Rapid determination of losartan and losartan acid in human plasma by multiplexed LC-MS/MS.

A rapid LC-MS/MS method has been developed and validated for the determination of losartan (LOS) and its metabolite losartan acid (LA) (EXP-3174) in human plasma using multiplexing technique (two HPLC units connected to one MS/MS). LOS and LA were extracted from human plasma by SPE technique using Oasis HLB cartridge without evaporation and reconstitution steps. Hydroflumethiazide (HFTZ) was used as an internal standard (IS). The analytes were separated on Zorbax SB C-18 column. The mass transition [M-H] ions used for detection were m/z 421.0 --> 127.0 for LOS, m/z 435.0 --> 157.0 for LA, and m/z 330.0 --> 239.0 for HFTZ. The proposed method was validated over the concentration range of 2.5-2000 ng/mL for LOS and 5.0-3000 ng/mL for LA with correlation coefficient > or = 0.9993. The overall recoveries for LOS, LA, and IS were 96.53, 99.86, and 94.16%, respectively. Total MS run time was 2.0 min/sample. The validated method has been successfully used to analyze human plasma samples for applications in 100 mg fasted and fed pharmacokinetic studies.

Tumor estrogen content and clinico-morphological and endocrine features of endometrial cancer.

OBJECTIVES: To compare estrogen concentrations in endometrial cancer tissue with those in macroscopically normal endometrium and with certain morphological characteristics of the tumor and endocrine parameters in patients. METHODS: The estradiol content was evaluated by radioimmunoassay after homogenization and extraction in 78 adenocarcinomas (61 from postmenopausal patients). RESULTS: Higher concentrations of estradiol in tumor tissue samples than in macroscopically normal endometrium were found in patients of both reproductive and postmenopausal age. This difference was the same in patients with either endometrial carcinoma type I or type II. No association between tumor steroid receptor levels, estradiol concentrations in blood serum, and timing of menopause with intratumoral estradiol contents was discovered. Estradiol concentrations in tumor tissues correlated positively with the clinical stage of disease and rate of tumor invasion (in patients with peripheric/lower type of fat topography), and negatively with tumor differentiation stage (in patients with central/upper type of fat topography) and the percentage of intact double-stranded DNA in normal endometrium. CONCLUSIONS: Tumor estrogen content in endometrial cancer has clinical significance that is modified in the presence of certain endocrine characteristics related to insulin resistance. The role of local estrogen production (aromatase activity) in this setting deserves special study.

The effect of anti-hypertensive therapy on urinary albumin excretion in Nigerian hypertensives.

The effect of blood pressure control on urinary protein excretion was assessed in 24 benign essential hypertensive subjects (12 males and 12 females). There were 23 controls (11 males and 12 females). Mean ages were 55 and 53 years respectively. Twenty-four hours urine was collected from each subject before and after control of blood pressure, while the controls had only one 24 h urine sample collected. 24 h urinary albumin excretion was assessed using the Bromocresol Green Method. Control of blood pressure in the subjects took an average of eight weeks. Subjects were either given hydroflumethiazide, alpha-methyldopa and/or prazosin as required. Blood pressure was measured in the right arm at each visit and pill counting was used to assess the compliance with therapy. The average urinary albumin excretion was significantly higher in the hypertensive subjects than the normotensive controls (P < 0.05). The average urinary albumin excretion after control of blood pressure was also significantly lower than, before control of blood pressure (P < 0.02). There was no correlation between SBP, DBP, MAP, and 24 h urinary albumin excretion in both subjects and controls. This study has shown that control of blood pressure can reduce or reverse urinary albumin excretion in Nigerian hypertensives.

A modified residual method to estimate the zero-order absorption rate constant in a one-compartment model.

The objective of this work was to develop a simple residual method to estimate the rate constant for actual or apparent zero-order absorption into a one-compartment model. The method is based on the fact that, in theory, a plot of residuals versus e-Kt is linear for a zero-order absorption process, where K represents the elimination rate constant governing the terminal phase of the concentration-time profile. The apparent absorption rate constant (K0) can be calculated from the slope and intercept of the residual plot. Simulated concentration-time data with superimposed random error (CV = 5, 10, 15%, n = 8), as well as data sets from the literature for hydroflumethiazide and theophylline were analyzed with the proposed method of residuals. Parameters derived with the new technique were compared to both the nonlinear least-squares regression and the Wagner-Nelson method, all of which yield comparable K0 estimates. These results indicate that the proposed method of residuals represents a simple approach for estimating the apparent zero-order absorption rate constant analogous to classic residual analysis for first-order absorption.

Phototoxicity to sulphonamide-derived oral antidiabetics and diuretics: investigations in hairless mice.

The oral antidiabetics glibenclamide, glipizide, glymidine, tolazamide and tolbutamide and the diuretics bemetizide, bendroflumethiazide, benzylhydrochlorothiazide, bumetanide, butizide, furosemide, hydrochlorothiazide, hydroflumethiazide and trichlormethiazide were investigated for phototoxic effects in hairless mice. The back of the animals (hr/hr-c3H/TifBom) was covered with Duoderm dressing, and at the site of two punched out holes 0.05 ml of the test substances at 0.25 mol/l concentration and the solvent alone as control were injected intradermally, respectively. Both test and control sites were irradiated with 6-12 J/cm2 of longwave UVA light from a "Bluelight 2000" apparatus (Hönle, Martinsried, Germany). Skin reactions were read at 24 and 48 h. Compared to the solvent alone, all of the test substances induced reactions (necrosis or oedema)--most frequently seen by macroscopic and histologic investigation and by measurements with a thickness gage. Injection of the test substance or solvent alone without or with subsequent UVA irradiation, as well as UVA alone, did not induce measurable skin changes in this model. Three oral antidiabetics and four diuretics, not yet described to induce photosensitivity in vitro nor in vivo, were detected as potential photosensitizers using our animal model.

Photoaugmentation effects demonstrated in vitro.

A number of sulphonamide-derived oral antidiabetics and diuretics were investigated for phototoxic properties, using different sources of light, by means of a photohemolysis test. Photohemolysis was obtained after irradiation with UVA, visible light and solar simulating irradiation. No phototoxic properties were seen when the test samples were exposed to UVB alone. The most prominent hemolysis was induced by solar simulating irradiation. When exposing the the test samples to UVB prior to the subsequently applied UVA, visible light or solar simulating irradiation, a significantly higher hemolysis was detected compared with the previous tests, suggesting photoaugmentation effects in this model.

Do thiazides reduce intestinal oxalate absorption?: a study in vitro using rabbit colon.

1. The purpose of the present study was to examine the effects of various diuretics on intestinal oxalate transport. Transmural oxalate fluxes were measured across isolated, short-circuited tissue segments removed from rabbits and placed in Ussing chambers. 2. The net absorptive flux of oxalate across the distal colon was significantly reduced in the presence of trichlormethiazide at 10(-4) mol/l. In contrast, this diuretic had no effect on oxalate transport in the other intestinal segments examined. Several of the thiazide diuretics tested had some inhibitory effect on colonic oxalate absorption, but at higher concentrations of 10(-3) mol/l or 10(-2) mol/l. 3. We conclude that the previously reported hypooxaluric effects of hydrochlorothiazide and chlorthalidone are most likely not the result of an exclusive or primary effect on intestinal oxalate transport. It is suggested that the reduction in colonic oxalate absorption that was observed with the thiazides probably involves the transport system responsible for oxalate efflux across the basolateral membrane of the colonocyte.

Metabolic effects of propranolol and hydroflumethiazide treatment in Kenyans with mild to moderate essential hypertension.

In a prospective single-blind comparative trial, sixty newly diagnosed mild to moderate hypertensives were randomly assigned to either propranolol or hydroflumethiazide monotherapy. Baseline fasting serum glucose lipid profiles, serum uric acid and potassium levels, were determined at the beginning of the trial. Repeat levels were determined at completion of twelve weeks of treatment. Propranolol treatment significantly reduced HDL-cholesterol (p < 0.02) and increased both VLDL and total serum triglycerides (p < 0.01). Hydroflumethiazide significantly increased total and LDL-chole-sterol, fasting serum glucose and uric acid levels (p < 0.01); potassium levels were significantly lowered (p < 0.01). Treatment with either propranolol or hydroflumethiazide is associated with significant metabolic side-effects which require regular monitoring and intervention as appropriate.

Randomised double-blind comparative study of efficacy and safety of hydroflumethiazide and reserpine and chlortalidone and atenolol in the treatment of mild to moderate hypertension in black patients.

This randomised, double-blind study compared the efficacy and safety of a fixed combination of hydroflumethiazide 50 mg and reserpine 0.125 mg (H-R) and chlortalidone 12.5 mg and atenolol 50 mg (C-A) in adult black patients with mild to moderate hypertension (a resting supine diastolic blood pressure (DBP) between 95 and 115 mmHg after a two week placebo washout period). If the DBP did not reach 90 mmHg after four weeks, the dosage was doubled. There were 27 patients in the H-R group and 22 in the C-A group who completed the study. In the H-R group, supine systolic and diastolic BP were reduced from 156.5 (95% confidence intervals 150.1-162.9) and 102.0 (97.5-106.5) mmHg to 137.0 (130.6-143.4) and 87.4 (83.0-91.9) mmHg, respectively. The corresponding values in the C-A group were 154.1 (147.0-161.2) and 103.4 (98.5-108.4) mmHg to 136.4 (129.3-143.5) and 91.2 (86.2-96.1) mmHg, respectively. Normalisation, response and control of DBP was achieved in 88.9, 92.6 and 100% of patients, respectively, in the H-R group, and in 81.8, 95.5 and 95.5% of patients in the C-A group, respectively. The dose was doubled in 14.8% of patients on H-R and 40.1% on C-A. No clinically significant abnormalities in laboratory variables and no serious adverse effects were encountered. Both drugs have been shown to be efficacious and safe in the treatment of mild to moderate hypertension in black patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative study of the efficacy and tolerability of hydroflumethiazide versus propranolol in Africans with mild to moderate hypertension.

Sixty patients with Diastolic Blood Pressure (DBP) of 100-110mmHg matched for age, sex and Bp levels were randomly assigned to propranolol 80mg daily or Hydroflumethiazide (HFM) 50mg daily. HFM causes a significant reduction in systolic blood pressure (SBP) and DBP within 4 weeks compared both with baseline and propranolol (SBP 143.7 +/- 12.3 vs 158.1 +/- 10.9mmHg, P < 0.05; DBP 92.0 +/- 4.5 vs 102.4 +/- 5.1mmHg, P < 0.05), (SBP 143.7 +/- 12.2 vs 152 +/- 11.0mmHg P < 0.05; DBP 92.0 +/- 4.5 vs 101.1 +/- 6.1mmHg, P < 0.05), respectively. Propranolol produced no significant difference from the baseline at 4 weeks (SBP 152.0 +/- H.0 vs 154.1 +/- 11.5mmHg NS; DBP 101.1 +/- 6.1 vs 102.2 +/- 5.6mmHg, NS). Reduction in BP by HFM was maintained after 8 and 12 weeks with further reduction but which did not achieve statistical significance. Increased dose of propranolol (160mg daily) after 4 weeks caused significant reduction in BP by 8 week (SBP 146.8 +/- 11.8 vs 152.0 +/- 11.0mmHg, P < 0.05; DBP 95.9 +/- 4.4 vs 101.1 +/- 6.1mmHg P < 0.05), which was maintained upto 12 weeks. The values however remained higher than in the HFM group. More patients in the HFM group achieved target BP (< 140/90), SBP 53.8% vs 29.6% P < 0.05, DBP 69.2% vs 14.8% P < 0.01. Incidence of side effects was similar and will be discussed. Thiazides are superior to B'blockers as initial monotherapy in black hypertensives.

Effect of thiazides on colonic NaCl absorption: role of carbonic anhydrase.

The mechanisms by which the benzothiadiazide class of diuretics inhibit electroneutral NaCl absorption are not fully understood. We studied the mechanisms of thiazide action in perfused loops of distal colon in anesthetized male Sprague-Dawley rats. Hydroflumethiazide (1 mM) reversibly inhibited greater than 40% of Na, Cl, and water absorption. Prior exposure of the colon to the carbonic anhydrase inhibitor methazolamide (0.1 mM) prevented the effects of hydroflumethiazide and metolazone, a thiazide-like drug, on colonic absorption. In Ussing flux chambers, addition of hydroflumethiazide to both the mucosal and serosal bathing solutions (but not to the mucosal solution alone) caused marked decreases in Na and Cl absorption. Such inhibition only occurred at concentrations of hydroflumethiazide (0.1 and 1.0 mM) that inhibited greater than 90% of carbonic anhydrase activity in homogenized colonic mucosa. We conclude that an important mechanism by which thiazides inhibit NaCl absorption in the rat distal colon is by inhibition of mucosal carbonic anhydrase. In tissues containing this enzyme, this mechanism of thiazide effect on ion flux must be considered.

Comparative effects of LN 5330 and diazoxide on insulin release and 86Rb+ permeability in perifused rat islets of Langerhans.

The benzothiadiazine derivative LN 5330 (chloro-7 trifluoromethyl-6 benzothiadiazine-1,2,4-dioxide-1,1) has been shown to inhibit insulin secretion and calcium uptake. The present study was carried out to investigate the effects of LN 5330 on insulin release and 86Rb- efflux from perifused rat pancreatic islets; a comparison was made with the structural analogue diazoxide. In the presence of 8.3 mM glucose, LN 5330 (100 microM) accelerated 86Rb+ efflux while reducing insulin output from the islets. LN 5330 induced a dose-dependent acceleration of 86Rb+ efflux and appeared to be a more potent activator of 86Rb+ efflux than diazoxide. The stimulatory effect of LN 5330 on 86Rb+ efflux persisted in the absence of extracellular calcium. In the absence of glucose, 86Rb+ permeability also increased, LN 5330 being again significantly more efficient than diazoxide at an equimolar concentration. These data indicate that the benzothiadiazine derivative LN 5330 inhibits insulin secretion by increasing the potassium permeability of the plasma membrane. It is suggested that, like diazoxide, this drug could activate the ATP-sensitive K+ channel.

NaCl absorption in the rabbit ileum. Effect of acid-base variables.

In vivo and in vitro studies suggest that acid-base variables regulate ion transport in the rabbit ileum. The relative importance of these variables on active Na+ and Cl- absorption has not been defined. Isolated, stripped ileal segments were studied under short-circuited conditions in the Ussing flux chamber. Unidirectional 22Na and 36Cl fluxes were measured after changes in bathing solution pH, PCO2, and/or [HCO3-]. When pH was decreased from 7.6 to 7.1, net flux of Na+ increased from 0.1 +/- 0.7 to 2.6 +/- 0.7 mu Eq/cm2 per hour and net flux of Cl- increased from -2.0 +/- 0.9 to 1.3 +/- 0.6 mu Eq/cm2 per hour. These changes were rapid in onset, completely reversible, and accounted for by changes in mucosal-to-serosal fluxes of these ions. They were accompanied by small decreases in short-circuit current, but there were no changes in residual flux. These pH effects were not inhibited by the presence of luminal bumetanide (1 mmol/L), furosemide (1 mmol/L), hydroflumethiazide (1 mmol/L), or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (1 mmol/L), or by the carbonic anhydrase inhibitor methazolamide (1 mmol/L). When data from all combinations of acid-base conditions were combined and analyzed by linear regression, pH was the only variable that correlated with mucosal-to-serosal fluxes (r = -0.84) and net flux (r = -0.85) for Na+, mucosal-to-serosal fluxes (r = -0.96) and net flux (r = -0.99) for Cl-, and short-circuit current (r = 0.97). These findings suggest that extracellular pH modulates active Na+ and Cl- absorption in the rabbit ileum.

Interaction of some benzothiadiazine diuretics with beta-cyclodextrin.

The interaction of three structurally related benzothiadiazine diuretics, viz., hydroflumethiazide, bendrofluazide and cyclopenthiazide wit beta-cyclodextrin was investigated. The study aimed at enhancing the solubility of these poorly soluble drugs via inclusion complexation with beta-cyclodextrin, Solubility measurements revealed that complexes were formed between each of three drugs and beta-cyclodextrin, The complexes were found to have 1:1 stoichiometric ratios and stability constants of 165.4, 55.7 and 27.9 M-1 for cyclopenthiazide, bendrofluazide and hydroflumethiazide, respectively. UV spectrophotometric analysis of the formed complexes agreed with the solubility results. The variation in the extent of binding between each of the three drugs and beta-cyclodextrin was attributed to their variation in molecular volume, partition coefficient and electrostatic properties of the binding sites.

Severe hypertension and haemospermia--a case report.

Haemospermia is an uncommon disorder of diverse aetiology, which includes diseases of the urethra, prostate and hypertension. There is no report of haemospermia in this environment and this communication describes a case associated with severe hypertension.

Effect of acute potassium depletion and thiazide treatment on blood glucose in the normal rat.

The effect of potassium depletion and a thiazide diuretic on glucose metabolism was investigated in male rats. Potassium depletion was induced by potassium-deficient diet and ion-exchange resin by stomach tube for 5 days. Plasma and muscle potassium was reduced by 30% (P less than 0.001), and fasting blood glucose concentration was elevated by 20% (P greater than 0.025) by potassium depletion. Hydroflumethiazide 10 mg/kg given orally once daily for 8 days produced no further alteration in any parameter. We conclude that acute potassium depletion in the rat may increase fasting blood glucose concentration, whereas addition of short-term thiazide treatment does not seem to potentiate this effect.

Antihypertensive treatment: long-term reversal of progression of albuminuria in incipient diabetic nephropathy. A longitudinal study of renal function.

This study was undertaken to clarify whether antihypertensive treatment has any effect on the rate of progression of kidney disease in patients with incipient diabetic nephropathy. Six insulin-dependent diabetic men with incipient nephropathy (urinary albumin excretion above 15 micrograms/min and total protein excretion below 0.5 g/24 h) were first given metoprolol (200 mg daily) with the subsequent addition of hydroflumethiazide. At the start of antihypertensive treatment, mean patient age was 32 +/- 4.2 years (SD) and mean duration of diabetes was 18 +/- 1.2 years. The patients were followed with repeated measurements of urinary albumin excretion for a mean of 5.4 +/- 3.1 years prior to, and for 4.7 +/- 1.3 years (SD) during treatment. Mean arterial blood pressure declined significantly during treatment, e.g., the values at 6 months before initiation of treatment being compared with values during the last 6 months of treatment fell from 107 mmHg +/- 7.6 to 93 +/- 3.8 (2p = 1.5%). Albumin excretion decreased from 131.0 micrograms/min X/divided by 2.9 (geometric mean X/divided by tolerance factor) to 41.7 micrograms/min X/divided by 2.9 (2p = 1.2%). Albumin clearance in per cent of glomerular filtration rate decreased from a mean of 0.0030 +/- 0.0019% (SD) to 0.0011 +/- 0.0010% (2p = 4.6%). The mean yearly increase in urinary albumin excretion before treatment was 18.0 +/- 17.0% (mean +/- SD); during treatment urinary albumin excretion decreased 19 +/- 10% per year (2p = 0.7%). No changes were seen in renal plasma flow (516 +/- 31.0 ml/min to 520 +/- 66 ml/min (n = 5)).(ABSTRACT TRUNCATED AT 250 WORDS)