Total cholesterol in serum determined by isotope dilution/mass spectrometry, with liquid-chromatographic separation

We describe an accurate, precise method for determination of total serum cholesterol by isotope dilution/mass spectrometry (IDMS) with liquid-chromatographic separation. After adding [3,4-13C]cholesterol to serum and hydrolyzing the cholesterol esters, we extract the total cholesterol. "High-performance" liquid chromatography (HPLC) is used to separate the extracted cholesterol for measurement by electron-impact mass spectrometry with use of a direct-insertion device. To evaluate the specificity and the accuracy of this method, we also studied the conventional IDMS method, which involves converting cholesterol to the trimethylsilyl ether and assay by gas chromatography-mass spectrometry with use of a capillary column. The coefficient of variation for the HPLC method was a little larger than that for the conventional method, but mean values by each method agreed within 1% for all sera tested.     

Impact of turbulent blood flow in the aortic root on de novo aortic insufficiency during continuous-flow left ventricular-assist device support

Severe aortic insufficiency (AI) after implantation of continuous-flow left ventricular-assist device (LVAD) affects device performance and outcomes. However, the mechanism for the occurrence and progression of AI has not been elucidated. We investigated the impact of nonphysiological retrograde blood flow in the aortic root on AI after LVAD implantation. Blood flow pattern was analyzed in patients with and without AI (n = 3 each) who underwent LVAD implantation, by computational fluid dynamics with patient-specific geometries, which were reproduced using electrocardiogram-gated 320-slice computed tomographic images. The total volume of retrograde blood flow during one cardiac cycle (716 ± 88 mL) was higher and the volume of slow blood flow (<0.1 cm/s) (0.16 ± 0.04 cm³) was lower in patients with AI than in those without AI (360 ± 111 mL, P = .0495, and 0.49 ± 0.08 cm³, P = .0495, respectively). No significant difference in wall shear stress on the aortic valve was observed between the groups. Patients with AI had a perpendicular anastomosis at the distal ascending aorta and the simulation in the modified anastomosis model of patients with AI showed that the retrograde blood flow pattern depended on the angle and position of anastomosis. Computational fluid dynamics revealed strong retrograde blood flow in the ascending aorta and aortic root in patients with AI after LVAD implantation. The angle and position of LVAD outflow anastomosis might impact retrograde blood flow and de novo AI after LVAD implantation.     

Very Short-Term Effects of the Dipeptidyl Peptidase-4 Inhibitor Sitagliptin on the Secretion of Insulin,Glucagon, and Incretin Hormones in Japanese Patients with Type?2 Diabetes Mellitus: Analysis of Meal Tolerance Test Data

Background

Sitagliptin inhibits dipeptidyl peptidase-4, which inactivates the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide. To assess its antidiabetic potency, we used meal tolerance tests (MTTs) to determine the very short-term effects of sitagliptin on plasma concentrations of insulin and glucagon.

Methods

On day 1, patients with newly diagnosed or uncontrolled type 2 diabetes mellitus started a calorie-restricted diet. On day 2, the first MTT was performed, before treatment with sitagliptin 50 mg/day started later the same day. On day 5, a second MTT was performed. Area under the concentration–time curves (AUCs) of relevant laboratory values were calculated [AUC from time zero to 2 h (AUC_0–2h) and from time zero to 4 h (AUC_0–4h)].

Results

Fifteen patients were enrolled. AUCs for postprandial plasma glucose were decreased after 3 days of sitagliptin treatment [AUC_0–2h 457 ± 115 mg/dL·h (25.4 ± 6.4 mmol/L·h) to 369 ± 108 mg/dL·h (20.5 ± 6.0 mmol/L·h); AUC_0–4h 896 ± 248 mg/dL·h (49.7 ± 13.8 mmol/L·h) to 701 ± 246 mg/dL·h (38.9 ± 13.7 mmol/L·h); both p < 0.001]. AUC_0–2h and AUC_0–4h for postprandial plasma glucagon also decreased: 195 ± 57 to 180 ± 57 pg/mL·h (p < 0.05) and 376 ± 105 to 349 ± 105 pg/mL·h (p < 0.01), respectively. The AUC_0–2h [median with quartile values (25 %, 75 %)] for active GLP-1 increased: 10.5 (8.5, 15.2) to 26.4 (16.7, 32.4) pmol/L·h (p = 0.03).

Conclusions

Very short-term (3-day) treatment with sitagliptin decreases postprandial plasma glucose significantly. This early reduction in glucose may result partly from suppression of excessive glucagon secretion, through a direct effect on active GLP-1. Improvement in postprandial plasma glucose, through suppression of glucagon secretion, is believed to be an advantage of sitagliptin for the treatment of patients with type 2 diabetes.     

Threonine-17 phosphorylation of phospholamban: a key determinant of frequency-dependent increase of cardiac contractility

Multiple studies have shown that phospholamban (PLN) plays a key role in regulation of frequency-dependent increase of cardiac contraction, a hallmark of the contractile reserve in myocardium. However, the mechanisms underlying this relationship remain elusive. Phosphorylation of PLN occurs on residues: serine-16 (Ser(16)) and threonine-17 (Thr(17)) in vivo. In isolated wild-type cardiomyocytes, we found that increases of stimulation frequency from 0.5 to 5 Hz were associated with increased Thr(17) phosphorylation of PLN and cardiac contractility. To further delineate the role of PLN phosphorylation in the frequency-dependent increases of cardiac function, three transgenic mouse models, expressing wild-type, Ser16Ala (S16A), or Thr17Ala (T17A) mutant PLN in the null background were generated. Transgenic lines expressing similar levels of wild-type or mutant PLN were selected and isolated cardiomyocytes were paced from 0.5 to 5 Hz. Upon increases in pacing frequency, the fractional shortening (FS) and rates of contraction (+dL/dt) and relaxation (-dL/dt) increased in wild-type and S16A mutant PLN cardiomyocytes. In contrast, in myocytes expressing the T17A mutant PLN, there were no increases in FS and +/-dL/dt upon increasing the frequency of stimulation. The time to 50% peak shortening (TTP(50)) and to 50% relaxation (TTR(50)) were also abbreviated to a much higher extent (two-fold) in wild-type and S16A mutant compared to T17A mutant PLN cardiomyocytes. These results indicate that Thr(17) phosphorylation of PLN is the major contributor to frequency-dependent increases of contractile and relaxation parameters in mouse cardiomyocytes, although some increases in these parameters occur even in the absence of PLN phosphorylation. Thus, the positive force-frequency relationship in cardiomyocytes is mechanistically and mainly related to PLN phosphorylation.     

Acute myocardial infarction associated with systemic lupus erythematosus documented by coronary arteriograms

A 19-year-old man with untreated systemic lupus erythematosus had an acute myocardial infarction. A coronary arteriogram five hours after the onset of symptoms revealed total occlusion of the left anterior descending coronary artery. Reperfusion was achieved by coronary thrombolytic therapy with urokinase. Four weeks later, a coronary arteriogram showed only minimal luminal irregularities at the original site of occlusion, where significant reduction in diameter could be induced by ergonovine maleate. This case suggests that coronary arterial involvement in systemic lupus erythematosus may be related to coronary arterial spasm.     

Altered platelet alpha 2 adrenoreceptor in acute myocardial infarction and its relation to plasma catecholamine concentrations.

Changes in platelet alpha 2 adrenoreceptors and their relation to plasma catecholamine concentrations were studied in 11 patients with acute transmural myocardial infarction. A radiolabelled alpha 2 adrenoreceptor antagonist, [3H]-yohimbine, was used to assay alpha 2 adrenoreceptors on platelet membranes, and plasma catecholamine concentrations were measured by high performance liquid chromatography. The number of platelet alpha 2 adrenoreceptors, the dissociation constant, and plasma noradrenaline and adrenaline concentrations were studied 6.6 (3.3) (mean (SD)) hours after the onset of acute myocardial infarction and one month later. The mean (SD) number of adrenoreceptors increased significantly from 94.5 (50.5) fmol/mg protein immediately after infarction to 157.0 (65.7) fmol/mg protein one month later. The dissociation constant, however, did not change significantly (4.33 (1.40) nmol/l vs 4.37 (1.22) nmol/l). Raised noradrenaline (5.60 (4.37) nmol/l) and adrenaline (0.28 (0.14) nmol/l) concentrations had fallen significantly to normal values (1.21 (0.67) and 0.09 (0.05) nmol/l respectively) a month after infarction. The decrease in the number of alpha 2 adrenoreceptors soon after infarction may be beneficial because such a change will reduce the strength of various reactions to catecholamines, such as vasoconstriction.     

Serum thioredoxin levels as a predictor of steatohepatitis in patients with nonalcoholic fatty liver disease

BACKGROUND/AIMS: Thioredoxin (TRX) is a stress-inducible thiol-containing protein. The aim of this study was to evaluate the clinical significance of serum TRX in patients with nonalcoholic steatohepatitis (NASH) or simple steatosis. METHODS: Serum TRX levels were determined using an enzyme-linked immunosorbent assay kit in 25 patients with NASH, 15 patients with simple steatosis, and 17 healthy volunteers. RESULTS: Serum TRX levels (medians and (ranges), ng/ml) were significantly elevated in patients with NASH (60.3 (17.6-104.7)), compared to those in patients with simple steatosis (24.6 (16.6-69.7), P=0.0009) and in healthy controls (23.5 (1.3-50.7), P<0.0001). Serum ferritin levels in patients with NASH were also significantly higher than the levels in patients with simple steatosis. The receiver operating characteristic curve confirmed that serum TRX and ferritin levels were predictors for distinguishing NASH from simple steatosis. Higher grades of histological iron staining were observed in NASH than in simple steatosis. Serum TRX tended to increase in accordance with hepatic iron accumulation and the histological severity in patients with NASH. CONCLUSIONS: The pathogenesis of NASH may be associated with iron-related oxidative stress. The serum TRX level is a parameter for discriminating NASH from simple steatosis as well as a predictor of the severity of NASH.     

Early result of heart transplantation in Japan: Osaka University experience

Since the new organ transplantation law was established in 1997, 17 heart transplantations have been performed in Japan, 7 of which were carried out at Osaka University Hospital. Recipient diagnosis was dilated cardiomyopathy in 2, dilated phase of hypertrophic cardiomyopathy in 4, and post-myocarditis cardiomyopathy in 1. Ages ranged from 8 to 49 years with a mean of 35.3 years. Five patients were bridged with a left ventricular assist device. The waiting period was 182-977 days (mean, 643 days). There was no early or late death during follow-up of 1-4.8 years. Under a standard triple-drug regimen using mycophenolate, there were 3 rejection episodes greater than grade 3 in 2 patients, and humoral rejection requiring plasmapheresis in one. A young boy whose donor was a hemodynamically compromised adult developed neurological sequelae after resuscitation following ventricular tachycardia. All patients were discharged and went back to work or their regular daily life. Although the donor shortage is still severe in Japan, the resumption of heart transplantation has been satisfactory, and left ventricular assist devices have played a crucial role.