Journal of Cardio-diabetes and metabolic disorders

REVIEW ARTICLES
Year
: 2022  |  Volume : 2  |  Issue : 1  |  Page : 1--8

The interpretation of biochemical investigations in the management of metabolic bone disorders


Roop B Kalia, Sajid Ansari, Anil Regmi 
 Department of Orthopaedics, All India Institute of Medical Sciences, Rishikesh, Uttarakhand, India

Correspondence Address:
Roop B Kalia
Department of Orthopedics, Level 6, All India Institute of Medical Sciences, Rishikesh, Uttarakhand
India

Abstract

A bone is basically a combination of the organic matrix, inorganic minerals (calcium phosphate), and vitamins that make the structural framework. The two counteracting processes, bone formation and bone resorption, make the bone a metabolically active tissue that undergoes continuous remodeling. The laboratory evaluation of serological and urinary markers is important in the diagnosis of suspected bone disease such as osteoporosis, rickets/osteomalacia, fluorosis, and primary hyperparathyroidism, which are common metabolic bone diseases (MBD), whereas a few rare MBDs include Paget’s disease, fibrous dysplasia, osteogenesis imperfecta, tumor-induced osteomalacia, etc. Calcium and phosphate level in serum and urine reflects the status of metabolism of bone. Markers of one formation include: alkaline phosphatase (ALP), osteocalcin (OCn), and procollagen I peptides: the amino (N-) terminal propeptide (PINP) and the carboxy (C-) terminal propeptide (PICP). Markers of bone resorption include hydroxyproline (OHP), hydroxylysine (HYL), deoxypyridinoline (DPD), pyridinoline (PYD), bone sialoprotein (BSP), osteopontin (OP), tartrate-resistant acid phosphatase 5b (TRAP 5b), carboxy-terminal crosslinked telopeptide of type 1 collagen (CTX-1), amino-terminal crosslinked telopeptide of type 1 collagen (NTX-1), cathepsin K (CTSK), urinary calcium, and acid phosphatase. Novel biochemical markers such as periostin, cathepsins, RANK-L, secreted frizzled-related proteins (sFRP), Wnt inhibitory factor-1 (WIF1), Dickkopfs (Dkk) 1–4, sphingosine-1-phosphate (S1P), sclerostin, fibroblast growth factor (FGF)-23, and miRNA are also the markers of bone metabolism. Biochemical markers of bone metabolism provide a potentially important clinical tool for assessing and monitoring MBD. These markers are quick to appear after any derangement in physiology. Still, we must keep in mind that the characteristics of any marker are at present primarily a function of the assay used for the assessment of the marker. That continued efforts aimed at improving the analysis and interpretation of markers that are known today must continue.



How to cite this article:
Kalia RB, Ansari S, Regmi A. The interpretation of biochemical investigations in the management of metabolic bone disorders.J Cardio Diabetes Metab Disord 2022;2:1-8


How to cite this URL:
Kalia RB, Ansari S, Regmi A. The interpretation of biochemical investigations in the management of metabolic bone disorders. J Cardio Diabetes Metab Disord [serial online] 2022 [cited 2022 Nov 29 ];2:1-8
Available from: http://www.cardiodiabetic.org/text.asp?2022/2/1/1/349194


Full Text



 Introduction



Bone is a dynamic living organ that provides structural and mechanical support and plays a central role in the mineral homeostasis of the body. Bone is basically a combination of the organic matrix, inorganic minerals (calcium phosphate), and vitamins that make the structural framework. Type I collagen forms approximately 94% of the organic bone matrix.[1] The two counteracting processes, bone formation and bone resorption, make the bone a metabolically active tissue that undergoes continuous remodeling. This process is also called bone turnover. Bone turnover is necessary for preserving the mechanical integrity of the skeleton and regulating calcium and phosphorus homeostasis. The overall bone mass depends on the balance between resorption and formation. Cancellous bone accounts for 20% of bone mass and 80% bone surface area and is metabolically more active and undergoes more rapid remodeling. Every year, 25% of cancellous and 2%–3% of compact bone undergo remodeling. The cells responsible for resorption and formation are osteoclasts and osteoblasts, respectively, which are guided by numerous factors (systemic and local) that regulate the function of these cells types.[2]

Many biochemical markers have been studied to monitor the various bone diseases and reflect the treatment’s effect. Broadly, they are conventionally divided into bone formation and bone resorption markers. An ideal biochemical marker should be specific for one of the metabolic processes in the bone so as to be clinically useful. Its complete physiology, including the mode of clearance, metabolism, and plasma half-life, should be known, and it should be easily measurable and stable in serum or urine. In reality, no such ideal marker exists and hence there is a need for multiple markers.[3]

Osteoporosis, rickets/osteomalacia, fluorosis, and primary hyperparathyroidism are some of the common metabolic bone diseases (MBD), whereas a few rare MBDs include Paget’s disease, tumor-induced osteomalacia, fibrous dysplasia, osteogenesis imperfecta, etc.[4] The laboratory evaluation of serological and urinary markers is essential in diagnosing suspected bone disease, some of them summarized in [Table 1].{Table 1}

Serum calcium

Calcium values may vary between laboratories; its normal plasma calcium level ranges from 9.1 to 10.7 mg/I00 mL.[5] Methods that can estimate calcium involve the precipitation of calcium as the oxalate complexing with ethylenediaminetetraacetic acid (EDTA). Nowadays newer method is in use that depends on emission, or absorption, spectrophotometry.[6] More than 50% of plasma calcium is ionized; the rest of the calcium is bound to protein. In ultrafiltrates, such as the renal glomerular filtrate, the calcium concentration is about 58% of the plasma calcium concentration. The ionized calcium is physiologically the most significant fraction, which is under hormonal control. Protein binding of calcium varies with protein concentration and with pH. The plasma calcium concentrations are incomplete when the total protein concentration is not accounted for. Plasma calcium concentration and plasma proteins are influenced by venous stasis during venipuncture, changing in position from the supine to upright, diurnal variation.[6] Neglect to account for this may diminish the value of sequential studies of plasma calcium. Parathyroid hormone and calcitonin are the main hormones to regulate calcium homeostasis. Hypercalcemia is due to a regulation defect of parathyroid hormone secretion, increased renal tubular reabsorption of calcium, and increased absorption from the gut, or increased bone destruction may lead to hypercalcemia. Hypoparathyroidism or end-organ insensitivity to parathyroid hormone, as in pseudohypoparathyroidism, may cause hypocalcemia. Hyperparathyroidism or hypoparathyroidism can be discovered by repeated determinations of plasma calcium or by challenging the homeostatic mechanisms—chlorthalidone, or EDTA infusions, and by simultaneous determination of plasma parathyroid hormone levels.[7]

Phosphate

Phosphorus homeostasis is regulated by a complex process that involves the interplay between parathormone (PTH) and vitamin D endocrine system [Figure 1].[8] Recent studies have shown that apart from vitamin D, the chief regulator of phosphate, several factors regulate phosphate homeostasis. These have been collectively known as the “phosphatonins,” some of which are fibroblast growth factor 23 (FGF-23), secreted frizzled-related protein 4 (sFRP-4), FGF-7, and matrix extracellular phosphoglycoprotein. Their role has been associated with various hypophosphatemic and hyperphosphatemic disorders, such as oncogenic osteomalacia, X-linked hypophosphatemic rickets, autosomal-dominant hypophosphatemic rickets, autosomal recessive hypophosphatemia, and tumoral calcinosis.[9]{Figure 1}

The evaluation of urinary excretion of phosphate is done as the initial analysis for hypophosphatemia to determine if the process involves a normal or abnormal renal response to hypophosphatemia. Low serum phosphate, under normal conditions, should lead to an increased reabsorption of phosphate and low urine excretion.[10] A normal renal response involves two pathways, namely decreased intestinal absorption and intracellular shifting of phosphate. To determine the renal response, the measurement of urinary phosphate is required. It can be obtained with a 24-h urine phosphate collection or calculated with the formula for fractional excretion of filtered phosphate (FEPO4) using random urine and plasma phosphate and random urine and serum creatinine values [FEPO4 = (UPO4 × PCr × 100)/(PPO4 × UCr)]. An appropriate renal response is determined by a 24-h urinary phosphate of less than 100 mg/day or a FEPO4 that is less than 5%.[11] There is a considerable variation in plasma phosphate ranging from 2.4 to 4.4 mg/100 mL in healthy adults. Many factors influence the plasma phosphate levels with the transient reduction seen after food intake and lower levels in renal tubular defects, potassium depletion, phosphate depletion, hyperparathyroidism, and hypercorticism. Higher than normal values are seen in conditions such as hypoparathyroidism, thyrotoxicosis, growth hormone excess, hypocorticism, and in some patients with Paget’s disease.

Bone formation biomarkers

Alkaline phosphatase

Alkaline phosphatase (ALP) is an enzyme produced largely in the liver and found in the blood. It is involved in the transport of substances from the intracellular compartment to the extracellular region across the membrane. In bone, ALP may also be involved in the breakdown of pyrophosphate, which is a potent inhibitor of calcium phosphate deposition at the extracellular level.[12] All ALP assays are based on serum with numerous estimation methods, including heat denaturation, electrophoresis, precipitation, selective inhibition, and, more recently, immunoassays. Of these methods, immunoassay is considered to be the method of choice because these assays have a better analytical sensitivity.[13] Newer immunoassays allow simple and rapid quantitation of either enzyme activity or enzyme mass. However, the specificity of immunoassays for the measurement of the bone fraction of ALP is decreased by many factors, for example, the same gene codes the liver and bone fractions, and therefore, the epitopes for discriminating between these isoforms are due solely to posttranslational differences. There is also cross-reactivity of the liver isoform. Therefore, in subjects with high liver alkaline phosphatase (ALP), results of bone ALP measurements may be artificially high, leading to false-positive results.[14]

Osteocalcin

It is a small protein exclusively synthesized by osteoblast, odontoblasts, and hypertrophic chondrocytes. OCn synthesis is dependent on vitamin K, which posttranslationally modifies the gene product with gamma-carboxyglutamate (Gla) residues; due to this modification, osteocalcin (OCn) is also known as bone Gla protein. Much of the newly synthesized protein is incorporated into the bone matrix, where it functions in binding calcium. During bone resorption, OCn is degraded; however, a majority, up to 70%, also enters circulation.[15] OCn is considered a specific marker of osteoblast function, and serum levels of immunoreactive OCn have been shown to correlate well with the bone formation rate. Several OCn assays have been reported, and the interpretation of OCn values is a function of its true physiological characteristics and the method utilized for analysis.[16] OCn is also incorporated into the bone matrix, and hence some studies have suggested that OCn fragments may be released even during bone resorption. This may be especially true for some smaller N-terminal fragments of OCn, which are found in individuals with high bone turnover. The ensuing heterogeneity of OCn fragments in serum results in considerable limitations in the clinical application of this highly specific marker.[17],[18] Some immunoassays utilize antibodies that detect the intact OCn molecule. However, only one-third of the total OCn serum pool represents the entire OCn. To circumvent this problem, newer assays measure the largest degradation product of OCn, the 1–43 (N-terminal/mid-molecule) fragments. After drawing, quick processing of the blood sample is essential for most assays since a loss of reactivity is noted within a few hours at room temperature. The same applies to sera subjected to multiple thawing or prolonged storage at temperatures above −25°C.

Procollagen I peptides

These are derivatives of the collagen type 1, which are in abundance in bone. Preprocollagens are precursor molecules that are characterized by short terminal extension peptides: the amino (N-) terminal propeptide (PINP) and the carboxy (C-) terminal propeptide (PICP) and, once secreted into the extracellular space, are cleaved and released into the circulation. Specific immunoassays and studies that can measure both these peptides have shown a good correlation between serum PICP levels and the rate of bone formation.[19],[20] Although the clinical relevance of PICP in evaluating MBD is still viewed with skepticism, serum PINP appears to be of greater diagnostic validity.[21],[22]

Markers of bone resorption

Hydroxyproline

Hydroxyproline (OHP) is formed intracellularly from the posttranslational hydroxylation of proline and constitutes 12%–14% of mature collagen’s total amino acid content. Most OHP is oxidized in the liver, but a small proportion (~10%) is excreted in the urine. Approximately 90% of collagen-derived OHP excretion is in the form of peptides resulting from collagen degradation. The remainder is in the form of fragments of the N- and C-terminal procollagen peptides cleaved during collagen synthesis. When bone resorption is increased, urinary OHP excretion rises. Still, when bone turnover is low, urinary OHP is not a reliable index of bone resorption since the other sources (for example, C1q complement component) can contribute up to 50% of the total urinary OHP.[18],[23] For this reason, inflammatory conditions can cause dramatic increases in urine excretion of hydroxyproline. Another issue is that the PINP extension peptide, cleaved from procollagen during bone formation, also contains a collagenous region that is degraded to OHP. It can be measured in a 24-h urine collection or, more conveniently, in fasting, second voided morning samples, expressed as a ratio to the creatinine concentration.[24]

Hydroxylysine

Hydroxylysine (HYL) is derived from lysine by posttranslational hydroxy modification. It is released into the circulation during collagen degradation. It has two forms: galactosyl hydroxylysine (GHYL) and glucosylgalactosyl-hydroxylysine (GGHYL), among which GHYL is more specific for bone biomarkers because it is only derived from bone resorption. In contrast, GGHYL can be found in skin and complement molecules of C1Q.[25] The high levels of GHYL, which can be measured without preanalytical hydrolysis and the extraction by high-performance liquid chromatography (HPLC) in fracture patients, suggest a possible defect in bone collagen.[26] The study is still unclear about the use of GHYL to assess osteoporosis and the evaluation of osteoporotic treatment. Its clinical application is still limited because of the absence of facile routine methods for its measurement.[27]

Deoxypyridinoline

Deoxypyridinoline (DPD) is formed by the proteolytic breakdown of crosslinked collagen during bone resorption and then released in circulation and excreted in the urine.[21] It mechanically stabilizes collagen by crosslinking between individual collagen peptides. It is used as a specific biomarker for bone resorption, as most DPDs are found in the bone and dentin. As DPD is excreted in the urine in free (40%) and peptide-bound (60%) forms, so previously, the DPD has been pretreated with preanalytical hydrolysis and extraction before HPLC analysis.[26] Recently, for the direct detection of urinary-free DPD, automated chemiluminescence immunoassay and enzyme immunoassay have been developed. In monitoring patients with bone pathology and MBD, the immunoassay approaches measurements of free DPD in urine have provided the possibility for its clinical application.[27]

Pyridinoline

Pyridinoline (PYD) is the degradation product of mature collagens formed during the extracellular maturation of fibrillar collagens and released into circulation.[27] PYD is also found in cartilage, bone, ligaments, and blood vessels, so it is a nonspecific biomarker of bone resorption compared to DPD. PYD exhibits a long-term chemical stability in both the free and conjugated forms by HPLC analyses.[28]

Bone sialoprotein

Bone sialoprotein (BSP) is the extracellular matrix component of mineralized tissues such as bone, dentin, cementum, and calcified cartilage, which forms approximately 8% of all noncollagenous proteins found in bone and cementum. It is composed of phosphorylated glycoprotein with a molecular weight of 60–80 kDa.[29] As osteoblasts, odontoblasts, and osteoclasts generate BSP, it is considered an essential factor for cell-matrix adhesion processes and stimulation of osteoclast-mediated bone resorption. Many studies have developed immunoassays for the measurement of BSP in serum. A significant correlation between bone-specific alkaline phosphatase and OCn has been demonstrated by serum levels of BSP in osteoporosis patients. It shows a great potential as a bone resorption biomarker for osteoporotic assessment.[27]

Osteopontin

Osteopontin (OP) is expressed by transformed cells, macrophages, activated T-lymphocytes, specialized epithelial cells, and bone cells as a phosphorylated glycoprotein.[30] In recent studies, overexpression of OP reveals less resistance to postmenopausal osteoporosis than women with normal OP levels.[31] In treating intermittent parathyroid hormone in menopausal osteoporosis, plasma OP level can be used as a biomarker.[27]

Tartrate-resistant acid phosphatase 5b

Osteoclasts secrete tartrate-resistant acid phosphatase 5b (TRAP 5b) during bone resorption.[32] It is metabolized in the liver and then excreted in the urine after hydrolysis. It has high specificity and high sensitivity in comparison with other bone biomarkers. It also signifies as a reference for osteoclast activity and numbers. It can be specifically detected in serum by immunoassays. Its serum level is evaluated to identify limited or extensive bone metastasis in breast cancer patients and to monitor alendronate treatment efficiency.[33]

Carboxy-terminal crosslinked telopeptide of type 1 collagen

Carboxy-terminal crosslinked telopeptide of type 1 collagen (CTX-1) and amino-terminal crosslinked telopeptide of type 1 collagen (NTX-1) are released during collagen degradation. These are investigated and used as bone resorption biomarkers. Enzyme-linked immunoassay (ELISA) is used to measured CTX-1 with a monoclonal antibody against an octapeptide sequence (linear beta-8AA octapeptides, EKAHD-β-GGR) in the α-1 (I) chain of the β-isoform. It also indicates the response to bisphosphonate therapy for postmenopausal osteoporosis.[34] It is a specific and sensitive biomarker of bone resorption, which can be rapidly done. However, serum CTX-1 is influenced by food intake, and blood withdrawal must occur in the fasting state because food intake substantially decreases the levels of CTX-1.[35]

Amino-terminal crosslinked telopeptide of type 1 collagen

The urinary NTX-1 has been used as a bone resorption biomarker to assess fracture risk in postmenopausal women.[35] NTX-1 is stable in urine at room temperature for up to 24 h and is usually quantified by ELISA with a urine sample. The urinary NTX-1 is selected as the preferred biomarker compared with serum CTX-1 for practical application because it is not affected by food intake and prevents blood withdrawal.[27]

Cathepsin K

Cathepsin is a member of the cysteine protease family, and it has 11 isoforms. Osteoclasts secrete cathepsin K (CTSK) by degrading bone matrix proteins in bone resorption defects.[36] Type 1 collagen, OP, and osteonectin are its degradation products. CTSK is an essential factor in bone resorption as they are expressed at the ruffled border of actively resorbing osteoclasts. Increased bone loss occurs in postmenopausal women and patients with osteoporosis, Paget’s disease, rheumatoid arthritis, and ankylosing spondylitis.[37],[38],[39],[40] After treatment with the bisphosphonate alendronate, its levels decline.[41]

The serum level of CTSK is a potential biomarker for fracture prediction and bone-mineral density.[42]

Urinary calcium

Fasting urine calcium concentration has been used for assessing the skeletal loss in either a 24-h sample or in a spot or first-morning specimen corrected for creatinine.[43] As urinary calcium is substantially affected by diet, renal function and handling, and excesses in hormones including PTH and estrogen, it lacks diagnostic sensitivity and specificity. But the level of urine calcium and its negative predictive value indicate high bone turnover. Low urine calcium values (0.15 mg/mg creatinine) are unlikely in patients with increased bone turnover, except when the skeleton is poorly mineralized because of calcitriol and calcidiol deficiencies or increased PTH.[44]

Acid phosphatase

Five isoenzymes of the lysosomal enzyme acid phosphatase are found in the blood. Its significant sources are bone, prostate, platelets, erythrocytes, and spleen. The bone isoenzyme is derived from osteoclasts.[45] It is present in high concentration and excreted into the microenvironment between the membrane sealing zone and the bone matrix. TRAP is the acid phosphatase that retains its activity after treatment from bone and other tissues.[46]

Novel biological markers of bone metabolism

The biochemical markers of bone metabolism have some limitations, such as being protein-based, reflecting the function of osteoblasts and osteoclasts rather than osteocytes and having unspecific metabolic activity between various skeletal compartments; new biological markers are now introduced as a marker of bone metabolism[47],[48] [Table 2].{Table 2}

Periostin: The periostin (POSTN) protein may serve as a marker of periosteal metabolism, as suggested by its name. The protein is a matricellular Gla-containing protein that may serve as a biomarker for periosteal tissue metabolism and reflects endosteal bone remodeling.[49] Additionally, POSTN regulates collagen crosslinking, which could also affect bone strength by regulating bone formation and bone mineral density (BMD). Several ELISAs have been developed, mainly in-house assays, that can detect it in peripheral blood.[50]

RANK-L: One of the main regulators of osteoclast formation and function is the receptor activator of the NF-*B ligand (RANKL)/RANK/osteoprotegerin system. Before glucocorticoid therapy, serum RANK-L levels were increased in patients with systemic autoimmune diseases.[51]

The Wingless (Wnt) signaling molecules: The Wnt signaling pathway plays a crucial role in osteoblast differentiation and activity.[52] A number of secreted proteins prevent ligand-receptor interactions, including soluble FRP-related proteins (sFRP), Wnt inhibitory factor-1 (WIF1), and Dickkopfs (Dkk) 1–4. The Wnt signaling pathway and its regulatory molecules, including Dkk-1 and sFRP, are implicated in bone turnover abnormalities associated with osteoporosis, arthritis, multiple myeloma, and bone metastases from prostate and breast cancer.[53]

Sphingosine-1-phosphate (S1P): A lipid mediator, S1P, acts on different cell functions via G protein-coupled receptors (SP1R1, SP1R2, and SP1R3).[54] Serum S1P levels were associated with high levels of bone resorption—but not bone formation—markers, low BMD, and a higher risk of prevalent vertebral fractures in postmenopausal women.[55]

Sclerostin: Several osteocyte signaling pathways regulate bone formation as a result of sclerosing dysplasias, sclerosteosis, and van Buchem disease.[56] Healthy adult women and men with normal bone mineral density and bone structure have higher serum levels of sclerostin, which reflect local bone production appropriately. In order to detect changes in sclerostin levels in blood, different immunoassays have been developed, including three commercially available tests.[57] Sclerostin levels are slightly higher in girls before puberty, tend to decrease in both sexes during puberty, but remain significantly higher in boys after puberty. Sclerostin levels were found to be significantly higher in postmenopausal women.[58]

FGF-23: It is a predominantly circulating factor found in osteoclasts; it negatively regulates the levels of inorganic phosphorous and 1,25-dihydroxyvitamin D3 in the blood.[59] A C-terminal assay that detects both intact FGF-23 and C-terminal fragments can be used to measure circulating FGF-23 using immunoassays detecting only the intact molecule.[59]

MicroRNAs (miRNAs): These are single-stranded, small RNA molecules (*22 nucleotides in length). By binding to specific sequences in the 3′ untranslated regions of the target mRNA, they either induce translational repression or cleavage of the target mRNA.[60] Patients with osteoporosis had upregulated levels of nine miRNAs. The expression of five of these miRNAs (miR21, miR23a, miR24, miR25, miR100, mir125b) was also significantly increased in hip bone tissue.[61] Besides regulating the normal function of osteoblasts and osteoblasts, miRNA can also be affected by bone diseases, such as osteoporosis. It is possible to measure miRNA levels in PBMCs, which are circulating reservoirs of osteoclast precursors.[62]

 Conclusion



Biochemical markers of bone metabolism provide a potentially important clinical tool for assessing and monitoring MBD. These markers are quick to appear after any derangement in physiology. Still, we must keep in mind that the characteristics of any marker are at present largely a function of the assay used for the assessment of the marker. That continued efforts aimed at improving the analysis and interpretation of markers that are known today must continue.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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