Bone mineral density and intervertebral disc height in type 2 diabetes

Journal of Diabetes and Its Complications xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M

Bone mineral density and intervertebral disc height in type 2 diabetes, Rachel Agius, Raymond Galea, Stephen Fava ⁎ Diabetes and Endocrine Centre, Mater Dei Hospital, Msida, Malta

a r t i c l e

i n f o

Article history: Received 11 December 2015 Received in revised form 26 January 2016 Accepted 27 January 2016 Available online xxxx Keywords: Osteoporosis Intervertebral disc height Intervertebral disc degeneration Type 2 diabetes Bone mineral density

a b s t r a c t Background: Studies of the effect of type 2 diabetes (T2D) on bone mineral density (BMD have produced conflicting results, possibly due to failure to adjust for potential confounding factors. Nonetheless, T2D has consistently been associated with increased fracture risk, suggesting that other factors might play a role. Objective: This study assesses the relationship between T2D and BMD at the femoral neck and spine in diabetic and non-diabetic subjects, after adjusting for multiple covariates which may affect BMD. Intervertebral disc height was also investigated in view of its possible relation to fracture risk. Methods: A cross-sectional study of 100 patients with T2DM of at least 5 years duration and 86 non-diabetic subjects was carried out. Results: There were no significant differences in T scores in either the spine or femoral neck after adjustment for potential confounding variables between T2D subjects and controls. Diabetic patients had a statistically lower intervertebral disc height between the 2nd and 3rd lumbar vertebrae (D3) after adjustment for potential confounders (p = 0.004). Urinary albumin:creatinine ratio, total cholesterol, LDL-cholesterol and cigarette smoking were independently associated with lower height of D3 in diabetic subjects. Conclusions: There is no significant independent association between T2D and BMD. However we found a novel association of significantly lower disc height in patients with T2D. This may contribute to the increased vertebral fracture risk in subjects with T2D. Further studies are needed to investigate the relationship of disc height, T2D and fracture risk. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Both type 2 diabetes (T2D) and osteoporosis are common chronic debilitating conditions associated with significant morbidity and mortality (Sealand, Razavi, & Adler, 2013). There is strong evidence in the literature that type 1 diabetes (T1D) is an independent risk factor for osteoporosis in both men and women, with a number of studies reporting reduced bone mineral density (BMD) (Kurra & Siris, 2011; Sealand et al., 2013). However, studies in subjects with T2D have reported more conflicting results, with some authors reporting that there is either a decrease or no difference in BMD when compared to non-diabetic subjects whilst others observed an increase in BMD when measured using dual-energy X-ray absorptiometry (DXA) (Kurra & Siris, 2011; Ma, Oei, Jiang, et al., 2012; Oei et al., 2013; Rakic, Davis, Chubb, et al., 2006; Sealand et al., 2013). A recently published meta-analysis concluded that BMD in both genders with T2D was significantly higher than in non-diabetic subjects indepen-

Funding sources: none Conflicts of interest: none ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (S. Fava).

dent of the skeletal site measured, age, body mass index (BMI) or medication use (Ma et al., 2012). However, the strength of a meta-analysis depends on the robustness of the individual studies that it draws upon. It is therefore pertinent to note that many studies did not adjust for a number of potential confounders. In contrast to the conflicting results on the possible association between T2D and BMD, there is a growing body of evidence showing that incidence of fragility fractures is increased in T2D especially at the femoral neck (Hothersall, Livingstone, Looker, et al., 2014; Nicodemus & Folsom, 2001; Oei et al., 2013). This has been confirmed in two meta-analyses (Janghorbani, Van Dam, Willett, & Hu, 2007; Vestergaard, 2007), both of which reported that T2D is associated with a significantly increased risk of fractures, although to a lesser degree than T1D. The aim of this study was to assess the possible association of BMD at the spine and femoral neck with T2D after correcting for multiple potential confounders which are known, or suspected, to affect BMD. These included age, gender, body mass index (BMI), waist circumference, alcohol intake, smoking status, coffee and tea consumption, use of calcium supplements, renal function (as assessed by the estimated glomerular filtration rate [eGFR]), thyroid status, serum sodium, serum lipid levels and presence of cardiovascular disease (CVD).

http://dx.doi.org/10.1016/j.jdiacomp.2016.01.021 1056-8727/© 2016 Elsevier Inc. All rights reserved.

Please cite this article as: Agius, R., et al., Bone mineral density and intervertebral disc height in type 2 diabetes, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.01.021

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R. Agius et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx

In view of the recently reported association of decreased vertebral disc height with increased vertebral fractures (Muscat Baron, Brincat, Galea, et al., 2007), we also investigated differences in disc height between T2D and controls. Furthermore, we performed a predetermined subgroup analysis of diabetic subjects in order to assess determinants of BMD and disc height in this group.

2. Materials and methods 2.1. Subjects and study population This study was a cross-sectional, case control study. One hundred Maltese patients with documented type 2 diabetes of at least 5 years duration and with an age range of between 42 and 79 were recruited from the diabetes out-patient clinics at Mater Dei Teaching Hospital in Malta between 2013 and 2014. Further inclusion criteria required the patients to be fully mobile at the time of the study and to have no recent history of prolonged period of inactivity. T2D status was ascertained by reviewing the patients' case notes, ensuring there was no history of prior ketosis, and that they had been on oral hypoglycaemic agents for at least 1 year in their treatment regime. Eighty-six non-diabetic sex-matched Maltese controls were also recruited either from non-diabetic general medical clinics or by being the spouse of the diabetic subject recruited or from hospital employees mostly consisting of either nursing or portering staff or their spouses. Exclusion criteria for both diabetic and control subjects included conditions which are known to influence bone metabolism (such as hyperparathyroidism, thyroid dysfunction, Cushing's syndrome, rheumatoid arthritis or any other connective tissue disorder, any form of malignancy and malabsorptive states); use of drugs which could influence bone turnover such as glucocorticoids, hormone replacement therapy (oestrogen or androgen preparations) or already on any form of treatment for osteoporosis (such as bisphosphonates, strontium, denosumab or calcitonin). Furthermore none of our diabetic patients were taking thiazolidinediones as oral hypoglycaemic agents since these are thought to have an association with bone loss and fracture risk (Loke, Singh, & Furberg, 2009). Data pertaining to smoking, alcohol intake, drug history, tea and coffee consumption, and use of calcium supplements were captured by use of a questionnaire. Information on presence of CVD was ascertained by review of case notes. Coronary artery disease (CAD) was defined as history of previous angina, acute coronary syndrome or a positive exercise stress test. The presence of peripheral vascular disease (PVD) was defined as the absence of pedal pulses, presence of a monophasic waveform on Doppler studies, or a prior history of bypass grafting or angioplasty in the lower limbs. Cerebrovascular disease (CrVD) was defined as previous stroke or transient ischemic attack. Height and weight were measured with the subject wearing light indoor clothing without shoes using a calibrated balance with stadiometer to the nearest 0.1 cm and 0.1 kg respectively. Body mass index (BMI) was calculated using the following equation: BMI = body weight (kg)/[height] 2. Waist circumference (WC) was calculated by measuring the waist halfway between the bottom of the rib cage and the superior iliac crest. Blood samples were withdrawn from both diabetic and control subjects for measurements of serum creatinine, calcium and phosphorus, sodium, total cholesterol, high density lipoprotein–cholesterol (HDL-C) and low density lipoprotein–cholesterol (LDL-C) as well as serum free tri-iodothyronine (T3), free tetra-iodothyronine (T4) and thyroid stimulating hormone (TSH). eGFR was calculated using the Modification of Diet in Renal Disease (MDRD) formula, which has been well-validated including in diabetic subjects (Levey et al., 1999). Furthermore in diabetic subjects the HbA1c (using high performance liquid chromatography) and urinary albumin:creatinine ratio (ACR) was also measured.

2.2. Dual-energy X-ray absorptiometry Bone mineral density was evaluated in both diabetic and control subjects using the DXA technique (Norland Bone Densitometer [DEXA 586]) because of its high accuracy and the fact that it is still considered to be the best method to measure this parameter (Cummings, Black, & Nevitt, 1993). BMD was measured at the lumbar spine (L2 to L4) and femoral neck and expressed as T-scores (this is the standard deviation [SD] from the mean value obtained in a 30-year old subject). The intervertebral discs measured were those between the 12th thoracic vertebra and the 3rd lumbar vertebra, with D1 representing the disc between the 12th thoracic vertebra and first lumbar vertebra, and D2 and D3 representing the next two discs respectively. This was carried out by the same operator at all times and measurement was done manually by applying the cursors to the edges of the discs and the distance between two consecutive vertebrae measured in the poster-anterior plane, representing the height of the annulus fibrosus as described by Muscat Baron et al. (2007). All subjects gave their informed consent stating willingness to participate in this study as well as to undergo biochemical testing and bone mineral density assessment via DXA. Ethical approval for this study was obtained from the Malta Medical School Ethics Committee. 2.3. Statistical analysis Sample size was determined so as to have 90% statistical power to detect a moderate effect size (Cohen's d = 0.5) at probability level of α = 0.05. Normality of distribution of all continuous variables was assessed using the Kolmogorov Smirnov test. Continuous data are presented as mean ± standard deviation (SD) for normally distributed variables, whilst non-normally distributed variables as median [interquartile range]. Categorical data are presented as number (percentage). Comparisons of means were made using the t-test or Fisher's exact test for normally distributed data. For non-normally distributed data, the Mann–Whitney test was used. Comparison of proportions was done using the Z-test. Thereafter, the difference in disc height and BMD between T2 diabetic subjects and controls was also assessed after adjustment for potential confounders using stepwise regression analysis (with bone mineral density and disc heights as the dependent variables and entered as continuous variables). In the first regression model, adjustment was made for age, gender and BMI. The second model was additionally adjusted for WC, tea and coffee consumption, smoking status, eGFR, use of calcium supplements, use of diuretic therapy, total cholesterol, HDL-C, LDL-C, thyroid stimulating hormone, free tri-iodothyronine (fT3), free tetra-iodothyronine (fT4), serum sodium level and presence of CVD. Furthermore, a predetermined subgroup analysis of diabetic subjects was carried out using Pearson's correlation analyses to evaluate the univariate relationship between BMD/disc height and risk factors. Multiple linear regression analysis was performed for co-variants which were statistically significant or quasi-significant (p b 0.1) in univariate analysis. All statistical calculations were carried out using Minitab statistical package. A p-value of ≤ 0.05 was taken to mean statistical significance. ‘p’ values were corrected for multiple analyses using the false discovery rate method where appropriate. 3. Results 3.1. Patient characteristics Table 1 shows the clinical characteristics of patients with T2D and the control group. In total, 100 patients with T2D and 86 controls were recruited. The study and control groups were matched for gender; however diabetic subjects were older than the control subjects: mean ± standard deviation [SD] 63 ± 7 vs. 59 ± 6 years respectively (p = 0.002). They were also significantly heavier (83 ± 15 vs.

Please cite this article as: Agius, R., et al., Bone mineral density and intervertebral disc height in type 2 diabetes, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.01.021

R. Agius et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx

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Table 1 Baseline patient demographic characteristics.

b

Age (years) Males (%)c Weight (kg)b BMI (kg/m2)d WC (cm)b History of osteoporotic fracturec History of traumatic fracturec Calcium supplementsc Diuretic usec Smokersd Tea consumptiond Coffee consumption (cups/week)d CADc PVDc CrVDc Any form of CVDc Postmenopausal (females)c

T2 DM (n = 100)

Controls (n = 86)

Uncorrected p-value

Corrected p-valuea

62.63 ± 7.82 53 (53%) 83.4 ± 15 31.54 [28.01–35.12] 102.4 ± 12.2 3 (3) 7 (7) 6 (6) 35 (35) 9 (9) 7 [0–21] 7 [0–20.25] 23 (23) 6 (6) 3 (3) 32 (32) 36 (80)

59.19 ± 6.36 36 (41.8%) 75.7 ± 13.3 29.0 [26.46–32.13] 92.8 ± 12.1 0 1 (1.1) 9 (10.4) 5 (5.8) 13 (15.1) 12 [3–21] 7 [0–20.75] 2 (2.3) 0 0 2 34 (68)

0.001 0.633 b0.001 0.002 b0.001 0.25 0.071 0.272 b0.001 0.203 0.496 0.878 b0.001 0.031 0.765 b0.001 0.62

0.002 0.707 b0.001 0.004 b0.001 0.25 0.071 0.369 0.002 0. 321 0.628 0.888 b0.001 0.059 0.808 b0.001 0.707

T2 DM: type 2 diabetes; BMI: body mass index; WC: waist circumference; CAD: coronary artery disease; PVD: peripheral vascular disease; CrVD: cerebro-vascular disease; CVD: cardiovascular disease; MTF: metformin; SU: sulphonylurea. a p-Values shown have been corrected for multiple testing by the false discovery method. b Unadjusted means ± SD c Number (%). d Median [interquartile range].

75 ± 13 kg; p b 0.001), had a higher BMI (31.54 [interquartile range, IQR, 28.01–35.12] vs. 29.0 [IQR 26.46–32.13] Kg/m 2; p = 0.004) and a larger WC (102 ± 12 vs. 93 ± 12 cm; p b 0.001) than controls. None of the patients had previous fractures. There were no statistically significant differences in smoking status, use of calcium supplements or in consumption of tea or coffee. T2D subjects were more likely to have CAD or any form of CVD and to be taking diuretics. Postmenopausal status in females was similar in both groups (36 T2D subjects vs. 34 controls). Mean duration of diabetes within the type 2 diabetic group was 9.82 years (range 5–35). Nineteen percent of diabetic patients were on insulin, 86% on metformin and 43% on sulphonylureas.

adjustment for potential confounders. BMD T-score in T2D subjects was slightly higher at the third lumbar vertebra in monovariate analysis, but this difference disappeared after correction for multiple testing and also after adjustment for potential confounders. There were no differences in hip or other vertebral T-scores between the diabetic and control subjects either in monovariate analysis or after adjustment for potential confounders. Disc height at the D3 level was significantly lower after adjustment for potential confounders in type 2 diabetic subjects when compared to controls (by 0.051 cm); however differences in D1 and D2 did not achieve statistical significance.

3.2. Biochemical analysis

3.4. Diabetic subgroup analysis

Table 2 summarises the biochemical data for both diabetic and control subjects. Diabetic subjects had lower eGFR values as well as lower total cholesterol and LDL-C values (p b 0.001). Mean HbA1c was 67 mmol/mol and ACR value was 11.69 mg/mmol in the diabetic group.

Correlation analyses in the diabetic subgroup to evaluate the univariate relationship between BMD/disc height and risk factors are summarised in Table 4. With respect to disc height, D3 was negatively correlated with ACR, cigarette smoking, total cholesterol and LDL-C (p value ≤0.05 in all cases). D2 was positively correlated with alcohol consumption, total cholesterol and LDL-C (all p values b 0.05). There were no significant correlations in D1. On the other hand BMD T-score of the lumbar spine showed a positive correlation with BMI (r = 0.27, p value = 0.01) and negative associations with total cholesterol and HDL-C (p values b0.05 in both cases). Femoral neck T-score was

3.3. Bone mineral density and disc height in T2 diabetic subjects and controls BMD T-scores and disc height for the intervertebral discs between T12 and L3 are summarised in Table 3. Fig. 1 shows differences after Table 2 Biochemical profile of both diabetic and control subjects.

2 b

eGFR (ml/min/1.73 m ) TSH (mIU/L)c FT4 (pmol/L)b TC (mmol/L)b HDL-C (mmol/L)c LDL-C (mmol/L)b Sodium (mmol/L)c HbA1C (%)b ACR (mg/g)b

T2 DM (n = 100)

Controls (n = 86)

Uncorrected p-value

Corrected p-valuea

82.5 ± 28.3 1.70 [1.23–2.1] 13.55 ± 1.85 4.75 ± 1.27 1.315 [1.09–1.5] 2.67 ± 1.05 141.0 [138.75–143] 8.38 ± 2.06 11.69 [6.4–20.61]

90.8 ± 16.7 1.79 [1.29–2.29] 12.67 ± 2.08 5.76 ± 0.9575 1.315 [1.09–1.63] 3.693 ± 0.953 142.0 [140–143]

0.016 0.4599 0.003 b0.001 0.553 b0.001 0.966

0.028 0.643 0.007 b0.001 0.645 b0.001 0.966

eGFR: estimated glomerular filtration rate; TSH: thyroid stimulating hormone; FT4: free thyroxine; TC: total cholesterol; HDL-C high density lipoprotein cholesterol; LDL-C: low density lipoprotein cholesterol; HbA1c: haemoglobin A1c; ACR: urinary albumin creatinine ratio. a p-Values shown have been corrected for multiple testing by the false discovery method. b Unadjusted mean ± SD. c Median [interquartile range].

Please cite this article as: Agius, R., et al., Bone mineral density and intervertebral disc height in type 2 diabetes, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.01.021

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R. Agius et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx

Table 3 Bone mineral density and disc heights measured by dual energy X-ray absorptiometry.

T-score (hip)b T-score (spine)c D1c (cm) D2c (cm) D3c (cm)

T2 DM (n = 100)

Controls (n = 86)

Uncorrected p-value

Corrected p-valuea

−1.53 [−0.58 to 2.00] 0.08 ± 1.25

−1.45 [−0.76 to 2.00] −0.29 ± 1.24

0.853

0.943

0.049

0.122

0.39 ± 0.10 0.42 ± 0.10 0.41 ± 0.09

0.244 0.943 0.042

0.4 0.943 0.122

0.40 ± 0.12 0.42 ± 0.12 0.39 ± 0.093

D1: disc height between 12th thoracic and 1st lumbar vertebra; D2: disc height between 1st lumbar and 2nd lumbar vertebra; D3: disc height between 2nd lumbar and 3rd lumbar vertebra. a p-Values shown have been corrected for multiple testing by the false discovery method. b Median [interquartile range]. c Unadjusted mean ± SD.

strongly and positively correlated with BMI, WC and eGFR (p values all b 0.05) but negatively correlated with age (r = − 0.34 p = b 0.001) and presence of CVD (r = − 0.26; p = 0.009). Further analysis was done on the diabetic group using multiple linear regression on variables that were significant or quasi-significant (p b 0.1) with BMD or disc height as the dependent variable (Tables 5 and 6). With respect to disc height, urinary ACR, cigarette smoking, total cholesterol and LDL-C all remained independently and negatively associated with disc height at D3 and only HbA1c showed 0.03 0.02

0.017

0.013

Table 4 Correlation analysis between all covariates and the intervertebral discs (D1, D2, D3) and spinal and femoral neck T-scores in type 2 diabetic subjects using Pearson correlation coefficient, Pearson's r. Covariate

Age (years) Sex BMI Waist circumference HbA1c Diabetes duration ACR Alcohol consumption No. of cigarettes Tea consumption Coffee consumption Serum TSH Serum free T4 Serum total cholesterol Serum HDL-C Serum LDL-C Serum sodium eGFR CVD

D1

D2

D3

T-score spine

T-score femoral neck

r

r

r

r

r

0.12 −0.10 −0.03 0.08

0.16 0.00 −0.09 0.05

−0.11 0.03 0.27⁎⁎ 0.16

−0.35 0.12 0.34⁎⁎ 0.21⁎

0.08 0.00 −0.08 0.04

0.07 −0.01 −0.18 0.22⁎

0.19 0.06 −0.19⁎ −0.02

0.01 −0.03 −0.06 −0.131

−0.16 −0.16 −0.15 −0.04

−0.01 0.10 0.10

−0.04 −0.03 0.01

−0.21⁎ −0.05

0.006 0.05 −0.05

0.002 0.07 −0.02

−0.06 −0.03 0.06

0.05 −0.02 −0.23⁎

0.03 −0.06 −0.22⁎

0.08 0.09 −0.91⁎

−0.04 −0.04 −0.06

0.09 −0.04 0.01 0.08 0.081

−0.06 −0.23⁎ 0.10 −0.03 −0.043

0.02 −0.23⁎ −0.01 −0.04 0.163

−0.17 −0.12 0.13 0.04 −0.074

−0.08 0.01 0.01 0.225⁎ −0.260⁎⁎

0.03 0.07 0.07 0.16

BMI: body mass index; HbA1c: haemoglobin A1c; ACR: urinary albumin creatinine ratio; CVD: any form of cardiovascular disease; TSH: thyroid stimulating hormone; FT4: free thyroxine; HDL-C high density lipoprotein cholesterol; LDL-C: low density lipoprotein cholesterol; eGFR: estimated glomerular filtration rate. ⁎ p b 0.05. ⁎⁎ p b 0.01.

0.01 0 0.001

0.01

D1 D2

0.013

0.02

D3

0.03

0.028*

0.04 0.05 0.051**

0.06 Model1

4. Discussion

Model2

0.25 0.22

0.21

0.2

0.15 Femoral neck T score Spine T score

0.1

an independent positive association with D3. Only ACR was negatively associated with disc height at D2. With respect to BMD, BMI was positively associated with T-scores at both the lumbar spine and femoral neck. Femoral neck BMD was also negatively associated with age and presence of CVD. Since there were no significant or quasi-significant correlations with regard to D1, no regression analysis was performed.

This present study shows no difference in hip or vertebral BMD T-score between diabetic subjects and controls either in univariate analysis or after adjustment for potential confounders. Previously reported associations between BMD and T2D may have been due to failure to adjust for potential confounders, which may also explain in the inconsistency of results of previous studies. We included a number of such potential confounders including age, gender, BMI, WC, tea and coffee consumption, smoking status, eGFR, use of calcium

0.05

0.05

Table 5 Multiple linear regression analyses of determinants in T2 diabetic subjects with the dependent variables being disc heights D3 and D2.

0.02

0 Model1

Model2

Fig. 1. Adjusted mean difference in disc heights (upper panel) and BMD (lower panel) between the diabetic and control groups. In model 1 differences were adjusted for age, gender and body mass index. Model 2 was additionally adjusted for waist circumference, tea and coffee consumption, smoking status, eGFR, use of calcium supplement, use of diuretic therapy, presence of cardiovascular disease total cholesterol, high-density-cholesterol, low density cholesterol, thyroid stimulating hormone and free thyroxine index and serum sodium level. *p b 0.05; **p = 0.004. All other differences were not statistically significant. ‘p’ values are corrected for multiple testing by the false discovery method).

HbA1c ACR TC LDL-C Cigarettes smoking

Dependent variable: D3

Dependent variable: D2

β coefficient (SE)

p-Value

β coefficient (SE)

p-Value

0.01 (0.004) −0.0003 (0.0001) −0.01 (0.013) −0.01 (0.01) −0.002 (0.001)

0.004 0.0001 0.013 0.016 0.022

−0.003 (0.000) −0.01 (0.02) −0.01 (0.02)

0.036 0.593 0.593

HbA1c: haemoglobin A1c; ACR: urinary albumin creatinine ratio; TC: total cholesterol; LDL-C: low density lipoprotein cholesterol.

Please cite this article as: Agius, R., et al., Bone mineral density and intervertebral disc height in type 2 diabetes, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.01.021

R. Agius et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx Table 6 Multiple linear regression analyses of determinants of bone mineral density in type 2 diabetic subjects with the dependent variables being lumbar spine and femoral neck T-score.

Age BMI WC eGFR TC HDL-C CVD

Dependent variable: lumbar spine T-score

Dependent variable: femoral neck T-score

β coefficient (SE)

β coefficient (SE)

p-Value

−0.034 0.067 0.002 0.007

0.049 0.034 0.915 0.145

p-Value

0.06 (0.02)

0.008

−0.48 (0.32) −0.48 (0.32)

0.140 0.140 −0.67

(0.017) (0.03) (0.014) (0.005)

0.03

BMI: body mass index; eGFR: estimated glomerular filtration rate; TC: total cholesterol; HDL-C: high density lipoprotein cholesterol; HbA1c: haemoglobin A1c; ACR: albumin creatinine ratio.

supplement, diuretic therapy use and thyroid status. All these factors are well-established as influencing BMD. Serum sodium concentration has also been linked to osteoporosis. Recently there have been several reports of an association of serum lipids with BMD (Kim et al., 2013), increased bone turnover (Jeong, Lee, Choi, et al., 2014) and increased fracture risk (Yamauchi et al., 2015). Furthermore, dyslipidaemia and cardiovascular disease have also been linked to intervertebral disc disease (Longo et al., 2011). For this reason we adjusted for all these factors as well. Our study therefore suggests that T2D is not independently associated with abnormal BMD. However, it has been consistently shown that subjects with T2D have an increased fracture risk, and that this risk in out of proportion to their BMD (Hothersall et al., 2014; Janghorbani et al., 2007; Ma et al., 2012; Oei et al., 2013; Vestergaard, 2007). This suggests that there are other factors which increase fracture risk in T2D. Interestingly, we found a lower D3 in diabetic subjects compared to controls in both regression models. To our knowledge, this has not been previously reported. It cannot be explained by the reported association of disc height with BMD (Kwok et al., 2012), since the latter was similar in the two groups. Decreased disc height has been previously shown to increase the risk of vertebral fractures (Adams, Pollintine, Tobias, Wakley, & Dolan, 2006; Muscat Baron et al., 2007), possibly as a result of abnormal distribution of compressive forces. In fact, disc degeneration has been reported to result in concentration of compressive load-bearing onto the neural arch and the posterior vertebral body in upright postures (Adams et al., 2006; Pollintine, Dolan, Tobias, & Adams, 2004) and on the anterior vertebral body in forward bending (Pollintine et al., 2004). Disc degeneration, as evidenced by low disc height may therefore explain, at least in part, the increased vertebral fracture risk observed in T2D (Rathmann & Kostev, 2015; Zhukouskaya et al., 2015). Our data are consistent with the finding of disc degeneration in rats with streptozocin-induced diabetes (Jiang, Zhang, Zheng, et al., 2013). Disc degeneration in diabetes may be mediated by abnormal disc protein glycation and accumulation of advanced glycation endproteins (Jiang et al., 2013; Tsung-Ting, Ho, Lin, et al., 2014). We postulate that abnormal glycation of disc proteins may lead to a reduction in the shock-absorbing properties of the disc and thus an increased propensity to fracture, as occurs in senescence (Muscat Baron et al., 2007). This is supported by data from animal models of type 2 diabetes (Fields et al., 2015). The intervertebral discs may also be particularly susceptible to the vasculopathy associated with diabetes, since they are dependent on diffusion of nutrients and oxygen from capillaries at their margins (reviewed by Grunhagen, Shirazi-Adl, Fairbank, & Urban, 2011). The discs are dependent on small capillaries which penetrate the subchondral plate of the vertebral body to form terminal loops at the end plate (Cheng et al., 2013; Kong, Park, Kim, & Park, 2014). Nutrients and oxygen therefore

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need to diffuse relatively long distances from these capillaries. Microvascular disease may hence impair intervertebral disc cell viability. Additionally, diabetes may also be associated with decreased endplate porosity (Fields et al., 2015), increased disc cell autophagy (Kong et al., 2014) and increased disc matrix degradation as a result of overactivity of the polyol pathway (Cheng et al., 2013). Our diabetic subgroup multivariate analysis may give some clues as to the possible reasons for lower disc height in T2D. We found a statistically strong independent negative association of disc height with ACR and an independent positive association with HbA1c. ACR is a very good marker of generalised microvascular disease. This therefore strengthens the possibility of microvascular disease playing a role in disc degeneration in T2D. The positive association of disc height with HbA1c is unclear. However, it should be noted that HbA1c only reflects very recent glycaemic control (less than 3 months). The result may possibly be due to lowering of HbA1c as a result of intensification of treatment in previously uncontrolled subjects with complications. It is likely that ACR is a better marker of total life-time glycaemic exposure than a single measure of HbA1c, since the development and progression of albuminuria are known to be linked to long-term glycaemic control (Stratton, Adler, Neil, et al., 2000). We also found smoking, total cholesterol and LDL-cholesterol to be significantly and independently associated with lower disc height. This is consistent with finding that these factors are associated with disc degeneration, as reported by authors (Battié et al., 1991; Longo et al., 2011). Both dyslipidaemia and smoking may adversely affect the blood supply and oxygenation of the disc. It is probable that there are other factors, not investigated in the present study, which contribute to the increased fracture risk in T2D. These may include abnormal bone architecture independent of BMD; trabecular bone health (not measured by BMD); sarcopaenia; decreased bone turnover (causing impaired healing of microfractures) and increased falls. Loss of proprioception, visual loss, reduced muscle strength and hypoglycaemia all predispose to falls in subjects with T2D (Kurra & Siris, 2011). In the diabetic subgroup, we found an independent positive association of spine and femoral neck BMD with BMI in multivariate analysis as well as independent negative association of femoral neck BMD with age and presence of CVD. These results replicate the findings of other authors in both the general population as well as in diabetic subjects (Lloyd, Alley, Hawkes, Hochberg, et al., 2014; Värri et al., 2014). Possible explanations of the link between low BMD and CVD include low grade inflammation and oxidative stress, which predispose to both low BMD and CVD (Danesh, Wheeler, Hirschfield, et al., 2004; Ding, Parameswaran, Udayan, Burgess, & Jones, 2008), and common genetic factors (Fodor et al., 2013). Osteoprotegerin may also mediate the association between low BMD and CVD. Although it inhibits bone absorption, some authors have reported a negative association with BMD (Pobeha, Petrasova, Tkacova, & Joppa, 2014). High osteoprotegerin have also been linked to increased cardiovascular risk (Schoppet, Sattler, Schaefer, et al., 2003), offering a possible explanation of the association of low BMD with cardiovascular disease. 4.1. Strengths and limitations The sample size was limited but it was similar to that of other cross-sectional studies done in type 2 diabetic subjects and was determined so as to have 90% statistical power to detect a moderate effect. Other strengths include the accurate capturing of patient demographic data and other diabetes-related parameters via a standardized questionnaire and most of the data being ascertained after reviewing the patients' hospital records ensuring high data quality. In view of this, we were able to adjust for multiple confounders. To our knowledge our study adjusted for the largest number of such variables, although there are undoubtedly other

Please cite this article as: Agius, R., et al., Bone mineral density and intervertebral disc height in type 2 diabetes, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.01.021

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residual confounders that we could not adjust for. Measurement of disc height (which is observer dependent) was carried out by only one person (the first author) in order to limit any potential observer bias. None of the diabetic subjects in this study were on the thiazolidinedione class of oral hypoglycaemic agents which are known to be associated with bone loss (Yaturu, Bryant, & Jain, 2007) and thus was not a confounding issue in this study. One limitation is the significant difference in age between the two groups, even though this was adjusted for in the statistical analysis. Another factor regarding the control group is that some patients were recruited from general medical outpatient clinics and while every care was taken to ensure that all inclusion and exclusion criteria were met, they might not be ‘true’ controls in that they might not be truly representing the general population. There were only about 25% of subjects recruited form general medical clinics with the remaining 75% being recruited from the general portering or nursing staff or their spouses. 5. Conclusion To our knowledge this is the first report of decrease in intervertebral disc height in diabetic subjects when compared to controls. We found no difference in lumbar spine or hip T score between diabetic and non-diabetic subjects after adjusting for these confounders. Our data therefore suggest that the inconsistency of previous reports in the literature is most likely to be due to failure to adjust for potential confounders. Our data further suggest that increased fracture risk in subjects with T2D must be mediated through factors other than BMD. One such contributory factor may be intervertebral disc degeneration resulting in reduction in height. We therefore suggest that, if our results are confirmed by other authors, disc height should be routinely measured in addition to BMD in T2D in order to better assess fracture risk. Disclosures The authors have no disclosures to declare. References Adams, M. A., Pollintine, P., Tobias, J. H., Wakley, G. K., & Dolan, P. (2006). Intervertebral disc degeneration can predispose to anterior vertebral fractures in the thoracolumbar spine. Journal of Bone and Mineral Research, 21, 1409–1416. Battié, M. C., Videman, T., Gill, K., Moneta, G. B., Nyman, R., Kaprio, J., & Koskenvuo, M. (1991). 1991 Volvo Award in clinical sciences. Smoking and lumbar intervertebral disc degeneration: an MRI study of identical twins. Spine (Phila Pa 1976), 16(9), 1015–1021. Cheng, X., Ni, B., Zhang, Z., Liu, Q., Wang, L., Ding, Y., & Hu, Y. (2013). Polyol pathway mediates enhanced degradation of extracellular matrix via p38 MAPK activation in intervertebral disc of diabetic rats. Connective Tissue Research, 54(2), 118–122. Cummings, S. R., Black, D. M., & Nevitt, M. C. (1993). Bone density at various sites for prediction of hip fractures. Lancet, 341, 72–75. Danesh, J., Wheeler, J. G., Hirschfield, G. M., et al. (2004). C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease. New England Journal of Medicine, 350(14), 1387–1397. Ding, C., Parameswaran, V., Udayan, R., Burgess, J., & Jones, G. (2008). Circulating levels of inflammatory markers predict change in bone mineral density and resorption in older adults: a longitudinal study. Journal of Clinical Endocrinology and Metabolism, 93(5), 1952–1958. Fields, A. J., Berg-Johansen, B., Metz, L. N., Miller, S., La, B., Liebenberg, E. C., ... Lotz, J. C. (2015). Alterations in intervertebral disc composition, matrix homeostasis and biomechanical behavior in the UCD-T2DM rat model of type 2 diabetes. Journal of Orthopaedic Research, 33(5), 738–746. Fodor, D., Bondor, C., Albu, A., Popp, R., Pop, I. V., & Poanta, L. (2013). Relationship between VKORC1 single nucleotide polymorphism 1173C N T, bone mineral density & carotid intima-media thickness. Indian Journal of Medical Research, 137(4), 734–741. Grunhagen, T., Shirazi-Adl, A., Fairbank, J. C., & Urban, J. P. (2011). Intervertebral disk nutrition: a review of factors influencing concentrations of nutrients and metabolites. The Orthopedic Clinics of North America, 42, 465–477.

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Please cite this article as: Agius, R., et al., Bone mineral density and intervertebral disc height in type 2 diabetes, Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.01.021