Macrophages possess both neutral and acidic protease activities toward low density lipoproteins.

Low density lipoproteins (LDL) have been strongly implicated in the pathogenesis of atherosclerosis. We have studied the proteolytic degradation of these lipoproteins by macrophages, which are a major cellular constituent of atherosclerotic lesions. Mouse peritoneal macrophages contained both an acidic and a less active but distinct neutral/alkaline protease activity toward human 125I-labelled LDL. The acidic activity had a pH optimum of 4.5 and the neutral/alkaline activity one of 8-8.5. The acidic activity started to plateau with increasing lipoprotein concentrations whereas the neutral activity was directly proportional to the lipoprotein concentration up to at least 150 micrograms of protein/ml. The acidic protease activity had a complex time course whereas the neutral activity was directly proportional to the time of incubation up to at least 48 h. Leupeptin (35 microM) and pepstatin (5 microM) inhibited the acidic activity by about 70% individually and almost entirely in combination, indicating that cathepsins B and D are important in the degradation of LDL by lysosomal cathepsins. In contrast, there was little, if any, inhibition of the neutral protease activity by leupeptin or pepstatin. The acidic protease activity was increased by both DL-dithiothreitol (5 mM) and disodium EDTA (1 mM) whereas the neutral protease activity was increased by dithiothreitol but inhibited partially by EDTA. The possible significance of macrophage neutral and acidic protease activities toward LDL in atherosclerosis needs to be assessed.

Analysis of polycyclic aromatic hydrocarbons in UK total diets.

Analysis of UK total-diet samples for polycyclic aromatic hydrocarbons was carried out using a simplified sample clean-up and a high-performance liquid chromatography dual fluorescence detector system. The results indicate that cereals and oils/fats contribute the major part (approximately one third each) of the polycyclic aromatic hydrocarbons in these total diets. Fruit, sugars and vegetables provide much of the remainder (approximately one quarter) while meat, fish, milk and beverages make relatively minor contributions. These results are compared with others in the current literature on polycyclic aromatic hydrocarbons in foods. The levels in the UK diet seem to be at least as low as those found elsewhere.

The effects of acetylated low-density lipoproteins on fluid-phase pinocytosis by macrophages.

A simple method has been set up to measure the rate of fluid-phase pinocytosis in resident mouse peritoneal macrophages in culture. The method uses 125I-labelled polyvinylpyrrolidone as a nondegradable marker of fluid-phase pinocytosis. The accumulation of 125I-labelled polyvinylpyrrolidone by the cells was directly proportional to its concentration in the culture medium up to at least 200 micrograms/ml. The estimates of the rate of fluid-phase pinocytosis were reproducible within each experiment (coefficient of variation 8.5%) but varied between individual experiments. Fluid-phase pinocytosis was undetectable at 4 degrees C and reduced greatly at 37 degrees C by metabolic inhibitors and 1 mM ZnSO4. High concentrations of human acetylated low-density lipoproteins, which are taken up rapidly by macrophages, decreased the rate of fluid-phase pinocytosis by up to about 70%. The inhibition was seen after only 2 h of incubation. Unmodified low-density lipoproteins, which are taken up only slowly by macrophages, did not usually inhibit fluid-phase pinocytosis (in fact, they sometimes increased it). Modified low-density lipoprotein uptake, leading to massive lipid accumulation in macrophages in the arterial wall, has been postulated to be involved in the pathogenesis of atherosclerosis. This study raises the possibility that the rate of fluid-phase pinocytosis in these lipid-laden arterial macrophages may be reduced.

An investigation of apparent total N-nitroso compounds in beer.

The concentration of apparent total N-nitroso compounds (ATNC) in beer has been investigated using a group-selective procedure based on chemical denitrosation with hydrogen bromide and chemiluminescence detection of the released nitric oxide. In a survey of samples of 40 brands of beer and lager, detectable levels of ATNC were present in 17 samples at concentrations of 20-100 micrograms N-NO/kg in 11 and 100-500 micrograms N-NO/kg in six. To determine the origin of ATNC in beer the production of a commercial batch was examined in detail. ATNC levels were below the detection limit in the sweet wort (aqueous extract of malt), bitter wort (malt extract boiled with hops) and also at the start of fermentation, but during the course of fermentation the concentration of ATNC increased appreciably and that of inorganic nitrate decreased; detectable, though transitory, levels of inorganic nitrite were observed. None of the brewing ingredients contained sufficiently high enough levels of ATNC to account for the concentration of these compounds present in the beer after fermentation. These findings suggest that the presence of detectable levels of ATNC in some beers is a result of N-nitrosation reactions occurring in the fermenting wort with the nitrosating species derived from reduction of nitrate, due probably to the presence of microbial species with nitrate reductase activity.

Ammonia caramels: specifications and analysis.

Twenty three UK commercially produced ammonia caramels and eight experimentally produced ammonia caramels have been analysed by a range of physical and chemical tests, which include solids content, nitrogen levels, colour intensity and pH. A statistical treatment of the results is reported.

Surveillance of potentially hazardous chemicals in food in the United Kingdom.

Surveillance of chemical contaminants in food plays an important role in helping to ensure a safe food supply in those countries that undertake it. This paper reviews the methods used in the UK as a means of highlighting the essential elements required by any food chemical surveillance programme. The following topics have been covered: quantifying food consumption, setting priorities in food surveillance, developing a common approach to the surveillance of different chemicals in the food supply (including the use of Total and Duplicate Diet Studies), estimating human intakes of chemicals from the diet, developing suitably sensitive and reliable methods of analysis, obtaining representative samples, and assessing and managing risk.

N-nitrosamine analysis in foods: N-nitrosoamino acids by high-performance liquid chromatography/thermal energy analysis and total N-nitroso compounds by chemical denitrosation/thermal energy analysis.

The total N-nitroso content of foods can be measured by chemical denitrosation and chemiluminescent detection of the eliminated nitric oxide. Appropriate procedures substantially reduce the 'system response' to the denitrosating agent, so that N-nitroso group contents down to 10 micrograms/kg can be measured on a one-gram sample. Using N-nitrosamine standards added to beer, the coefficients of variation are approximately 10% and 5% at N-nitroso contents of 19 and 94 micrograms/kg, respectively. In cured meats, the coefficient of variation for unidentified N-nitroso compounds is 26% for a 0.3-g sample containing 600 micrograms/kg. Some interference from non-nitroso compounds is possible, but, in some commodities at least, these interfering compounds are not detectable. Conditions have been established that allow measurement of N-nitrosoamino acids in foods using a high-pressure liquid chromatograph interfaced to a Thermal Energy Analyzer, without the need for prior derivatization. After extraction of lipids with hexane, nitrosoamino acids are extracted with ethyl acetate and subjected to appropriate clean-up stages prior to high pressure liquid chromatography on Microbondapak CN with a hexane:ethanol:acetic acid mobile phase and Thermal Energy Analyzer detection. Recoveries from cured meat are in the 55-75% range for N-nitrososarcosine, N-nitrosoproline and N-nitrosohydroxyproline; elution is complete within seven minutes.

Estimation of nitropolycyclic aromatic hydrocarbons in foods.

A method is described for the sample clean-up and estimation of nitropolycyclic aromatic hydrocarbons (nitro-PAH) in foods. The analysis involves the novel use of a coupled capillary gas chromatograph/thermal energy analyser and provides a detection limit for 1-nitropyrene of 12 pg (equivalent to 0.02 micrograms/kg for a 50-g sample). Only 3 out of 24 samples contained detectable quantities of nitro-PAHs; these were in the range 0.2-2.0 micrograms/kg. In contrast to the widespread occurrence of the parent PAHs in foodstuffs, the presence of nitro-derivatives appears to be minimal.

The application of a chemical denitrosation and chemiluminescence detection procedure for estimation of the apparent concentration of total N-nitroso compounds in foods and beverages.

The apparent total N-nitroso content of foods can be measured by a procedure based on chemical denitrosation and chemiluminescent detection of the eliminated nitric oxide. Procedures have been established which substantially reduce the 'apparatus blank' response to the denitrosating agent and allow total nitroso contents down to 10 micrograms (N-NO)/kg to be measured reproducibly on a 1-g sample. Typically, duplicate analyses of samples containing 10-1000 micrograms (N-NO)/kg differ by less than 15% of their mean. Potentially the method can be subject to some interference from compounds other than N-nitroso compounds, but at least in some commodities these interfering compounds do not exist in measurable amounts.

A survey of the occurrence of aflatoxin M1 in UK-produced milk for the period 1981-1983.

A UK survey for the occurrence of aflatoxin M1 in bulked dried milks (totalling 277 samples) obtained at monthly intervals from a number of commercial creameries in the UK over a two-year period (1981-1983), showed 98% of the samples to have levels below 0.03 micrograms/kg. For liquid milks sampled from individual farms over the same period (totalling 409 samples), 94% of the samples had aflatoxin M1 levels below 0.01 micrograms/kg. All samples were initially screened by a two-dimensional thin layer chromatographic method and quantification of positive results was by reverse phase h.p.l.c. with fluorescence detection. The results of this survey show that UK milk is largely free of aflatoxin M1 contamination, the incidence and levels, where observed, being significantly lower than for other European countries, which demonstrates the effectiveness of UK legislative action controlling feedstuff contamination.

Total N-nitroso group analysis of foods. II. Further studies on the precision and sensitivity of the assay.

The total N-nitroso content of foods can be measured by chemical denitrosation with hydrogen bromide and chemiluminescence detection of the cleaved nitric oxide radical. The denitrosation reagent itself causes a significant detector response which has limited the application of the technique to trace analysis. A procedure is described in which the errors associated with this interference are minimized. Application of this method to the trace analysis of aqueous and solid samples is reported together with an investigation of the effects of sample size on the accuracy and sensitivity of the assay as applied to aqueous analytes. The magnitude and significance of the false-positive response from nitrate is discussed in relation to the analysis of cured meats.