Determinants of Doppler indexes of left ventricular diastolic function in normal subjects (the Framingham Heart Study).

Normative Doppler values and determinants of left ventricular (LV) diastolic function in healthy subjects have not been fully elucidated. Subjects from the Framingham Heart Study were examined to describe reference values and determinants of echocardiographic Doppler indexes of diastolic function. One hundred twenty-seven randomly selected, rigorously defined, normal subjects, approximately evenly distributed by sex and age from the third through the eighth decades were studied by Doppler echocardiography. Normative values for 7 frequently used Doppler indexes of LV diastolic function are presented. Doppler indexes of LV diastolic function change dramatically with age; the peak velocity of early filling divided by late filling (peak velocity E/A) ranges from a mean of 2.08 +/- 0.55 for subjects in their third decade to 0.84 +/- 0.29 for those in their eighth decade. A peak velocity E/A ratio less than 1 is abnormal in subjects aged less than 40 years, but occurs in most subjects aged greater than or equal to 70 years. The high correlations between age and Doppler indexes of LV diastolic function are not greatly attenuated after adjustment for other clinical parameters associated with diastolic function; the multivariate partial correlation coefficient between age and peak velocity E/A is -0.80 (p less than 0.0001). Heart rate, PR interval, LV systolic function, sex and systolic blood pressure are minor determinants of Doppler indexes of diastolic function. Body mass index, left atrial diameter, and LV wall thickness, internal dimension and mass have little or no association with Doppler indexes in healthy subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitral annular calcification and the risk of stroke in an elderly cohort.

BACKGROUND: Previous clinical studies have suggested that there is an association between mitral annular calcification and the risk of stroke, but it is unclear whether this association is independent of the traditional risk factors for stroke. We examined the relation between mitral annular calcification and the incidence of stroke in a population-based study. METHODS: Subjects in the Framingham Study receiving a routine examination underwent M-mode echocardiography to determine the presence and severity (thickness in millimeters) of mitral annular calcification. The incidence of stroke during eight years of follow-up was analyzed with a proportional-hazards model adjusting for the calcification, age, sex, systolic blood pressure, diabetes mellitus, cigarette smoking, atrial fibrillation, and coronary heart disease or congestive heart failure. RESULTS: Among 1159 subjects whose echocardiograms could be assessed for mitral annular calcification and who had no history or current evidence of stroke at the index examination (51 percent of all subjects), the prevalence of mitral annular calcification was 10.3 percent in the men and 15.8 percent in the women. Multivariate analysis demonstrated that the presence of mitral annular calcification was associated with a relative risk of stroke of 2.10 (95 percent confidence interval, 1.24 to 3.57; P = 0.006). There was a continuous relation between the incidence of stroke and the severity of mitral annular calcification; each millimeter of thickening as shown on the echocardiogram represented a relative risk of stroke of 1.24 (95 percent confidence interval, 1.12 to 1.37; P less than 0.001). Furthermore, even when subjects with coronary heart disease or congestive heart failure were excluded from the analysis, subjects with mitral annular calcification still had twice the risk of stroke. CONCLUSIONS: In an elderly, longitudinally followed population-based cohort, mitral annular calcification was associated with a doubled risk of stroke, independently of traditional risk factors for stroke. Whether such calcification contributes causally to the risk of stroke or is merely a marker of increased risk because of its association with other precursors of stroke remains unknown.

Separation of neutral lipid, free fatty acid and phospholipid classes by normal phase HPLC.

Normal phase high performance liquid chromatography methods are described for the separation of neutral lipid, fatty acid and five phospholipid classes using spectrophotometric detection at 206 nm. Separations were accomplished in less than 10 min for each lipid class. A mobile phase consisting of hexane/methyltertiarybutylether/acetic acid (100:5:0.02) proved effective in separating cholesteryl ester and triglyceride with recoveries of 100% for radiolabeled cholesteryl oleate and 98% for radiolabeled triolein. Free fatty acid and cholesterol were separated by two different mobile phases. The first, hexane/methyltertiarybutylether/acetic acid (70:30:0.02) effectively separated free fatty acids and cholesterol, but did not separate cholesterol from 1,2-diglyceride. A mobile phase consisting of hexane/isopropanol/acetic acid (100:2:0.02) effectively separated free fatty acid, cholesterol, 1,2-diglyceride and 1,3-diglyceride. Recoveries of oleic acid and cholesterol were 100% and 97%, respectively. Five phospholipid classes were separated using methyltertiarybutylether/methanol/aqueous ammonium acetate (pH 8.6) (5:8:2) as the mobile phase. The recoveries of phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine were each greater than 96%.

Rapid separation of neutral lipids, free fatty acids and polar lipids using prepacked silica Sep-Pak columns.

A method is described for the separation of neutral lipid, free fatty acid and polar lipid classes using small (600 mg), prepacked silica Sep-Pak columns. Combinations of hexane and methyltertiarybutylether were used to progressively elute cholesteryl ester first then triglyceride from the column. After column acidification, fatty acids were eluted followed by cholesterol. Recoveries of these lipids were 96% or greater. Polar lipids were eluted from the column using combinations of methyltertiarybutylether, methanol and ammonium acetate. Phospholipid classes could not be separated completely from each other. Phosphatidylethanolamine and phosphatidylinositol eluted together, whereas the more polar phosphatidylcholine, sphingomyelin and lysophosphatidylcholine were eluted as a second fraction. Recoveries of each phospholipid was greater than 98%.

Effects of cholesterol and 25-hydroxycholesterol on smooth muscle cell and endothelial cell growth.

Auto-oxidation products of cholesterol may play a role in atherogenesis. In order to determine whether cholesterol or 25-hydroxycholesterol, a cholesterol auto-oxidation product, affected growth of vessel wall cells, sparse and confluent cultures of rabbit thoracic aorta smooth muscle cells and human umbilical vein endothelial cells were exposed to these compounds for 88 hr. The compounds were administered at 10(-4), 10(-5), 10(-6) or 10(-7) M in either ethanol or fetal bovine serum (FBS) vehicle. Cells were counted electronically, and the results were expressed as the percent growth in experimental vs control wells. Cholesterol did not inhibit cell growth under any experimental condition. 25-Hydroxycholesterol had the following effects: inhibited confluent smooth muscle cell growth at 10(-4) M in ethanol vehicle only; inhibited sparse smooth muscle cell growth in a dose-related manner at 10(-4), 10(-5) and 10(-6) M in ethanol vehicle, but in FBS vehicle inhibited at only 10(-4) and 10(-5) M; inhibited confluent human umbilical vein endothelial cells at 10(-4) M in ethanol vehicle only; and inhibited sparse human umbilical vein endothelial cell growth at 10(-4) and 10(-5) M in ethanol vehicle only. Thus, rabbit aortic smooth muscle cell growth was more sensitive to inhibition by 25-hydroxycholesterol than human umbilical vein endothelial cell growth was.

Sebum-suppressing activity of the nonpolar arotinoid Ro 15-0778 in rodents.

Retinoids are known to modulate sebaceous gland activity in humans and animals. The nonpolar arotinoid Ro 15-0778 [(E)-1,2,3,4-tetrahydro-1,1,4,4-tetramethyl-6-(1-methyl-2-phenylethen yl) naphthalene] does not contain a polar end group and is devoid of the classical retinoid side effects of hypervitaminosis A. The favorable toxicological profile stimulated the evaluation of this arotinoid in animal models of sebum production. In castrated, testosterone-stimulated male rats, Ro 15-0778 is 50 times more potent than 13-cis-retinoic acid in inhibiting the production and subsequent secretion of sebum. The oral ED50 value of Ro 15-0778 is 30 micrograms/kg, while an oral dose of 0.5 mg/kg inhibited sebum secretion nearly 100%. In testosterone-stimulated female rats, Ro 15-0778 inhibits sebum secretion significantly with an oral ED50 of 140 micrograms/kg and an s.c. ED50 of 75 micrograms/kg. Ro 15-0778 was also evaluated for its ability to prevent testosterone induction of the immature hamster flank organ. The topical ED50 is 0.53 mg/kg and the oral ED50 is 38 mg/kg. This arotinoid is similarly active in mature male hamsters without testosterone treatment. In addition, the retinoid is active topically and orally in reducing the size of the gerbil abdominal sebaceous gland. The compound exhibits no antiandrogenic activity when tested in ventral prostrate and seminal vesicle assays in rats. Additionally, the compound does not have estrogenic activity when tested in the rat uterine weight assay. High doses of Ro 15-0778 in humans did not demonstrate significant sebum-suppressing activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection.

Normal phase, isocratic high-performance liquid chromatography methods are described for the separation of neutral lipid and fatty acid classes using low wavelength detection. Prior to high-performance liquid chromatography, methods were developed and are described for the separation of phospholipids from neutral lipids and fatty acids using small (600 mg) silica Sep-PaksTM. Recoveries of cholesteryl esters, triglycerides, fatty acids, and phospholipids from the silica columns were greater than 95%. Two mobile phases are described for lipid class separation by high-performance liquid chromatography. The first mobile phase, hexane-2-propanol-acetic acid 100:0.5:01, resulted in incomplete separation of cholesteryl ester and triglyceride but excellent separations of fatty acids and cholesterol. The second mobile phase, hexane-n-butyl chloride-acetonitrile-acetic acid 90:10:1.5:0.01, resulted in complete separation of the four lipid classes. This mobile phase also separated individual triglycerides and fatty acids based on the number of double bonds. Recoveries of radiolabeled lipids for the four lipid classes from high-performance liquid chromatography was greater than 95% with both mobile phases.

Effects of diet and obesity on brown adipose metabolism.

The effect of diet-induced obesity on interscapular brown adipose tissue (IBAT) was assessed after feeding male Sprague-Dawley rats a high-fat diet for 3-5 mo beginning at 3 mo of age. IBAT pads in 6-mo-old obese rats were heavier (22%), had more lipid (71%), and larger unilocular cells (38%) than chow-fed controls. Mitochondrial morphology, beta-adrenergic receptor binding ([ 125I]iodocyanopindolol), and norepinephrine-stimulated lipolysis were similar in IBAT from obese and control rats. When 8-mo-old chow-fed rats were switched to the high-fat diet for 7-14 days, IBAT pads became hypercellular without cell hypertrophy and with a 70% increase in norepinephrine-induced lipolysis. However, when 8-mo-old obese rats that had been on the high-fat diet for 5 mo were switched to chow for 3 days, IBAT cellularity was unchanged, but norepinephrine-induced lipolysis was increased 70%. Therefore, in lean and obese 6- to 8-mo-old rats, short-term dietary manipulation led to metabolic activation, whereas chronic diet-induced obesity on a stable diet was associated with a return of IBAT metabolism to control levels.

Effect of phenoxybenzamine on development of adipose tissue in lean and obese Zucker rats.

Young male Zucker lean (Fa/-) and obese (fa/fa) rats were fed the alpha-adrenergic blocking agent phenoxybenzamine as a dietary admixture for 35 days. In lean and obese rats, phenoxybenzamine treatment decreased significantly body weight gain, food consumption, grams of carcass fat, and grams of carcass protein. Lean rats exhibited reduced fat cell size and number in retroperitoneal, epididymal, and inguinal fat depots. Obese rats treated with phenoxybenzamine exhibited significantly decreased numbers of fat cells in the retroperitoneal, epididymal, and inguinal fat depots and a small decreased cell size in the inguinal fat depot only. The levels of carcass fat and protein and fat cell number in obese and lean rats treated with phenoxybenzamine for 35 days were similar to pretreatment values in agreement with the lack of body weight gain. Although values in agreement with the lack of body weight gain. Although rats exhibited marked decreases in fat accumulation during phenoxybenzamine treatment, fat cell size and number returned to control values during the posttreatment period with a marked hyperplasia occurring particularly in the retroperitoneal fat depot of obese rats. Serum levels of insulin were suppressed and free fatty acid levels increased in obese rats during phenoxybenzamine treatment, suggesting a stimulation of the sympathoadrenal system. This study shows that despite severe restrictions in fat cell proliferation during the rapid-growth phase of the obese Zucker rat, the mechanisms for cellular proliferation and fat deposition remain intact.

Abnormal brown adipose composition and beta-adrenoceptor binding in obese Zucker rats.

The composition, morphology, beta-adrenergic receptor binding, and in vitro lipolysis were examined in lean and obese, 5- to 6-mo-old male Zucker rat interscapular brown adipose tissue (IBAT). IBAT pads from obese rats were heavier (283%), had more lipid (700%), and more (75%)( and larger (83%) adipocytes than those from lean rats. Also, IBAT from obese rats had no multiloculated cells, and 50% of their IBAT adipocytes were the size of white fat cells. High affinity binding for (-)-[3H]dihydroalprenolol (KD, 15-18 nM), as well as the estimated KD values for binding and the 1/2 Vmax values for adrenergic agonist-induced lipolysis were similar in isolated IBAT cells from lean and obese rats. However, adipocytes from IBAT in obese rats had 75% fewer high affinity beta-adrenergic binding sites per cell (Bmax) compared to those in lean rats. These findings are most compatible with the infiltration of IBAT by white adipocytes. Such infiltration would be expected to reduce the overall thermogenic capacity of IBAT in obese Zucker rats and thereby contribute to the maintenance of their obesity.

Phenethylamine inhibitors of partially purified rat and human pancreatic lipase.

Methodology for the preparation of rat and human pancreatic lipase (EC 3.1.1.3) is described, which resulted in good yield of partially purified, stable enzyme useful for kinetic studies. Apparent Km values for the rat (6.5 mM) and human (3.5 mM) enzyme were determined with triolein as the substrate. Several compounds of the phenethylamine class were found to be inhibitors of both rat and human pancreatic lipase. The structural feature in the phenethylamine series tested, which appeared to be necessary for lipase inhibition, was a halogenated substituent on the 3 or 4 position of the aromatic ring as in flutiorex, fenfluramine, N-benzyl-beta-methoxy-3-(trifluoromethyl)phenethylamine (I), chlorphentermine and p-chloroamphetamine. A chloro group at the 2 position was ineffective (chlortermine). Alterations in the ethylamine portion of the molecule did not cause significant changes in the inhibitory properties of the active phenethylamines.

Metabolic and sympatho-adrenal abnormalities in the obese Zucker rat: effect of chronic phenoxybenzamine treatment.

The obese Zucker rat manifests a number of physiologic and metabolic abnormalities which are controlled or modulated by the sympatho-adrenal system. The interrelationship of these was examined by subjecting 3-4 month old male, homozygous lean and obese Zucker rats to various stresses which are known to activate the sympatho-adrenal system, and by chronic (16-19 days) phenoxybenzamine (PBZ) treatment to block alpha-adrenergic receptors. Both obese and lean PBZ treated rats gained only 1% and 10% of the body weight of their respective control rats during the treatment period, while only the lean rats had a significant reduction (20%) in food intake. Control obese rats failed to maintain rectal temperature after 4 hr at 7 degrees C and their relative output of plasma catecholamines (CA) to cold stress, as measured from indwelling atrial cannulae, was decreased. PBZ treatment did not alter this rectal temperature response although it was associated with increased baseline norepinephrine levels (at ambient temperature 21-22 degrees C) and relative output of CA in the obese rats, suggesting tat sympathetic neural activity was increased under these circumstances. No abnormalities of sympatho-adrenal function, as reflected in plasma CA levels, were found in treated or control obese rats immobilization for 1 hr followed by decapitation. Simultaneously obtained baseline plasma glucose levels were similar in untreated lean and obese rats, but insulin and glycerol levels in the obese rat were 1350% and 213% of lean values, respectively. During sequential stresses, the obese rats became markedly hyperglycemic and hyperglycerolemic compared to the lean rats, while insulin levels were decreased more in the obese than lean rats (12-15% versus 34-35% of controls, respectively). PBZ affected insulin levels only in the obese rats, reducing their baseline levels by 4-fold and stressed induced levels to those seen in the lean control rats. These results suggest that some of the metabolic and physiologic abnormalities of the obese Zucker rat which are modulated by the sympatho-adrenal system can be normalized by procedures which increase sympatho-adrenal activity.

Antiobesity activity of pluronic L-101.

Pluronic L-101, a hydrophobic surface-active agent, was a potent in-vitro inhibitor of human pancreatic lipase. When administered as a 1 percent or 3 percent dietary admix to meal-fed rats, pluronic L-101 produced a significant and dose-dependent decrease in body-weight gain while not affecting food consumption. Excretion of dietary fat in the feces was enhanced significantly in a dose-dependent manner during pluronic L-101 treatment. Pluronic F-68, a hydrophilic surface-active agent which was a poor in-vitro inhibitor of human pancreatic lipase, did not produce decreases in body-weight gain or increase fecal-fat excretion in rats. The decreased body-weight gain produced by pluronic L-101 was reflected in a decreased percentage of carcass fat; the percentage of carcass protein was unchanged. Liver wet weight was reduced significantly only at the 3 percent pluronic L-101 level. Serum levels of cholesterol, triglyceride and glucose remained unchanged. During pluronic L-101 treatment no overt signs of toxicity were observed.

Caloric compensatory responses to diets containing either nonabsorbable carbohydrate or lipid by obese and lean Zucker rats.

The effects on food intake and body weight in lean and obese Zucker rats were evaluated following substitution in the diet with either 1) poorly absorbable lipid (hydrogenated soybean oil) for corn oil, or 2) nonabsorbable carbohydrate (fiber) for glucose. Lean Zucker rats compensated for the reduced caloric availability of the high-fiber and hydrogenated oil diets by increasing food consumption. In contrast, obese rats did not respond significantly to these dietary alterations and failed to attain caloric balance during the 16-day study. These differences in caloric compensatory responses were reflected in body weight gains. There were no differences in the amount of weight gained by lean rats fed either the high-or low-fiber diets because of compensatory hyperphagia in the high-fiber group. Lean rats fed the hydrogented oil diets gained less weight than controls fed corn oil diets, even in the presence of compensatory hyperphagia, because of the enhanced fecal excretion of water and metabolites caused by the poorly absorbed fat diet. As a result of a delayed and incomplete response to reduced caloric availability, obese rats fed the high-fiber and hydrogenated oil diets gained significantly less weight than the obese rats fed low-fiber and corn oil diets.

Pharmacological treatment of obesity.

Obesity results when the ingestion of energy exceeds its utilization, leading to an excessive expansion of the adipose tissue mass. Current pharmacological therapy for the obese patient focuses primarily on reducing energy intake. Anorectic agents reduce food consumption by modifying central systems in the brain which are involved in appetite regulation. These agents are reviewed in terms of mechanism of action, and clinical safety and efficacy in suppressing appetite and promoting weight loss. Newer anorectic agents which are being evaluated currently in clinical and animal studies are described. Clinical assessments of therapeutic regimens utilizing the thyroid hormones and human chorionic gonadotropin are evaluated. Finally, an overview of novel pharmacological approaches to the treatment of obesity is presented.

Analyses of renal medullary lipid droplets from normal, hydronephrotic, and indomethacin treated rabbits.

Lipid droplets isolated from rabbit renal medullary tissue were analyzed and found to be composed of triglyceride and free fatty acids in a ratio of 2.9:1. These triglycerides were unique when compared to triglycerides of other rabbit tissues examined, in that they contained high percentages of octadecanoic acid (stearic acid, 9.8%), 5,8,11,14-eicosatetraenoic acid (arachidonic acid, 6.8%), and 7,10,13,16-docosatetraenoic acid (adrenic acid, 10%). Lipid droplet triglycerides were found to increase during experimental hydronephrosis and after administration of indomethacin, a prostaglandin synthetase and phosphodiesterase inhibitor. From gas liquid chromatography of fatty acid methyl esters of these triglycerides, it was determined that they were enriched further in their percent composition of 9,12-octadecadienoic acid (linoleic acid) and arachidonic acid, a prostaglandin precursor. The inverse relationship between lipid droplets and prostaglandin content in the inner medulla suggested a significant role of lipid droplet triglycerides as storage pools for prostaglandin precursors.