Ethics statement
The study protocol (No. 2016–11–009-019) was approved by the institutional review board of this institute. Study procedures were performed following Declaration of Helsinki principles. Written informed consent was obtained from each participant.
Study design and population
In this prospective study, participants were enrolled at a hospital in South Korea between January 2018 and June 2019. Only participants 55–80 years of age were included in the study. Exclusion criteria were patients with a history of malignancy, active infection, auto-immune disease, or previous fracture or surgery of structures in or around the shoulder. No patient had megaloblastic anemia or neuropathy associated with clinically established Vit B12 deficiency.
The study group was divided into the RC tear group and the non-RC tear group. For inclusion in the RC tear group, a patient was required to have a full-thickness RC tear involving the supraspinatus or other RC tendons, or both, and admission to the study institute for arthroscopic RC repair. A non-operative treatment protocol was followed for at least 3 months before surgery. The non-operative treatment protocol included stopping offending activities, physiotherapy (manipulation, stretching, ultrasound, electrical stimulation, and strengthening exercises), corticosteroid injections, anti-inflammatory agents, and extracorporeal shock wave therapy. Only patients with degenerative RC tear were included in the RC group. The condition was confirmed by detailed history taking including the significant trauma history around the shoulder with a physical examination and magnetic resonance imaging (MRI) (Signa Architect 3.0 T; General Electronic Healthcare) to exclude the traumatic RC tear. MRI was used to measure tear size (width and length). During the study period, 75 patients had a diagnosis of full-thickness RC tear and underwent surgical treatment. Fourteen of these patients refused to participate in the study. Thirteen patients were excluded from the study were because the data were incomplete. One patient was excluded because of auto-immune disease (rheumatoid arthritis), malignancy (2 patients), or a history of fracture (3 patients) or surgery in the affected shoulder (2 patients). A total of 40 patients were assigned to the RC tear group.
To be eligible for inclusion in the non-RC tear group, a patient had no RC tear or associated symptoms or clinical signs (e.g., loss of shoulder motion, signs of impingement, tenderness), and visited the institute’s orthopedic clinic. Eligible patients visited the orthopedic clinic during the study period because of minor trauma (dressing superficial lacerations, contusions, and abrasions) of any area except the shoulder region. None previously underwent surgery relating to RC tear. A total of 47 healthy participants who met these criteria and agreed to participate in the study were assigned to the non-RC tear group (Fig. 1).
Variables and data collection
General information collected for each participant included demographic data and medical history. Data on biochemical markers including glucose, magnesium (Mg), calcium (Ca), phosphorus (P), zinc (Zn), homocysteine (HCY), vitamin D (Vit D), folate, and Vit B12 were evaluated. To measure biochemical markers, approximately 10-ml venous blood samples were collected in vacuum-sealed blood tubes in the outpatient or inpatient clinic after an overnight fast. Each sample was centrifuged (3000 rpm, 15 min) and then stored at − 80 °C. Serum electrolyte levels were obtained using a Roche COBAS E701 electrolyte analyzer. Serum Vit B12 was determined via electrochemiluminescence immune assay (ELISA) (Roche Elecsys 2010 analyzer; Switzerland). The remaining components were analyzed using a Siemens Atellica auto-analyzer. All samples were analyzed at the hospital’s laboratory services in the department of clinical pathology at our institution. All staff who performed the analyses were blinded to the study group assignments [13].
Bone mineral density (BMD, g/cm2) was also measured because study findings indicate that BMD is associated with RC tear development [14]. Dual-energy X-ray absorptiometry (Hologic QDR-4500, USA; software version 9.30.044) was used to measure lumbar spine (L1 - L4) and proximal femur (femur neck and total hip) areal BMD.
Arthroscopic examination and RC repair
The same surgeon performed each arthroscopic procedure with the patient in the lateral decubitus position. After the surgeon accessed the subacromial space, partial bursectomy and debridement were used to visualize torn RC tendon margins. When the torn cuff was visible, the RC repairs were performed based on tear size [15].
Sample size calculation and statistical analyses
The results were presented as mean (± standard deviation) values within groups. Variables with P-values < 0.05 were considered statistically significant. The minimum sample size was calculated to ensure sufficient power to analyze the primary outcome variable (RC tear). The parameters included in the sample size estimate were: two-sided t-test, alpha = 0.05, power = 0.80, assumed difference in Vit B12 levels of 21.2% between non-RC tear (744 ± 239 pg/ml) and RC tear (587 ± 215 pg/ml) groups. The values used were based on results of preliminary studies. The calculated minimum sample size was 34 for each group.
To identify correlations between nominal variables and RC tear, Chi-square tests were performed between the RC tear group and non-RC tear group for diabetes mellitus, hypertension, and stroke history. Independent sample t-tests were performed for continuous variables. Continuous variables included glucose, Mg, Ca, P, Zn, Vit D, Vit B12, folate, HCY and BMD.
Logistic regression analysis (backward elimination) was performed to identify independent predictors of RC tear while adjusting for demographic and clinical variables. The variables were retained or deleted based on their statistical contribution. After this process, we were able to find out following 8 variables: Sex, Age, DM, Glucose, P, Vit D, Vit B12, and BMD.
A bivariate analysis was done to compare Vit B12 by RC tear using log-binomial regression to calculate relative risk. We split the Vit B12 according to the quartiles into 4 groups: samples divided by the cut-off point 25th (460.1 pg/mL), 50th (559.0 pg/mL), and 75th (695.5 pg/mL) percentile. Unadjusted and adjusted relative risks and 95% confidence intervals were reported. Adjusted analyses were controlled for eight variables selected by backward elimination regression previously.
Analysis of variance (ANOVA) was used for the analysis of differences in Vit B12 concentrations according to the ranges of retraction (length) and anteroposterior extension (width). These statistical analyses were performed using G*Power software and R version 3.5.2 for Windows.