The effects of catechol-O-methyltransferase inhibition on estrogen metabolite and oxidative DNA damage levels in estradiol-treated MCF-7 cells.

Many of the major identified risk factors for breast cancer are associated with exposure to endogenous estrogen. In addition to the effects of estrogen as a growth factor, experimental and epidemiological evidence suggest that catechol metabolites of estrogen also contribute to estrogen carcinogenesis by both direct and indirect genotoxic mechanisms. O-Methylation catalyzed by catechol-O-methyltransferase (COMT) is a Phase II metabolic inactivation pathway for catechol estrogens. We and others have found that a polymorphism in the COMT gene, which codes for a low activity variant of the COMT enzyme, is associated with an increased risk of developing breast cancer; therefore, the goal of the current study was to investigate the role of decreased COMT activity on estrogen catechol levels and on oxidative DNA damage, as measured by 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) levels. MCF-7 cells were pretreated with dioxin as a means to increase estrogen metabolism to catechol estrogens, then treated with estradiol (E2) +/- Ro 41-0960, a COMT-specific inhibitor. After extraction from culture medium, estrogen metabolites were separated using an high-performance liquid chromatography-electrochemical detection method. As expected, dioxin dramatically increased E2 oxidative metabolism, primarily to its 2-OH and 2-methoxy metabolites. The COMT inhibitor blocked 2-methoxy E2 formation. This was associated with increased 2-hydroxy E2 (2-OH E2) and 8-oxo-dG levels. In the presence of COMT inhibition, increased oxidative DNA damage was detected in MCF-7 cells exposed to as low as 0.1 microM E2, whereas in the absence of COMT inhibition, no increase in 8-oxo-dG was detected at E2 concentrations < or =10 microM. This study is the first to show that O-methylation of 2-OH E2 by COMT is protective against oxidative DNA damage caused by 2-OH E2, a major oxidative metabolite of E2.

Physiological predictors of increasing total and central adiposity in aging men and women.

BACKGROUND: Increasing levels of total and central body fat with advancing age contribute to the development of cardiovascular and metabolic disease. We examined gender-related differences and physiological predictors of the rate of increase in total and central body fat in men and women. METHODS: We studied 427 healthy men (age range, 17 to 90 years) and 293 women (age range, 18 to 88 years). We measured body fatness by hydrostatic weighing, central adiposity from the waist circumference, peak volume of oxygen utilization (VO2) from a treadmill test, leisure time physical activity (LTA) from a questionnaire, resting metabolic rate and respiratory quotient from indirect calorimetry, and energy intake from 3-day food diaries. RESULTS: Fat mass increased with age, and the rate was greater in women (r = .61; slope = 0.25 kg/y; P < .01) than in men (r = .43; slope = 0.16 kg/y; P < .01). Increasing fat mass in men and women was most strongly associated with declines in peak VO2 and LTA. Controlling for these variables reduced the increase in fat mass from 17% to 3% per decade in men and from 26% to 5% per decade in women. The increase in waist circumference with age was also greater in women (r = .53; slope = 0.28 cm/y) than in men (r = .39; slope = 0.18 cm/y; P < .01). Increasing waist circumference with age in men and women was most strongly associated with declines in LTA and peak VO2, respectively. Control for these variables reduced the age-related increase in waist circumference from 2% to 1% per decade in men and from 4% to 1% per decade in women. We observed no independent contribution of resting metabolic rate, respiratory quotient, menopause status, energy, or macronutrient intake to the age-related increase in fat mass and waist circumference. CONCLUSIONS: Our findings suggest that (1) the age-related increase in fat mass and waist circumference is greater in women than in men and (2) the physiological characteristics that reflect a decline in physical activity-related energy expenditure, rather than resting energy expenditure, are important predictors of the increases in total and central fatness. Lifestyle changes that increase the level of physical activity may be advantageous in blunting age-related increases in total and central body fatness.

Relationship between physical activity and HDL-cholesterol in healthy older men and women: a cross-sectional and exercise intervention study.

We used cross-sectional and exercise intervention studies to examine whether physical activity levels or increases in peak aerobic capacity (peak VO2) explain variation in high density lipoprotein cholesterol (HDL-C) levels in older men and women. In the cross-sectional study, 307 older individuals (169 men; 138 women; 67 +/- 7 years) were characterized for HDL-C, leisure time physical activity, peak VO2, body composition, body fat distribution and dietary intake. HDL-C was 19% higher (P < 0.001) in women (57 +/- 14 mg/dl) versus men (48 +/- 14 mg/dl). Thirty-two percent of the variation in HDL-C in older men was explained by the waist circumference (r2 = 16%), percent dietary intake of alcohol (r2 = 11%), and carbohydrate (r2 = 6%). Waist circumference was also the best predictor of HDL-C in older women, (r2 = 7%); with percent dietary intake of carbohydrate adding an additional 6% to the model. Neither peak VO2 nor leisure time physical activity were independent predictors of HDL-C. Statistical control for the aforementioned variables diminished, but did not abolish gender differences in HDL-C. Thirty-seven older individuals (23 men; 14 women) participated in a 2-month exercise program in which individuals by week eight were expending approximately 900 kcal per week in exercise energy expenditure. Subjects were maintained in energy balance throughout the exercise program. Endurance training significantly increased peak VO2 by 15% in both men and women, and by design, body composition and body fat distribution did not change. No changes in HDL-C levels were noted. In conclusion, variations in leisure time physical activity or increases in peak VO2 are not independent predictors of HDL-C levels in healthy older men and women. Instead, central adiposity, as estimated by the waist circumference, and to a lesser extent, dietary intake of carbohydrate and alcohol, are significant predictors of variation in plasma HDL-C levels. Furthermore, short-term exercise training, generating less than 900 kcal per week in exercise energy expenditure, in the absence of weight loss, fails to influence HDL-C levels.

Sarcopenia in aging humans: the impact of menopause and disease.

We examine the association of the menopause transition, congestive heart failure, and Parkinson's disease on body composition and energy expenditure. We present evidence suggesting that the normal menopausal transition is associated with accelerated loss of fat-free mass, a decline in resting metabolic rate, and increased central body fatness. Second, we show that the cardiac cachexia associated with heart failure is partially due to an elevated level of energy expenditure. Despite having a lower quantity of fat-free mass, congestive heart failure patients have a higher resting metabolic rate (approximately 283 kcal/d) for their metabolic size than healthy elderly. The elevated level of resting energy expenditure probably contributes to their unexplained weight loss. Parkinson's patients experience muscular rigidity and tremor which could contribute to inappropriately high levels of energy expenditure and difficulty in maintaining body weight and composition. We examined resting metabolic rate and body composition in eight Parkinson's patients and 34 healthy age-matched controls. Parkinson's patients showed lower levels of fat-free mass (approximately 6 kg), but similar resting metabolic rates (1601 +/- 250 kcal/d) versus healthy controls (1671 +/- 212 kcal/d), suggesting a hypermetabolic state. A re-examination of daily energy needs and the metabolic factors contributing to periods of energy imbalance during the menopausal transition and in several disease states may be a prerequisite to offsetting accelerated sarcopenia.

Opposite effects of metallothionein I and spermine on mitochondrial function.

Previously, we reported that the stress-induced protein metallothionein I (MT) modulated the oxygen consumption (VO2) of isolated rat liver mitochondria [Life Sci. 55 221-226, 1994]. We now present confirmation of this finding, and the additional observations that in rat liver mitochondria, MT caused swelling and depolarization. These actions of MT were inhibited by the aliphatic polyamine, spermine. Our findings suggest that mitochondrial function could be influenced by the balance between MT and spermine.

Relationship between physical activity recall and free-living daily physical activity in older claudicants.

The purposes of this study were to determine the relationship between the physical activity values obtained from the peripheral arterial disease-physical activity recall (PAD-PAR) questionnaire and (1) the free-living daily physical activity obtained from the doubly labeled water technique and (2) clinical measures of PAD severity. Fifty-one older PAD patients (age= 70 +/- 6 years) were recruited from the Vascular Clinic at the Baltimore Veterans Affairs Medical Center and from radio and newspaper advertisements. Energy expenditure of physical activity (EEPA) was determined by using doubly labeled water and indirect calorimetry techniques. PAD severity was measured by ankle/brachial index (ABI) and walking distance to maximal claudication pain determined during a graded treadmill test. In addition, patients were also characterized on body composition and total daily energy expenditure. The physical activity values obtained from the PAD-PAR questionnaire (113 +/- 37 MET-hr/wk) were not related to EEPA (542 +/- 260 kcal/day; r= -0.057, p=0.690), ABI (0.64 +/- 0.19; r=0.032, p=0.826), or distance to maximal claudication pain (376 +/- 229 m; r=-0.054, p=0.731). The authors conclude that the PAD-PAR questionnaire is not an accurate measurement of free-living daily physical activity when compared to EEPA by use of the criterion method of doubly labeled water, and the activity questionnaire measures were poorly correlated with clinical measures of PAD severity.

Relationship between free-living daily physical activity and ambulatory measures in older claudicants.

The purpose of this study was to determine the relationship between free-living daily physical activity and ambulatory measurements in peripheral arterial occlusive disease (PAOD) patients with intermittent claudication. Thirty-four older, nonsmoking PAOD patients with intermittent claudication (age=69.0 +/- 6.0 years, ankle/brachial index [ABI] =0.63 +/- 0.18) were recruited from the Vascular Clinic at the Baltimore Veterans Affairs Medical Center and from radio and newspaper advertisements. Energy expenditure of physical activity (EEPA) was determined by using doubly labeled water and indirect calorimetry techniques. Patients were also characterized on claudication distances and peak oxygen uptake during a graded treadmill test, 6-minute walking distance, weight, body mass index, and percent body fat. The claudication patients were sedentary, as EEPA was 362 +/- 266 kcal/day. EEPA was related to the 6-minute walk distance (369 +/- 68 meters; r=0.629, P<0.001), to the number of steps taken during 6 minutes (605 +/- 99 steps; r=0.485, P=0.008), to the treadmill distance to maximal claudication (313 +/- 131 meters; r=0.470, P=0.010), and to the time to relief of pain (6:21 +/- 3:57 min:sec; r=-0.417, P=0.017). None of the other ambulatory and body composition measurements were correlated with EEPA. In conclusion, a reduction in free-living daily physical activity was associated with a decrease in ambulatory ability and with more severe intermittent claudication in older PAOD patients.

Relationship between free-living daily physical activity and peripheral circulation in patients with intermittent claudication.

The purpose of this study was to determine the relationship between free-living daily physical activity and peripheral circulation under resting, reactive hyperemia, and maximal exercise conditions in peripheral arterial occlusive disease (PAOD) patients with intermittent claudication. Sixty-one PAOD patients (age = 70 +/- 6 years, ankle/brachial index [ABI] = 0.57 +/- 0.24) were recruited from the Vascular Clinic at the Baltimore Veterans Affairs Medical Center and from radio and newspaper advertisements. Free-living daily physical activity was measured as the energy expenditure of physical activity (EEPA), determined from doubly labeled water and indirect calorimetry. Patients also were characterized on ankle/brachial index, calf blood flow, calf transcutaneous oxygen tension (TcPO2), and calf transcutaneous heating power (TcHP). ABI and calf blood flow served as markers of the macrocirculation of the lower extremity, while TcPO2 and TcHP served as markers of the microcirculation. The claudication patients were sedentary, reflected by a mean EEPA value of 486 +/- 274 kcal/day. EEPA was related to calf TcHP at rest (282 +/- 24 mW; r = -0.413, p = 0.002), after postocclusion reactive hyperemia (275 +/- 22 mW; r = -0.381, p = 0.004), and after maximal exercise (276 +/- 20 mW; r = -0.461, p<0.001). ABI, calf blood flow, and calf TcPO2 were not related to EEPA under any condition. In conclusion, higher levels of free-living daily physical activity were associated with better microcirculation of the calf musculature in older PAOD patients with intermittent claudication.

Immobilized enzyme assay of creatine kinase with amperometric detection.

Serum creatine kinase-MB isoenzyme was determined by an immunoinhibition-amperometric technique in a coupled reaction scheme in which ADP and ATP are recycled and hydrogen peroxide is a product of the reaction. Two enzymes in the scheme, glycerokinase and glycerol-3-phosphate oxidase, were co-immobilized on collagen membrane at the surface of a platinum electrode polarized at 700 mV. The rate of production of hydrogen peroxide in the reaction was measured amperometrically at the electrode and related to the activity of creatine kinase. Calibration curves prepared with human creatine kinase-MB isoenzyme standards were linear from 10 to 500 U liter-1 with a detection limit of 8 U liter-1. Total creatine kinase in serum was also determined by the present method. The precision of the method was 3.1% relative standard deviation at the 10 U liter-1 level of creatine kinase. Interference from adenylate kinase was eliminated by the addition of diadenosine-5'-pentaphosphate to samples.

Exercise, substrate utilization and energy requirements in the elderly.

We consider the impact of endurance and resistance exercise on energy expenditure and substrate utilization in the elderly. We present data to show that endurance exercise increases resting metabolic rate in elderly men and women and these changes appear to be sympathetically mediated. Preliminary data also shows that vigorous endurance exercise causes a compensatory decline in energy expenditure during the non-exercising portion of the day, suggesting that this type of exercise prescription may actually be counter-productive to promoting fat loss. Several studies using resistance training interventions in older individuals suggests that this exercise mode also increases resting metabolic rate and basal sympathetic nervous system activity. The impact of age and endurance exercise on fat mobilization and oxidation are briefly considered. The major findings are that whole body fat oxidation decreased with advancing age and this decrease was primarily related to the age-associated decrease in fat-free mass. Furthermore, endurance training altered basal utilization patterns in the elderly by shifting disposal of fatty acids from nonoxidative to oxidative pathways and that increases in fat oxidation were related to the increase in norepinephrine appearance rate. Exercise interventions that preserve fat-free mass and/or enhance aerobic capacity may increase or maintain fat oxidation and possibly blunt the tendency towards increasing adiposity in older individuals.

Development and comparative evaluation of immunoblot assays for detecting autoantibodies to Scl 70 and Jo 1 antigens in serum.

We developed rapid 24-h immunoblot assays for detecting autoantibodies to Scl 70 and Jo 1 antigens in serum. In comparative studies, we evaluated the analytical sensitivity of the immunoblot assays and commercial immunodiffusion assays for anti-Scl 70 and anti-Jo 1 autoantibodies with the use of positive control sera, and compared the frequencies of positive and negative results in a group of 116 sera, including specimens from 34 healthy controls and 82 patients with various connective-tissue diseases. The immunoblot assays were greater than 100-fold more sensitive than immunodiffusion for detecting both autoantibodies. Despite greater analytical sensitivity, there were no false-positive results by the immunoblot assay for anti-Scl 70 or anti-Jo 1 autoantibodies in sera from either the controls or the patients. The diagnostic sensitivity of the immunoblot assay for anti-Scl 70 autoantibodies in patients with scleroderma was greater than that of the immunodiffusion assay, 70% vs 20%, and was equivalent in patients with polymyositis, 43%. We conclude that rapid immunoblot assays for anti-Scl 70 and anti-Jo 1 autoantibodies are superior to immunodiffusion assays for clinical use and are suitable for routine use in the clinical laboratory.

Body fatness and waist circumference are independent predictors of the age-associated increase in fasting insulin levels in healthy men and women.

OBJECTIVE: Elevated levels of fasting insulin increase the risk for coronary artery disease. In this study, we considered the independent contributions of age, total body fatness, abdominal fat distribution, peak aerobic capacity, leisure time physical activity, dietary intake, and fasting glucose levels to the age-associated increase in fasting insulin levels in healthy men and women. DESIGN: A cross-sectional analysis. SUBJECTS: A total of 427 healthy men and 293 healthy women age 18 to 90 years. MEASUREMENTS: Plasma glucose and insulin levels were measured in the fasted state and subjects were characterized for body fatness from underwater weighting, abdominal fat distribution from the waist circumference, peak VO2 from a treadmill test to exhaustion, leisure time physical activity from a structured interview and dietary intake from a 3-day food diary. The independent predictors of fasting insulin levels were determined by stepwise multiple regression analysis. RESULTS: Fasting insulin concentrations increased with age in both men (0.09 +/- 0.02 pmol/yr, unadjusted slope +/- SEE) and women (0.14 +/- 0.03 pmol/yr). In men, the major independent predictors of fasting insulin levels were percent body fat (r2 = 28%, P < 0.001) and waist circumference (r2 = 3% P < 0.001). Age, glucose concentration, and peak VO2 were small (1% each), but independent contributors to the variance in insulin levels. In women, the waist circumference (r2 = 18%, P < 0.001) and the fasting glucose concentration (r2 = 7%, P < 0.001) were the independent correlates of fasting insulin concentrations. CONCLUSIONS: Our data suggest that total adiposity and central body fat distribution are significant determinants of the increase in fasting insulin levels, whereas age, dietary intake, and levels of aerobic fitness and physical activity appear to be less important contributors to the variation in fasting insulin concentrations in healthy men and women.

Total daily energy expenditure in free-living older African-Americans and Caucasians.

Low rates of daily energy expenditure, increased energy intake, or a combination of both contribute to obesity in African-Americans. We examined whether African-Americans have lower rates of free-living daily energy expenditure than Caucasians. One hundred sixty-four (> 55 yr) volunteers (37 African-American women, 52 Caucasian women, 28 African-American men, and 47 Caucasian men) were characterized for total daily energy expenditure, resting metabolic rate, and physical activity energy expenditure from the doubly labeled water method and indirect calorimetry. Absolute total daily energy expenditure was lower in women than men but was not different between African-Americans and Caucasians. However, we found race and gender differences in total daily energy expenditure after controlling for differences in fat-free mass. Total daily energy expenditure was 10% lower (P < 0.01) in African-Americans compared with Caucasians due to a 5% lower resting metabolic rate (P < 0.01) and 19% lower physical activity energy expenditure (P = 0.08). Moreover, total daily energy expenditure was 16% lower (P < 0.01) in women compared with men due to a 6% lower resting metabolic rate (P = 0.09) and a 37% lower physical activity energy expenditure (P = 0.06). Low rates of energy expenditure may be a predisposing factor for obesity, particularly in African-American women.