Evaluation of 4β-Hydroxycholesterol and 25-Hydroxycholesterol as Endogenous Biomarkers of CYP3A4: Study with CYP3A-Humanized Mice

Cytochrome P450 3A (CYP3A) enzymes metabolize approximately half of all drugs on the market. Since the endogenous compounds 4β-hydroxycholesterol (4β-HC) and 25-hydroxycholesterol (25-HC) are generated from cholesterol via CYP3A enzymes, we examined whether the plasma levels of 4β-HC and 25-HC reflect hepatic CYP3A4 activity by using a CYP3A-humanized mouse model, in which the function of endogenous Cyp3a was genetically replaced by human CYP3A. CYP3A-humanized mice have great advantages for evaluation of the relationship between hepatic CYP3A protein levels and plasma and hepatic levels of 4β-HC and 25-HC. Levels of CYP3A4 protein in the liver microsomes of CYP3A-humanized mice were increased by treatment with pregnenolone-16α-carbonitrile, a CYP3A inducer. Hepatic and plasma levels of 4β-HC and 25-HC normalized by cholesterol were significantly correlated with hepatic CYP3A4 protein levels. In addition, in vitro studies using human liver microsomes showed that the formation of 4β-HC was strongly inhibited by a CYP3A inhibitor, while the inhibitory effect of the CYP3A inhibition on the formation of 25-HC was weak. These results suggested that CYP3A mainly contributed to the formation of 4β-HC in human liver microsomes, whereas other factors may be involved in the formation of 25-HC. In conclusion, the in vivo studies using CYP3A-humanized mice suggest that plasma 4β-HC and 25-HC levels reflect hepatic CYP3A4 activity. Furthermore, taking the results of in vitro studies using human liver microsomes into consideration, 4β-HC is a more reliable biomarker of hepatic CYP3A activity.     

Optimal cannula positioning of HeartMate 3 left ventricular assist device

Cannula position in HeartMate II and HeartWare left ventricular assist devices (LVADs) is associated with clinical outcome. This study aimed to investigate the clinical implication of the device positioning in HeartMate 3 LVAD cohort. Consecutive patients who underwent HeartMate 3 LVAD implantation were followed for one year from index discharge. At index discharge, chest X-ray parameters were measured: (a) cannula coronal angle, (b) height of pump bottom, (c) cannula sagittal angle, and (d) cannula lumen area. The association of each measurement of cannula position with one-year clinical outcomes was investigated. Sixty-four HeartMate 3 LVAD patients (58 years old, 64% male) were enrolled. In the multivariable Cox regression model, the cannula coronal angle was a significant predictor of death or heart failure readmission (hazard ratio 1.27 [1.01-1.60], P = .045). Patients with a cannula coronal angle ≤28° had lower central venous pressure (P = .030), lower pulmonary capillary wedge pressure (P = .027), and smaller left ventricular size (P = .019) compared to those with the angle >28°. Right ventricular size and parameters of right ventricular function were also better in the narrow angle group, as was one-year cumulative incidence of death or heart failure readmission (10% vs. 50%, P = .008). Narrow cannula coronal angle in patients with HeartMate 3 LVADs was associated with improved cardiac unloading and lower incidence of death or heart failure readmission. Larger studies to confirm the implication of optimal device positioning are warranted.     

Application of a quantitative 1H-NMR method for the determination of amygdalin in Persicae semen, Armeniacae semen, and Mume fructus

A quantitative ¹H-NMR method (qHNMR) was used to measure the amygdalin content of Persicae semen, Armeniacae semen, and Mume fructus, in each of which amygdalin constitutes a major component. The purity of amygdalin was calculated from the ratio of the intensity of the amygdalin H-2 signal at δ 6.50 ppm in pyridine-d ₅ to that of the hexamethyldisilane (HMD) signal at 0 ppm. The HMD concentration was corrected by the International System of Units (SI) traceability with certified reference material (CRM)-grade bisphenol A. qHNMR revealed the amygdalin contents to be 2.72 and 3.13 % in 2 lots of Persicae semen, 3.62 and 5.19 % in 2 lots of Armeniacae semen, and 0.23 % in Mume fructus. Thus, we demonstrated the utility of this method for the quantitative analysis of crude drugs.     

Intermittent hypoxia-induced increases in reactive oxygen species activate NFATc3 increasing endothelin-1 vasoconstrictor reactivity

Sleep apnea (SA), defined as intermittent respiratory arrest during sleep, is associated with increased incidence of hypertension, peripheral vascular disease, stroke, and sudden cardiac death. We have shown that intermittent hypoxia with CO2 supplementation (IH), a model for SA, increases blood pressure and circulating ET-1 levels, upregulates lung pre-pro ET-1 mRNA, increases vasoconstrictor reactivity to ET-1 in rat small mesenteric arteries (MA) and increases vascular reactive oxygen species (ROS). NFAT activity is increased in the aorta (AO) and MA of mice exposed to IH in an ET-1-dependent manner, and the genetic ablation of the isoform NFATc3 prevents IH-induced hypertension. We hypothesized that IH causes an increase in arterial ROS generation, which activates NFATc3 to increase vasoconstrictor reactivity to ET-1. In support of our hypothesis, we found that IH increases ROS in AO and MA. In vivo administration of the SOD mimetic tempol during IH exposure prevents IH-induced increases in NFAT activity in mouse MA and AO. We found that IH causes an NFATc3-dependent increase in vasoconstrictor reactivity to ET-1, accompanied by an increase in vessel wall [Ca²⁺]. Our results indicate that IH exposure causes an increase in arterial ROS to activate NFATc3, which then increases vasoconstrictor reactivity and Ca²⁺ response to ET-1. These studies highlight a novel regulatory pathway, and demonstrate the potential clinical relevance of NFAT inhibition to prevent hypertension in SA patients.     

Classification of human immunodeficiency virus-infected patients with chronic kidney disease using a combination of proteinuria and estimated glomerular filtration rate

Background

In 2012, the Kidney Disease: Improving Global Outcomes (KDIGO) updated the 2002 Kidney Disease Outcomes Quality Initiative (KDOQI) clinical practice guideline for chronic kidney disease (CKD). The 2012 KDIGO guideline elaborated the identification and prognosis of CKD by combining albuminuria with estimated glomerular filtration rate (eGFR). Identification of CKD with a high risk for a poor prognosis was investigated in human immunodeficiency virus (HIV)-infected individuals by applying the new guideline.

Methods

A total of 1,447 HIV-infected patients (1,351 male, 96 female; mean age 44.4 ± 11.5 years) were classified using a combination of eGFR and dipstick proteinuria, as a convenient alternative to albuminuria. Proteinuria was classified into 3 grades—(A1) – and +/? , (A2) 1+ and 2+ , and (A3) 3+ and 4+. eGFR was classified into 6 grades—(G1) ≤90, (G2) 60–89, (G3a) 45–59, (G3b) 30–44, (G4) 15–29, and (G5) <15 mL/min/1.73 m².

Results

Mean CD4 cell count was 487 ± 214 /μL, with 80.7 % of patients having an undetectable HIV-RNA level. The prevalence of CKD stage ≤2 and stage ≥3 classified according to KDOQI staging was 93.4 and 6.6 %, respectively. Using the new KDIGO classification, the prevalence of CKD with either a low (green) or moderately increased (yellow) risk was 96.9 %, while the prevalence for a high (orange) and very high (red) risk was 3.1 %.

Conclusion

The use of the new KDIGO classification may reduce the prevalence of HIV-infected CKD individuals who are at high risk for a poor prognosis by nearly a half.     

Superficial elevated-type early gallbladder carcinoma treated by laparoscopic cholecystectomy

A 60-year-old woman was admitted to our department for detailed examination of a polypoid lesion of the gallbladder detected at the time of a mass survey by ultrasound. Endoscopic ultrasonography (EUS) demonstrated a broad-based mass lesion, about 10 mm in size, with an irregular surface, at the peritoneal side of the body of the gallbladder. The layer structure of the gallbladder wall had not been destroyed by the mass. Computed tomography showed no direct invasion of the liver or other evidence of metastasis. Type-IIa (superficial elevated-type) early gallbladder cancer was suspected and laparoscopic cholecystectomy was performed. Histologically, the tumor proved to be a papillo-tubular adenocarcinoma, 9×8 mm in size, confined to the mucosa and without lymphatic permeation, vascular involvement, perineural invasion, or other signs of metastasis. Laparoscopic cholecystectomy for gallbladder cancer can be indicated only when a lesion is a pedunculated protruded-type (type-Ip) cancer, or a broad-based cancer 10 mm or less in size located on the peritoneal side with no destruction of the layer structure of the wall demonstrated by EUS. This strategy is justified only with precise evaluation of the lesion by EUS.