Amperometric determination of serum total cholesterol with nanoparticles of cholesterol esterase and cholesterol oxidase

We describe the preparation of glutaraldehyde cross-linked and functionalized cholesterol esterase nanoparticles (ChENPs) and cholesterol oxidase nanoparticles (ChOxNPs) aggregates and their co-immobilization onto Au electrode for improved amperometric determination of serum total cholesterol. Transmission electron microscope (TEM) images of ChENPs and ChOxNPs showed their spherical shape and average size of 35.40 and 56.97 nm, respectively. Scanning electron microscope (SEM) studies of Au electrode confirmed the co-immobilization of enzyme nanoparticles (ENPs). The biosensor exhibited optimal response at pH 5.5 and 40 °C within 5 s when polarized at +0.25 V versus Ag/AgCl. The working/linear range of the biosensor was 10–700 mg/dl for cholesterol. The sensor showed high sensitivity and measured total cholesterol as low as 0.1 mg/dl. The biosensor was evaluated and employed for total cholesterol determination in sera of apparently healthy and diseased persons. The analytical recovery of added cholesterol was 90%, whereas the within-batch and between-batch coefficients of variation (CVs) were less than 2% and less than 3%. There was a good correlation (r = 0.99) between serum cholesterol values as measured by the standard enzymic colorimetric method and the current method. The initial activity of ENPs/working electrode was reduced by 50% during its regular use (200 times) over a period of 60 days when stored dry at 4 °C.     

Dietary fat and cholesterol and serum cholesterol in the gerbil

Groups of gerbils were fed purified diets containing either 10 or 20% of safflower, olive, or coconut oil. Each diet was fed without cholesterol and with 0.1 and 0.2% of added cholesterol. The animals were bled after 2, 4, and 8 wk for the determination of the level of serum cholesterol. The major factors affecting the level of serum cholesterol were the kind of dietary oil, the amount of dietary cholesterol, and the length of time the diet was fed. The level of safflower oil had a statistically significant effect but the level of olive or coconut oil had no significant effect. Various other statistically significant interactions were observed which make simple interpretations of the data difficult. The levels of serum cholesterol achieved in the gerbils fed the different oils with no or very low levels of dietary cholesterol were similar to those seen in men fed the same oils. Although the gerbil is apparently resistant to the development of atherosclerosis, it may be a useful model for studying the effect of dietary fats upon cholesterol metabolism.     

Membrane cholesterol dynamics: cholesterol domains and kinetic pools

Nonreceptor mediated cholesterol uptake and reverse cholesterol transport in cells occur through cellular membranes. Thus, elucidation of cholesterol dynamics in membranes is essential to understanding cellular cholesterol accumulation and loss. To this end, it has become increasingly evident that cholesterol is not randomly distributed in either model or biologic membranes. Instead, membrane cholesterol appears to be organized into structural and kinetic domains or pools. Cholesterol-rich and poor domains can even be observed histochemically and physically isolated from epithelial cell surface membranes. The physiologic importance of these domains is 2-fold: (i) Select membrane proteins (receptors, transporters, etc.) are localized in either cholesterol-rich or cholesterol-poor domains. Consequently, the structure and properties of the domains rather than of the bulk lipid may selectively affect the function of proteins residing therein. (ii) Kinetic evidence suggests that cholesterol transport through and between membranes may occur through specific domains or pools. Regulation of the size and properties of such domains may be controlling factors of cholesterol transport or accumulation in cells. Recent technologic advances in the use of fluorescent sterols have allowed examination of cholesterol domain structure in model and biologic membranes. These techniques have been applied to examine the role of high-density lipoprotein, cholesterol lowering drugs, and intracellular lipid transfer proteins in membrane sterol domain structure and sterol movement between membranes.     

The interaction of cholesterol absorption and cholesterol synthesis in man

The total miscible pool of cholesterol in the body is determined largely by the interaction of cholesterol absorption and synthesis. In the present study we have examined the net effects of this interplay in one normal and five hypercholesteremic subjects when various amounts of cholesterol were made available for absorption. Feeding large amounts of cholesterol to the normocholesteremic patient caused an expansion of body pools by as much as 20 g before the amount of cholesterol re-excreted as fecal neutral steroids each day came into balance with the cholesterol absorbed from the diet. There was no detectable decrease in total body synthesis of cholesterol nor any increase in conversion of cholesterol into bile acids. However, feedback control of cholesterol synthesis was demonstrable when large quantities of plant sterols were fed: in the hypercholesteremic patients thus studied, the absorption of both endogenous and exogenous cholesterol was then greatly reduced, and a compensatory increase in synthesis occurred. Thus, the plant sterol experiments, but not the cholesterol feeding experiment, demonstrated that feedback control by dietary cholesterol does occur in man. That feedback control by dietary cholesterol is relatively unimportant in man seems to be due to the fact that in the metabolic "steady state" the absorption mechanism is essentially saturated by the large amounts of endogenous cholesterol available for reabsorption. These findings demonstrate that there are important differences between man and various laboratory animals in regard to the interaction of absorption and synthesis as factors controlling the size of tissue pools of cholesterol.     

A relation between high-density-lipoprotein cholesterol and bile cholesterol saturation.

The association of cholesterol gall stones with coronary artery disease is controversial. To investigate this possible relation at the biochemical level, bile cholesterol saturation and the plasma concentrations of triglycerides, total cholesterol, and high-density-lipoprotein cholesterol (HDL cholesterol) were measured in 25 healthy, middle-aged women. Bile cholesterol saturation index was negatively correlated with HDL cholesterol. It was positively correlated with plasma triglycerides and with total cholesterol minus HDL cholesterol. These findings provide a biochemical basis for a positive association in women between cholesterol gall stones and coronary artery disease.     

Hepatic cholesterol metabolism in cholesterol gallstone disease

Hepatic cholesterol metabolism was examined in 27 Swedish patients with cholesterol gallstone disease and in 13 patients free of gallstones operated for roentgenographically suspect polyps in the gallbladder. All 40 patients underwent cholecystectomy, and a liver biopsy and gallbladder bile were obtained at surgery. The cholesterol saturation of gallbladder bile was significantly higher in patients with gallstones compared to the gallstone-free controls (131 +/- 13 vs. 75 +/- 5%, P less than 0.001). Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, governing cholesterol synthesis, did not differ between gallstone and gallstone-free patients (104 +/- 11 vs. and 109 +/- 22 pmol/min per mg protein, respectively). The activity of cholesterol 7 alpha-hydroxylase, catalyzing the catabolism of cholesterol to bile acids, was not significantly decreased in gallstone patients (6.2 +/- 1.1 vs. 8.0 +/- 2.0 pmol/min per mg protein). The capacity to esterify cholesterol, judged by the activity of acyl coenzyme A:cholesterol acyltransferase (ACAT), was similar in gallstone and gallstone-free patients (5.4 +/- 0.4 vs. 6.7 +/- 1.1 pmol/min per mg protein). In the presence of exogenous cholesterol, ACAT activity increased by more than fourfold in both groups. No correlation was found between the saturation of gallbladder bile and any of the mentioned enzyme activities in gallstone patients. It is concluded that distinct abnormalities in cholesterol metabolizing enzymes are not of major importance for development of gallstones in Swedish patients with cholesterol gallstone disease. The results support the contention that the etiology of cholesterol gallstones is multifactorial.     

Another cholesterol hypothesis: cholesterol as antioxidant

Current emphasis on cholesterol as agency if not cause of human atherosclerosis and subsequent cardiovascular disease ignores the essentiality of cholesterol in life processes. Additionally ignored is the ubiquitous presence of low levels of oxidized cholesterol derivatives (oxysterols) in human blood and select tissues, oxysterols also implicated in atherosclerosis. Whereas such oxysterols may be regarded putatively as agents injurious to the aorta, an alternative view of some of them is here proposed: that B-ring oxidized oxysterols of human blood represent past interception of blood and tissue oxidants in vivo by cholesterol as an ordinary aspect of oxygen metabolism. Such interception and subsequent efficient hepatic metabolism of oxysterols so formed, with biliary secretion and fecal excretion, constitute as in vivo antioxidant system. Whether cholesterol, oxysterols, oxidized lipoproteins, or oxidants in blood, singly or in concert, cause or exacerbate human atherosclerosis remains to be understood.     

Fractional cholesterol esterification rate and muscle cholesterol of healthy young men

A group of fourteen healthy young male volunteers was examined to define more exactly the relations between lecithin cholesterol acyltransferase activity (LCAT), fractional cholesterol esterification rate (FER), total cholesterol (TC) and its free and esterified fractions (FC, CE) in skeletal muscles under physiological conditions. The mean values (+/- S.D.) of LCAT activity (95.4 +/- 16.3 mumol .1(-1) per hour), and FER (7.45 +/- 1.54% per hour) corresponded to published data on normolipidaemic healthy men of normal body weight. The mean value of TC in muscles was 332 +/- 83 micrograms per 100 mg of non-collagen protein, of which 14 +/- 7.4 per cent was formed by cholesterol esters. There was positive correlation between TC in muscles and age. Significant positive correlations between FER and the content of esterified cholesterol in muscles, and between FER and the proportion of esterified to total muscle cholesterol were found. These results suggest a close interrelation of cholesterol ester metabolism in the plasma and in slow pool tissues.     

A novel alkyne cholesterol to trace cellular cholesterol metabolism and localization

Cholesterol is an important lipid of mammalian cells and plays a fundamental role in many biological processes. Its concentration in the various cellular membranes differs and is tightly regulated. Here, we present a novel alkyne cholesterol analog suitable for tracing both cholesterol metabolism and localization. This probe can be detected by click chemistry employing various reporter azides. Alkyne cholesterol is accepted by cellular enzymes from different biological species (Brevibacterium, yeast, rat, human) and these enzymes include cholesterol oxidases, hydroxylases, and acyl transferases that generate the expected metabolites in in vitro and in vivo assays. Using fluorescence microscopy, we studied the distribution of cholesterol at subcellular resolution, detecting the lipid in the Golgi and at the plasma membrane, but also in the endoplasmic reticulum and mitochondria. In summary, alkyne cholesterol represents a versatile, sensitive, and easy-to-use tool for tracking cellular cholesterol metabolism and localization as it allows for manifold detection methods including mass spectrometry, thin-layer chromatography/fluorography, and fluorescence microscopy.     

Lipoprotein cholesterol and atherosclerosis

Progressive accumulation of cholesterol in the arterial wall causes atherosclerosis, the pathologic process underlying most heart attacks and strokes. Low density lipoprotein (LDL), the major carrier of blood cholesterol, has been implicated in the buildup of cholesterol in atherosclerotic plaques. Endothelial cells that line arteries function to transport LDL into the vessel wall. Models for the mechanism of cholesterol accumulation in atherosclerotic plaques emphasize increased LDL uptake into the vessel wall or increased retention of LDL that has entered the vessel wall. This article reviews the pathways of cholesterol entry and removal, the metabolism, and the physical changes of cholesterol in the vessel wall. How these processes are believed to contribute to cholesterol buildup in atherosclerotic plaques is discussed.     

Phytosterols and cholesterol metabolism

PURPOSE OF REVIEW: Phytosterols are plant sterols structurally similar to cholesterol that act in the intestine to lower cholesterol absorption. Because they have very low systemic absorption and are already present in healthy diets, increasing the intake of phytosterols may be a practical way to reduce coronary heart disease with minimum risk. RECENT FINDINGS: Phytosterols displace cholesterol from intestinal micelles, reducing the pool of absorbable cholesterol, but they are also rapidly taken up by enterocytes and increase expression of the adenosine triphosphate-binding cassette A1 sterol transporter. Phytosterol esters dissolved in food fat reduce LDL-cholesterol by 10% at a maximum effective dose of 2 g/day. However, this work probably understates the true effectiveness of phytosterols because it does not account for those naturally present in baseline diets. Single meal studies show that phytosterols in intact foods are bioactive at doses as low as 150 mg. The potential effectiveness of phytosterols has been improved in several ways. Individuals most likely to respond have been identified as having high cholesterol absorption and low cholesterol biosynthesis. Phytosterols can be emulsified with lecithin and delivered in non-fat or low-fat foods and beverages, and the amount of fat in fat-based preparations can be reduced substantially with the retention of bioactivity. SUMMARY: Phytosterols effectively reduce LDL-cholesterol when given as supplements, and the smaller amounts in natural foods also appear to be important. Future work will focus on the better delivery of phytosterols in natural foods and supplements and on further defining the mechanisms of action.