The use of alkaline phosphatase as a bone turnover marker after spinal cord injury: A scoping review of human and animal studies

Background: Serum alkaline phosphatase (ALP) is measured as an indicator of bone or liver disease. Bone-specific alkaline phosphatase (B-ALP) is an isoform of ALP found in the bone tissue which can predict fractures and heterotopic ossification. Objective: The aim of this scoping review was to explore the current use of ALP and B-ALP in studies using humans or animal models of SCI, and to identify ways to advance future research using ALP and B-ALP as a bone marker after SCI. Results: HUMAN STUDIES: 42 studies were included. The evidence regarding changes or differences in ALP levels in individuals with SCI compared to controls is conflicting. For example, a negative correlation between B-ALP and total femur BMD was observed in only one of three studies examining the association. B-ALP seemed to increase after administration of teriparatide, and to decrease after treatment with denosumab. The effects of exercise on ALP and B-ALP levels are heterogeneous and depend on the type of exercise performed. ANIMAL STUDIES: 11 studies were included. There is uncertainty regarding the response of ALP or B-ALP levels after SCI; levels increased after some interventions, including vibration protocols, curcumin supplementation, cycles in electromagnetic field or hyperbaric chamber. Calcitonin or bisphosphonate administration did not affect ALP levels. Conclusion: Researchers are encouraged to measure the bone-specific isoform of ALP rather than total ALP in future studies in humans of animal models of SCI.


Introduction
Fracture risk is elevated after spinal cord injury (SCI) and it is closely associated with bone mineral density (BMD) and duration of injury. 1,2 BMD can be a surrogate outcome for bone strength, and it is part of the main tools for the assessment of risk of fracture. 3,4 However, 30-50% of fragility fractures occur among individuals with SCI who have a femoral neck T-score above the osteoporotic threshold. 5 Moreover, there are not fracture risk calculators specifically designed for people with SCI, and there is a lack of agreement between the available tools in the SCI population. 6 In the general population, bone mass progressively decreases throughout life: less than 1% of bone tissue is lost every year after the fourth decade, 7 while women in the first five to ten years after menopause may lose up to 2-4% of bone per year. 8,9 In the acute phase post-SCI, bone is lost at a rate of 4% per month, 10 and a slower ongoing bone loss at the tibial diaphysis (∼0.5% per month) is detectable as late as ten years after the injury. 1 Bone metabolism is a dynamic process that undergoes daily changes regardless of variations in BMD. 11 The levels of circulating bone turnover markers (BTMs) reflect the metabolic state of the bone, and have been used as surrogate measures of bone resorption and formation. Even though BTMs alone are not suitable to estimate bone loss or select the appropriate pharmacological intervention, they have been used to monitor the efficacy of treatments. 12 Alkaline phosphatase (ALP) is an enzyme that is found in several tissues in the human body, with the highest concentration in bone and liver. ALP is higher during childhood and puberty; 13 women older than 50 years have higher values than women 20-50 years, while there appears to be no agerelated differences in adult men. 14 Therefore, serum ALP is measured as an indicator of bone or liver disease, and in the general population, is associated with obesity, fatty liver disease, diabetes and chronic kidney disease, 15,16 which are also frequent in people with SCI. [17][18][19][20] The presence of obesity alone increased the risk of elevated liver enzymes, including ALP, by a factor two to three. 21 The authors acknowledge that there are many non-hepatic etiologies of elevated ALP in the SCI population that may warrant investigation, which may include healing fracture, Paget's Disease, vitamin D insufficiency, heart or kidney failure, thyroid or parathyroid disease, that are outside the scope of this review.
Serum ALP is a common BTM for screening for heterotopic ossification (HO), even though HO may also present with normal serum ALP levels, 22 and some studies did not show associations between serum ALP levels and HO. [23][24][25][26] Conversely, low levels of ALP represent a hallmark of hypophosphatasia, a rare disorder that alters mineralization of bone and teeth, causing low BMD, joint pain and loss of secondary teeth. 27 Bone-specific alkaline phosphatase (B-ALP) is an isoform of ALP found in the bone tissue, which degrades the mineralization inhibitor pyrophosphate with alkaline pH conditions, and its presence on the osteoblasts' membrane is required for bone mineralization. 28 B-ALP is an appropriate marker of bone formation given its slow clearance, low intra-individual variability and role in the calcification process. [29][30][31] High levels of serum B-ALP predict spine and nonspine fracture in able-bodied individuals, 32 while the evidence on hip fractures is conflicting. 33,34 However, there is uncertainty regarding the response of B-ALP following SCI, and the evidence regarding changes in B-ALP after SCI and its role in bone loss is heterogeneous and conflicting. Furthermore, there is the need to explore what has been done, to date, in terms of populations and methodologies used to study B-ALP across the SCI population.
The SCI Rehabilitation Translational Continuum (ReCon) Team met in Toronto, ON, Canada in February 2018 and August 2018 to define the central concepts, current state of the field, target population, and outcomes of interest. There was consensus on the need to explore the current use of bone biomarkers in SCI research to identify optimal biomarkers, stimulate the standardization of use and reporting practices, and suggest the abandonment of ineffective biomarkers in both humans and animals. Rodents are biologically similar to humans and thus predisposed to many similar health conditions. Also, they have a short duration of life and so can easily be studied throughout their whole lifespan, and the environment around the animal can be easily controlled by the researcher, allowing observational studies under ideal conditions and the performance of efficacy trials. Therefore, a series of systematic and scoping reviews was performed to explore the current use of bone biomarkers in studies using animal or human models of SCI. The research question of this scoping review was: "What is the current use of ALP and B-ALP in studies using animal or human models of SCI?". Since serum ALP is associated with different pathological conditions, a secondary aim was to identify research gaps and ways to advance future research using B-ALP as a bone marker after SCI.

Methods
The ReCon Team is composed of 29 early, mid and senior career investigators and their trainees, and consumer advisors. The team has wide expertise in muscle and bone physiology in human and animal models of SCI.
We followed the Joanna Briggs Institute Scoping Review Methods Group 35 to guide the scoping review process, and the Preferred Reporting Items for Systematic Reviews and Meta-analyses extension for Scoping Reviews -PRISMA ScR 36 to guide the methodological guidance and best practice reporting of the scoping review publication. 37 In brief, this process involved defining a research question, selecting relevant studies, extracting data and summarizing results. The methodology recommends including both quantitative and qualitative research, as well as evidence from expert opinion sources to answer questions of effectiveness, appropriateness, meaningfulness, and feasibility of health practices and delivery methods.

Study eligibility criteria
Electronic search for identification of studies Web-based systematic searches were conducted on: EMBASE (MEDLINE and PubMed), Cochrane Database of Systematic Reviews, Cochrane Central, CINAHL, and Emcare. All searches were conducted up to August 15, 2018. A common search strategy was adopted for all the scoping reviews, and the following Medical Subject Headings (MeSH) indexing search terms were used: "spinal cord injuries", "bone and bones", and "biomarkers", with manual inclusion of the main bone biomarkers reported in the literature: adiponectin (ADP), adrenocorticotropic hormone (ACTH), alkaline phosphatase (ALP), C-telopeptide (CTX), calcium, cathepsin, collagen, Dickkopf-related protein 1 (DDK-1), estradiol, estriol, estrone, interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-17 (IL-17), leptin, lysine, N-telopeptide (NTX), osteocalcin (OC), osteopontin (OPN), Carboxy-terminal propeptide of type 1 procollagen (P1CP), procollagen type 1 N-terminal propeptide (P1NP), parathyroid hormone (PTH), proline, receptor activator of nuclear factor kappa-B ligand (RANKL), sclerostin (SOST), testosterone, tumor necrosis factor-alpha (TNF-α), and vitamin D (Vit D). When appropriate, concepts were searched using the Boolean Operators (i.e. AND, OR). Index terms were searched using truncation and phrase symbols when appropriate to ensure accurate and ample results. Each database was searched using the same methodology with no time limitations and duplicates were removed. The final detailed version of the search strategy for each database is available in the Supplementary Material.

Types of study participants
The eligibility criteria for human studies were adults (18 years and older) with acute or chronic traumatic SCI, including men and/or women with American Spinal Injury Association Impairment Scale (AIS) A-D. For studies using animal models, we included any model (e.g. rat, mice, pig, monkey), adults, males and females, with traumatic injuries. Traumatic injuries are physical injuries of sudden onset and severity which require immediate medical attention. […] Traumatic injuries are the result of a wide variety of blunt, penetrating and burn mechanisms. They include motor vehicle collisions, sports injuries, falls, natural disasters and a multitude of other physical injuries which can occur at home, on the street, or while at work and require immediate care. 38

Types of bone turnover markers included
We included studies that measured alkaline phosphatase (ALP) or the bone-specific isoform (B-ALP) with no limitations concerning the measurement technique. Studies that did not report measurement procedures were included. We included studies that measured ALP or B-ALP with diagnostic purposes or to monitor the effects of both pharmacological and nonpharmacological interventions.

Types of studies included
Articles were included if published in the peer-reviewed, scientific literature in English in a full-text version (e.g. no abstracts were included). We included randomized controlled trials (RCTs), quasi-experimental/non-randomized, longitudinal single-group trials, prospective or cross-sectional observational studies, case series (n > 3 for human studies; n > 1 for longitudinal animal studies), that included at least ALP or B-ALP. Studies that did not fit these designs (e.g. clinical commentaries, reviews, editorials, interviews, lectures, letters, newspaper articles, patient education handouts, conference abstracts, or unpublished literature) or did not fit the above criteria were excluded.

Data collection and analysis
Selection of studies Two team members (EN and EP) independently reviewed title, abstract and descriptions of each study considering the eligibility criteria (Level 1 screening). Level 2 screening involved two members (EN and EP) of the team applying the inclusion criteria to all fulltext articles included during Level 1. Any conflicts during Level 1 and Level 2 screening were solved by a

Data extraction/management
Two independent members of the team (EN and EP) extracted data from each study that passed Level 1 and 2 screening to Microsoft Excel using the data extraction elements specified in Table  1. Disagreements regarding data extraction were resolved by a third-party reviewer (MJW).

Data analysis
A narrative synthesis was used to summarize the outcomes of each included manuscript according to the methodology for scoping reviews. Data charting was performed based on population, purpose and technique for the measurement of ALP or B-ALP, BTMs commonly measured with ALP or B-ALP and study design. Human and animal data were grouped separately due to the nature of methodologies and outcomes.

Studies with human participants
Participants Thirty-five studies recruited participants with both paraplegia and tetraplegia, 10,[40][41][42][43][44][46][47][48][49][50][51][52][53][55][56][57][58][59][60][61][62][63][64][65][66][67][70][71][72][73][74][75][76][77] six studies included only people with paraplegia 1,39,54,68,69,78 and one study only individuals with tetraplegia. 44 Twenty-seven studies included people with both complete and incomplete lesions, 10 59 Scandinavian method, 60 spectrophotometry, 72 and standard radioimmunoassay procedures (BM/ Hitachi 747, Boehringer Mannheim, Sydney, Australia). 48 One study performed immunoextraction and then used a kit from QUIDEL Corporation (San Diego, CA, USA), 64 and one study used a kit from IDS (London, UK). 39 Fourteen studies did not report the methods of ALP measurement. 42  and serum ALP. Serum ALP levels do not explain lumbar-hip, lumbar-radius and hip-radius T-score discordances, 39 but Gifre et al. 53 observed a negative correlation between B-ALP and total femur BMD (r = −0.63, P = 0.001). In a cohort of people with acute SCI, baseline B-ALP > 14 ng/mL was a risk factor for the development of osteoporosis (relative risk ratio = 2.83, P = 0.041). 51 Conversely, ALP was not associated with age, sex, height and weight, time from injury, injury level and completeness. 73 Similarly, Reiter et al. 71 did not observe significant differences in B-ALP between people with a SCI > 5 years and people <1 year post-injury. Zehnder et al. 1 did not find any relationships between age and ALP, but plasma ALP decreased with time after injury (97 participants, 0-30 years after SCI, r = 0.376, P < 0.0001). Two studies showed a decrease within the first three months in ALP and B-ALP, respectively, 40,64 but only Kostovski et al. 64 found a statistically significant decrease (−20%, P = 0.008), with no further changes in the following nine months. Conversely, in a 6month prospective study, Roberts et al. 72 detected a slight increase in ALP levels until week sixteen, with statistically significant increases at weeks 3 ( p < 0.02), 4 (P < 0.05), 6 (P < 0.02), 8 (P < 0.05) and 16 (P < 0.05) compared to baseline. Accordingly, Broholm et al. 44 noticed an 8% increase in ALP levels (P = 0.05) over 30 months among women 3-49 years (median 11) after SCI. Finally, three studies did not show differences in B-ALP between baseline and 6,12,24 months. 49,50,61 Findings from intervention studies Six studies measured ALP or B-ALP after a pharmacological intervention. Three RCTs did not find statistically significant between-group differences in ALP or B-ALP when comparing those who received alendronate, 55 dichloroethylene diphosphonate 69 or zoledronic acid 76 to placebo, while another RCT showed increases in B-ALP (+56.7%, P = 0.005) after a 12-month teriparatide intervention, 47 and two studies showed decreases in B-ALP levels in 14 osteoporotic patients treated with denosumab. 49,52 A pre-post study noticed statistically significant increases in B-ALP after a 12month intervention including 6 months of roboticassisted stepping and teriparatide, followed by 6 months of teriparatide alone. 56 One RCT showed increases in B-ALP (+21.9%, P = 0.005) after a 12month intervention with vibration protocol and teriparatide administration. 46 One RCT found statistically significant decreases in ALP (−25.1%, P < 0.0001) in 29 paraplegic individuals compared to 26 controls after a 24-month alendronate and calcium intervention. 78 A prospective intervention-drug study did not detect any variations in ALP levels after a 3-month daily administration of 2000 IU vitamin D3 (i.e. cholecalciferol) and 3.25 g calcium carbonate, 40 while one RCT detected a statistically significant difference in B-ALP levels (P < 0.05) after a 6-month intervention consisting in 110mg/kg curcumin administered daily compared to control. 57 The effects of exercise on ALP and B-ALP levels are heterogeneous and depend on the type of activity performed. A 6-month treadmill and neuromuscular electrical stimulation (NMES) protocol increased B-ALP and osteocalcin in 81% of the participants affected by paraplegia compared to 20% of the individual in the control group. 45 A pilot RCT compared two different intensities of cycling with functional electric stimulation (FES cycling), and there were no statistically significant between-group differences in B-ALP. 58 However, participants allocated to the protocol at a lower cadence showed a statistically significant within-group increase in B-ALP (=15.5%, P = 0.02), while the within-group change in ALP in participants allocated to the higher cadence protocol was not statistically significant. Another pre-post study did not detect any statistically significant within-or betweengroup differences in ALP after a FES cycling intervention. 43 Studies using animal models  79 and one other study used guinea pig SCI models. 83 Two studies created severe but incomplete contusive SCI models by using Allen's method (motor incomplete), 83,84 while the remaining nine studies performed a thoracic spinal cord transection (motor complete). [79][80][81][82][85][86][87][88][89] Two studies used the enzyme-linked immunosorbent assay (ELISA) 79,85 and a standard colorimetric procedure, 80,86 one study used a plasma ALP kit, 89 7170A Hitachi auto-biochemistry analyzer, 84 QUIDEL, 83 ALP B-test Wako kit 82 and spectrophotometry, 81 while to studies did not report the measurement technique utilized. 87,88 The map of the animal studies is shown in Fig. 3.
Purpose for the use of alkaline phosphatase Findings from observational studies Two studies showed lower ALP levels in SCI mice compared to control two weeks post-surgery. 83, 89 Manjhi et al. 86 showed a significant decrease in B-ALP in femur and tibia 8 weeks after SCI (P < 0.001). Similarly, Ding et al. 87 observed lower ALP levels in the SCI mouse models compared to control one, two and four weeks after femoral shaft fracture (P < 0.05). However, Li et al. 84 detected higher ALP levels in SCI rats one and two weeks after surgery compared to control (P < 0.01). Three other studies did not observe any statistically significant differences in B-ALP levels between SCI rats and control at eight and twelve days, one, two, three, six and eight weeks, and six months after SCI. 80,81,87 Findings from intervention studies Liu et al. 85 showed significantly higher levels of B-ALP after cycles of hyperbaric chamber treatment beginning 3 h after SCI, compared to 12 h after SCI, SCI with no hyperbaric chamber treatment and control. Furthermore, the group that began the treatment cycles 12 h after injury showed higher B-ALP compared to SCI group with no treatment.
Manjhi et al. 86 analyzed femur and tibia lyophilized bone powder with plasma ALP kit, and showed significantly higher B-ALP levels were significantly in the SCI rats that underwent cycles in an electromagnetic field compared who SCI rats who did not, while no differences were observed between the electromagnetic field group and the control. Minematsu et al. 87 showed an increase in ALP in SCI rats after a 2-week wholebody vibration protocol compared to control (P < 0.05). Jiang et al. 82 noticed lower B-ALP levels in the SCI mice compared to a hindlimb immobilization group after a 48-hour mechanic cyclic strain program. Yang et al. 88 demonstrated an increase in B-ALP after 2 weeks of curcumin supplementation in SCI rats compared to control (P < 0.05), while 30 days of calcitonin or diphosphonates administration did not affect ALP levels. 88 Ponzano et al. The use of alkaline phosphatase as a bone turnover marker after spinal cord injury

Discussion
The present review highlights the potential utility of ALP and its bone-specific isoform (B-ALP) in assessing changes in bone turnover in response to interventions in humans living with SCI, but the evidence in animal models is less compelling. The literature regarding changes in ALP or B-ALP levels after SCI versus people without SCI is conflicting, and, at this time, ALP or B-ALP should not be used to estimate bone mineral density, bone mineral loss or fracture risk. The association between ALP and comorbidities like chronic kidney disease, diabetes or adipose infiltration in the liver should be further investigated in the SCI population before making recommendations on the use of ALP for diagnostic or prognostic purposes. Finally, the current evidence in animal models does not provide support for the utility of ALP or B-ALP to evaluate bone status in rodent models of SCI.

Studies with human participants
ALP does not represent an accurate marker to assess bone formation after SCI. The response of ALP in humans with acute SCI was not consistent: Kaya et al. 62 reported an increase, but other studies did not find statistically significant differences in ALP levels between acute and chronic SCI. 70,71,73 Furthermore, in the same studies, calcitonin and osteocalcin levels, two markers of bone formation, have not consistently been found to be significantly higher in SCI versus control or in acute versus chronic SCI. 62,70 Moreover, increases in ALP have been reported by studies that measured total serum ALP, 54,62,68,70 while studies that measured B-ALP did not notice any differences in people with SCI compared to the control group. 41,53,[65][66][67] Therefore, elevated levels of ALP might indicate an increased metabolic activity after SCI, rather than increased bone formation. Heterogeneity is still present when the longitudinal changes in ALP after SCI are examined. Roberts et al. 72 and Broholm et al. 43 noticed an increase in serum ALP over four and 30 months, respectively. However, high levels of serum ALP might be a result of low vitamin D levels, a common condition after SCI. One study in humans showed a decrease in B-ALP in the first three months after SCI 64 ; however, future studies are required to determine the relationship between B-ALP and duration of SCI and to link them with the degree of bone loss and/or fracture risk. Finally, three studies of four did not show any associations between ALP and BMD, 39,46,77 suggesting that we should avoid using ALP to estimate bone formation or BMD in individuals with SCI. Further, there were no observed differences in ALP and B-ALP between people with motor complete and incomplete injuries. Thus, the existing literature does not allow us to draw conclusions on the differences in ALP or B-ALP between people with complete and incomplete injuries, tetraplegia and paraplegia, or acute versus chronic injuries.
High levels of serum B-ALP predict major osteoporotic fractures in non-disabled postmenopausal women, 32 but such an association, along with the relationship between ALP or B-ALP and HO, warrants further exploration in the SCI population. Therefore, new studies focusing on the bone-specific isoform instead of the total serum ALP might provide evidence in favor of the use of B-ALP to assess bone formation and estimate fracture risk after SCI.
Preliminary evidence indicates that serum ALP and the bone-specific isoform might be used to assess changes in bone turnover in response to pharmacological interventions, but serum ALP did not change after antiresorptive therapies. Antiresorptive medications do not increase bone formation, at least initially, and this might explain the absence of variation in serum ALP level. Our findings are consistent with the concept of B-ALP as bone formation marker, and with findings from clinical trials in the non-SCI population. Two RCTs among postmenopausal women with and without osteoporosis reported an increase in ALP only in the group treated with teriparatide compared to antiresorptive therapy with raloxifene or placebo. 90,91 Another trial among women at high risk of fracture ( previous fracture or family history of fracture) showed increases in B-ALP levels after teriparatide administration. 92 The existing evidence advocates for the measurement of B-ALP when assessing bone status after pharmacologic therapies in the SCI population; however, clinical decisions should not be based exclusively on B-ALP, and further studies are warranted to determine what clinically meaningful changes in B-ALP are. On the other hand, the evidence showing changes in ALP after exercise interventions is heterogeneous. Only one study showed increases in ALP after a NMES and treadmill protocol, 45 while another trial observed higher ALP levels than baseline only in the lower intensity cycling group. 58 The effects of aerobic training on ALP are uncertain also in the non-SCI population. Conversely, B-ALP levels appear to increase after progressive resistance training in both younger and older adults; however, to date, no studies have investigated B-ALP response after progressive resistance training in individuals with SCI. B-ALP may be useful to monitor response to pharmacological therapy (and ALP specifically for anti-resorptive therapy), but further research is needed to evaluate the utility of B-ALP as a marker of bone formation that is responsive to non-pharmacological interventions, as well as its levels in presence of diseases like diabetes, adipose infiltration in the liver or chronic kidney disease. Furthermore, ALP may be problematic to interpret in people with evolving neurogenic obesity and liver dysfunction. 93 Studies using animal models The number of animal studies is too limited and the evidence too heterogeneous to drive conclusions regarding the response of ALP after SCI. The evidence regarding ALP or B-ALP changes is also conflicting in animal models of osteoporosis. One study noticed a decrease in total ALP among tail-suspension mice models of osteoporosis; 93 however, the absence of mechanical stimuli hinders osteoblast activity and formation and, considering that the bone-specific isoform of ALP is found in the osteoblast, this might explain the decrease in ALP observed in the study. Therefore, it is possible that the decrease in ALP or B-ALP observed in a few animal models of SCI results from the absence of loading. Another study in mice with retinoid-induced osteoporosis showed a reduction in ALP compared to the control group, 94 but it is possible that the decrease is a result of the inhibitory effect of retinoids on ALP. 95 Indeed, serum ALP and B-ALP commonly increase in mice who underwent ovariectomy or oophorectomy. Conversely, the heterogeneity in the response of ALP after SCI is not explained by the technique used to generate the injury. Animal models of complete SCI are less clinically relevant, but it is rather difficult to achieve an incomplete injury that causes robust bone loss and muscle wasting in rats, because they regain weight support at stance. However, the findings are also inconsistent when the same technique to generate the injury is used, suggesting that ALP is not indicated to assess bone metabolism in animal models of SCI.
Only a limited number of studies with different types of intervention measured ALP in animal models of SCI; therefore, based on the available evidence, it is not possible to make recommendations regarding the use of ALP to assess the effectiveness of interventions. The positive effects of a hyperbaric chamber protocol on B-ALP observed by Liu et al. 85 can be explained by the osteogenic effect of hyperbaric oxygen therapy, which may increase osteoblast differentiation in presence of osteonecrotic bone loss. 96 Manjhi et al. 86 observed statistically-significant increases in B-ALP levels of the SCI rats that underwent cycles in an electromagnetic field compared who SCI rats who did not. One study evaluating the effect of static magnetic fields in rat mesenchymal stem cells noticed higher but non-statistically significant levels of ALP in the exposed versus control group. 97 However, neuroregenerative effects of electromagnetic fields have been observed in rat models of SCI. 98 Minematsu et al. 87 showed an increase in B-ALP in SCI rats after a 2week whole-body vibration protocol compared to control. However, in a more recent study among growing rats, the same authors did not detect increases in B-ALP despite improvements in other trabecular bone parameters. 99 Yang et al. 89 demonstrated an increase in B-ALP after 2 weeks of curcumin supplementation, in line with previous evidence of the osteogenic effect with a consequent increased ALP activity of curcumin at the mesenchymal stem cells level. 100 Therefore, more studies are required to clarify whether variations in B-ALP reflect actual changes in bone parameters in animal models of SCI.

Limitations and future directions
Only 24 of 42 human studies and six of eleven studies on animal models measured the bone-specific isoform of ALP. In humans, ALP can be classified into at least four tissue-specific isoforms: placental ALP, intestinal ALP, liver/bone/kidney ALP, and germ cell ALP. Therefore, elevated levels of total serum ALP may not necessarily reflect changes in bone metabolism and, even though B-ALP corresponds to 50% of the total ALP, conclusions from studies that did not assess the bone-specific isoform of ALP might be misleading. The International Osteoporosis Foundation and the International Federation of Clinical Chemistry and Laboratory Medicine recommend the use of CTX (Cterminal telopeptide of type I collagen) as bone resorption marker and P1NP ( procollagen type 1 N propeptide) as bone formation marker. However, B-ALP and P1NP showed similar responses to interventions; therefore, considering its ability to predict fractures in the non-SCI population, future studies should explore the response of B-ALP after SCI and its association with BMD, with the ultimate goal to determine if B-ALP can be used to estimate bone metabolic activity and BMD after SCI. Moreover, only Singh et al. 77 measured BMD at the proximal tibia; hence, future studied should assess the association between B-ALP and BMD at the distal femur and proximal tibia, where the largest bone loss occurs, and the fracture risk is highest in people with SCI. Most of the human studies recruited individuals with both paraplegia and tetraplegia, and only one study was conducted exclusively in subjects with tetraplegia; therefore, we are not able to determine whether B-ALP responses vary by the level of the lesion. Similarly, only seven studies included people with both complete and incomplete lesions. Notwithstanding the challenges of recruiting people with incomplete lesions, studies in individuals with some residual motor function below the neurological level may help understand whether ambulation or adapted resistance training exercises stimulate osteoblastogenesis and, consequently, B-ALP activity in the lower limbs. The limited number of studies and the high heterogeneity of the results suggests uncertainty regarding the responsiveness or validity of B-ALP as a measure to assess the bone metabolic activity in animal models of SCI or the effects of interventions.
Finally, 14 human studies and two animal studies did not describe the procedures used to measure ALP levels. Although the methodology of scoping reviews does not include a quality assessment of the included studies, this is of concern when findings from different studies are compared. ALP and B-ALP may be responsive to pharmacological, exercise or multimodal interventions in humans living with SCI, but, based upon the available evidence, they should not be used to estimate BMD or bone status after SCI. The evidence in animal models of SCI does not permit conclusions about the use of B-ALP. In future studies on bone health, researchers are encouraged to measure the bone-specific isoform of ALP rather than total ALP. However, although ALP and B-ALP have been shown to predict vertebral fractures in the non-SCI population, the evidence on the viability of circulating ALP or B-ALP for predicting bone loss or fracture risk after SCI is inconclusive; therefore, inferences should be made with caution. Ye G., Zariffa J. Only the main authors are responsible for the content of this manuscript.

Availability of data and material
The data being reported are accurate and are coming from the official source.