Abstract
Zinc (Zn) is a cofactor of numerous enzymes and is necessary for the stability of cell membranes and DNA structure.1 Thiazide diuretics2,3-5 and angiotensin-converting enzyme inhibitors (ACE-I)6,7,-9 have been shown to increase urinary Zn excretion and cause Zn depletion. Thiazide use may also result in hypomagnesemia (by increased urinary losses of magnesium [Mg]),10,-12 which may exacerbate hypertension.
The effects of ACE-I and angiotensin receptor blockers (ARB) on electrolyte metabolism are often similar. Losartan, an ARB, can cause taste abnormalities typical of Zn deficiency,13-15 and a single dose of losartan has been shown to increase urinary Mg excretion in healthy volunteers.16 The effects of long-term treatment with losartan on Zn and Mg metabolism in hypertensive patiens have not been studied.
Because a combination of losartan and hydrochlorothiazide is commonly used for the treatment of hypertension, their effect on Zn and Mg metabolism may be additive, resulting in a more pronounced Zn and Mg depletion and potentially blunting their antihypertensive effect.
In addition, Zn is a cofactor of the free-radical scavenger enzyme superoxide dismutase (SOD).17 Therefore, Zn depletion may result in increased oxidative stress and decreased nitric oxide (NO) bioavailability.
The aim of our study was to examine the effect of losartan and losartan/hydrochlorothiazide combination on Zn, Mg, and NO metabolism in hypertensive patients.
Patients and methods
Patients
The study was approved by the local ethics committee and patients gave informed consent. Patients with mild to moderate hypertension (140 to 160 / 90 to 100 mm Hg) and normal renal function (serum creatinine <1.5 mg/dL) were recruited from the local hypertension clinic. Patients with diabetes mellitus, known hypersensitivity to the study medications, or currently using diuretics, ACE-I, or ARB were excluded. Patients were also excluded if their blood pressure (BP) was normalized after losartan treatment.
Study design
In an open study, patients were treated with losartan 50 mg (Ocsaar, MSD Co., Petah-Tikva, Israel) once daily for 4 weeks followed by a fixed combination of 50 mg losartan and 12.5 mg hydrochlorothiazide (losartan/hydrochlorothiazide 50/12.5; Ocsaar plus, MSD Co.) for another 4 weeks. Patients were examined on three occasions: at baseline and at the end of each treatment period (ie, after 4 and 8 weeks). During each visit, sitting BP and heart rate were measured after 10 min rest, and patients were interviewed by a trained dietician who assessed Zn and Mg intake through the patients’ self-reported diaries and by recall analysis. During the first interview with the dietitian, patients were asked about any multivitamins or food supplements that they were taking and were required to stop using these throughout the study.
Dietary intake of Zn and Mg were calculated using computerized analysis (DOS-based magic program “MANA,” specially adapted for data entry and analysis of food intake records). This program was developed by the Israeli Food and Nutrition Administration based on the Food Intake Analysis System (FIAS) of the United States Department of Agriculture18 and especially adapted to meet the needs of the Israeli population. It is used for the Continuing Survey of Food Intakes by Individuals (CSFII) and for specific laboratory results regarding Israeli food items and reports from the Israeli food industry. This is the most comprehensive database used for the analyses of the Israeli National Health and Nutrition Surveys (“MABAT”).19
Laboratory measurements
Blood and urine were collected during each visit. Baseline blood and urinary collections were performed after a 2-week washout of all antihypertensive medications. After an overnight fast, blood was drawn for deterimining sodium, potassium, urea, creatinine, calcium, uric acid, Zn, and Mg levels. Urine was collected over 24 h for the measurement of Zn, Mg, and nitric oxide metabolites (NOx = NO2+ NO3). The urine samples were refrigerated throughout the 24-h collection and stored immediately after arrival at the laboratory at −20°C until analysis. Serum levels of sodium, potassium, urea, creatinine, calcium and uric acid were measured using an automatic analyzer (Hitachi 917, Tokyo, Japan). Serum Zn and Mg concentrations were determined using an atomic absorption spectrophotometer (Spectra AA 800, Varian, Australia) by a standard procedure. Our normal range for serum Zn is 66 to 110 μg/dL and for urinary Zn excretion 140 to 800 μg/24 h. For Mg, serum levels are 1.5 to 2.3 mg/dL and for urinary Mg excretion 73 to 122 mg/24 h.20 Urinary data are corrected for urinary creatinine excretion. Fractional excretion of Zn (FEZn) was calculated by the following formula: ([urinary zinc × serum creatinine]/ [urinary creatinine × serum zinc]) × 100.
Determination of Zn and Mg in peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque gradient, as previously described.8 Cells were counted in a hemocytometer in 4% Türk solution and viability assessed by 0.1% eosin exclusion. Only samples with viability ≥95% were used for the study. Cell samples were stored in 1 mL PBS at −30° until further analyzed. All samples were finally analyzed at the same time, when PBMC samples were defrosted and the cells were digested in 1 mL concentrated HNO3. The digestion was completed during 1-h incubation at 90°C. Both Zn and Mg levels were measured using the atomic absorption spectrophotometer (Spectra AA, Varian, Australia). Total protein of cell samples was determined using the Bradford assay. The results were subsequently calculated as concentrations per milligram of protein.
Urinary NOx determination
The NOx concentrations were determined in 24-h urine collections using a specific colorimetric kit (Sigma Chemical, St. Louis, MO) according to the manufacturer’s instructions. The assay is based on a rapid conversion of NO to nitrate and nitrite in any aqueous solution and on subsequent conversion of the nitrate fraction to nitrite using NADH-dependent nitrate reductase. The resultant nitrite is a stable compound that can be quantified using the Griess reagent.21 Results are within sensitivity of 1 pmol/L NO per milliliter of any aqueous solution, including urine.
Statistical analysis
Data are presented as mean ± SEM. The effect of treatment on mean serum, urine, and PBMC levels of Mg and Zn was assessed by a general linear model repeated-measures analysis of variance with post hoc comparisons when appropriate. The SPSS statistical software program, version 10.0 (SPSS Inc., Chicago, IL), was used for all analyses. A P value <.05 was considered as statistically significant.
Results
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Twenty-one patients were recruited to the study. Four patients were withdrawn from the final analysis: one patient attained normal BP after 4 weeks of treatment with losartan; one patient had baseline hypercalcemia; in one patient there was a violation of the study protocol (the addition of hydrochlorothiazide to losartan in the first study period by the treating physician); and in one patient urine collections were unreliable. Data of the remaining 17 patients are presented.
Patients demographic and baseline hemodynamic characteristics are presented in Table 1.
Both study medications were well tolerated; however, one patient complained of mild taste changes during losartan therapy that were unchanged until the end of both study periods.
Systolic BP was significantly reduced by treatment (P = .002). After losartan treatment, systolic BP decreased from 158 ± 5 mm Hg at baseline to 146 ± 3 mm Hg (P = .0004), and decreased further to 141 ± 5 mm Hg after losartan/hydrochlorothiazide (P = .02 compared with treatment with losartan). Diastolic BP was reduced also (P = .04 repeated-measures analysis of variance), but the difference from baseline reached significance only after treatment with losartan/hydrochlorothiazide (from 95 ± 3 mm Hg at baseline to 88 ± 3 mm Hg). Losartan treatment, however, had no significant effect on diastolic BP (from 95 ± 3 mm Hg at baseline to 93 ± 2 mm Hg, P = .14). Heart rate did not change significantly throughout the study (data not shown).
The effects of treatment on Zn and Mg metabolism are shown in Table 2.
Dietary intake of Zn and Mg did not change significantly throughout the study (Table 2). Treatment resulted in a significant increase in urinary Zn excretion in all patients (P = .001). After losartan treatment, urinary Zn excretion increased from 0.020 ± 0.004 μg/mg creatinine at baseline to 0.034 ± 0.005 μg/mg creatinine (P = .02). Losartan/hydrochlorothiazide treatment further increased urinary Zn excretion from 0.034 ± 0.005 μg/mg creatinine after losartan to 0.053 ± 0.008 μg/mg creatinine (P = .03). The FEZn increased significantly after losartan and losartan/hydrochlorothiazide: from 0.02% ± 0.005% at baseline to 0.04% ± 0.006% and 0.06% ± 0.01% (P = .05, 0.05 and 0.004 for baseline v losartan, losartan v losartan/hydrochlorothiazide, and baseline v losartan/hydrochlorothiazide, respectively).
Serum Zn concentrations gradually decreased during the study (P = .014), from 80.0 ± 3.7 μg/dL at baseline to 76.0 ± 3.7 μg/dL after losartan and 74.0 ± 3.0 μg/dL after losartan/hydrochlorothiazide (P = .007 v baseline).
Four patients had hypozincemia at baseline, and their serum Zn levels remained unchanged and below the normal range after both treatment periods. Only two patients with initially normal serum Zn levels developed hypozincemia by the end of the study. There was a significant correlation between baseline serum Zn levels and the degree of change in serum Zn after losratan/hydrochlrohiazide treatment (R = 0.55, P = .02). Thus, patients with higher baseline serum Zn levels had more pronounced decreases in serum Zn after treatment.
There was a positive correlation between decreases in systolic BP and decrease in serum Zn levels after treatment with losartan (R = 0.5, P = .05). However, there was no correlation between the change in BP and urinary Zn/creatinine ratio (R = 0.05, P = .8). Further decrease in systolic BP after losartan/hydrochlorothiazis therapy did not correlate with either serum or urinary Zn.
There was a trend toward decrease in PBMC Zn levels after treatment, but this was not statistically significant (P = .06) (Table 2).
There was no significant correlation between serum Zn levels and body mass index.
Serum Mg levels, urinary excretion of Mg, and PBMC Mg concentrations remained unchanged after both treatments (P = .1 and 0.17 respectively) (Table 2).
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There was no effect of treatment on urinary NOx/creatinine ratio: from 0.14 ± 0.03 μg/mg at baseline to 0.15 ± 0.03 μg/mg after losartan and 0.17 ± 0.03 μg/mg after losartan/hydrochlorothiazide (P = .4).
There was a mild but significant change in serum creatinine during treatment (P = .03), mainly due to an increase in serum creatinine after losartan/hydrochlorothiazide (from 0.86 ± 0.04 mg/dL after losartan to 0.93 ± 0.06 mg/dL after losartan/hydrochlorothiazide, P = .03).
Discussion
In the current study, we evaluated the effects of the angiotensin receptor blocker losartan and the fixed losartan/hydrochlorothiazide combination on Zn, Mg, and NO metabolism in hypertensive patients. We found that losartan treatment resulted in Zn depletion, mediated by an increase in urinary Zn excretion. Treatment with losartan/hydrochlorothiazide had an additive effect on urinary Zn losses. Both Mg and NO metabolism, however, were unaffected.
Thiazide diuretics have been reported to increase urinary Zn excretion and to induce hypozincemia.2-5 These effects are probably caused by the blockage of distal tubular absorption of Zn.5
The mechanism by which losartan causes Zn depletion is unknown. Studies in both animals22,23 and humans6,7-9 have shown that ACE-I can cause Zn depletion by increasing urinary losses of Zn. We have previously reported that the ACE-I captopril and enalapril can cause a decrease in intramonocytic Zn concentrations, and captopril also induces an increase in urinary Zn excretion.6,8 Similar effects were reported by Peczkowka.9 It has been suggested that captopril may act as a chelating agent due to its sulphydryl group causing excessive urinary Zn losses.24 However, the fact that other ACE-I6,8 and losartan had similar effects argues for a class effect possibly related to hemodynamic or metabolic effects of angiotesin II. Renal Zn handling involves both proximal and distal reabsorbtion of the filtered Zn. In the proximal tubule, Zn is bound to a specific Zn binding protein at the brush border and is then transported to the blood by the divalent cation transporter (DCT1).25 In the distal tubule, a different transporter with high affinity to Zn (DCT2) has recently been identified.26 In both cases, Zn absorption is facilitated by proton excretion to the lumen by the Na/H antiporter. Angiotensin II is a potent inducer of the Na/H antiporter. Blocking agiotensin II effects with either ACE-I or ARB could potentially decrease renal Na/H antiporter activity followed by decreased tubular absorption of Zn and resulting zincuria. This mechanism, however, has not yet been validated. Mild volume contraction caused by hydrochlorothiazide therapy could theoretically increase proximal reabsorption of water and electrolytes, thereby affecting Zn absorption. However, in an animal study, no effect of dehydration or hyperfiltration (achieved by manitol infusion) on renal Zn handling was found.27
In a recent study, Baykal et al28 examined the effect of different antihypertensive medications on Zn homeostasis. Consistent with our results, erythrocyte Zn content decreased significantly after treatment with the ARB valsartan and remained unchanged after treatment with β-blockers (metoprolol), α-blockers (doxazosin), and calium-channel blockers (amlodipine). Treatment with the ACE-I ramipril also resulted in a decrease in erythrocyte Zn, although this change was not statistically significant.
Recent studies have suggested that plasma Zn concentrations are higher in untreated hypertensive patients than in control subjects,29-31 and intestinal Zn absorption in patients with hypertension is increased as compared with that in control subjects.24 The decrease in Zn in patients treated with ACE-I or ARB may therefore merely represent normalization in body Zn content; however, this is probably not the case. There are several case reports of losartan-treated patients experiencing characteristic adverse effects attributed to Zn deficiency, ie, taste disturbances.13-15 Moreover, Baykal et al28 found that erythrocyte Zn levels in hypertensive patients treated with the ARB valsartan were consistent with a mild deficiency state, whereas treatment with other antihypertensive agents (resulting in a similar degree of BP reduction) had no effect on Zn levels.
Obesity is another factor that may affect serum Zn levels and has been associated with hypozincemia, increased oxidative stress, and decreased SOD activity in humans.32 Although some of our patients were obese and tended to have lower baseline levels of Zn, there was no correlation between body mass index and the magnitude of changes in serum Zn and urinary Zn excretion after treatment.
Zinc deficiency may have clinical implications in hypertensive patients, especially those treated by ARB. Zinc is a cofactor of the free-radical scavenger enzyme SOD.17 In animal models, Zn deficiency resulted in decreased SOD activity,33,34 increased renal vascular resistance,34 and aggravation of hypertension,33 all reversed by treatment with the SOD-mimetic tempol. Enhanced NO inactivation by the resulting increased oxidative stress was implicated as the causative mechanism. Chronic inhibition of SOD in animals also resulted in impaired NO-mediated vasodilation due to increased NO inactivation and increased oxidative stress.35 Decreased SOD activity has also been reported in human hypertension28,31 and may have a role in the increased oxidative stress found in hypertensive patients.36 The ARB have been shown to decrease oxidative stress (by inhibiting angiotesin II-mediated activation of NADPH oxidase),37 increase NO bioavailabilty, and improve endothelial function.38 Thus, Zn depletion and increased oxidative stress may attenuate part of the beneficial effects of ARB.
However, we found no effect of treatment on NO metabolism as measured by urinary NO metabolites. Although this finding does not exclude a possible effect of Zn depletion on SOD activity in our patients, it may reflect the operation of other effective antioxidative mechanisms. Alternatively, the magnitude of Zn depletion in our study may have been milder than in experimental Zn defficiency in humans and in animal models. In experimental studies of Zn deficiency in humans, after 28 weeks of a low-Zn diet, symptoms appeared after a decrease of 25% or more in plasma Zn.39 In a recent study examining Zn metabolism and SOD activity in hypertensive patients,28 a 25% decrease in plasma Zn (from 88.4 to 65.8 μg/dL) was associated with a significant change in SOD activity after treatment with valsartan. Animal studies of Zn deficiency and increased oxidative stress were associated with a >40% decrease in plasma Zn (104 v 65 μg/dL).34 In our study, serum Zn levels decreased significantly but in a lower magnitude of 7% ± 2%. Longer periods of treatment and higher doses of losartan and hydrochlorothiazide could, however, result in more profound Zn deficiency and effects on NO meabolism.
Depletion of Mg is a known metabolic effect of thiazide diuretics and a previous report has shown that a single dose of losartan increased urinary Mg excretion in healthy subjects.16 Thus, the combination of losartan and hydrochlorothiazide may have a synergistic effect, resulting in a more profound Mg depletion. Our data indicate, however, that neither losartan nor losartan/hydrochlorothiazide had any effect on Mg metabolism. One possible explanation may be the relatively small dose of hydrochlorothiazide (12.5 mg) used in our study.
A limitation of our study is that all patients had hydrochlorothiazide added to losartan. Therefore, the further Zn depletion after hydrochlorothiazide treatment could be attributed to either the effect of losartan over time or the additive effect of hydrochlorothiazide. However, several studies have shown thiazide diuretics to increase urinary Zn losses and induce Zn deficiency.2-5 It is therefore plausible that hydrochlorothiazide contributed, at least in part, to the negative Zn balance.
In conclusion, we found that losartan therapy resulted in a Zn depletion in hypertensive patients that was further exacerbated by the addition of hydrochlorothiazide. Losartan/hydrochlorothiazide had no effect on Mg or NO metabolism. The mechanism of Zn depletion induced by ACE-I and ARB warrants further investigation.
References
Author notes
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