Species-specific in vitro pharmacological effects of the cannabinoid receptor 2 (CB2) selective ligand AM1241 and its resolved enantiomers.

BACKGROUND AND PURPOSE: Racemic (R,S) AM1241 is a cannabinoid receptor 2 (CB(2))-selective aminoalkylindole with antinociceptive efficacy in animal pain models. The purpose of our studies was to provide a characterization of R,S-AM1241 and its resolved enantiomers in vitro and in vivo. EXPERIMENTAL APPROACH: Competition binding assays were performed using membranes from cell lines expressing recombinant human, rat, and mouse CB(2) receptors. Inhibition of cAMP was assayed using intact CB(2)-expressing cells. A mouse model of visceral pain (para-phenylquinone, PPQ) and a rat model of acute inflammatory pain (carrageenan) were employed to characterize the compounds in vivo. KEY RESULTS: In cAMP inhibition assays, R,S-AM1241 was found to be an agonist at human CB(2), but an inverse agonist at rat and mouse CB(2) receptors. R-AM1241 bound with more than 40-fold higher affinity than S-AM1241, to all three CB(2) receptors and displayed a functional profile similar to that of the racemate. In contrast, S-AM1241 was an agonist at all three CB(2) receptors. In pain models, S-AM1241 was more efficacious than either R-AM1241 or the racemate. Antagonist blockade demonstrated that the in vivo effects of S-AM1241 were mediated by CB(2) receptors. CONCLUSIONS AND IMPLICATIONS: These findings constitute the first in vitro functional assessment of R,S-AM1241 at rodent CB(2) receptors and the first characterization of the AM1241 enantiomers in recombinant cell systems and in vivo. The greater antinociceptive efficacy of S-AM1241, the functional CB(2) agonist enantiomer of AM1241, is consistent with previous observations that CB(2) agonists are effective in relief of pain.

Altered cellular magnesium responsiveness to hyperglycemia in hypertensive subjects.

Previous studies by our group have identified ionic aspects of insulin resistance in hypertension, in which cellular responses to insulin were influenced by the basal intracellular ionic environment-the lower the cytosolic free magnesium (Mg(i)), the less Mg(i) increased following insulin stimulation. To investigate whether this ionic insulin resistance represents a more general abnormality of cellular responsiveness in hypertension, we studied Mg(i) responses to nonhormonal signals such as hyperglycemia (15 mmol/L) and used (31)P-nuclear magnetic resonance (NMR) spectroscopy to measure Mg(i) in erythrocytes from normal (NL, n=14) and hypertensive (HTN, n=12) subjects before and 30, 60, 120, and 180 minutes after in vitro glucose incubations. Basal Mg(i) levels were significantly lower in HTN subjects than in NL subjects (169+/-10 versus 205+/-8 micromol.L(-1), P<0.01). In NL cells, hyperglycemia significantly lowered Mg(i), from 205+/-8 micromol.L(-1) (basal, T=0) to 181+/-8, 162+/-6, 152+/-7, and 175+/-9 micromol.L(-1) (T=30, 60, 120, and 180, respectively; P<0.005 versus T=0 at all times). In HTN cells, maximal Mg(i) responses to hyperglycemia were blunted, from 169+/-10 micromol.L(-1) (basal, T=0) to 170+/-11, 179+/-12, 181+/-14, and 173+/-15 micromol.L(-1) (T=30, 60, 120, and 180, respectively; P=NS versus T=0 at all times). For all subjects, Mg(i) responses to hyperglycemia were closely related to basal Mg(i) levels: the higher the Mg(i), the greater the response (n=26, r=0.620, P<0.001). Thus, (1) erythrocytes from hypertensive vis-à-vis normotensive subjects are resistant to the ionic effects of extracellular hyperglycemia on Mg(i) levels, and (2) cellular ionic responses to glucose depend on the basal Mg(i) environment. Altogether, these data support a role for altered extracellular glucose levels in regulating cellular magnesium metabolism and also suggest the importance of ionic factors in determining cellular responsiveness to nonhormonal as well as hormonal signals.

Insulin-mimetic action of vanadate: role of intracellular magnesium.

The insulin-mimetic effect of vanadate is well established, and vanadate has been shown to improve insulin sensitivity in diabetic rats and humans. Although the exact mechanism(s) remain undefined, we have previously demonstrated a direct relation of intracellular free magnesium (Mg(i)) levels to glucose disposal, to insulinemic responses following glucose loading, and to insulin-induced ionic effects. To investigate whether the insulin-mimetic effects of vanadate could similarly be mediated by Mg(i), we utilized (31)P-nuclear magnetic resonance spectroscopy to measure Mg(i) in erythrocytes from normal (NL, n=10) and hypertensive (HTN, n=12) subjects, before and after incubation with insulin and with different doses of sodium vanadate. In NL, vanadate elevated Mg(i) levels, with maximum efficacy at 50 7 micromol/L (186+/-6 to 222+/-6 7micromol/L, P>0.01), as did physiologically maximal doses of insulin, 200 7microU/mL (185+/-6 to 222+/-8 7micromol/L, P<0.01). In HTN, only vanadate, but not insulin, increased Mg(i) (insulin: 173+/-7 to 180+/-9 7micromol/L, P=NS; vanadate: 170+/-7 to 208+/-10 7micromol/L, P<0.01). Mg(i) responses to insulin (r=0.637, P<0.001), but not to vanadate (r=0.15, P=NS), were closely and directly related to basal Mg(i) levels. We conclude that (1) both vanadate and insulin stimulate erythrocyte Mg(i) levels; (2) cellular Mg(i) responses to insulin, but not to vanadate, depend on basal Mg(i) content-the lower the basal Mg(i), the less the Mg(i) response to insulin. As such, (3) Mg(i) responses to vanadate were equivalent among HTN and NL, whereas HTN cells exhibited blunted Mg(i) responses to insulin, and (4) the ability of vanadate to improve insulin sensitivity clinically may be mediated, at least in part, by its ability to increase Mg(i) levels, which in turn, helps to determine cellular insulin action.

Relation of cellular potassium to other mineral ions in hypertension and diabetes.

To investigate the role of intracellular potassium (K(i))and other ions in hypertension and diabetes, we utilized (39)K-, (23)Na-, (31)P-, and (19)F-nuclear magnetic resonance (NMR) spectroscopy to measure K(i), intracellular sodium (Na(i)), intracellular free magnesium (Mg(i)), and cytosolic free calcium (Ca(i)), respectively, in red blood cells of fasting normotensive nondiabetic control subjects (n=10), untreated (n=13) and treated (n=14) essential hypertensive subjects, and diabetic subjects (n=5). In 12 subjects (6 hypertensive and 6 normotensive controls), ions were also measured before and after the acute infusion of 1 L of normal saline. Compared with those in controls (K(i)=148+/-2.0 mmol/L), K(i) levels were significantly lower in hypertensive (132.2+/-2.9 mmol/L, sig=0.05) and in type 2 diabetic subjects (121.2+/-6.8 mmol/L, sig=0.05). K(i) was higher in treated hypertensives than in untreated hypertensives (139+/-3.1 mmol/L, sig=0.05) but was still lower than in normals. Although no significant relation was observed between basal K(i) and Na(i) values, saline infusion elevated Na(i) (P<0.01) and reciprocally suppressed K(i) levels (142+/-2.4 to 131+/-2.2 mmol/L, P<0.01). K(i) was strongly and inversely related to Ca(i) (r=-0.846, P<0.001), and was directly related to Mg(i) (r=0.664, P<0.001). We conclude that (1) K(i) depletion is a common feature of essential hypertension and type 2 diabetes, (2) treatment of hypertension at least partially restores K(i) levels toward normal, and (3) fasting steady-state K(i) levels are closely linked to Ca(i) and Mg(i) homeostasis. Altogether, these results emphasize the similar and coordinate nature of ionic defects in diabetes and hypertension and suggest that their interpretation requires an understanding of their interaction.

Assessment of sequential same arm agreement of blood pressure measurements by a CVProfilor DO-2020 versus a Baumanometer mercury sphygmomanometer.

OBJECTIVE: The aim of this study was to assess the accuracy of sequential same arm blood pressure measurement by the mercury sphygmomanometer with the oscillometric blood pressure measurements from a device that also determines arterial elasticity. METHODS: A prospective, multicentre, clinical study evaluated sequential same arm blood pressure measurements, using a mercury sphygmomanometer (Baumanometer, W. A. Baum Co., Inc., Copiague, New York, USA) and an oscillometric non-invasive device that calculates arterial elasticity (CVProfilor DO-2020 Cardiovascular Profiling System, Hypertension Diagnostics, Inc., Eagan, Minnesota, USA). Blood pressure was measured supine in triplicate, 3 min apart in a randomized sequence after a period of rest. RESULTS: The study population of 230 normotensive and hypertensive subjects included 57% females, 51% Caucasians, and 33% African Americans. The mean difference between test methods of systolic blood pressure, diastolic blood pressure, and heart rate was -3.2 +/- 6.9 mmHg, +0.8 +/- 5.9 mmHg, and +1.0 +/- 5.7 beats/minute. For systolic and diastolic blood pressure, 60.9 and 70.4% of sequential measurements by each method were within +/- 5 mmHg. Few or no points fell beyond the mean +/- 2 standard deviations lines for each cuff bladder size. CONCLUSION: Sequential same arm measurements of the CVProfilor DO-2020 Cardiovascular Profiling System measures blood pressure by an oscillometric method (dynamic linear deflation) with reasonable agreement with a mercury sphygmomanometer.

Arterial elasticity among normotensive subjects and treated and untreated hypertensive subjects.

OBJECTIVE: The aim of this study was to determine arterial elasticity in normotensive and hypertensive individuals. BACKGROUND: In addition to blood pressure, other parameters serve as markers for vascular disease. Arterial elasticity is one parameter that can be determined by a modified Windkessel model of the circulation. This model estimates, from a computerized pulse contour analysis, the proximal (capacitive) elasticity of the large arteries and the distal (reflective) elasticity of the small arteries. METHODS: A prospective, multi-center, controlled clinical study evaluated large-artery and small-artery elasticity indices in four groups: (1) normotensives without a family history of hypertension; (2) normotensives with a family history of hypertension; (3) treated and controlled hypertensives; and (4) untreated and uncontrolled hypertensives. Blood pressure, using a mercury manometer, and arterial elasticity, using a CVProfilor DO-2020 CardioVascular Profiling System (Hypertension Diagnostics, Inc., Eagan, MN, USA), were measured supine in triplicate 3 min apart in a randomized sequence. RESULTS: There were 212 evaluable subjects of mean age 46 years; 57% were women, 51% Caucasian and 33% African-American. Comparing normotensives without a family history and untreated hypertensives, both large-artery and small-artery elasticity indices were significantly different (P < 0.0001). After controlling for age and body surface area, a significant linear trend (P = 0.0001) across the four groups was detected for both large- and small-artery elasticity indices. CONCLUSION: As the hypertension status worsened, large- and small-artery elasticity indices decreased, suggesting a potential for the diagnostic use of arterial elasticity determinations.

Pulse waveform analysis of arterial compliance: relation to other techniques, age, and metabolic variables.

To assess the physiologic and clinical relevance of newer noninvasive measures of vascular compliance, computerized arterial pulse waveform analysis (CAPWA) of the radial pulse was used to calculate two components of compliance, C1 (capacitive) and C2 (oscillatory or reflective), in 87 normotensive (N1BP, n = 20), untreated hypertensive (HiBP, n = 21), and treated hypertensive (HiBP-Rx, n = 46) subjects. These values were compared with two other indices of compliance, the ratio of stroke volume to pulse pressure (SV/PP) and magnetic resonance imaging (MRI)-based aortic distensibility; and were also correlated with demographic and biochemical values. The HiBP subjects displayed lower C1 (1.34 +/- 0.09 v. 1.70 +/- 0.11 mL/mm Hg, significance [sig] = .05) and C2 (0.031 +/- 0.003 v 0.073 +/- 0.02 mL/mm Hg, sig = .005) than N1BP subjects. This was not true for C1 (1.64 +/- 0.08 mL/mm Hg) and C2 (0.052 +/- 0.005 mL/mm Hg) values in HiBP-Rx subjects. The C1 (r = 0.917, P < .0001) and C2 (r = 0.677, P < .0001) were both closely related to SV/PP, whereas C1 (r = 0.748, P = .002), but not C2, was significantly related to MRI-determined aortic distensibility. Among other factors measured, age exerted a strong negative influence on both C1 (r = -0.696, P < .0001) and C2 (r = -0.611, P < .0001) compliance components. Positive correlations were observed between C1 (r = 0.863, P = .006), aortic distensibility (r = 0.597, P = .19) and 24-h urinary sodium excretion, and between C1- and MR spectroscopy-determined in situ skeletal muscle intracellular free magnesium (r = 0.827, P = .006), whereas C2 was inversely related to MRI-determined abdominal visceral fat area (r = -0.512, P = .042) and fasting blood glucose (r = -0.846, P = .001). Altogether, the close correspondence between CAPWA, other compliance techniques, and known cardiovascular risk factors suggests the clinical relevance of CAPWA in the assessment of altered vascular function in hypertension.

Factors affecting blood pressure responses to diet: the Vanguard study.

To study physiologic factors affecting the blood pressure (BP) response to nonpharmacologic maneuvers, fasting blood glucose, insulin, lipid and mineral levels, urinary mineral excretion, and the calcium regulating hormones parathyroid hormone (PTH) and 1,25 dihydroxyvitamin D (1,25 (OH)2D) were measured in 71 unmedicated hypertensive (26 hypertensive only [HT], 45 hypertensive hyperlipidemic [HTHL]), and 87 normotensive hyperlipidemic (NTHL) control subjects before and during a 10-week multicenter, randomized controlled trial comparing a prepared meal plan (CCNW) with a self-selected diet (SSD) based on nutritionist counseling. Blood pressure fell to a greater extent in hypertensive versus normotensive subjects (-8+/-1/-5+/-1 v -2+/-1/-2+/-1 mm Hg, P < .0001/P < .0001), and on CCNW versus SSD diets (delta systolic BP [SBP]/delta diastolic BP [DBP], P = .033/P = .002). Diet-induced weight change was the strongest correlate of changes in BP (SBP: r = 0.360, P < .0001; DBP: r = 0.414, P < .0001), which, on multivariate analysis for deltaSBP, could partly be accounted for by diet-induced changes in fasting glucose (r = 0.215, P = .009) and cholesterol (r = 0.219, P = .006) levels. Independently of weight, diet-induced changes in SBP also were significantly related to concomitant changes in urinary excretion of potassium (r = -0.285, P = .001), magnesium (r = -0.254, P = .003), and calcium relative to sodium (r = -0.200, P = .021), but not to sodium per se; and to changes in serum potassium (r = -0.249, P = .002), phosphorus (r = -0.279, P = .001), PTH (r = 0.288, P = .0006), and 1,25 D (r = 0.202, P = .017). We conclude that the ability of diet to lower BP successfully may result from the additive contributions of multiple components. Independently of weight loss and the associated changes in circulating glucose and cholesterol, BP is influenced by the increasing provision of minerals such as potassium, magnesium, and calcium, perhaps by virtue of their suppressive effects on circulating vasoactive calcium regulating hormones.

Cellular ionic alterations with age: relation to hypertension and diabetes.

BACKGROUND: Cytosolic free calcium (Cai) and magnesium (Mgi) are vital to cellular homeostasis and function. OBJECTIVE: To evaluate cellular divalent cations in normal subjects at different ages and their relationship to ion levels in essential hypertension and diabetes. DESIGN: A cross-sectional study. SETTING: A university hospital in New York. PARTICIPANTS: A total of 103 subjects (32 older, 71.1 +/- 1.2 y/o, and 71 young/middle aged subjects, 51.1 +/- 2.3 y/o). INTERVENTION: Oral glucose tolerance test. MEASUREMENTS: 19F and 31P NMR spectroscopy were used to measure Cai and Mgi levels in erythrocytes from normal (>65 y/o, n = 11; <65 y/o, n = 26), hypertensive (EH) (>65 y/o, n = 9; <65 y/o, n = 30), and type 2 diabetic (DM) (>65 y/o, n = 12; <65 y/o, n = 15) subjects; these levels were also compared with glucose and insulin levels before and after oral glucose loading. RESULTS: Fasting Mgi levels were lower (207 +/- 7.8 vs 236 +/- 7.5 microM; P < .05) and Cai higher (32.2 +/- 3.0 vs 20.3 +/- 1.8 nM; P < .05) in older than in younger normal subjects. For all normal subjects, the greater the age, the higher the Cai (r = 0.622, P = .004) and the lower the Mgi (r = -0.423; P = .011). However, no significant (P = NS) differences in Mgi or Cai levels were observed between older normal and young/middle-aged subjects with EH (Mgi = 189.7 +/- 5.9 vs 182.6 +/- 9.8 microM; Cai = 33.8 +/- 4.9 vs 35.6 +/- 4.0 nM) or DM (Mgi = 182.8 +/- 10.9 vs 180.8 +/- 8.1 microM; Cai = 33.6 +/- 4.3 vs 39.7 +/- 5.9 nM). Significant relationships were also found between cellular ion content, blood pressure, and glycemic indices. CONCLUSIONS: Aging is associated with the onset of altered Cai and Mgi levels, indistinguishable from those observed in hypertension and diabetes, independent of age. We suggest that these ionic changes may be clinically significant, underlying the predisposition of older subjects to cardiovascular and metabolic diseases.

A randomized trial of improved weight loss with a prepared meal plan in overweight and obese patients: impact on cardiovascular risk reduction.

OBJECTIVE: To assess the long-term effects of a prepackaged, nutritionally complete, prepared meal plan compared with a usual-care diet (UCD) on weight loss and cardiovascular risk factors in overweight and obese persons. DESIGN: In this randomized multicenter study, 302 persons with hypertension and dyslipidemia (n = 183) or with type 2 diabetes mellitus (n = 119) were randomized to the nutrient-fortified prepared meal plan (approximately 22% energy from fat, 58% from carbohydrate, and 20% from protein) or to a macronutrient-equivalent UCD. MAIN OUTCOME MEASURES: The primary outcome measure was weight change. Secondary measures were changes in blood pressure or plasma lipid, lipoprotein, glucose, or glycosylated hemoglobin levels; quality of life; nutrient intake; and dietary compliance. RESULTS: After 1 year, weight change in the hypertension/dyslipidemia group was -5.8+/-6.8 kg with the prepared meal plan vs -1.7+/-6.5 kg with the UCD plan (P<.001); for the type 2 diabetes mellitus group, the change was -3.0+/-5.4 kg with the prepared meal plan vs -1.0+/-3.8 kg with the UCD plan (P<.001) (data given as mean +/- SD). In both groups, both interventions improved blood pressure, total and low-density lipoprotein cholesterol levels, glycosylated hemoglobin level, and quality of life (P<.02); in the diabetic group, the glucose level was reduced (P<.001). Compared with those in the UCD group, participants with hypertension/dyslipidemia in the prepared meal plan group showed greater improvements in total (P<.01) and high-density lipoprotein (P<.03) cholesterol levels, systolic blood pressure (P<.03), and glucose level (P<.03); in participants with type 2 diabetes mellitus, there were greater improvements in glucose (P =.046) and glycosylated hemoglobin (P<.02) levels. The prepared meal plan group also showed greater improvements in quality of life (P<.05) and compliance (P<.001) than the UCD group. CONCLUSIONS: Long-term dietary interventions induced significant weight loss and improved cardiovascular risk in high-risk patients. The prepared meal plan simultaneously provided the simplicity and nutrient composition necessary to maintain long-term compliance and to reduce cardiovascular risk.

Concentration-targeted phase I trials of atevirdine mesylate in patients with HIV infection: dosage requirements and pharmacokinetic studies. The ACTG 187 and 199 study teams.

RATIONALE: To determine the dosage requirements and pharmacokinetics of atevirdine, a non-nucleoside reverse transcriptase inhibitor and its N-dealkylated metabolite (N-ATV) during phase I studies in patients receiving atevirdine alone or in combination with zidovudine. DESIGN: Two open label, phase I studies conducted by the adult AIDS Clinical Trials Group (ACTG) in which atevirdine was administered every 8 h with weekly dosage adjustments to attain targeted trough plasma atevirdine concentrations. SETTING: Five Adult AIDS Clinical Trials Units. PATIENTS: Fifty patients (ACTG 199; n = 20 and ACTG 187; n = 30) with HIV-1 infection and < or =500 CD4+ lymphocytes/mm3. INTERVENTION: ACTG 199; 12 weeks of therapy with atevirdine (dose-adjusted to achieve plasma trough atevirdine concentrations of 5-10 microM) and zidovudine (200 mg every 8 h). ACTG 187: 12 weeks of atevirdine monotherapy with atevirdine doses adjusted to achieve escalating, targeted trough plasma concentration ranges (5-13, 14-22, and 23-31 microM). MEASUREMENTS: ACTG 199: atevirdine, N-ATV and zidovudine trough determinations weekly (all patients) and intensive pharmacokinetics (selected patients) prior to and at 6 and 12 weeks during combination therapy. ACTG 187: atevirdine and N-ATV trough concentrations over a 12 week period. Intensive pharmacokinetic studies were conducted prior to and at 4 and/or 8 weeks during atevirdine monotherapy in female patients. RESULTS: Atevirdine plasma concentrations demonstrated considerable interpatient variability which was minimized by the adjustment of maintenance doses (range: 600-3900 mg/day) to achieve the desired trough concentrations. In ACTG 187, the mean number of weeks to attain the target value, and the percentage of patients who attained the target, was group I (5-11 microM): 2.7+/-2.4 weeks (92%); group II (12-21 microM): 2.6+/-1.8 (64%); and group III (22-31 microM): 7.0+/-5.6 weeks (27%). In ACTG 199 it was 3.2+/-5.2 weeks (95%) to achieve a 5-10 microM trough. Atevirdine demonstrated a mono- or bi-exponential decline among most of the patients studied after the first dose. During multiple-dosing a number of patterns of atevirdine disposition were observed including; rapid absorption with Cmax at 0.5-1 h, delayed absorption with Cmax at 3-4 h; minimal Cmax to Cmin fluctuation and Cmax to Cmin ratios of > 4. N-ATV (an inactive metabolite) patterns were characterized on day one by rapid appearance of the metabolite which peaked at 2-3 h after the dose and declined in a mono- or bi-exponential manner. At steady-state N-ATV patterns demonstrated minimal Cmax to Cmin fluctuations with some of the patients having more stable plasma N-ATV concentrations, while others had greater fluctuations week to week. CONCLUSIONS: Considerable interpatient variability was noted in the pharmacokinetics of atevirdine. The variation in drug disposition was reflected in the range of daily doses required to attain the targeted trough concentrations. Atevirdine metabolism did not appear to reach saturation during chronic dosing in many of our patients, as reflected by the pattern of N-ATV/ATV ratios in plasma and saturation was not an explanation for the variation in dosing requirements. No apparent differences were noted between males and females, and atevirdine did not appear to influence zidovudine disposition.

Effects of aging on serum ionized and cytosolic free calcium: relation to hypertension and diabetes.

Elevated cytosolic free calcium (Ca(i)) and reciprocally reduced, extracellular ionized calcium (Ca-ion) levels are observed in both hypertension and non-insulin-dependent diabetes mellitus (NIDDM). Because the changes of vascular function and insulin sensitivity in these conditions resemble the changes associated with "normal" aging, we wondered to what extent similar alterations in calcium metabolism occur with aging per se in the absence of overt hypertension or diabetes. We therefore measured platelet Ca(i) levels by spectrofluorometry and serum Ca-ion levels in normotensive, nondiabetic, healthy, normal, elderly (>65 years old) subjects, mean age +/-SEM, 72.2+/-1.5 years old (n=11); in healthy, normal, young (<65 years old) adults, 46.1+/-2.3 years old (n=12); in 10 young adult hypertensives, 48.6+/-1.9 years old; and in 10 normotensive NIDDM subjects, 49.2+/-1.6 years old. Platelet Ca(i) levels were higher (104.5+/-4.9 versus 80.2+/-1.8 nmol/L, P<0.01) and Ca-ion levels lower (1.212+/-0.010 versus 1.236+/-0.011 mmol/L, P<0.05) in normal elderly compared with young control subjects, but normal elderly Ca(i) and Ca-ion levels were indistinguishable from those in hypertensive (Ca(i) 107.5+/-3.6 nmol/L, Ca-ion 1.210+/-0.009 mmol/L) and NIDDM (Ca(i) 110.7+/-4.7 nmol/L, Ca-ion 1.204+/-0.014 mmol/L) subjects. In normal subjects, significant correlations were found between platelet Ca(i) levels and age (r=0.655, P<0.01) and between Ca(i) levels and systolic blood pressure (r=0.733, P<0.001). We conclude that aging is associated with alterations of Ca(i) and Ca-ion levels resembling those changes present at any age in hypertension and type 2 diabetes. We hypothesize that these alterations of calcium metabolism underlie the predisposition to the alterations of blood pressure and insulin sensitivity characteristic of "normal" aging. The data also suggest that studies of the aging process should be limited to subjects with normal blood pressure and glucose tolerance.

Protective effects of captopril against ischemic stress: role of cellular Mg.

Magnesium (Mg) deficiency enhances tissue sensitivity to ischemic damage, an effect reversed not only by Mg, but also by sulfhydryl (SH)-containing compounds. We therefore created an in vitro model of red blood cell ischemia to investigate whether the protective effects of these compounds might be related to effects on intracellular free Mg (Mg(i)) content. (31)P-nuclear magnetic resonance (NMR) spectroscopy was used to measure the high-energy metabolites ATP and 2,3-diphosphoglycerate (DPG) and Mg(i) and inorganic phosphate (P(i)) levels in erythrocytes before and for 6 hours after progressive oxygen depletion in the presence or absence of SH-compounds, including captopril, N-acetyl-L-cysteine (NAC), penicillamine, and N-(2-mercaptopropionyl)-glycine (MPG). Under basal aerobic conditions, captopril increased Mg(i) in a dose- and time-dependent fashion (174.5+/-5.3 to 217.1+/-5.1 micromol/L, P<0. 05 at 100 micromol/L, 60 minutes). The SH compounds NAC, penicillamine, and MPG but not the non-SH compound enalaprilat also significantly raised Mg(i) in erythrocytes (P<0.05). With oxygen deprivation, a consistent decrease occurred in both ATP and 2,3-DPG levels associated with a rise in P(i) and in the P(i)/2,3-DPG ratio used as an index of high-energy metabolite depletion. Captopril, compared with control, retarded the rise in P(i) and reduced the P(i)/2,3-DPG ratio (P<0.008 and P<0.025 at 4 and 6 hours, respectively). Furthermore, the higher the initial Mg(i) and the greater the captopril-induced rise in Mg(i), the greater the metabolite-protective effect (r=0.799 and r=0.823, respectively; P<0. 01 for both). Altogether, the data suggest that Mg influences the cellular response to ischemia and that the ability of SH compounds such as captopril to ameliorate ischemic injury may at least in part be attributable to the ability of such compounds to increase cytosolic free Mg levels.

Effects of vitamin E and glutathione on glucose metabolism: role of magnesium.

Vitamin E is an antioxidant that has been demonstrated to improve insulin action. Glutathione, another natural antioxidant, may also be important in blood pressure and glucose homeostasis, consistent with the involvement of free radicals in both essential hypertension and diabetes mellitus. Our group has recently suggested that the effects of reduced glutathione on glucose metabolism may be mediated, at least in part, by intracellular magnesium levels (Mg([i])). Recent evidence suggests that vitamin E enhances glutathione levels and may play a protective role in magnesium deficiency-induced cardiac lesions. To directly investigate the effects of vitamin E supplementation on insulin sensitivity in hypertension, in relation to the effects on circulating levels of reduced (GSH) and oxidized (GSSG) glutathione and on Mg([i]), we performed a 4-week, double-blind, randomized study of vitamin E administration (600 mg/d) versus placebo in 24 hypertensive patients and measured whole-body glucose disposal (WBGD) by euglycemic glucose clamp, GSH/GSSG ratios, and Mg([i]) before and after intervention. The relationships among WBGD, GSH/GSSG, and Mg([i]) in both groups were evaluated. In hypertensive subjects, vitamin E administration significantly increased WBGD (25.56+/-0.61 to 31.75+/-0.53 micromol/kg of fat-free mass per minute; P<0.01), GSH/GSSG ratio (1.10+/-0.07 to 1.65+/-0.11; P<0.01), and Mg([i]) (1.71+/-0.042 to 1.99+/-0.049 mmol/L; P<0.01). In basal conditions, WBGD was significantly related to both GSH/GSSG ratios (r=0.58, P=0.047) and Mg([i]) (r=0.78, P=0.003). These data show a clinical link between vitamin E administration, cellular magnesium, GSH/GSSG ratio, and tissue glucose metabolism. Further studies are needed to explore the cellular mechanism(s) of this association.

The cellular ionic basis of hypertension and allied clinical conditions.

Two central concepts of human hypertensive disease remain poorly understood: (1) elevated blood pressure as merely one component of an underlying systemic condition, characterized by multiple defects in diverse tissues (eg, "Syndrome X"), and (2) the heterogeneity of hypertension, in which different and even opposite clinical responses to different dietary and drug therapies are routinely observed among equally hypertensive subjects. To help explain these clinical phenomena, a unifying "ionic hypothesis" is proposed, in which steady-state elevations of cytosolic free calcium and suppressed intracellular free magnesium levels, characteristic features of all hypertension, concomitantly alter the function of many tissues. In blood vessels this causes vasoconstriction, arterial stiffness, and/or hypertension; in the heart, cardiac hypertrophy; in platelets, increased aggregation and thrombosis; in fat and skeletal muscle, insulin resistance; in pancreatic beta cells, other endocrine tissues, and sympathetic neurons, potentiated stimulus-secretion coupling resulting in hyperinsulinemia, increased sympathetic nerve activity, and so on. Furthermore, an analysis of cellular biochemical, dietary-nutrient, and hormonal factors that normally regulate steady-state levels of these intracellular ions suggests an ionic equivalent to Laragh's volume-vasoconstriction analysis of hypertension. This provides a cellular-based explanation for the heterogeneity of hypertension and a rational basis for individualizing dietary and drug recommendations among different hypertensive subjects.

Increased dietary micronutrients decrease serum homocysteine concentrations in patients at high risk of cardiovascular disease.

BACKGROUND: Elevated blood homocysteine is a risk factor for cardiovascular disease. A 5-micromol/L increase is associated with an approximately 70% increase in relative risk of cardiovascular disease in adults. For patients with established risk factors, this risk is likely even greater. OBJECTIVE: Effects of increased dietary folate and recommended intakes of vitamins B-12 and B-6 on serum total homocysteine (tHcy) were assessed in individuals at high risk of cardiovascular disease. DESIGN: This trial was conducted at 10 medical research centers in the United States and Canada and included 491 adults with hypertension, dyslipidemia, type 2 diabetes, or a combination thereof. Participants were randomly assigned to follow a prepared meal plan (PMP; n = 244) or a self-selected diet (SSD; n = 247) for 10 wk, which were matched for macronutrient content. The PMP was fortified to provide >/=100% of the recommended dietary allowances for 23 micronutrients, including folate. RESULTS: Mean folate intakes at 10 wk were 601 +/- 143 microgram/d with the PMP and 270 +/- 107 microgram/d with the SSD. With the PMP, serum tHcy concentrations fell from 10.8 +/- 5.8 to 9.3 +/- 4.9 micromol/L (P < 0.0001) between weeks 0 and 10 and the change was associated with increased intakes of folate, vitamin B-12, and vitamin B-6 and with increased serum and red blood cell folate and serum vitamin B-12 concentrations. tHcy concentrations did not change significantly with the SSD. CONCLUSIONS: The PMP resulted in increased intakes and serum concentrations of folate and vitamin B-12. These changes were associated with reduced serum tHcy concentrations in persons at high risk of cardiovascular disease.

Nutritionally complete prepared meal plan to reduce cardiovascular risk factors: a randomized clinical trial.

OBJECTIVE: To compare a nutritionally complete prepared meal plan that meets national dietary guidelines to usual-care dietary therapy for hypertension, dyslipidemia, and glycemic control. DESIGN: Randomized, controlled trial. SUBJECTS/SETTING: Outpatients with hypertension, dyslipidemia, or diabetes mellitus (n = 251) were recruited at 6 medical centers in the United States and Canada. INTERVENTION: The prepared meal plan, which was developed by university-based nutrition and cardiovascular scientists and food technologists at Campbell's Center for Nutrition & Wellness (CCNW), provided the optimal levels of macronutrients and micronutrients recommended for cardiovascular risk reduction in a variety of prepackaged meals and snacks. After a 4-week pretrial period to assess baseline state, participants were randomized to the CCNW plan or "usual-care" diet for 10 weeks. MAIN OUTCOME MEASURES: Blood pressure, carbohydrate metabolism, lipoproteins, homocysteine, weight, nutrient intake, compliance. STATISTICAL ANALYSES PERFORMED: Repeated measures analysis of variance. RESULTS: Lipoproteins, carbohydrate metabolism, blood pressure, and weight improved on both plans. Mean differences (+/- standard deviation) between baseline and follow-up for the CCNW plan and the usual-care plan, respectively, were total cholesterol, -0.41 +/- 0.64 and -0.20 +/- 0.50 mmol/L (between-group P < .01); plasma glucose, -0.7 +/- 1.7 and -0.3 +/- 1.3 mmol/L (P < .05); systolic blood pressure, -5.2 +/- 10.0 and -4.7 +/- 9.0 mm Hg (P = .67), diastolic blood pressure, -3.8 +/- 5.9 and -2.2 +/- 5.5 mm Hg (P < .05); and homocysteine, -1.3 +/- 3.8 and 0.2 +/- 3.4 mumol/L (P < .01). The CCNW plan led to greater weight loss than the usual-care diet (-5.5 +/- 3.8 kg vs -3.0 +/- 3.2 kg, P < .0001). APPLICATIONS/CONCLUSION: The nutritionally complete CCNW plan offers greater improvements in lipids, blood sugars, homocysteine, and weight loss than usual-care diet therapy. This prepackaged comprehensive nutrition program can augment both the prescription and practice of optimal dietary therapy.

Effects of glutathione on red blood cell intracellular magnesium: relation to glucose metabolism.

Recent evidence suggests that the endogenous antioxidant glutathione may play a protective role in cardiovascular disease. To directly investigate the role of glutathione in the regulation of glucose metabolism in hypertension, we studied the acute effects of in vivo infusions of this antioxidant (alone or in combination with insulin) on whole body glucose disposal (WBGD) using euglycemic glucose clamp and the effects on total red blood cell intracellular magnesium (RBC-Mg) in hypertensive (n=20) and normotensive (n=30) subjects. The relationships among WBGD, circulating reduced/oxidized glutathione (GSH/GSSG) levels, and RBC-Mg in both groups were evaluated. The in vitro effects of glutathione (100 micromol/L) on RBC free cytosolic magnesium (Mg(i)) were also studied. In vivo infusions of glutathione (15 mg/minx120 minutes) increased RBC-Mg in both normotensives and hypertensives (1.99+/-0.02 to 2.13+/-0.03 mmol/L, P<0.01, and 1.69+/-0.03 to 1.81+/-0.03 mmol/L, P<0.01, respectively). In vitro GSH but not GSSG increased Mg(i) (179+/-3 to 214+/-5 micromol/L, P<0.01). In basal conditions, RBC-Mg values were related to GSH/GSSG ratios (r=0.84, P<0.0001), and WBGD was directly, significantly, and independently related to both GSH/GSSG ratios (r=0.79, P<0.0001) and RBC-Mg (r=0.89, P<0.0001). This was also true when hypertensive and control groups were analyzed separately. On multivariate analysis, basal RBC-Mg (t=6.81, P<0.001), GSH/GSSG (t=3. 67, P<0.02), and blood pressure (t=2.89, P<0.05) were each independent determinants of WBGD, with RBC-Mg explaining 31% of the variability of WBGD. These data demonstrate a direct action of glutathione both in vivo and in vitro to enhance intracellular magnesium and a clinical linkage between cellular magnesium, GSH/GSSG ratios, and tissue glucose metabolism.

Multicenter randomized trial of a comprehensive prepared meal program in type 2 diabetes.

OBJECTIVE: To evaluate the clinical effects of a comprehensive prepackaged meal plan, incorporating the overall dietary guidelines of the American Diabetes Association and other national health organizations, relative to those of a self-selected diet based on exchange lists in free-living individuals with type 2 diabetes. RESEARCH DESIGN AND METHODS: A total of 202 women and men (BMI < or = 42 kg/m2) whose diabetes was treated with diet alone or an oral hypoglycemic agent were enrolled at 10 medical centers. After a 4-week baseline period, participants were randomized to a nutrient-fortified prepared meal plan or a self-selected exchange-list diet for 10 weeks. On a caloric basis, both interventions were designed to provide 55-60% carbohydrate, 20-30% fat, and 15-20% protein. At intervals, 3-day food records were completed, and body weight, glycemic control, plasma lipids, and blood pressure were assessed. RESULTS: Food records showed that multiple nutritional improvements were achieved with both diet plans. There were significant overall reductions in body weight and BMI, fasting plasma glucose and serum insulin, fructosamine, HbA1c, total and LDL cholesterol, and blood pressure (P < 0.001 or better for all). In general, differences in major end points between the diet plans were not statistically significant. CONCLUSIONS: Glycemic control and cardiovascular risk factors improve in individuals with type 2 diabetes who consume diets in accordance with the American Diabetes Association guidelines. The prepared meal program was as clinically effective as the exchange-list diet. The prepared meal plan has the additional advantages of being easily prescribed and eliminating the complexities of meeting the multiple dietary recommendations for type 2 diabetes management.