October - December 2017 | Vol 3 | Issue 4 | Page : 47-52
1 Founder and Head Researcher, OPTM Research Institute, 145 Rashbehari Avenue, Kolkata – 700029, India
Backgrounds: The disease risk factor prediction with calcium-phosphorus ratio (CPR) and/or parathyroid hormone (PTH) levels are suitable biomarkers. The aim of the present study was to investigate the functional instability of these biomarkers in the blood on the risk of osteoarthritic disorder (OAD) by using receiver operating characteristic (ROC) curves. Methods: Separate evaluations were performed for subjects 132 with OAD and 109 without OAD symptoms using questionnaires, standardized physical and radiographic examinations, and risk factor identification (hypoparathyroidism, hypocalcaemia and hyperphosphatemia diseases). The blood levels of PTH, calcium, and phosphorus were measured by using appropriate kits. ROC curve and logistic regression analyses were performed for the PTH and CPR levels. Results: The area under the ROC curve (AUC), 95% CI for the AUC, for the OAD compared with the non-OAD cohorts were 0.985, 0.969-1.000, and P<0.001 for the PTH analysis and 0.579, 0.506-0.652, and P<0.05 for the CPR analysis. In the OAD cohort, the AUC and the PTH risk were higher for men than for women; AUC=1.000 for men, and AUC=0.977 for women, with both AUC values highly significant (P<0.001). The CPR risk was higher for men (AUC=0.614, 95% CI=0.483-0.746, P=0.079) than for women (AUC=0.516, 95% CI=0.419-0.613, P=0.736) but was not statistically significant in either sex. Conclusion: A functional instability risk that is higher in male than female OAD cohorts causes lower PTH and CPR levels during OADs, which can be considered one of the OAD diagnostic protocols besides radiological images.
Keywords:Osteoarthritic disorders, Diagnostic protocol, Calcium-phosphorus ratio, Parathyroid hormone, Degenerative changes, Receiver operating characteristics
Parathyroid hormone (PTH) is secreted by four, tiny, rice-grain-sized parathyroid glands located in the neck region behind the thyroid gland. PTH controls the calcium and phosphorus levels in the blood, which are the most and second most abundant minerals in the body, respectively. PTH very powerfully influences bone cells, causing them to release their calcium into the blood stream. However, when bones release their calcium into the blood under the influence of PTH at too high a rate, retaining too little calcium, the diseases osteopenia and then osteoporosis develop. The PTH test is also very helpful to detect diseases such as hyper-or hypoparathyroidism. A blood PTH level that is too high or too low can cause problems not only with bones but also with the kidneys as well as changes in the body levels of calcium and vitamin D. A proper calcium balance is crucial for the function of the nervous, muscular and skeletal systems, which assist nerve impulse conduction and muscle contraction. Biochemically PTH exerts a major in?uence over the systemic rate of bone resorption, which is assisted by osteoclasts (bone resorbing cells). The researchers have documented that PTH increases the restoratives activity of pre-existing osteoclasts through a primary hormonal interaction with cells of the osteoblastic lineage, which possess PTH receptors and responsiveness.[3-5] Fuller et al. stated that for osteoclast formation from osteoclast precursors, a close relationship and dependency on a direct action of 1,25(OH)2D3 and/or PTH exists. Interestingly, the association between endogenous PTH and knee osteoarthritis was examined in an animal model study that showed disease modification by PTH; however, no data were obtained in humans to show a significant association between PTH and radiographic knee osteoarthritis.
Several studies have also revealed an association between lowering the calcium-phosphorus ratio (CPR) and disease as well as between PTH induction and disorders. Estimates of the CPR and PTH levels have already been established during various diseases, such as renal failure, heart diseases, neurological disorders, rheumatic arthritis, osteoarthritis disorders and others.[7-12] However, no one has analyzed these risk factors through the statistically relevant data of lowered CPR and lowered PTH levels (hypoparathyroidism) in the blood of patients with osteoarthritic disorders (OADs).
Additionally, the estimated blood CPR is known to be a suitable marker during bone formation. As stated earlier, any disease leads to oxidative stress and the existence of free radical generation in different tissues. Several researchers have studied whether inflammation and rheumatoid arthritis cause oxidative stress and a decrease in the blood CPR.[13-15] Calcium and phosphorus are well-known micronutrients that researchers have established as participants in the regulation of different physiological functions. Calcium regulates vascular contraction, vasodilatation, glandular secretion, muscular contraction, glycogen metabolism, neurotransmission and, finally, the maintenance of bone health and mineralization;[9, 16] while phosphorus facilitates mineral metabolism, cellular signal transduction, the exchange of energy and, along with calcium, bone development.[9, 17] A decreased blood calcium level causes bone deformation, renal disease and hypoparathyroidism, among other problems.[18-19] Thomas and Endres and Rude have documented that increased total calcium enhances hyperparathyroidism as well as other diseases, and that phosphorus, also found in serum, is an important bone mineral, along with calcium.[18-19]
Osteoarthritic disorders (OADs) belong to a painful, inflammatory and degenerative disease involving the muscles and bones of any joint, including the knee, ankle, hip and vertebral column, to name a few.[8,20-23] Several diagnostic protocols based on such disciplines as radiology, anatomy, biochemistry and haematology have already been established.[24-28] In patients with OADs, numerous biochemical markers have shown an increasing trend in various tissues, such as blood, cartilage, bone and synovial fluid.[28-35] According to several researchers, statistical exploration through receiver operating characteristic (ROC) curves and logistic regression analyses can be used to confirm the sensitivity and specificity of the biomarker data obtained from clinical research.[36-38] Moreover, ROC curves describe the sensitivity of the prediction rule in relation to one minus the specificity. Basically, a ROC curve is a risk-prediction-based model for disease diagnosis[37-39] whereas the use of logistic regression to analyze biomarkers included as continuously-valued covariates yields a model that is implicitly based on the assumption that the biomarker follows a normal (Gaussian) distribution among individuals who experience the disease (experimental group) as well as those who have no disease (control group).[38,41-42] The odds ratio is obtained in logistic regression analyses to determine whether patients are highly susceptible or relatively immune to disease.
The routine biochemical testing of patients for disease biomarkers is a common phenomenon but an approach with a statistical interpretation for several risk factors identified for other diseases that has been found by few research studies to be an accurate diagnostic classification.[6, 43-46] Nevertheless, considerable research has documented bone deformities through radiographic gradations as well as hyperthyroidism in OADs. However, an approach with CPR and PTH markers is lacking for the detection of such risk factors as hypo-parathyroidism, hypocalcaemia and hyperphosphatemia by the analysis of statistically relevant ROC curves, logistic regression analyses and respective 95% CIs in subjects with and without OAD. The present study analyzed hypoparathyroidism, hypocalcemia and hyperphosphatemia as OAD risk factors through statistically relevant data on markers using a lowered calcium-phosphorus ratio (CPR) and lowered parathyroid hormone (PTH) level in the serum of patients with osteoarthritic disorders.
Recruitment of patients
In all, 351 participants (239 females and 112 males) with an average age of 57.03±6.89 years were evaluated in this study; the participants were treated at OPTM Health Care (P) Ltd. centers in Kolkata, Delhi, and Mumbai, India, from January 2016 to March 2017. The study protocol was evaluated and approved by the OPTM Research Institute Ethics Committee. This institute is registered with the Indian government. An institutional-review-board- approved consent form for physical examinations, blood sample collections and cervical spine, lumbar spine and hip and knee joint images (X-rays, CT scan or MRI) was required for the study and was signed by all the patients during the first phase of the screening procedure.
One hundred and ten (78 females and 32 males) of the 351 participants were excluded due to any concomitant disease, severe metabolic disorder, addiction or psychiatric problem, surgery or arthroscopy within the three months prior to inclusion, oncological condition or severe bone or joint deformation. The following additional exclusion criteria were considered: multiple drug dependence, a pacemaker, chronic liver and heart diseases, a history of cancer, severe neurological disease and a patient unwillingness to agree to a physical evaluation.
After applying the exclusion criteria, 132 (94 females and 38 males) of the remaining 241 subjects composed the experimental group (OAD group); these subjects had complaints of pain or visual inflammation and signs of OADs in the cervical or lumber region, hip joints, knee joints or any body part, as evidenced by X-ray, CT scan or MRI reports. The remaining 109 subjects (67 females and 42 males) composed the control group (non-OAD group) and had no complaints of pain or visual inflammation and no signs of OAD in anybody region, according to the radiological analysis.
Specific biochemical parameters in blood
A 5-ml blood sample was collected from each subject with or without OAD in a plain vial. The blood samples were then centrifuged at 1000×g for 10 min at 4oC to obtain serum. Finally, the serum contents of calcium, phosphorus and PTH were analyzed for both the experimental and the control subjects. The calcium levels (mg/dl) were quantitatively assessed at a wavelength of 650 nm via a photometric test method using arsenazo III provided by the Calcium AS FS kit (DiaSys Diagnostic Systems GmbH, Germany). The kit was developed based on the methods reported by Michaylova and Ilkova and Bauer. The phosphorus levels (mg/dl) were quantitatively assessed at a wavelength of 340 nm using a photometric test method provided by the Phosphate FS kit (DiaSys Diagnostic Systems GmbH, Germany). The kit was developed based on the methods reported by Thomas. The PTH levels (pg/ml) were quantitatively assessed using an immunoassay available in an intact-PTH ELISA kit (Biomerica Inc., U.S.A, Ref 7022). The kit was developed based on the methods reported by Raisz et al., Mallette and Kruger et al.
The continuous variables, such as the serum CPR and PTH levels of the OAD subjects, are expressed as means, standard deviations (SD) and 95% confidence intervals (CIs) of differences and were compared between the experimental and control groups using the Mann-Whitney U test, because the data do not follow a normal distribution. A non-parametric receiver operating characteristic (ROC) curve analysis was performed to evaluate the prediction accuracy of the various measurements taken in the experimental group, indicated by the area under the curve (AUC). The AUCs for the CPR and PTH measurements were calculated separately. The ROC curves indicate the relationship between the true positive (sensitivity) and false positive (1- specificity) cases and were constructed for the CPR and PTH markers. An AUC of 1 indicates a unique test with a sensitivity and specificity of 100%, while an AUC of 0.5 or less demonstrates that the diagnostic test is less useful. A binary logistic regression analysis was used to determine the odds ratio of different markers in predicting the OAD propensity of the experimental group. All the analyses were performed the statistical software IBM SPSS (version 20). An alpha level of 5% was used i.e., the P-values less than 0.05 were considered statistically significant.
The risk factors and the values for the biochemical markers, such as the PTH and CPR levels, were evaluated in the serum samples of the experimental subjects suffering from OAD and the control subjects without OADs. Table 1 shows that the PTH levels (mean ±SD) were lower for the 132 OAD subjects (17.11±6.17 pg/ml) than for the control subjects (51.49±14.30 pg/ml), as were the CPR levels (2.53±0.38 for the OAD and 2.67±0.45 for the non-OAD subjects). The respective mean difference (md) and its 95% confidence interval (CI) were 34.38 and 31.67- 37.09 pg/ml for PTH, a highly significant value (P<0.001), as well as 0.14 and 0.03-0.25 for CPR, a highly significant value statistically (P<0.01). Furthermore, the means ± SD for the PTH levels were lower in the 94 females with OAD (17.51±7.05 pg/ml) than in the 67 females without OAD (50.35±14.99 pg/ml), as were the CPR levels (2.54±0.27 for the OAD females and 2.59±0.40 for the non-OAD females). The respective mean difference (md) and its 95% CI were 32.84 and 29.35 -36.33 pg/ml for PTH, a highly significant value (P<0.001), and 0.05 and 0.05-0.15 for CPR, not a significant value (P= 0.345). Moreover, the means ± SD for both the PTH and CPR levels were lower in the 38 male OAD subjects than in the 42 male non-OAD subjects: 16.12 ± 2.96 compared with 53.32±13.09 pg/ml for PTH and 2.51±0.58 compared with 2.80±0.50 for the CPR level, respectively. Their mds (and 95% CIs) were 37.20 (32.87-42.53) pg/ml for PTH, a highly significant value (P<0.001), and 0.29 (0.05-0.53) for CPR, a highly significant value statistically (P<0.05) but one without practical significance. ROC graphs and statistics
A ROC analysis graph relating the sensitivity and 1-specificity values for the PTH and CPR levels in the 132 OAD compared with the 109 non-OAD subjects of both sexes is shown in Fig 1. The AUC and 95% CI for PTH were 0.985 and 0.969-1.000, respectively, a highly significant AUC value (P<0.001), whereas those for the CPR marker were 0.579 and 0.506-0.652, respectively, a highly significant value statistically (P<0.05). As shown in Table 1 for PTH, the results of the logistic regression analyses regarding the coefficient, odds ratio (OR) and 95% CI were -0.260, 0.771, and 0.712-0.836, respectively, a highly significant outcome (P<0.001). Table 1 also presents the regression coefficient, OR and 95% CI for the CPR variable: -0.793, 0.452, and 0.091-2.257, respectively, an outcome without statistical significance (P=0.333).
The ROC analysis curves relating to sensitivity and 1-specificity values for the PTH and CPR levels in 94 women with OAD and 67 women without OAD are exhibited in Fig 2. The AUC and 95% CI values for PTH were 0.977 and 0.953-1.000, respectively, with a highly significant value (P<0.001), whereas those for the CPR marker were 0.516 and 0.419-0.613, respectively, without a significant value (P=0.736). As shown in Table 1 for PTH, the results of the logistic regression analyses for the coefficient, OR, and 95% CI were -0.218, 0.804, and 0.746-0.867, respectively, with a highly significant value (P<0.001). For the CPR marker, the corresponding logistic regression parameters were -0.870, 0.419, and 0.049-3.591, respectively, without a significant value (P=0.428), as shown in Table 1.
A ROC analysis graph relating the sensitivity and 1-specificity values for the PTH and CPR levels in 38 men with OAD and 42 men without OAD is shown in Fig.3. The AUC and 95% CI for PTH were 1.000 and 1.000 to1.000, respectively, with a highly significant value (P<0.001; whereas those for the CPR level were 0.614 and 0.483-0.746, respectively, without a significant value (P=0.079). As shown in Table 1 for PTH, the results of the logistic regression analysis for the coefficient, OR, and 95% CI were -20.742, <0.001, and <0.001 to <0.001, respectively, without a significant value (P=0.964). For the CPR level, the corresponding logistic regression results were -6.79, 0.001, and <0.001 to <0.001, respectively, without a significant value (P=0.993).
Table 1: Means, standard deviations, ROC curves and logistic regression analyses for 132 OAD and 109 non- OAD subjects
Figure 1: Receiver operating characteristic curves for the PTH and the CPR of 132 OAD and 109 non-OAD combined-sex subjects. The area under the curve for PTH was 0.985 (95% CI = 0.969 – 1.000 with the P-value<0.001) and for the CPR was 0.579 (95% CI = 0.506 – 0.652, with the P- value=0.034).
Figure 2: Receiver operating characteristic curves for the PTH and the CPR of 94 OAD and 67 non-OAD female patients. The area under the curve for PTH was 0.977 (95% CI= 0.953 – 1.000, with the P-value <0.001) and for the CPR was 0.516 (95% CI =0.419 -0.613, with the P-value =0.736).
Figure 3: . Receiver operating characteristic curves for the PTH and the CPR of 38 OAD and 42 non-OAD male patients. The area under the curve for PTH was 1.000 (95% CI = 1.000 -1.000, with the P-value <0.001) and for CPR was 0.614 (95% CI = 0.483 -0.746, with the P- value=0.079).
In this present cohort study, the biomarker evaluation results showed lower PTH and CPR levels in the serum of experimental subjects (combined-sex, female-only and male-only analyses) with osteoarthritic disorders compared with those without the disorders. The area under the curve that was derived from the ROC curve analysis, was 0.985 for combined-sex group, 0.977 for the females alone and 1.000 for the males alone in the PTH analysis and 0.579 for the combined-sex group, 0.516 for the females and 0.614 for the males in the CPR analysis. These results, suggest that these two biomarkers can be identified as potential high-risk factors. When the calcium level decreases and the phosphorous level increases, the diseases called hypocalcemia with hyperphosphatemia are well known to develop. The present results clearly indicated signs of hypoparathyroidism accompanying OAD (Table 1 and Figs. 1 – 3).
Generally, the parathyroid gland secretes more PTH when the blood calcium level falls too low, and this increased blood PTH level sends a signal to bone for the release of excess calcium into the blood as a feedback mechanism.[3-5,52] Even so, PTH primarily regulates the blood calcium level by maintaining an adequate calcium level during times of very low calcium concentrations.[3-5] In the biochemical pathway regulating the body calcium and phosphate levels, these two minerals react antagonistically: calcium is known to increase in blood as the blood phosphorous level decreases. Ultimately, these two minerals are controlled by PTH in the blood.[9,16-17] Additionally, nearly 99% of the body calcium is well known to reside in the skeletal system, whereas 85% of the phosphorous is located in the bones and teeth. The remaining calcium is extracellular and plays a role in nerve conduction, muscle contraction, blood clotting and immune system activation. That calcium binds with phosphate and is finally deposited in the tissues has been established. A build-up of these deposits causes tissue calcification. In the present data set, calcium was observed to show relatively low values, while phosphorous showed higher values, resulting in the lower observed CPR and PTH levels in the subjects with OADs compared to those without OADs (Table 1).
The present study provides evidence to support the supposition that hypoparathyroidism is a risk factor for OADs. According to Norman, hypoparathyroidism is based on a reduction in the secretion of PTH or in its activity, possibly to a non-functional level, which additionally triggers a decline in the blood calcium level, causing hypocalcaemia, while increasing the blood phosphorus levels causing hyperphosphatemia. These abnormal features are only very rarely found, except in pseudo-hypoparathyroidism, a deficiency of calcium and vitamin D that also occurs after parathyroid gland surgery. Researchers have reported elevated PTH and phosphorus as risk factors for kidney disease,[11,53] but no one has previously attempted to diagnose lower calcium and PTH together with higher phosphorous in blood as risk factors for OADs. A heightened intestinal absorption of calcium has also been found, which is fully regulated by vitamin D. However, in this present study, the risk factors, such as lowered PTH and calcium together with increased phosphorous, as well as the statistically relevant data analyzed through the ROC curves and logistic regression analyses for combined-sex, female-only and male-only subject groups, clearly revealed that certain abnormal features of other diseases are risk factors for OADs and that males are more likely than combined-sex or female groups to show both biomarkers with a P<0.001 odds ratio for the risk factors (Table 1). Moreover, data representation with ROC curves and odds ratios is suitable for evaluating the sensitivity and one minus specificity values to establish methods for the proper diagnosis of particular diseases and additional risk factors.[6,54-55] Therefore, the present study confirms as a diagnostic protocol that the progression of OADs leads to a high risk of hypoparathyroidism, hypocalcemia, and hyperphosphatemia.
The present results led to the conclusion that the impact of functional instability risk causes lower PTH and CPR levels during OAD and these risks were higher in the male than in the female OAD cohorts. This study has confirmed a novel diagnostic protocol for hypoparathyroidism, hypocalcemia, and hyperphosphatemia during the development of OADs. The current research was an endeavor to report for the first time a predictive risk via the statistical analysis of ROC curves and `logistic regression analyses. As such, no symptoms have been detected that would be useful to track the lowering of PTH and calcium in blood during OADs. Individual research studies on knee osteoarthritis have documented the PTH level as a risk factor[6,8] or as the mechanism of low or high calcium, phosphorus and PTH during several diseases, such as cardiovascular, renal and rheumatic and arthritic illness, among others.[7-11,17] Further research is underway to estimate the vitamin D levels and to elucidate the mechanism by which calcium and PTH are lowered during OADs.