Millions of people in the U.S. are affected by osteoporosis and the number is expected to rise as the population ages. Postmenopausal women are most often affected. Also at risk are elderly males and individuals with calcium or vitamin D deficiency, hypogonadism, hyperparathyroidism, hyperthyroidism, Paget’s disease, malignancy, gastrointestinal disease, and connective tissue disease, as well as those who smoke, abuse alcohol, or take certain medications. Afflicted individuals are at significantly increased risk for bone fracture (wrist, spine, proximal femur, humerus) leading to chronic pain, disability, and even death. Treatment options include vitamin D and calcium supplementation, bisphosphonates (eg, alendronate, risedronate), calcitonin, hormone replacement therapy (HRT), parathyroid hormone (PTH, amino acids 1-34) and raloxifene, a selective estrogen receptor modulator (SERM).

Osteoporosis is a preventable disorder. Since patients are asymptomatic in the early stages, current guidelines recommend evaluating fracture risk in postmenopausal women and in men once they reach 50 years of age.¹ The World Health Organization (WHO) has developed the FRAX™′  tool, which is available online, for this risk assessment.² Factors used in the tool are listed in Table 1.

Bone mineral density (BMD), which is the basis of osteoporosis diagnosis, is recommended for women ≥65 years of age and men ≥70 years. When indicated by their risk profile, BMD is recommended for postmenopausal women and for men between 50 and 70 years old. Furthermore, BMD is recommended for patients who have suffered a fracture,¹ and it should be accompanied by a thorough evaluation for secondary osteoporosis in the young, as secondary causes account for 64% of all osteoporosis in young men and women.³ Additionally, secondary causes account for 20% of osteoporosis in postmenopausal women.³ The National Osteoporosis Foundation (NOF) recommends consideration of secondary causes in all postmenopausal women and in men ≥50 years of age.¹

Screening for secondary causes begins with a history and physical examination followed by routine laboratory testing (chemistry profile, CBC, sedimentation rate or C-reactive protein, TSH, and 24-hour urine calcium). Individuals with suspected vitamin D deficiency (eg, those with persistent, nonspecific musculoskeletal pain; the elderly; housebound individuals; those with malabsorptive syndromes; those treated with anticonvulsants; etc.) should also be tested for 25-hydroxyvitamin D concentration. If abnormal results are obtained, further evaluation is recommended. The causes of secondary osteoporosis are listed in the NOF guidelines,¹ and testing for the most common of these is listed in Table 2.

Bone turnover markers (formation and resorption) are helpful in predicting the rate of postmenopausal bone loss^4,5 and in assessing the risk for bone fracture.^6-8 When assessing risk, the combined use of BMD and bone turnover markers is more effective than use of either risk factor alone (Fig. 1). Baseline levels (ie, pretreatment levels) of some markers may predict those who will benefit most from selected therapy and/or may predict the amplitude of response. For example, Chestnut et al found that the odds of BMD gain from HRT increased by a factor of 5.0 for every 30 nmol BCE/mmol creatinine increase in baseline urine NTx level.⁹ Either bone formation or bone resorption markers may be used for therapeutic monitoring. After 3 to 6 months of antiresorptive therapy, bone markers predict BMD response and changes in fracture risk.^8-12 Thus, they provide a more real-time assessment of therapeutic response than BMD measurements (ie, 3 to 6 months vs 1 to 2 years for BMD).^9,13 Since evidence of therapeutic response is rapidly available, bone markers may assist in rapid optimization of drug dosage and in encouraging patient compliance.

Various guidelines address the use of bone turnover markers (Table 3). Multiple factors that contribute to variability in measurement must be considered when using them, however. These factors include patient age, gender, and menopausal status; diurnal variation; food intake; certain medications; and lack of assay standardization. Variability can be minimized by 1) use of age-, gender-, and menopausal-specific reference ranges; 2) collection of samples at the same time of day to minimize diurnal variation when monitoring therapy; 3) overnight fasting to minimize dietary affects; 4) use of a washout period for any drugs that affect bone or calcium metabolism prior to sample collection for baseline measurements; and 5) use of the same method and laboratory to circumvent assay standardization issues.

• Individuals being evaluated for secondary osteoporosis

• Individuals with decreased BMD and/or adult bone fracture

• Individuals who are about to begin therapy

• Individuals who are currently receiving therapy

Secondary Osteoporosis

The list of secondary causes of osteoporosis is long; thus, a large number of laboratory tests are available. As mentioned previously, the primary screening tests include a chemistry profile, CBC, sedimentation rate (ESR) or C-reactive protein (CRP), TSH, 24-hour urine calcium, and 25-hydroxy-vitamin D in at-risk individuals. Additional tests are listed in Table 2.

• Osteocalcin: This immunoradiometric assay (IRMA) measures the level of human osteocalcin (bone Gla protein or BGP) in serum. Both the intact molecule (amino acids 1-49) and the N-midfragment (amino acids 1-43) are detected.

• Alkaline Phosphatase, Bone Specific (BSAP): This immunochemiluminescence assay (ICMA) measures the level of the bone-specific isoenzyme of alkaline phosphatase in serum. Crossreactivity with other alkaline phosphatase isoenzymes is 15% for liver, 5% for intestinal, and <1% for placental alkaline phosphatase.¹⁶ BSAP provides a general index of bone formation and a specific index of total osteoblast activity.

• C-telopeptide (CTx): This electrochemiluminescence assay (ECLIA) measures the level of collagen type I C-telopeptide in serum.

• N-telopeptide (NTx): This enhanced ICMA measures the level of type I collagen cross-linked aminoterminal peptide in urine.

• Pyridinium Collagen Cross-Links: This high performance liquid chromatography (HPLC) method measures urinary levels of total pyridinoline (Pyr, hydroxylysylpyridinoline) and total deoxypyridinoline (D-Pyr, lysylpyridinoline) collagen crosslinks.

• Tartrate Resistant Acid Phosphatase (TRAP): This enzymatic method measures the serum level of acid phosphatase that is resistant to tartrate inhibition. TRAP includes acid phosphatase from bone and other sources such as alveolar and monocyte-derived macrophages, placenta, spleen, and erythrocytes.

• Hydroxyproline: This colorimetric method measures the urinary level of total, free, or total and free hydroxyproline.

Tests that can be used to screen for or rule out secondary osteoporosis are listed in Table 2. For example, abnormalities in FSH, LH, estrogen, and testosterone can establish sex hormone deficiency as a secondary cause of osteoporosis. Calcium and vitamin D concentrations can point to a host of nutritional and absorption disorders. An abnormal CBC can suggest benign or malignant hematologic disorders, and an ESR or CRP can be used to screen for inflammatory disorders. TSH can be used to screen for thyrotoxicosis and PTH for parathyroid disorders, while a chemistry profile and CBC can be used to screen for alcohol abuse and other disorders.

Glucocorticoid excess (endogenous or exogenous) inhibits bone formation, leading to bone loss and fracture. Patients with a disorder characterized by a high blood cortisol concentration and those receiving long-term glucocorticoid therapy should be monitored with osteocalcin to assess the degree of bone formation inhibition or with BMD.

BSAP and osteocalcin are the most effective markers of bone formation¹⁷ and are particularly useful for monitoring bone formation therapies. They can be utilized to monitor patients on antiresorptive therapies as well. Osteocalcin and BSAP levels are generally concordant in osteoporosis. Contraindications include renal disease (osteocalcin) and hepatic disease (BSAP).

NTx (Osteomark^®′) and CTx (CrossLaps^®′) are the preferred markers of bone resorption. These markers are more specific to bone than other bone turnover markers. Although an HPLC test for both total pyridinoline (Pyr) and deoxypyridinoline (D-Pyr) is available, D-Pyr is more specific to bone than Pyr and, therefore, the better marker.

Studies indicate that baseline levels of NTx are useful for predicting BMD response to HRT, and serial measurements reflect response to HRT and bisphosphonates (alendronate).^18,19 The magnitude of NTx change during and following therapy provides an advantage when monitoring short-term responses.^20,21

Studies have shown that baseline CTx levels are useful for assessing fracture risk in postmenopausal women and indicating response to HRT and bisphosphonate therapy as early as 3 months after initiation of therapy.^22-24 Such response predicted an increase in BMD²³ or a reduced fracture risk.²⁴

The newer resorption markers detailed above are more reliable than TRAP and hydroxyproline. TRAP is adversely affected by enzyme inhibitors in serum, has limited stability and analytical sensitivity, and lacks specificity for bone. The assays for urinary hydroxyproline are tedious, and levels are affected by diet and crossreaction with the C1q fraction of complement. Hydroxyproline is useful only when careful patient preparation and sample collection guidelines are followed.

Primary screening test results for some of the disorders causing secondary osteoporosis are listed below.

Alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), bilirubin, and alkaline phosphatase may be increased in alcoholism. Since ALT is less sensitive to alcoholism than AST, an increased AST:ALT ratio is likely. Uric acid and the mean corpuscular volume (MCV) may also be increased.

Celiac disease may manifest with anemia associated with decreased iron or folic acid; increased ESR or CRP; decreased calcium, vitamin D, albumin, sodium, and potassium; and increased alkaline phosphatase.

Inflammatory bowel disease may manifest with anemia (decreased hemoglobin), increased platelet and white blood cell (WBC) counts, and increased ESR or CRP. The severity of anemia and the magnitude of increased ESR correlate with severity of the disease.

Primary biliary cirrhosis is initially detected by a 2-fold or greater elevation in the alkaline phosphatase concentration; GGT is also elevated. In the initial stages, bilirubin is normal and ALT and AST are minimally increased. Levels of these analytes increase as the disease progresses.

Low calcium levels are consistent with low dietary intake, inadequate gastrointestinal absorption such as occurs in Celiac disease, and vitamin D deficiency. Elevated calcium levels are consistent with hyperparathyroidism (PTH also increased), leukemia, lymphoma, multiple myeloma, thyrotoxicosis, and idiopathic hypercalciuria.

The CBC with differential may reveal an increased WBC count (except in hairy cell leukemia), decreased red blood cell (RBC) count, decreased platelet count, decreased hemoglobin, and immature WBCs.

Plasma cell proliferation leads to bone resorption, leading to increased blood calcium levels. Serum creatinine, urea nitrogen, and uric acid levels may also be elevated, but alkaline phosphatase is usually normal. The CBC with differential is likely to indicate normochromic, normocytic anemia; some patients have megaloblastic anemia. The ESR will be elevated.

A CBC with differential may reveal decreased RBC count, decreased hemoglobin, and sickle cells in those with sickle cell anemia. Anemia, decreased WBC count, decreased platelets, and a decreased percentage of lymphocytes are consistent with systemic mastocytosis. Some patients may have increased WBC count and/or increased percentage of eosinophils, basophils, monocytes, or platelets. A low hemoglobin, hematocrit, and MCV along with anisocytosis, poikilocytosis, target cells, and microcytic, hypochromic, nucleated RBCs are consistent with thalassemia.

FSH, LH, and estradiol blood levels are high, while testosterone levels are low, in Klinefelter’s syndrome. Low blood levels of FSH, LH, estrogen, and testosterone are consistent with panhypopituitarism that may lead to secondary osteoporosis. An elevation in FSH and LH levels is consistent with premature ovarian failure. Increased FSH and LH levels, along with decreased testosterone and dihydrotestosterone, are consistent with Turner syndrome.

Thyroid hormone excess leads to increased bone resorption. A low TSH level, along with high free T4 and free T3 levels, suggests thyrotoxicosis. Alkaline phosphatase and serum calcium levels may be elevated.

Low levels of vitamin D (25-hydroxy) are consistent with low dietary intake and inadequate gastrointestinal absorption such as occurs in Celiac disease.

In adults, elevated levels of bone turnover markers (both formation and resorption markers) may be associated with increased bone loss, decreased BMD, and increased risk for bone fractures. The higher the rate of bone turnover, the greater the rate of bone loss, particularly at or soon after menopause. Independent of BMD, elevated levels may predict future decreases in BMD and an increased risk for fractures.^10,25 Excessive rates of bone turnover are associated with primary osteoporosis as well as secondarily with hyperthyroidism, glucocorticoid excess, hyperparathyroidism, clinical use of gonadotropin-releasing hormone (GN-RH) agonists, or deficient skeletal growth and maturation.

During or post therapy, a 30% to 60% decrease in bone turnover levels (into the premenopausal range) is indicative of therapeutic response (Table 4). Levels of bone resorption markers decrease within 1 to 3 months of therapy, whereas bone formation marker values decrease later (within 3 to 9 months).^9,13 Such decreases predict an increase in BMD over 2 years.¹³ The absence of decreasing levels post therapy may be due to patient noncompliance, inadequate dosage, or ineffective therapeutic agent.

Levels of bone turnover markers are affected by marker specificity for bone collagen and metabolic clearance (liver uptake, renal excretion, etc.), and thus are of unequal sensitivity and specificity. Estimates of net excess of bone resorption over bone formation may be misleading; however, either bone formation or bone resorption markers will reflect the overall rate of bone turnover. Bone marker levels are also affected by the timing of sample collection due to diurnal variation (early morning peak and afternoon nadir). Intra-individual biological variations range from a low of 20% in NTx to a high of 63% in deoxypyridinoline by HPLC.²⁶ Interpretation is highly dependent on the specific marker and method of analysis used.

Osteocalcin is the major non-collagenous matrix protein of bone. Serum levels are increased in primary and secondary hyperparathyroidism, secondary osteosarcoma, healing bone fractures, hyperthyroidism, Paget’s disease, acromegaly, impaired renal glomerular function and renal failure, and in response to coumarin anticoagulants, slow-release sodium fluoride, phenytoin and 1,25-dihydroxyvitamin D. Osteocalcin is also increased in elderly men (>60 years) and postmenopausal women. Levels in Mexican-Americans are generally increased relative to non-Hispanic Caucasians.²⁷

Osteocalcin decreases in response to successful osteoporosis therapy. It is decreased in hypoparathyroidism, hypothyroidism, Cushing’s syndrome, and sometimes in multiple myeloma and malignant hypercalcemia. Decreased levels may also be attributed to hemolysis, lipidemia, and excessive freezing and thawing of the sample. There is a 10% to 30% difference between the peak and nadir levels.

C-telopeptide (CTx): Increased CTx results indicate an increased rate of bone resorption. Increased levels are associated with osteoporosis, osteopenia, celiac disease, Paget’s disease, primary hyperthyroidism, rheumatoid arthritis, and non-adult onset growth hormone deficiency. A decreased level following HRT or bisphosphonate therapy suggests favorable response and may predict an increase in BMD or a decrease in fracture risk.^23,24

A CTx result within the premenopausal reference range does not rule out osteoporosis nor the need for therapy.

N-telopeptide (NTx): Increased NTx results indicate an increased rate of bone resorption. Increased urinary levels predict a rapid decrease in BMD; the greater the NTx elevation, the greater the decrease in BMD.²⁹ Increased NTx may also be observed in osteopenia, osteoporosis, celiac disease, Paget’s disease, primary hyperparathyroidism, acromegaly, rheumatoid arthritis, growth hormone deficiency (non-adult onset), and malignant metastases to bone. Early postmenopausal women (<3 years postmenopausal) with urinary baseline NTx levels ≥67 nmol/mmol experience the greatest benefit from hormone replacement therapy (HRT).⁹ A ≥30% decline in urinary N-telopeptide concentration following 6 months of HRT or alendronate is indicative of a positive therapeutic response in postmenopausal women.⁹ Eighty-eight percent of women with such a decline were shown to have maintained or increased their bone mineral density (BMD) at 1 year.⁹ Similar declines in NTx and similar correlation with BMD was observed post alendronate therapy.¹¹

An NTx result within the premenopausal reference range does not rule out osteoporosis nor the need for therapy.

Pyridinium Collagen Cross-Links (pyridinoline [Pyr] and deoxypyridinoline [D-Pyr]) are absent from most tissues, including skin. D-Pyr is more specific for bone than Pyr since the ratio of D-Pyr to Pyr is high in bone relative to other tissues. Levels correlate with bone histomorphometry and are increased at menopause and in osteoporosis, primary hyperparathyroidism, Paget’s disease, rheumatoid arthritis, hyperthyroidism, and bone metastases. Decreases are associated with response to estrogen therapy and bisphosphonate therapy and correlate with increased BMD.

Tartrate Resistant Acid Phosphatase (TRAP) results are not specific to bone; erythrocytic acid phosphatase released during in vitro clotting may falsely increase values as well as other non-bone sources. Levels are increased in osteoporosis, osteomalacia, Paget’s disease, primary hyperparathyroidism, metastatic cancer, advanced renal failure, and growing children.

Hydroxyproline is a modified amino acid that is present in all collagen. Urinary levels are increased significantly in Paget’s disease and to a lesser degree in primary and secondary hyperparathyroidism. It is also increased in osteoporosis, hypo- and hyperthyroidism, burns, psoriasis, acromegaly, inborn errors of metabolism, hydroxyprolinemia, familial aminoglycinuria, and possibly lymphomas, mammary carcinoma, and bone metastatic prostate carcinoma. Substantial increases may also occur in inflammatory conditions due to crossreactivity with complement factor C1q. Diets high in gelatin may cause falsely elevated results. Hydroxyproline is poorly correlated with bone resorption assessed by calcium kinetics and with histomorphometry. Decreased levels, however, virtually exclude increased bone turnover.³⁰

Osteoporosis is a preventable disorder and patient management is moving toward a scenario in which postmenopausal women and elderly men will be evaluated for osteoporosis and fracture risk.^1,14,15 BMD should be offered to at-risk individuals as well as elderly women (≥65 years of age) and men (≥70 years of age).¹ Counseling regarding life-style changes and possible preventive therapy including HRT or bisphosphonates is encouraged. Although BMD remains the primary tool for diagnosis and monitoring, bone turnover markers are useful for assessing fracture risk, predicting bone loss, and monitoring antiresorptive therapy.^1,14