Not performing an OGTT results in significant underdiagnosis of (pre)diabetes in a high risk adult Caucasian population.

OBJECTIVE: Type 2 diabetes (T2DM) is known to be underdiagnosed. Tests for diagnosis include fasting plasma glucose (FPG), oral glucose tolerance test (OGTT) and HbA1c. HbA1c can be tested in non-fasting conditions. Therefore, general practitioners almost no longer execute OGTT's. We evaluated the performance of OGTT versus HbA1c in a population consisting of overweight and obese subjects, which can be considered a 'high risk' population. RESEARCH DESIGN AND METHODS: A total of, 1241 overweight and obese subjects without a history of diabetes (male/female: 375/866, age 44±13 years, body mass index 38.0±6.1 kg m-2) were tested for glucose tolerance status using FPG, OGTT and HbA1c. RESULTS: Exactly, 46.8% were found to have prediabetes and 11.9% were newly diagnosed with T2DM (male/female=18.9/8.9%) using ADA criteria. Testing only HbA1c would have resulted in 78 subjects being diagnosed with T2DM, but 47.3% of newly diagnosed patients would have been missed if OGTT would not have been done. Exactly 581 subjects were diagnosed with prediabetes, 1.4% subjects had impaired fasting glucose (IFG) 30.5% had impaired glucose tolerance (IGT), 5.1% subjects had a combined IFG+IGT, and 9.8% had an isolated elevated HbA1c (5.7-6.4%). Of the 581 subjects with prediabetes, 257 had an HbA1c <5.7%. Therefore, 44.2% subjects would have been missed when OGTT would not have been done. CONCLUSION: In a population with only overweight and obese adult subjects, 46.8% were diagnosed with prediabetes and 11.9% were newly diagnosed with diabetes. Exactly, 5.6 and 20.7% of total population met the diagnostic criteria of the OGTT for diabetes and prediabetes, respectively, but did not meet the diagnostic criteria of the HbA1c. These data suggest that not performing an OGTT results in significant underdiagnose of T2DM in an overweight and obese adult population.

Screening for type 2 diabetes mellitus in overweight and obese subjects made easy by the FINDRISC score.

AIM: To evaluate the use of the FINDRISC score in an overweight and obese population to predict glucose status. METHODS: In 651 overweight/obese subjects (M/F: 193/458, age 43±13 y, BMI 38.2±6.1kg/m(2)) glucose status was tested using OGTT and HbA1c. Furthermore, the FINDRISC questionnaire and CT visceral fat (VAT) and subcutaneous fat (SAT) were examined. RESULTS: Exactly 50.4% were found to have prediabetes and 11.1% were newly diagnosed with type 2 diabetes (T2DM) (M/F=22.2/8.8%). Subjects without T2DM had a FINDRISC score of 11±3, those with pre-DM 13±4, and subjects with de novo T2DM 15±5. The aROC of the FINDRISC for detecting T2DM was 0.76 (95% CI 0.72-0.82), with 13 as cutoff point. The FINDRISC score correlated with VAT (r=0.34, p<0.001) and VAT/SAT ratio (r=0.39, p<0.001). The aROC of the FINDRISC to detect excess VAT was 0.79 (95%CI 0.72-0.84). CONCLUSIONS: In a large group of overweight and obese subjects, 50.4% were found to have pre-DM and 11.1% were newly diagnosed with T2DM. The FINDRISC score increased with worsening of glucose tolerance status and proved to be an independent predictor of T2DM status, as did HOMA-B, HOMA-S and VAT. The FINDRISC can also function as a good tool to predict visceral obesity.

No conclusive evidence for association of polymorphisms in the adiponectin receptor 1 gene, AdipoR1, with common obesity.

AdipoR1 is one of the adiponectin receptors which are important for adiponectin signaling. Because adiponectin is a candidate gene for common obesity, it is also hypothesized that variations in AdipoR1 may be involved in the development of complex obesity. Therefore, we designed an association study for the AdipoR1 gene. We performed a case-control association study including 1,021 obese subjects (mean age 42 ± 12 years; mean BMI 38.2 ± 6.2 kg/m²) and 226 lean, healthy individuals (mean age 36 ± 7 years; mean BMI 22.1 ± 1.7 kg/m²). Nine tagSNPs were selected to cover the entire AdipoR1 gene and surrounding 7 kb region (based on HapMap data). TagSNPs were genotyped using AcycloPrime-Fluorescence Polarization (FP) SNP Detection kits and TaqMan Pre-Designed SNP Genotyping assays according to manufacturer's protocols. We found that the rs1075399 non-reference allele decreases obesity risk by 45 % in men only [odds ratio (OR) = 0.55, 95 % CI 0.35-0.87, nominal P = 0.010]. However, after Bonferroni correction for multiple testing, this association is lost. None of the other tagSNPs were associated with obesity when studying the entire population, nor when looking at men and women separately. Quantitative analysis of the effect of each SNP on height, weight, and BMI revealed that none of the tagSNPs are associated with weight or BMI. We report here that we found no decisive evidence for association between AdipoR1 tagSNPs and complex obesity in our Belgian Caucasian population.

Visceral adiposity and insulin resistance are independent predictors of the presence of non-cirrhotic NAFLD-related portal hypertension.

INTRODUCTION: We previously demonstrated in an animal model that steatosis, in the absence of fibrosis, induces a significant rise in portal pressure, indicating substantial changes in liver hemodynamics. As assessment of portal pressure is an invasive procedure, non-invasive parameters are needed to identify patients at risk. AIMS: To study the portal pressure in nonalcoholic fatty liver disease patients and to identify factors that are possibly related to steatosis-induced changes in liver hemodynamics. MATERIALS AND METHODS: Patients presenting with a problem of overweight or obesity, and in whom non-invasive investigations showed signs of liver involvement, were proposed for transjugular hepatic vein catheterization and liver biopsy. The biopsy was scored according to the Nonalcoholic Steatohepatitis Clinical Research Network Scoring System. RESULTS: A total of 50 consecutive patients were studied. Their mean age was 47.9 ± 1.8 years; 31 (62%) were female. Hepatic venous pressure gradient was normal in 36 (72%) and elevated in 14 (28%) patients. The degree of steatosis was the only histological parameter that differed significantly between the two groups (P=0.016), and was a predictor of the presence of portal hypertension (PHT) in regression analysis (P=0.010). Comparing normal versus portal hypertensive patients, waist circumference (117 ± 2 versus 128 ± 4 cm, P=0.005), waist-hip ratio (0.96 ± 0.06 versus 1.04 ± 0.03, P=0.003), visceral fat (229 ± 15 versus 292 ± 35 cm(2), P=0.022), fasting insulin (15.4 ± 1.7 versus 21.8 ± 2.4 µU ml(-1), P=0.032), fasting c-peptide (1.22 ± 0.06 versus 1.49 ± 0.09 nmol l(-1), P=0.035) and homeostasis model assessment-insulin resistance (HOMA IR) (3.28 ± 0.29 versus 4.81 ± 0.57, P=0.019) were significantly higher. Age, gender, liver enzymes, ferritin and high-sensitive C-reactive protein were not significantly different. In regression analysis, waist circumference (P=0.008) and HOMA IR (P=0.043) were independent predictors of PHT. CONCLUSIONS: Estimates of both visceral adiposity and IR are predictors for the presence of PHT, related to the degree of steatosis, and may help in identifying patients who are at risk of developing steatosis-related complications.

Visceral adipose tissue and inflammation correlate with elevated liver tests in a cohort of overweight and obese patients.

OBJECTIVE: To study the relationship between elevated liver tests and high sensitive C-reactive protein (hs-CRP), as potential markers of liver inflammation and non-alcoholic steatohepatitis (NASH), with anthropometric and laboratory parameters in overweight patients, especially the relationship with visceral adipose tissue (VAT). METHODS: Patients presenting to the obesity clinic were prospectively included. Detailed anthropometry, computed tomography (CT)-measured VAT, liver tests (aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT)) and hs-CRP were assessed, along with an extended series of biochemical parameters. RESULTS: All 480 patients (gender distribution male (M)/female (F) (10/90%)) with complete data were included. Mean age was 39+/-13 years, mean BMI 34.5+/-6.0 kg m(-2). In 37.3% of the patients one or more of the liver tests were elevated. VAT was positively related to AST (r=0.18, P<0.001), ALT (r=0.29, P<0.001), ALP (r=0.16, P<0.01) and GGT (r=0.39, P<0.001). Comparing subjects with high (VAT>or=113 cm(2)) vs low (VAT<113 cm(2)) VAT levels, significant differences were noted for AST (26+/-12 vs 24+/-12 U l(-1), P=0.003), ALT (37+/-21 vs 31+/-21 U l(-1), P<0.001), ALP (76+/-20 vs 71+/-18 U l(-1), P=0.008), GGT (33+/-20 vs 25+/-15 U l(-1), P<0.001) and hs-CRP (0.62+/-0.43 vs 0.52+/-0.48 mg dl(-1), P<0.001). After correction for BMI the difference in AST and ALP between the high vs low VAT group disappeared. The differences for ALT and GGT remained significant (P=0.008 and P<0.001 respectively). After correction for hs-CRP the four different liver tests remained significantly higher in the high VAT group. A stepwise multiple regression analysis revealed that every single liver test has his own most important determinant; VAT and hs-CRP for AST, insulin resistance calculated with homeostasis model assessment (HOMA-IR) and hs-CRP for ALT and ALP, and triglycerides and VAT for GGT. CONCLUSION: In overweight and obese patients, liver tests, especially ALT and GGT, are associated with visceral fat mass. After correction for BMI and hs-CRP, ALT and GGT are significantly higher in patients with increased VAT, thereby supporting evidence for a potential key role of VAT in the pathogenesis of non-alcoholic fatty liver disease (NAFLD).

Is there an increased incidence of surgically removed thyroid carcinoma in Belgium ten years after Chernobyl? A study of hospital discharge data.

In order to provide some answers to the much debated subject of the consequences of the Chernobyl accident, this study attempts to measure the incidence of surgically removed thyroid cancers in Belgium ten years following the explosion. The analysis was made from the hospital discharge data between 1993 and 1998. It offers the advantage of national coverage in spite of certain validity limits. The results show an increase in surgically removed thyroid cancers, which is not, however, evident in the more susceptible younger generation who were involved at the time of the accident. Furthermore, the geographic distribution of the incidence is more marked in the south of the country, unaffected by the radioactive iodine contamination of 1986, which was more prevalent in the east of the country. The study of the type of surgery involved shows a rise in the proportion of total thyroidectomies. These findings are in favour of the hypothesis of a causal effect linking the increased incidence of thyroid cancers to medical practice and surgery in particular and not to the consequence of the possible contamination.

Expression of inhibitor of apoptosis protein Livin in renal cell carcinoma and non-tumorous adult kidney.

The antiapoptotic Livin/ML-IAP gene has recently gained much attention as a potential new target for cancer therapy. Reports indicating that livin is expressed almost exclusively in tumours, but not in the corresponding normal tissue, suggested that the targeted inhibition of livin may present a novel tumour-specific therapeutic strategy. Here, we compared the expression of livin in renal cell carcinoma and in non-tumorous adult kidney tissue by quantitative real-time reverse transcription-PCR, immunoblotting, and immunohistochemistry. We found that livin expression was significantly increased in tumours (P=0.0077), but was also clearly detectable in non-tumorous adult kidney. Transcripts encoding Livin isoforms alpha and beta were found in both renal cell carcinoma and normal tissue, without obvious qualitative differences. Livin protein in renal cell carcinoma samples exhibited cytoplasmic and/or nuclear staining. In non-tumorous kidney tissue, Livin protein expression was only detectable in specific cell types and restricted to the cytoplasm. Thus, whereas the relative overexpression of livin in renal cell carcinoma indicates that it may still represent a therapeutic target to increase the apoptotic sensitivity of kidney cancer cells, this strategy is likely to be not tumour-specific.

Targeted inhibition of Livin resensitizes renal cancer cells towards apoptosis.

Cancer cells are typically characterized by apoptosis deficiency. In order to investigate a possible role for the anti-apoptotic livin gene in renal cell cancer (RCC), we analyzed its expression in tumor tissue samples and in RCC-derived cell lines. In addition, we studied the contribution of livin to the apoptotic resistance of RCC cells by RNA interference (RNAi). Livin gene expression was detected in a significant portion of RCC tumor tissue specimens (13/14, 92.9%) and tumor-derived cell lines (12/15, 80.0%). Moreover, targeted inhibition of livin by RNAi markedly sensitized RCC cells towards proapoptotic stimuli, such as UV irradiation or the chemotherapeutic drugs etoposide, 5-fluorouracil, and vinblastine. These effects were specific for livin expressing tumor cells. We conclude that livin can contribute significantly to the apoptosis resistance of RCC cells. Targeted inhibition of livin could represent a novel therapeutic strategy to increase the sensitivity of renal cancers towards pro-apoptotic agents.

Among inflammation and coagulation markers, PAI-1 is a true component of the metabolic syndrome.

OBJECTIVE: To investigate whether leukocyte count, fibrinogen, von Willebrand factor (vWF) and plasminogen activator inhibitor-1 activity (PAI-1) are increased in subjects with the metabolic syndrome as defined by the National Cholesterol Education Program-Adult Treatment Panel III (NCEP-ATPIII) and the World Health Organisation (WHO). DESIGN: Cross-sectional study. SUBJECTS: A total of 520 overweight and obese subjects: 379 women and 141 men, visiting the weight management clinic of a University Hospital. SUBJECTS AND MEASUREMENTS: Waist circumference, triglycerides, HDL cholesterol, blood pressure and fasting glucose were determined, and the presence or absence of the metabolic syndrome according to the NCEP-ATPIII criteria was assessed. In 349 subjects, data on the waist-to-hip ratio (WHR) and albumin excretion rate were available and the WHO criteria were applied. Insulin resistance was defined using the HOMA-IR index. RESULTS: Subjects with the metabolic syndrome according to the NCEP-ATPIII criteria had significantly higher levels of leukocyte count (P < 0.001) and PAI-1 (P < 0.001), while no significant differences were found for fibrinogen or vWF (P > 0.05). Using the WHO criteria, similar results were found except for vWF, where higher levels were found in subjects with the metabolic syndrome. When subjects were classified according to the number of components of the metabolic syndrome, levels of leukocyte count, vWF and PAI-1 activity were significantly different (P < 0.05). In logistic regression analysis PAI-1, gender and leukocyte count were independent determinants of the metabolic syndrome (P < 0.001). CONCLUSION: Evidence for being a true component of the metabolic syndrome is strong for PAI-1, less for leukocyte count and weak for vWF and fibrinogen.

Screening for melanocortin-4 receptor mutations in a cohort of Belgian morbidly obese adults and children.

OBJECTIVE: To investigate whether pathogenic melanocortin-4 receptor (MC4R) mutations are a common cause of obesity in Belgium. DESIGN: Cross-sectional mutation analysis. SUBJECTS: In total, 95 morbidly obese adults (mean age 44.02+/-11.35 years; mean BMI 47.87+/-4.17 kg/m2 and 123 obese children and adolescents were screened for mutations in MC4R (mean age 16.56+/-2.58 years; BMI>95th percentile for age and sex; mean % overweight 170.86 +/- 23.63). MEASUREMENTS: A series of anthropometric (e.g. weight, height, waist, hip), biochemical and clinical measurements were performed on all subjects. The entire coding region of MC4R was screened using DHPLC, a highly sensitive and specific method for mutation analysis. Direct sequencing was performed when the chromatogram deviated from the WT pattern. RESULTS: Mutation screening of a cohort of Belgian obese adults and children did not detect any pathogenic mutations as only the previously described polymorphisms Val103Ile, Thr112Met and Ile251Leu were detected. CONCLUSION: Pathogenic mutations in MC4R are not a common cause of obesity in a Belgian population of obese adults, children and adolescents.

Characterization of an RFamide-related peptide orphan GPCR in C. elegans.

We cloned and characterized an orphan FMRFamide-related peptide (FaRP) GPCR in Caenorhabditis elegans. We synthesized numerous structurally different FaRPs that were found in the C. elegans genome by bioinformatic analysis and used them to screen cells expressing the C26F1.6 receptor. Two peptides ending in M(orL)VRFamide elicited a calcium response in receptor-expressing mammalian Chinese hamster ovary cells. The response was dose-dependent and appeared to be very specific; that is, none of the other FaRPs were active, not even closely related peptides also ending in M(orL)VRFamide, which are encoded by the same peptide precursor. Pharmacological profiling with a truncated series of the most active peptide revealed that the full peptide sequence is necessary for receptor activation.

Obesity, haemostasis and the fibrinolytic system.

Obese patients are at risk for the development of cardiovascular diseases, which can in part be explained by disturbances in the haemostatic and fibrinolytic systems. Indeed, obese subjects tend to have higher values of fibrinogen, factor VII, factor VIII, von Willebrand factor and plasminogen activator inhibitor compared to non-obese subjects. Abdominal obesity, in particular, has been shown to be associated with disturbances in fibrinogen, factor VIII and von Willebrand factor, while less consistent results have been found for factor VII. Recently it has been demonstrated that the adipocyte itself is able to produce plasminogen activator inhibitor-1, possibly explaining the high levels found in obesity. Different studies have investigated the association between haemostatic and fibrinolytic parameters and the insulin resistance syndrome, often present in obese subjects. Fibrinogen has been found to be related to insulin, but it has been suggested that this relationship is not independent of the accompanying inflammatory reaction. Results from studies on the relationship between insulin resistance and factor VII, factor VIII and von Willebrand factor levels are inconsistent. In contrast, plasminogen activator inhibitor-1 has been found to correlate with all components of the insulin resistance syndrome, and can be considered as a true component of this metabolic syndrome. Weight loss seems to have a beneficial effect on factor VII--probably mediated through a reduction in triglycerides. Data on factor VIII and von Willebrand factor are scarce but weight loss does not seem to have an effect. Fibrinogen does not seem to be reduced by modest weight loss and a more substantial weight loss seems necessary to lower fibrinogen levels. In contrast, both modest and substantial weight loss have been found to significantly reduce plasminogen activator inhibitor-1 levels. In conclusion, the increased cardiovascular risk observed in obesity could in part be explained by the association between insulin resistance and components of the fibrinolytic and haemostatic systems. Whether this relationship is truly causal or indirect needs to be elucidated further.

Quantitative baseline renography 48 hours after renal transplantation predicts long-term graft survival.

Technetium-99m mercaptoacetylglycine (99mTc-MAG3) renography is a successful non-invasive method for the evaluation of renal transplants. In this study we prospectively studied 256 patients after renal transplantation, using the MAG3 uptake capacity in the first 10 min (MUC10), which is an index of renal function representing kidney activity as a fraction of the injected dose. Renal scintigraphy was performed in all patients within 48 h of transplantation. Renograms were obtained over 20 min, after bolus injection of +/-100 MBq 99mTc-MAG3. MUC10 ranged between 0.0 and 94.9. The patients were divided into five groups: group 1, MUC10 <1; group 2, 1< or =MUC10 < 5; group 3, 5 < or = MUC10 <10; group 4, 10 < or = MUC10 < 30; group 5, MUC10 > or =30. In 235 patients, follow-up for 5 years was completed. Considering all the renal transplants, 30% of the grafts ceased functioning within the first 5 years. Six grafts showed a MUC10 of < 1, and none survived the first year. After 5 years, 43% of the grafts with a MUC10 of 1-5 still functioned, as compared with 63% of those with a MUC10 of 5-10, 72% of those with a MUC10 of 10-30 and 83% of those with a MUC10 of > or =30. Kaplan-Meier survival analysis showed significant differences between the five different patient groups based on MUC10 in terms of the percentage of kidneys that ceased to function within 5 years. The period of cold ischaemia and the donor age were known in 178 patients. In contrast to cold ischaemia time, the MUC10 and the donor age showed a significant effect (P<0.05) on graft survival. This study indicates that the MUC10 early after renal transplantation is useful as a prognostic factor in the evaluation of kidney transplants and that a MUC10 of <1 indicates a dismal prognosis for the renal transplant.

Peptide aptamers targeting the hepatitis B virus core protein: a new class of molecules with antiviral activity.

A substantial proportion of the worldwide liver cancer incidence is associated with chronic hepatitis B virus (HBV) infection. The therapeutic management of HBV infections is still problematic and novel antiviral strategies are urgently required. Using the peptide aptamer screening system, we aimed to isolate new molecules, which can block viral replication by interfering with capsid formation. Eight peptide aptamers were isolated from a randomized expression library, which specifically bound to the HBV core protein under intracellular conditions. One of them, named C1-1, efficiently inhibited viral capsid formation and, consequently, HBV replication and virion production. Hence, C1-1 is a novel model compound for inhibiting HBV replication by blocking capsid formation and provides a new basis for the development of therapeutic molecules with specific antiviral potential against HBV infections.

Visceral fat is a determinant of PAI-1 activity in diabetic and non-diabetic overweight and obese women.

Plasminogen activator inhibitor type 1 (PAI-1), an inhibitor of fibrinolysis and an important and independent cardiovascular risk factor, has been shown to be elevated in obesity and type 2 diabetes. Recent study results have suggested that adipose tissue--visceral fat in particular--could play an important role in the fibrinolytic process.In order to assess the specific role of this fat distribution, we measured PAI-1 activity (AU/ml) and visceral fat (CT-scan at level L4-L5) in 2 groups of 30 overweight and obese diabetic and overweight and obese non-diabetic women. Subjects were matched for age, weight, body mass index, fat mass and total abdominal fat. Visceral adipose tissue and PAI-1 were significantly higher in diabetic women (p = 0.022 and p = 0.004 respectively) than in non-diabetic patients. Visceral fat correlated significantly with PAI-1 activity, even after correction for insulin and triglycerides (r = 0.28, p = 0.034). Stepwise regression analysis showed visceral fat as the most important determinant factor for PAI-1 in the whole group and in the non-diabetic group. In the diabetic group, fasting insulin was the most important determinant. These results show that visceral fat is more important than BMI or total body fat in the determination of PAI-1 levels. Furthermore, the increased amount of visceral fat in type 2 diabetics may contribute to the increase of PAI-1 activity levels and the subsequent increased risk for thrombovascular disease, regardless of BMI and total fatness.

Peptide aptamers: new tools to study protein interactions.

The ability to specifically interfere with the function of proteins of pathological significance has been a goal for molecular medicine for many years. Peptide aptamers comprise a new class of molecules, with a peptide moiety of randomized sequence, which are selected for their ability to bind to a given target protein under intracellular conditions. They have the potential to inhibit the biochemical activities of a target protein, can delineate the interactions of the target protein in regulatory networks, and identify novel therapeutic targets. Peptide aptamers represent a new basis for drug design and protein therapy, with implications for basic and applied research, for a broad variety of different types of diseases.

Captopril renography and the relevance of abnormal but bilateral identical curves in the diagnosis of renal artery stenosis.

BACKGROUND: Captopril renography (CR) has been shown to improve the effectiveness of renal scintigraphy in renovascular hypertension, by inhibiting angiotensin-converting enzyme. CR is particularly sensitive and specific in unilateral renal artery stenosis (RAS), but results in patients with bilateral RAS are less favourable. The aim of this study was to investigate the meaning of abnormal but identical renographic curves in the diagnosis of RAS. PATIENTS AND METHODS: One hundred and fifty-eight patients clinically suspected for renovascular hypertension underwent CR, using 50 MBq (99)Tc(m)-mercaptoacetyltriglycine ((99)Tc(m)-MAG(3)), prior to performing renal angiography. CR was performed 1 h after captopril administration. Renograms were analysed according to the consensus criteria. All patients underwent angiography, considered as the "gold standard" in the detection of the presence of RAS (stenosis >50% was defined as significant). All kidneys were categorized into three groups, scintigraphically as well as angiographically: no stenosis, unilateral stenosis and bilateral stenosis. RESULTS: Out of 158 patients 100 (63%) showed a RAS on angiography (58 (37%) unilateral, 42 (26%) bilateral). The sensitivity and specificity of CR evaluated by patient was 83% and 75%, respectively. Thirty patients with completely identical curves were identified, 21 patients with normal curves and nine patients with abnormal identical curves. All but one patient showed no RAS on the angiogram. In this single patient a unilateral stenosis was found. CONCLUSION: Identical curves on the renogram generally suggest no RAS and are probably due to intrinsic parenchymal disease.

Preparation and certification of a reference material on PCBs in pig fat and its application in quality control in monitoring laboratories during the Belgian "PCB-crisis".

In this article, the production and validation of a new certified reference material "PCBs in animal fat" for the control of the maximum level of 200 ng/g setup by the European Communities for veterinary products from Belgium is described. Three materials are established: a blank, one material with about 100 ng/g and one with about 200 ng/g (sum of seven PCBs). Data on the production and certification are given. Additionally, this material was used as an unknown test material in the quality assurance program of the Belgium meat monitoring system (before the certification of the material). While the certification was performed with an uncertainty of less than 10%, the round robin exhibited larger deviations. However, these deviations were less than 20% for most of the 30 participating laboratories. Only two had significantly higher deviations.