.
    5. Parfitt AM. What is the normal rate of bone remodeling? Bone 2004;35:1–3.
    6. Seibel MJ. Clinical use of markers of bone turnover in metastatic bone disease. Nat Clin Pract Oncol 2005;2:504–17.
    7. Stokstad E. Bone quality fills holes in fracture risk. Science 2005;308:1580–81.
    8. Tortolani PJ, McCarthy EF, Sponseller PD. Bone mineral density deficiency in children. J Am Acad Orthop Surg 2002;10:57–66.
    9. U.S. Department of Health and Human Services. Bone Health and Osteoporosis: A Report of the Surgeon General. Rockville, MD: U.S. Department of Health and Human Services, Office of the Surgeon General, 2004.
    10. Worsfold M, Powell DE, Jones TJW, Davie MWJ. Assessment of urinary bone markers for monitoring treatment of osteoporosis. Clin Chem 2004;50: 2263–70.

Catherine Hammett-Stabler, PhD, DABCC, FACB is an associate professor in the Department of Pathology and Laboratory Medicine at the University of North Carolina–Chapel Hill. She serves as the director of the Clinical Toxicology, Clinical Pharmacology and Pediatric Metabolism Laboratories and as an associate director of the Core Laboratory for the McLendon Laboratories at UNC Hospitals.

The author has a relationship with the following companies that, in the context of this article, could be perceived as a real or potential conflict of interest: Roche Diagnostics and Ortho-Clinical Diagnostics.

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