Assessment of osteoporosis knowledge, awareness, and risk factors among premenopausal and postmenopausal women from Jordan: a cross-sectional study.

RATIONALE: Lack of information about osteoporosis knowledge and awareness among premenopausal compared to postmenopausal women in Jordan. MAIN RESULT: Women had an average-poor knowledge and awareness about osteoporosis. SIGNIFICANCE: This study highlights the need to improve women's knowledge about osteoporosis, its consequences, potential risk factors, preventive measures, and treatment options. PURPOSE: To assess osteoporosis knowledge, awareness, and risk factor profile among premenopausal and postmenopausal women from Jordan. METHODS: This was a cross-sectional study that involved 490 premenopausal and 488 postmenopausal women from the general population of Jordan. Face-to-face interviews were conducted to collect the sociodemographic and clinical data and to complete the Osteoporosis Knowledge Assessment Tool (OKAT) questionnaire. RESULTS: Premenopausal and postmenopausal women had an average-poor level of knowledge and awareness regarding osteoporosis, with a total mean score of 51.3 and 50.9, respectively, out of the total OKAT score of 100. More than 50% of premenopausal women correctly answered 11 questions, while >50% of postmenopausal women correctly answered 9 questions out of 20 in OKAT, which are related to knowledge and awareness about osteoporosis. The participants' marital status (being married), higher educational level, and higher economic status were significantly associated with better knowledge and awareness about osteoporosis (p-values < 0.05). Postmenopausal women had higher osteoporosis risk profile including older age, higher body mass index, less regular exercise, and less exposure to sunlight versus premenopausal women. CONCLUSION: Premenopausal and postmenopausal women from Jordan had an average-poor level of knowledge and awareness about osteoporosis. Higher educational levels and higher income are associated with better knowledge and awareness about osteoporosis. It is therefore crucial to improve the knowledge of women in Jordan about osteoporosis and its consequences, as well as the potential risk factors, preventive measures, and treatment options. Conducting periodic osteoporosis awareness and educational campaigns are necessary to spread the awareness of the disease.

Hot Flashes in Adolescence and Young Adult Females: a Link to Vitamin D and Calcium.

Background: Menopause is a stage in life when a woman stops having menstruation and the ovaries produce less estrogen. Hot flashes (HFs) are the classical symptoms for menopausal transition and cessation of menses. Increased anxiety had been reported as a significant risk factor of HFs. Vitamin D deficiency and low daily dietary calcium intake may be associated with the occurrence of hot flashes (HFs) in adolescents and young females that are not related to hormonal changes of menopausal transition. Objective: The aim of this study is to validate this hypothesis. Methods: A case-control study was conducted. Thirty-eight females (38) with HFs aged 18-40 years, and 38 age-matched healthy controls with no HFs were involved. Participants answered questions about HFs symptoms. Psychological symptoms were assessed using the Hospital Anxiety and Depression Scale (HADS). Serum vitamin D, Follicle Stimulating Hormone, Estradiol, and Prolactin were measured. Results: Vitamin D deficiency, psychological symptoms, and Musculoskeletal pain (MSP) were more prevalent in cases versus controls. About 73.68% of females had HFs on a daily basis, 73.7% of them reported that their HFs associated with excessive sweating. Spearman correlation revealed that number of daily HFs were correlated positively and significantly with anxiety scores (r2= -0.278, p=0.045), and average MSP pain (r2=-0.536, p=<0.001). Binary logistic regression showed that, Anxiety score and vitamin D status, (OR=1.33(1.104-1.7), p=0.02, and OR=0.89(0.79-0.99, p=0.03) respectively were the predictors for HFs. Conclusion: This study showed that adolescents and young females may experience HFs that are not related to hormonal changes of menopausal transition. The predictors for HFs were vitamin D deficiency and anxiety.

Predictors of age at menopause and psychiatric symptoms among postmenopausal females in Jordan.

OBJECTIVE: This study evaluated the impact of various factors on age at natural menopause as well as psychiatric symptoms including anxiety and depression among postmenopausal women in Jordan. METHODS: A cross-sectional study was conducted and included females with natural menopause (n = 450). A structured interview-based questionnaire was used to collect data about subjects' sociodemographics, health, reproductive and environmental factors. Hospital anxiety and depression scale (HADS) was used to assess psychiatric symptoms. Factors associated with age at natural menopause, depression, or anxiety were identified. RESULTS: The mean age at natural menopause was 49.5 ± 4.8 years. Mothers' age at menopause, the regularity of cycles, age at last pregnancy and diabetes were significant positive predictors of age at menopause (p < 0.05). The mean anxiety and depressive scores were 6.52 ± 4.26 and 6.77 ± 3.44 respectively. Age, high school education, being nonsmoker and breastfeeding history were inversely associated with anxiety but only cycle length and multiparous were positively associated with anxiety (p < 0.05). While education and being nonsmoker were negatively associated with depression, hypertension was a positive predictor. CONCLUSIONS: The results reveal several environmental, health, and reproductive predictors of age at menopause or psychiatric symptoms among postmenopausal women in Jordan.

Nightmares and bad dreams among individuals with musculoskeletal pain: a link to vitamin D and calcium.

Nightmares are frightening or disturbing dreams that awaken sleepers while bad dreams are disturbing dreams that do not awaken sleepers. Both types are known to be associated with psychological symptoms including anxiety and depression. Chronic pain is often comorbid with psychological symptoms and vitamin D deficiency increases risk of chronic musculoskeletal pain (MSP), which in turn is associated with increased risk of anxiety and depression. We aimed to investigate associations between types of dreams, psychological symptoms, vitamin D, and calcium intake in individuals with MSP. The study included 191 outpatients with MSP and 191 age/gender matched healthy controls. Psychological symptoms were assessed using Hospital Anxiety and Depression Scale. Serum vitamin D was measured and daily calcium intake was estimated. Participants were asked about types of their dreams (normal, bad, or nightmares) during the past month. Binary logistic regression was used to find predictors of MSP and bad dreams and nightmares. Bad dreams and nightmares, vitamin D deficiency, low calcium intake, anxiety, and depression were more prevalent in cases versus controls (Ps<0.001). Chi-square analyses showed that types of dreams were associated with anxiety, depression, and MSP (Ps<0.001). Participants with normal dreams had higher vitamin D (P<0.01) and calcium intake (P<0.001) and lower anxiety and depression scores (Ps<0.001) compared to participants with bad dreams and nightmares. Anxiety, depression and MSP were predictors for bad dreams and nightmares. Further studies are required to assess if vitamin D supplementation and increasing calcium intake may improve MSP, psychological symptoms and thus prevent nightmares and bad dreams.

Association of asthma with low serum vitamin D and its related musculoskeletal and psychological symptoms in adults: a case-control study.

There are complex potential inter-relationships between the chronic inflammation of asthma and poor control, vitamin D deficiency, musculoskeletal pain and anxiety and depression. The aim was to investigate associations between vitamin D and these possible co-morbidities. This case-controlled study involved 75 adults with asthma and 75 controls. Serum 25-hydroxyvitamin D (25(OH)D) was measured, levels of anxiety, depression, musculoskeletal pain, and asthma control were assessed. Participants with asthma had lower 25(OH)D and higher anxiety scores and higher measures of musculoskeletal pain compared to controls. Binary logistic regression showed that asthma was associated with decreased 25(OH)D (Odds ratio (OR) = 0.86), general weakness (OR = 13.29), complaint of musculoskeletal pain (OR = 13.73), and increased intensity of musculoskeletal pain (OR = 0.61) and number of painful sites (OR = 2.58). Asthma was not associated with anxiety or depression. Further studies are required to investigate if vitamin D supplementation can improve asthma symptoms and musculoskeletal pain.

Self-reported sleep bruxism is associated with vitamin D deficiency and low dietary calcium intake: a case-control study.

BACKGROUND: Sleep bruxism may result in deleterious effects including loss of tooth enamel, fracture of teeth or restorations, teeth hypersensitivity or pain, and headache. The aim was to study the link between sleep bruxism, low serum vitamin D, low consumption of dietary calcium, psychological symptoms, and frequent headache. METHODS: This case-controlled study included 50 individuals with sleep bruxism and 50 age and gender matched controls. 25-hydroxyvitamin D was measured in serum. Hospital Anxiety and Depression Scale was used to measure anxiety and depression. Data about dietary calcium and frequent headache were self-reported. RESULTS: Participants with sleep bruxism had lower 25-hydroxyvitamin D and higher scores of anxiety and depression compared to controls (p < 0.05). Vitamin D deficiency, abnormal scores of anxiety and depression, low calcium consumption (< 323 mg/day), and frequent headache were reported in higher % of individuals with sleep bruxism compared to controls (p < 0.05). Binary logistic regression showed that sleep bruxism was significantly associated with vitamin D deficiency (OR = 6.66, p = 0.02), low consumption of dietary calcium (OR = 5.94, p = 0.01), and frequent headache (OR = 9.24, p < 0.001). Multiple linear regression showed that anxiety was significantly associated with decreased 25-hydroxyvitamin D (p = 0.03), increased scores of depression (p < 0.001) and female sex (p = 0.01). Binary logistic regression also showed that frequent headache was significantly associated with sleep bruxism (OR = 5.51, p < 0.01). CONCLUSIONS: Sleep bruxism was associated with vitamin D deficiency and low consumption of calcium and was also associated with increased scores of anxiety and depression. Further investigations should be performed to check if vitamin D and calcium supplementation could relieve sleep bruxism.

High prevalence of low dairy calcium intake and association with insomnia, anxiety, depression and musculoskeletal pain in university students from Jordan.

OBJECTIVE: To assess dairy Ca intake and investigate its relationship with insomnia and other common co-morbidities including anxiety, depression and musculoskeletal pain (MSP) among university students. DESIGN: Cross-sectional study. SETTING: University, Irbid, Jordan. PARTICIPANTS: Male and female individuals (n 1000), aged 20·87 ± 2·69 years. RESULTS: Low dairy Ca intake (<1000 mg/d) was reported by 96·5 % of participants, and moderate to severe insomnia reported by 15·6 % of participants. Abnormal anxiety and depression scores were reported by 26·2 and 18·0 % of participants, respectively. MSP was reported by 42·9 % of participants. Participants with moderate to severe insomnia had lower dairy Ca, higher anxiety and depression scores and higher measures of MSP compared to participants with no insomnia (P-values < 0·05). Dairy Ca was weakly inversely correlated with Insomnia Severity Index (ISI) score, depression score and measures of MSP (P-values < 0·05). Regression analysis indicated that insomnia was predicted by low dairy Ca, anxiety, depression, MSP and smoking (P-values < 0·05). Both anxiety and depression were predicted by increased ISI score (P-values < 0·05), while depression alone was predicted by low dairy Ca (P-value < 0·01). MSP was predicted by increased ISI and anxiety scores (P-values < 0·05). CONCLUSIONS: Low dairy Ca was highly prevalent and associated with insomnia and depression among university students. Individuals should be advised to increase dietary Ca intake to achieve the recommended daily amount. Further research is required to investigate a potential causal relationship between low Ca and both insomnia and its related co-morbidities.

Poor sleep quality among young adults: The role of anxiety, depression, musculoskeletal pain, and low dietary calcium intake.

PURPOSE: This study examined the relationships between sleep quality, anxiety, depression, musculoskeletal pain (MSP), and calcium intake. DESIGN AND METHODS: In this cross-sectional study (N = 1422), sleep was assessed using the Pittsburgh Sleep Quality Index, anxiety and depression were assessed using the Hospital Anxiety and Depression Scale, and calcium intake and MSP were assessed by self-reporting. FINDINGS: Poor sleep quality was reported by 62.66% of the participants. The participants with poor sleep quality reported lower calcium intake, higher anxiety and depression levels, more severe MSP, and multisite pain. Anxiety, depression, low calcium intake, and multisite pain were significant predictors of poor sleep quality. Anxiety was predicted by poor sleep quality, depression, multisite pain, and sex (ie, female). Depression was predicted by anxiety, poor sleep quality, and low calcium intake. PRACTICAL IMPLICATIONS: The findings underscore the role of low calcium intake in the development of sleep problems, anxiety, depression, and MSP. Individuals with these conditions should be advised to increase their calcium intake.

High Prevalence of Vitamin D Deficiency and Correlation with Cystatin-C and Other Cardiovascular and Renal Risk Biomarkers in Patients with Type 2 Diabetes Mellitus Complicated with Hypertension.

BACKGROUND: Vitamin D is increasingly investigated as having a role in Type 2 Diabetes Mellitus (T2DM) and its cardiovascular and renal complications. OBJECTIVE: This study aimed to investigate the association between 25-hydroxyvitamin D (25-OHD) and biomarkers of cardiovascular and renal complications, including cystatin-C. METHODS: This cross-sectional study involved 117 participants with T2DM that was not complicated with cardiovascular or renal diseases except hypertension. 25-OHD was measured by electrochemiluminescence immunoassay, while cystatin-C was measured by enzyme-linked-immunosorbent-assay. Other biomarkers, including lipids, creatinine, urea and glycemic measures, were determined by the routine biochemistry assays. RESULTS: The prevalence of vitamin D deficiency was 74.36%. There was no significant difference in cardiovascular and renal biomarkers, including glucose, HbA1c, lipids, urea, creatinine and cystatin-C between participants with adequate and deficient vitamin D (p-values>0.05). Participants with adequate vitamin D were older in age, more obese and having lower eGFR (p-values<0.05). 25-OHD was weakly correlated with age, duration of DM, urea, creatinine and inversely correlated with eGFR (rvalues< 0.32, p-values<0.05). Although creatinine and cystatin-C were directly correlated (r=0.42, pvalue< 0.001), cystatin-C and 25-OHD were not correlated (p-value>0.05). Hypertensive participants were more obese, having a longer duration of DM and higher urea and cystatin-C compared to nonhypertensive participants (p-values<0.05). Binary logistic regression analysis revealed that hypertension could be predicted from increased BMI. CONCLUSION: 25-OHD was not found to be correlated with cardiovascular risk biomarkers, but it was correlated with renal biomarkers, including urea, creatinine and eGFR. Cystatin-C and 25-OHD were not observed to be correlated to each other, but both were correlated to renal function. Obesity was a significant predictor of hypertension.

Impaired awareness of hypoglycemia in children and adolescents with type 1 diabetes mellitus in north of Jordan.

BACKGROUND: Hypoglycemia is a common complication of insulin therapy in patients with Type 1 Diabetes Mellitus (DM). Awareness of hypoglycemic symptoms helps patients to recognize hypoglycemia and initiate self-treatment. Impaired Awareness of Hypoglycemia (IAH) exposes patients to severe hypoglycemia, which could be associated with seizures and unconsciousness. This study aimed to assess IAH, frequency of hypoglycemia, severe hypoglycemia and intensity of hypoglycemic symptoms among children and adolescents with Type 1 DM in North of Jordan. METHODS: Data were collected from 94 children and adolescents with Type 1 DM. Clarke's and Edinburgh surveys were used to assess IAH and individual symptoms of hypoglycemia, respectively. Frequency of hypoglycemia and other related information were obtained by self-reporting or from medical records. RESULTS: 16.0% of participants were having IAH, 66.0% of participants reported recurrent hypoglycemia (>once/month) and 18.0% of participants developed ≥1 severe hypoglycemia during the previous year. IAH was not associated with age, gender, duration of DM, HbA1c, insulin regimen, adherence to insulin or development of severe hypoglycemia (p-values> 0.05). Instead, IAH was associated with frequency of hypoglycemia during the previous 6 months (p-value< 0.01). Hunger, tiredness, dizziness, drowsiness, inability to concentrate, trembling and weakness were the most common symptoms felt by participants when they develop hypoglycemia. Hunger was the only common symptom that was significantly higher in children compared to adolescent (p-value < 0.01). CONCLUSIONS: This study has reported low prevalence of IAH in children and adolescents with Type 1 DM in North of Jordan. IAH was more common in subjects with more frequent hypoglycemia.

Waterpipe tobacco smoke distresses cardiovascular biomarkers in mice: alterations in protein expression of metalloproteinases, endothelin and myeloperoxidase.

Introduction: Tobacco use is a major risk factor of cardiovascular diseases (CVD) and atherosclerosis in particular. The use of waterpipe smoking (WPS) is increasing due to the misperception that it is less harmful than cigarette smoking due to its flavor and the use of water as a filter. Thus, research that investigates toxic effects of WPS is essential. The aim of this study was to investigate the effect of WPS on major cardiovascular biomarkers that may develop atherosclerosis in mice. Methods: BALB/c mice were exposed to WPS for either two weeks (acute exposure) or eight weeks (chronic exposure). Then, the heart tissue homogenates were analyzed to elucidate the effects of WPS on matrix metalloproteinase (MMPs: isoforms 1, 3, and 9), metallopeptidase inhibitor (TIMP1), endothelin-1 (ET-1) and myeloperoxidase (MPO) using ELISA technique. Results: Current data showed that acute exposure to WPS significantly enhanced the levels of MMP-3, MMP-9, and MPO (p < 0.05) compared to their corresponding control. However, the body was capable to restore the increased levels of these parameters following chronic exposure to WPS for 8 weeks (p > 0.05). Additionally, the levels of ET-1 were significantly higher upon chronic exposure to WPS compared to both control and acute exposure groups (p < 0.05). Conclusions: Waterpipe exposure has a significant negative effect on the cardiovascular system. The enhancement of the atherosclerotic biomarkers (MMP-3, MMP-9, MPO, and ET-1) might represent an early scavenger of compensatory efforts to maintain cardiovascular function after WPS exposure.

Association of 25-hydroxyvitamin D with HDL-cholesterol and other cardiovascular risk biomarkers in subjects with non-cardiac chest pain.

BACKGROUND: Chest pain is a serious symptom that is routinely investigated as a sign of coronary artery disease. Non-cardiac chest pain (NCCP) is indistinguishable from ischemic chest pain and both are considered serious and receive similar medical investigations. Although NCCP is not associated with cardiovascular diseases (CVDs), patients with NCCP may become anxious and frightened from developing coronary events. So, it will be valuable to improve modifiable cardiovascular risk factors in such subjects to reduce fear from CVDs. Because vitamin D deficiency was considered as a possible modifiable cardiovascular risk factor, our aim was to investigate association between serum vitamin D and cardiovascular risk variables in subjects with NCCP. METHODS: A cross-sectional study involved 104 subjects who underwent cardiac catheterization that did not reveal any cardiac origin for their chest pain. 25-hydroxyvitamin D was measured by electrochemiluminescence immunoassay, glucose was measured by hexokinase method, hemoglobin A1c (HbA1c) was measured by turbidimetric inhibition immunoassay and lipid profile was measured by enzymatic colorimetric assays. RESULTS: High density lipoprotein cholesterol (HDL-C) was significantly higher in subjects with sufficient vitamin D compared to those with insufficient or deficient vitamin D (p-value< 0.01). 25-hydroxyvitamin D was positively associated with HDL-C (p-value< 0.01) and inversely associated with HbA1c (p-value = 0.02). 25-hydroxyvitamin D was not significantly correlated with other cardiovascular biomarkers including blood pressure, glucose, and other components of lipid profile (p-values> 0.05). CONCLUSIONS: low serum vitamin D could be involved in reducing HDL-C and increasing HbA1c and thus it may increase cardiovascular risk in subjects with NCCP.

Impaired Awareness of Hypoglycaemia in Insulin-treated Type 2 Diabetes Mellitus.

BACKGROUND: Patients with Type 2 Diabetes Mellitus (T2DM) may develop hypoglycemia as an adverse effect of insulin therapy. Hypoglycemia has dangerous consequences that result from neuroglycopenia and hypersecretion of counter-regulatory hormones. Patients who recognize early symptoms of hypoglycemia can initiate self-treatment and rectify the situation. Impaired Awareness of Hypoglycemia (IAH) predisposes patients to severe hypoglycemia and unconsciousness. OBJECTIVE: To assess the prevalence of IAH, the intensity of hypoglycaemic symptoms, the frequency of severe hypoglycemia and factors associated with IAH in patients with insulin-treated T2DM. METHODS: This is a cross-sectional study that used Clarke's and Gold's surveys to assess IAH and Edinburgh survey to assess the intensity of hypoglycemic symptoms in patients with insulin-treated T2DM (n= 388). The frequency of hypoglycemia and other data were collected by self-reporting or from medical records. RESULTS: The prevalence (95% confidence interval) of IAH was 17.01% (13.27%-20.75%) as determined by Clarke's method and 5.93% (3.58-8.28) by Gold's method (Odds= 3.25, p-value<0.00001). Drowsiness, hunger, sweating, tiredness, trembling and weakness, were the most intense hypoglycaemic symptoms, and 6.19% of participants reported at least one episode of severe hypoglycaemia within the past year. Regardless of classification method used, IAH is not dependent on age, gender, duration of T2DM or duration of insulin therapy (p-values>0.05). Instead, IAH is positively associated with frequency of hypoglycaemia during the previous six months (p-value<0.05) and development of severe hypoglycaemia within the past year (p-value <0.05). CONCLUSION: This study highlights large variability in IAH depending on the method used for assessment. Increased hypoglycaemia frequency may increase the prevalence of IAH and thus the development of severe hypoglycemia.

Non-cardiac Chest Pain and Anxiety: A Possible Link to Vitamin D and Calcium.

This study was performed to check the hypothesis that low serum vitamin D and reduced calcium intake may contribute to the comorbidity of psychological symptoms (anxiety and depression) and non-cardiac chest pain (NCCP). The design was a case-control study that involved 40 subjects with NCCP and 40 age and gender-matched healthy controls. Serum vitamin D was assessed using electrochemiluminescence immunoassay; anxiety and depression symptoms were assessed using Hospital Anxiety and Depression Scale, and dietary calcium intake was assessed by self-reporting. Subjects with NCCP had higher anxiety and depression scores and lower vitamin D and dietary calcium intake compared to healthy controls (p < .05). NCCP was associated with anxiety score (odds ratio = 1.40, p < .01). Prevalence of abnormal anxiety score was much higher in subjects with NCCP (47.5%) compared to healthy controls (7.5%). Anxiety score was inversely correlated with vitamin D level and dietary calcium intake (p < .01). Anxiety score was inversely associated with vitamin D level (R2 = .32, p < .05). In conclusion, development of NCCP can be predicted from increased anxiety score which in turn can be predicted from low vitamin D levels. This suggests physicians to consider anxiety and vitamin D deficiency as possible causes for NCCP.

Association between serum 25-hydroxyvitamin D, hemoglobin A1c and fasting blood glucose levels in adults with diabetes mellitus.

In the present study, the aim was to investigate the association between serum 25-hydroxyvitamin D concentration and measures of glycemic control including hemoglobin A1c (HbA1c) and fasting blood glucose (FBG) in adult patients with diabetes mellitus (DM) from the north of Jordan. Another aim was to compare serum levels of 25-hydroxyvitamin D between patients with good glycemic control and patients with uncontrolled DM. This was a cross-sectional study that included 261 participants with DM. The concentration of 25-hydroxyvitamin D was measured using electrochemiluminescence immunoassay, HbA1c was measured using turbidimetric inhibition immunoassay and FBG was measured using the hexokinase method. Data regarding other clinical variables were obtained from medical records or by self-reporting. Participants with good glycemic control exhibited significantly higher levels of 25-hydroxyvitamin D compared with participants with uncontrolled DM (P=0.03). Participants with sufficient vitamin D status (>30 ng/ml in serum) exhibited significantly lower HbA1c level compared with participants with deficient vitamin D (<20 ng/ml) status (P=0.02). Correlation analysis determined significant inverse correlations between 25-hydroxyvitamin D levels and HbA1c and FBG levels (r=-0.23 and -0.17, respectively, both P<0.01). There were also significant correlations between duration of DM and HbA1c and FBG levels (both r=0.21, P<0.01). HbA1c level was also inversely correlated with participants' age (r=-0.19, P<0.01). Further multiple linear regression analysis revealed an inverse significant association between HbA1c and 25-hydroxyvitamin D levels (F=12.95, R2=0.48, P<0.01) but did not identify a similar association between FBG and 25-hydroxyvitamin D levels. These findings may encourage further research to identify if vitamin D supplementation may improve measures of glycemic control, and how vitamin D may affect glucose homeostasis in patients with DM.

Prevalence of musculoskeletal pain in association with serum 25-hydroxyvitamin D concentrations in patients with type 2 diabetes mellitus.

The aim of the present study was to investigate the prevalence of musculoskeletal pain in patients with type 2 diabetes mellitus (T2DM) in association with 25-hydroxyvitamin D levels, anxiety, depression and neuropathy. A cross-sectional study was conducted involving a total of 124 T2DM patients. Musculoskeletal pain was determined by self-reporting of painful body sites. Pain intensity was assessed using a scale of 0-10. Anxiety and depression were assessed using the Hospital Anxiety and Depression Scale. Neuropathy was assessed using the PainDETECT questionnaire. The concentration of 25-hydroxyvitamin D was measured using liquid chromatography-tandem mass spectrometry. Fasting blood sugar (FBS) was determined using the hexokinase method and glycated hemoglobin (HbA1c) level was determined using turbidimetric inhibition immunoassay. The neck, lower back and head were reported as the most common painful sites (affected in 60.5, 60.5 and 56.5% of patients, respectively). Pain in the lower extremities, including the knees, lower legs and feet, was more common compared with pain in the upper extremities. The pain measurements of number of painful sites and pain intensity did not differ significantly among patients with sufficient (>30 ng/ml), insufficient (20-30 ng/ml) and deficient (<20 ng/ml) vitamin D levels (P>0.05). The pain measurements were identified to have no correlation with age, body mass index, FBS, HbA1c level, 25-hydroxyvitamin D concentration, anxiety or depression (P>0.05). However, the pain measurements were correlated with duration of T2DM and neuropathy score (P<0.05). Further regression analysis demonstrated that the pain measurements were significantly associated with the neuropathy score (P<0.05). In conclusion, musculoskeletal pain in patients with T2DM was not associated with 25-hydroxyvitamin D concentration, but was associated with neuropathy score. This may encourage further investigations to assess the etiology of musculoskeletal pain in T2DM, and whether vitamin D supplementation and management of neuropathy would be of value as pain relief treatment.

Strategies for enumeration of circulating microvesicles on a conventional flow cytometer: Counting beads and scatter parameters.

Enumeration of circulating microvesicles (MVs) by conventional flow cytometry is accomplished by the addition of a known amount of counting beads and calculated from the formula: MV/µl = (MV count/bead count) × final bead concentration. We sought to optimize each variable in the equation by determining the best parameters for detecting 'MV count' and examining the effects of different bead preparations and concentrations on the final calculation. Three commercially available bead preparations (TruCount, Flow-Count and CountBright) were tested, and MV detection on a BD FACSCanto was optimized for gating by either forward scatter (FSC) or side scatter (SSC); the results were compared by calculating different subsets of MV on a series of 74 typical patient plasma samples. The relationship between the number of beads added to each test and the number of beads counted by flow cytometry remained linear over a wide range of bead concentrations (R2 ≥ 0.997). However, TruCount beads produced the most consistent (concentration variation = 3.8%) calculated numbers of plasma CD41+/Annexin V+ MV, which were significantly higher from that calculated using either Flow-Count or CountBright (p < 0.001). The FACSCanto was able to resolve 0.5 µm beads by FSC and 0.16 µm beads by SSC, but there were significantly more background events using SSC compared with FSC (3113 vs. 470; p = 0.008). In general, sample analysis by SSC resulted in significantly higher numbers of MV (p < 0.0001) but was well correlated with enumeration by FSC for all MV subtypes (ρ = 0.62-0.89, p < 0.0001). We conclude that all counting beads provided linear results at concentrations ranging from 6 beads/µl to 100 beads/µl, but TruCount was the most consistent. Using SSC to gate MV events produced high background which negatively affected counting bead enumeration and overall MV calculations. Strategies to reduce SSC background should be employed in order to reliably use this technique.

High Prevalence of Vitamin D Deficiency and Correlation of Serum Vitamin D with Cardiovascular Risk in Patients with Metabolic Syndrome.

BACKGROUND: Metabolic syndrome (MetS) identifies subjects with increased risk of cardiovascular disease when they have a combination of insulin resistance, obesity, hypertension, hyperglycemia, low high-density lipoprotein cholesterol (HDL-C), and elevated triglycerides (TGs). Increasing evidence suggests that vitamin D deficiency could be associated with diabetes and MetS. The aim is to assess if 25-hydroxyvitamin D (25-OHD) is correlated with cardiovascular risk components of MetS. METHODS: A cross-sectional study involved 124 diabetic patients with MetS according to International Diabetes Federation definition. 25-OHD was measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fasting insulin, lipid profile, glucose, and hemoglobin-A1c (HbA1c) were determined using routinely standard laboratory methods. Insulin resistance was assessed using homeostatic model assessment (HOMA). RESULTS: 59.68% and 27.42% of patients have vitamin D deficiency and insufficiency, respectively. Systolic blood pressure (SBP) was significantly higher in patients with vitamin D deficiency compared to patients with sufficient vitamin D (P < 0.05). Serum log (25-OHD) was inversely correlated with SBP, HbA1c, low-density lipoprotein cholesterol (LDL-C), TGs, and total cholesterol and directly correlated with pancreatic ß cell function (HOMA-ß) (P < 0.05). Multiple linear regression analysis has shown that SBP can be predicted from log (25-OHD) (B = -9.388, P < 0.05), while HbA1c, LDL-C, TGs, total cholesterol, and HOMA-ß cannot be predicted from log (25-OHD), (P > 0.05). CONCLUSIONS: Vitamin D deficiency was very prevalent among patients with MetS. 25-OHD was inversely correlated with glycemic control and cardiovascular risk components of MetS except HDL-C, insulin resistance, and obesity. SBP was the only cardiovascular risk component that can be predicted from vitamin D concentrations.

Bio-maleimide-stained plasma microparticles can be purified in a native state and target human proximal tubular HK2 cells.

Plasma microparticles (MPs) are heterogeneously sized submicron extracellular vesicles that originate from the cell membrane as a result of cell activation or apoptosis. Circulating MPs express cell-specific molecules that reflect their cell of origin and they are increasingly investigated for their potential role in intercellular communication. The aim of the current study was to determine if size exclusion chromatography could be used to purify fluorescent-labeled MPs in sufficient concentrations to be used experimentally in cell binding assays. Bio-maleimide was used to stain plasma MPs in platelet free plasma before applying to size exclusion chromatography. Collected fractions were analyzed for protein content and MPs were enumerated by flow cytometry. Fractions were ultracentrifuged and MPs further confirmed by western blotting for the putative diabetic marker, cluster of differentiation (CD)36 and platelet-specific CD41 proteins. Fractions that contained MPs were incubated with HK2 cells to determine MP-cell binding. Bio-maleimide-stained MPs were detected across various fractions of size exclusion, and pellets of these fractions confirmed positivity for the MP markers, CD41 and CD36. The addition of the isolated MPs to HK2 renal tubular cells and analysis by epi-fluorescent imaging demonstrated that, in principle, the labeled MPs are able to bind to cells in vitro. Notably, only the first eluted MP fraction bound HK2 cells indicating a possible association between MP size and cell-targeting properties.

Low simvastatin concentrations reduce oleic acid-induced steatosis in HepG2 cells: An in vitro model of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is an inflammatory condition caused by hepatic lipid accumulation that is associated with insulin resistance, diabetes and metabolic syndrome. Although statins should be used with caution in liver diseases, they are increasingly investigated as a possible treatment for NAFLD. The present study recreated an in vitro model of NAFLD using HepG2 cells exposed to oleic acid (OA), which was used to quantify OA-induced lipid accumulation in HepG2 cells treated with various concentrations of simvastatin. In addition, the effect of simvastatin on HepG2 cell morphology and microparticle generation as a marker of cell apoptosis was assessed. OA-induced lipid accumulation was quantified by Oil Red O staining and extraction for optical density determination. Stained lipid droplets were visualized using phase contrast microscopy. Furthermore, HepG2 cell-derived microparticles were counted by flow cytometry subsequent to staining for Annexin V. HepG2 cells treated with 0-1 mM OA showed dose-dependent lipid accumulation. Treatment of HepG2 cells with increasing concentrations of simvastatin followed by treatment with 1 mM OA showed that low simvastatin concentrations (4-10 µM) were able to reduce lipid accumulation by ~40%, whereas high simvastatin concentrations (20 and 30 µM) induced apoptotic changes in cell morphology and increased the production of Annexin V+ microparticles. This suggests that low simvastatin doses may have a role in preventing NAFLD. However, further investigations are required to confirm this action in vivo and to determine the underlying mechanism by which simvastatin reduces hepatic steatosis.