Unveiling extracellular inorganic phosphate signals from blood in human cardiac 31P NMR spectra.

31P NMR spectra of the human heart are usually contaminated by signals that originate from blood. The main blood signals are 2,3-diphosphoglycerate (2,3-DPG), which overlap and sometimes obscure the signal of myocardial inorganic phosphate used to calculate intracellular pH and to monitor metabolic changes in the heart. In this work we demonstrate, first, that even without proton decoupling the resolution of such spectra can be high enough to evaluate intracellular inorganic phosphate of myocardium in about 70% of the spectra and, second, that extracellular inorganic phosphate from blood contributes a signal in the chemical shift region of the 2-phosphate signal of 2,3-DPG.

[Pathogenesis of coronary disease]. / Zur Pathogenese der koronaren Herzerkrankung.

Being overweight (OW) was recognized very early as a risk factor for coronary heart disease (CHD). Its significance in the pathogenesis of CHD has been strengthened by observations showing that OW is responsible for the development of diabetes, hypertension and lipid disorders due to its induction of insulin resistance (IR). Its key role has been underlined further by recent studies indicating that OW causes endothelial dysfunction via elevated serum fatty acids, which initiates the molecular events that further the process of CHD. It is, therefore, of the utmost importance to determine its roots. The most probable reason for its high incidence is due to the genetic outfit of most people which does not permit adequate adaptation of the cerebral cortex according to the environmental changes which have occurred since the early days.

Augmented sympathetic response to bradykinin in the diabetic heart before autonomic denervation.

We studied whether diabetes mellitus affects the bradykinin (BK)-induced release of norepinephrine (NE) from rat cardiac sympathetic endings in situ. Three groups were studied. Group A (n=12) was rendered diabetic with streptozotocin (STZ), group B (n=13) received STZ and insulin, and group C (n=14) received citrate buffer only. NPH insulin was given to group B from day 7 after STZ. Atria were paced (3Hz) with rectangular voltage pulses at mechanical threshold intensity (0.15 V/cm). The release of NE was assessed through its effects on contractile force in the presence of atropine (1 micromol/L). Intensifying the field stimulation above the neural threshold ( approximately 0.4 V/cm) produced a graded positive inotropic effect that was due to the release of NE from sympathetic nerve endings. The additional effect of 0.1 micromol/L BK on the force of contraction was determined at half-maximal neural stimulation (ie, at approximately 0.65 V/cm). Then, after washing out BK and lowering the stimulation intensity to mechanical threshold, a cumulative dose-response curve for added NE was generated, allowing the positive inotropic effects of neural stimulation (with or without BK) to be expressed in terms of an equivalent inotropic concentration of added NE ([NE(eq)]). Neural stimulation, in the absence of BK, gave an [NE(eq)] of 32+/-3 nmol/L in group A, 44+/-6 nmol/L in group B, and 37+/-6 nmol/L in group C. BK increased [NE(eq)] by a factor of 6.2+/-0.9 in group A, 4.5+/-0.5 in group B, and 3.7+/-0.3 in group C. This factor was greater in group A than in group C but indistinguishable in groups B and C. Atria from normal and diabetic rats were incubated in (3)[H]NE for 60 minutes. Excess tracer was removed, and atria were stimulated during a series of 1-minute episodes at half-maximal neural stimulation to cause exocytotic (3)[H]NE release. BK augmented (3)[H]NE release in normal (n=4) and in diabetic (n=4) atria. This BK-induced increase of (3)[H]NE overflow (expressed as a fraction of tissue (3)[H]NE radioactivity) was 4 times greater in diabetic than in normal preparations. The response to BK in releasing sympathetic neurotransmitter is augmented in diabetic rats, recovering in a manner dependent on insulin.

Insulin enhances the bradykinin response in L8 rat skeletal myoblasts.

Inhibitors of ACE/kininase II enhance insulin sensitivity, an action that is mediated in part by bradykinin (BK). We investigated whether insulin interacts with the BK receptor signaling to modulate the inositol 1,4,5-trisphosphate (IP3) response to BK in L8 rat skeletal myoblasts. Stimulation of the cultures with BK (10 nmol/l) for 15 s increased IP3 from a basal level of 75.2 +/- 7.6 to 200.2 +/- 15.7 pmol/mg protein. Treatment of the cultures with 1, 2, and 20 nmol/l of insulin for 90 min before adding BK increased IP3 formation by the same BK dose to 328.2 +/- 19, 434.5 +/- 18, and 460.8 +/-21.3 pmol/mg protein, respectively. When wortmannin was administered to inhibit phosphatidylinositol (PI) 3-kinases at lower concentration (1 nmol/l), it increased IP3 formation stimulated by BK only when insulin was present. At a higher concentration (100 nmol/l), wortmannin significantly enhanced BK-induced IP3 formation in the absence of insulin. Genistein and tyrphostin A-23, tyrosine kinase inhibitors, completely reversed the elevated IP3 formation by BK and insulin. The IP3 response to 10 nmol/l BK was 223.3 +/- 11.8 pmol/mg protein in the absence of insulin and 402.2 +/- 12.0 pmol/mg protein in the presence of 2 nmol/l insulin. However, when exposing the cultures to 1 nmol/l genistein or tyrphostin A-23, the IP3 response to BK in the presence of insulin decreased to 211.8 +/- 46.7 and 187.7 +/- 19.9 pmol/mg protein. Tyrphostin A-1, the inactive analog, was ineffective. Exposing the cells to 1 micromol/ 3,4,5-trimethoxybenzoic acid 8-[diethylamino]octyl ester, an intracellular Ca2+ antagonist, did not change the potentiation by insulin. But, exposing them to 0.1 micromol/l n-[6-aminohexyl]-5-chloro-1-naphthalene-sulfonamide, a calmodulin antagonist, resulted in enhanced IP3 response to BK alone to 292.2 +/- 18.5 pmol/mg protein and to BK in the presence of 1, 2, and 20 nmol/l insulin to 488 +/- 22.2, 625.5 +/- 11.6, and 665.2 +/- 15.9 pmol/mg protein, respectively. In conclusion, insulin potentiates BK-induced IP3 production in L8 rat skeletal myoblasts, and this action of insulin involves a tyrosine kinase. Inhibition of PI 3-kinases potentiated BK-induced IP3 formation in the presence of insulin. Calmodulin blocked the action of insulin. These results support a modulatory effect of insulin on the BK signaling system via a tyrosine kinase in L8 rat skeletal myoblasts that results in increased IP3 formation. Because BK release from skeletal muscle increases during contractions, this action of insulin is likely to play a role in the modulation of the excitation-contraction coupling process of the skeletal muscle.

Artifacts in CSI-measurements caused by the drift of the static magnetic field.

In chemical shift resolved spectroscopic imaging (CSI) temporal changes in the static magnetic field (drift) can lead to distortions of the phase encoding process. This can result in localization artifacts. The extent of the artifact depends on the size of the drift, the number of acquisitions, as well as on the combination of the size of the field of view and the number of phase encoding gradient steps. Furthermore, it is affected by the succession of the phase encoding gradients. Precautions are described which allow substantial minimization of the artifact.

[On pathogenesis of coronary heart disease]. / Zur Pathogenese der koronaren Herzerkrankung.

Being overweight (OW) was recognized very early as a risk factor for coronary heart disease (CHD). Its significance in the pathogenesis of CHD has been strengthened by observations showing that OW is responsible for the development of diabetes, hypertension and lipid disorders due to its induction of insulin resistance (IR). Its key role has been underlined further by recent studies indicating that OW causes endothelial dysfunction via elevated serum fatty acids, which initiates the molecular events that further the process of CHD. It is, therefore, of the utmost importance to determine its roots. The most probable reason for its high incidence is due to the genetic outfit of most people which does not permit adequate adaptation of the cerebral cortex according to the environmental changes which have occured since the early days.

Potential pitfall in the determination of free [Mg2+] by 31P NMR when using the beta/alpha-ATP peak height ratio method.

Recently, Clarke et al. (Clarke K, Kashiwaya Y, King MT, Gates D, Keon CA, Cross HR, Radda GK, Veech RL. The beta/alpha peak height ratio of ATP. A measure of free [Mg2+(free)] using 31P NMR, J. Biol. Chem. 1996;271:21142 21150.) reported a new method to noninvasively determine the concentration of intracellular free magnesium ([Mg2+(free)]) based on the measurement of the peak height ratio h(beta/alpha) of the beta- and alpha-ATP signals in 31P NMR spectra. h(beta/alpha) varies with [Mg2+(free)], however, the study presented here shows that h(beta/alpha) also strongly depends on the homogeneity of the static magnetic field. For this reason, we performed at a magnetic field strength of 1.5 T 31P NMR measurements of solutions that mimic intracellular medium. The magnetic field homogeneity was varied by changing the currents in the shim coils, and the effect on hbeta/alpha is demonstrated with and without proton decoupling. In both cases, h(beta/alpha) strongly depends on the magnetic field homogeneity and can therefore lead to a pitfall in the determination of [Mg2+(free)].

Oral administration of RAC-alpha-lipoic acid modulates insulin sensitivity in patients with type-2 diabetes mellitus: a placebo-controlled pilot trial.

Alpha-lipoic acid (ALA), a naturally occuring compound and a radical scavenger was shown to enhance glucose transport and utilization in different experimental and animal models. Clinical studies described an increase of insulin sensitivity after acute and short-term (10 d) parenteral administration of ALA. The effects of a 4-week oral treatment with alpha-lipoic acid were evaluated in a placebo-controlled, multicenter pilot study to determine see whether oral treatment also improves insulin sensitivity. Seventy-four patients with type-2 diabetes were randomized to either placebo (n = 19); or active treatment in various doses of 600 mg once daily (n = 19), twice daily (1200 mg; n = 18), or thrice daily (1800 mg; n = 18) alpha-lipoic acid. An isoglycemic glucose-clamp was done on days 0 (pre) and 29 (post). In this explorative study, analysis was done according to the number of subjects showing an improvement of insulin sensitivity after treatment. Furthermore, the effects of active vs. placebo treatment on insulin sensitivity was compared. All four groups were comparable and had a similar degree of hyperglycemia and insulin sensitivity at baseline. When compared to placebo, significantly more subjects had an increase in insulin-stimulated glucose disposal (MCR) after ALA treatment in each group. As there was no dose effect seen in the three different alpha-lipoic acid groups, all subjects receiving ALA were combined in the "active" group and then compared to placebo. This revealed significantly different changes in MCR after treatment (+27% vs. placebo; p < .01). This placebo-controlled explorative study confirms previous observations of an increase of insulin sensitivity in type-2 diabetes after acute and chronic intravenous administration of ALA. The results suggest that oral administration of alpha-lipoic acid can improve insulin sensitivity in patients with type-2 diabetes. The encouraging findings of this pilot trial need to be substantiated by further investigations.

ACE inhibition and glucose transport in insulinresistant muscle: roles of bradykinin and nitric oxide.

Acute administration of the angiotensin-converting enzyme (ACE) inhibitor captopril enhances insulin-stimulated glucose transport activity in skeletal muscle of the insulin-resistant obese Zucker rat. The present study was designed to assess whether this effect is mediated by an increase in the nonapeptide bradykinin (BK), by a decrease in action of ANG II, or both. Obese Zucker rats (8-9 wk old) were treated for 2 h with either captopril (50 mg/kg orally), bradykinin (200 micrograms/kg ip), or the ANG II receptor (AT(1) subtype) antagonist eprosartan (20 mg/kg orally). Captopril treatment enhanced in vitro insulin-stimulated (2 mU/ml) 2-deoxyglucose uptake in the epitrochlearis muscle by 22% (251 +/- 7 vs. 205 +/- 9 pmol. mg(-1). 20 min(-1); P < 0.05), whereas BK treatment enhanced this variable by 18% (249 +/- 15 vs. 215 +/- 7 pmol. mg(-1). 20 min(-1); P < 0.05). Eprosartan did not significantly modify insulin action. The BK-mediated increase in insulin action was completely abolished by pretreatment with either the specific BK-B(2) receptor antagonist HOE 140 (200 micrograms/kg ip) or the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (50 mg/kg ip). Collectively, these results indicate that the modulation of insulin action by BK likely underlies the metabolic effects of ACE inhibitors in the insulin-resistant obese Zucker rat. Moreover, this modulation of insulin action by BK is likely mediated through B(2) receptors and by an increase in nitric oxide production and/or action in skeletal muscle tissue.

The beta2-adrenergic modulator celiprolol reduces insulin resistance in obese Zucker rats.

Essential hypertension is associated with an increased incidence of insulin resistance of skeletal muscle glucose transport. The present study determined if celiprolol, an antihypertensive agent with selective beta1-adrenoceptor antagonist and additional beta2-agonistic properties, administered by gavage either acutely (3 hr) or chronically (14 d), had a direct effect on improving glucose tolerance and insulin-stimulated glucose transport activity (using 2-deoxyglucose (2-DG) uptake) in isolated epitrochlearis muscles of the insulin-resistant obese Zucker rat. The effects of a selective beta1-blocker, metoprolol, were also assessed. Acute administration of celiprolol, but not metoprolol, increased insulin-stimulated 2-DG uptake in muscle by 22% (p<0.05). Chronic celiprolol treatment significantly lowered fasting plasma insulin (22%) and free fatty acids (40%) in comparison to obese control values. Moreover, chronic celiprolol administration decreased the glucose-insulin index (calculated as the product of the glucose and insulin areas under the curve during an oral glucose tolerance test), by 32% (p<0.05) compared to obese controls, indicating that peripheral insulin action was increased. Indeed, insulin-stimulated skeletal muscle 2-DG uptake was enhanced by 49% (p<0.05) in these celiprolol-treated obese animals. Metoprolol was without significant effect on any of these variables following chronic administration. These findings indicate that, in this animal model of insulin resistance, the beta1-antagonist/beta2-agonist celiprolol has a specific effect of improving insulin-stimulated skeletal muscle glucose transport that is independent of any hemodynamic alterations.

31P nuclear magnetic resonance spectroscopy: a noninvasive tool to monitor metabolic abnormalities in left ventricular hypertrophy in human.

31p nuclear magnetic resonance (NMR) spectroscopy represents a unique instrument to noninvasively monitor myocardial metabolism in humans. The technique has been used to study the metabolism in myocardial hypertrophy in humans with hypertension, aortic stenosis, aortic incompetence, mitral regurgitation, and hypertrophic cardiomyopathy, as well as after maintenance dialysis or long-term physical exercise in elite cyclists. A primary aim is the determination of the phosphocreatine (PCr)/adenosine triphosphate (ATP) ratio, which reflects the energetic state of the myocardium. Recent investigations take advantage of proton decoupling in 31p NMR spectroscopy, which, besides the PCr/ATP ratio, also allows the determination of the inorganic phosphate/ PCr and the phosphomonoester/PCr ratios as additional indicators for alterations in myocardial metabolism. Abnormal myocardial metabolism was found in humans with aortic stenosis, mitral regurgitation, hypertrophic cardiomyopathy, and in patients who undergo maintenance dialysis. A trend toward a lower PCr/ATP ratio was reported in hypertension and aortic incompetence patients. Several studies have revealed a dependence of the metabolic abnormalities on the degree of heart failure, and one study claimed that a correlation with the extent of hypertrophy exists. No metabolic abnormalities were found in elite cyclists.

31P/1H WALTZ-4 broadband decoupling at 1.5 T: different versions of the composite pulse and consequences when using a surface coil.

Two derivatives of the wideband alternating-phase low-power technique for zero-residual splitting (WALTZ)-4 decoupling sequence for broadband decoupling named WALTZ-4a and WALTZ-4b were compared for their proton decoupling performance in 31P nuclear magnetic resonance (NMR) spectroscopy using a Siemens Magnetom SP 1.5 T whole-body imager. Version WALTZ-4a originally implemented by the manufacturer doubles and triples the transmitter amplitude of the 90 degrees pulse to achieve the 180 degrees and 270 degrees flip angle required for one composite pulse R in the WALTZ sequence. WALTZ-4b follows the sequence reported from Shaka et al. and leaves the transmitter amplitude constant but increases the durations of the 180 degrees and 270 degrees pulses. The decoupling performance of WALTZ-4b is superior because it requires less transmitter power and, therefore, it is advantageous in all in vivo studies where a low specific absorption rate is desired. When WALTZ-4 is used in combination with a surface coil for transmission the theoretically required flip angles cannot be achieved in the entire sensitive volume of the coil. The decoupling performance was therefore investigated at lower and higher flip angles. Again, WALTZ-4b is advantageous and provides, in certain ranges that are off-resonant from the decoupling frequency, a good decoupling quality even for flip angles that are only 60% of the theoretically required.

Effect of chronic bradykinin administration on insulin action in an animal model of insulin resistance.

The nonapeptide bradykinin (BK) has been implicated as the mediator of the beneficial effect of angiotensin-converting enzyme inhibitors on insulin-stimulated glucose transport in insulin-resistant skeletal muscle. In the present study, the effects of chronic in vivo BK treatment of obese Zucker (fa/fa) rats, a model of glucose intolerance and severe insulin resistance, on whole body glucose tolerance and skeletal muscle glucose transport activity stimulated by insulin or contractions were investigated. BK was administered subcutaneously (twice daily at 40 microg/kg body wt) for 14 consecutive days. Compared with a saline-treated obese group, the BK-treated obese animals had significantly (P < 0.05) lower fasting plasma levels of insulin (20%) and free fatty acids (26%), whereas plasma glucose was not different. During a 1 g/kg body wt oral glucose tolerance test, the glucose and insulin responses [incremental areas under the curve (AUC)] were 21 and 29% lower, respectively, in the BK-treated obese group. The glucose-insulin index, the product of the glucose and insulin AUCs and an indirect index of in vivo insulin action, was 52% lower in the BK-treated obese group compared with the obese control group. Moreover, 2-deoxyglucose uptake in the isolated epitrochlearis muscle stimulated by a maximally effective dose of insulin (2 mU/ml) was 52% greater in the BK-treated obese group. Contraction-stimulated (10 tetani) 2-deoxyglucose uptake was also enhanced by 35% as a result of the BK treatment. In conclusion, these findings indicate that in the severely insulin-resistant obese Zucker rat, chronic in vivo treatment with BK can significantly improve whole body glucose tolerance, possibly as a result of the enhanced insulin-stimulated skeletal muscle glucose transport activity observed in these animals.

Detection of phosphomonoester signals in proton-decoupled 31P NMR spectra of the myocardium of patients with myocardial hypertrophy.

Proton-decoupled 31P NMR spectroscopy at 1.5 T of the anterior left ventricular myocardium was used to monitor myocardial phosphate metabolism in asymptomatic patients with hypertrophic cardiomyopathy (HCM, n = 14) and aortic stenosis (AS, n = 12). In addition to the well-known phosphorus signals a phosphomonoester (PME) signal was detected at about 6.9 ppm in 7 HCM and 2 AS patients. This signal was not observed in the spectra of normal controls (n = 11). We suggest that in spectra of patients with myocardial hypertrophy the presence of a PME signal reflects alterations in myocardial glucose metabolism.

31P NMR spectroscopy detects metabolic abnormalities in asymptomatic patients with hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) often causes sudden, unexpected death in adolescents and young adults. Alterations in myocardial metabolism are considered to be causes for contractile dysfunction. We examined the question of whether metabolic abnormalities antedate the manifestation of symptoms in patients with HCM. METHODS AND RESULTS: Proton-decoupled 31P NMR spectroscopy of the anterior left ventricular wall of the heart of 14 young, asymptomatic patients with HCM was performed with a 1.5-T whole-body imager. Spectra of the phosphate metabolites were compared with those of normal control subjects. The patients exhibited a significantly reduced (P<0.02) ratio of phosphocreatine (PCr) to ATP of 1.98+/-0.37 (mean+/-SD), compared with 2.46+/-0.53 obtained in 11 normal control subjects. In addition, the group of patients with severe hypertrophy of the interventricular septum (n=8) showed a significantly increased (P<0.05) Pi-to-PCr ratio, with a Pi x 100/PCr of 20.0+/-8.3 versus 9.7+/-7.2 in control subjects. Both abnormalities are similar to those found in ischemic myocardium. This view is also supported by a significantly increased (P<0.01) phosphomonoester (PME)-to-PCr ratio, with a PME x 100/PCr of 20.7+/-11.2 compared with 8.4+/-6.7 in control subjects, indicating altered glucose metabolism. CONCLUSIONS: 31P NMR spectroscopy detects alterations of myocardial metabolism in asymptomatic patients with HCM. These alterations may contribute to the understanding of the pathophysiology and natural history of the disease.

Proton-decoupled myocardial 31P NMR spectroscopy reveals decreased PCr/Pi in patients with severe hypertrophic cardiomyopathy.

Disturbed myocardial energy metabolism may occur in patients with primary hypertrophic cardiomyopathy (HCM). A noninvasive way to gain insight into cardiac energy metabolism is provided by in vivo 31P nuclear magnetic resonance (NMR) spectroscopy. 31P NMR spectroscopy with proton decoupling was performed in 13 patients aged 13-36 years with HCM on a 1.5 T Magnetom with a double resonant surface coil. A 2D chemical shift imaging (CSI) sequence in combination with slice selective excitation was used to acquire spectra of the anteroseptal region of the left ventricle (volume element: 38 mL). The chemical shifts of the phosphorus metabolites, intracellular pHi, and coupling constants J(alphabeta) and J(gammabeta) were calculated. Peak areas of 2,3-diphosphoglycerate (DPG), Pi, and adenosine triphosphate (ATP) were determined and corrected for blood contamination, saturation, and differences in nuclear Overhauser enhancements (NOE). The maximum thickness of the interventricular septum (IVSmax) was determined from tomographic long-axis images and expressed as number of standard deviations above the mean of the normal population (Z score). The patients were then divided into 2 groups: 6 patients with moderate HCM (HCMm, Z score < or = 5) and 7 patients with severe HCM (HCMs, Z score > 5). No differences between both groups and a control group of healthy volunteers (n = 16) were found with respect to phosphocreatine (PCr)/gamma-ATP ratio, pHi, or the coupling constants. Only the PCr/Pi ratio differed significantly from the control group (HCM(all), alpha < 0.05, HCMs, alpha < 0.02, 2-sided U test). The decrease of the PCr/Pi ratio in patients with HCM is probably caused by ischemically decreased oxygen supply in the severely hypertrophied myocardium.