High-performance liquid chromatographic determination of furosemide in plasma and urine and its use in bioavailability studies

A sensitive, selective and efficient reversed-phase high-performance liquid chromatographic (HPLC) method is reported for the determination of furosemide in human plasma and urine. The method has a sensitivity limit of 5 ng/ml in plasma, with acceptable within- and between-day reproducibilities and good linearity (r²>0.99) over a concentration range from 0.05 to 2.00 μg/ml. The one-step extract of furosemide and the internal standard (warfarin) from acidified plasma or urine was eluted through a μBondapak C₁₈ column with a mobile phase composed of 0.01 M potassium dihydrogenphosphate and acetonitrile (62:38, v/v) adjusted to pH 3.0. Within-day coefficients of variation (C.V.s) ranged from 1.08 to 8.63% for plasma and from 2.52 to 3.10% for urine, whereas between-day C.V.s ranged from 4.25 to 10.77% for plasma and from 5.15 to 6.81% for urine at three different concentrations. The minimum quantifiable concentration of furosemide was determined to be 5 ng/ml. The HPLC method described has the capability of rapid and reproducible measurement of low levels of furosemide in small amounts of plasma and urine. This method was utilized in bioavailability/pharmacokinetic studies for the routine monitoring of furosemide levels in adults, children and neonate patients.     

Determination and pharmacokinetics of a furosemide–amiloride drug combination

The study presents an accurate and precise HPLC assay for the determination of furosemide and amiloride in human specimens. Both drugs were extracted from human plasma with ethyl acetate; furosemide was extracted at pH 1 and amiloride at pH 12. While chromatographic separation conditions, i.e., column, mobile phase and flow-rate were the same for both investigated drugs, furosemide was detected using a UV absorbance detector, whereas amiloride, because of its very low therapeutic range, was detected with a spectrofluorimetric detector. The linearity of the furosemide and amiloride assays were confirmed over the range of 30–3000 ng/ml and 0.5–30 ng/ml, respectively. These concentrations correspond well with the therapeutic ranges of both drugs. The extraction recoveries, depending on concentration, exceed 80% for furosemide and 74% for amiloride. The reported methods were applied to pharmacokinetic investigations of the two compounds taken in form of a drug combination.     

呋塞米联合硝普钠注射液治疗顽固性心力衰竭的临床效果研究

目的:探讨呋塞米与硝普钠注射液联合治疗顽固性心力衰竭的临床效果及安全性。方法:选择2016年5月至2018年5月在我院进行治疗的90例顽固性心力衰竭患者,按照随机数字表法分为观察组和对照组。对照组给予基础治疗,观察组以对照组为基础加用呋塞米联合硝普钠注射液治疗。治疗后,观察和比较两组的临床疗效、治疗前后血清B型脑钠肽(BNP)、C-反应蛋白(CRP)水平及心功能[左室射血分数(LVEF)、左室舒张末容积指数(LVEDVI)、左室收缩末容积指数(LVESVI)、左室舒张早期与晚期充盈速度比值(E/A)]的变化。结果:治疗后,观察组总有效率(91.1%)明显高于对照组(66.7%)(P0.05)。与治疗前相比,两组患者治疗后的血清BNP、CRP、LVEDVI、LVESVI水平均明显低于治疗前,LVEF、E/A明显高于治疗前(P0.05);与对照组相比,观察组治疗后血清BNP、CRP、LVEDVI、LVESVI水平均明显降低,LVEF、E/A显著升高(P0.05)。两组治疗期间均未发生严重不良反应。结论:与常规治疗相比,呋塞米联合硝普钠注射液治疗顽固性心力衰竭患者可更有效改善其心功能,提高其临床疗效,且安全性高。     

Decrease in the Time-Dependent Difference in Urinary Excretion of Furosemide with Age

The influence of age on the time-dependent difference in urinary excretion of furosemide, a loop diuretic agent, was examined in this longitudinal study. Male Wistar rats were maintained under conditions of light from 07:00 to 19:00 h and dark from 19:00 to 07:00 h. Furosemide (30 mg/kg) was given orally at 12:00 h (day trial) or 00:00 h (night trial) to rats at 3 months of age, and urine was collected for 8 h after dosage. Thereafter, the identical protocol was repeated using the same animals at 6, 9, 12, 15, 18, and 21 months of age. The urinary excretion of furosemide was significantly greater in the day than in the night trial at 3 months of age. Such a time-dependent difference was observed for up to 15 months, but disappeared at 18 and 21 months of age. The time-dependent difference in urinary excretion of furosemide (day trial — night trial) decreased gradually throughout the observation period of the study. These results suggest that the time-dependent difference in the urinary excretion of furosemide diminishes during the aging process and disappears by 18 months of age in male Wistar rats.     

Effect of ouabain upon diuretic-sensitive K+ transport in cultured cells evidence for separate modes of operation of the transporter

(1) Unidirectional K⁺ (⁸⁶Rb) influx and efflux were measured in subconfluent layers of MDCK renal epithelial cells and HeLa carcinoma cells. (2) In both MDCK and HeLa cells, the furosemide-inhibitable and chloride-dependent component of K⁺ influx/efflux was stimulated 2-fold by a 30 min incubation in 1 · 10^?3 M ouabain. (3) Measurements of net K⁺ loss and Na⁺ gain in ouabain-treated cells at 1 h failed to show any diuretic sensitive component, confirming the exchange character of the diuretic-sensitive fluxes. (4) Prolonged incubations for 2.5 h in ouabain revealed a furosemide- and anion-dependent K⁺ (Cl^?) outward net flux uncoupled from net Na⁺ movement. Net K⁺ (Cl^?) outward flux was half-maximally inhibited by 2 μM furosemide. (5) After 2.5 h ouabain treatment, the anion and cation dependence of the diuretic-sensitive K⁺ influx/efflux were essentially unchanged when compared to untreated controls.     

50%葡萄糖静注联合呋塞米治疗顽固性心力衰竭疗效分析

目的:探讨50%葡萄糖静注联合持续泵入呋塞米治疗顽固性心力衰竭临床效果。方法:62例顽固性心力衰竭患者随机分成联合治疗组和对照组,每组31例,联合治疗组采取50%葡萄糖100 m L静注联合呋塞米100 mg持续静脉泵入,对照组采取呋塞米300 mg间断注射。记录两组患者临床治疗效果,比较两组患者治疗前后24小时尿量、脑利钠肽、心脏指数(CI)、左心室舒张期前后径(LVEDD)和左室射血分数(LVEF)变化情况。结果:联合治疗组患者临床效果总有效率90.3%,远高于对照组64.5%(P0.05);治疗后,两组患者24 h尿量均有所增加,BNP浓度均有所减少,差异均具有统计学意义(均P0.05),联合治疗组患者24 h尿量多于对照组,BNP浓度少于对照组,差异均具有统计学意义(均P0.05),两组患者CI和LVEF均较治疗前提高(均P0.05),而LVEDD较治疗前差异无统计学意义(P0.05),联合治疗组患者CI和LVEF提高程度均高于对照组(均P0.05)。结论:50%葡萄糖静注联合持续泵入呋塞米治疗顽固性心力衰竭短期临床效果显著,能够明显减少水钠潴留,减轻水肿,改善心脏功能。     

Backdoor phosphorylation of basolateral plasma membranes of small intestinal epithelial cells: characterization of a furosemide-induced phosphoprotein related to the second sodium pump

Enterocyte has two different Na+-stimulated ATPases, the ouabain-sensitive Na+/K+ ATPase and a furosemide-inhibitable Na+ ATPase. To identify the polypeptide associated with the Na+-ATPase, 32Pi phosphorylation into basolateral membranes of enterocyte was investigated. Both, ouabain and furosemide induced Mg2+-dependent, vanadate-sensitive 32Pi incorporation into a 100kDa polypeptide. K(m) for Pi was 17.7+/-1.82 microM and 16.8+/-0.69 microM for ouabain-induced and furosemide-induced phosphorylation, respectively. K(m) for furosemide was 1.3+/-0.21 mM. Furosemide-induced 32Pi incorporation was sensitive to alkaline pH and hydroxylamine suggesting an acyl-phosphate bond. Na+ and K+ inhibited 32Pi incorporation induced by ouabain. In contrast, Na+ stimulated furosemide-induced phosphorylation with a K(m) of 16.5+/-5.59 mM while K+ had no effect. Purified Na+/K+ ATPase only presented ouabain-induced phosphoprotein, indicating that furosemide-induced phosphorylation is not related to this enzyme and appears to correspond to a new member of P-type ATPases associated with the second Na+ pump.     

Evaluation of the diuretic and urinary electrolyte effects of methanolic extract of Peganum harmala L. in Wistar albino rats

The use of traditional medicines as a diuretic agent has been increasing in recent years. The diuretic activity of a number of plant extracts used as diuretic agents in ethnomedicine has been confirmed in experimental animals. However, despite the widespread use of Peganum harmala in traditional medicine, there is a paucity of data supporting its use as a diuretic agent. Therefore, the present study aimed to envisage the true effect and magnitude of diuresis of methanolic extract of P. harmala (MEPH) in comparison with a well-known diuretic drug furosemide using Wistar albino rats. MEPH was administered orally in three different doses (150, 300 and 450 mg/kg) to experimentally dehydrated rats. Furosemide (10 mg/kg orally) was used as a reference drug. The diuretic effect of the MEPH was evaluated by measuring urine volume, urine pH, urinary electrolyte levels, natriuretic and saliuretic effects. The urine volume (in mL) measured at 5 h and 24 h and electrolyte excretion (Na⁺, K⁺, and Cl⁻) at 24 h duration were measured. The urine output and urinary electrolyte excretion were found to be significantly higher in rats treated with MEPH as compared to normal rats in a dose dependent manner (P < 0.05). The results of our study were comparable to furosemide drug. Based on observed results, we can recommend that P. harmala may be an effective diuretic, however, toxicity studies should be conducted before administration.     

Molecular mechanism of an adverse drug–drug interaction of allopurinol and furosemide in gout treatment

Gout patients receiving a combination of allopurinol and furosemide require higher allopurinol doses to achieve the target serum urate (SU) of <6 mg/dl (Stamp et al., 2012) [1]. Our study aimed to identify the molecular basis for this observation. We used a fluorimetric assay to determine the impact of furosemide and oxypurinol (the active metabolite of allopurinol) on xanthine oxidase (XO) activity. Immunoblot analysis quantified expression of XO and AMP-kinase (AMPK) in drug-treated human liver (HepG2) and primary kidney (HRCE) cells. In silico analysis identified miR-448 as a potential XO-regulator, whose expression level in HepG2 cells was examined by qPCR.     

Occurrence of passive furosemide-sensitive transmembrane potassium transport in cultured cells

Furosemide ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) inhibits a proportion of the total passive (ouabain-insensitive) K⁺ influx into primary chick heart cell cultures (85%), BC₃H1 cells (75%), MDCK cells (40%) and HeLa cells (57%). This action of furosemide upon K⁺ influx is independent of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{pump}$ inhibition since the furosemide-sensitive component of the K⁺ influx is identical in the presence and absence of ouabain ($\text{1 · 10}{}^{\mathrm{?3}}\text{M}$). For HeLa cells the passive, furosemide-sensitive component of K⁺ influx is markedly dependent upon the external K⁺, Na⁺ and Cl^? content. Acetate, iodide and nitrate are ineffective as substitutes for Cl^?, whereas Br^? is partially effective. Partial Cl^? replacement by NO₃^? gave an apparent affinity of 100 mM [Cl]. Na⁺ replacement by choline⁺ abolishes the furosemide-sensitive component, whereas Li⁺ replacement reduces this component by 48%. Partial Na⁺ replacement by choline⁺ gives an apparent affinity of 25 mM [Na⁺]. Variation in the external K⁺ content gives an affinity for the furosemide-sensitive component of approx. 1.0 mM. Furosemide inhibition of the passive K⁺ inflúx is of high affinity, half-maximal inhibition being observed at $\text{5 · 10}{}^{\mathrm{?6}}\text{M}$ furosemide. Piretanide ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) and phloretin ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) inhibit the same component of passive K⁺ influx as furosemide; ethacrynic acid and amiloride ($\text{both}\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) partially so. The stilbene, SITS ($\text{1 · 10}{}^{\mathrm{?6}}\text{M}$), was ineffective as an inhibitor of the furosemide-sensitive component.     

Water deprivation in lactating rats: Changes in nucleolar dry mass of paraventricular and supraoptic neurones

Summary The separation of function between and within the paraventricular (PV) and supraoptic (SO) nuclei was investigated in the rat. Nucleolar dry mass of PV and SO neurones was measured to detect increased synthetic activity after water deprivation for 3 days, lactation for 8 days or water deprivation during days 5 to 8 of lactation. Lactation or water deprivation increased nucleolar dry mass in both PV and SO neurones. These stimuli caused similar nucleolar changes in PV neurones, but water deprivation caused greater changes in SO neurones than lactation. The effects of lactation and water deprivation were additive for both SO and PV neurones. Furosemide was used to intensify the dehydration stimulus to determine whether such intensification could have caused the greater nucleolar changes when lactation and water deprivation were combined. For PV neurones this was not the case, but remained a possibility for SO neurones. Measurements of serum osmolality in the experimental groups were ranked as follows: water deprivation + furosemide > lactation + water deprivation > water deprivation > lactation = virgin control. Loss of body weight was similar in the first two groups but less during water deprivation alone. Although milk yield fell, milk was obtained by the litters of lactating animals throughout the period of water deprivation.     

沙库巴曲缬沙坦联合呋塞米治疗慢性心力衰竭的疗效研究

目的:探究沙库巴曲缬沙坦联合呋塞米治疗慢性心力衰竭的临床疗效。方法:选择2017年1月-2018年12月于我院诊治的慢性心力衰竭患者60例,随机将其分为两组。其中,对照组给予呋塞米进行治疗,研究组在对照组基础上联合沙库巴曲缬沙坦进行治疗,对比两组患者的治疗总有效率、治疗前后心功能、血清N端B型脑钠肽(NT-proBNP)、醛固酮(ALD)、细胞间黏附分子-1(ICAM-1)水平的变化及不良反应的发生情况。结果:治疗后,研究组患者的治疗总有效率[96.7%(29/30)]显著高于对照组[80.0%(24/30)](P0.05)。两组患者治疗后的左心室射血分数(LVEF)均较治疗前显著升高,左心室舒张末期内径(LVEDD)、左心室收缩末期内径(LVESD)均较治疗前明显降低(P0.05),且研究组LVEF显著高于对照组,而LVEDD和LVESD明显低于对照组(P0.05);两组患者治疗后的血清NT-proBNP、ALD、ICAM-1水平均较治疗前显著降低(P0.05),且研究组以上指标均显著低于对照组低(P0.05)。对照组患者不良反应发生率[10.0%(3/30)]与研究组[13.3%(4/30)]比较无显著性差异(P0.05)。结论:沙库巴曲缬沙坦联合呋塞米治疗慢性心力衰竭的效果显著优于单用呋塞米治疗,其可有效改善患者的心功能且安全性较高,可能与其明显改善患者血清NT-proBNP、ALD、ICAM-1水平有关。     

穴位注射速尿治疗肝硬化腹水的疗效观察

目的：观察穴位注射速尿治疗肝硬化腹水的临床效果,寻求治疗肝硬化腹水的理想的治疗方式。方法：取穴足三里、三阴交、肾俞注射速尿20-40mg。结果：疗效显著经。统计学处理P〈0．05据有显著差异。讨论：穴位注射速尿治疗肝硬化腹水,操作简单且用药剂量小,副作用小,安全性高疗效显著。     

穴位注射速尿治疗肝硬化腹水的疗效观察

目的:观察穴位注射速尿治疗肝硬化腹水的临床效果,寻求治疗肝硬化腹水的理想的治疗方式。方法:取穴足三里、三阴交、肾俞注射速尿20-40mg。结果:疗效显著经。统计学处理P<0.05据有显著差异。讨论:穴位注射速尿治疗肝硬化腹水,操作简单且用药剂量小,副作用小,安全性高疗效显著。     

左西孟旦联合呋塞米治疗顽固性心力衰竭的疗效分析

目的:探讨左西孟旦联合呋塞米治疗顽固性心力衰竭的疗效。方法:选取了80例顽固性心力衰竭患者,按随机数字表法分为两组对照组(39例)给予左西孟旦注射液,观察组(41例)给予左西孟旦联合呋塞米治疗后24 h,通过观察并记录治疗后的疗效,治疗前后心搏量(SV),左室射血分数(LVEF)和N末端B型利钠肽原(NT-proBNP)水平及随访3 d内不良反应情况,评价左西孟旦联合呋塞米治疗顽固性心力衰竭的疗效。结果:治疗后观察组患者心功能改善1级以上患者共37例,对照组28例,观察组有效率明显高于对照组(P0.05),治疗前,两组SV,LVEF,NT-proBNP无统计学差异(P0.05),治疗后,两组SV和LVEF水平均增加,且观察组高于对照组治疗后,两组NT-proBNP水平均降低,且观察组明显低于对照组(P0.05),随访3d,两组不良反应率相比,无统计学差异(P0.05)。结论:采用左西孟旦联合呋塞米对顽固性心力衰竭具有较好的治疗效果,能有效增强心脏收缩力,提高心输出量,减轻心力衰竭程度。