Article title: Correlativity between osteocalcin and coronary artery calcification in middle-aged and elderly population Author names:Na

Background and Objective: With the increase of aging, osteoporosis and cardiovascular diseases are common diseases in middle-aged and elderly people. The purpose of this study is to explore the correlation between osteocalcin and coronary calcification. Methods: A total of 150 middle-aged and elderly physical examination subjects who had undergone coronary artery CT angiography (CTA) were selected as the research objects. The calcification score of the coronary artery was calculated according to the Agatston (AS) integral method, and the total score was divided into the coronary artery calcification group and the non-calcification group. The indexes of N-terminal midfragment of osteocalcin (N-MID), routine biochemical indexes and basic conditions of each patient were recorded. Results: In the comparison of general data between the non-calcification group and the calcification group, the differences in age(P<0.001), pressure difference (P=0.01)and low density lipoprotein cholesterol (LDL-C)(P<0.001)were statistically significant, the differences in smoking history (P<0.001)and drinking history (P< 0.001)were statistically significant, and the differences in N-MID (P<0.001)were statistically significant. Coronary artery calcification score (CACS) was correlated with age, pressure difference, LDL-C, N-MID, smoking history and drinking history. The four variables, age, LDL, N-MID, and smoking history, have certain effects on coronary artery calcification (CAC). Conclusions: In middle-aged and elderly people undergoing physical examination, NMID is correlated with CAC and can be used as a predictive indicator. Abbreviations: AS: Agatston BGLAP: Bone glutamate protein BMI: Body mass index CABG: Coronary artery bypass grafting CAC: Coronary calcification calcification CACS: Coronary artery calcification score CAD: Coronary artery disease CHD: Coronary heart disease CKD: Chronic renal failure cOC: Carboxylated osteocalcin CTA: Coronary artery CT angiography CVD: Cardiovascular disease FBG: Fasting blood glucose GGCX: Glutamate γ‐carboxylase HDL-C: High density lipoprotein cholesterol HR: Heart rate LDL-C: Low density lipoprotein cholesterol N-MID: N-terminal midfragment of osteocalcin OC: Osteocalcin TC: Total cholesterol


Background
By the end of 2017, there were 2.409 million elderly people over the age of 60 in the country 1 . With the increase of aging, the health problems of the elderly become more and more prominent. Osteoporosis and cardiovascular disease (CVD) are the two most common diseases among the elderly, and some studies have suggested that there may be a link between the two [2][3][4][5] . As people get older, the calcium lost from bones is deposited in the cardiovascular system 6 . Vascular calcification (VC) is a recognized risk factor for cardiovascular morbidity and mortality 7 and is currently considered an active process [8][9][10][11] .VC has some similarities to bone mineralization and is related to the transformation of vascular smooth muscle cells (VSMCs) into osteoblast-like phenotypes 12 .
There are 17 types of vitamin k-dependent proteins (VKDPs), including osteocalcin (OC) [also known as bone glutamate protein (BGLAP)]. OC is divided into two kinds: carboxylated osteocalcin (cOC) and uncarboxylated osteocalcin (uOC). uOC is converted into cOC under the action of glutamate γ-carboxylase (GGCX) and vitamin K 13 . OC is the most abundant non-collagenous protein in the bone mineralization matrix 14 . The characteristic of atherosclerosis is associated with circulating OC 15 . OC mediates calcification of VSMCs 16 . These studies show that OC is correlated with VC.
At present, the relationship between OC and VC has not been determined.
Research on cOC is still lacking. In this paper, one of the indexes of bone metabolism -N-terminal midfragment of osteocalcin (N-MID) was included to investigate its relationship with coronary artery calcification (CAC).

Study population
A total of 150 middle-aged and elderly physical examination subjects who had undergone coronary artery CT angiography (CTA) were selected as the research objects.
The exclusion criteria for all selected candidates are: diseases that affect bone metabolism, such as hyperthyroidism, hyperparathyroidism, chronic renal failure or malignancy; or use drugs that affect bone metabolism, such as glucocorticoids, estrogens, or bisphosphonates；patients with incomplete clinical data; previous history of fracture；acute heart, liver, kidney, lung and other important organ complications occurred within the last 3 months. All participants in this study agreed in writing.

General data
Retrospective data analysis was used to record the subjects' gender、age、body mass index (BMI)、systolic blood pressure、diastolic blood pressure、differential blood pressure、heart rate (HR)、fasting blood glucose (FBG)、triglyceride (TG)、total We used the third generation N-MID assay kit to detect the whole 1-49 of cOC and 1-43 of degraded cOC fragments by enzyme-linked immunoassay.

Measurement of CAC score
Multi-slice spiral CT was used to detect the calcification score of coronary arteries 17 ，The calcification integral was calculated by the Agatston (AS) integral method，AS calcification integral condition：CT value ≥130HU, calcification area ≥1mm², calcification score = calcification area × calcification peak score(130~199Hu=1， 200~299Hu=2， 300~399Hu=3， ≥400Hu=4). The coronary artery was divided into four parts: left main trunk, left anterior descending branch, left circumflex branch, and right coronary artery, and calcification scores were calculated for each part. The integral results of each part are added to obtain a total integral 18 .
According to the total score, this study was divided into the coronary artery calcification group and the non-calcification group (calcification group: > 10; non-calcification group: ≤10).

Statistical analysis
Statistical software SPSS19.0 was used for statistical analysis. The measurement data is expressed in mean ± standard deviation ( s x ± ), the counting data is represented by probability, and the grade data by frequency. For comparison of measurement data, the independent data t test is used if the data conforms to normal distribution and uniform variance, and the Wilcoxon rank sum test is used if data does not conform to normal distribution or uneven variance. The comparison of counting data was conducted by χ² test, and Spearman rank correlation analysis was used for correlation analysis. Binary Logistic regression was used to analyze the correlation between multiple variables and coronary artery calcification score. The test level was α=0.05, and P < 0.05 was considered statistically significant. Table 1. Comparison of the general data between the two groups showed statistically significant differences in age (P <0.001), pressure difference (P = 0.01) and LDL-C (P <0.001), and differences in smoking history (P ＜ 0.001) and drinking history (P ＜ 0.001)were statistically significant. There was no statistically significant difference in gender, height, weight, BMI, systolic blood pressure, diastolic blood pressure, HR, FBG, TG, TC and HDL-C (P> 0.05).

The relationship between CAC and N-MID is shown in table 2. The relationship
between CAC and N-MID was statistically significant when comparing without gender distinction (P <0.001). When comparing the relationship between CAC and N-MID in male (P=0.005) and female (P＜0.001), the difference was still statistically significant.
Tab.2 The relationship between N-MID and CAC was discussed by gender stratification.  20 .
Vascular calcification is a marker of atherosclerosis 21 . In calcified atherosclerotic lesions, VSMCs express a variety of osteogenic differentiation markers 22 , similar to the osteogenic mineralization process 23 . VC is caused by the imbalance of vascular wall mineralization promoters 24 and inhibitors and is a serious health problem with serious clinical consequences 25 . CAC is an independent predictor of coronary heart disease(CHD) / CVD 26 . Compared with other risk markers, it has better identification and risk reclassification ability [27][28][29] , which is related to the degree of coronary artery disease (CAD) 30 . Studies have shown that in the early stage of dialysis patients with chronic renal failure(CKD), CAC is closely related to the incidence of CVD events and all-cause mortality 31 .
There are many risk factors for CVD, including age, diabetes, hypertension, hyperlipidemia and so on. The results of this study showed that age, LDL and other general data were significantly higher in the calcified group than in the non-calcified group. In this study, differential pressure was added, and the results showed that the differential pressure was significantly different between the two groups and correlated with the CACS.
Some studies have found that the progressive calcification of atherosclerotic plaque is accompanied by the accumulation of insignificant calcitonin and osteocalcin, and the concentration of OC is significantly increased during the calcification process 32 .
A meta-analysis 33 evaluated the relationship between circulating OC(total OC, uOC, and there is no difference in cOC levels between the two groups 34 . A prospective cohort study examining the association of various bone markers with CVD risk in type 2 diabetes found no association between OC and CVD risk 35 . Studies have found that there is no difference in serum OC levels between stroke patients and healthy controls 36 .
A meta-analysis found no clear association between OC and VC or the degree of atherosclerosis 14 .
OC is a non-specific collagen produced by osteoblasts in the bone and consists of 49 amino acids. Previous studies have not come to a uniform conclusion. This study included the N-MID indicator, we used the third generation n-mid assay kit to detect the whole 1-49 of cOC and 1-43 of degraded cOC fragments by enzyme-linked immunoassay. The metabolism of bone tissue can be understood by detecting the level of n-mid in blood. In this paper, the relationship between N-MID and CAC was compared. We found that compared with the non-calcification group, the value of N-MID in the calcification group was higher, and the difference was statistically significant. This article also proves that N-MID is related to CAC and has a certain impact on CAC. This paper also discusses the relationship between n-mid and CAC through gender stratification, and finds that there is still a correlation between the two,

Conclusion
In middle-aged and elderly people undergoing physical examination, N-MID is correlated with CAC and can be used as a predictive indicator.

Declarations:
Ethics approval and consent to participate: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the Declaration of Helsinki and have been approved by Ethics committee of Shandong university Qilu hospital.
Written informed consent was obtained from individual or guardian participants.

Consent for publication: Not applicable.
Availability of data and materials: All data generated or analyzed during this study are included in this published article.

Competing interests:
The authors declare that they have no competing interests.
Funding：Major science and technology innovation project of Shandong province (2019JZZY011110). To pay for the experiment and the manuscript.

Authors' contributions: Na Li collected a variety of clinical information and analyzed
it to reach conclusions. Na Li was a major contributor in writing the manuscript. Zhi -Mian Zhang provided this topic and conducted a clinical study. All authors read and approved the final manuscript.