Does hyperphenylalaninemia induce brain glucose hypometabolism? Cerebral spinal fluid findings in treated adult phenylketonuric patients.

Despite numerous studies in human patients and animal models for phenylketonuria (PKU; OMIM#261600), the pathophysiology of PKU and the underlying causes of brain dysfunction and cognitive problems in PKU patients are not well understood. In this study, lumbar cerebral spinal fluid (CSF) was obtained immediately after blood sampling from early-treated adult PKU patients who had fasted overnight. Metabolite and amino acid concentrations in the CSF of PKU patients were compared with those of non-PKU controls. The CSF concentrations and CSF/plasma ratios for glucose and lactate were found to be below normal, similar to what has been reported for glucose transporter1 (GLUT1) deficiency patients who exhibit many of the same clinical symptoms as untreated PKU patients. CSF glucose and lactate levels were negatively correlated with CSF phenylalanine (Phe), while CSF glutamine and glutamate levels were positively correlated with CSF Phe levels. Plasma glucose levels were negatively correlated with plasma Phe concentrations in PKU subjects, which partly explains the reduced CSF glucose concentrations. Although brain glucose concentrations are unlikely to be low enough to impair brain glucose utilization, it is possible that the metabolism of Phe in the brain to produce phenyllactate, which can be transported across the blood-brain barrier to the blood, may consume glucose and/or lactate to generate the carbon backbone for glutamate. This glutamate is then converted to glutamine and carries the Phe-derived ammonia from the brain to the blood. While this mechanism remains to be tested, it may explain the correlations of CSF glutamine, glucose, and lactate concentrations with CSF Phe.

Sustainable manufacturing through application of reconfigurable and intelligent systems in production processes: a system perspective.

Sustainable production aims at creating products from processes that minimize environmental impact, energy consumption and natural resources. Customers and markets are ever more leaning towards digital, custom, and flexible solutions with lower environmental impact. Hence, Industry 4.0 (I4.0) solutions are increasingly including social and environmental sustainability aspects. We focus on the realization of an infrastructure integrating industrially relevant application modules by combining system reconfigurability and artificial intelligence, towards sustainable production. To meet the final goal of sustainable production, we address four challenges considering flexibility and sustainability in production in a holistic way: (1) developing infrastructural and methodological tools to support companies to explore the potential of I4.0 towards sustainable production; (2) managing the configurability and customization possibilities of products; (3) effectively handling the flexibility provided by a production system with rapid reconfiguration capabilities; (4) integrating hardware and software flexibility by using reconfigurable robotics and machine learning methods. By developing and connecting different application modules, we obtain a physical demonstrator which represents on the one hand an exemplary scenario of reconfigurable and flexible production system; on the other, it enables new research activities and insights with a see, touch & feel approach for industrial and research realities.

Phenylketonuria (PKU) Urinary Metabolomic Phenotype Is Defined by Genotype and Metabolite Imbalance: Results in 51 Early Treated Patients Using Ex Vivo 1H-NMR Analysis.

Phenylketonuria (PKU) is a rare metabolic disorder caused by mutations in the phenylalanine hydroxylase gene. Depending on the severity of the genetic mutation, medical treatment, and patient dietary management, elevated phenylalanine (Phe) may occur in blood and brain tissues. Research has recently shown that high Phe not only impacts the central nervous system, but also other organ systems (e.g., heart and microbiome). This study used ex vivo proton nuclear magnetic resonance (1H-NMR) analysis of urine samples from PKU patients (mean 14.9 ± 9.2 years, n = 51) to identify the impact of elevated blood Phe and PKU treatment on metabolic profiles. Our results found that 24 out of 98 urinary metabolites showed a significant difference (p < 0.05) for PKU patients compared to age-matched healthy controls (n = 51) based on an analysis of urinary metabolome. These altered urinary metabolites were related to Phe metabolism, dysbiosis, creatine synthesis or intake, the tricarboxylic acid (TCA) cycle, end products of nicotinamide-adenine dinucleotide degradation, and metabolites associated with a low Phe diet. There was an excellent correlation between the metabolome and genotype of PKU patients and healthy controls of 96.7% in a confusion matrix model. Metabolomic investigations may contribute to a better understanding of PKU pathophysiology.

Ex vivo proton spectroscopy (1 H-NMR) analysis of inborn errors of metabolism: Automatic and computer-assisted analyses.

There are about 1500 genetic metabolic diseases. A small number of treatable diseases are diagnosed by newborn screening programs, which are continually being developed. However, most diseases can only be diagnosed based on clinical symptoms or metabolic findings. The main biological fluids used are urine, plasma and, in special situations, cerebrospinal fluid. In contrast to commonly used methods such as gas chromatography and high performance liquid chromatography mass spectrometry, ex vivo proton spectroscopy (1 H-NMR) is not yet used in routine clinical practice, although it has been recommended for more than 30 years. Automatic analysis and improved NMR technology have also expanded the applications used for the diagnosis of inborn errors of metabolism. We provide a mini-overview of typical applications, especially in urine but also in plasma, used to diagnose common but also rare genetic metabolic diseases with 1 H-NMR. The use of computer-assisted diagnostic suggestions can facilitate interpretation of the profiles. In a proof of principle, to date, 182 reports of 59 different diseases and 500 reports of healthy children are stored. The percentage of correct automatic diagnoses was 74%. Using the same 1 H-NMR profile-targeted analysis, it is possible to apply an untargeted approach that distinguishes profile differences from healthy individuals. Thus, additional conditions such as lysosomal storage diseases or drug interferences are detectable. Furthermore, because 1 H-NMR is highly reproducible and can detect a variety of different substance categories, the metabolomic approach is suitable for monitoring patient treatment and revealing additional factors such as nutrition and microbiome metabolism. Besides the progress in analytical techniques, a multiomics approach is most effective to combine metabolomics with, for example, whole exome sequencing, to also diagnose patients with nondetectable metabolic abnormalities in biological fluids. In this mini review we also provide our own data to demonstrate the role of NMR in a multiomics platform in the field of inborn errors of metabolism.

Start Selective and Rigidify: The Discovery Path toward a Next Generation of EGFR Tyrosine Kinase Inhibitors.

The epidermal growth factor receptor (EGFR), when carrying an activating mutation like del19 or L858R, acts as an oncogenic driver in a subset of lung tumors. While tumor responses to tyrosine kinase inhibitors (TKIs) are accompanied by marked tumor shrinkage, the response is usually not durable. Most patients relapse within two years of therapy often due to acquisition of an additional mutation in EGFR kinase domain that confers resistance to TKIs. Crucially, oncogenic EGFR harboring both resistance mutations, T790M and C797S, can no longer be inhibited by currently approved EGFR TKIs. Here, we describe the discovery of BI-4020, which is a noncovalent, wild-type EGFR sparing, macrocyclic TKI. BI-4020 potently inhibits the above-described EGFR variants and induces tumor regressions in a cross-resistant EGFRdel19 T790M C797S xenograft model. Key was the identification of a highly selective but moderately potent benzimidazole followed by complete rigidification of the molecule through macrocyclization.

Association Between Vascular Cell Adhesion Molecule 1 and Atrial Fibrillation.

Importance: Accumulating evidence links inflammation and atrial fibrillation (AF). Objective: To assess whether markers of systemic and atrial inflammation are associated with incident AF in the general population. Design, Setting, and Participants: The Bruneck Study is a prospective, population-based cohort study with a 20-year follow-up (n = 909). The population included a random sample of the general community aged 40 to 79 years. Levels of 13 inflammation markers were measured at baseline in 1990. Findings were replicated in a case-control sample nested within the prospective Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) study (n = 1770). Data analysis was performed from February to May 2016. Exposures: Levels of 13 inflammation markers. Main Outcomes and Measures: Incident AF over a 20-year follow-up period in the Bruneck Study. Results: Of the 909 participants included in the Bruneck Study, mean [SD] age was 58.8 (11.4) years and 448 (49.3%) were women. Among the 880 participants free of prevalent AF (n = 29) at baseline, 117 developed AF during the 20-year follow-up period (incidence rate, 8.2; 95% CI, 6.8-9.6 per 1000 person-years). The levels of soluble vascular cell adhesion molecule 1 (VCAM-1) and osteoprotegerin were significantly associated with incident AF (hazard ratio [HR], 1.49; 95% CI, 1.26-1.78; and 1.46; 95% CI, 1.25-1.69, respectively; P < .001 with Bonferroni correction for both), but osteoprotegerin lost significance after age and sex adjustment (HR, 1.05; 95% CI, 0.87-1.27; P > .99 with Bonferroni correction). Matrix metalloproteinase 9, metalloproteinase inhibitor 1, monocyte chemoattractant protein-1, P-selectin, fibrinogen, receptor activator of nuclear factor-κB ligand, high-sensitivity C-reactive protein, adiponectin, leptin, soluble intercellular adhesion molecule 1, and E-selectin all fell short of significance (after Bonferroni correction in unadjusted and age- and sex-adjusted analyses). The HR for a 1-SD higher soluble VCAM-1 level was 1.34 (95% CI, 1.11-1.62; Bonferroni-corrected P = .03) in a multivariable model. The association was of a dose-response type, at least as strong as that obtained for N-terminal pro-B-type natriuretic peptide (multivariable HR for a 1-SD higher N-terminal pro-B-type natriuretic peptide level, 1.15; 95% CI, 1.04-1.26), internally consistent in various subgroups, and successfully replicated in the SAPHIR Study (age- and sex-adjusted, and multivariable odds ratios for a 1-SD higher soluble VCAM-1 level, 1.91; 95% CI, 1.24-2.96, P = .003; and 2.59; 95% CI, 1.45-4.60; P = .001). Conclusions and Relevance: Levels of soluble VCAM-1, but not other inflammation markers, are significantly associated with new-onset AF in the general community. Future studies should address whether soluble VCAM-1 is capable of improving AF risk classification beyond the information provided by standard risk scores.

Very-Low-Density Lipoprotein-Associated Apolipoproteins Predict Cardiovascular Events and Are Lowered by Inhibition of APOC-III.

BACKGROUND: Routine apolipoprotein (apo) measurements for cardiovascular disease (CVD) are restricted to apoA-I and apoB. Here, the authors measured an unprecedented range of apolipoproteins in a prospective, population-based study and relate their plasma levels to risk of CVD. OBJECTIVES: This study sought to measure apolipoproteins directly by mass spectrometry and compare their associations with incident CVD and to obtain a system-level understanding of the correlations of apolipoproteins with the plasma lipidome and proteome. METHODS: Associations of 13 apolipoproteins, 135 lipid species, and 211 other plasma proteins with incident CVD (91 events), defined as stroke, myocardial infarction, or sudden cardiac death, were assessed prospectively over a 10-year period in the Bruneck Study (N = 688) using multiple-reaction monitoring mass spectrometry. Changes in apolipoprotein and lipid levels following treatment with volanesorsen, a second-generation antisense drug targeting apoC-III, were determined in 2 human intervention trials, one of which was randomized. RESULTS: The apolipoproteins most significantly associated with incident CVD were apoC-II (hazard ratio per 1 SD [HR/SD]: 1.40; 95% confidence interval [CI]: 1.17 to 1.67), apoC-III (HR/SD: 1.38; 95% CI: 1.17 to 1.63), and apoE (HR/SD: 1.31; 95% CI: 1.13 to 1.52). Associations were independent of high-density lipoprotein (HDL) and non-HDL cholesterol, and extended to stroke and myocardial infarction. Lipidomic and proteomic profiles implicated these 3 very-low-density lipoprotein (VLDL)-associated apolipoproteins in de novo lipogenesis, glucose metabolism, complement activation, blood coagulation, and inflammation. Notably, apoC-II/apoC-III/apoE correlated with a pattern of lipid species previously linked to CVD risk. ApoC-III inhibition by volanesorsen reduced plasma levels of apoC-II, apoC-III, triacylglycerols, and diacylglycerols, and increased apoA-I, apoA-II, and apoM (all p < 0.05 vs. placebo) without affecting apoB-100 (p = 0.73). CONCLUSIONS: The strong associations of VLDL-associated apolipoproteins with incident CVD in the general community support the concept of targeting triacylglycerol-rich lipoproteins to reduce risk of CVD.

Successful intrauterine treatment of a patient with cobalamin C defect.

Cobalamin C (cblC) defect is an inherited autosomal recessive disorder that affects cobalamin metabolism. Patients are treated with hydroxycobalamin to ameliorate the clinical features of early-onset disease and prevent clinical symptoms in late-onset disease. Here we describe a patient in whom prenatal maternal treatment with 30 mg/week hydroxycobalamin and 5 mg/day folic acid from week 15 of pregnancy prevented disease manifestation in a girl who is now 11 years old with normal IQ and only mild ophthalmic findings. The affected older sister received postnatal treatment only and is severely intellectually disabled with severe ophthalmic symptoms. This case highlights the potential of early, high-dose intrauterine treatment in a fetus affected by the cblC defect.

Heme oxygenase-1 gene promoter microsatellite polymorphism is associated with progressive atherosclerosis and incident cardiovascular disease.

OBJECTIVE: The enzyme heme oxygenase-1 (HO-1) exerts cytoprotective effects in response to various cellular stressors. A variable number tandem repeat polymorphism in the HO-1 gene promoter region has previously been linked to cardiovascular disease. We examined this association prospectively in the general population. APPROACH AND RESULTS: Incidence of stroke, myocardial infarction, or vascular death was registered between 1995 and 2010 in 812 participants of the Bruneck Study aged 45 to 84 years (49.4% males). Carotid atherosclerosis progression was quantified by high-resolution ultrasound. HO-1 variable number tandem repeat length was determined by polymerase chain reaction. Subjects with ≥32 tandem repeats on both HO-1 alleles compared with the rest of the population (recessive trait) featured substantially increased cardiovascular disease risk (hazard ratio [95% confidence interval], 5.45 [2.39, 12.42]; P<0.0001), enhanced atherosclerosis progression (median difference in atherosclerosis score [interquartile range], 2.1 [0.8, 5.6] versus 0.0 [0.0, 2.2] mm; P=0.0012), and a trend toward higher levels of oxidized phospholipids on apolipoprotein B-100 (median oxidized phospholipids/apolipoprotein B level [interquartile range], 11364 [4160, 18330] versus 4844 [3174, 12284] relative light units; P=0.0554). Increased cardiovascular disease risk in those homozygous for ≥32 repeats was also detected in a pooled analysis of 7848 participants of the Bruneck, SAPHIR, and KORA prospective studies (hazard ratio [95% confidence interval], 3.26 [1.50, 7.33]; P=0.0043). CONCLUSIONS: This study found a strong association between the HO-1 variable number tandem repeat polymorphism and cardiovascular disease risk confined to subjects with a high number of repeats on both HO-1 alleles and provides evidence for accelerated atherogenesis and decreased antioxidant defense in this vascular high-risk group.

Signature of circulating microRNAs in osteoarthritis.

BACKGROUND: Osteoarthritis is the most common form of arthritis and a major socioeconomic burden. Our study is the first to explore the association between serum microRNA levels and the development of severe osteoarthritis of the knee and hip joint in the general population. METHODS: We followed 816 Caucasian individuals from 1995 to 2010 and assessed joint arthroplasty as a definitive outcome of severe osteoarthritis of the knee and hip. After a microarray screen, we validated 12 microRNAs by real-time PCR in the entire cohort at baseline. RESULTS: In Cox regression analysis, three microRNAs were associated with severe knee and hip osteoarthritis. let-7e was a negative predictor for total joint arthroplasty with an adjusted HR of 0.75 (95% CI 0.58 to 0.96; p=0.021) when normalised to U6, and 0.76 (95% CI 0.6 to 0.97; p=0.026) after normalisation to the Ct average. miRNA-454 was inversely correlated with severe knee or hip osteoarthritis with an adjusted HR of 0.77 (95% CI 0.61 to 0.97; p=0.028) when normalised to U6. This correlation was lost when data were normalised to Ct average (p=0.118). Finally, miRNA-885-5p showed a trend towards a positive relationship with arthroplasty when normalised to U6 (HR 1.24; 95% CI 0.95 to 1.62; p=0.107) or to Ct average (HR 1.30; 95% CI 0.99 to 1.70; p=0.056). CONCLUSIONS: Our study is the first to identify differentially expressed circulating microRNAs in osteoarthritis patients necessitating arthroplasty in a large, population-based cohort. Among these microRNAs, let-7e emerged as potential predictor for severe knee or hip osteoarthritis.

Carotid atherosclerosis and incident atrial fibrillation.

OBJECTIVE: Atrial fibrillation (AF) and atherosclerotic vascular disease are closely entangled disorders and often coexist. Whether atherosclerosis predisposes to the development of AF has not been fully elucidated. APPROACH AND RESULTS: This study was performed within the framework of the Bruneck Study, a population-based survey with near-complete participation (932 of 1000), long-term follow-up (1990-2010), and thorough assessment of AF. The carotid arteries served as a window to systemic atherosclerosis and were scanned every 5 years. Pooled logistic regression and multistate proportional hazards models were used to identify risk predictors of incident AF and effects of AF on mortality. During follow-up, 118 new cases of AF were detected (incidence per 1000 person-years of 8.1; 95% confidence interval, 6.8-9.6). Individuals with atherosclerosis were more likely to develop AF than individuals without (odds ratio, 1.8; 95% confidence interval, 1.1-3.1; P=0.021). This finding applied to women and men and to both baseline and incident atherosclerosis during follow-up. Subjects with atherosclerosis and AF were significantly more likely to die than those with either condition alone (P=0.0034), and mortality in this group was ≈ 4-fold compared with individuals free of atherosclerosis and AF (hazard ratio, 4.2; 95% confidence interval, 2.6-6.8; P<0.0001). CONCLUSIONS: We found that subjects with carotid atherosclerosis are at high risk of developing AF.

Blockade of receptor activator of nuclear factor-κB (RANKL) signaling improves hepatic insulin resistance and prevents development of diabetes mellitus.

Hepatic insulin resistance is a driving force in the pathogenesis of type 2 diabetes mellitus (T2DM) and is tightly coupled with excessive storage of fat and the ensuing inflammation within the liver. There is compelling evidence that activation of the transcription factor nuclear factor-κB (NF-κB) and downstream inflammatory signaling pathways systemically and in the liver are key events in the etiology of hepatic insulin resistance and ß-cell dysfunction, although the molecular mechanisms involved are incompletely understood. We here test the hypothesis that receptor activator of NF-κB ligand (RANKL), a prototypic activator of NF-κB, contributes to this process using both an epidemiological and experimental approach. In the prospective population-based Bruneck Study, a high serum concentration of soluble RANKL emerged as a significant (P<0.001) and independent risk predictor of T2DM manifestation. In close agreement, systemic or hepatic blockage of RANKL signaling in genetic and nutritional mouse models of T2DM resulted in a marked improvement of hepatic insulin sensitivity and amelioration or even normalization of plasma glucose concentrations and glucose tolerance. Overall, this study provides evidence for a role of RANKL signaling in the pathogenesis of T2DM. If so, translation to the clinic may be feasible given current pharmacological strategies to lower RANKL activity to treat osteoporosis.

Asymmetric and symmetric dimethylarginines are of similar predictive value for cardiovascular risk in the general population.

OBJECTIVES: Asymmetric dimethylarginine (ADMA) has raised considerable interest, as it is an endogenous inhibitor of nitric oxide synthesis. While increased plasma levels of ADMA have been reported in different cardiovascular disease states, its association with symmetric dimethylarginine (SDMA) has not been evaluated in a prospective population-based study. METHODS AND RESULTS: We performed a mass spectrometry-based analysis of ADMA and SDMA in the plasma of 572 participants of the Bruneck study. Levels of ADMA and SDMA were significantly correlated with each other (r=0.189, p<0.001). Age and parameters of renal function, however, showed a stronger influence on SDMA than on ADMA. Both ADMA and SDMA were predictive of cardiovascular disease in multivariate analysis and the associated hazard ratios over the 5-year observation period were of similar strength: 3.86 (1.36-10.9) and 7.91 (1.94-32.3) for ADMA and SDMA, respectively (p=0.011 and 0.004). Separate analyses focused on quintile groups of SDMA revealed that the increase in cardiovascular risk was mainly confined to the top category (>0.80 micromol/L). CONCLUSION: This study argues against an exclusive ADMA effect in mediating cardiovascular risk. Instead, SDMA, its supposedly inactive counterpart, has similar diagnostic value in this large prospective cohort.

Lipoprotein-associated phospholipase A2 activity, ferritin levels, metabolic syndrome, and 10-year cardiovascular and non-cardiovascular mortality: results from the Bruneck study.

AIMS: To identify factors that influence plasma levels and assess the prognostic value of lipoprotein-associated phospholipase A2 (Lp-PLA2) activity in a prospective, population-based survey of the epidemiology and pathogenesis of atherosclerosis. METHODS AND RESULTS: The Bruneck study is a prospective, population-based survey initiated in 1990. Lp-PLA2 activity and baseline variables for the current analysis were measured in 765 subjects aged 45-84 years in 1995. Incident cardiovascular disease (CVD) (cardiovascular death, myocardial infarction, stroke, and transient ischaemic attack) and rates of non-CVD mortality were assessed between 1995 and 2005. Subjects with incident CVD had higher levels of Lp-PLA2 activity (884 +/- 196 vs. 771 +/- 192 micromol/min/L, P < 0.001). Increased Lp-PLA2 activity was significantly related to incident CVD [age- and sex-adjusted hazard ratio (95%CI) 2.9 (1.6-5.5); third vs. first tertile group; P < 0.001] and with vascular mortality but not with non-CVD mortality. Lp-PLA2 activity was enhanced in subjects with the metabolic syndrome and showed highly significant positive associations with LDL-C, apoB-100, ferritin, and HOMA-IR, and inverse associations with HDL-C and anti-oxidant levels. CONCLUSION: Increased Lp-PLA2 activity is associated with metabolic syndrome and incident fatal and non-fatal CVD, but not with non-CVD mortality. Furthermore, Lp-PLA2 activity is strongly influenced by ferritin levels, LDL-C, and apoB-100 supporting its integral role in lipid peroxidation. Clinical utility of Lp-PLA2 activity for prediction of cardiovascular risk has to be explored in future studies.

Soluble receptor activator of nuclear factor-kappa B ligand and risk for cardiovascular disease.

BACKGROUND: Overexpression of receptor activator of nuclear factor-kappaB ligand (RANKL) is a prominent feature of vulnerable atherosclerotic lesions prone to rupture and was thought to contribute to the transition from a stable to an unstable plaque phenotype in both human and murine atherosclerosis because of its ability to promote matrix degradation, monocyte/macrophage chemotaxis, and vascular calcification. METHODS AND RESULTS: The Bruneck Study is a prospective, population-based survey of men and women 40 to 79 years of age at the 1990 baseline examination. Levels of soluble RANKL and other variables were assessed in 909 subjects (1990). All cases of cardiovascular disease were carefully recorded between 1990 and 2005. During follow-up, cardiovascular disease (defined as ischemic stroke and transient ischemic attack, myocardial infarction, and vascular death) manifested in 124 of the 909 subjects. Baseline serum level of RANKL emerged as a highly significant predictor of vascular risk (adjusted hazard ratio per 1-unit increase in soluble RANKL, 1.27; 95% confidence interval, 1.16 to 1.40; P<0.001). Predictive significance was independent of that afforded by the classic vascular risk factors, C-reactive protein, osteoprotegerin concentration, and severity of carotid atherosclerosis. Findings were internally consistent and robust in a variety of sensitivity analyses. Notably, soluble RANKL was not associated with carotid or femoral artery atherosclerosis. CONCLUSIONS: Our study lends large-scale epidemiological support to a role for RANKL in cardiovascular disease. In the absence of a significant association between RANKL and atherosclerosis, the idea that RANKL promotes plaque destabilization and rupture is a highly appealing concept.

Insulin resistance as estimated by homeostasis model assessment predicts incident symptomatic cardiovascular disease in caucasian subjects from the general population: the Bruneck study.

OBJECTIVE: The purpose of this study was to evaluate whether insulin resistance is associated to cardiovascular disease (CVD) and to understand whether this association can be explained by traditional and novel CVD risk factors associated with this metabolic disorder. RESEARCH DESIGN AND METHODS: We examined a sample representative of the population of Bruneck, Italy (n = 919; aged 40-79 years). Insulin-resistant subjects were those with a score in the top quartile of the homeostasis model assessment (HOMA) for insulin resistance (HOMA-IR). Risk factors correlated with insulin resistance included BMI, A1C, HDL cholesterol, triglycerides, blood pressure, high-sensitivity C-reactive protein (hsCRP), fibrinogen, oxidized LDL, vascular cell adhesion molecule-1 (VCAM-1), and adiponectin. Subjects without CVD at baseline were followed up for 15 years for incident CVD, a composite end point including fatal and nonfatal myocardial infarction and stroke, transient ischemic attack, and any revascularization procedure. RESULTS: During follow-up, 118 subjects experienced a first symptomatic CVD event. Levels of HOMA-IR were higher at baseline among subjects who developed CVD (2.8) compared with those remaining free of CVD (2.5) (P < 0.05). Levels of HOMA-IR also were significantly correlated (P < 0.05) with most CVD risk factors we evaluated. In Cox proportional hazard models, insulin-resistant subjects had an age-, sex-, and smoking-adjusted 2.1-fold increased risk (95% CI 1.3-3.1) of incident symptomatic CVD relative to non-insulin-resistant subjects. After sequential adjustment for physical activity and classic risk factors (A1C, LDL cholesterol, and hypertension) as well as BMI, HDL cholesterol, triglycerides, and novel risk factors, including fibrinogen, oxidized LDL, hsCRP, VCAM-1, and adiponectin, the association between HOMA-IR and incident CVD remained significant and virtually unchanged (hazard ratio 2.2 [95% CI 1.4-3.6], P < 0.001). CONCLUSIONS: HOMA-estimated insulin resistance is associated with subsequent symptomatic CVD in the general population independently of all classic and several nontraditional risk factors. These data suggest that insulin resistance may be an important target to reduce CVD risk.

Genetic and acquired inflammatory conditions are synergistically associated with early carotid atherosclerosis.

BACKGROUND AND PURPOSE: If chronic inflammation plays a causal role in atherogenesis, individuals with proinflammatory gene variants would be expected to develop more atherosclerosis. We recently found a synergistic association between 3 functional proinflammatory gene polymorphisms/haplotypes and smoking on carotid intima-media thickness (IMT). We replicated this finding in a second large population and extended the analysis by inclusion of other inflammatory conditions (chronic infection and obesity/abnormal glucose tolerance). METHODS: Common carotid and femoral artery IMT was determined in the Bruneck Study population (n=810). Proinflammatory variants were determined in 3 genes (IL-6 [-174C, -572G, -597A haplotype], IL-1-receptor antagonist [VNTR *2], and endotoxin receptor CD-14 [-159C]). RESULTS: There was a significant relationship between gene-variant score and carotid IMT: age- and sex-adjusted mean IMT in subjects with 0, 1, and >or=2 gene variants was 936, 987 and 1047 microm, respectively (P=0.001), and synergistic effects of gene-variant score and smoking on IMT measurements (P=0.040). Analogous findings were obtained for obesity/abnormal glucose tolerance and chronic infection. Interactive effects of gene-variant score and a risk factor score composed of the acquired inflammatory conditions were highly significant (P<0.001 each). Results were similar for femoral artery IMT. CONCLUSIONS: These results provide support for a causal role of inflammation in carotid atherosclerosis, and emphasize the importance of gene-gene and gene-environment interactions in this pathogenic pathway. This may help to explain the substantial variability of disease expression in subjects with proinflammatory risk factors such as smoking, diabetes and chronic infection.

Long-term treatment of patients with mild and classical phenylketonuria by tetrahydrobiopterin.

Tetrahydrobiopterin (BH4), the natural cofactor of phenylalanine hydroxylase (EC 1.14.16.1), can reduce blood phenylalanine (Phe) in BH4 sensitive patients with hyperphenylalaninemia (McKuisick 261600). We report on the long-term treatment of eight patients with mild and classical phenylketonuria (blood Phe levels maximum blood Phe levels between 771 and 1500 micromol/L) using BH4 at a dosage of 8-12 mg/kg BW per day. In all patients reduction of blood Phe was >30% after BH4 loading test. Three patients were treated from birth by BH4 only, five after initial low Phe dietary treatment. Seven of them continue to be on BH4 treatment only, one has a relaxed low protein diet. No side effects could be observed (longest observation time 5 years), somatic and psychomotor development were normal. The main problem of BH4 treatment is finding an optimal dosage at different ages and an under special conditions like infectious diseases. There is evidence that in some patients BH4 treatment may allow a more relaxed low protein diet showing positive effects on weight gain and quality of life. Further controlled studies are necessary not only to rule out any side effects but also for optimizing treatment strategies with BH4 treatment in mild phenylketonuria.

Population-based incidence rates and risk factors for type 2 diabetes in white individuals: the Bruneck study.

Incidence rates and risk factors for type 2 diabetes in low-risk populations are not well documented. We investigated these in white individuals who were aged 40-79 years and from the population of Bruneck, Italy. Of an age- and sex-stratified random sample of 1,000 individuals who were identified in 1990, 919 underwent an oral glucose tolerance test (OGTT) and an assessment of physiological risk factors for diabetes, including insulin resistance (homeostasis model assessment, HOMA-IR), and postchallenge insulin response (Sluiter's Index). Diabetes at baseline by fasting or 2-h OGTT plasma glucose (World Health Organization criteria, n = 82) was excluded, leaving 837 individuals who were followed for 10 years. Incident cases of diabetes were ascertained by confirmed diabetes treatment or a fasting glucose >or=7.0 mmol/l. At follow-up, 64 individuals had developed diabetes, corresponding to a population-standardized incidence rate of 7.6 per 1,000 person-years. Sex- and age-adjusted incidence rates were elevated 11-fold in individuals with impaired fasting glucose at baseline, 4-fold in those with impaired glucose tolerance, 3-fold in overweight individuals, 10-fold in obese individuals, and approximately 2-fold in individuals with dyslipidemia or hypertension. Incidence rates increased with increasing HOMA-IR and decreasing Sluiter's Index. As compared with normal insulin sensitivity and normal insulin response, individuals with low insulin sensitivity and low insulin response had a sevenfold higher risk of diabetes. Baseline impaired fasting glucose, BMI, HOMA-IR, and Sluiter's Index were the only independent predictors of incident diabetes in multivariate analyses. We conclude that approximately 1% of European white individuals aged 40-79 years develop type 2 diabetes annually and that "subdiabetic" hyperglycemia, obesity, insulin resistance, and impaired insulin response to glucose are independent predictors of diabetes.

Soluble RANKL and risk of nontraumatic fracture.

CONTEXT: The receptor activator of nuclear factor kappaB ligand (RANKL) is essential for osteoclast and, possibly, osteoblast activation and may represent a key link between bone formation and resorption. OBJECTIVE: To determine the relationship between serum level of RANKL and the risk of nontraumatic fracture. DESIGN, SETTING, AND PARTICIPANTS: As part of a prospective population-based study conducted in Bruneck, Italy, we recorded all fractures that occurred between 1990 and 2000 in 906 participants and classified them as traumatic (n = 115) or nontraumatic (n = 31). Serum levels of RANKL and osteoprotegerin and characteristics of bone metabolism and lifestyle were assessed in 1990 and at follow-up in 1995 and 2000. MAIN OUTCOME MEASURE: Incident nontraumatic fracture by levels of RANKL. RESULTS: Levels of RANKL did not differ between sexes and were not related to age, menopausal status, lifestyle characteristics, or data from bone ultrasound at the heel. However, RANKL emerged as a significant predictor of nontraumatic fracture. In pooled logistic regression analysis, the relative risks of nontraumatic fracture in the lowest and middle vs highest tertile for RANKL were 10.0 (95% confidence interval [CI], 2.3-43.1) and 3.9 (95% CI, 0.8-19.0) (P<.001 for trend), respectively. Patients in the highest-tertile group had a low risk of fracture even in the presence of other predisposing factors, whereas women aged 60 years or older in the lowest tertile had a 5-year rate of nontraumatic fracture greater than 7%. CONCLUSIONS: A low level of RANKL is an independent predictor of nontraumatic fracture. This finding is consistent with the hypothesis of an important role of RANKL in human bone turnover and if confirmed in future investigations may gain relevance for assessment of fracture risk.