|Year : 2015 | Volume
| Issue : 3 | Page : 269-275
Changes in biochemical markers in blood and urine in case of malunion and nonunion after fracture of long bones
Soumi Das1, Soumya Ghosh2, Keya Pal1, Arunima Chaudhuri3, Soma Datta4
1 Department of Biochemistry, Burdwan Medical College, Burdwan, West Bengal, India
2 Department of Orthopedics, Burdwan Medical College and Hospital, Burdwan, West Bengal, India
3 Department of Physiology, Burdwan Medical College and Hospital, Burdwan, West Bengal, India
4 Department of Pathology, Burdwan Medical College and Hospital, Burdwan, West Bengal, India
|Date of Web Publication||2-Sep-2015|
Department of Physiology, Burdwan Medical College and Hospital, Krishnasayar South, Borehat, Burdwan - 713 102, West Bengal
Background: Biochemical markers provide a dynamic view of the remodeling process of bone. Aims: To monitor biochemical markers in long bone fractures as they reflect the actual status of fracture healing. Materials and Methods: The present study after taking Institutional ethical clearance and informed consent of the subjects. Patients with normal fracture union served as controls (50); Patients with impaired fracture union served as cases. They were further divided into two groups: Fracture nonunion (20) and fracture malunion (30). Serum calcium, serum alkaline phosphatase (ALP), urinary calcium to creatinine ratio, and urinary total and free hydroxyproline were measured. Results: Serum ALP, serum calcium, urinary total and free hydroxyproline, and urinary calcium to creatinine ratio were increased in all patients after fracture. Serum ALP, urinary total and free hydroxyproline decreased after treatment in nonunion patients. Serum ALP reached to lower level of reference interval in this group. But urinary total and free hydroxyproline decreased to less than normal level. Serum calcium increased up to 2 weeks following treatment and then reduced to normal level within 1-month in fracture normal union and malunion groups. Serum calcium reduced at a very slow rate after treatment in nonunion patients and remained within reference interval. Significant differences were found between normal union and nonunion groups in case of serum ALP, urinary total and free hydroxyproline levels after treatment. Significant differences were found between normal union and nonunion groups in serum calcium level after treatment on day 7 and 14. Conclusions: Serial monitoring of biochemical markers of bone turnover reflect the actual status of bone resorption and bone formation, respectively. Thus, they can be used as an adjunct to clinical and radiological evidence of fracture healing.
التغيرات في المؤشرات الكيميائية الحيوية في الدم و البول في مرضي عدم الالتئام و سوء الالتئام في كسور العظام الطويلة
خلفية البحث: توفر العلامات الكيميائية الحيوية وجهة نظر ديناميكية لعملية إعادة تشكيل العظام.
الأهداف: رصد المؤشرات الكيمايئة الحيوية في كسور العظام الطويلة ؛ لأنها تعكس الحالة الفعلية لالتئام الكسور.
الطرق والمواد: أجريت هذه الدراسة بعد أخذ الموافقة المؤسسية والأخلاقية من المرضى واختيرت عينة الدراسة من 50 مريضا من الذين تعرضوا لكسور التحمت بطربقة عادية وهم يشكلون العينة الضابطة. واختيرت مجموعة مرضى قسمت إلى مجموعتين تكونت المجموعة الأولى من 20 مريضا يمثلون مجموعة عدم الالتحام، و مجموعة من 30 مريضا يمثلون مجموعة سوء الالتحام. و تم قياس نسبة الكالسيوم وانزيم الفوسفات القلوي فى مصل الدم بالاضافة الى نسبة الكالسيوم إلى نسبة الكرياتنين في البول ، وكمية البول ، ونسبة الهايدروكسيبرولين الحر.
النتائج: ارتفعت نسبة الفوسفات القلوي ونسبة الكالسيوم فى مصل الدم ، وزادت كمية البول وزادت ايضا نسبة الهيدروكسيبرولين الحر ونسبة الكالسيوم إلى نسبة الكرياتنين في البول في كل المرضى الذين لديهم كسور. ولكن لوحظ انخفاضا فى معدل انيزم الفوسفات القلوي الحر في هذه المجموعة. وقد انخفضت ايضا كمية البول ونسبة كسيبرولين الحر ولوحظ ايضا ان نسبة الكالسيوم قد ارتفعت بعد انقضاء اسبوعين من العلاج ثم انخفضت بعد مرور شهر في المرضي من العينة الضابطة وفى الذين كانوا يعانون من عدم الالتحام. وفى المقابل تدنى مستوى الكالسيوم ببطء ملحوظ بعد انقضاء فترة العلاج فى مجموعة عدم الالتحام الى ان بقى فى المستوى العادى. وسجلت فروقات ملحوظة فى نسبة انزيم الفوسفات القلوي وكميه البول ونسبة الهيدروكسوبرولين فى مرضى الالتحام العادى وفى مجموعة عدم الالتام بعد انقضاء فترة العلاج وقد وجدت فروقات ذات دلاله احصائيه بين مرضي الالتحام العادي و مجموعة سوءالالتحام و مجموعة عدم الالتحام في مستوي الكالسيوم في الدم بعد أسبوع او أسبوعين من العلاج.
الاستنتاجات : الرصد المتسلسل للمؤشرات الكميائية الحيوية لدورة العظم تعكس الوضع الفعلي لامتصاص العظام وبنائها على التوالي. وعليه يمكن استخدام المؤشرات سالفة الذكر كعاملا مساعدا يدلل على التئام الكسور بالاضافة الى الدلائل السريرية والاشعاعية.
Keywords: Biochemical marker, fracture long bone, healing
|How to cite this article:|
Das S, Ghosh S, Pal K, Chaudhuri A, Datta S. Changes in biochemical markers in blood and urine in case of malunion and nonunion after fracture of long bones. Saudi J Sports Med 2015;15:269-75
|How to cite this URL:|
Das S, Ghosh S, Pal K, Chaudhuri A, Datta S. Changes in biochemical markers in blood and urine in case of malunion and nonunion after fracture of long bones. Saudi J Sports Med [serial online] 2015 [cited 2019 May 21];15:269-75. Available from: http://www.sjosm.org/text.asp?2015/15/3/269/164304
| Introduction|| |
Biochemical markers provide a dynamic view of the remodeling process of bone. ,,, Biochemical markers of bone turnover are generally divided into two subclasses: Bone resorption and bone formation markers. The bone resorption markers are related to osteoclast resorption of matrix and include tartrate-resistant acid phosphatase and degradation products of type I collagen in protein matrix especially hydroxyproline, telopeptides, etc. Bone formation markers are osteocalcin and bone-specific alkaline phosphatase (S-bone ALP) produced from osteoblasts. Serum calcium originates from bone resorption. Bone resorption markers are increased soon after a fracture event, and bone formation markers increased gradually thereafter. Also, changes in bone resorption and bone formation markers for patients with nonunion of long bones have been reported. ,,,,,,
Bone remodeling (BR) plays an integral role in the union of fractures which consists of removal of older bone tissue followed by callus formation. ,,,,, Clinical examination and radiological assessment are the cornerstone of fracture union. Other approaches for the clinical evaluation of bone status include study of bone mineral density (BMD), Radionucleotide scan, bone histomorphometry, and biochemical markers. While X-ray, BMD, and radionucleotide scan provide information primarily about the bone macrostructure, integrity, quantity, and ultimate outcome of healing, only biochemical markers provide a dynamic picture about the underlying process of BR including its turnover, pathogenesis and can differentiate between normal union and nonunion. ,,,,,,
In the process of normal fracture healing, S-bone ALP may be associated with osteoblastic activity during the early stage of fracture healing, whereas osteocalcin may be associated with mineralization of the woven bone during the late stage of the fracture healing process. ,,,,,, Alkaline phosphatase (ALP) and S-bone ALP activities may serve as markers of the course and rate of bone healing after sustained fractures. Increased production of collagen is also found soon after fracture and associated with an increase in the hydroxyproline. These alterations are reflected in changes in levels of ALP in plasma and total and free hydroxyproline excretion in urine. Hydroxyproline found mainly in collagen, imparts stability to the molecule and represents about 13% of amino acid content of collagen. It is regarded as a marker of bone resorption. ,,,, Calcium in serum mainly comes from bone and thus increases in bone resorption. Thus, excretion of calcium in urine increases with bone resorption. Calcium to creatinine ratio of random urine specimens may be used to detect hypercalciuria in patients suspected of having metabolic bone disease or other abnormalities of calcium metabolism with normal renal function. ,,,,,
So the present study was conducted to measure serum ALP, serum calcium, urinary total and free hydroxyproline, and urinary calcium to creatinine ratio as these factors reflect actual status of fracture healing and may be used as an adjunct in patient monitoring to get better results.
| Materials and Methods|| |
The present study was conducted in Burdwan Medical College in a time span of 3 years after taking Institutional ethical clearance and informed consent of the subjects.
Patients aged >21 years and <50 years with clinically and radiologically confirmed cases of diaphyseal fracture of long bone of lower extremity.
Patients with pathological conditions that prevent proper union such as sepsis, bone tumor, and vascular compromises were excluded. Patients were also screened to rule out the presence of any other factor (e.g., bile duct obstruction, pregnancy, growing children) that may contribute to the rise in ALP levels. Patients with normal urea and creatinine levels were only included. Polytrauma patients, patients given calcium supplementation, and patients in the extremes of age were excluded. Patients included in the study were asked to avoid diets rich in collagen.
All the fractures were treated operatively within 4-6 days of the injury.
The observers were blinded for the samples that is periodic results of the biochemical analysis were not known to those who observed radiological healing in X-rays or not until the final stage of completion of the study. There is a good chance of bias in interpretation of radiographs. There is a chance of bias if the worker who does the clinical assessment has the knowledge as to which case showed radiological union. Thus, it becomes important if the samples blinded until the end of this study. There is a chance of bias as to which case showed radiological union to those who assess the levels of ALP and urinary hydroxyproline. So the samples were blinded until the end of the study.
In the present study, two groups of populations were included:
- Patients with normal fracture union served as controls (50)
- Patients with impaired fracture union served as cases. They were further divided into 2 groups: Fracture nonunion (20) and fracture malunion (30).
Serum calcium level (ortho cresolphthalein complex method), serum ALP (IFCC kinetic method using DEA as buffer without PLP), urinary calcium to creatinine ratio (Modified Jaffe's kinetic method), and urinary total and free hydroxyproline (level Bergman and Loxley method). ,,
Centrifuge machine (REMI T8), Auto-analyzer (XL-600), Semi- auto-analyzer (CHEM 5 PLUS V2), Double-beam spectrophotometer (Double-Beam UV-Vis Spectrophotometer UV5704SS), Micropipettes and tips, disposable syringe, and glass tubes.
Collection and processing of samples
Five milliliters fasting venous blood was collected (on day 0, 7, 14, 90). The collected blood was then centrifuged at 1500 RPM speed for 5 min for separation of serum. 24 h urinary sample was collected in sterile container without any preservative from each case and control. pH of urine was checked, and the samples were neutralized by adding alkali or acid.
Serum total calcium measurement
Calcium in an alkaline medium combines with O-cresolphthalein complex one to form a purple colored complex. Intensity of the color formed is directly proportional to the amount of calcium present in the sample. The color measured using 578 nm filters.
Contents of reagent
Ortho cresolphthalein complex (color) reagent; DEA buffer; calcium standard, 10 mg/dL.
All reagents brought to room temperature. The working reagent prepared by mixing equal parts of the color reagent and the buffer reagent. This is stable for 7 days at 2-8°C. Normal reference values: Serum/plasma: 8.5-11.0 mg/dL.
Serum alkaline phosphatase measurement
Alkaline phosphatase at an alkaline medium hydrolyses p-nitrophenyl phosphate to form p-nitrophenol (PNP) and phosphate. The rate of formation of PNP is measured as an increase in absorbance that is proportional to the ALP activity in the sample.
A clean dry test tube labeled as test (T) taken.
Contents of the tube are mixed well. Initial absorbance after 1 min (Å) and absorbance after every 1-3 min measured at 405 nm wavelength. Mean absorbance change per minute (ΔA/min) calculated.
Alkaline phosphatase activity in IU/L= ΔA/min × 2754.
In our auto-analyzer (XL 600), we have got direct value of serum ALP in IU/L as the system parameters for ALP were programmed in the instrument.
Measurement of urinary calcium to creatinine ratio
Urinary calcium and creatinine values are measured separately in mg/dL and then ratio is calculated at 37°C.
Urinary calcium measurement
Urinary calcium measured in the same way by same reagent as serum calcium measurement except the sample. Urine is diluted 10 times with normal saline and the result multiplied by the dilution factor. Normal value of urine calcium: 100-300 mg/24 h urine.
Urinary creatinine measurement
Picric acid in alkaline medium reacts with creatinine and forms an orange colored complex with the alkaline picrate. Intensity of the color formed during the fixed time is directly proportional to the amount of creatinine present in the sample.
Reference values of urinary creatinine
Males: 1.1-3.0 g/24 h urine; females: 1.0-1.8 g/24 h urine.
Pipette into clean dry test tubes labeled standard (S) and test (T). Contents of the tubes are mixed well. Initial absorbance A1 of both the S and T measured after exactly 30 s. Another absorbance A2 of both S and T measured exactly 60 s later.
Change in absorbance ΔA for both S and T calculated. For standard ΔAS = A2S−A1S;
For test ΔAT = A2T−A1T.
In our auto-analyzer (XL 600), urine creatinine measured.
Measurement of 24 h urinary total and free hydroxyproline
Total and free urinary hydroxyproline was estimated from the 24 h urinary samples according to the method of Bergman and Loxley, 1970 (43). First urinary hydroxyproline was measured. This is free hydroxyproline. Then urine sample incubated between 100°C and 120°C for about 24 h. In this way hydroxyproline, which is bound to protein molecules, becomes free. Then urinary hydroxyproline measurement was repeated again. Later values give total hydroxyproline level in 24 h urine sample.
4-hydroxyproline standard: Stock solution: 2 mg/ml or 2000 μg/ml.
Stock solution diluted 10 times to the concentration of 0.2 mg/ml or 200 μg/ml.
- A 7% w/v aqueous solution of chloramine T made up daily in present work
- An acetate/citrate buffer of pH 6 is made up by dissolving 57 g sodium acetate (3H 2 O), 37.5 g tri-sodium citrate (2H 2 O), 5.5 g citric acid (H 2 O) and 385 ml isopropanol in water and made 1 L with water. This solution is stable indefinitely.
Solution 1 (a) and 1 (b) mixed in ratio of 1:4 just before start of each series of determinations.
Ehrlich's reagent solution
- p-dimethylamino-benzaldehyde dissolved in 60% perchloric acid (S.G. 1.54) in proportions of 2 g aldehyde and 3 ml acid. Though the solution is stable for several weeks in a dark bottle, it is made up fresh in present work
Solution (a) and (b) mixed in 3:13 ratio.
Clean dry 30 ml test tubes are taken.
One milliliter portion of each neutral/faintly acidic urine sample had pipetted into each test tube. Two milliliter isopropanol added from a pipette and mixed. One milliliter oxidant solution added to each tube and all are well mixed and allowed to stand for 4 (±1) min at room temperature (17-21°C). Thirteen milliliters Ehrlich's reagent solution added to each tube and well mixed. Tubes are heated for 25 min at 60°C in a water bath. Then tubes are cooled for 2-3 min in running tap water. Then solution of each tube diluted to 50 ml in a flask with isopropanol. The absorbance measured within 4 h in a 1 cm cuvette at 558 nm against water blank in a double-beam spectrophotometer (Double-Beam UV-Vis Spectrophotometer UV5704SS).
A hydroxyproline standard curve is prepared before samples are tested using 4-hydroxyproline standard stock solution and working solutions are prepared by dissolving hydroxyproline standard in distilled water. Serial dilutions of working solution made with distilled water to get hydroxyproline solutions of different concentrations.
4-hydroxyproline solutions of different concentrations are used as samples. Their absorbances are measured, and the standard curve is prepared. After measurement of absorbances of test sample values of hydroxyproline are obtained in μg/ml by comparing with the standard curve. The values are multiplied by the total volume of 24 h urine (in milliliter) to get the 24 h urinary hydroxyproline in mg/day. Reference interval of urinary total hydroxyproline: 19-36 mg/day.
The computer software "Statistical Package for the Social Sciences (SPSS) version 16 (SPSS Inc., released 2007. SPSS for Windows, version 16.0. SPSS Inc., Chicago, USA).
| Results|| |
The average age of the patient in the study was 35.43 years, and 87% were males, and 13% females. Serum ALP, serum calcium, urinary total and free hydroxyproline, and urinary calcium to creatinine ratio were increased in all patients after fracture. Serum ALP, urinary total and free hydroxyproline increased after treatment up to 8 th week and then decreased to normal reference interval until BR was complete in case of fracture normal union and malunion groups. Serum ALP, urinary total and free hydroxyproline decreased after treatment in nonunion patients. Serum ALP reached to lower level of reference interval in this group. But urinary total and free hydroxyproline decreased to less than normal level. Serum calcium increased up to 2 weeks following treatment and then reduced to normal level within 1-month in fracture normal union and malunion groups. Serum calcium reduced at a very slow rate after treatment in nonunion patients and remained within reference interval. Urinary calcium to creatinine ratio reduced in all patient groups after treatment. No significant difference was found between normal union and malunion groups in case of all the parameters on all test days.
Significant differences were found between normal union and nonunion groups in case of serum ALP, urinary total and free hydroxyproline levels after treatment. Significant differences were found between normal union and nonunion groups in serum calcium level after treatment on day-7 and 14. No significant difference was found between normal union and nonunion groups in case of urinary calcium to creatinine ratio [Table 1], [Table 2], [Table 3], [Table 4] and [Table 5]. A positive correlation existed between serum ALP and urinary total hydroxyproline in normal united group. Calcium to creatinine ratio showed positive correlation with other bone turnover markers that is serum ALP, urinary total and free hydroxyproline in both case groups only after fracture that is before treatment but not after treatment.
|Table 1: Means and SD of all parameters in the three groups of patients on test day-0|
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|Table 2: Means and SD of all parameters in the three groups of patients on test day-7|
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|Table 3: Mean and SD of all parameters in the three groups of patients on test day-14|
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|Table 4: Means and SD of all parameters in the three groups of patients on test day-90|
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| Discussion|| |
This study was undertaken in the Department of Biochemistry of Burdwan Medical College and Hospital in collaboration with the Department of Orthopedics of Burdwan Medical College and Hospital. It focuses on the changes in levels of bone turnover markers during fracture healing process.
Serum biochemical markers of bone formation such as ALP activity may be clinically useful in evaluating the progress of healing. Changes in serum ALP activity were studied by Komnenou et al.  in 83 dogs with closed long bone diaphyseal fractures treated surgically. Group A dogs (n = 35) developed a medium-sized callus that led to bone union within 2 months. Group B dogs (n = 36) had a hypertrophic callus and delayed union, within 3-5 months. Group C dogs (n = 12) had slow progress in fracture healing, with minimal callus formation during a 2-month period. Changes in mean serum ALP activity followed the same pattern in Groups A and B, reaching a maximum level on day 10. Group A values returned to normal within 2 months, at which point bone union was complete, whereas Group B values remained increased and returned to normal within 3-5 months, thus correlating with delayed union. In Group C, mean serum ALP activities showed no significant changes during the 2-month follow-up period, consistent with failure of bone union (nonunion). Serum P and Ca changes followed a proportional and inverse pattern to ALP changes, respectively.
In a study by Muljacic et al.,  the activity of S-bone ALP was measured in the serum of 41 patients with long bone fractures. The activity of S-bone ALP was assessed every 7 days over a period of 4 weeks. The increase of ALP correlated with an increase of S-bone ALP levels. In addition, changes in ALP levels on days 7 and 14 as compared to those on day 1 postinjury were associated with changes in S-bone ALP levels on the same day. Likewise, the callus volume correlated with the decrease, no change or increase in the levels of ALP and S-bone ALP in the same way.
Thirty-three patients with a fresh vertebral fracture were enrolled by Ohishi et al. in 2008.  Urinary type I collagen C-terminal telopeptide, pyridinoline, deoxypyridinoline, serum C-terminal telopeptide, and N-midportion of osteocalcin (OC N-mid ) were determined at the time of hospital admission and at 2, 4, 12, 24, and 48 weeks. Subjects were divided into two groups according to the results of magnetic resonance images taken 48 weeks after fracture. Twenty-four were normally united (Group N) and nine had delayed or nonunion (Group D) of the spine. No differences between values of bone resorption markers in Group N and Group D were observed at any time. Serum OC N-mid in Group N started to increase at 2 weeks and reached the peak value at 24 weeks (180%); serum OC N-mid in Group D increased at most 120% from baseline to 4 weeks. Values of serum OC N-mid in Group N were higher at 24 and 48 weeks than those in Group D.
Thiry-six patients of long bone fracture were randomly allocated for the study by Mukhopadhyay et al. in 2011.  On day 0 after fracture, serum and urinary samples were collected, and X-ray of the affected part was taken. Subsequent samples were collected, and X-ray taken just after management (either operative or conservative), after 3 rd , 5 th , 8 th , and 12 th week. The patients were divided into two groups that progressed to proper union or malunion. The levels of serum ALP, urinary total and free hydroxyproline levels were measured. A statistically significant positive correlation between total urinary hydroxyproline excretion and serum ALP indicated progress toward satisfactory union.
Similar results were also observed in our study. Thus, serial monitoring of biochemical markers of bone turnover such as urinary hydroxyproline, serum ALP reflect the actual status of bone resorption, and bone formation, respectively, over a short period.
| Conclusions|| |
Serial monitoring of biochemical markers of bone turnover like urinary hydroxyproline, serum ALP reflect the actual status of bone resorption, and bone formation respectively over a short period. Thus, they can be used as an adjunct to clinical and radiological evidence of fracture healing. A statistically significant positive correlation between total urinary hydroxyproline excretion and serum ALP indicate progress toward fracture union. Normal union and malunion can be differentiated by clinical examinations and radiological investigations. Indications of nonunion obtained from the biochemical markers could be helpful in performing early interventional procedures.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]