Acute stress potentiates brain response to milkshake as a function of body weight and chronic stress.

OBJECTIVE: Stress is associated with an increased intake of palatable foods and with weight gain, particularly in overweight women. Stress, food and body mass index (BMI) have been separately shown to affect amygdala activity. However, it is not known whether stress influences amygdala responses to palatable foods, and whether this response is associated with chronic stress or BMI. DESIGN: A total of 14 overweight and obese women participated in a functional magnetic resonance imaging (fMRI) scan as they consumed a palatable milkshake during script-driven, autobiographical, guided imagery of stressful and neutral-relaxing scenarios. RESULTS: We report that a network including insula, somatomotor mouth area, ventral striatum and thalamus responds to milkshake receipt, but none of these areas are affected by stress. In contrast, whereas the left amygdala responds to milkshake irrespective of condition, the right amygdala responds to milkshake only under stressful conditions. Moreover, this right amygdala response is positively associated with basal cortisol levels, an objective measure of chronic stress. We also found a positive relationship between BMI and stress-related increased response to milkshake in the orbitofrontal cortex(OFC). CONCLUSION: These results demonstrate that acute stress potentiates response to food in the right amygdala and OFC as a function of chronic stress and body weight, respectively. This suggests that the influence of acute stress in potentiating amygdala and OFC responses to food is dependent upon individual factors like BMI and chronic stress. We conclude that BMI and chronic stress play a significant role in brain response to food and in stress-related eating.

Amygdala response to sucrose consumption is inversely related to artificial sweetener use.

Controversy exists over whether exposure to artificial sweeteners degrades the predictive relationship between sweet taste and its post-ingestive consequences. Here we tested whether brain response to caloric sucrose is influenced by individual differences in self-reported artificial sweetener use. Twenty-six subjects participated in fMRI scanning while consuming sucrose solutions. A negative correlation between artificial sweetener use and amygdala response to sucrose ingestion was observed. This finding supports the hypothesis that artificial sweetener use may be associated with brain changes that could influence eating behavior.

Evidence for an integrated oral sensory module in the human anterior ventral insula.

Taste, which is almost always accompanied by other oral sensations, serves to identify potential nutrients and toxins. The present study was designed to determine the influence of sensory modality (chemesthetic vs. gustatory) and physiological significance (potentially nutritive vs. potentially harmful) on insular response to oral stimulation. Sixteen subjects underwent functional magnetic resonance imaging scanning while receiving 2 potentially nutritive solutions (sucrose and NaCl), 2 potentially harmful solutions (quinine and capsaicin, a chemesthetic stimulus), and a tasteless control solution. We identified a region of anterior ventral insula that responded to oral stimulation irrespective of modality or physiological significance. However, when subjects tasted a potentially nutritive stimulus, the connectivity between the insula and a feeding network including the hypothalamus, ventral pallidum, and striatum was greater than when tasting a potentially harmful stimulus. No differential connectivity was observed as a function of modality (gustatory vs. chemesthetic). These results support the existence of an integrated supramodal flavor system in the anterior ventral insula that preferentially communicates with the circuits guiding feeding when the flavor is potentially nutritive.

Individual differences in the neurophysiology of reward and the obesity epidemic.

The obesity epidemic has unfolded in a matter of decades, not millennia, and therefore cannot be attributed to a drift in the genome. Rather, the temporal characteristics of the epidemic more closely track environmental and lifestyle changes, such as reduced physical activity, increased availability of palatable and energy-dense foods and drinks, and increased acceptance of eating outside of meal time (among others). One important observation is that not everyone is becoming obese. This suggests that individual factors interact with recent environmental changes to predispose some to overeat. One hypothesis that has been gaining traction in the neuroscience community is that individual differences in the neural encoding of foods may predispose some to overeat in the presence of a surplus of energy-dense, palatable foods and drinks. The aim of this review is to highlight several possible ways by which individual differences in the neurophysiology of food reward may lead to overeating.

Thermal transitions in human plasma low density lipoproteins.

Thermal analysis of human plasma low density lipoproteins reveals a broad reversible transition encompassing body temperature. The calorimetric and x-ray scattering data identify this transition as a cooperation, liquid-crystalline to liquid phase change involving the cholesterol esters in the lipoprotein. This behavior requires the presence of a region rich in cholesterol ester within the lipoprotein.

The physicochemical basis of cholesterol gallstone formation in man.

The concentrations of bile salt, lecithin, and cholesterol were determined on each of 66 samples of gall bladder bile from patients with cholesterol gallstones and 25 samples of normal gall bladder bile. When these three constituents were plotted simultaneously on triangular coordinates, a complete separation of the normal and "abnormal" bile was achieved. This separation was the result of an increase in the quantity of cholesterol relative to the amounts of bile salts and lecithin contained in the bile from patients with cholesterol gallstones. An in vitro model system was constructed (on triangular coordinates) that allows prediction of the maximum amount of cholesterol that can be solubilized in solutions containing varying proportions of bile salt and lecithin. When the bile data were compared with the solubility of cholesterol derived from the model system, normal biles were found to be less than saturated with cholesterol, whereas biles from patients with cholesterol gallstones were saturated and in some cases contained insoluble cholesterol in the form of microcrystals. It is suggested that the physical state of bile (i.e., the presence or absence of insoluble cholesterol) is determined by the relative concentrations of bile salt, lecithin, and cholesterol, and the other biliary constituents do not appear to significantly effect the solubility of cholesterol in bile.

Fatty acid distribution in systems modeling the normal and diabetic human circulation. A 13C nuclear magnetic resonance study.

A nonperturbing 13C nuclear magnetic resonance (NMR) method was used to monitor the equilibrium distribution of carboxyl 13C-enriched fatty acids (FA) between distinct binding sites on human serum albumin, native human lipoproteins, and/or phospholipid model membranes, under conditions that mimic the normal and diabetic human circulation. Two variables pertinent to the diabetic circulation were examined: FA/albumin mole ratio (as elevated in insulin deficiency and/or nephrosis) and pH (as decreased in acidosis). 13C NMR spectra for samples containing carboxyl 13C-enriched palmitate, human serum albumin, and phospholipid vesicles or native lipoproteins (all samples at pH 7.4, 37 degrees C) exhibited up to six carboxyl NMR resonances corresponding to FA bound to distinct binding sites on albumin and nonalbumin components. When the sample FA/albumin mole ratio was 1, three FA carboxyl resonances were observed (182.2, 181.8, and 181.6 ppm; designated peaks beta, gamma, and beta', respectively). These resonances corresponded to FA bound to three distinct high-affinity binding sites on human serum albumin. When the sample mole ratio value exceeded 1, additional carboxyl resonances corresponding to FA bound to phospholipid vesicles (179.0 ppm, peak phi), lipoproteins (180.7 ppm, peak sigma), and lower affinity sites on albumin (183.8 ppm, peak alpha; 181.9 ppm, peak gamma'), were observed. The intensity of peaks phi and sigma increased with increasing mole ratio or decreasing pH. Using Lorentzian lineshape analysis, the relative mole quantities of FA bound to albumin and nonalbumin binding sites were determined. Plots of the fraction of FA associated with nonalbumin components as a function of FA/albumin mole ratio were linear and extrapolated to the abscissa at a mole ratio value of 1. This pattern of FA distribution was observed regardless of the type of nonalbumin acceptor used (phospholipid vesicles, human high- or low-density lipoproteins) or the type of FA used (palmitate, oleate, or stearate), and provided evidence for negative cooperativity for human serum albumin upon binding of 1 mol of FA per mole albumin. These in vitro NMR results suggest that the threshold FA/albumin mole ratio value for alterations in FA distributions in the human circulation may be 1, rather than 3, as previously held. The pathophysiological implications of these findings are discussed.

The physical chemistry of cholesterol solubility in bile. Relationship to gallstone formation and dissolution in man.

We determined the maximum solubilities of cholesterol in aqueous conjugated bile salt-egg lecithin-cholesterol systems as a function of several physical-chemical variables including those of physiological importance employing phase equilibria techniques. Equilibration rates are influenced by time and the method of sample preparation in that metastable supersaturation is readily induced at high bile salt: lecithin ratios, and equilibrium saturation by dissolution is achieved sluggisly at low bile salt:lecithin ratios. Equilibrium values for cholesterol saturation vary with the bile salt species, bile salt: lecithin ratio, temperature, ionic strength, and, in particular, with the total concentration of biliary lipids. Within physiological bile salt:lecithin ratios at 37 degreesC the influence of bile salt type and ionic strength is small, whereas the effects of bile salt:lecithin ratio and the total lipid concentration are major factors. We plotted on triangular coordinates a family of cholesterol solubility curves for each total lipid concentration (0.30--30 g/dl) and computed fifth-degree polynomial equations for each curve. With both the curves and the polynomial equations the "per cent cholesterol saturation" of fasting gallbladder and hepatic biles from patients with and without gallstones was calculated and both methods gave similar values. These results deomonstrate that by employing cholesterol saturation values appropriate to the total lipid concentration (range 0.2--24.9 g/dl) of individual biles, all cholesterol stone patients have supersaturated gallbladder biles, (mean, 132% [normal weight individuals], and 199% [morbidly obese individuals]). With controls and pigment stone patients the mean values were 95 and 98%, respectively, and in both approximately 50% of biles were supersaturated. Fasting hepatic biles were significantly more supersaturated than gallbladder biles (means 228--273%). Cholesterol monohydrate crystals were found in the majority of gallbladder (83%) and hepatic (58%) biles of cholesterol gallstone patients but were not observed in pigment stone patients or controls. We conclude that of the several factors in addition to the bile salt:lecithin ratios which can influence the cholesterol saturation of bile the total lipid concentration is the predominant determinant physiologically. Our results demonstrate that (a) metastable supersaturation is frequent in both normal and abnormal biles, (b) cholesterol gallstone patients have supersaturated gallbladder and hepatic biles without exception, and (c) the predominant driving force for cholesterol precipitation appears to be the absolute degree of cholesterol supersaturation.

Quantitation of the transfer of surface phospholipid of chylomicrons to the high density lipoprotein fraction during the catabolism of chylomicrons in the rat.

Small chylomicrons (CM) labeled with cholesterol, cholesterol ester, phospholipid, and, in some cases, protein, were used to study the fate of these constituents as the CM are catabolized in the circulations of the hepatectomized and intact rat. In the hepatectomized animal after (1/2) h, CM are greatly reduced in volume, surface area, and diameter. During this period, the CM lost >92% of the mass of their triacylglycerol, >77% of the mass of their phospholipid, and >39% of their protein. Compared to the injected CM, the chemically altered particles, called CM "remnants," have a reduction in volume of 96% and in surface area of 88%. The labeled cholesterol esters remain with the CM remnants but, strikingly, a major fraction of the labeled phospholipids and labeled soluble apoproteins leave the CM and are found in the high density lipoprotein (HDL) fraction. The chemical composition of this HDL fraction contains relatively more phospholipid and less cholesterol ester than normal rat HDL. Because of the difference in composition of HDL between normal rats and those given CM, we estimate that the HDL phospholipid pool increased by congruent with25% by the infusion of congruent with 4-5 mg of CM phospholipid. Approximately 5 mg of phospholipid is secreted on CM by a fed rat in 1 h. The findings in hepatectomized rats indicate that a major fraction of the phospholipid and a minor fraction of the protein (soluble non-B apoproteins) of newly secreted CM are transferred from the CM to the HDL fraction during remnant formation. The same process probably occurs in intact rats except that the remnant particles are rapidly removed from the plasma by the liver and a smaller fraction of the surface of the CM enters the HDL fraction.

Primate biliary physiology. 8. The effect of phenobarbital upon bile salt synthesis and pool size, biliary lipid secretion, and bile composition.

Phenobarbital, by inducing liver microsomal enzymes, may affect bile acid synthesis from cholesterol and thus alter the secretion of biliary lipids and the composition of bile. We, therefore, determined the effects of phenobarbital on bile flow, biliary lipid secretion, bile acid synthesis, and bile-acid pool size. Using an experimental preparation that allows controlled interruption of the enterohepatic circulation (1), we administered 5 mg/kg per day of phenobarbital to healthy Rhesus monkeys for 1-2 wk to achieve steady-state conditions. Three animals were studied with an intact enterohepatic circulation and three with a total bile fistula, each animal served as its own control. Total bile flow and secretion of bile salt, phospholipid, and cholesterol were measured every 24 h during steady-state conditions. Further, under conditions of an intact enterohepatic circulation bile-acid synthetic rate was measured in three animals and pool size estimated in two animals during both control and drug treatment periods. Phenobarbital at doses of 5 mg/kg per day increased bile flow 30-50% in all animals (P < 0.001). The increased bile flow resulted from both an increased "bile-salt independent fraction" and an increased bile-salt secretion rate. Phenobarbital significantly increased bile salt (P < 0.01) and phospholipid secretion (P < 0.05) by about 30% but cholesterol secretion was not significantly changed. Consequently, the concentration of cholesterol relative to bile salt and phospholipid was decreased (P < 0.001). Phenobarbital significantly enhanced the maximal rate of bile acid synthesis 25-30% in all three monkeys with total bile fistulas (P < 0.05) and also augmented bile acid synthesis and pool size in animals with intact enterohepatic circulations despite the fact that the rates of bile salt returning to the liver in these animals would have inhibited bile acid synthesis in control animals. Thus, phenobarbital not only increases the maximal rate of bile acid synthesis but also alters the normal control mechanisms by which bile salts returning to the liver inhibit bile salt synthesis. The fact that phenobarbital treatment results in increased synthesis of bile salt and unchanged secretion of cholesterol is consistant with the view that the drug augments conversion of hepatic cholesterol to bile salt. The resulting decrease in relative cholesterol content in bile may have therapeutic implications for cholesterol gallstone therapy.

Biliary lipid secretion in cholesterol gallstone disease. The effect of cholecystectomy and obesity.

Cholesterol gallstone disease is initiated in a liver which produces abnormal bile with excess cholesterol relative to bile salts and phospholipid. To define the responsible secretory mechanism(s), the rate of biliary lipid secretion was measured by a duodenal marker perfusion technique, while the bile salt pool was simultaneously estimated by isotope dilution. Two groups of control patients expected to have normal biliary lipid composition--14 subjects without hepatobiliary disease and 6 patients with pigment gallstones, were compared to two experimental groups expected to have abnormal bile--10 nonobese patients with cholesterol gallstones and 7 obese subjects without gallstones. Both control groups had nearly identical biliary lipid secretion rates, and a corresponding low relative molar concentration of cholesterol. Two different secretory mechanisms were found to be responsible for the abnormal bile in the experimental groups. In the nonobese patients with cholesterol gallstones, bile salt and phospholipid secretion rates were both significantly reduced. Conversely, the grossly obese subjects had an increased cholesterol secretion. To determine how cholecystectomy improves biliary lipid composition, three groups of gallstone patients --6 with pigment stones, 4 grossly obese with cholesterol stones, and 13 nonobese with cholesterol stones --were all examined after full recovery from surgery. In the nonobese patients with cholesterol gallstones, both bile salt and phospholipid secretion significantly increased, causing a definite improvement in bile composition. Cholecystectomy produced a similar but less marked trend in the obese patients with cholesterol stones, and in the patients with pigment stones. Cholesterol secretion, however, was unaffected by surgery. The bile salt pool was definitely small in the nonobese patients with cholesterol gallstones and became slightly smaller after cholecystectomy. The pool was significantly reduced by cholecystectomyin the patients with cholesterol gallstones. Removal of the gallbladder in all three groups caused a greater fraction of the pool to cycle around the enterohepatic circulation each hour. This more rapid cycling produced the increase in bile salt and phospolipid secretion, and was responsible for the improved composition found after cholecystectomy.

Effects of controlled interruption of the enterohepatic circulation of bile salts by biliary diversion and by ileal resection on bile salt secretion, synthesis, and pool size in the rhesus monkey.

The effects of controlled interruption of the enterohepatic circulation (EHC) of bile salts by biliary diversion on bile volume, bile salt secretion and synthesis rates, bile salt pool size, and the relationship to fecal fat excretion were studied in 16 rhesus monkeys. Bile from a chronic bile fistula was returned to the intestine through an electronic stream-splitter which, by diverting different percentages of bile to a collecting system, provided graded and controlled interruption of the EHC. The increase in hepatic bile salt synthesis in response to interruption of the EHC was limited and reached a maximum rate at 20% interruption of the EHC. Up to this level of biliary diversion, the increased hepatic synthesis compensated for bile salt loss so that bile salt secretion and pool size were maintained at normal levels. With diversion of 33% or more, there was no further increase in hepatic bile salt synthesis to compensate for external loss, and as a result there was diminished bile salt secretion, a reduction in bile salt pool size, and steatorrhea was observed. The effects of interruption of the EHC by the streamsplitter were compared with those produced by resection of the distal one-third or two-thirds of small bowel. While ileal resection appreciably reduced bile salt secretion, the EHC was by no means abolished. Bile salt reabsorption from the residual intestine was greater after one-third than after two-thirds small bowel resection. These observations suggest that jejunal reabsorption of bile salts occurs and may well contribute to the normal EHC.

Biliary lipid secretion and bile composition after acute and chronic interruption of the enterohepatic circulation in the Rhesus monkey. IV. Primate biliary physiology.

Bile salts and phospholipids are both required to solubilize biliary cholesterol. Since interruption of the enterohepatic circulation (EHC) depletes bile of bile salts, we have examined in the rhesus monkey the effects of controlled interruption of the EHC on biliary secretion of bile salt, phospholipid, and cholesterol and on the relative proportions of these components in bile. Immediately after complete interruption of the EHC, bile secretion and bile composition remained normal for 2-3 hr. During the next 3 hr, however, secretion of all components decreased. Bile salt decreased to a greater extent than phospholipid and cholesterol, and the bile was now supersaturated with cholesterol. 12-24 hr after interruption of the EHC, a new steady state was reached in which there was a relative deficiency of bile salt and a relative increase in phospholipid and cholesterol. The resulting bile, although somewhat more saturated with cholesterol, was not supersaturated with cholesterol but was stable with respect to cholesterol solubility. Thus, bile instability conducive to gallstone formation occurs transiently within hours after interruption of the EHC. Prolonged large interruptions in the steady state animal also produce a relative bile salt deficiency, but in this situation cholesterol remains soluble in the bile of these animals because there occurs a concomitant relative increase in phospholipid. When the EHC was only partially interrupted, secretion rates and the relative concentration of bile salt, phospholipid, and cholesterol did not change significantly from control values until more than 20% of the bile was diverted. Modest changes in the relative composition of bile occurred when 33 and 66% of the bile was diverted, and these changes were very similar to those produced by resection of the distal small bowel.

Interaction of collagen with the lipids of tendon xanthomata.

To determine the physical state of lipids in tendon xanthomata, six specimens surgically removed from three patients with familial hypercholesterolemia were studied by microscopy, calorimetry, and x-ray diffraction. The major constituents of the xanthomata were lipid (33% of dry weight) and collagen (24% of dry weight). The principal lipids were cholesterol ester and cholesterol. Light microscopy and thin-section electron microscopy showed occasional clusters of foam cells separated by masses of extracellular collagen. Polarized light microscopy of fresh, minced tissue showed rare droplets of free cholesterol ester. When heated, the tissue shrank abruptly at approximately equal to 70 degrees C and, consequently, a large amount of cholesterol ester was released. Scanning calorimetry of fresh pieces of xanthoma showed a single, broad, reversible liquid crystalline transition of cholesterol ester with peak temperature from 32 to 38 degrees C. The enthalpy (0971 +/- 0.07 cal/g) was reduced compared with the isolated cholesterol ester from each xanthoma (1.1+/-0.01 cal/g). There was a large irreversible collagen denaturation endotherm (peak temperature = 67 degrees C; enthalpy 9.9 cal/g collagen) that corresponded to the tissue shrinkage noted by microscopy. After the collagen denaturation, the sample displayed double-peaked reversible liquid crystalline transitions of cholesterol ester, of enthalpy 1.18 +/- 0.1 cal/g, that were identical to transitions of isolated cholesterol ester. Fibers dissected fron xanthomata were examined by X-ray diffraction at temperatures below and above the cholesterol ester transition. At 20 degrees C there was a weakly oriented equatorial reflection of Bragg spacing 36A, which corresponded to the smectic phase of cholesterol ester, and a series of oriented collagen reflections. At 42 degrees C the cholesterol ester reflection disappeared. Stretched fibers examined at 10 degrees C showed good orientation of collagen and cholesterol ester reflections, and in addition, meridional spacings which indicated oriented crystallization of cholesterol ester. These studies suggest that a major component of tendon xanthomata is extracellular cholesterol ester which displays altered melting and molecular orientation as a result of an interaction with collagen. At xanthoma temperatures, the cholesterol ester is in a smectic liquid crystalline state, probably layered between collagen fibrils, with the long axis of the cholesterolester molecules perpendicular to the axis of the collagen fiber. Such collagen-cholesterol ester interactions may favor the extracellular deposition of cholesterol ester derived either from intracellular sources or directly from plasma lipoproteins.

Effects of taurodihydrofusidate, a bile salt analogue, on bile formation and biliary lipid secretion in the rhesus monkey.

Bile salts play a major role in bile formation and biliary lipid secretion. Sodium taurodihydrofusidate (TDHF), a derivative of the antibiotic fusidic acid, closely resembles bile salts in terms of structure, micellar characteristics, and capacity ot solubilize otherwise insolbule lipids. We have therefore studied the biliary secretion of this bile salt analogue and its influence on bile formation and biliary lipid secretion in primates. Alert, unanesthetized female rhesus monkeys prepared with a total biliary fistula were allowed to reach a steady bile salt secretion rate before each study. In three animals (group I),[14C]TDHF was infused intravenously. Most of the compound was secreted rapidly in bile chemically unchanged. The biliary secretion of this drug produced a twofold increase in bile flow; however, the bile salt output was markedly reduced during the infusion. In spite of this reduction, the phospholipid output remained essentially unchanged whereas the cholesterol output increased almost twofold. In five other animals (group II), the effect of TDHF on the bile salt secretion was further investigated by an intravenous infusion of [14C]taurocholate followed by a combined infusion of [14C]taurocholate and TDHF. When TDHF was added to the infusate, a reduction in the [14C]taurocholate output and a progressive rise in the plasma [14C]taurocholate concentration were observed in each animal. An analysis of the data in both groups indicates that (a) the most likely explanation to account for the decreased bile salt output is that the bile salt analogue, TDHF, interfered with bile salt secretion into the biliary canaliculi; (b) TDHF induces a greater secretion of biliary water than was observed with bile salts, an effect consistent with a stimulation of the bile salt-independent canalicular flow; (c) at similar 3alpha-hydroxysteroid secretion rates TDHF caused a significant increase in cholesterol secretion compared to that induced by bile salt. This finding suggests that TDHF affects cholesterol metabolism or secretion in a way distinct from bile salts. Thus, the solubilization of biliary lipids in mixed micelles, although essential, is only one of the factors which determine their secretion into bile.

Physical chemistry of the lipids of human atherosclerotic lesions. Demonstration of a lesion intermediate between fatty streaks and advanced plaques.

95 individual human atherosclerotic lesions from 26 persons were classified into three groups under the dissecting microscope: fatty streaks, fibrous plaques, and gruel (atheromatous) plaques. Each lesion was isolated by microdissection, its lipid composition determined by chromatography, and the physical states of the lipids identified by polarizing microscopy and in some cases by X-ray diffraction. The composition of each lesion was plotted on the in vitro phase diagram of the major lipids of plaques: cholesterol, cholesterol ester, and phospholipid. The observed physical states were compared with those predicted by the location of the lipid composition on the phase diagram. The most severe lesions (gruel plaques) had an average lipid composition of cholesterol 31.5+/-1.9%, cholesterol ester 47.2+/-2.3%, and phospholipid 15.3+/-0.5%. Their compositions fell within the three-phase zone of the phase diagram, predicting the lipids to be separated into a cholesterol crystal phase, a cholesterol ester oily phase and a phospholipid liquid crystalline phase. In addition to the phospholipid liquid crystalline phase of membranes and myelin-like figures demonstrable by electron microscopy, polarizing microscopy revealed the other two predicted phases, isotropic cholesterol ester-rich droplets and cholesterol crystals. X-ray diffraction studies verified the identity of the crystals as cholesterol monohydrate. Fibrous plaques also had an average lipid composition within the three-phase zone of the phase diagram. Polarizing microscopy revealed the presence of cholesterol monohydrate crystals and lipid droplets in all of these lesions; the droplets were predominately isotropic in 28 of the 31 fibrous plaques. Although these lesions had less free cholesterol and more cholesterol ester than gruel plaques, they were otherwise similar. Fatty streaks had compositions within both the two- and three-phase zones of the phase diagram. Compared with gruel plaques, the fatty streaks within the two-phase zone, defined as "ordinary," had more cholesterol ester, less free cholesterol, a higher cholesteryl oleate/cholesteryl linoleate ratio, a lower sphingomyelin/lecithin ratio, more anisotropic lipid droplets, and rare or no cholesterol crystals. Those lesions within the three-phase zone had many chemical and physical features intermediate between ordinary fatty streaks and gruel plaques. Moreover, 68% of these "intermediate" lesions had no cholesterol crystals by polarizing microscopy in spite of their compositions being within the three-phase zone, indicating the cholesterol ester oily phase or the phospholipid phase or both were supersaturated with cholesterol. Identification of this group of intermediate lesions provides evidence that some fatty streaks may be precursors of advanced plaques.

Physicochemical and histological changes in the arterial wall of nonhuman primates during progression and regression of atherosclerosis.

To identify the temporal changes occurring during progression and regression of atherosclerosis in nonhuman primates, we have studied the physicochemical and histological characteristics of arterial wall lesions during a 30-mo progression period of diet-induced hypercholesterolemia and during a 12-mo period of regression. Three groups of cynomolgous monkeys (Macaca fascicularis) were studied. Control groups were fed a basal chow diet for 18, 24, and 30 mo and were compared with progression groups that were fed a high-cholesterol-containing diet for up to 30 mo. Regression groups were fed a high-cholesterol diet for 18 mo to induce atherosclerosis and then fed monkey chow for up to 12 mo. The progression group monkeys were killed at 6, 12, 18, 24, and 30 mo, and the regression animals were killed at 24 and 30 mo (i.e., after 6 and 12 mo of being fed a noncholesterol-containing chow diet). Histology and morphometry, physical microscopy for cholesterol monohydrate crystals, foam cell and droplet melting points and chemical composition studies were completed on a large number of individual arterial lesions. Control animals had very little cholesterol ester, rare foam cells, and no extracellular cholesterol ester droplets or cholesterol crystals. During progression, the arteries first increased cholesterol ester content to produce high melting (approximately 45 degrees C) foam cell-rich lesions essentially devoid of cholesterol crystals. With time, the number of cholesterol crystals increased so that by 30 mo large numbers were present. Foam cells decreased with time but their melting temperature remained high while that of extracellular droplets fell to approximately 38 degrees C. Between 18 and 30 mo necrosis appeared and worsened. After 6-mo regression, unexpected changes occurred in the lesions. Compared with 24-mo progression, the chemical composition showed a relative increase in free cholesterol, a decrease in cholesterol ester and microscopy revealed large numbers of cholesterol crystals. Concomitantly, foam cells decreased and the melting temperature of both intra- and extracellular cholesterol ester markedly decreased. After 12-mo regression cholesterol decreased, cholesterol crystals and necrosis diminished and collagen appeared increased. Thus, during progression there is initially an increase in the number of foam cells containing very high-melting intracellular cholesterol ester droplets. By 30 mo, cholesterol crystals and necrosis dominate and high-melting foam cells appear only at lesion margins, suggesting that the initial process continues at the lesion edge. The lower melting point of extracellular esters indicates a lipid composition different from intracellular droplets. Thus, the changes observed in these animals generally reflect those predicted for progression of human atherosclerosis. During the initial 6 mo of regression, necrosis remains, the number of foam cell decreases, and cholesterol ester content decreases; however the relative proportion of free cholesterol content increases, and large numbers of cholesterol content are formed. Thus, large and rapid decreases in serum cholesterol concentration to produce regression in fact may result in the precipitation of cholesterol monohydrate and an apparent worsening of the lesions. More prolonged regression (12-mo) tends to return the lipid composition of the artery wall towards normal, partially reduces cholesterol crystals, and results in an improved but scarred intima.

Studies on the structure of low density lipoproteins isolated from Macaca fascicularis fed an atherogenic diet.

Cynomolgus monkeys, Macaca fascicularis, fed cholesterol-containing saturated-fat diets develop increased levels of high molecular weight plasma low density lipoproteins (LDL), associated with accelerated atherosclerosis. To study the composition and structure of these abnormal particles, LDL from monkeys, fed atherogenic and control diets, were characterized chemically and examined by differential scanning calorimetry and low-angle X-ray scattering. LDL from animals on the experimental diet showed an increase in molecular weight (4.0 to 7.0 x 10(6), experimental diet compared with 3.0 to 3.7 x 10(6), control diet) associated with a large increase in cholesterol ester content and concomitant smaller increases in protein, phospholipid, and free cholesterol. There was a strong positive correlation between molecular weight and the number of saturated and monounsaturated cholesterol esters in the particle. In contrast, particle content of polyunsaturated cholesterol esters remained constant despite large changes in total particle cholesterol esters.When examined by calorimetry and X-ray scattering, LDL from monkeys on both diets diplayed a reversible transition of cholesterol esters from an ordered smeticlike (layered) structure to a more disordered state. For all animals on the experimental diet, the peak temperature of the cholesterol-ester transition (42-48 degrees C) was above body temperature (39 degrees C), but below body temperature on the control diet (34-38.5 degrees C). In the experimental group, the transition temperature was correlated with the LDL molecular weight. However, after thermal disruption of LDL, liquid-crystalline transitions of LDL cholesterol esters were observed in the same temperature range as in the intact lipoprotein, which shows that changes in particle size had little effect on the cholesterol-ester transition temperature. Rather, the transition temperature was determined by the degree of saturation of the LDL cholesterol ester fatty acids and the LDL cholesterol ester: triglyceride ratio, both of which correlated with increased LDL molecular weight.The existence of smectic-like cholesterol ester in LDL at body temperature was clearly a discriminating feature between monkeys on control and experimental diets. Diet-induced changes in the lipid composition of precursor lipoproteins of LDL appeared to lead to the existence of smectic-like cholesterol ester in LDL above body temperature. The altered composition and structure of the core lipids of high molecular weight LDL probably account, in part, for the previously documented correlation between increased LDL molecular weight and atherosclerosis in this species.

The storage lipids in Tangier disease. A physical chemical study.

The physical states and phase behavior of the lipids of the spleen, liver, and splenic artery from a 38-yr-old man with Tangier disease were studied. Many intracellular lipid droplets in the smectic liquid crystalline state were identified by polarizing microscopy in macrophages in both the spleen and liver, but not in the splenic artery. The droplets within individual cells melted sharply over a narrow temperature range, indicating a uniform lipid composition of the droplets of each cell. However different cells melted over a wide range, 20-53 degrees C indicating heterogeneity of lipid droplet composition between cells. Furthermore, most of the cells (81%) had droplets in the liquid crystalline state at 37 degrees C. X-ray diffraction studies of splenic tissue at 37 degrees C revealed a diffraction pattern typical of cholesterol esters in the smectic liquid crystalline state. Differential scanning calorimetry of spleen showed a broad reversible transition from 29-52 degrees C, with a maximum mean transition temperature at 42 degrees C, correlating closely with the polarizing microscopy observations. The enthalpy of the transition, 0.86+/-0.07 cal/g of cholesterol ester, was quantitatively similar to that of the liquid crystalline to liquid transition of pure cholesterol esters indicating that nearly all of the cholesterol esters in the tissue were free to undergo the smectic-isotropic phase transition. Lipid compositions of spleen and liver were determined, and when plotted on the cholesterol-phospholipid-cholesterol ester phase diagram, fell within the two phase zone. The two phases, cholesterol ester droplets and phospholipid bilayers were isolated by ultracentrifugation of tissue homogenates. Lipid compositions of the separated phases approximated those predicted by the phase diagram. Extracted lipids from the spleen, when dispersed in water and ultracentrifuged, underwent phase separation in a similar way. Thus (a) most of the storage lipids in the liver and spleen of this patient were in the liquid crystalline state at body temperature, (b) the phase behavior of the storage lipids conformed to that predicted by lipid model systems indicating lipid-lipid interactions predominate in affected cells, (c) lipid droplets within individual cells have similar compositions, whereas droplet composition varies from cell to cell, and (d) cholesterol ester does not accumulate in the splenic artery. Since Tangier patients lack high density lipoprotein, we conclude that high density lipoprotein-mediated cholesterol removal from cells is essential only for those cells which have an obligate intake of cholesterol (macrophages).

Filamentous, helical, and tubular microstructures during cholesterol crystallization from bile. Evidence that cholesterol does not nucleate classic monohydrate plates.

Precipitation of cholesterol in gallbladder bile is believed to produce platelike cholesterol monohydrate crystals directly. We report complementary time-lapse microscopic studies of cholesterol crystallization from model bile that reveal initial assembly of filamentous cholesterol crystals covered by a monomolecular layer of lecithin. Over a few days, the filaments evolved through needle, helical, and tubular microstructures to form classical platelike cholesterol monohydrate crystals. Similar crystallization phenomena were observed in human gallbladder biles from cholesterol but not pigment stone patients. Synchrotron x-ray diffraction of the earliest filaments suggested a cholesterol monohydrate polymorph or admixture with an anhydrous cholesterol precursor. However, density gradient centrifugation of filamentous crystals revealed that their density was 1.032 g/ml, consistent with anhydrous cholesterol. Conventional x-ray diffraction of transitional crystalline forms was consistent with pure cholesterol monohydrate crystals, as were the equilibrium platelike crystals. These novel findings suggest that crystalline cholesterol in bile may not be completely mature or hydrated initially, but undergoes a series of transformations to become thermodynamically stable monohydrate plates. These observations have important implications for understanding the control of cholesterol crystallization in bile, as well as explaining putative crystal cytotoxicity during gallstone formation.