Malcolm Whiting

The need for a large-scale trial of fibrate therapy in diabetes : the rationale and design of the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study

BackgroundFibrates correct the typical lipid abnormalities of type 2 diabetes mellitus, yet no study, to date, has specifically set out to evaluate the role of fibrate therapy in preventing cardiovascular events in this setting.MethodsSubjects with type 2 diabetes, aged 50–75 years, were screened for eligibility to participate in a long-term trial of comicronized fenofibrate 200 mg daily compared with matching placebo to assess benefits of treatment on the occurrence of coronary and other vascular events. People with total cholesterol levels 3.0–6.5 mmol/L plus either a total-to-HDLc ratio >4.0 or triglyceride level >1.0 mmol/L with no clear indication for lipid-modifying therapy were eligible.ResultsA total of 9795 people were randomized into the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial. All received dietary advice, followed by a 6-week single-blind placebo run-in, then a 6-week active run-in period before randomization. Participants are being followed up every 6 months for outcome events and safety assessments. The study is designed to yield at least 500 coronary events (primary endpoint: first nonfatal myocardial infarction or coronary death) over 5 years, to have 80% power to identify as statistically significant at 2P = 0.05 a 22% reduction in such events, using intention-to-treat methods.ConclusionsType 2 diabetes is the most common endocrine disorder worldwide, and its prevalence is increasing. The current evidence about use of fibrates in type 2 diabetes, from around 2000 people treated, will increase with FIELD to evidence from around 12000. FIELD will establish the role of fenofibrate treatment in reducing cardiovascular risk in people with type 2 diabetes. The main results are expected to be available in late 2005.Background Fibrates correct the typical lipid abnormalities of type 2 diabetes mellitus, yet no study, to date, has specifically set out to evaluate the role of fibrate therapy in preventing cardiovascular events in this setting. Methods Subjects with type 2 diabetes, aged 50–75 years, were screened for eligibility to participate in a long-term trial of comicronized fenofibrate 200 mg daily compared with matching placebo to assess benefits of treatment on the occurrence of coronary and other vascular events. People with total cholesterol levels 3.0–6.5 mmol/L plus either a total-to-HDLc ratio >4.0 or triglyceride level >1.0 mmol/L with no clear indication for lipid-modifying therapy were eligible. Results A total of 9795 people were randomized into the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial. All received dietary advice, followed by a 6-week single-blind placebo run-in, then a 6-week active run-in period before randomization. Participants are being followed up every 6 months for outcome events and safety assessments. The study is designed to yield at least 500 coronary events (primary endpoint: first nonfatal myocardial infarction or coronary death) over 5 years, to have 80% power to identify as statistically significant at 2P = 0.05 a 22% reduction in such events, using intention-to-treat methods. Conclusions Type 2 diabetes is the most common endocrine disorder worldwide, and its prevalence is increasing. The current evidence about use of fibrates in type 2 diabetes, from around 2000 people treated, will increase with FIELD to evidence from around 12000. FIELD will establish the role of fenofibrate treatment in reducing cardiovascular risk in people with type 2 diabetes. The main results are expected to be available in late 2005.

Sequence variation in DDAH1 and DDAH2 genes is strongly and additively associated with serum ADMA concentrations in individuals with type 2 diabetes

Background Asymmetric dimethylarginine (ADMA), present in human serum, is an endogenous inhibitor of nitric oxide synthase and contributes to vascular disease. Dimethylarginine dimethylaminohydrolase (DDAH) is an ADMA degrading enzyme that has two isoforms: DDAHI and DDAHII. We sought to determine whether serum ADMA levels in type 2 diabetes are influenced by common polymorphisms in the DDAH1 and DDAH2 genes. Methodology/Principal Findings Relevant clinical parameters were measured and peripheral whole blood obtained for serum and genetic analysis on 343 participants with type 2 diabetes. Serum ADMA concentrations were determined by mass spectroscopy. Twenty six tag SNPs in the DDAH1 and 10 in the DDAH2 gene were genotyped in all subjects and tested for association with serum ADMA levels. Several SNPs and haplotypes in the DDAH genes were strongly associated with ADMA levels. Most significantly in the DDAH1 gene, rs669173 (p = 2.96×10−7), rs7521189 (p = 6.40×10−7), rs2474123 (p = 0.00082) and rs13373844 (p = 0.00027), and in the DDAH2 gene, rs3131383 (p = 0.0029) and the TGCCCAGGAG haplotype (p = 0.0012) were significantly associated with ADMA levels. Sub-analysis by diabetic retinopathy (DR) status revealed these variants were associated with ADMA levels predominantly in participants without DR. Combined analysis of the most strongly associated SNPs in DDAH1 (rs669173) and DDAH2 (rs3131383) revealed an additive effect (p = 1.37×10−8) on ADMA levels. Conclusions/Significance Genetic variation in the DDAH1 and 2 genes is significantly associated with serum ADMA levels. Further studies are required to determine the pathophysiological significance of elevated serum ADMA in type 2 diabetes and to better understand how DDAH gene variation influences ADMA levels.

Simultaneous measurement of urinary metanephrines and catecholamines by liquid chromatography with tandem mass spectrometric detection

Background The measurement of catecholamines and metanephrines in urine is an important diagnostic test in biochemical screening for phaeochromocytoma. Tandem mass spectrometry (MSMS) has the potential to be used in a profiling method for simultaneous assay of these analytes. Methods Optimal conditions were established for the MSMS detection of catecholamines (noradrenalin, adrenalin and dopamine) and metanephrines (normetanephrine and metanephrine), including commercially available isotopically labelled compounds for use as internal standards. Chromatographic separation of all five polar biogenic amines was achieved under solvent conditions that were compatible with MSMS and multiple reaction monitoring. Several types of solid-phase extraction cartridge were used to investigate clean-up conditions for urine, and acid-hydrolysates of urine, prior to LC-MSMS. Results Total catecholamines and metanephrines from acid-hydrolysed urines, or free catecholamines and free metanephrines from native urines, were complexed with diphenyl-boronate and recovered in high yield from polymer cartridges after elution with formic acid. Direct injection of eluates into the LC-MSMS system allowed quantitation of catecholamines and metanephrines with a run time of 6 min per sample. Biogenic amine concentrations for patient urines and quality assurance programme samples, and assay imprecision, were similar to values obtained with high-performance liquid chromatography methods, which used electrochemical detection. In normal urines, the ratio of free to total catecholamines was around three-fold higher than the ratio of free to total metanephrines. Conclusion The assay of urinary catecholamines and metanephrines can be achieved simultaneously using one LC-MSMS method, which is rapid and reduces labour and consumable costs for routine application.

Relationships of HDL Cholesterol, ApoA-I, and ApoA-II With Homocysteine and Creatinine in Patients With Type 2 Diabetes Treated With Fenofibrate

Objective—The purpose of this study was to determine fenofibrate-induced changes in plasma high-density lipoprotein cholesterol (HDL-C), apoliprotein (apo) A-I, and apolipoprotein (apo) A-II and how they relate to changes in plasma homocysteine and creatinine. Methods and Results—FIELD was a double-blind placebo-controlled trial done in Australia, New Zealand, and Finland. All FIELD subjects were included except those who started statin therapy or permanently discontinued fenofibrate. Patients were randomized to receive daily micronised fenofibrate (200 mg) or matching placebo and were followed up for a median of 5 years. Plasma HDL-C, apoA-I, apoA-II, homocysteine, and creatinine were measured. There was an inverse relationship between baseline homocysteine levels and HDL-C in the placebo (P=0.07 for linear trend) and fenofibrate groups (P<0.0001) and apoA-I (P<0.001, both groups). The increases in homocysteine and creatinine in the fenofibrate group correlated positively (P<0.0001). The greater the increase in homocysteine induced by fenofibrate, the smaller the increases in HDL-c and apoA-I (P<0.0001 for linear trends). There was a highly significant and positive relationship between fenofibrate-induced changes in homocysteine and apoA-II levels. Conclusions—PPARα agonists that have a more robust effect on HDL-C and apoA-I would be desirable.

The correlation between third-trimester maternal and newborn-serum 25-hydroxy-vitamin D in a selected South Australian group of newborn samples

Background Although vitamin D insufficiency is prevalent in the community, only a few population-based studies have measured serum 25-hydroxy-vitamin D (25OHD) levels during pregnancy and in newborns. Maternal vitamin D deficiency has been linked to pregnancy complications, as well as hypocalcaemia and rickets in the newborn. Here, the authors report third-trimester maternal and newborn-serum 25OHD concentrations in 101 neonates whose serum samples were sent for testing. Methods The newborn 25OHD levels were correlated with the third-trimester maternal serum 25OHD levels using a least-square regression analysis. All samples were measured using an enzyme immunoassay (EIA). Ten randomly selected newborn serum samples were also measured using liquid chromatography/tandem mass spectrometry LC-MSMS, and correlated with the EIA method. Results Out of 99 mothers of the newborns, only 19, 42 and 68 had their 25OHD level measured in the first, second and third trimester respectively. The mean maternal age was 30 years, while the mean maternal third-trimester 25OHD concentration was 48 nmol/l. Of the newborns, 53% were female, and 85% were term deliveries. The mean newborn-serum 25OHD was 68 nmol/l. Neonatal 25OHD was related to maternal third-trimester levels measured by EIA (r=0.3; newborn 25OHD=0.42(maternal 25OHD)+44.2; p=0.02). EIA and LC-MSMS concentrations for newborns correlated significantly over a range between 20 and 103 nmol/l by EIA (r=0.9; EIA=1.04(LCMSMS)+10.1; p<0.00 (slope); p=0.18 (intercept)). The mean 25OHD concentration in women who suffered pre-eclampsia and premature rupture of membranes were 45 and 39 nmol/l respectively. Conclusions Newborn-serum 25OHD concentrations depend on the maternal circulating plasma 25OHD level at least during the third trimester. Neonatal 25OHD levels obtained by EIA correlated well with LC-MSMS. Although the EIA values for neonates were greater than LC-MSMS values, this difference was not statistically significant.

Diabetic Retinopathy Is Associated With Elevated Serum Asymmetric and Symmetric Dimethylarginines

OBJECTIVE Asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and l-arginine directly influence nitric oxide production. Our objective was to test whether serum ADMA, SDMA, or l-arginine levels correlate with diabetic retinopathy subtype or severity. RESEARCH DESIGN AND METHODS A total of 162 subjects with type 1 diabetes and 343 with type 2 diabetes, of whom 329 subjects had no diabetic retinopathy, 27 had nonproliferative diabetic retinopathy (NPDR), 101 had proliferative diabetic retinopathy (PDR), and 107 had clinically significant macular edema (CSME), were recruited. Blinding diabetic retinopathy was defined as severe NPDR, PDR, or CSME. Serum ADMA, SDMA, and l-arginine concentrations were determined by mass spectroscopy. RESULTS In multivariate analysis, blinding diabetic retinopathy, PDR, and nephropathy were associated with significantly increased serum levels of ADMA (P < 0.001), SDMA (P < 0.001), and l-arginine (P = 0.001). Elevated ADMA (P < 0.001) and SDMA (P < 0.001) were also significantly associated with CSME. CONCLUSIONS Severe forms of diabetic retinopathy are associated with elevated serum ADMA, SDMA, and l-arginine. Further investigation is required to determine whether these findings are of clinical relevance.

Plasma homocysteine and the risk of venous thromboembolism: insights from the FIELD study.

Abstract Background: The lipid-lowering effect of fenofibrate is accompanied by a rise in plasma homocysteine (HCY), a potential risk factor for venous thromboembolism (VTE). This study investigated the relationship between HCY and the risk of VTE in patients treated with fenofibrate. Methods: The relationship between HCY and deep-vein thrombosis or pulmonary embolism was investigated in 9522 participants of the 5-year Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial. All subjects received fenofibrate during a 6-week active run-in phase before randomization. A Cox proportional-hazards model was used to assess the effect of HCY on risk of venous thromboembolic events. Results: During active-drug run-in, HCY rose on average by 6.5 μmol/L, accompanied by a substantial rise in plasma creatinine (+12%). Fenofibrate-induced changes in HCY and creatinine were fully reversible in the placebo group but persisted in the treatment group until reversing at the end of therapy. During follow-up, 1.8% had at least one episode of deep-vein thrombosis or pulmonary embolism: 103 on fenofibrate and 68 on placebo (log-rank p=0.006). In multivariate analysis, every 5 μmol/L higher baseline HCY was associated with 19% higher risk of VTE. Fenofibrate treatment was associated with 52% higher risk, but the change in HCY with fenofibrate was not significantly associated with VTE after adjustment for baseline HCY. Conclusions: Hyperhomocysteinemia is prospectively associated with VTE. Fenofibrate may predispose individuals with high pretreatment HCY towards VTE. The fenofibrate-induced increase in HCY did not, however, explain the risk associated with fenofibrate therapy.

Evaluation of 25-hydroxy vitamin D assay on the immunodiagnostic systems iSYS analyser.

Background We evaluated the recently released chemiluminescence assay for 25-hydroxy vitamin D (25-OHD) on the Immunodiagnostic Systems iSYS (IDS-iSYS) automated analyser. Methods The IDS-iSYS comparison was performed using patient samples previously measured for 25-OHD by a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method (n = 119) and an IDS enzyme immunoassy (IDS-EIA) method (n = 64). Limit of detection and limit of quantification were determined from a precision profile. Imprecision was assessed using quality control material and pooled serum. External QAP material (Vitamin D External Quality Assessment Scheme, UK) was analysed to establish inaccuracy. Linearity was assessed by two dilution studies. Cross-reactivity was determined by three serial dilution studies of patient samples with known 25-OHD2 concentrations. Results The IDS-iSYS correlated well with both established methods (iSYS = 1.03LC-MS/MS − 6.53, R 2 = 0.82 and iSYS = 1.07IDS-EIA − 1.61, R 2 = 0.86). Imprecision of the iSYS assay for IDS control material was 13.4% at 32 nmol/L, 10% at 78 nmol/L, 9.4% at 161 nmol/L, and for the pooled material 9.3% at 72 nmol/L and 5.6% at 158 nmol/L. The evaluation found the assay to be highly accurate (IDS-iSYS = 0.93ALTM + 3.79, R 2 = 0.94) and linear (obs1 = 0.93exp1 − 5.05, R 2 = 0.99 (P = 0.256); and obs2 = 0.97exp2 + 6.07, R 2 = 0.97 (P = 0.654); ALTM, all-laboratory trimmed mean). Cross-reactivity studies demonstrated no significant difference to the calculated total 25-OHD as measured by LC-MS/MS. Conclusions Even though the imprecision of the iSYS was found to be greater than that of the LC-MS/MS and EIA methods, the performance characteristics of the IDS-iSYS 25-OHD assay are suitable for routine diagnostic purposes on a high throughput automated analyser.

Measurement of Plasma LDL Cholesterol in Patients with Diabetes

OBJECTIVE To assess the accuracy of plasma LDL cholesterol concentrations estimated by the Friedewald formula and a direct immunoseparation method by comparison with a reference ultracentrifugation procedure in patients with diabetes. RESEARCH DESIGN AND METHODS Fasting plasma samples with triglyceride concentrations < 4.5 mmol/l were collected from 100 patients with diabetes (28 type I and 72 type II) and LDL cholesterol concentrations were compared by the three methods. RESULTS LDL cholesterol values determined by the reference β-quantitation procedure were highly correlated with both the Friedewald formula (r = 0.96) and a direct immunoseparation method (r = 0.92). Calculated (Friedewald) LDL cholesterol coincided with the reference method with < 10% error in 74% of the total diabetic group (82% of type I and 68% of type II diabetic patients). However, agreement between the direct LDL cholesterol and reference methods was significantly less (P = 0.02), with only 44% of patients having an error of < 10% (52% of type I and 41% of type II diabetic patients). The direct immunoseparation method for LDL cholesterol showed a positive bias with increasing triglyceride concentrations, particularly for patients with type II diabetes. CONCLUSIONS In the group of diabetic patients studied with plasma triglyceride concentrations < 4.5 mmol/l, the Friedewald formula provided an accurate estimation of LDL cholesterol. The direct immunoseparation method significantly overestimated LDL cholesterol at triglyceride levels between 2 and 4.5 mmol/l.

Elevation of serum asymmetrical and symmetrical dimethylarginine in patients with advanced glaucoma.

PURPOSE Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are the dimethylated isomeric derivatives of the amino acid L-arginine. ADMA is an endogenous inhibitor of nitric oxide synthase (NOS), while SDMA is a competitive inhibitor of cellular uptake of L-arginine, the substrate for NOS. As such, these metabolites are associated with endothelial dysfunction. As the nitric oxide pathway and endothelial dysfunction have been implicated in glaucoma, the aim of this study was to investigate serum ADMA, SDMA, and L-arginine levels in individuals with advanced glaucoma compared with normal controls. In addition, we have investigated genetic variation in the DDAH1 and DDAH2 genes, encoding the enzymes responsible for degradation of ADMA, for association with ADMA level in glaucoma patients and controls. METHODS Two hundred eleven patients with advanced glaucoma and 295 normal controls were recruited. Liquid chromatography-tandem mass spectrometry was used to measure the serum ADMA, SDMA, and L-arginine levels of participants. Single nucleotide polymorphisms in the DDAH1 and DDAH2 genes reportedly associated with ADMA level were genotyped in all individuals. RESULTS A significant increase in both serum ADMA and SDMA concentration was detected in advanced glaucoma cases compared with controls (P ≤ 0.0001). No significant change was detected in serum L-arginine concentration. No association of polymorphisms in DDAH1 and DDAH2 with either ADMA level or glaucoma was detected. CONCLUSIONS The serum levels of two dimethylarginines, ADMA and SDMA, are associated with advanced glaucoma. These data further implicate the nitric-oxide pathway in glaucoma pathogenesis.