[Secular trends in smoking according to educational level between 1986 and 1996: The MONICA study. Catalonia]. / Tendencias seculares del tabaquismo según el nivel educativo entre 1986 y 1996: estudio MONICA-Cataluña.

OBJECTIVE: To describe trends from 1986-1996 in the prevalence of cigarette smoking between ages 25 to 64 in the MONICA-Catalonia study, according to educational level, and to validate these trends biochemically. METHODS: Three cross-sectional surveys in independent random samples of the general population of central Catalonia area carried out in 1986-88, 1990-92 and 1994-96 following the World Health Organization's MONICA protocol. Serum thiocyanate was determined by the Bowler method. RESULTS: A total of 2,571, 2,934 and 3,485 men and women were examined with response rates of 74, 67 and 72% in each survey respectively. The age-adjusted cigarette smoking prevalence decreased in men by 5.1% (95% CI: 1.5 to 8.7) and increased in women by +8.5% (95% CI: +5.6 to +11.4). The prevalence was 46.5% in men and 23.9% in women in 1994-96. The greatest decrease was in men aged 55-64 (9.9%) and the greatest increase was in women aged 35-44 (+14.8%). These trends were confirmed by serum thiocyanate levels, which decreased from 78.9 to 73.9 µmol/l (p = 0.07) in men and increased from 43.7 to 49.8 µmol/l (p < 0.01) in women during the study period. Cigarette smoking increased in less educated women (+10%) and decreased in university women (6%), while cigarette smoking in men decreased irrespective of educational level. Serum thiocyanate levels confirmed the relationship with educational level. CONCLUSIONS: The prevalence of cigarette smoking decreased in men between 1986 and 1996 but remained high in 1996. Women showed a pattern of progressive adoption of smoking, especially those with lower educational level.

ApoA-II expression in CETP transgenic mice increases VLDL production and impairs VLDL clearance.

Apolipoprotein (apo)A-II is a major high density lipoprotein (HDL) protein; however, its role in lipoprotein metabolism is largely unknown. Transgenic (Tg) mice that overexpress human apoA-II present functional lecithin: cholesterol acyltransferase deficiency, HDL deficiency, hypertriglyceridemia and, when fed an atherogenic diet, increased non-HDL cholesterol and increased susceptibility to atherosclerosis. In contrast to humans, mice do not present cholesteryl ester transfer protein (CETP) activity in plasma. To study the in vivo interaction of these two proteins, we crossbred human apoA-II and CETP-Tg mice. CETP x apoA-II-Tg mice fed an atherogenic diet, compared with CETP-Tg mice presented a 2-fold decrease in HDL cholesterol and a quantitatively similar increase in total plasma cholesterol and percentage of free cholesterol, non-HDL cholesterol, and free fatty acids, together with a remarkable 112-fold increase in plasma triglycerides. Plasma triglycerides in CETP x apoA-II-Tg mice were mainly associated with very low density lipoproteins (VLDL), which were also enriched in protein content, and resulted from a combination of higher production rate compared with both of their progenitors and non-Tg control mice, and decreased catabolism compared only with CETP-Tg mice. These results show CETP x apoA-II-Tg mice to be a good model with which to study mechanisms leading to VLDL overproduction and suggest that CETP and, in particular apoA-II, may play a role in the regulation of VLDL metabolism.

Tendencias seculares del tabaquismo según el nivel educativoentre 1986 y 1996: estudio MONICA-Cataluña / Secular trends in smoking according to educational level between 1986 and 1996: The MONICA study. Catalonia (Spain)

Objetivo: Describir las tendencias entre 1986 y 1996, en el estudio MONICA-Cataluña, de la prevalencia de fumadores de cigarrillos entre 25 y 64 años de edad, según el nivel educativo, y validarlas bioquímicamente. Métodos: Tres encuestas-exámenes de salud transversales en muestras aleatorias independientes de la población general del centro de Cataluña, realizadas en los años 1986-1988,1990-1992 y 1994-1996, siguiendo el protocolo del estudio MÓNICA de la Organización Mundial de la Salud. El tiocianatosérico se determinó por el método de Bowler. Resultados: Se examinaron 2.571, 2.934 y 3.485 varones y mujeres con tasas de respuesta del 74, 67 y 72% en cada examen, respectivamente. La prevalencia de fumadores de cigarrillos ajustada por edad descendió un 5,1% (IC del 95%:–1,5 a –8,7) en varones y aumentó un 8,5% (IC del 95%: +5,6a +11,4) en mujeres. En 1994-1996, la prevalencia fue del46,5% en varones y del 23,9% en mujeres. El mayor descenso (..) (AU)

Objective: To describe trends from 1986-1996 in the prevalence of cigarette smoking between ages 25 to 64 in the MONICA-Catalonia study, according to educational level, and to validate these trends biochemically. Methods: Three cross-sectional surveys in independent random samples of the general population of central Catalonia area carried out in 1986-88, 1990-92 and 1994-96 following the World Health Organization’s MONICA protocol. Serum thiocyanate was determined by the Bowler method. Results: A total of 2,571, 2,934 and 3,485 men and women were examined with response rates of 74, 67 and 72% in each survey respectively. The age-adjusted cigarette smoking prevalence decreased in men by –5.1% (95% CI: –1.5 to –8.7)and increased in women by +8.5% (95% CI: +5.6 to +11.4).The prevalence was 46.5% in men and 23.9% in women in (..) (AU)

Increased production of very-low-density lipoproteins in transgenic mice overexpressing human apolipoprotein A-II and fed with a high-fat diet.

We investigated the mechanisms that lead to combined hyperlipidemia in transgenic mice that overexpress human apolipoprotein (apo) A-II (line 11.1). The 11.1 transgenic mice develop pronounced hypertriglyceridemia, and a moderate increase in free fatty acid (FFA) and plasma cholesterol, especially when fed a high-fat/high-cholesterol diet. Post-heparin plasma lipoprotein lipase and hepatic lipase activities (using artificial or natural autologous substrates), the decay of plasma triglycerides with fasting, and the fractional catabolic rate of the radiolabeled VLDL-triglyceride (both fasting and postprandial) were similar in 11. 1 transgenic mice and in control mice. In contrast, a 2.5-fold increase in hepatic VLDL-triglyceride production was observed in 11. 1 transgenic mice in a period of 2 h in which blood lipolysis was inhibited. This increased synthesis of hepatic VLDL-triglyceride used preformed FFA rather than FFA of de novo hepatic synthesis. The 11.1 transgenic mice also presented reduced epididymal/parametrial white adipose tissue weight (1.5-fold), increased rate of epididymal/parametrial hormone-sensitive lipase-mediated lipolysis (1.2-fold) and an increase in cholesterol and, especially, in triglyceride liver content, suggesting an enhanced mobilization of fat as the source of preformed FFA reaching the liver. Increased plasma FFA was reverted by insulin, demonstrating that 11.1 transgenic mice are not insulin resistant. We conclude that the overexpression of human apoA-II in transgenic mice induces combined hyperlipidemia through an increase in VLDL production. These mice will be useful in the study of molecular mechanisms that regulate the overproduction of VLDL, a situation of major pathophysiological interest since it is the basic mechanism underlying familial combined hyperlipidemia.

Prospective evaluation of the contribution of K-ras mutational analysis and CA 19.9 measurement to cytological diagnosis in patients with clinical suspicion of pancreatic cancer.

The aim of this study was to prospectively evaluate the diagnostic contribution of the detection of K-ras mutation and measurement of serum CA 19.9 concentrations to cytological diagnosis in patients with clinical suspicion of pancreatic cancer. These patients had either the presence or absence of a pancreatic mass as determined by imaging procedures. A total of 156 consecutive patients with clinical suspicion of pancreatic cancer or for confirmation and follow-up of their chronic pancreatitis disease were included: 84 patients presenting a pancreatic mass (group 1) and 72 patients without a pancreatic mass (group 2). K-ras mutations were detected by a restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) method and CA 19.9 by an immunoluminometric assay. When a pancreatic mass was present, cytology offered a high sensitivity, but with a significant number of inconclusive results and K-ras mutational analysis offered a highly specific test. In the absence of a pancreatic mass, CA 19.9 (cut-off 100 U/ml) increased the sensitivity of the diagnosis by cytology and K-ras mutational analysis did not add significant information. Thus both tests contribute to the clinical decision process when pancreatic cancer is clinically suspected and the cytological report is not conclusive.

Comparison of the Abbott IMx and a high-performance liquid chromatography method for measuring total plasma homocysteine.

The determination of the total concentration of plasma homocysteine is of interest in a variety of clinical circumstances, especially, in the evaluation of the risk of cardiovascular disease. However, most of the methods available to date, many of them chromatographic, are not well suited for the majority of clinical laboratories. Several automated methods are now or will be, shortly, commercially available. We have compared one of them, the fluorescence polarization immunoassay (FPIA) adapted to the IMx analyzer (Abbott Laboratories), with the high-performance liquid chromatography (HPLC) method with fluorescent detection currently used in our laboratory. The results show that the FPIA-IMx method is less imprecise and slightly more sensitive than the HPLC. The comparison of 67 clinical plasma specimens indicated that there is a proportional error disagreement between FPIA-IMx and HPLC (FPIA=1.19 HPLC + 0.92; confidence region for slope and y-intercept were, respectively, from 1.06 to 1.31 and from -0.06 to 2.32). The nature of this error is not explained by the experiments performed to study the inaccuracy of both methods, which included the investigation of dilution parallelism, analytical recovery and cross-reactivity. The different results of homocysteine concentration obtained with FPIA-IMx and HPLC must be taken into account when a change of methodology is under consideration.

Expression of human apolipoprotein A-II in apolipoprotein E-deficient mice induces features of familial combined hyperlipidemia.

Familial combined hyperlipidemia (FCHL) is a common inherited hyperlipidemia and a major risk factor for atherothrombotic cardiovascular disease. The cause(s) leading to FCHL are largely unknown, but the existence of unidentified "major" genes that would increase VLDL production and of "modifier" genes that would influence the phenotype of the disease has been proposed. Expression of apolipoprotein A-II (apoA-II), a high density lipoprotein (HDL) of unknown function, in transgenic mice produced increased concentration of apoB-containing lipoproteins and decreased HDL. Here we show that expression of human apoA-II in apoE-deficient mice induces a dose-dependent increase in VLDL, resulting in plasma triglyceride elevations of up to 24-fold in a mouse line that has 2-fold the concentration of human apoA-II of normolipidemic humans, as well as other well-known characteristics of FCHL: increased concentrations of cholesterol, triglyceride, and apoB in very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL), reduced HDL cholesterol, normal lipoprotein lipase and hepatic lipase activities, increased production of VLDL triglycerides, and increased susceptibility to atherosclerosis. However, FCHL patients do not have plasma concentrations of human apoA-II as high as those of apoE-deficient mice overexpressing human apoA-II, and the apoA-II gene has not been linked to FCHL in genome-wide scans. Therefore, the apoA-II gene could be a "modifier" FCHL gene influencing the phenotype of the disease in some individuals through unkown mechanisms including an action on a "major" FCHL gene. We conclude that apoE-deficient mice overexpressing human apoA-II constitute useful animal models with which to study the mechanisms leading to overproduction of VLDL, and that apoA-II may function to regulate VLDL production.

Determinants of plasma homocyst(e)ine in patients with nephrotic syndrome.

Hyperhomocyst(e)inemia is an independent risk factor for atherothrombosis in several clinical settings in which renal function is impaired, but its prevalence in the nephrotic syndrome has not been investigated in detail, even though this syndrome provides an excellent model in which to study a possible link between albuminuria, proteinuria, and hyperhomocyst(e)inemia. We obtained plasma and urine from 27 patients with biopsy-confirmed membranous glomerulonephritis presenting nephrotic syndrome and 27 matched controls and determined the concentrations of homocyst(e)ine and proteins considered putative markers of glomerular and tubular function. Hyperhomocyst(e)inemia, defined as the mean +SD of the plasma homocyst(e)ine concentration of the controls [plasma homocyst(e)ine concentration >10.8 micromol/l] was present in 26% of the patients with nephrotic syndrome but in only 7.4% of the controls. Furthermore, the degree of hyperhomocyst(e)inemia was more severe in the nephrotic patients than in the controls. The existence of renal failure, tubular damage, and, interestingly, relatively well conserved glomerular function barrier were the main predictors of increased levels of plasma homocyst(e)ine. In conclusion, hyperhomocyst(e)inemia is a frequent cardiovascular risk factor present in patients with nephrotic syndrome and renal failure, but it is not directly associated with proteinuria.

A highly sensitive method for K-ras mutation detection is useful in diagnosis of gastrointestinal cancer.

Detection of molecular features such as K-ras mutations has been used to evaluate potential tumour markers in a wide variety of clinical samples. Here we have applied a recently developed highly sensitive method for detection of K-ras codon 12 mutations to colorectal and pancreatic cancer diagnosis. We analysed 67 faecal samples from patients undergoing diagnostic colonoscopy under suspicion of colorectal cancer. PCR products were obtained in 62 of 67 (93%) faecal samples. Mutations were detected in exfoliated cells in 6 of 22 (27%) of the adenomas and in 6 of 11 (55%) of adenocarcinomas. No false positives were observed. Agreement between faecal samples and corresponding tissues was 100% for adenocarcinomas and 65% for adenomas. Mutations were also analysed in 61 pancreatic fine-needle aspirates. Mutations were detected in 36 of 45 (80%) of the pancreatic aspirates diagnosed as pancreatic cancer without false positives. Our findings suggest that, when colorectal cancer is suspected, detection of K-ras codon 12 mutations in faecal samples using this new method is specific for colorectal tumours. Additionally, this technique is a good alternative for evaluation of pancreatic masses.

Beta 2-microglobulin and immunoglobulins are more useful markers of disease progression in HIV than neopterin and adenosine deaminase.

Reference change values of six biochemical quantities (beta 2-microglobulin, neopterin, adenosine deaminase and immunoglobulins IgA, IgG and IgM) have been established in asymptomatic human immunodeficiency virus (HIV)-infected patients following the method described by Harris and Yasaka in 1983. Patients included in the evaluation were classified as A1, A2 or A3 according to the classification of the Centers for Disease Control (CDC) (January 1993). All patients were followed-up quarterly, with a minimum of four samples each available for statistical analysis. The main objective of this paper was to study whether differences found to be greater than calculated reference change values could predict clinical or immunological worsening in patients' status. Retrospective analysis was made in asymptomatic patients (n = 256) included in an HIV infection protocol carried out in our hospital. Of these patients, 179 showed clinical or immunological worsening during the study period and 77 maintained their clinical and immunological status. Changes in beta 2-microglobulin showed the greatest sensitivity to detect clinical or immunological worsening (43.0%), whereas changes in adenosine deaminase showed the lowest (21.8%). Clinical or immunological worsening in 169 of the 179 patients was detected by one of the six biochemical quantities evaluated. Ten patients showed clinical or immunological worsening, although differences between measurements were lower than the reference change values calculated. Of 77 patients whose clinical state did not deteriorate, there was a change in biochemical analytes greater than the reference value calculated in 29 patients (a period of 12 months had elapsed since detection). In 48 patients, no increases greater than calculated reference change values were detected. The sensitivity obtained using the six analytes was 94.4% and the specificity was 62.3%.

Effect of simvastatin treatment on the electronegative low-density lipoprotein present in patients with heterozygous familial hypercholesterolemia.

Most described modifications of low-density lipoprotein (LDL) cholesterol share an increase in its negative electric charge; in fact, an electronegative form of LDL can be identified and isolated from plasma. Although the exact nature of the chemical modification of electronegative LDL is still controversial, its toxicity on endothelial cells has been demonstrated. Statins have protective effects against cardiovascular disease that are independent of their lipid-lowering action and which could be due, at least in part, to the prevention of LDL modification. We evaluated the effect of 6 months of simvastatin therapy (40 mg/day) on electronegative LDL proportion and LDL susceptibility to in vitro induced oxidation in 21 patients with heterozygous familial hypercholesterolemia (FH). Eleven normolipemic subjects were analyzed as a control group. Total cholesterol as well as LDL and very low density lipoprotein cholesterol, triglycerides, and apoprotein B decreased 30% after the first month of therapy, with no further decreases thereafter. LDL susceptibility to oxidation was similar in FH patients and controls and did not change throughout the treatment. Electronegative LDL proportion was 35.1 +/- 9.9% in FH patients and 9.1 +/- 2.4% in control subjects (p <0.0001) but, in contrast to total LDL cholesterol and the rest of lipid parameters, it decreased to 28.6 +/- 9.1% in the third month and to 21.2 +/- 7.7% in the sixth month of therapy. The decrease in these cytotoxic particles may be a relevant mechanism by which simvastatin protects against cardiovascular disease.

Free cholesterol deposition in the cornea of human apolipoprotein A-II transgenic mice with functional lecithin: cholesterol acyltransferase deficiency.

We have developed several lines of transgenic animals that overexpress different levels of human apolipoprotein A-II (apoA-II). The 11.1 transgenic line has human apoA-II in plasma at threefold the level in normolipidemic humans and a functional lecithin:cholesterol acyltransferase (LCAT) deficiency. The latter is a biochemical phenotype similar to that of fish-eye disease (FED), which is characterized by free cholesterol (FC) and phospholipid accumulation in the cornea, leading to opacity and impaired vision. To assess whether the metabolic alterations in these mice also lead to lipid accumulation in the cornea, we fed them on a long-term regular chow or high-fat/high-cholesterol (HF/HC) diet. The 11.1 transgenic mice showed a moderate accumulation of FC in the cornea, but only when fed the regular chow diet. This FC accumulation was less severe than the accumulation described in FED, which may explain the lack of corneal opacity in these mice. Electron microscopy and immunoblotting analysis of the cornea of 11.1 transgenic mice in comparison to control mice showed (1) a mild but nevertheless more intense intracytoplasmatic lipid particle deposition in the epithelial cells and (2) a decrease of immunoreactive apoA-I in the area of Bowman's layer and at the superficial stroma. The serum capacity to cause cholesterol efflux from rat fibroblasts was decreased in 11.1 transgenic mice, but only in those fed a regular chow diet. We conclude that 11.1 human apoA-II transgenic mice may be a useful model for studies of early lipid deposition in the cornea and its possible prevention.

K-ras gene mutations in the diagnosis of fine-needle aspirates of pancreatic masses: prospective study using two techniques with different detection limits.

Detection of K-ras mutations may be useful in the evaluation of pancreatic cancer. The aim of this study was to assess, in a prospective design, the diagnostic utility of K-ras mutation analysis in 62 consecutive fine-needle aspirates of pancreatic masses, using two PCR-based techniques-standard and enriched-with detection limits of a mutant allele in the presence of 10(2) or 10(3) wild-type alleles, respectively. Cytology alone offered a diagnostic sensitivity of 75%. The enriched higher sensitivity detection technique, in combination with cytology, offered a diagnostic sensitivity of 91% without false positives. The molecular analysis would have contributed to diagnosis in an additional 14 cases of pancreatic cancer. The standard technique contributed to diagnosis in an additional 9 cases. These results strongly support the use of the enriched method of detecting K-ras mutations as a complement to cytology in the evaluation of pancreatic masses.

Molecular diagnosis of lecithin: cholesterol acyltransferase deficiency in a presymptomatic proband.

We report the molecular diagnosis of a lecithin : cholesterol acyltransferase deficiency in a 12-year old proband with a high-density lipoprotein deficiency. The increased percentage of free cholesterol in plasma and high-density lipoprotein indicated an inherited lecithin : cholesterol acyltransferase deficiency as the underlying cause. This diagnosis was confirmed by a low plasma lecithin : cholesterol acyltransferase activity and a combination of genetic analyses which demonstrated compound heterozygosity for two mutations in the lecithin : cholesterol acyltransferase gene of the proband. One was a previously unreported 2 bp deletion leading to a stop signal in codon 77 and the other a point mutation causing Arg 135-->Gln transition. To our knowledge, this is the first diagnosis of lecithin : cholesterol acyltransferase deficiency in a pre-symptomatic patient. Whether the proband will develop signs of complete lecithin : cholesterol acyltransferase deficiency or the milder form (Fish Eye Disease) is uncertain, although the former possibility is more likely. The risk of premature atherosclerosis conferred by lecithin : cholesterol acyltransferase deficiency is not well established. The proband will need to be carefully monitored in the future.

Ascorbic acid inhibits the increase in low-density lipoprotein (LDL) susceptibility to oxidation and the proportion of electronegative LDL induced by intense aerobic exercise.

BACKGROUND: We have previously reported the finding of an acute increment in the susceptibility of low-density lipoprotein (LDL) to oxidation and in the proportion of electronegative LDL [LDL(-)] after intense exercise. We have now studied the effect of oral supplementation with 1 g ascorbic acid, immediately before a 4-h athletic race, on the susceptibility of LDL to oxidation, the proportion of LDL(-), and the alpha-tocopherol and lipid peroxides content in LDL, in order to inhibit such deleterious changes, and to confirm the oxidative nature of modifications of LDL induced by exercise. METHODS: We studied seven highly trained runners who received a supplement of 1 g ascorbic acid and a control group of seven who did not receive the supplement. The susceptibility of LDL to oxidation was assessed by measurement of conjugated dienes after CuSO4-induced oxidation, the proportion of LDL(-) was determined by anion exchange chromatography, alpha-tocopherol was quantified by reverse-phase high performance liquid chromatography, and lipid peroxides were measured by the thiobarbituric acid-reactive substances (TBARS) method. RESULTS: After exercise, in the control group there was an increase in both the susceptibility of LDL to oxidation (change in lag phase from 51.4 +/- 4.7 min to 47.0 +/- 4.6 min, P < 0.05) and the proportion of LDL(-) (from 11.1 +/- 1.4% to 13.0 +/- 2.2%, P < 0.05), but these did not occur in the ascorbic acid group (change in lag phase from 49.7 +/- 2.3 min to 50.4 +/- 4.2 min, and in LDL(-) from 9.7 +/- 1.7% to 10.1 +/- 1.7%). No significant changes in the absolute amount of LDL alpha-tocopherol were observed after exercise (ascorbic acid group: 6.65 +/- 0.94 mol/mol apoB before the race, 7.13 +/- 0.88 mol/mol apoB after the race; control group: 7.34 +/-0.69 mol/mol apoB before the race, 7.06 +/- 0.69 mol/mol apoB after the race), but significant differences were found when increments or decrements of alpha-tocopherol were tested (alpha-tocopherol increased 9.9 +/- 11.5% in the ascorbic acid group, and decreased 0.6 +/- 7.3% in the control group; P < 0.018). TBARS did not change after exercise. CONCLUSIONS: We conclude that 1 g ascorbic acid inhibits the increase in LDL susceptibility to oxidation after exercise, preventing this acute pro-atherogenic effect. In addition, the observation that LDL(-) enhancement is prevented by ascorbic acid supports the hypothesis that at least some of the circulating LDL(-) originates from oxidative processes.