Lipid and non-lipid cardiovascular risk factors in postmenopausal type 2 diabetic women with and without coronary heart disease.

BACKGROUND: Coronary heart disease (CHD) is the leading cause of death in diabetic women. In addition to hyperglycemia, other factors may contribute to the excessive cardiovascular risk. AIM: In this study we evaluated common and emerging risk factors in a selected group of postmenopausal type 2 diabetic women with (n = 36) and without CHD (n = 59), not taking lipid-lowering medications. METHODS: Clinical and lifestyle data were collected, and metabolic and lipid profile, as well as fasting plasma levels of total homocysteine (tHcy), folate, vitamin B12, C-reactive protein (hsCRP), interleukin 6 (IL-6), and vascular cell adhesion molecule-1 (VCAM-1) were measured in all participants. RESULTS: Age, menopause and diabetes duration, family history for cardiovascular disease, prevalence of hypertension and current insulin use were greater in diabetic women with than without CHD (P < 0.05 for all comparisons). CHD women also showed higher levels of triglycerides, small dense LDL (sdLDL), remnant-like particle cholesterol, tHcy, and VCAM-1, and a lower creatinine clearance (P < 0.05 all). Conversely, the two groups were comparable for BMI, waist circumference, smoking habit, fasting plasma glucose, HbA1c, total cholesterol, low-density lipoprotein cholesterol (LDL-C), HDL cholesterol, folate, vitamin B12, hsCRP and IL-6 levels. At multivariate analysis, lower creatinine clearance (OR = 0.932, P = 0.017) and higher sdLDL serum concentration (OR = 1.224, P = 0.037) were the strongest risk factors associated with CHD in this population, whereas no significant association was noted with LDL-C. CONCLUSIONS: Our data suggest that beyond LDL-C, a lower creatinine clearance and more subtle alterations of LDL particles, together with a constellation of several well known and emerging cardiovascular risk factors, are stronger contributors to the high CHD risk of diabetic women.

Resistin levels and inflammatory and endothelial dysfunction markers in obese postmenopausal women with type 2 diabetes mellitus.

BACKGROUND: Obesity-associated coronary heart disease (CHD) risk is higher in women than in men with type 2 diabetes (T2DM). Resistin, an adipokine secreted by adispose tissue, may contribute to this higher risk. AIMS: To explore the relationships among resistin levels and common inflammatory and endothelial dysfunction markers and CHD risk in obese post-menopausal T2DM women. METHODS: Serum levels of resistin, hsCRP, IL-6, Soluble vascular cell adhesion molecule (sVCAM), homocysteine (tHcy), HOMA-IR and metabolic parameters were determined in a group of 132 T2DM women with and without documented CHD and in 55 non-diabetic women. RESULTS: Resistin, sVCAM, IL-6 and tHcy levels were comparable in T2DM and controls. CHD women showed higher resistin, sVCAM and tHcy levels than those without CHD, and for resistin this difference remained significant after age-adjustment (P = 0.013); conversely hsCRP were ~ 2X higher in T2DM women than in controls (P = 0.0132) without any difference according to CHD history. At univariate analysis resistin levels were significantly associated with age, waist circumference, hypertension, tHcy, hsPCR, sVCAM, IL-6, HDL-cholesterol, triglycerides and creatinine levels, but only creatinine, triglycerides, hsCRP, IL-6 and sVCAM were independently associated to resistin levels at stepwise regression analysis. Resistin levels were independently associated to CHD, increasing the risk by 1.15 times (0.986-1.344 95% CI), together with age, tHcy, LDL-C and hypertension. CONCLUSIONS: Circulating resistin levels were comparable in obese/overweight T2DM and control women. In T2DM women, resistin levels correlated with markers of renal function, systemic inflammation and endothelial dysfunction and were independently associated with a higher CHD risk.

Menopause modulates homocysteine levels in diabetic and non-diabetic women.

High total homocysteine (tHcy) plasma levels may contribute to the increased cardiovascular risk of Type 2 diabetic women. However, to date, data on factors modulating tHcy concentration in this population are scarce. Fasting tHcy, vitamin B12, folate plasma levels, and the methylene tetrahydrofolate reductase (MTHFR) C677T genotype as well as clinical, biochemical, and lifestyle variables were compared in 91 Type 2 diabetic and 91 matched non-diabetic women (40 pre- and 51 post-menopausal, in each group). Fasting tHcy concentration did not differ between diabetic and control women, even after multivariable adjustment. In both groups, tHcy levels increased after menopause, but the differences were weakened after multivariable adjustment. The MTHFR genotype distribution was in accordance with the Hardy-Weinberg equilibrium, with a similar TT frequency in diabetic (22.2 %) and control women (19.8%). Overall, tHcy plasma concentration was higher in TT homozygous compared to other genotypes. We found a menopause-genotype interaction on tHcy levels (p=0.068 for menopause*genotype interaction); overall, the increase of tHcy concentration in TT subjects was limited to pre-menopause (p<0.0001; adjusted p=0.024), and this was confirmed after considering diabetic and control women separately (p=0.001 and p=0.01, respectively). At multivariate analysis, menopause was an independent correlate of tHcy concentration, together with creatinine, folate and MTHFR genotype. Our data show that menopause has a strong influence on tHcy concentration even in Type 2 diabetic women and demonstrate, for the first time, that it may modulate the association between tHcy and the common MTHFR polymorphism both in diabetic and non-diabetic women.

Two-dimensional electrophoresis of plasma lipoproteins: recognition of new apo A-I-containing subpopulations.

Two-dimensional electrophoresis has been used to resolve 12 distinct apo A-I-containing high-density lipoprotein (HDL) subpopulations in human plasma. The subpopulations were quantitated by 125I-labeled, monospecific antibody and phosphor-imaging. Modification and standardization of the agarose electrophoresis (first dimension) enabled us to recognize new HDL subpopulations. Lipoprotein mobilities in agarose were expressed relative to the mobility of the sample's endogenous albumin. We demonstrated the presence of lipoproteins with mobilities faster than and similar to albumin, as well as subpopulations with mobilities slower than albumin. We refer to these as pre alpha, alpha and pre beta, respectively. Lipoprotein molecular sizes were determined with a non-denaturing polyacrylamide gradient gel electrophoresis (PAGE) (2% to 36%) in the second dimension. Internal standard of 125I-labeled proteins of known molecular size was run simultaneously in each gel permitting accurate size determination. We have demonstrated that ultracentrifugally-isolated lipoproteins are different from the native apo A-I-containing subpopulations. The major difference observed was the loss of pre beta 1 and pre beta 2 particles from the d < 1.21 g/ml fractions to the d > 1.21 g/ml fractions. Possible physiologic and pathologic implications of these findings are also discussed.

Comparison of apo A-I-containing subpopulations of dog plasma and prenodal peripheral lymph: evidence for alteration in subpopulations in the interstitial space.

To study in vivo reverse cholesterol transport, dog plasma and lymph apo A-I-containing subpopulations were compared by two-dimensional electrophoresis. Charge and size of subpopulations were similar in plasma and lymph, but the distribution of subpopulations varied considerably. An increase in pre-beta and pre-alpha particles in lymph suggests these changes are a reflection of in vivo reverse cholesterol transport.

Distribution of ApoA-I-containing HDL subpopulations in patients with coronary heart disease.

High density lipoproteins (HDLs) and their subspecies play a role in the development of coronary heart disease (CHD). HDL subpopulations were measured by 2-dimensional nondenaturing gel electrophoresis in 79 male control subjects and 76 male CHD patients to test the hypothesis that greater differences in apolipoprotein (apo)A-I-containing HDL subpopulations would exist between these 2 groups than for traditional lipid levels. In CHD subjects, HDL cholesterol (HDL-C) was lower (-14%, P<0.001), whereas total cholesterol and the low density lipoprotein cholesterol/HDL-C ratio were higher (9% [P:<0.05] and 21% [P:<0.01], respectively) compared with control levels. No significant differences were found for low density lipoprotein cholesterol, triglyceride, and apoA-I levels. In CHD subjects, there were significantly (P:<0.001) lower concentrations of the large lipoprotein (Lp)A-I alpha(1) (-35%), pre-alpha(1) (-50%), pre-alpha(2) (-33%), and pre-alpha(3) (-31%) subpopulations, whereas the concentrations of the small LpA-I/A-II alpha(3) particles were significantly (P:<0.001) higher (20%). Because alpha(1) was decreased more than HDL-C and plasma apoA-I concentrations in CHD subjects, the ratios of HDL-C to alpha(1) and of apoA-I to alpha(1) were significantly (P:<0.001) higher by 36% and 57%, respectively, compared with control values. Subjects with low HDL-C levels (35 mg/dL). Therefore, we stratified participants according to HDL-C concentrations into low and normal groups. The differences in lipid levels between controls and HDL-C-matched cases substantially decreased; however, the significant differences in HDL subspecies remained. Our research findings support the concept that compared with control subjects, CHD patients not only have HDL deficiency but also have a major rearrangement in the HDL subpopulations with significantly lower alpha(1) and pre-alpha(1-3) (LpA-I) and significantly higher alpha(3) (LpA-I/A-II) particles.

Subpopulations of high density lipoproteins in homozygous and heterozygous Tangier disease.

Tangier disease (TD) is characterized by severe high-density lipoproteins (HDL) deficiency, hypercatabolism of HDL constituents, impaired cellular cholesterol efflux, and mutations in the gene of ATP-binding cassette 1 (ABC-1). In the present study, we determined plasma lipid and apolipoprotein levels, and HDL subpopulations, in 110 subjects from a large TD kindred in which the proband was homozygous for an A-->C missense mutation at nucleotide 5338 of the ABC-1 transcript. In the proband HDL-C, apoA-I, and apoA-II concentrations were 2, 1, and 2 mg/dl, respectively, apoA-I was present only in prebeta(1), while apoA-II was found free of apoA-I in two distinct alpha mobility subpopulations with different sizes. The smaller size particles contained only apoA-II while the larger one contained apoA-II and apo(a). Relative to unaffected male relatives (n=30), male heterozygotes (n=21) had significant reductions (P<0.001) in plasma HDL-C (-45%), apoA-I (-34%), apoA-II (-59%), apoA-IV (-40%), Lp(a) (-62%), and apoB (-55%) concentrations, and a significant increase (P<0.05, +33%) in plasma apoC-III levels. Female heterozygotes (n=11) similarly had significant reductions (P<0.001) in the concentrations of plasma HDL-C (-42%), apoA-I (-27%), apoA-II (-52%), Lp(a) (-27%), and (P<0.01) apoA-IV (-28%), apoB (-13%), and a significant increase (P<0.05) in plasma apoE levels (+29%) as compared to unaffected female relatives (n=41). Large size HDL subpopulations, especially the two LpA-I particles: alpha(1) and prealpha(1) were dramatically reduced in both male and female heterozygotes relative to their unaffected family members. Since apoA-II decreased more than apoA-I in both male and female heterozygotes, the ratios of apoA-I/apoA-II were significantly (P<0.01) increased. The prevalence of CHD was 60% higher in the 32 heterozygotes than in the 71 unaffected relatives even though the latter group was on average 7 years older. We conclude that TD homozygotes have only prebeta(1) apoA-I-containing HDL subpopulations, while heterozygotes have HDL that is selectively depleted in the large alpha(1), prealpha(1), and alpha(2), prealpha(2) subpopulations, resulting in HDL particles that are small in size, poor in cholesterol, but relatively enriched in apoA-I compared to those of their unaffected relatives. These abnormalities appear to result in a higher risk of CHD in heterozygotes than in unaffected controls.

Improved allocation of costs through analysis of variation in data: planning of laboratory studies.

BACKGROUND: When developing a new laboratory test for study of human diseases, it is important to identify and control internal and external sources of variation that affect test results. It is also imperative that the precision of the test not only meets pre-established requirements and not exceed allowable total error, but also that these objectives are reached without undue expenditure of either time or financial resources. METHODS: This study applies statistical principles in designing a cost-effective experimental approach for determining the analytical precision of a new test. This approach applies the statistical concept of variance components to the problem of balancing a pre-established level of analytical precision against expenses incurred in achieving this precision. RESULTS: We demonstrated (1) estimation of variance components, (2) use of these estimates for improving allocation of costs within the experiment, and (3) use of these estimates for determining the optimal number of replicate measurements. CONCLUSIONS: Although elimination of all sources of variation that can affect laboratory test results is unlikely, the application of analysis of variance (ANOVA) statistical techniques can lead to a cost-effective allocation of resources for estimating the precision of a laboratory test.

Comparison of effects of paraquat and methidation on enzyme activity and tissue necrosis of carp, following exposure to the pesticides singly or in combination.

Under aquarium conditions, treatment with the herbicide paraquat (PQ) and with the insecticide methidation (MD) caused cell damage and stress in carp (Cyprinus carpio L.), as shown by increases in glutamate-dehydrogenase (GIDH, EC 1.4.1.2), glutamate-oxalacetate transaminase (GOT, EC 2.6.1.1) and lactate dehydrogenase (LDH, EC 1.1.1.27) activities and in blood sugar levels. PQ proved synergistic with MD in certain cases as regards the harmful effect exerted. On combined treatment, dilated extracellular spaces were visible by light microscope in the liver, while electronmicroscopic studies revealed signs indicative of cell autolysis in the same organ.

Fluid dynamic characteristics of monopivot magnetic suspension blood pumps.

A monopivot magnetic suspension blood pump is a centrifugal pump under development with a magnetic suspension and a ceramic pivot to support the impeller with minimum contact. The pump size has been reduced by implementing a direct impeller drive mechanism in place of a magnetic coupling and motor. Flow visualization studies revealed that high shear, which seems to be closely related to hemolysis, concentrates in boundary layers near the walls. This implies that fluid dynamic shear can be reduced not by widening the gap, but by reducing the impeller velocity. Therefore, compared with the results of the previous semi-open curved vane impeller model, impeller velocity was reduced by 30% with a closed impeller having radial straight vanes, and smaller impeller/housing gaps. The volute shape around the impeller tip was also changed such that the outflow from the impeller enters along the center plane of the volute. To examine the effect of the improvements, hemolysis testing was conducted and found that the newly developed closed impeller model generated a lower level of hemolysis than the previous semi-open impeller model.

The effect of an organophosphorus pesticide on the enzymes of carp (Cyprinus carpio L.).

1. The effect of 1.5, 2.0 and 5.0 ppm methidation was studied on the ASAT, ALAT, LDH and AChE enzymes, as well as the blood sugar and adrenaline levels in carp. 2. According to our studies methidation implies a potential hazard on the normal biochemical processes of fishes: causing tissue necrosis indicated by the increase ASAT, ALAT and LDH activities, inducing continual stress effect reflected by the increased blood sugar and adrenaline levels, inhibiting AChE activity in the various organs, the consequence under acute effects being fish kill.

A rapid microbiotest for the detection of cyanobacterial toxins.

Cyanobacteria occur widely in lakes, reservoirs, ponds, and slow flowing rivers. Many species are known to produce toxins (cyanotoxins), a number of which are of concern for health. Cyanotoxins vary in chemical structure and may be found intracellular or released into water. There is not only a wide variation in the toxicity of known cyanotoxins but a substantial number of toxins have to date not been identified chemically. Chemical analysis of cyanotoxins is nowadays not used for routine monitoring because it is time consuming, it requires specialized equipment and expertise, and is hence expensive. There is hence an urgent need for rapid tests in surface waters to detect cyanobacterial toxins because of the need for safe drinking water and safe natural bathing waters, which may be burdened by cyanobacterial blooms or scums. Previous investigations have already shown that larvae of the anostracan crustacean Thamnocephalus platyurus are quite sensitive to neurotoxic and hepatotoxic cyanotoxins. The present paper reports on the sensitivity comparison of the (1 h) Rapidtoxkit (based on a sublethal endpoint) and the (24 h) Thamnotoxkit microbiotest (based on mortality). Both assays make use of larvae of T. platyurus. The Rapidtoxkit is a new microbiotest that determines the decrease of ingestion of colored particles by the crustacean larvae, which are stressed by a short exposure to toxicants. Fifteen cyanobacterial samples composed of laboratory strains and natural bloom samples were tested by both microbiotests. All samples were also analyzed concurrently by HPLC for microcystins and cylindrospermopsin. The correlation coefficient between the two microbiotests (r = 0.82) showed the very good correspondence between the sublethal and the lethal effects. No known toxins could be detected in some samples, although the latter were found highly toxic to the test organisms in both bioassays. These results point to the presence of unknown toxin(s) produced by some cyanobacteria such as e.g., the Cylindrospermopsis raciborskii strain isolated from Lake Balaton in Hungary. This comparative study clearly showed that the 1 h Rapidtoxkit is an attractive rapid alternative to the Thamnotoxkit microbiotest.

Effect of pesticides on the LDH activity and isoenzyme pattern of carp (Cyprinus carpio L.) sera.

Copper sulphate (CuSO4), paraquat (PQ) and methidathion (MD) all caused tissue necrosis as demonstrated by increased levels of lactate dehydrogenase (LDH, EC 1.1.1.27) in blood sera. The main target of damage is shown by the isoenzymes appearing in the blood sera. The CuSO4 treatment damaged mainly the heart and skeletal muscles. This is indicated by the increased LDH level and the M and H subunits appearing in the isoenzyme pattern of the serum. PQ damaged first of all the gills and methidathion had an effect on the skeletal muscles.

Clinical significance of lipoprotein size and risk for coronary atherosclerosis.

Correlation between coronary heart disease and lipoprotein size and composition is well documented. Within the low-density lipoprotein (LDL) family the small LDL particles are associated with increased risk of coronary heart disease. These particles also have increased apolipoprotein (apo) B content. The appearance of these small LDL particles is the manifestation of complex alteration of plasma lipoprotein metabolism. The LDL size is influenced by genetic, endocrine, and environmental factors. Within the high-density lipoprotein (HDL) family the decrease of larger HDL2 particles is associated with coronary heart disease. HDLs can also be separated according to their apoprotein composition into particles containing lipoprotein (Lp)A-I only and particles containing LpA-I and LpA-II. Most studies have shown that the concentration of LpA-I-only particles decreases in coronary heart disease. HDLs are remodeled in the circulation and this remodeling continues in vitro after the blood is taken. Therefore adequate preservation of blood samples is necessary.

Presence and formation of 'free apolipoprotein A-I-like' particles in human plasma.

The influence of dilution on apolipoprotein (apo) A-I-containing subpopulations was studied in human plasma. Agarose electrophoresis and two-dimensional agarose nondenaturing gradient polyacrylamide gel electrophoresis were used. Both in one- and two-dimensional electrophoresis, an increase of charge was observed that resulted in an increase of subpopulations with pre-alpha mobility. Dilution of plasma also resulted in a decrease in the size of apo A-I-containing pre-beta 1 subpopulations. The existence of smaller pre-beta 1 particles was confirmed by subjecting undiluted and 8x diluted plasma to 3% to 16% nondenaturing gradient gel electrophoresis for 4 hours. In addition to the generally observed pre-beta 1 subpopulations, smaller particles similar in size to the free apo A-I were detected even in the undiluted plasma. During dilution, the proportion of larger pre-beta 1 particles decreased while the smaller ones increased, and in 8x diluted plasma, almost all the pre-beta 1 was present in smaller sizes. Using 3% to 35% nondenaturing polyacrylamide gels run for 24 hours, no pre-beta 1 particles could be detected in 8x diluted plasma because the small pre-beta 1 electrophoresed out. These studies show that pre-beta 1 particles can be converted to smaller ones during dilution. It also was demonstrated that "free apo A-I-like" pre-beta 1 particles are present in undiluted plasma. The presence of these particles may have important physiological and pathophysiological functions.