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 Table of Contents  
Year : 2021  |  Volume : 5  |  Issue : 3  |  Page : 245-251

A review on biomarkers in clinical osteoporosis - Significance of hydroxyproline

1 Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Mysuru, Karnataka, India
2 Department of Orthopedics, JSS Medical College and Hospital, JSS Academy of Higher Education and Research, Mysuru, Karnataka, India

Date of Submission16-May-2021
Date of Acceptance20-Jun-2021
Date of Web Publication7-Sep-2021

Correspondence Address:
Dr. Gurupadayya Bannimath
Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Mysuru - 570 015, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_91_21

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Osteoporosis is a chronic disorder in both men and women, where it decreases the bone quality and quantity, which may lead to a fall in bone mass density. It mostly occurs because of reduced peak bone mass or excessive formation or resorption during bone remodeling. Various degradation products of bone collagen are pyridinoline (Pyr), deoxypyridinoline, N-terminal telopeptides, and hydroxyproline (Hyp), which are excreted in the urine. Among that, Hyp is a crucial biomarker used to correlate osteoporosis condition. An overview of all the biomarkers and the signed note of Hyp with different analytical methods are provided in the current piece of work. To quantify amounts in biological samples, various methods have been developed for Hyp analysis, such as the colorimetric technique, gas chromatography, gas chromatography-mass spectrometry, and widely used high-performance liquid chromatography tests to detect the concentration of Hyp in different matrices. This review states the importance of biomarkers and Hyp's significance for detecting osteoporosis, clinical manifestation, and different analytical methods for Hyp. The study indicates that hydroxyproline is a unique biomarker used to detect osteoporosis. It is also showing the clinical manifestation of osteoporosis and discussed different analytical methods for hydroxyproline.

Keywords: Bone mass density, high-performance liquid chromatography, hydroxyproline, osteoporosis

How to cite this article:
Adugani S, Bannimath G, Sastry P. A review on biomarkers in clinical osteoporosis - Significance of hydroxyproline. Biomed Biotechnol Res J 2021;5:245-51

How to cite this URL:
Adugani S, Bannimath G, Sastry P. A review on biomarkers in clinical osteoporosis - Significance of hydroxyproline. Biomed Biotechnol Res J [serial online] 2021 [cited 2022 Oct 7];5:245-51. Available from: https://www.bmbtrj.org/text.asp?2021/5/3/245/325618

  Introduction Top

Osteoporosis is a systemic skeletal condition in which bone mass or bone tissue loss can further lead to invoice and low bone mass density (BMD) (density of bone mass) and reduced bone strength. According to World Health Organization (WHO) statistics, more than 8.9 million fractures occur worldwide each year. Osteoporosis can affect both men and women, but this condition is a more severe issue for postmenopausal women and older people at the age of 45. BMD can then assess the magnitude of this problem from the frequency of fractures that lead to reduced BMD (osteoporosis). There are multiple measures to diagnose fractures and osteoporosis, which are T-score, BMD test (DXA), and FRAX.[1] A significant way to classify an individual with an elevated risk of fracture is to diagnose by 2.5 standard deviations < bone mineral density in the hip and spine is compared with the bone mass of healthy and adult individuals, as calculated by dual energy X-ray absorptiometry (DXA).[2] Different biomarkers are used to diagnose the disease's state, which is the detectable predictor of a biological condition. The biochemical bone formation and bone resorption markers are mostly used and detailed in [Table 1].
Table 1: Biochemical markers

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  Pathogenesis of Osteoporosis Top

Bone strength represents two primary components, bone strength and bone quality.[2] Bone strength depends on BMD, whereas bone grade refers to the totality of properties and features that impair the bone's ability to withstand fractures. Bone metabolism is a continuous remodeling process that is generally kept in a tight balance between old or injured bone resorption (modeling) and new bone modification (remodeling). It is influenced by bone mass density (BMD) and is significantly correlated with bone capacity.[3] The bone is modeled and remodeled from birth to early adulthood and at the age of 30 years reaches peak bone mass. The process of modeling and remodeling consists of different cellular events on the bone surface. The peak bone mass is strongly affected by heritable and environmental factors that can be reduced by nutritional deficiency, hormonal imbalance (female estrogen deficiency), physical activity, and sex. The three primary cells involved in bone remodeling are osteoclasts, osteoblasts, and osteocytes. From these, Osteoclasts play membrane infolding, Osteoblasts helps in the resorption process, and osteocytes secrete collagenase and other enzymes respectively. Stem cells are used for resorbing bone which is derived from osteoclasts. Osteoblasts (bone-remodeling cells) are derived from mesenchymal precursors. Osteocytes are adult osteoblasts trapped within the calcified bone which are interlinked by long dendritic processes. In the ossification process, osteoclasts (bone resorption cells) play a critical role in bone resorption.[3] It is assumed that they provide a contact network to relay data about mechanical forces that can change the bone structure and bone resorption. The removal of damaged bone plays a crucial role in adult osteocytes or bone-forming cells, signaling the need for adaptive remodeling and regeneration of new bone growth. The density of bone mass reduces as bone turnover is higher than the rate of bone formation. There would be osteocyte depletion due to a rising age of 50 years of postmenopausal women and increased resorption levels, leading to osteoporosis. [Figure 1] shows the detailed mechanism of osteogenesis and bone turnover.
Figure 1: General mechanism of osteogenesis and bone turnover

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  Clinical Manifestation Top

Bone strength and bone quality can be described that bone mineral density shows the number of minerals in a specific bone volume (mostly Ca2+ and P3−). It measures osteoporosis (a condition marked by decreased bone mass where the density of bone is <2.5 standard deviations from normal). In clinical settings, the single photon absorptiometry (SPA) measurement of BMD was originally designed for the calculation of bone mass, and dual photon absorptiometry can calculate density in the subsidiary skeleton and the axial skeleton. Later, it is represented as grams per cubic centimeter (g/cm3) utilizing dual X-ray absorptiometry (DXA) which can quantify the areal BMD.[4] An alternative method for measuring bone density in the axial skeleton is quantitative computed tomography (QCT). Instead of areal bone density (g/cm2) measured by DXA, QCT can calculate the actual volumetric bone density in g/cm3. Cortical and trabecular elements of the vertebral body can be studied using QCT.[4] DXA has many benefits relative to DXA and SPA methods. It can measure the bone density in the spinal, proximal femur, distal radius regions, and complete body BMD. It generates both high and low protons to calculate X-ray penetration into the body.[4] DXA scan can occur at three significant hips, lumbar spine, or forearm sites. The best indicator of potential hip fracture is hip bone density measurement. BMD expressed between 2 standards: T score (BMD comparison with a healthy person) and Z score (BMD comparison is performed between the healthy individuals of the same age and sex). T score is the number of standard deviations from which the effects of the patient's measurements are calculated to reach (positive T score) or fall below (negative T score). T score standards are used by older age and postmenopausal women.[5] The WHO declared osteoporosis as a T score of less or equal to −2.5 and a T score between −1.0 and −2.5 for osteopenia as shown in [Figure 2].
Figure 2: Measurement of bone density by T score differentiation between normal bone, osteopenia, and osteoporosis

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  Availability of Various Biomarkers for Detection of Osteoporosis Top

Bone metamorphosis is a complex and constant remodeling mechanism between the damaged bone and new bone development in a tightly coupled equilibrium. Quantitative changes in markers represent the complex mechanism of bone biotransformation. For example, osteogenesis and bone turnover markers are substantially higher in postmenopausal women than in premenopausal women, indicating bone loss with estrogen deficiency.[2] Biomarkers important to bone biotransformation include bone formation markers, bone resorption markers, and bone metabolism markers' modulation. Biomarkers are a detectable indication of some biological situation or disorder that the processes of underlying bone disorders may be used to diagnose.

Urinary Ca2+

Ca2+ ions play a fundamental role and provide the skeleton with rigidity. Urinary Ca2+ homeostasis is regulated in the gastrointestinal tract (GIT) and bone by parathyroid hormone (PTH). The vast predominance (99%) is found in the bones. The total amount of Ca2+ stored by adults is around 1–2 kg generally between 8.5 and 10.5 mg/dL. Standard values are collected on urine samples over 24 h, with average between 100 and 250 mg of Ca2+. Urinary Ca2+ elimination is inexpensive to measure and represents the association between glomerular filtration levels and tubular reabsorption rates and the amount of Ca2+ released during bone resorption which is composite by calciotropic factors, such as PTH and Vitamin D.[6] Most of this disseminated Ca2+ is bound to albumin. Because of this, increases in the serum protein levels can affect overall Ca2+ levels in the blood. Via absorption from the intestine and turnover from the bone, Ca2+ reaches the extracellular liquid. It is eliminated through absorption into the GIT, urine, sweat loss, and bone deposition. Urine Ca2+ levels will reflect dietary intake. High urinary Ca2+ (hypercalciuria) is due to overconsumption of dietary Na+ intake, increased intestinal absorption of Ca2+, defective renal tubular absorption of Ca2+, and high bone resorption. The number of urinary calcium releases in different ages is shown in [Table 2].
Table 2: Analysis of urinary Ca2+

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In extracellular fluid, only a portion of the Ca2+ contained is present and used in the form of ionized Ca2+. PTH tightly regulates ionized Ca2+. High concentrations of Ca2+ (>300 mg/24 h) in the urine are often signs of an overactive parathyroid gland. PTH is released in response to serum Ca2+ levels: Parathyroid Ca2+-sensing receptors induce PTH to release when serum Ca2+ levels are less. PTH acts to raise the level of serum Ca2+, increase Ca2+ resorption in the renal tubule, and decrease phosphorus resorption at the same time. PTH also activates Ca2+ resorption from the bone and increases the Vitamin D synthesis, promoting the absorption of Ca2+ from the intestine. Hyperparathyroidism results in abnormal absorption and elevated serum Ca2+ concentrations, leading to hypercalcemia, hypercalciuria, and hyperphosphaturia, for the reasons mentioned above. Thus, Ca2+ levels in the urine are gradually increased in the formation of hyperparathyroidism. The concentration levels of Ca2+ by parathyroid cells and its effect on bone resorption are shown in [Figure 3].
Figure 3: Mechanism of increased concentration levels of Ca2+

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Hydroxyproline (Hyp) is an altered amino acid produced by the posttranslational hydroxylation of type 1 collagen integral proline residues. Hyp and its metabolites are biologically and biochemically unique amino acids. It comprises one-third of the amino acids in the proteins of collagen. It aids in the stabilization of collagen, whereas elastin, the second main containing protein, is about 2% of Hyp.[7] It is also a vast extracellular component in connective tissues (skin, tendon, cartilage, vascular system vessels, and bone). In clinical trials, measures of the normal total biotransformation of urinary Hyp and the ratio of excretion of Hyp to creatinine have been commonly used in both metabolic bone dysfunction and changes arising during average development, menopause, and aging. During the age 50–60, there is a degradation of bone and the stored Hyp in the collagen is released and it is not reutilized; 90% is degraded to the free amino acid form and passes through the glomerular tubules where Hyp is almost wholly resorbed and catabolized in the liver and transformed to urea and carbon dioxide.[2] The remaining 10% of Hyp is released in small polypeptide chains that pass through the glomerulus and are filtered and excreted in the urine without further metabolism. Urinary Hyp peptide excretion would be most useful in analyzing disease of connective tissues at the clinical level. It represents the quantitatively significant fraction of Hyp released by collagen breakdown (1 h).

Acid phosphatase

An enzyme acid phosphatase is localized in different organs mainly in lysosomes. It is resistant to l (+)-tartrate. In the blood, tartrate-resistant acid phosphatase and five isoenzymes are found in bone, platelet cells, red blood cells, and the spleen respectively. The isoenzyme bone is obtained from osteoclasts, where it is located in large amounts, and is bio-transformed between the membrane sealing region and the osteoid in the microenvironment. Acid phosphatase is a dynamic enzyme that plays a significant role in the process of bone turnover.[8] It is released into circulation by “leakage,” during bone turnover and after detachment of the osteoclast's sealing zone. Due to its size, assays for acid phosphatase are serum or plasma-based – average values of acid phosphatase range between 0 and 0.8 U/L. Abnormal levels of acid phosphatase in the blood may indicate black water illness, hyperparathyroidism, and myeloma.[9]

N-telopeptide and C-telopeptide

During bone turnover, the collagen fragments with attached cross-links of amino and carboxy-terminal are released. These are called telopeptides. Bone resorption biomarkers such as N-telopeptides (NTX-1) and C-telopeptides (CTX-1) are widely investigated and used in the urine. These are Type 1 collagen. NTX are measured by immunoassay with an antibody to the N-telopeptide fragment's alpha-2 chain. CTX are calculated by a monoclonal antibody immunoassay (enzyme-linked immunosorbent assay) against an octapeptide sequence in the β-isoform alpha-1 chain. The urinary NTX-1 is chosen for practical use as the preferred biomarker relative to serum CTX-1 as it is not affected by food consumption and inhibits the removal of blood.[10]

Pyridinoline and deoxypyridinoline cross-links

The nonreducible pyridinium cross-links, pyridinoline (Pyr) and deoxypyridinoline (D-Pyr), are produced by the posttranslational modification of lysine and hydroxylysine, which stabilize the mature collagen. Approximately 3:1 ratio of Pyr and D-Pyr is released from the bone and D-Pyr is bone-specific. Most of the bone and dentin contain DPD, and it is used as a primary biomarker for bone resorption. During bone resorption, the cross-linked collagens are proteolytically broken down, and then, the DPD is released into the bloodstream and excreted by the urine.[10] In both free and conjugated forms, Pyr shows long-term stability by high-performance liquid chromatography (HPLC) analysis. During resorption, nearly 60% of the cross-links are released and bound to proteins and the other 40% are free. Pyr is present in soft tissues such as ligaments, tendons, and articular cartilage, and D-Pyr is bone-specific. The HPLC or immunoassay methods are used for the analysis of nonmetabolized pyridinium cross-links, which are not metabolized in urine either before or after hydrolysis. The overall summary of the detection of bone markers is shown in [Table 3].
Table 3: Summary of detection techniques for bone resorption biomarkers

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  Discussion Top

Significance of hydroxyproline-A urinary biomarker for the detection of osteoporosis from other biomarkers

The primarily formed structural protein of bones that obtains about 90% of the organic bone matrix is Type I collagen. C-terminal telopeptide or Hyp type I collagen (CTx-I) is released into circulation as it is broken down during the bone resorption process. Hyp [Figure 4] is a modified amino acid produced from the posttranslational hydroxylation of type 1 collagen integral proline residues.

Apart from other biomarkers, Hyp is said to be an essential and collagen-rich biomarker. Collagen fibers provide bone with tensile strength. It consists of 14% of the total amino and imino content released during the bone breakdown. Some breakdown of procollagen product molecules that are not absorbed into mature collagen creates a limited percentage of urinary Hyp.[11] When the rate of bone resorption is high, there is an elevated rate of Hyp excretion. Hyp urinary estimation is used in normal and abnormal situations for the turnover of bone collagen. Hyp excretion can be measured in a 24-h urine sample, expressed in fasting as a ratio to creatinine concentration.[12] The amino acid sequence in the collagen is abundant in proline. Around 50% of proline side chains are posttranslationally hydroxylated to form Hyp since it is excreted in the form of peptides. The proline-Hyp dipeptide (Php) also acts as a biomarker in nonhydrolyzed urine.[13] A significant product of collagen breakdown is Hyp. Hyp can manipulate signaling pathways to control disease progression associated with collagen,[14] as depicted in [Figure 5]. During the inflammation process, the t-Hyp present in the collagen is released to diagnose associated bone diseases and tumors.[14] Bone degradation will occur during collagen breakdown or other bone fractures, and this accumulated Hyp will be removed from the bone. Still, it will not be recycled to create new collagen. This released Hyp is excreted by urine and transferred to the liver by the remaining 10%–20%.[15] It is common to test for Hyp in the urine and serum. The spectrum of Hyp in recent research differs according to various age groups and gender, as shown in [Table 4].
Figure 4: Structure of hydroxyproline

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Figure 5: Signaling pathway to control disease progression

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Table 4: Estimation of hydroxyproline

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Paget's syndrome, hyperparathyroidism, osteomyelitis, hyperthyroidism, and skeletal metastases can be found with elevated Hyp levels. However, other disorders, including rheumatoid arthritis, Kussmaul's disease, abnormalities in the musculoskeletal system (Marfan syndrome), enlargement of peripheral body parts (acromegaly), monosomy X, and pregnancy, can also cause a rise in this marker

  Clinical Applications on Bone Biomarkers Top

Finding out the individuals at increased higher risk of fracture

According to many prospective studies, BMD measurements are given above or below threshold ranges than healthy women (T score [−2.5] SD).[16] Diagnosing osteoporosis at a higher risk of fracture is important, while biochemical bone turnover markers are useful to test bone loss.[17] In this condition, markers of bone turnover can be used to define the rate of bone degradation and assess the risk of fractures. Levels of bone markers decrease rapidly with antiresorptive therapies. The levels reached after 3–6 months of treatment have been more strongly associated with breakdown outcomes than BMD changes. In osteoporosis (alendronate, risedronate, and raloxifene) treatment trials, bone turnover markers tend to tightly associate with fracture risk reduction than with bone mineral density reduction.[18]

Predicting the advantages of care that patients undergo

Various studies have been reported that estrogen-, calcitonin-, and biphosphates-treated women with higher pretreatment levels of bone resorption. Conceptually, patients with increased bone resorption levels are more likely to respond to anticatabolic agents, while patients with low bone resorption are more likely to benefit from anabolic therapy.[19] However, the average number of patients expected to be treated to prevent a single fracture over 1–3 years of risedronate therapy was slightly smaller in patients relative to high baseline turnover in low baseline women.

Promoting adherence to therapy

Early improvements in bone turnover markers can indicate changes in BMD and fracture frequency in response to therapy.[20] Sixty-five postmenopausal women were randomized into three separate groups: no surveillance, nurse visit monitoring, whether bone turnover markers are effective in detecting and monitoring bone marker measurement daily the tracked groups (nurses or markers) showed dramatically (57%) improved combined commitment to therapy after 1 year of treatment with raloxifene relative to no surveillance, although there was no disparity between the forms of monitoring.[21] Effect trial of oral risedronate; “reinforcement” was the expression of a doctor's input based on the findings of the urinary NTX bone marker. The subgroup of patients who got a supportive message (based on a substantial reduction in urinary NTX) saw a significant (57%) improvement in overall commitment to therapy relative to no monitoring compared to people in the no-reinforcement group. There was no distinction, however, between the monitoring forms.

Clinical risk assessments for osteoporosis

Fractures and clinical risk aspects are the relevant significant sequelae of osteoporosis. Several interfering aspects contribute to the risk of brittle bone, including clinical, medical, genetic, and nourishing factors which cause osteoporosis.[22] A woman with postmenopausal or with the age of 50–60 years who undergo loss in body weight and low percentage of body fat content are the independent risk factors to postmenopausal osteoporosis addition to the history of hyperthyroidism and the risk of hip fracture in older women are shown [Table 5].
Table 5: Clinical risk factors in osteoporosis

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  Methods Top

Different analytical methods for quantification of hydroxyproline

The amount of Hyp in osteoporotic patients is observed in serum and urine analysis. There are various methods developed for the study of Hyp such as gas chromatography-mass spectrometry, liquid chromatography, and liquid chromatography with mass spectrometry. Hyp was also detected using HPLC with an electrogenerated chemiluminescence detector by tris(2,2′-bipyridyl) ruthenium (II) as a reagent. A recent literature survey reveals that HPLC analysis is performed using derivatives like phenylisothiocyanate, and the colorimetric method was determined mostly. The HPLC approach has been commonly used in different matrices to assess the Hyp and derivatization process for fluorescence detection because there is a lack of chromophore or fluorophore in its chemical structure. The detailed conditions for the analytical method development for Hyp are shown in [Table 6].
Table 6: Analytical methods for hydroxyproline

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  Conclusion Top

In recent years, there has been tremendous progress in understanding the use of biomarkers and their osteoporosis applications. These biomarkers are the essential markers in the diagnosis of the diseased condition in this research. Hyp is primarily found in connective tissue proteins such as collagen, aside from the other biomarkers, and its hydroxyl groups help stabilize the collagen fiber. Hyp is not recycled during collagen breakdown; 90% is degraded to the free form of amino acid and passes into the glomerulus; in comparison, Hyp is almost exclusively resorbed and catabolized into urea and carbon dioxide in the liver and biotransformed through urine. The various analytical methods used to detect the concentration of Hyp and other biomarkers in urine samples are indicated in this study. HPLC is a unique easy technique widely used in different matrices for the determination of Hyp. There are other tools, such as gas chromatography and colorimetric detection, including HPLC. The clinical manifestation addresses the osteoporotic disease and the diagnosis of BMD trials and numerous approaches for its diagnosis. If biomarker changes exist, they are not disease-specific and represent the overall skeleton metabolism. Among all the bone resorption biomarkers, Hyp has shown great potential as a sensitive and stable bone biomarker for early detection of osteoporosis. Hyp has a versatile range of abilities to manipulate signaling pathways to control disease progression associated with collagen.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]


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