Sustainable production and degradation of plastics using microbes.

Plastics are indispensable in everyday life and industry, but the environmental impact of plastic waste on ecosystems and human health is a huge concern. Microbial biotechnology offers sustainable routes to plastic production and waste management. Bacteria and fungi can produce plastics, as well as their constituent monomers, from renewable biomass, such as crops, agricultural residues, wood and organic waste. Bacteria and fungi can also degrade plastics. We review state-of-the-art microbial technologies for sustainable production and degradation of bio-based plastics and highlight the potential contributions of microorganisms to a circular economy for plastics.

Metabolic engineering for sustainability and health.

Bio-based production of chemicals and materials has attracted much attention due to the urgent need to establish sustainability and enhance human health. Metabolic engineering (ME) allows purposeful modification of cellular metabolic, regulatory, and signaling networks to achieve enhanced production of desired chemicals and degradation of environmentally harmful chemicals. ME has significantly progressed over the past 30 years through further integration of the strategies of synthetic biology, systems biology, evolutionary engineering, and data science aided by artificial intelligence. Here we review the field of ME from its emergence to the current state-of-the-art, highlighting its contribution to sustainable production of chemicals, health, and the environment through representative examples. Future challenges of ME and perspectives are also discussed.

Escherichia coli as a platform microbial host for systems metabolic engineering.

Bio-based production of industrially important chemicals and materials from non-edible and renewable biomass has become increasingly important to resolve the urgent worldwide issues including climate change. Also, bio-based production, instead of chemical synthesis, of food ingredients and natural products has gained ever increasing interest for health benefits. Systems metabolic engineering allows more efficient development of microbial cell factories capable of sustainable, green, and human-friendly production of diverse chemicals and materials. Escherichia coli is unarguably the most widely employed host strain for the bio-based production of chemicals and materials. In the present paper, we review the tools and strategies employed for systems metabolic engineering of E. coli. Next, representative examples and strategies for the production of chemicals including biofuels, bulk and specialty chemicals, and natural products are discussed, followed by discussion on materials including polyhydroxyalkanoates (PHAs), proteins, and nanomaterials. Lastly, future perspectives and challenges remaining for systems metabolic engineering of E. coli are discussed.

Allopurinol ameliorates high fructose diet induced hepatic steatosis in diabetic rats through modulation of lipid metabolism, inflammation, and ER stress pathway.

Excess fructose consumption contributes to development obesity, metabolic syndrome, and nonalcoholic fatty liver disease (NAFLD). Uric acid (UA), a metabolite of fructose metabolism, may have a direct role in development of NAFLD, with unclear mechanism. This study aimed to evaluate role of fructose and UA in NAFLD and explore mechanisms of allopurinol (Allo, a UA lowering medication) on NAFLD in Otsuka Long-Evans Tokushima Fatty (OLETF) rats fed a high fructose diet (HFrD), with Long-Evans Tokushima Otsuka (LETO) rats used as a control. There were six groups: LETO, LETO-Allo, OLETF, OLETF-Allo, OLETF-HFrD, and OLETF-HFrD-Allo. HFrD significantly increased body weight, epididymal fat weight, and serum concentrations of UA, cholesterol, triglyceride, HbA1c, hepatic enzymes, HOMA-IR, fasting insulin, and two hour-glucose after intraperitoneal glucose tolerance tests, as well as NAFLD activity score of liver, compared to the OLETF group. Allopurinol treatment significantly reduced hepatic steatosis, epididymal fat, serum UA, HOMA-IR, hepatic enzyme levels, and cholesterol in the OLETF-HFrD-Allo group. Additionally, allopurinol significantly downregulated expression of lipogenic genes, upregulated lipid oxidation genes, downregulated hepatic pro-inflammatory cytokine genes, and decreased ER-stress induced protein expression, in comparison with the OLETF-HFrD group. In conclusion, allopurinol ameliorates HFrD-induced hepatic steatosis through modulation of hepatic lipid metabolism, inflammation, and ER stress pathway. UA may have a direct role in development of fructose-induced hepatic steatosis, and allopurinol could be a candidate for prevention or treatment of NAFLD.

Pancreatic fat accumulation is associated with decreased ß-cell function and deterioration in glucose tolerance in Korean adults.

AIMS: This study was designed to investigate the association between pancreatic fat content (PFC) and insulin secretory capacity as well as glucose tolerance in Korean adults. MATERIALS: A total of 39 participants (mean age 49.9 years, 53% males) without a previous history of diabetes, or those previously diagnosed as having diabetes but with less than 10 years of disease duration and no medication history were included. They were stratified according to the results of the oral glucose tolerance test (OGTT): normal glucose tolerance, prediabetes, and diabetes. METHODS: All participants underwent the proton magnetic resonance spectroscopy (1 H-MRS) to assess PFC. Insulin sensitivity and ß-cell function were measured by the frequently sampled intravenous glucose tolerance tests (FSIVGTT) and OGTT-derived indices. RESULTS: As glucose tolerance deteriorated, parameters such as Stumvoll index, oral glucose insulin sensitivity index, homeostatic model assessment (HOMA)-ß, insulinogenic index and oral disposition index from the OGTT, and acute insulin response to glucose (AIR) and disposition index (DI) from the FSIVGTT were decreased. PFC increased with deterioration in glucose tolerance (NGT: 12.0%, prediabetes: 23.7%, and diabetes: 31.9%). Correlation analysis indicated that glucose levels at 60 and 120 min during the OGTT were positively correlated with PFC. Also, there was a significant negative correlation between PFC and DI as well as AIR derived from the FSIVGTT. CONCLUSIONS: PFC evaluated by 1 H-MRS in Korean adults was higher in those diagnosed with diabetes than those with normal glucose tolerance status. PFC also showed a significant negative correlation with indices reflecting beta cell function.

Microbial Polyhydroxyalkanoates and Nonnatural Polyesters.

Microorganisms produce diverse polymers for various purposes such as storing genetic information, energy, and reducing power, and serving as structural materials and scaffolds. Among these polymers, polyhydroxyalkanoates (PHAs) are microbial polyesters synthesized and accumulated intracellularly as a storage material of carbon, energy, and reducing power under unfavorable growth conditions in the presence of excess carbon source. PHAs have attracted considerable attention for their wide range of applications in industrial and medical fields. Since the first discovery of PHA accumulating bacteria about 100 years ago, remarkable advances have been made in the understanding of PHA biosynthesis and metabolic engineering of microorganisms toward developing efficient PHA producers. Recently, nonnatural polyesters have also been synthesized by metabolically engineered microorganisms, which opened a new avenue toward sustainable production of more diverse plastics. Herein, the current state of PHAs and nonnatural polyesters is reviewed, covering mechanisms of microbial polyester biosynthesis, metabolic pathways, and enzymes involved in biosynthesis of short-chain-length PHAs, medium-chain-length PHAs, and nonnatural polyesters, especially 2-hydroxyacid-containing polyesters, metabolic engineering strategies to produce novel polymers and enhance production capabilities and fermentation, and downstream processing strategies for cost-effective production of these microbial polyesters. In addition, the applications of PHAs and prospects are discussed.

Microbial production of fatty acids and derivative chemicals.

Microbial production of fatty acids and derivatives from non-edible biomass has attracted much attention as an alternative to their production from plant oils and animal fats. Fatty acids and some of their derivatives are ubiquitous metabolites synthesized for membrane biosynthesis and other metabolic purposes in microorganisms. These compounds, however, are rarely produced beyond cellular demands, frequently resulting in low titers even after metabolic engineering. Recently, more advanced metabolic engineering strategies including systems metabolic engineering allowed improved production of fatty acids and their derivatives by employing non-oleaginous and oleaginous microorganisms. Here, we review metabolic engineering strategies developed for the production of fatty acids and derivative chemicals by non-oleaginous and oleaginous microorganisms in recent years.

Metabolic engineering for the synthesis of polyesters: A 100-year journey from polyhydroxyalkanoates to non-natural microbial polyesters.

As concerns increase regarding sustainable industries and environmental pollutions caused by the accumulation of non-degradable plastic wastes, bio-based polymers, particularly biodegradable plastics, have attracted considerable attention as potential candidates for solving these problems by substituting petroleum-based plastics. Among these candidates, polyhydroxyalkanoates (PHAs), natural polyesters that are synthesized and accumulated in a range of microorganisms, are considered as promising biopolymers since they have biocompatibility, biodegradability, and material properties similar to those of commodity plastics. Accordingly, substantial efforts have been made to gain a better understanding of mechanisms related to the biosynthesis and properties of PHAs and to develop natural and recombinant microorganisms that can efficiently produce PHAs comprising desired monomers with high titer and productivity for industrial applications. Recent advances in biotechnology, including those related to evolutionary engineering, synthetic biology, and systems biology, can provide efficient and effective tools and strategies that reduce time, labor, and costs to develop microbial platform strains that produce desired chemicals and materials. Adopting these technologies in a systematic manner has enabled microbial fermentative production of non-natural polyesters such as poly(lactate) [PLA], poly(lactate-co-glycolate) [PLGA], and even polyesters consisting of aromatic monomers from renewable biomass-derived carbohydrates, which can be widely used in current chemical industries. In this review, we present an overview of strain development for the production of various important natural PHAs, which will give the reader an insight into the recent advances and provide indicators for the future direction of engineering microorganisms as plastic cell factories. On the basis of our current understanding of PHA biosynthesis systems, we discuss recent advances in the approaches adopted for strain development in the production of non-natural polyesters, notably 2-hydroxycarboxylic acid-containing polymers, with particular reference to systems metabolic engineering strategies.

Biocatalytic synthesis of polylactate and its copolymers by engineered microorganisms.

Poly(lactate), also called poly(lactic acid) or poly(lactide) [PLA], has been one of the most attractive bio-based polymers since it possesses desirable material properties for its use in general performance plastics in addition to biodegradability and biocompatibility. PLA has been produced by biological and chemical hybrid process comprising microbial fermentation for lactate (LA) production followed by purification and chemical polymerization process of LA. Recently, the direct one-step fermentative processes for production of PLA and several LA-containing polyesters have been developed by employing metabolically engineered microorganisms. Since natural microorganisms cannot produce the LA-containing polymers, several engineering strategies have been employed together based on the polyhydroxyalkanoate (PHA) biosynthesis system. In this chapter, we summarize strategies and procedures on developing the engineered microorganisms producing PLA and its copolymers, cultivating the cells, and extracting the polymers from the cells. Focuses were given on construction of enzymatic polymerization process of LA: design of metabolic pathway for PLA by mimicking PHA biosynthetic pathway, examination of possible enzymes, and engineering of the enzymes for better performances. This synthetic pathway has been established in a microorganism producing LA that enabled one-step fermentative production of LA-containing polyesters from carbohydrates derived from renewable biomass. Polymer production has been further enhanced by implementing strain engineering to concentrate the metabolic fluxes toward PLA formation. In addition, various monomers such as glycolate, 2-hydroxybutyrate, and phenyllactate have been copolymerized with LA by the microbial system. These fermentative production systems developed by using the engineered microorganisms can be versatile and sustainable platforms for the production of LA-containing polyesters and other non-natural polymers.

Efficacy and safety of Gelidium elegans intake on bowel symptoms in obese adults: A 12-week randomized double-blind placebo-controlled trial.

BACKGROUND/AIMS: Gelidium elegans (GE) is known to have antiobesity effects and beneficial effects on functional bowel symptoms in preclinical studies. The aim of this study was to determine the efficacy and safety of GE intake on bowel symptoms in obese human adults. METHODS: This 12-week single-center randomized double-blind placebo-controlled study was performed from September 2016 to May 2017. Consecutive obese subjects were randomly assigned (1:1) to either GE (1 g) or placebo (1 g) once daily group for 12 weeks. Patients' bowel symptoms were evaluated using the Bristol Stool Form Scale, Constipation Scoring System (CSS), and Patient Assessment of Constipation-Symptoms (PAC-SYM) questionnaire. RESULTS: The stool symptom score of PAC-SYM significantly improved in the GE group compared with the placebo group after the 12-week treatment (P = .041). Abdominal discomfort score of CSS significantly decreased at 12 weeks compared to that at baseline in the GE group (P = .003), but not in the placebo group (P = .398). In addition, abdominal discomfort score of CSS slightly decreased in the GE group compared with the placebo group after the 12-week treatment (P = .060). However, stool consistency, total CSS score, and PAC-SYM score did not change significantly in both GE group and the placebo group over the 12-week treatment period. CONCLUSIONS: GE treatment for 12 weeks improved the stool symptom score on the PAC-SYM and abdominal discomfort score on the CSS in obese adults. However, further research is needed in large-scale human studies.

Statins for primary prevention in adults aged 75 years and older: A nationwide population-based case-control study.

BACKGROUND AND AIMS: There is inadequate evidence to establish statin treatment for primary prevention in the elderly. This study evaluated whether statins are beneficial for primary prevention of cardiovascular disease (CVD) and all-cause death in adults aged ≥ 75 years. METHODS: A nationwide, nested case-control study was conducted in Korea. Individuals who developed CVD, including myocardial infarction (MI), stroke, or death from all causes, were matched to controls based on duration of follow-up, age, and sex at the index date. The statin administration data from both groups were retrospectively collected from the index date to five years before. Odds ratios (ORs) and 95% confidence intervals (CIs) for composite and individual outcomes associated with statin treatment were estimated by conditional logistic regression analyses. RESULTS: In total, 11,017 cases were matched to 55,085 control subjects. Current use of statins was significantly associated with a reduced risk of composite outcome (adjusted OR [AOR] 0.77; 95% CI 0.71-0.84), compared with non-users. Current use of statin also reduced the risk of stroke (AOR 0.74; 95% CI 0.61-0.89) and of all-cause death (AOR 0.73; 95% CI 0.66-0.81), but not of MI. However, former use of statins had no effect on CVD or all-cause death. There were significant decreasing trends in the incidence of composite outcomes and individual stroke or all-cause death with longer duration of statin treatment. CONCLUSIONS: Current statin treatment has a beneficial effect as primary prevention for composite outcomes and individual event of stroke or all-cause death in Koreans aged ≥75 years.

Exendin-4, a glucagon-like peptide-1 receptor agonist, reduces hepatic steatosis and endoplasmic reticulum stress by inducing nuclear factor erythroid-derived 2-related factor 2 nuclear translocation.

Activation of endoplasmic reticulum (ER) stress is involved in the development of nonalcoholic fatty liver disease. Glucagon-like peptide-1 (GLP-1) has been reported to reduce hepatic steatosis, but the underlying mechanism has not been fully elucidated. Here, we investigated whether exendin-4 (EX-4), a GLP-1 receptor analogue, improves hepatic steatosis through ER stress reduction. Furthermore, we explored which ER stress pathway is involved in this process, with a focus on the protein kinase RNA-like ER kinase (PERK)-nuclear factor erythroid-derived 2-related factor 2 (Nrf2) pathway. EX-4 treatment reduced hepatic lipid accumulation by suppressing the expression of lipogenic genes and restoring the expression of ß-oxidation genes in palmitate-treated HepG2 cells and high fat diet (HFD)-fed mice. In addition, EX-4 treatment suppressed hepatic ER stress activation in HFD-fed mice and tunicamycin-treated mice. In particular, EX-4 treatment restored HFD- and tunicamycin-induced Nrf2 nuclear translocation to control levels. Inhibition of Nrf2 by siRNA enhanced phosphorylation of PERK and eukaryotic translation initiation factor 2α (eIF2α), as well as other substrates of the PERK pathway. Nrf2 knockdown also inhibited the protective effects of EX-4 against lipid accumulation, ER stress activation, and cell death in palmitate-treated HepG2 cells. EX-4 treatment prevents hepatic steatosis and improves cell survival by regulating hepatic lipid metabolism and reducing ER stress activation, and Nrf2 plays an essential role in the protective effect of GLP-1 on hepatic steatosis.

Markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida.

Pseudomonas putida has gained much interest among metabolic engineers as a workhorse for producing valuable natural products. While a few gene knockout tools for P. putida have been reported, integration of heterologous genes into the chromosome of P. putida, an essential strategy to develop stable industrial strains producing heterologous bioproducts, requires development of a more efficient method. Current methods rely on time-consuming homologous recombination techniques and transposon-mediated random insertions. Here we report a RecET recombineering system for markerless integration of heterologous genes into the P. putida chromosome. The efficiency and capacity of the recombineering system were first demonstrated by knocking out various genetic loci on the P. putida chromosome with knockout lengths widely spanning 0.6-101.7 kb. The RecET recombineering system developed here allowed successful integration of biosynthetic gene clusters for four proof-of-concept bioproducts, including protein, polyketide, isoprenoid, and amino acid derivative, into the target genetic locus of P. putida chromosome. The markerless recombineering system was completed by combining Cre/lox system and developing efficient plasmid curing systems, generating final strains free of antibiotic markers and plasmids. This markerless recombineering system for efficient gene knockout and integration will expedite metabolic engineering of P. putida, a bacterial host strain of increasing academic and industrial interest.

Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation.

Plastics, including poly(ethylene terephthalate) (PET), possess many desirable characteristics and thus are widely used in daily life. However, non-biodegradability, once thought to be an advantage offered by plastics, is causing major environmental problem. Recently, a PET-degrading bacterium, Ideonella sakaiensis, was identified and suggested for possible use in degradation and/or recycling of PET. However, the molecular mechanism of PET degradation is not known. Here we report the crystal structure of I. sakaiensis PETase (IsPETase) at 1.5 Å resolution. IsPETase has a Ser-His-Asp catalytic triad at its active site and contains an optimal substrate binding site to accommodate four monohydroxyethyl terephthalate (MHET) moieties of PET. Based on structural and site-directed mutagenesis experiments, the detailed process of PET degradation into MHET, terephthalic acid, and ethylene glycol is suggested. Moreover, other PETase candidates potentially having high PET-degrading activities are suggested based on phylogenetic tree analysis of 69 PETase-like proteins.

Differential association between sarcopenia and metabolic phenotype in Korean young and older adults with and without obesity.

OBJECTIVE: To determine whether sarcopenia was associated with metabolic phenotype in subjects with and without obesity. METHODS: A total of 6,021 participants (2,592 men, 3,429 women) aged 30 to 93 years were assessed using data from the 2009 Korea National Health and Nutrition Examination Survey. Sarcopenia was defined as appendicular skeletal muscle mass divided by weight (%) that is <1 SD below the sex-specific mean for young adults. Metabolically unhealthy was defined as ≥2 components of metabolic syndrome or the presence of hypertension, diabetes, or cardiovascular disease. Obesity was defined as body mass index ≥25.0 kg/m2 . RESULTS: Sarcopenia was associated with a metabolically unhealthy phenotype in nonobese men independent of age, smoking, regular physical activity, daily energy intake, total body fat, fasting insulin, non-HDL cholesterol, white blood cell count, ferritin level, and 25(OH) vitamin D level (OR per 1 SD increment (95% CI) 1.88 (1.28-2.75), P < 0.01), but this association was confounded by and not independent of total body fat in nonobese women. Sarcopenia was not associated with a metabolically unhealthy phenotype in subjects with obesity. CONCLUSIONS: Sarcopenia was independently associated with a metabolically unhealthy phenotype in nonobese men, but this association was not evident in nonobese women or subjects with obesity.

Aster spathulifolius Maxim extract reduces body weight and fat mass in obese humans.

Aster spathulifolius Maxim (AS), a perennial herb of the genus Aster within the family Asteraceae, induced weight loss in a rat model of diet-induced obesity. We hypothesized that AS could also reduce body weight in obese humans. Therefore, we performed a randomized, double-blind, placebo-controlled clinical trial in Korea to evaluate the effect of AS extract (ASE) on body weight and fat mass and its safety in obese humans. Forty-four obese participants (body mass index [BMI], 25-30 kg/m(2)) aged ≥20 years were randomly assigned to the placebo or ASE group (700 mg/d of ASE) and were instructed to take a once-daily pill for 12 weeks. Weight, BMI, waist circumference, fat mass (measured using bioimpedance, dual-energy X-ray absorptiometry, and computed tomography), and laboratory tests were assessed at baseline and at 12 weeks. Body weight significantly decreased after 12 weeks of treatment in the ASE group (placebo vs ASE: -0.08 ± 2.11 kg vs -3.30 ± 3.15 kg, P < .05), and so did body fat mass (placebo vs ASE; bioimpedance method: -0.51 ± 1.89 kg vs -2.38 ± 2.30 kg, P < .05; dual-energy X-ray absorptiometry: 0.38 ± 1.59 kg vs -2.26 ± 2.37 kg, P < .05). Changes in lipid profiles, fasting plasma glucose, and hemoglobin A1c did not differ between the 2 groups. No drug-related adverse events were observed during the study. In conclusion, ASE significantly decreases body weight and fat mass in obese humans, suggesting that ASE may be a potential therapeutic candidate for reducing obesity.

Metabolic Engineering of Escherichia coli for the Production of 3-Hydroxypropionic Acid and Malonic Acid through ß-Alanine Route.

Escherichia coli was metabolically engineered to produce industrially important platform chemicals, 3-hydroxypropionic acid (3-HP) and malonic acid (MA), through the ß-alanine (BA) route. First, various combinations of downstream enzymes were screened and BA pyruvate transaminase (encoded by pa0132) from P. aeruginosa was selected to generate malonic semialdehyde (MSA) from BA. This platform strain was engineered by introducing E. coli MSA reductase (encoded by ydfG) to reduce MSA to 3-HP. Replacement of native promoter of the sdhC gene with the strong trc promoter in the genome increased 3-HP production to 3.69 g/L in flask culture. Introduction of E. coli semialdehyde dehydrogenase (encoded by yneI) into the platform strain resulted in the production of MA, and additional deletion of the ydfG gene increased MA production to 0.450 g/L in flask culture. Fed-batch cultures of final engineered strains resulted in the production of 31.1 g/L 3-HP or 3.60 g/L MA from glucose.

Effects of C-reactive protein on bone cells.

AIMS: Inflammatory processes are involved in bone remodeling. C-reactive protein (CRP) is an acute phase reactant that reflects different degrees of inflammation. Accumulating evidence suggests that CRP is an inflammatory marker and a direct cause of disease. Therefore, we examined the direct effects of CRP on bone cells. MAIN METHODS: We used RAW 264.7 cells to evaluate the direct effects of CRP on osteoclast differentiation. We carried out alkaline phosphatase (ALP) and bone nodule formation assays using MC3T3-E1 cells to evaluate osteoblast differentiation. Expression of osteoclast-specific and osteoblast-specific genes and effects on cell signaling pathways associated with cell differentiation were analyzed by reverse transcription polymerase chain reaction and Western blotting. KEY FINDINGS: CRP significantly and dose-dependently inhibited TRAP-positive multinucleated cell formation in RANKL-induced RAW 264.7 cell cultures. We observed suppression of p38, ERK and AKT mitogen-activated protein kinases induced by RANKL in Western blots after CRP treatment of RAW 264.7 cells. CRP also suppressed ALP activity and mineralization by Alizarin red S staining of MC3T3-E1 cell cultures. CRP suppressed osteoclast-specific and osteoblast-specific genes. Furthermore, CRP increased interferon beta (IFN-ß) mRNA expression and protein levels in RAW 264.7 and MC3T3-E1 cells, and these effects were suppressed by oxPAPC, an inhibitor of Toll-like receptor (TLR) signaling. SIGNIFICANCE: These data indicated that CRP may have a direct role on osteoclast and osteoblast differentiation via TLR signaling pathways.

Association between the circulating total osteocalcin level and the development of cardiovascular disease in middle-aged men: a mean 8.7-year longitudinal follow-up study.

AIM: Recent studies have suggested that the serum osteocalcin level is associated with various cardiovascular risk factors. The aim of this study was to determine whether the serum total osteocalcin level is associated with the development of cardiovascular disease (CVD). METHODS: A total of 1,290 men 40-78 years of age were enrolled. The subjects were followed regularly at the Health Promotion Center on an outpatient basis and during hospitalization for a mean of 8.7 years, and the incidence of CVD (coronary heart disease [CHD] and stroke) was determined. RESULTS: At baseline, the body mass index, body fat percentage, fasting glucose, homeostasis model assessment-insulin resistance, triglyceride and non-high density lipoprotein (HDL) cholesterol levels were inversely and the HDL cholesterol levels were positively associated with the serum osteocalcin levels. In addition, the prevalence of diabetes or metabolic syndrome decreased as the osteocalcin tertile increased. However, no differences were observed in the prevalence of hypertension across the osteocalcin tertiles. Incident CVD occurred in 74 (5.7%) of the study subjects (29 patients with CHD and 47 patients with stroke). According to the Cox proportional hazards models, however, there were no statistical differences in the development of stroke, CHD or CVD across the osteocalcin tertiles after adjusting for other risk factors for CVD, including age, body mass index, current smoking, low-density lipoprotein cholesterol, diabetes, hypertension and the serum creatinine level. CONCLUSION: In conclusion, the serum total osteocalcin level was not associated with the development of CVD after adjusting for other risk factors for CVD in this cohort.