Vol 32 | Issue 1 | Jan – June 2017 | page: 32-37 | Sabir Ali, Ajai Singh, Manish Yadav, Abbas Ali Mahdi, Rajeshwer Nath Srivastava
Authors: Sabir Ali , Ajai Singh , Manish Yadav , Abbas Ali Mahdi , Rajeshwer Nath Srivastava 
 Department of Orthopaedic Surgery, K. G. Medical University, Lucknow, INDIA.
 Department of Biochemistry, K. G. Medical University, Lucknow, INDIA.
Address of Correspondence
Dr. Ajai Singh
Department of Orthopaedics Surgery,
K. G. Medical University, Lucknow, India.
Introduction: Predicting the fracture healing outcomes early is one of the most important and fundamental clinical determinations made in Orthopaedics. However, till date there are no methods yet.
Materials and methods: In this cohort study, after ethical clearance and informed consent formalities, total 107 patients were analysed. The serial estimation of total urine hydroxyproline was done at different follow-ups and further correlates it with the fracture healing progression measured using RUST score.
Results: As per 24th week of radiological follow-up, these patients were divided into two groups: Normal healing (n=91) and Impaired healing (n=16) groups. The total urinary hydroxyproline levels were measured at different follow-ups showed statistically significant difference between normal and impaired healing groups, except at baseline. A statistically significant positive correlation between total urinary hydroxyproline levels with fracture healing progression.
Conclusion: The serial monitoring of total urinary hydroxyproline levels can be used as an adjunct to clinical and radiological evidence of fracture healing to predict the outcomes early.
Keywords: Total Urinary Hydroxyproline, Fracture Healing, Impaired Healing.
Fractures are common orthopaedic problem occurs most in long bones. Amongst all long bones, shaft of tibia is one of the commonest bones that are prone to fractures. Also due to lesser soft tissue coverage on anterior aspect, such fractures have relatively higher incidence of impaired healing amounting to 2 – 10% [1-2]. Non-union fracture is a dreaded complication with devastating outcomes for the patients that have required involving multiple surgical procedures, pain, morbidity, prolonged hospital stay and functional and psychosocial disability . Ability in prompt identification of patients who are at high risk of non-union will enable early appropriate targeted treatment intervention leading to a successful outcome. Such approach would benefit not only the patient’s wellbeing but also the health care system in terms of the cost implications associated with long lasting treatment interventions and prolonged hospital stay.
Hydroxyproline is released by the breakdown of collagen in the tissues, especially during bone resorption, it degraded into free amino acid that circulates in plasma, and is almost entirely reabsorbed by the kidney. At last it completely oxidized in the liver and degraded to carbon dioxide and urea [4-5]. As the hydroxyproline released during degradation of collagen cannot be reutilized in collagen synthesis, these excreted in the urine and act as a definitive indicator of bone matrix turnover [6-8]. In the present study, we plan to analyzed the serial estimation of total urinary hydroxyproline throughout the initial phase of healing in patients with diaphyseal tibial fracture.
Materials and Methods
This is a prospective cohort study conducted between 2011 to 2015 at our institutional trauma center. After obtaining ethical clearance (Ref. Code: 55 E.C.M. IIB/P6) from institutional ethical review committee and informed consent formalities, the demographic data of all enrolled patients were collected.
As per inclusion-exclusion criteria, total 119 patients of both sexes of aged between 18 and 40 years with simple, fresh (less than 03 days) traumatic diaphyseal fractures of both bones leg managed conservatively were included in the study. Also the patients included in the study were suggested to avoid diets rich in collagen. All patients included in this study were managed conservatively (reduction -setting and above knee plaster cast under general/regional anesthesia). Prior to the management, the clinical and radiological examinations were done. They were admitted for next 24-48 hours and then discharged with a standard advice.
Total urinary hydroxyproline level were estimated from the 24 hours urinary samples according to the method of Bergman and Loxley, 1970  at 04th day (baseline) and 06th & 10th weeks of post-fracture follow-ups.
The clinico-radiological examination was done at 06th, 10th, 16th, 20th, 24th post fracture weeks. The radiological progression of healing was evaluated using RUST scoring system [10-11]. The x-rays for RUST score were examined separately by two orthopaedic surgeons blindly and findings were noted separately. The average of scores was taken for final decision/analysis. The clinic-radiological evaluation at 24th week was used to label the healing as normal or impaired. Patients with normal bony healing were defined with RUST score ≥ 7 by the end of 24th week along with painless (no tenderness), motionless (no abnormal mobility) with presence of transmitted movements at fracture site. Otherwise they were labeled as impaired healing [10-11]. The clinical & radiological status (RUST Score) of union based on 24th week was then analysed against total urinary hydroxyproline level.
Statistical analysis was performed using SPSS software (SPSS Inc., Chicago, IL, USA) for Windows program (15.0 version). The continuous variables were evaluated by mean (±standard deviation) or range value when required. For comparison of the means between the two groups, analysis by Student’s t-test (unpaired) with 95% confidence interval. The correlation analysis was done using Spearman correlation coefficient. A P < 0.05 or 0.001 was regarded as significant.
Out of 128 patients, 09 were excluded as per inclusion-exclusion criteria. Out of these 119 patients who were enrolled in our study, 12 patients were lost to follow-up. So, only 107 patients were analysed. The baseline characteristics of the patients showed in Table-1, which do not show any statistically significant difference.
As per clinico-radiological status of fracture healing at 24th week, these 107 patients were divided into: Normal Healing (n = 91) and Impaired Healing (n = 16) groups. Mean RUST score at 06th, 10th, 16th, 20th, and 24th weeks of post fracture follow-up were 6.32±0.49, 7.89±0.46, 8.41±0.60, 10.22±0.90 and 11.08±0.86 respectively in normal healing patients and 4.34±0.39, 4.65±0.43, 5.06±0.47, 5.62±0.46, 5.87±0.59 respectively in impaired healing patients. The mean time of healing in normal healing patients was 17.2 ± 3.7 weeks. Mean RUST score were significantly higher at each of the radiological follow-ups in normal healing as compare to impaired healing groups (p <0.0001) [Fig-1].
Total urinary hydroxyproline level at 04th day and 06th & 10th weeks after post-fracture follow-ups were 11.24±2.25, 13.62±3.11 and 10.63±2.79 respectively in normal healing patients and 10.89±2.08, 3.87±1.92 and 2.12±0.93 respectively in impaired healing patients.
The peak total urinary hydroxyproline level was obtained at 04th day (baseline) of post fracture. Significant positive correlation was found between the peak mean total urinary hydroxyproline level (at 04th day) with the fracture healing progression at all different follow up measured using RUST scoring [Table-3; Fig-2].
Bone turnover is probably faster than in soft tissues, whereas nearly half of human collagen remains in bone. Excretion of hydroxyproline in urine is regarded as a marker of bone resorption. Approximately 50% of urinary hydroxyproline is derived from bone collagen breakdown . Increased production of collagen is allied with increase in the hydroxyproline [13-14]. In the present study, we plan to analyzed the serial estimation of total urinary hydroxyproline throughout the initial phase of healing, hypothesizing that the total urinary hydroxyproline level might be altered and showed difference in level while compared between normal and impaired healing patients.
In the present study we observed that the total urinary hydroxyproline levels were measured at different follow-ups during the fracture healing progression showed statistically significant difference between normal and impaired healing groups, except at baseline value. Furthermore, statistically significant positive correlation between total urinary hydroxyproline levels with fracture healing progression (RUST score) were also observed.
Increase in the hydroxyproline also found soon after fracture that associated with an increased production of collagen. These modifications represent the changes in levels of total and free hydroxyproline excretion in urine [15-16]. In a recent study by Das., et al. , in patients with long bone fractures, significant differences were observed between normal union and nonunion groups in case of urinary hydroxyproline levels after treatment with a positive correlation between urinary hydroxyproline in normal united group. Mukhopadhyay., et al. in their study also observed significant difference in total urinary hydroxyproline between normal union and nonunion groups, showed positive correlation with fracture healing. Our study observations relies on the results obtained by Das et al.,  and Mukhopadhyay et al.,. Thus, concluded that the serial monitoring of urinary hydroxyproline reflect the actual status of bone turnover in real time . However, small sample size was the limitation of the present study.
Serial monitoring of biochemical marker of bone turnover like urinary hydroxyproline, reflect the actual status of fracture healing progression. Thus, quantification of total urinary hydroxyproline might be used as a complementary assessment tool to assess the fracture healing progression in parallel to clinic-radiological examination. By this, we can accurately assess the healing progression at real time and also may early predict the impaired healing, which could be helpful in performing early interventional procedures. Thus, this approach would benefit not only the patient’s wellbeing but also to the total health care system in terms of the substantial cost associated with long lasting treatment interventions and prolonged hospital stay. However, further multicentric studies were recommended.
This study was funded by Indian Council of Medical Research, New Delhi (No5/4-5/12/Trauma/2011-NCD-I).
Conflict of interest
The authors have no conflict of interests in this article.
This study was supported by Department of Biochemistry, King George’s Medical University, Lucknow, Uttar Pradesh, India and funded by funded by Indian Council of Medical Research, New Delhi.
1. Patel M., McCarthy J. J., Herzenberg J. Tibial Nonunion. Medscape Reference (2011).
2. Reed L. K., Mormino M. A. Distal tibia nonunions. Foot and Ankle Clinics 2008 13(4),725–735.
3. Marsh D. Concepts of fracture union, delayed union, and nonunion. Clinical Orthopaedics and Related Research 1998; (355, supplement),S22–S30.
4. Prockop DJ. Isotopic studies on collagen degradation and the urine excretion of hydroxyproline. Journal of Clinical Investigation 1964; 43(3): 453-460.
5. Lindstedt S and Prockop DJ. Isotopic studies on urinary hydroxyproline as evidence for rapidly catabolized forms of collagen in the young rat. Journal of Biological chemistry 1961; 236(5): 1399-1403.
6. Prockop DJ, Kivirikko KI, Tuderman L, Guzman NA. The biosynthesis of collagen and its disorders. New England Journal of Medicine 1979; 301(1): 77-85.
7. Mukhopadhyay M, Sinha R, Pal M, Bhattacharyya S, Dan A, Roy MM. Role of common biochemical markers for the assessment of fracture union. Indian Journal Clinical of Biochemistry 2011; 26(3): 274-278.
8. Jakob C, Zavrski I, Heider U, Brux B, Eucker J, Langelotz C, Sinha P, Possinger K, Sezer O Bone resorption parameters [carboxy-terminal telopeptide of type-I collagen (ICTP), amino-terminal collagen type-I telopeptide (NTx), and deoxypyridinoline (Dpd)] in MGUS and multiple myeloma. European Journal of Haematology 2002 69(1): 37-42.
9. Bergman I, Loxley R. The determination of hydroxyproline in urinary hydrolysates. Clin Chim Acta. 1979; 96:125–30.
10. Sabir Ali, Ajai Singh, Avinash Agarwal, Anit Parihar, A A Mahdi, R. N. Srivastava. Reliability of the RUST Score for the Assessment of Union in Simple Diaphyseal Tibial Fractures. Int J Biomed Res. 2014; 05 (05): 333-335.
11. Whelan DB, Bhandari M, Stephen D, Kreder H, McKee MD, Zdero R, Schemitsch EH. Development of the radiographic union score for tibial fractures for the assessment of tibial fracture healing after intramedullary fixation. Journal of Trauma and Acute Care Surgery 2010; 68(3):629–632.
12. Lowry M, Hall DE, Brosnan JT. Hydmxyproline metabolism by the rat kidney: distribution of renal enzymes of hydroxyproline catabolism and renal conversion of hydroxyproline to glycine and serine. Metabolism 1985; 34(10): 955-961.
13. Sjoerdsma A, Davidson J, Udenfriend S, Mitoma C. Increased excretion of hydroxyproline in Marfan’s syndrome. Lancet 1958; 2(7054): 994-994.
14. Block RJ, Weiss KW, Almquist HJ, Carroll DB, Gordon WG, Saperstein S. Amino Acid Handbook; Methods and Results of Protein Analysis. Springfield 1956; 3(record: 19571404492): 346.
15. Hodgkinson A and Thompson T. Measurement of the fasting urinary hydroxyproline: creatinine ratio in normal adults and its variation with age and sex. Journal of Clinical Pathology 1982; 35(8): 807-811.
16. Deacon AC, Hulme P, Hesp R, Green JR, Tellez M, Reeve J. Estimation of whole body bone resorption rate: a comparison of urinary total hydroxyproline excretion with two radioisotopic tracer methods in osteoporosis. Clinical Chemistry Acta 1987; 166(2-3): 297-306.
17. Soumi Das, Soumya Ghosh, Keya Pal, Arunima Chaudhuri, Soma Datta .Changes in biochemical markers in blood and urine in case of malunion and nonunion after fracture of long bones. Saudi Journal of Sports Medicine 2015; 15(3): 269-275.
18. Manish Yadav, Sabir Ali, Ajai Singh, Abbas Ali Mahdi. Hydroxyproline for the Assessment of Fracture Union. EC Orthopaedics 2016; 3(4): 368-376.
|How to Cite this Article: Ali S, Singh A, Yadav M, Mahdi A A, Srivastava R N. Role of Total Urinary Hydroxyproline for the Assessment of Fracture Union. Journal of Bone and Joint Diseases Jan – June 2017;32(1): 32-37.|