Physiologically relevant screening of polyphenol-rich commercial preparations for bioactivity in vascular endothelial cells and application to healthy volunteers: A viable workflow and a cautionary tale.

This study describes the screening of 13 commercially-available plant extracts for pharmacological activity modulating vascular function using an endothelial cell model. A French maritime pine bark extract (FMPBE) was found to have the greatest effect upon nitric oxide availability in control (181% ± 36% of untreated cells) and dysfunctional cells (132% ± 8% of untreated control cells). In healthy volunteers, the FMPBE increased plasma nitrite concentrations 8 h post-consumption compared to baseline (baseline corrected median 1.71 ± 0.38 (25% IQR) and 4.76 (75% IQR) µM, p < 0.05). This was followed by a placebo-controlled, healthy volunteer study, which showed no effects on plasma nitrite. It was confirmed that different batches of extract had been used in the healthy volunteer studies, and this second batch lacked bioactivity, assessed using the in vitro model. No difference in plasma catechin levels was seen at 8 h following supplementation between the studies (252 ± 194 nM versus 50 ± 64 nM, p > 0.05), however HPLC-UV fingerprinting showed that the new batch had a 5-15% in major constituents (including procyanidins A2, B1 and B2) compared to the original batch. This research describes a robust mechanism for screening bioactive extracts for vascular effects. It also highlights batch variability as a significant limitation when using complex extracts for pharmacological activity, and suggests the use of in vitro systems as a tool to identify this problem in future studies.

Perfluorinated alkyl acids in the serum and follicular fluid of UK women with and without polycystic ovarian syndrome undergoing fertility treatment and associations with hormonal and metabolic parameters.

Women with polycystic ovarian syndrome (PCOS) undergoing treatment for infertility could be a sensitive subpopulation for endocrine effects of exposure to perfluorinated alkyl acids (PFAAs), persistent organic pollutants with potential endocrine activity. Women with, PCOS (n = 30) and age- and BMI-matched controls (n = 29) were recruited from a UK fertility clinic in 2015. Paired serum and follicular fluid samples were collected and analysed for 13 PFAAs. Sex steroid and thyroid hormones, and metabolic markers were measured and assessed for associations with serum PFAAs. Four PFAAs were detected in all serum and follicular fluid samples and concentrations in the two matrices were highly correlated (R2 > 0.95): perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonate (PFHxS), and perfluorononanoic acid (PFNA). Serum PFOS was positively associated with age (1 ng/mL per yr, p < 0.05) and was higher in PCOS cases than controls (geometric mean [GM] 3.9 vs. 3.1 ng/mL, p < 0.05) and in women with irregular vs. regular menstrual cycles (GM 3.9 vs. 3.0 ng/mL, p = 0.01). After adjustment for confounders, serum testosterone was significantly associated with PFOA, PFHxS, PFNA, and the molar sum of the four frequently detected serum PFAAs (approximately 50 percent increase per ln-unit) among controls but not PCOS cases. HbA1c in PCOS cases was inversely associated with serum PFOA, PFHxs, and sum of PFAAs (2-3 mmol/mol per ln-unit). In controls, fasting glucose was positively associated with serum PFOA and sum of PFAAs (0.25 nmol/L per ln-unit increase in PFAAs). Few other associations were observed. The analyses and findings here should be considered exploratory in light of the relatively small sample sizes and large number of statistical comparisons conducted. However, the data do not suggest increased sensitivity to potential endocrine effects of PFAAs in PCOS patients.

Soy isoflavones improve cardiovascular disease risk markers in women during the early menopause.

BACKGROUND: Hormone replacement therapy may be beneficial for cardiovascular disease risk (CVR) in post-menopausal women. Soy isoflavones may act as selective estrogen receptor modulators. The aim of this study was to evaluate whether soy isoflavones had an effect on CVR markers. METHODS: The expected 10-year risk of cardiovascular disease and mortality were calculated as a secondary endpoint from a double blind randomised parallel study involving 200 women (mean age 55 years, Caucasian, Hull, UK, 2012) in the early menopause who were randomised to 15 g soy protein with 66 mg isoflavone (SPI) or 15 g soy protein alone (depleted of all isoflavones; SP) given as a snack bar between meals daily for 6 months. Age, diabetes, smoking, blood pressure and lipid profiles were used to calculate CVR using the Framingham CVR engine. RESULTS: SPI treatment resulted in a significant reduction in the metabolic parameters and systolic blood pressure compared to SP (p < 0.01). There were no changes in fasting lipid profile and diastolic blood pressure with either treatment. At 6 months, changes in these parameters with SPI treatment were reflected in a calculated 27% (p < 0.01) reduction in 10 year coronary heart disease risk, a 37% (p < 0.01) reduction in myocardial infarction risk, a 24% (p < 0.04) reduction in cardiovascular disease and 42% (p < 0.02) reduction in cardiovascular disease death risk. CONCLUSIONS: Supplementation with soy protein with isoflavones for 6 months significantly improved CVR markers and calculated CVR at 6 months during early menopause compared to soy protein without isoflavones. ISRCTN REGISTRY: ISRCTN34051237.

Platelet function following induced hypoglycaemia in type 2 diabetes.

AIM: Strict glycaemic control has been associated with an increased mortality rate in subjects with type 2 diabetes (T2DM). Here we examined platelet function immediately and 24hours following induced hypoglycaemia in people with type 2 diabetes compared to healthy age-matched controls. METHODS: Hyperinsulinaemic clamps reduced blood glucose to 2.8mmol/L (50mg/dl) for 1hour. Sampling at baseline; euglycaemia 5mmol/L (90mg/dl); hypoglycaemia; and at 24 post clamp were undertaken. Platelet function was measured by whole blood flow cytometry. RESULTS: 10 subjects with T2DM and 8 controls were recruited. Platelets from people with T2DM showed reduced sensitivity to prostacyclin (PGI2, 1nM) following hypoglycaemia. The ability of PGI2 to inhibit platelet activation was significantly impaired at 24hours compared to baseline in the T2DM group. Here, inhibition of fibrinogen binding was 29.5% (10.3-43.8) compared to 50.8% (36.8-61.1), (P<0.05), while inhibition of P-selectin expression was 32% (16.1-47.6) vs. 54.4% (42.5-67.5) (P<0.05). No significant changes in platelet function were noted in controls. CONCLUSION: Induced hypoglycaemia in T2DM enhances platelet hyperactivity through impaired sensitivity to prostacyclin at 24hours.

The repeatability of the abbreviated (4-h) Oral Fat Tolerance Test and influence of prior acute aerobic exercise.

PURPOSE: The Oral Fat Tolerance Test (OFTT) is regarded as a repeatable measure used to assess postprandial triglyceride (TAG) levels, with higher levels observed in cardio-metabolic disorders. Acute aerobic exercise intervention before OFTT reduces the TAG response, but the repeatability of this effect is unknown. The aim of this study was to determine the repeatability of the abbreviated 4-h OFTT with and without immediate prior aerobic exercise. METHODS: On four separate days, healthy adult male participants underwent two 4-h OFTT (n = 10) and another two 4-h OFTT with 1-h of standardised moderate intensity aerobic exercise performed immediately before meal ingestion (n = 11). The OFTT meal composition included 75.4 g total fat, 21.7 g carbohydrate and 13.7 g protein. Venous blood was sampled at baseline and hourly up to 4 h after the OFTT meal ingestion, and TAG area under the curve (AUC) was calculated. RESULTS: Nonparametric Bland-Altman analysis of 4-h TAG AUC revealed that 9 of 10 repeat measurements fell within ±15 % of the median TAG AUC for the OFTT. By contrast, two of 11 repeat measurements fell within ±15 % of the median TAG AUC for the OFTT undertaken with 1-h prior aerobic exercise. CONCLUSIONS: The 4-h OFTT is a repeatable test of postprandial TAG responses in healthy men. However, aerobic exercise performed immediately before OFTT considerably increases the variability of TAG AUC. These findings have implications for interpretation of research studies investigating exercise intervention performed immediately before OFTT. Future studies should also investigate the repeatability of exercise performed 8-24 h before OFTT.

Effect of soy on bone turn-over markers in men with type 2 diabetes and hypogonadism - a randomised controlled study.

Type 2 diabetes (T2DM) is associated with increased risk of fractures. Soy supplementation has been shown to have a beneficial effect on bone turnover markers (BTM) in postmenopausal women. However, the effect of soy supplementation on BTM in T2DM and particularly in men is unclear. We performed an analysis of a randomized double blind parallel study of 200 men with T2DM treated with soy, either with or without isoflavones. Outcome measures were type I collagen crosslinked beta C-telopeptide (ßCTX), and type 1 procollagen-N-propeptide (P1NP). The men, with a total testosterone <12 nmol/L, were treated with 15 g soy protein containing 66 mg of isoflavones (SPI) or 15 g soy protein alone without isoflavones (SP) daily for three months. There was a 15% reduction in ßCTX after three months of SPI compared to SP supplementation. There was no significant difference in P1NP with either SPI or SP supplementation. There was a significant linear correlation between the reduction in ßCTX in the SPI group with the reduction in HbA1c (r2 = 0.42; p = 0.04) and HOMA-IR (r2 = 0.54; p = 0.02). Our study indicates that there was a significant reduction in bone resorption following 3 months of SPI supplementation that correlated with an improvement of glycemic control in men with T2DM.

Effects of human recombinant growth hormone on exercise capacity, cardiac structure, and cardiac function in patients with adult-onset growth hormone deficiency.

Objective Epidemiological studies suggest that adult-onset growth hormone deficiency (AGHD) might increase the risk of death from cardiovascular causes. Methods This was a 6-month double-blind, placebo-controlled, randomised, cross-over trial followed by a 6-month open-label phase. Seventeen patients with AGHD received either recombinant human growth hormone (rGH) (0.4 mg injection daily) or placebo for 12 weeks, underwent washout for 2 weeks, and were then crossed over to the alternative treatment for a further 12 weeks. Cardiac magnetic resonance imaging, echocardiography, and cardiopulmonary exercise testing were performed at baseline, 12 weeks, 26 weeks, and the end of the open phase (12 months). The results were compared with those of 16 age- and sex-matched control subjects. Results At baseline, patients with AGHD had a significantly higher systolic blood pressure, ejection fraction, and left ventricular mass than the control group, even when corrected for body surface area. Treatment with rGH normalised the insulin-like growth factor 1 concentration without an effect on exercise capacity, cardiac structure, or cardiac function. Conclusion Administration of rGH therapy for 6 to 9 months failed to normalise the functional and structural cardiac differences observed in patients with AGHD when compared with a control group.

Increased expression of circulating miRNA-93 in women with polycystic ovary syndrome may represent a novel, non-invasive biomarker for diagnosis.

MicroRNAs (miRNA) are a novel class of small noncoding single-stranded RNA molecules that regulate gene expression. There is increasing evidence of their importance in polycystic ovary syndrome (PCOS). The objective was to determine if miRNA-93 and miRNA-223 are differentially expressed in the circulation of women with PCOS compared to age matched women. A case-control study comparing women with PCOS (n = 25) to age and weight matched controls (n = 24) without PCOS was performed. MiRNA-93 and miRNA-223 were determined by total RNA reverse transcription. Both miRNA-93 and miRNA-223 were significantly increased relative to the control group (p < 0.01, p = 0.029 respectively). In both groups there was no correlation of either miRNA-93 or miRNA-223 with insulin, HOMA-IR, HOMA-ß or testosterone levels. The area under the receiver operator characteristic curve for miR-223 and miR-93 was 0.66 and 0.72 respectively, suggesting miR-93 is a more efficient biomarker than miR-223 for diagnosis of PCOS. The combination of the two miRNAs together, tested using multiple logistic regression analysis, did not improve the diagnostic potential. In conclusion, circulating miRNA-93 and miRNA-223 were higher in women with PCOS compared to age and weight matched controls independent of insulin resistance and testosterone levels, and miR-93 may represent a novel diagnostic biomarker for PCOS.

LDL cholesterol variability in patients with Type 2 diabetes taking atorvastatin and simvastatin: a comparison of two formulae for LDL-C estimation.

BACKGROUND: A new formula was recently proposed by Cordovo et al. that was more highly correlated with low-density lipoprotein (LDL) measured directly than the Friedewald LDL formula. We conducted this prospective study to establish whether the new formula allows true variations in LDL within the same individual to be tracked more closely than that of the Friedewald formula. METHODS: A cross-over study of biological variation of lipids in 26 patients with Type 2 diabetes (T2DM) taking either a short half-life statin, simvastatin 40 mg (n=10), or a long half-life statin, atorvastatin 10 mg. After three months on one statin, fasting lipids were measured on 10 occasions over a five-week period. The same procedure was then followed for the other statin. The LDL was measured by a direct LDL immunoassay and was compared to the LDL estimated by the Friedewald and Cordova (0.7516) × (total cholesterol [TC]-high-density lipoprotein cholesterol [HDL-C]) formulae. RESULTS: As a group, the calculated or measured mean LDL was no different between statins. However, the biological coefficient of variation (CV) of directly measured LDL was far larger with simvastatin than atorvastatin. This difference was detected by Cordova LDL but not found with the Friedewald LDL formula. CONCLUSIONS: In contrast to Friedewald LDL, Cordova LDL estimation revealed LDL to be much more stable in T2DM patients taking atorvastatin rather than simvastatin that was in accord with LDL when measured directly. Therefore, Cordova LDL which is a measure of non-HDL-cholesterol is the simplest, cheapest and the most convenient measurement for assessment of response to statin treatment.

Biological variation of cardiovascular risk factors in patients with diabetes.

Biological variation refers to the natural fluctuations found when repeated measurements are made in a biological system. Generally, biological variation remains within narrow boundaries in health, but may differ in pathological states, with implications for the diagnosis and monitoring of disease processes. In disease, biological variation may alter such that any subsequent measurement may need to have a greater difference compared with a healthy control to be biologically relevant. Treatments such as insulin or anti-hypertensive therapy have been shown to reduce biological variability closer to normal levels and theoretically this may help prevent complication development or progression in conditions such as diabetes. This article reviews how biological variation can influence our identification and assessment of vascular risk factors in a person with diabetes. The role of biological variation in the diagnosis of diabetes (glucose and HbA1c) is then examined. Finally, the influence that common treatments in diabetes have in modifying biological variation is described.

The effect of atorvastatin and simvastatin on vitamin D, oxidative stress and inflammatory marker concentrations in patients with type 2 diabetes: a crossover study.

The pleiotropic effect of statins may be mediated in part through raising 25 hydroxy vitamin D (25OHD) concentrations. It has also been shown that an increase in oxidative stress and inflammatory markers are a feature of the patients with type 2 diabetes (T2DM). A cross-over study of 26 patients with T2DM taking either simvastatin 40 mg or atorvastatin 10 mg was undertaken. After 3 months on one statin, lipids, C-reactive protein (hsCRP), 25OHD and malondialdehyde (MDA) were measured repeatedly. The same procedure was then followed taking the other statin. Despite similar lipid-lowering, the mean 25OHD was higher on atorvastatin compared with simvastatin and the mean MDA and hsCRP levels lower, irrespective of which statin the patients were taking before crossover. The changes in 25OHD predicted changes in CRP and MDA levels. Thus, compared with simvastatin, atorvastatin shows apparently beneficial pleiotropic effects with respect to 25OHD concentrations as well as markers of oxidative stress and inflammation in patients with T2DM.

Recent advances in cardiovascular aspects of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) has been associated with increased cardiovascular risk (CVR) markers, but population studies have not clarified whether there is an increase in cardiovascular morbidity and mortality. Four different PCOS phenotypes resulted from the Rotterdam criteria that may differ in their CVR potential, thus introducing further complexity. This has led to studies using surrogate CVR markers including biomarkers in blood and imaging such as flow-mediated vasodilatation. In PCOS, both peripheral and central insulin resistance (IR) have been shown. Weight loss has been shown to improve IR and visceral fat, while insulin sensitizer therapies with metformin or thiazolidinediones improve IR and endothelial dysfunction. IR is also found in non-alcoholic fatty liver disease that in turn is very common in PCOS; studies have suggested that IR may be improved by treatment with metformin and omega-3 fish oils. PCOS patients have a more dyslipidemic phenotype that is worse in 'classical PCOS' associated with a higher CVR. Studies with atorvastatin and simvastatin have reported a decrease in the lipid parameters and an improvement in CVR indices including IR, but it is unclear whether this is due to their lipid-lowering action or a pleiotropic effect of the statin. In this expert opinion review, the relevant literature published during the last 2 years was considered. It focuses on some recent important data that has emerged while also exposing the gaps that remain in our knowledge that need to be addressed.

Endocrine disruptor & nutritional effects of heavy metals in ovarian hyperstimulation.

PURPOSE: There is increasing concern that environmental chemicals have a direct effect on fertility. Heavy metals such as mercury have been shown to affect various organ systems in humans including nervous system and skin, however they could also act as endocrine disrupting chemicals adversely affecting fertility. Metals such as zinc and selenium are essential micronutrients with diverse functions that may be important for reproductive outcomes. We measured mercury, zinc and selenium levels in the hair, a reliable reflection of long term environmental exposure and dietary status, to correlate with the outcome of ovarian hyperstimulation for in vitro fertilisation (IVF) treatment. METHODS: We analysed the hair of 30 subfertile women for mercury, zinc and selenium using inductively coupled mass spectrometry. Each woman underwent one cycle of IVF treatment. Correlation between the levels of these trace metals and treatment outcomes was investigated. RESULTS: Thirty women were recruited with mean (±SD) age of 32.7(4.4) years and BMI of 25.4(5.0)kg/m(2). Hair mercury concentration showed a negative correlation with oocyte yield (p < 0.05,ßcoefficient 0.38) and follicle number (p = 0.03,ß coefficient0.19) after ovarian stimulation. Zinc and selenium levels in hair correlated positively with oocyte yield after ovarian stimulation (p < 0.05,ß coefficient0.15) and (p = 0.03,ß coefficient0.21) respectively. Selenium levels in hair correlated significantly with follicle number following stimulation (p = 0.04, ßcoefficient0.22). There was no correlation between mercury, zinc and selenium in hair and their corresponding serum levels. CONCLUSION: These data suggest that mercury had a deleterious effect whilst there was a positive effect for zinc and selenium in the ovarian response to gonadotrophin therapy for IVF. Hair analysis offers a novel method of investigating the impact of long-term exposure to endocrine disruptors and nutritional status on reproductive outcomes.

Response at 3 months to insulin dose decisions made at exenatide initiation in the Association of British Clinical Diabetologists (ABCD) nationwide exenatide audit.

It is uncertain what should be done with insulin dose if starting exenatide. In the ABCD nationwide exenatide audit, many patients with type 2 diabetes had worsened glycaemia when insulin was stopped. If starting exenatide, insulin should not be stopped but weaned off only if there is significant glycaemic response.

Is there a role for immune and anti-in-flammatory therapy in type 2 diabetes?

There is a growing body of evidence to suggest that type 2 diabetes is associated with a generalised activation of the innate immune system, in which there is a chronic low-grade inflammation. Further evidence for roles of inflammation in type 2 diabetes comes from clinical studies using either anti-inflammatory approaches or biological agents that target specific proinflammatory cytokine pathways to improve parameters of glucose control especially with IL-1ß (Interleukin 1 ß) antagonism and salsalate (non-acetylated prodrug of salicylate) treatment. In this review, recent evidence implicating the pathological involvement of the immune system in type 2 diabetes is examined, together with the role of potential treatment modalities targeting the immune system in the management of type 2 diabetes.

Safety, efficacy and tolerability of exenatide in combination with insulin in the Association of British Clinical Diabetologists nationwide exenatide audit*.

AIM: To assess the extent, safety, efficacy and tolerability of reported off-licence exenatide use through a nationwide audit. METHODS: The Association of British Clinical Diabetologists hosted a password-protected, online collection of anonymized data of exenatide use in real clinical practice. Three hundred and fifteen contributors from 126 centres across UK provided data on 6717 patients. HbA1c and weight changes, exenatide discontinuation, adverse events and treatment satisfaction were compared between non-insulin and insulin-treated patients. RESULTS: Four thousand eight hundred and fifty-seven patients had baseline and follow-up treatment status with mean (±s.d.) baseline HbA1c 9.45 ± 1.69% and BMI 40.0 ± 8.2 kg/m(2) . Of the 4857 patients, 1921 (39.6%) used exenatide with insulin. Comparing patients who continued insulin with exenatide with non-insulin-treated patients, mean (±s.e.) latest HbA1c and weight reduction (median 26 weeks) were 0.51 ± 0.06 versus 0.94 ± 0.04% (p < 0.001) and 5.8 ± 0.2 versus 5.5 ± 0.1 kg (p = 0.278). Insulin-treated patients had higher rates of exenatide discontinuation (31.0 vs. 13.9%, p < 0.001), hypoglycaemia (8.9 vs. 6.1%, p < 0.001), gastrointestinal side effects (28.4 vs. 25.0%, p = 0.008) and treatment dissatisfaction (20.8 vs. 5.7%, p < 0.001). However, 34.2% of the patients continuing insulin still achieved HbA1c reduction ≥1%. There was significant insulin discontinuation, dose reduction and greater sulphonylurea discontinuation among insulin-treated patients. CONCLUSIONS: Addition of exenatide to obese, insulin-treated patients can improve glycaemia and weight. Adverse events were statistically but probably not clinically significantly higher, but combination treatment was less well tolerated. Overall, exenatide was less effective in lowering HbA1c among insulin-treated patients, although significant number of insulin-treated patients still achieved significant HbA1c, weight and insulin reductions. Further research into identifying obese, insulin-treated patients who will tolerate and benefit from exenatide treatment is urgently needed.

High-cocoa polyphenol-rich chocolate improves HDL cholesterol in Type 2 diabetes patients.

AIM: To examine the effects of chocolate on lipid profiles, weight and glycaemic control in individuals with Type 2 diabetes. METHODS: Twelve individuals with Type 2 diabetes on stable medication were enrolled in a randomized, placebo-controlled double-blind crossover study. Subjects were randomized to 45 g chocolate with or without a high polyphenol content for 8 weeks and then crossed over after a 4-week washout period. Changes in weight, glycaemic control, lipid profile and high-sensitivity C-reactive protein were measured at the beginning and at the end of each intervention. RESULTS: HDL cholesterol increased significantly with high polyphenol chocolate (1.16 ± 0.08 vs. 1.26 ± 0.08 mmol/l, P = 0.05) with a decrease in the total cholesterol: HDL ratio (4.4 ± 0.4 vs. 4.1 ± 0.4 mmol/l, P = 0.04). No changes were seen with the low polyphenol chocolate in any parameters. Over the course of 16 weeks of daily chocolate consumption neither weight nor glycaemic control altered from baseline. CONCLUSION: High polyphenol chocolate is effective in improving the atherosclerotic cholesterol profile in patients with diabetes by increasing HDL cholesterol and improving the cholesterol:HDL ratio without affecting weight, inflammatory markers, insulin resistance or glycaemic control.

Low density lipoprotein-cholesterol variability in patients with type 2 diabetes taking atorvastatin compared to simvastatin: justification for direct measurement?

AIM: The benefit of direct, as opposed to calculated, low density lipoprotein -cholesterol (LDL-C) measurement remains unclear. This study compared the biological variability of direct LDL in patients with type 2 diabetes (T2DM) on equivalent doses of the short half-life statin, simvastatin or the longer half-life statin, atorvastatin. METHODS: A cross-over study of biological variation of lipids in 26 patients with T2DM taking either simvastatin 40 mg (n = 10) or atorvastatin 10 mg. After 3 months on one statin, fasting lipids were measured on 10 occasions over a 5-week period. The same procedure was then followed on the other statin. The variability of LDL-C was established using a Beckman direct assay. RESULTS: As a group, mean LDL was no different between statins (mean +/- s.d.) (1.69 +/- 0.60 mmol/l simvastatin vs. 1.67 +/- 0.60 mmol/l atorvastatin, p = 0.19). However, in all patients, the intraindividual biological variability of LDL while taking simvastatin was markedly higher than with atorvastatin (average s.d. = 0.17 mmol /l simvastatin vs. 0.01 mmol/l, p < 0.0001). Friedewald calculated LDL variability was no different between statins (average s.d. = 0.34 mmol /l simvastatin vs. 0.21 mmol/l atorvastatin, p = 0.19). CONCLUSIONS: In contrast to calculated values, direct measurement revealed LDL to be much more stable (the s.d. being an order of magnitude) in T2DM patients taking atorvastatin rather than simvastatin. This means LDL targets can be consistently met with less lipid monitoring using atorvastatin rather than simvastatin. Direct LDL measurement may therefore have a particular role in monitoring patients on statin treatment.