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Biochemical Markers

Biochemical markers of bone turnover can be used as an adjunct to DXA in the assessment of bone health among patients with SCI. Serum and urine markers provide useful insight into bone metabolism at specific time points after injury and are an effective tool for selecting patients who would benefit from therapy and monitoring response to therapy. The current therapeutic utility of bone turnover markers is limited by day-to-day, diurnal, inter-individual, and inter-assay variability. For urine markers, results need to be corrected for creatinine (Reiter et al. 2007).

Markers of bone formation include bone-specific alkaline phosphatase (BALP), osteocalcin (OC), N-terminal propeptide of type I collagen (PINP), and C-terminal propeptide of type I collagen (PICP). Markers of bone resorption include urinary free and total pyridinoline (Pyr) and deoxypyridinoline (DPD) crosslinks, type 1 collagen C-telopeptide (CTX), and N-telopeptide (NTX). Pyr and DPD are molecules that provide stability to collagen and, along with CTX and NTX, are released when collagen is degraded during bone resorption (Brown et al. 2009).

Biochemical markers of bone turnover can be used as an adjunct to DXA in the assessment of bone health among patients with SCI. Serum and urine markers provide useful insight into bone metabolism at specific time points after injury and are an effective tool for selecting patients who would benefit from therapy and monitoring response to therapy. The current therapeutic utility of bone turnover markers is limited by day-to-day, diurnal, inter-individual, and inter-assay variability. For urine markers, results need to be corrected for creatinine (Reiter et al. 2007).

Markers of bone formation include alkaline phosphatase (ALP), bone-specific alkaline phosphatase (BALP), osteocalcin (OC), N-terminal propeptide of type I collagen (P1NP), and C-terminal propeptide of type I collagen (CINP). Markers of bone resorption include urinary free and total pyridinoline (PYD) and deoxypyridinoline (DPD) crosslinks, type 1 collagen C-telopeptide (CTX), and N-telopeptide (NTX). PYD and DPD are molecules that provide stability to collagen and, along with CTX and NTX, are released when collagen is degraded during bone resorption (Brown et al. 2009) (Table 5).

For a bone marker to be useful in assessing the rate of bone turnover and/or monitoring therapy effectiveness, the difference in the rate of bone turnover before and after SCI, as well as the early period versus the late period after SCI, needs to be discernible. Consensus regarding which biomarkers are best to monitor bone turnover is needed in the SCI community. Several authors have suggested candidate biomarkers including sclerostin (Morse et al. 2013) and adiponectin (Doherty et al. 2014). However, due to analytical discordance between the different assay kits) and biological variability (type 2 diabetes, estrogen level, parathyroid hormone etc.), diagnostic performance of these biomarkers has to be yet validated (Wheater et al. 2013, Liu et al. 2013, Durosier et al. 2013, Morris et al. 2017) (Tables 6-8).

The International Osteoporosis Foundation and the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC-IOF) Working Group for Standardization of Bone Marker Assays, and the National Bone Health Alliance (NBHA) recommend CTX and P1NP as reference bone markers to inform on fracture risk and efficacy of osteoporosis treatment (Vasikaran et al. 2011, Bauer et al. 2012, Johansson et al. 2014, Morris et al. 2017). This is due in part to their low inter-individual variability, relatively stable nature in serum at room temperature and current availability of reference intervals for these biomarkers for geographic regions and individual assays (Morris et al. 2017). In some clinical studies, however, the urine NTX marker could be preferred due to its lower sensitivity to circadian changes and food intake (Wheater et al. 2013).

Consensus regarding the choice of biomarker and the associated assay techniques are needed to cross-study comparison and future meta-analysis.

Table 5: Bone Formation and Resorption Markers

Author Year; Country
Score
Research Design
Total Sample Size
MethodsOutcome
Craven et. al, 2017

Canada

Randomized Controlled Trial

Level 1

N = 34

Population: 34 participants (26 men, 8 women) with chronic traumatic SCI; C2-T12; age: 55 years; TPI: 5 years; 13 AIS C, 20 AIS D.

 

Outcome Measures: OC was measured with radioimmunoassay, CTX with Roche Elecsys© 2010 immunoassay using electrochemiluminescence immunoassay (Roche Diagnostics GmbH, Indianapolis, IN, USA) and serum sclerostin was measured by BIOMEDICA sclerostin ELISA (Alpca Diagnostics, Salem, NH, USA). All markers were assessed at baseline, and 4 months. Treatment: 45 min, 3x/week, 4 months.

Control group (CONV): aerobic (20-25 min, 3-5 Borg; arm or leg bicycling, walking in parallel bars or on the treadmill) and resistance training (2–3 sets of 12–15 maximum repetitions for muscles capable of voluntary contraction).

FES-walking with body-weight support group: open-loop FES (8–125 mA, 250–300 µs pulse duration, 20–50 Hz) over the quadriceps, hamstrings, tibialis anterior and gastrocnemius while walking with body weight support.

1.        OC

Range of Values prior intervention [mean±SD]:

SCI CONV: 20.1 ± 8.3 ng/ml

SCI FES-walking:16.7 ± 6.5 ng/ml

Range of Values post-intervention [mean±SD]:

SCI CONV: 20.7 ± 8.6 ng/ml

SCI FES-walking: 17.8 ± 6.2 ng/ml

Normal Range: 24–70 ng/mL for men 18–30 years; 14–42 ng/mL for men 31–50 years; 14–46 ng/mL for men 51–70 years and

11–43 ng/mL for premenopausal women

15–46 ng/mL for postmenopausal women

MCID/LSC: CV was ~8%

 

2.      CTX

Range of values prior intervention [mean±SD]:

SCI CONV: 0.24 ± 0.21 ng/ml

SCI FES-walking: 0.26 ± 0.15 ng/ml

Range of Values post intervention [mean±SD]:

SCI CONV: 0.27 ± 0.18 ng/ml

SCI FES-walking: 0.24 ± 0.17 ng/ml

Normal value: 0.155– 0.873 ng/mL for men 18–30 years; 0.093–0.630 ng/ mL for men 31–50 years; 0.035–0.836 ng/mL for men 51–70 years and 0.025–0.573 ng/mL for premenopausal women and 0.104–1.008 ng/mL for postmenopausal women.

MCID/LSC: CV was < 10%

Important association: –

 

3.      Sclerostin

Range of values prior intervention [mean±SD]:

SCI CONV: 58.3 ± 12.4 ng/ml

SCI FES-walking: 52.9 ± 16.8 ng/ml

Range of Values post intervention [mean±SD]:

SCI CONV: 61.1 ± 13.5 ng/ml

SCI FES-walking: 54.3 ± 20.1 ng/ml

Normal value: 0 – 240 pmol/l

MCID/LSC: run replicates were <12.5%

Important association: –

 

 

Invernizzi et. al

2015

Italy

Case-Control Study

Level 3

N=43

Population: 28 participants (23 men, 5 women) with chronic SCI; AIS A-C; C5 – T12; age: 40.5 ± 7.1 years; TPI: 90.8 ± 53.1 months; 24 paraplegic, 4 tetraplegic, 22 motor complete and 6 motor incomplete SCI individuals. 15 healthy controls (5 men, 10 women; age: 28.4 ± 4.1 years).

 

Outcome Measures: alkaline phosphate (BALP), parathyroid hormone (PTH) and Beta-CrossLaps (Beta-CTX)

 

1.        Beta-CTX

Range of values [mean±SD]:

SCI: 461.7 ± 215.5 pg/ml

Controls: 399.2 ± 223.9 pg/ml

Normal value: –

MCID/LSC: –

Important association: –

 

2.      BALP

Range of values [mean±SD]:

SCI: 12.6 ± 4.0 mcg/l

Controls: 11.6 ± 6.0 mcg/l

Normal value: –

MCID/LSC: –

Important association: –

 

3.      PTH

Range of values [mean±SD]:

SCI: 43.9 ± 13.8 pg/ml

Controls: 29.7 ± 6.9 pg/ml

Normal value: –

MCID/LSC: –

Important association: –

 

 

Gaspar et. al

2014

Brazil

Cross-Sectional

Level 5

N= 46

 

Population: 29 sub-acute and chronic men with traumatic SCI; AIS A – B; T2 – T12; age: 32.7 ± 6.9 years; TPI: 5.3 years (range: 0.5 – 24). Control group: 17 non-disabled men (age: 31.9 ± 5.8 years).

 

Outcome Measures: collagen type I C-terminal telopeptide (CTX) was measured using commercial chemiluminescence immunoassays (Elecsys Analyzers, Roche), alkaline phosphate (BALP) was assessed by using a colorimetric method (ADVIA1650), PTH measured using an in-house electrochemiluminescence

immunoassay

 

 

1.        CTX

Range of values [mean±SD]:

SCI: 0.439 ± 0.212 ng/ml

Controls: 0.475 ± 0.556 ng/ml

 

Normal value: 0.2 – 0.7ng/ml

MCID/LSC: inter-assay CV was 4.7%, intra-assay CV of 4.6%

Important association: There was a significant inverse relationship between the CTX values and the duration of injury.

 

2.      BALP

Range of values [mean±SD]:

SCI: 58.1 ± 11.9

Controls: 89.2 ± 10.6

Normal value: –

MCID/LSC: –

Important association: –

 

3.      PTH

Range of values [mean±SD]:

SCI: 34.3 ± 18.2 pg/ml

Controls:29.8 ± 8.1 pg/ml

Normal value: –

MCID/LSC: inter-assay CV was 13.4% and intra-assay CV of 5%

Important association: –

 

 

 

Tan et. al, 2014

USA

Cross-sectional

Level 5

N = 27

Population: 27 men with SCI; age: 40.7±11.5 years; AIS A-C; C4 or lower; TPI: 13.2 ± 11.7 (range: 0.12 to 37.5) years; 19 paraplegic, 8 tetraplegic, 22 motor complete and 5 motor incomplete.

 

Outcome Measures: Total OC and CTX were measured by electrochemiluminescence immunoassay on a 2010 Elecsys autoanalyzer Roche Diagnostics, Indianapolis, IN).

 

1.        OC

Range of Values [mean±SD]:

SCI: 22.0 ± 7.5 ng/ml

Normal Range: value: –

MCID/LSC: CV was < 10%

Important Associations:

Maximal load was negatively associated with years’ post-injury and adiponectin while it was positively associated with lower extremity lean mass

2.      CTX

Range of values [mean±SD]:

SCI: 0.377 ± 0.223 ng/ml

Normal value: –

MCID/LSC: CV was < 10%

Important association: –

 

Table 6. Sclerostin and Myostatin – Normal range, responsiveness

Author Year; Country
Score
Research Design
Total Sample Size
MethodsOutcome
Invernizzi et. al

2015

Italy

Case-Control Study

Level 3

N=43

Population: 28 participants (23 men, 5 women) with chronic SCI; AIS A-C; C5 – T12; age: 40.5 ± 7.1 years; TPI: 90.8 ± 53.1 months; 24 paraplegic, 4 tetraplegic, 22 motor complete and 6 motor incomplete SCI individuals. 15 healthy controls (5 men, 10 women; age: 28.4 ± 4.1 years).

 

Outcome Measures: Serum sclerostin was measured by SOST Elisa Kit (Biomedica Gruppe, Vienna, Austria), myostatin quantified by the Elisa assay (MyBioSource, San Diego, CA, USA)

 

 

1.        Sclerostin

Range of values [mean ± SD]

SCI: 70 ± 30 pmol/l

Controls: 25 ± 5 pmol/l

Normal value: 0 – 240 pmol/l

MCID/LSC: –

Important association: serum sclerostin levels were statistically higher in individuals suffering from SCI compared with healthy controls

 

2.      Myostatin

Range of values [mean±SD]

SCI: 17 ± 6 ng/ml

Controls: 7 ± 6 ng/ml

Normal range: 0.625 – 20 ng/ml

MCID/LSC: –

Important association: myostatin serum levels are significantly higher in individuals with SCI than those in healthy controls; strong correlation with appendicular muscle mass index and moderate correlation with serum sclerostin in motor complete SCI

Alignment of the choice of biomarkers across future bone health studies is critical as it may allow for cross-study comparison, future meta-analyses, and to inform the development of SCI-specific normative datasets. Therefore, we suggest the use of CTX and P1NP bone turnover markers as a future minimum data set and harmonization of units for reporting CTX (ng/L) and P1NP (μg/L) as recommended by IFCC-IOF Bone Marker Standards Working Group (Morris et al. 2017). In addition, adipokines showed promising results; however, more studies are needed to determine their feasibility as primary biomarkers for bone health and osteoporotic fracture risk among individuals with SCI.

The selection of an appropriate analytic assay remains the main limitation of data harmonization as there is an apparent lack of comparability between particular assays, automated (Roche Elecsys/Cobas and IDSiSYS) or manual (Orion Diagnostica). To overcome this issue, the US Foundation of the National Institutes of Health is currently collecting data from all clinical trials in osteoporosis to perform an individual meta-analysis (http://www.fnih.org/what-we-do/current-research-programs/biomarkers-consortium-bone-quality-project) that would overcome the criticisms of inconsistent statistical methodology and small sample size (Morris et al. 2017).

Table 7: Vitamin D Deficiency

Author Year; Country
Score
Research Design
Total Sample Size
MethodsOutcome
Tan et. al, 2014

USA

Cross-sectional

N = 27

Population: 27 men with SCI; AIS A-C; C4 or lower; age: 40.7 years; TPI: 13 years; 19 paraplegics, 8 tetraplegics, 22 motor complete and 5 motor incomplete SCI individuals.

 

Outcome Measures: 25 OH vitamin D (25(OH)D) was quantified by enzyme immunoassay (Immunodiagnostic Systems Inc., Fountain Hills, AZ).

Range of values [mean±SD]:

SCI: 30.9 ± 9.8 ng/ml

Normal value: > 30 ng/ml

Deficiency (< 30 ng/ml): 55.6%

MCID/LSC: CV was < 10%

Important association: –

 

 

Invernizzi et. al

2015

Italy

Case-Control Study

N = 43

Population: 28 participants (23 men, 5 women) with chronic SCI; AIS A-C; C5 – T12; age: 40.5 ± 7.1 years; TPI: 90.8 ± 53.1 months; 24 paraplegic, 4 tetraplegic, 22 motor complete and 6 motor incomplete SCI individuals. 15 healthy controls (5 men, 10 women; age: 28.4 ± 4.1 years).

 

Outcome Measures: 25(OH) Vitamin D (25(OH)D).

 

Range of values [mean±SD]:

SCI: 12.3 ± 6.6 ng/ml

Controls: 20.5 ± 7.1 ng/ml

 

Normal value: > 30 ng/ml

Deficiency (< 30 ng/ml):

SCI: 100%

Controls: 80%

Deficiency (< 10 ng/ml):

SCI: 50%

Controls: 0%

MCID/LSC: –

Important association: 25(OH)D serum levels were also significantly higher in healthy controls compared with individuals with SCI.

 

Doubelt et. al

2015

Canada

Cross-sectional observational study

N = 42

Population: 34 participants (32 men, 2 women) with chronic SCI; age: 40.0 ± 10.9 years; TPI: 12.7 ± 9.0 years; AIS A-D; C1 – T12; 27 traumatic, 7 nontraumatic; 12 paraplegic, 22 tetraplegic; 17 motor complete and 17 motor incomplete. Control group: 8 matched non-disabled individuals.

 

Outcome Measures: plasma 25-hydroxyvitamin D using ultra-high-performance liquid chromatography-tandem mass spectrometry

 

Range of Values (min – max)

SCI: 18 – 120 nmol/L*

Controls: 50 – 115 nmol/L*

Range of Values [mean±SD]:

SCI: 69.3 ± 23.3 nmol/L*

Controls: 76.5 ± 19.8 nmol/L*

 

Normal value: > 75 nmol/L*

Deficiency SCI:

(<75 nmol/L*): 60%

(<30 nmol/L*): 10%

 

MCID/LSC: CV was <10%

Important associations:

 

*10 nmol/L = 3.145 ng/ml

Javidan et. al, 2014

Iran

Cross-sectional study

N = 148

Population: 148 participants; 116 men [age: 51 years (range 14 – 73)], 32 women [age: 43 years (range: 36 – 54)] with traumatic SCI who had no previous history of endocrine disorders and were not on specific medications.

 

Outcome Measures:

25-hydroxyvitamin D [25(OH)D] was assessed by a competitive protein-binding assay

Range of Values: –

 

Normal value: 30 – 74 ng/ml

Deficiency (<30 ng/ml): 64.7%

 

MCID/LSC: –

Important associations: –

 

Gaspar et. al

2014

Brazil

Cross-Sectional

N = 46

Population: 29 sub-acute and chronic men with traumatic SCI; AIS A – B; T2 – T12; age: 32.7 ± 6.9 years; TPI: 5.3 years (range: 0.5 – 24). Control group: 17 non-disabled men (age: 31.9 ± 5.8 years).

 

Outcome Measures: 25-hydroxyvitamin D [25(OH)D] were measured using chemiluminescence immunoassay technology (Liaison, DiaSorin).

 

Range of values [mean±SD]:

SCI: 22.2 ± 10.2 ng/ml

Controls: 205.8 ± 7.3 ng/ml

 

Normal value: >30 ng/ml

Deficiency (<30 ng/ml)

SCI: 44.4%

Controls: 23.5%

 

MCID/LSC: intra-assay CV was 4.6%, inter-assay CV of 8.2%

Important associations: There was a significant inverse relationship between the CTX values and the duration of injury. In the controls, the 25(OH)D level was positively correlated with the T and with the lumbar spine BMD, but these correlations were not observed in the individuals with SCI.

* All data expressed as mean±SD, unless expressed otherwise.

Table 8: Adipokines and Insulin

Author Year; Country
Score
Research Design
Total Sample Size
MethodsOutcome
Tan et. al, 2014

USA

Cross-sectional

N = 27

Population: 27 men with SCI; AIS A-C; C4 or lower; age: 40.7 years; TPI: 13 years; 19 paraplegic, 8 tetraplegic, 22 motor complete and 5 motor incomplete SCI individuals.

 

Outcome Measures: Plasma adiponectin level quantified by ELISA assay (Alpco Diagnostics,

Salem, NH).

1.        Adiponectin

Range of values [mean±SD]:

SCI in total: 4214 ± 1954 ng/ml

SCI with fractures: 5657±3003 ng/ml

SCI without fractures: 3802±1380 ng/ml

Normal range: –

MCID/LSC: CV was < 10%

Important association: adiponectin was inversely associated with axial stiffness and maximal load after adjusting for injury duration, and lower extremity lean mass and positively associated with lower extremity lean mass. Participants with osteoporotic fractures had significantly higher adiponectin levels compared to those without an osteoporotic fracture.

 

Doubelt et. al

2015

Canada

Cross-sectional observational study

N = 42

Population: 34 participants with chronic SCI (AIS A-D, C1 – T12, mean age: 40 years, mean TPI: 12.7 years), 12 paraplegics, 22 tetraplegics, 17 motor complete and 17 motor incomplete SCI individuals. 8 non-SCI individuals as comparison group – sex, age, waist circumference, and BMI matched

 

Outcome Measures: Serum adiponectin, leptin and insulin using the Milliplex Map Kit for Human Adipokine Magnetic bead Panel 2 (Millipore Corporation, Billerica, CA)

1.        Adiponectin

Range of values (min – max):

SCI: 8 – 200 ng/ml;

Controls: 7.2 – 39.9 ng/ml:

Range of values [mean±SD]:

SCI: 40.5 ± 44.0 ng/ml

Controls: 18.7 ± 10.5 ng/ml

Normal Range: –

MCID/LSC: –

Important association: Adiponectin was positively correlated with lumbar spine aBMD.

 

2.      Leptin

Range of values (min – max):

SCI: 0.21 – 180 ng/ml

Controls: 1 – 16 ng/ml

Range of values [mean±SD]:

SCI: 14.8 ± 31.4 ng/ml

Controls: 5.7 ± 5.2 ng/ml

 

Normal value: –

MCID/LSC: –

Important association: SCI cohort showed significant associations between aBMD at the femoral neck and lumbar spine with leptin.

 

3.      Insulin:

Range of values (min – max):

SCI: 58 – 1180 pg/L

Controls: 111 – 437 pg/L

Range of values [mean±SD]:

SCI: 288 ± 234 pg/L

Controls: 236 ± 116 pg/L

 

Normal value: –

MCID/LSC: –

Important associations: SCI cohort showed significant associations between aBMD at the femoral neck and lumbar spine with insulin.

 

Invernizzi et. al

2015

Italy

Case-Control Study

N =43

Population: 28 participants (23 men, 5 women) with chronic SCI; AIS A-C; C5 – T12; age: 40.5 ± 7.1 years; TPI: 90.8 ± 53.1 months; 24 paraplegic, 4 tetraplegic, 22 motor complete and 6 motor incomplete SCI individuals. 15 healthy controls (5 men, 10 women; age: 28.4 ± 4.1 years).

 

Outcome Measures: insulin-like growth factor I

 

1.        Insulin

Range of values [mean±SD]:

SCI: 177.6 ± 47.7 ng/ml

Controls: 241.1 ± 92.8 ng/ml

 

Normal value: –

MCID/LSC: –

Important associations: –