Mathematical estimation of the potential effect of vascular remodelling/dilatation on B-mode ultrasound intima-medial thickness.

BACKGROUND: Arterial diameter changes are known to impact wall thickness, but the clinical relevance of the changes is unclear. AIM: To use known mathematical relationships to estimate anticipated changes in arterial wall thicknesses occurring with enlargement of atherosclerotic regions. DESIGN: Mathematical relationships between a cylinder's diameter and its wall thickness were used to calculate the theoretical effect of diameter enlargement on the thickness of an atherosclerotic wall. METHODS: Equating the wall areas of two cylinders, one of smaller diameter than the other, allowed estimation of the degree of thickening that would be needed to maintain intima-medial thickness (IMT) after arterial remodelling. The difference in cylinder diameters was based on arterial diameter enlargement reported with atherosclerosis progression. Thus, the calculated wall changes estimate arterial changes which could go undetected if only IMT is measured by ultrasound. RESULTS: The expected IMT change for diameter enlargement is not a linear function of the diameter change, but varies depending upon initial size (diameter and IMT). Thus a 0.6 mm arterial diameter enlargement would be expected to cause a 0.039-0.235 mm change in IMT, depending on artery size. The estimated IMT change is similar to that associated with major atherosclerotic risk factors. DISCUSSION: The level of vascular remodelling reported with atherosclerosis could have a measurable impact on IMT, suggesting that indicators incorporating both diameter and IMT may be better disease indicators than IMT alone. Arterial diameters, as well as IMT, should be obtained in ultrasound studies of atherosclerosis.

Faecal tumour M2 pyruvate kinase: a new, sensitive screening tool for colorectal cancer.

Proliferating cells, especially tumour cells, express a special isoenzyme of pyruvate kinase, termed M2-PK, which can occur in a tetrameric form with a high affinity to its substrate, phosphoenolpyruvate (PEP), and in a dimeric form with a low PEP affinity. In tumour cells, the dimeric form is usually predominant and is therefore termed Tumour M2-PK. The levels of Tumour M2-PK within tumours and in EDTA-plasma correlate with staging and the ability of the tumour cells to metastasise. Since most colorectal tumours grow intraluminally, it appeared interesting to determine whether Tumour M2-PK is detectable in the faeces of tumour patients. Stool samples were tested by ELISA from controls without colorectal cancer and colorectal cancer patients. Whereas Tumour M2-PK levels were low in the control group (mean value+/-s.e.m.: 3.3+/-0.4, n=144), they were high in the case of colorectal cancer (56.1+/-15.3, n=60). At a cutoff value of 4 U ml(-1), the sensitivity was 73%. TNM and Dukes' classification of the tumours revealed a strong correlation between faecal Tumour M2-PK levels and staging. The determination of Tumour M2-PK in faeces provides a new promising screening tool for colorectal tumours.

Fuzzy logic-based tumor marker profiles improved sensitivity of the detection of progression in small-cell lung cancer patients.

Tumor markers were used for disease monitoring in small-cell lung cancer patients. The aim of this study was to improve diagnostic efficiency in the detection of tumor progression in small-cell lung cancer patients by using fuzzy logic modeling in combination with a tumor marker panel (NSE, ProGRP, Tumor M2-PK, CYFRA 21-1, and CEA). Thirty-three consecutive small-cell lung cancer patients were included in a prospective study. The changes in blood levels of tumor markers and their analysis by fuzzy logic modeling were compared with the clinical evaluation of response versus non-response to therapy. Clinical monitoring was performed according to the standard criteria of the WHO. Tumor M2-PK was measured in plasma with an ELISA, all other markers were measured in sera. At 90% specificity, clinically detected tumor progression was found by the best single marker, NSE, in 32% of all cases. A fuzzy logic rule-based system employing a tumor marker panel increased the sensitivity significantly (P>0.0001) in small-cell carcinomas to 67% with the threemarker combination NSE/ProGRP/Tumor M2-PK and to 56% with the best two-marker combination ProGRP/Tumor M2-PK, respectively. An improvement of sensitivity was also observed using the two-marker combination of ProGRP/NSE (sensitivity 49%) or NSE/Tumor M2-PK (sensitivity 52%). The fuzzy classifier was able to detect a higher rate of progression in small-cell lung cancer patients compared with the multiple logistic regression analysis using the marker combination NSE/ProGRP/Tumor M2-PK (sensitivity 44%; AUC=0.76). With the fuzzy logic method and different tumor marker panels (NSE, ProGRP and Tumor M2-PK), a new diagnostic tool for the detection of progression in patients with small-cell lung cancer is available.

Pyruvate kinase type M2: a crossroad in the tumor metabolome.

Cell proliferation is a process that consumes large amounts of energy. A reduction in the nutrient supply can lead to cell death by ATP depletion, if cell proliferation is not limited. A key sensor for this regulation is the glycolytic enzyme pyruvate kinase, which determines whether glucose carbons are channelled to synthetic processes or used for glycolytic energy production. In unicellular organisms pyruvate kinase is regulated by ATP, ADP and AMP, by ribose 5-P, the precursor of the nucleic acid synthesis, and by the glycolytic intermediate fructose 1,6-P2 (FBP), thereby adapting cell proliferation to nutrient supply. The mammalian pyruvate kinase isoenzyme type M2 (M2-PK) displays the same kinetic properties as the pyruvate kinase enzyme from unicellular organisms. The mammalian M2-PK isoenzyme can switch between a less active dimeric form and a highly active tetrameric form which regulates the channeling of glucose carbons either to synthetic processes (dimeric form) or to glycolytic energy production (tetrameric form). Tumor cells are usually characterized by a high amount of the dimeric form leading to a strong accumulation of all glycolytic phosphometabolites above pyruvate kinase. The tetramer-dimer ratio is regulated by ATP, FBP and serine and by direct interactions with different oncoproteins (pp60v-src, HPV-16 E7). In solid tumors with sufficient oxygen supply pyruvate is supplied by glutaminolysis. Pyruvate produced in glycolysis and glutaminolysis is used for the synthesis of lactate, glutamate and fatty acids thereby releasing the hydrogen produced in the glycolytic glyceraldehyde 3-phosphate dehydrogenase reaction.

Regulatory potential of n-3 fatty acids in immunological and inflammatory processes.

Over the last few years immunonutrition has gained increasing importance. Among other compounds lipids, especially n-3 polyunsaturated fatty acids, were shown to influence the immune response. The anti-inflammatory effects they exert can be induced by free fatty acids, triglyceride fatty acids, after incorporation into the membrane phopspholipid bilayer or following metabolism to eicosanoids. n-3 Fatty acids influence inflammatory cell activation processes from signal transduction to protein expression even involving effects at the genomic level. n-3 Fatty acid-mediated mechanisms decreased cytokine-induced adhesion molecule expression, thereby reducing inflammatory leucocyte-endothelium interactions and modified lipid mediator synthesis, thus influencing the transendothelial migration of leucocytes and leucocyte trafficking in general. Even the metabolic repertoire of specific immunocompetent cells such as cytokine release or proliferation is modified by n-3 fatty acids. Beyond this they regulate lipid homeostasis shifting the metabolic pathways towards energy supply thus optimizing the function of immune cells. Due to the regulatory impact on different processes of inflammatory and immune cell activation n-3 fatty acids provide positive effects on various states of immune deficiencies and diseases with a hyperinflammatory character, among which selected examples are presented.

[Brain emboli in the lungs of cattle]. / ZNS-Emboli in der Rinderlunge.

There is no information whether the BSE agent is introduced into the human food chain through contamination of the lungs of cattle with central nervous system tissue (CNS). Studies in the United Kingdom and in the USA showed that CNS tissue could contaminate the lungs after using pneumatic powered air injection stunners (e.g. "The Knocker") or after pithing. Thus, pithing was forbidden in the European Union since January 2001. In German abattoirs conventional cartridge-fired stunners (e.g. model by Schermer) are usually applied. Pithing was used up to December 2000 in approx. 75% of the German abattoirs. In the present study 323 lungs of cattle were analysed for CNS. The lungs were derived from cattle exclusive stunned by use of the knocker from Schermer. 60% of the lungs contained emboli which were tested with immuno chemistry as well as immuno histochemistry to detect CNS. Two of 108 pooled samples showed a faint immuno reaction in the anti-NSE and anti-GFAP immunoblot. Further two particles showed a faint reaction for NSE and GFAP in immuno histochemistry, thus suggesting the presence of CNS. Even though CNS tissue could not be shown in the histological investigation, we used our findings to estimate the worst case scenario for human BSE exposure risk (HER) by lung contaminated by CNS emboli. The content of CNS in the samples was estimated to be about 0.11% when the respective immuno reactions were calibrated against standards containing known brain concentrations. Under the assumption that only one lung in the pooled samples was contaminated with BSE-infected central nervous tissue, the HER was calculated to reach a maximum of 2.2 x 10(-5) CoID50/consumer after consumption of a sausage with a portion of 10% lung. The results of our study suggest that the contamination of the lung with CNS after using a conventional cartridge-fired stunner cannot be excluded, however, the incidence appears to be very low. In addition, presumed CNS emboli, if at all, are microscopically small. Furthermore the incidence of BSE in Germany is very low and lungs of cattle are usually not consumed. Thus we can judge the potential for human oral exposure after consumption of lungs of cattle which were stunned in Germany to be extremely low. A final assessment, however, is impossible as there is no knowledge about the minimum infectious dose for humans.

An immunoassay for canine pancreatic elastase 1 as an indicator for exocrine pancreatic insufficiency in dogs.

The detection of pancreatic elastase 1 in stool samples has become the noninvasive gold standard for the diagnosis of pancreatic insufficiency in humans. Accordingly, the development of a sandwich-ELISA specific for canine pancreatic elastase 1, based on monoclonal antibodies, is presented here. The test has a detection range of 4-240 microg canine pancreatic elastase l/g feces. The intraassay coefficient of variation is 7.4%, and the interassay coefficient of variation is 7.7%. Spiking experiments show that canine elastase 1 is quantitatively detectable in fecal samples. Interestingly, the range of the elastase 1 concentration in canine feces within several days is higher as compared with humans. As the proposed cutoff of 10 microg/g is below this variation range in 96.1% of the tested samples, the effect on the test specificity is negligible. Because the test detects neither human nor bovine and porcine elastase 1, pancreatic function can be monitored without interrupting an enzyme replacement therapy.

Metabolic cooperation between different oncogenes during cell transformation: interaction between activated ras and HPV-16 E7.

The metabolism of tumor cells (tumor metabolome) is characterized by a high concentration of glycolytic enzymes including pyruvate kinase isoenzyme type M2 (M2-PK), a high glutaminolytic capacity, high fructose 1,6-bisphosphate (FBP) levels and a low (ATP+GTP):(CTP+UTP) ratio. The sequence of events required for the establishment of the tumor metabolome is presently unknown. In non-transformed rat kidney (NRK) cells we observed a high glutaminolytic flux rate and a low (ATP+GTP):(CTP+UTP) ratio, whereas FBP levels and M2-PK activity are still extremely low. After stable expression of oncogenic ras in NRK cells a strong upregulation of FBP levels and of M2-PK activity was observed. Elevated FBP levels induce a tetramerization of M2-PK and its migration into the glycolytic enzyme complex. AMP levels increase whereas UTP and CTP levels strongly decrease. Thus, ras expression completes the glycolytic part of tumor metabolism leading to the inhibition of nucleic acid synthesis and cell proliferation. The HPV-16 E7 oncoprotein, which cooperates with ras in cell transformation, directly binds to M2-PK, induces its dimerization and restores nucleic acid synthesis as well as cell proliferation. Apparently, the combination of the different metabolic effects of ras and E7 constructs the perfect tumor metabolome as generally found in tumor cells.

Brain in human nutrition and variant Creutzfeldt-Jakob disease risk (vCJD): detection of brain in retail liver sausages using cholesterol and neuron specific enolase (NSE) as markers.

No information is available about the consumption of brain via meat products. With respect to the new variant of Creutzfeldt-Jakob disease (vCJD) and the presumed food-borne transmission of bovine spongiform encephalopathy (BSE) to humans, a preliminary survey for brain and/or spinal cord (tissues of the central nervous system, CNS) was conducted. We applied a previously developed integrated procedure using cholesterol and neuron specific enolase (NSE) as markers. Quantification of cholesterol had to be backed up by NSE immunochemistry in order to account for low specificity and relatively high variances. Out of 126 high-quality finely graded liver sausages, five samples (4 %) showed positive NSE immunoresponses. In four of these samples a transgression of the normal maximum cholesterol content was obtained. The identification of such a considerable number of CNS-positive sausages indicates that brain consumption is not as rare as previously assumed. Overall, the present integrated method could be successfully applied for the detection of CNS in heat-treated meat products. Its routine application in official food control would deter illegal practice and thus help to control transmissible spongiform encephalopathies.

Effects of the human papilloma virus HPV-16 E7 oncoprotein on glycolysis and glutaminolysis: role of pyruvate kinase type M2 and the glycolytic-enzyme complex.

Proliferating and tumour cells express the glycolytic isoenzyme, pyruvate kinase type M2 (M2-PK), which occurs in a highly active tetrameric form and in a dimeric form with low affinity for phosphoenolpyruvate. The switch between the two forms regulates glycolytic phosphometabolite pools and the interaction between glycolysis and glutaminolysis. In the present study, we show the effects of oncoprotein E7 of the human papilloma virus (HPV)-16 (E7)-transformation on two NIH 3T3 cell strains with different metabolic characteristics. E7-transformation of the high glycolytic NIH 3T3 cell strain led to a shift of M2-PK to the dimeric form and, in consequence, to a decrease in the cellular pyruvate kinase mass-action ratio, the glycolytic flux rate and the (ATP+GTP)/(UTP+CTP) ratio, as well as to an increase in fructose 1,6-bisphosphate (FBP) levels, glutamine consumption and cell proliferation. The low glycolytic NIH 3T3 cell strain is characterized by high pyruvate and glutamine consumption rates and by an intrinsically large amount of the dimeric form of M2-PK, which is correlated with high FBP levels, a low (ATP+GTP)/(CTP+UTP) ratio and a high proliferation rate. E7-transformation of this cell strain led to an alteration in the glycolytic-enzyme complex that correlates with an increase in pyruvate and glutamine consumption and a slight increase in the flow of glucose to lactate. The association of phosphoglyceromutase within the glycolytic-enzyme complex led to an increase of glucose and serine consumption and a disruption of the linkage between glucose consumption and glutaminolysis. In both NIH 3T3 cell lines, transformation increased glutaminolysis and the positive correlation between alanine and lactate production.

Identification of central nervous system tissue in retail meat products.

A procedure to detect tissues from the central nervous system that involved quantification of cholesterol and immunochemical detection of neuron-specific enolase and glial fibrillary acidic protein was used to analyze 402 samples of heat-treated meat products from various food outlets in Germany. The cholesterol content of 16 samples (4.0%) indicated the possible presence of central nervous system tissue because the levels exceeded the normal maximum cholesterol content of cooked sausages. In 7 of these 16 heat-treated meat products, immunoblotting of both neuron-specific enolase and glial fibrillary acidic protein confirmed the presence of CNS tissue. Repeated sampling by veterinary officials and analysis by both cholesterol quantification and immunoblotting confirmed these findings. Whereas all of the control samples (with and without added central nervous system tissue) were correctly classified by both cholesterol quantification and immunoblotting, negative results of immunoblotting must be carefully interpreted in the case of intensively heat-treated meat products. Thus, studies have yet to establish an increase in sensitivity of immunoblotting of neuron-specific enolase and glial fibrillary acidic protein. However, the detection of illegal use of central nervous system tissue in heat-treated retail meat products demonstrates the need for suitable analytical methods to control transmissible encephalopathies and to enforce labeling laws.

Quantitative detection of tumor M2-pyruvate kinase in plasma of patients with lung cancer in comparison to other lung diseases.

Lung cancer is one of the predominant causes of cancer death. The aim of this project is the development of a screening method in persons with high risk for developing lung cancer, based on the measurement of Tumor M2-pyruvate kinase (Tumor M2-PK). Tumor M2-PK is quantitatively detectable in ethylenediaminetetraacetic acid-plasma with a sensitive enzyme-linked immunosorbent assay. So far, 60 patients with newly diagnosed lung cancer were included. These were compared to 24 patients with acute inflammatory lung diseases, 56 patients with pneumoconiosis, 22 patients with obstructive airway diseases, and 28 healthy persons. Tumor patients and some individuals suffering from severe inflammatory lung diseases had significantly higher Tumor M2-PK concentrations in ethylenediaminetetraacetic acid-plasma than all the other groups. The histologic tumor type had no influence on the plasma levels of Tumor M2-PK. Tumor M2-PK concentrations correlate strongly with the tumor stage, showing significantly increasing concentrations with progressive tumor stages. The present data indicate that Tumor M2-PK could be a valuable tumor marker for the detection of lung cancer.

Comparison of the tumor markers tumor M2-PK, CEA, CYFRA 21-1, NSE and SCC in the diagnosis of lung cancer.

The study presents data comparing the new tumor marker Tumor M2-PK with CEA, CYFRA 21-1, NSE and SCC in the diagnosis of lung cancer. Tumor M2-PK is quantitatively detectable in EDTA-plasma with a sensitive ELISA. The results of the tumor marker test were compared with respect to the different histological tumor types and with the tumor staging. So far 144 newly diagnosed lung cancer patients were included. Significantly elevated tumor marker concentrations were found with progressive tumor stages. The best correlation with the tumor stage was observed for Tumor M2-PK and CYFRA 21-1. Comparison of the sensitivities in the detection of lung cancer indicated that the Tumor M2-PK-test (sensitivity: 58%) is more efficient than the CEA-Test (sensitivity: 39%) or CYFRA 21-1 (sensitivity: 48%). Generally higher sensitivity for non-small cell lung cancer only was shown for Tumor M2-PK (sensitivity: 65%), CEA (sensitivity: 42%) and CYFRA 21-1 (sensitivity: 58%). For small-cell lung cancer the marker NSE was more sensitive than all other markers. Initial follow-up studies indicate that Tumor M2-PK and CYFRA 21-1 can be used to monitor disease with tumor progression or regression during chemotherapy. The present data indicated that Tumor M2-PK could be a valuable tumor marker for the detection of lung cancer.

Tumor M2-PK and glutaminolytic enzymes in the metabolic shift of tumor cells.

The pyruvate kinase isoenzyme M2-PK is known to be associated with a metabolic shift that is characteristic for tumor cells. Meanwhile, the universal expression of this isoenzyme is the basis for the detection of various tumor diseases in human clinical diagnosis. Other enzymes which are known to be essential for this tumor specific metabolic shift in rat chemical carcinogenesis are the NADP-dependent enzymes malic enzyme, isocitrate dehydrogenase and glucose 6-phosphate dehydrogenase. To evaluate the role of these enzymes in human carcinogenesis, we compared their enzymatic activities in normal colon mucosa and tissues derived from primary colon tumors. Histochemical staining showed that the enzyme activities were restricted to mucosal colon cells and colon cancer cells. The enzymes were strongly but heterogeneously expressed in the tumor tissues, whereas staining of normal mucosa was weak. Tumor M2-PK showed the most prominent differences in normal colon mucosa and colon cancer cells.

The pyruvate kinase isoenzyme tumor M2 (Tu M2-PK) as a tumor marker for renal carcinoma.

Various isoforms of the glycolytic enzyme pyruvate kinase are expressed in different cell-types. One of these isoforms, the type Tu M2-PK, is strongly overexpressed by tumor cells and released into body fluids. The concentration of Tu M2-PK in body fluids can be quantitatively determined by a commercially available enzyme-linked immunosorbent assay (ELISA)-kit. Using this kit, the Tu M2-PK concentration was measured in EDTA-plasma of 64 patients with renal carcinoma and 10 patients suffering from nephritis. The ranges of the Tu M2-PK-concentrations of the two groups did not overlap, indicating a highly significant discrimination of renal carcinoma and benign renal diseases. Furthermore, the Tu M2-PK-concentration in EDTA-plasma correlates strongly with the Robson tumor stage of the 64 patients. The present results indicate that the Tu M2-PK might be the first tumor marker which could be an excellent complementation of the diagnostic program for renal carcinoma.

Quantitative detection of tumor M2-PK in serum and plasma.

The majority of tumors express an isoform of the glycolytic enzyme pyruvate kinase, the type tumor M2. The isoenzyme exists in an active tetrameric and a less active dimeric form. The dimeric form is strongly overexpressed in tumor cells and this new tumor marker is thus called tumor M2-PK. This isoenzyme is released from tumor cells and is quantitatively detectable in body fluids by a sensitive enzyme-linked immunosorbent assay (ELISA). To establish the ELISA for the routine diagnostic in a clinical laboratory, the sample stability was evaluated. Therefore, blood samples were collected in different ways from healthy donors. Reproducibility of tumor M2-PK detection in EDTA-plasma was not affected by the day to day 'stress' in a clinical routine (e.g. shaking, leaving the samples at room temperature for several hours without prior centrifugation). Similar results were obtained with citrate-plasma, whereas detection in serum and heparin-plasma was only reproducible when the blood samples were centrifuged within 2 hrs after collection. It appears that lymphocytes contain small amounts of the tumor M2-PK isoenzyme. They might release tumor M2-PK in heparin-plasma and serum samples, but not in EDTA-plasma samples. The results indicated that EDTA-plasma appears to be most appropriate for the routine diagnostic of tumor M2-PK as a tumor marker.

Alterations in the glycolytic and glutaminolytic pathways after malignant transformation of rat liver oval cells.

Oval cells are liver epithelial cells that proliferate during the early stages of hepatocarcinogenesis induced by a variety of chemicals. The oval cell lines OC/CDE 6 and OC/CDE 22 have been established in our laboratory at two time points (6 and 22 weeks) of the carcinogenic process and have been malignantly transformed by different procedures. During the transformation process, the glycolytic and glutaminolytic flux rates were consistently up-regulated and this process was accompanied by an overproportional increase in the activities of cytosolic hexokinase and 6-phosphogluconate dehydrogenase. In transformed oval cells, a strong correlation between the glycolytic flux rate and glutamine consumption as well as glutamate production was observed. Furthermore, the transport of glycolytic hydrogen, produced by the glyceraldehyde 3-phosphate dehydrogenase-catalyzed reaction, from the cytosol into the mitochondria by means of the malate-aspartate shuttle was enhanced, this being due to alterations in the activities of malate dehydrogenase and glutamate oxaloacetate transaminase. The up-regulation of the glycolytic hydrogen transport and the alterations in the glycolytic enzyme complex led to an enhanced pyruvate production at high glycolytic flux rates. Taken together, our data are further proof that a special metabolic feature (increased glycolysis and glutaminolysis) is characteristic for tumor cells and that the mechanisms by which this metabolic state is induced can be totally different.

The effect of ursodeoxycholic acid on the florid duct lesion of primary biliary cirrhosis.

The frequency with which florid duct lesions are seen in needle-biopsy specimens of the liver was assessed in patients with primary biliary cirrhosis (PBC) enrolled in a 2-year randomized, double-blind, placebo-controlled trial of ursodeoxycholic acid (UDCA) versus placebo. Paired biopsy specimens obtained at entry and after 2 years on medication were reviewed blindly and mostly simultaneously by a panel of 5 hepatopathologists who, earlier, had characterized the florid duct lesion, which has been well described in the pathology literature. Florid duct lesions at entry were identified in approximately 36%. Patients with earlier disease showed florid duct lesions much more frequently than those with more advanced disease. The prevalence of florid duct lesions in 60 patients receiving placebo medication fell from 38.3% to 21.7%, P =. 025, over the period of 2 years. The prevalence of florid duct lesions also decreased in the 55 patients receiving UDCA, from 32.7% to 18.2%, P =.046. The prevalences of these lesions in the placebo and UDCA patients at entry and at 2 years were not significantly different from each other. The findings suggest that UDCA does not prevent ongoing bile duct destruction in patients with PBC. Instead, they support the impression that UDCA exerts its beneficial effects by protecting against the consequences of bile duct destruction.

Expression of pyruvate kinase M2 in preneoplastic hepatic foci of N-nitrosomorpholine-treated rats.

The expression of the pyruvate kinase (PK) isoenzymes L and M2 was analysed in the livers of rats treated with the hepatocarcinogenic agent N-nitrosomorpholine (NNM) in the drinking water. In control animals L-PK expression was restricted to liver parenchymal cells, whereas M2-PK was detected in bile duct epithelial, blood vessel wall, endothelial and Kupffer cells. In rats treated with NNM proliferating oval cells were consistently L-PK negative and M2-PK positive, while the ductal cells of cholangiofibroses were clearly L-PK positive and coexpressed M2-PK. However, no morphological differentiation of ductal cells into hepatocyte-like cells was observed. In the clear and acidophilic cell foci storing glycogen in excess strong staining for L-PK was observed. In glycogen-poor foci induced by NNM a shift from L-PK to M2-PK expression takes place.

Development of an integrated procedure for the detection of central nervous tissue in meat products using cholesterol and neuron-specific enolase as markers.

The emergence of a new variant of Creutzfeldt-Jakob disease during the bovine spongiform encephalopathy epidemic has focused attention on the use of tissue from the central nervous system (CNS) in food. So far, the banning of CNS tissue could not be effectively controlled because procedures for detection were missing. With regard to preventive health protection and labeling law enforcement, we have developed an integrated procedure for the detection of CNS tissue in meat products. Herein, we show that antigenic characteristics of neuron-specific enolase (NSE) quantitatively survive technological treatment including severe homogenization and pressure heating. Using both poly- and monoclonal antibodies against NSE in the Western blot, bovine and porcine brain could be detected in sausages, albeit with varying sensitivity (1 to 4%). Sensitivity was increased after reduction of fat content (30 to 40%) of the samples by means of a soxhlet extraction. This made possible the detection of brain addition as low as 0.25% when using monoclonal antibodies. Immunohistology showed distribution of CNS tissue in heat-treated meat products to be homogeneous. Immunoreaction was not found to be bound to morphologically intact histological or cytological structures; however, it proved to be highly specific. The quantification of cholesterol provides a low-cost screening method for the rapid identification of meat products, suspicious with regard to CNS tissue addition. Cholesterol content increased by 26 mg per 100 g of fresh substance for each percentage of brain added to internally produced reference material. Using three different approaches (internal reference material, raw material, and field samples), a provisional cutoff point of normal cholesterol content was calculated for emulsion-type cooked sausages to be 115 mg/100 g (P < 0.05).