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An elevated plasma level of homocysteine (hyperhomocysteinemia) is thought to be an important risk factor for a variety of cardiovascular diseases including preeclampsia. Although clinical studies have reported a 2-3 fold elevation in plasma levels of homocysteine in women who developed preeclampsia, the importance of hyperhomocysteinemia in causing endothelial dysfunction and increases in arterial pressure during pregnancy is unknown.
Therefore, the purpose of this study was to determine the effects of a 2-3 fold elevation in plasma homocysteine levels on arterial pressure, chronic pressure natriuresis relationship, and endothelial factors during pregnancy in the rat. Homocysteine treatment for 4 weeks increased plasma homocysteine levels in pregnant rats from 7.1 ± 1.9 to 16.7 ± 2.3 μmol/L.
Homocysteine treatment decreased urinary nitrate/nitrite levels from 53 +/- 7 vs. 39 +/- 5 (umol/24 h/kg BW) in pregnant rats while having no effect on urinary excretion of endothelin. Homocysteine treatment had no effect on MAP in pregnant rats (104 +/- 2 vs. 107 +/- 3 mmHg) nor on the chronic pressure natriuresis relationship.
These results suggest that while hyperhomocysteinemia decreases nitric oxide production in pregnant rats, hyperhomocysteinemia does not affect MAP, the chronic pressure-natriuresis relationship, or urinary excretion of endothelin in pregnant rats. Moreover, the reported 2-3 fold elevation in plasma level of homocysteine in women with preeclampsia is unlikely to contribute to the hypertension of preeclampsia.
Preeclampsia is a multisystemic disorder of pregnancy estimated to affect 5% to 10% of all pregnancies in the United States.1 Preeclampsia is also one of the leading causes of maternal death and prenatal morbitity.1,2,3 Although widespread vascular dysfunction and increases in blood pressure occurs during preeclampsia, the mechanisms responsible for this disorder are unclear.1,2,3 Endothelial dysfunction, decreases in nitric oxide production, and enhanced endothelin production have all been suggested to be important factors in the pathophysiology of hypertension during preeclampsia.4,5,6,7,8
A number of clinical studies have shown that women with higher plasma homocysteine (hyperhomocysteinemia) levels early in pregnancy have a higher incidence of preeclampsia, intra-uterine growth restriction (IUGR), as well as first trimester miscarriages. 8,9,10,11,12 Elevated homocysteine levels, a demethylated derivative of methionine, has been suggested as a risk factor for a variety of cardiovascular diseases including coronary artery disease, stroke, and peripheral arterial disease.13,14 Hyperhomocysteinemia has been reported to cause endothelial dysfunction by scavenging or trapping nitric oxide.15 We and others have reported that nitric oxide (NO) plays an important role in mediating the cardiovascular and hemodynamic changes during pregnancy.5,16,17,18 In addition, a decrease in nitric oxide production and/or availability has been shown to increase endothelin production, reduce the renal pressure natriuresis relationship, and cause a salt-sensitive form of hypertension.19 Therefore, it is possible that hyperhomocysteinemia during pregnancy may decrease the nitric oxide levels during pregnancy and lead to hypertension. Although there is a strong correlation between hyperhomocysteinemia and preeclampsia there are no studies to our knowledge that demonstrate a physiological role for elevated homocysteine levels during pregnancy.20,21 Therefore, the purpose of this study was to assess the effect of hyperhomocysteinemia; comparable to plasma levels observed in women in preeclampsia, on arterial pressure, renal pressure natriuresis relationship, and nitric oxide and endothelin production during pregnancy.
All experimental procedures executed in this study were in accordance with National Institutes of Health guidelines for use and care of animals with approval of all protocols by the Animal Care and Use Committee at the University of Mississippi Medical Center. Female, virgin Sprague Dawley rats purchased from Harlan Sprague Dawley Inc. (Indianapolis, IN) were housed in a temperature-controlled room (23°C) with a 12:12 hour light/dark cycle with food and water available ad libitum.
Rats treated with homocysteine were administered DL-homocysteine (Sigma, St. Louis, MO) in the drinking water (0.67 mg/ml) one week prior to breeding and maintained on this treatment throughout the experiment to create a condition of hyperhomocysteinemia. On day 17 and 18 pregnancy, rats on a normal sodium diet were placed in individual metabolism cage for 24 hours for collection of urine. Control (n=7) and homocysteine treated (n=9) rats were surgically instrumented with femoral catheters on day 18 of pregnancy. Arterial pressure measurements and subsequent harvesting of kidneys were performed on day 19 - 20 of pregnancy.
To assess the chronic pressure natriuresis relationship, arterial pressure and sodium excretion were measured in four groups of pregnant rats on day 19 of pregnancy: pregnant control rats-normal 1% NaCl diet (n=7), pregnant control rats-high 8% NaCl diet (n=6), pregnant rats treated with homocysteine- normal 1% NaCl diet (n=9), and pregnant rats treated with homocysteine-high 8% NaCl diet (n=5).
The effects of hyperhomocysteinemia on arterial pressure and the chronic pressure natriuresis relationship were also examined in virgin rats: virgin control-normal sodium diet (n=12), virgin control-high sodium diet (n=6), virgin treated with homocysteine-normal sodium diet (n=10) and virgin treated with homocysteine-high sodium diet (n=6).
Rats were anesthetized with 5% isoflurane (W.A. Butler Co., Memphis, TN) delivered by an anesthesia apparatus (Vaporizer for Forane Anesthetic, Ohio Medical Products, Madison, WI). During isoflurane anesthesia, rats were surgically instrumented with catheters (PE 50 tubing) in the femoral artery for blood sampling and blood pressure monitoring. The arterial catheter was tunneled to the back of the neck and exteriorized. Arterial pressure was monitored with a pressure transducer connected to a recorder for continuous recording.
Total plasma homocysteine (tHcys) was measured by fluorescence HPLC analysis as described previously by Chen and Zou.22 Briefly, blood samples of 1 mL were collected into Vacutainer tubes containing sodium heparin (Becton Dickinson) and immediately centrifuged at 1000g for 10 minutes at 4°C. Plasma samples (100-μL) or solutions mixed with 10 μL internal standard, (2-mercaptoethylamine (ME) (2.0 mmol/L)), were treated with 10 μL of 10% tri-n-butylphosphine (TBP) in dimethylformamide at 4°C for 30 minutes. Subsequently, 100 μL of the supernatant was transferred into a solution containing 20 μL of 1.55 mol/L sodium hydroxide, 250 μL of 0.125 mol/L borate buffer (pH 9.5), and 100 μL of 1.0 mg/mL ABD-Fsolution. The resulting mixture was incubated at 60°C for 30 minutes to accomplish derivatization of plasma thiols. HPLC was performed with a Hewlett-Packard Model 1090 Series II system with an autosampler. Separation was carried out at ambient temperature on an analytical column, Supelco LC-18-DB (150×4.6 mm ID, 5 μm) with a Supelcosil LC-18 guard column (20×4.6 mm ID, 5 μm). Fluorescence intensities were measured spectrophotometrically (Hewlett-Packard Model 1046A) at an excitation wavelength of 385 nm and emission wavelength of 515 nm. The peak area of the chromatographs was quantified with a Hewlett-Packard 3392 integrator. The analytical column was eluted with 0.1 mol/L potassium dihydrogenphosphate buffer (pH 2.1) containing 6% acetonitrile (vol/vol) as mobile phase with a flow rate of 2.0 mL/min.
24-hour nitrite/nitrate excretion was used to determine whole body NO production. Animals were fed a low nitrite/nitrate diet (AIN76, ICN Biomedicals, Inc., Aurora, OH) starting 5 days prior to placement in the metabolism cages. Nitrite/nitrate excretion determination was described previously.23 Briefly, E. coli was the source of nitrate reductase for conversion of nitrate to nitrite and sodium nitrate will be used as the standard to verify that all nitrate is converted to nitrite. The concentration of nitrite was measured colorimetrically using the Griess reagent. Sodium nitrite was used as the standard with data expressed as μmole nitrate/nitrite excreted/24 h of the rat.
The urinary excretion of endothelin was measured in 24-hour urine samples. The samples were frozen and stored at −80°C until determination of endothelin concentration by radioimmunoassay kit (Amersham Internatal, Amersham, UK) utilizing competitive binding with a fixed quantity of 125I-labeled endothelin-1 (synthetic) for an endothelin-1 specific antibody.
Urinary excretion of protein was measured in animals with 24 hour urine collection and storage of final sample at −20°C. Protein concentration was determined using a Sigma Protein Determination kit (P5656, Sigma Chemical Co.)24
Sigma-STAT version 1.0 was used for all statistical analysis. All data are expressed as mean ± SEM. All treated group were compared to their respective controls. When comparisons were made between groups, an unpaired student t-test was used. A value of P<0.05 was considered statistically significant. A 3 way ANOVA and multiple comparison test (Holm-Sidak method) was utilized to compare the effects of Na diet and homocysteine in blood pressure between virgin and pregnant groups.
Figure 1 illustrates the effect of increasing plasma homocysteine levels on mean arterial pressure in pregnant rats on a normal sodium intake. Increasing plasma homocysteine from 7.1 ± 1.9 to 16.7 ± 2.3 μmol/ L (p<0.05) did not have an effect on mean arterial pressure in homocysteine treated pregnant rats (107± 3 mmHg) as compared to control pregnant rats (104± 2 mmHg).
Figure 2 depicts the effect of elevated homocysteine levels on urinary nitrate/nitrite, endothelin and protein excretion. Urinary nitrate/nitrite excretion was significantly reduced from 53 ± 7 to 39 ± 5ρg/day (p<0.05) when pregnant rats were treated with homocysteine. Homocysteine treatment did not have an effect on endothelin or protein excretion. Endothelin excretion averaged 1.2± 0.2 pg/day in pregnant rats and 1.3± 0.2 pg/day in homocysteine treated pregnant rats. Urinary protein excretion averaged 19.1 ± 9 mg/day in control pregnant rats and 29.1± 2 mg/day in homocysteine treated pregnant rats.
Figure 3 illustrates the effect of increasing homocysteine levels in virgin and pregnant rats on the slope of the pressure natriuresis relationship. As we previously reported the chronic pressure natriuresis relationship was shifted leftward in control pregnant rats as compared to control virgin rats. Increasing plasma homocysteine levels did not affect the chronic pressure natriuresis relationship in virgin or pregnant rats.
Clinical studies have shown that elevated plasma levels of homocysteine in early pregnancy increases the risk factor for preeclampsia.20,21 For example, Lopez-Quesada and colleagues reported that pregnant women with hyperhomocysteinemia in the third trimester of pregnancy have a 8-fold increased risk for developing preeclampsia.21 Normal plasma homocysteine levels for pregnant women are ~ 8.4 μmol/L and hyperhomocysteinemia is classified as a plasma level of homocysteine of ~ 10.5 μmol/L or higher.20,21 In this study we elevated the plasma levels of homocysteine one week before breeding and continued the homocysteine treatment throughout the three weeks of pregnancy in rats. Our pregnant rats had plasma homocysteine levels of of ~ 17 μmol/L on day 19 of pregnancy, a 2.5-fold increase over our control pregnant rats and comparable to the values observed in women with preeclampsia.
Experimental and clinical evidence support a correlation between plasma homocysteine levels and blood pressure.11,12,13,21 Increased homocysteine levels has been proposed to cause hypertension through arterial stiffening.25 Hyperhomocysteinemia has also been suggested to cause hypertension via an oxidative stress and endothelial dysfunction.11,12,13 However, most of the animal studies that have observed an effect of homocysteine treatment on blood pressure did not measure plasma homocysteine or utilized doses of homocysteine that would achieve concentrations far beyond those observed in preeclamptic women. In the present study we evaluated the effects of experimentally induced hyperhomocysteinemia on blood pressure in pregnant rats. Although we increased our plasma concentration of homocysteine to levels comparable to humans with preeclampsia, we found no significant effect on blood pressure in pregnant rats. These findings are consistent with a previous study where a 10 week homocysteine treatment in male rats resulted in a 4-5 fold increase in plasma homocysteine but no effect on blood pressure.26 In addition, Yang and colleagues recently reported that while the plasma homocysteine levels in male mice lacking the cystathionine γ-lyase (CSE) gene were nine times higher than that of wild type mice, blood pressure in the two genotypes was similar. Moreover, they found that female and male CSE-/- displayed similar blood pressures despite females having six times the plasma homocysteine levels in males.27
While our study did not demonstrate an effect of clinically relevant levels of homocysteine on blood pressure in pregnant rats, we cannot rule out the possibility that more long-term exposure to hyperhomocysteineimia may adversely affect blood pressure regulation during pregnancy or lead to other manifestations of preeclampsia. However, recent clinical trials where homocysteine levels were lowered with folic acid and vitamin B did not reduce the risk of major cardiovascular events in patients with vascular disease.28
We and others have reported that activation of NO plays an important role in mediating the cardiovascular and hemodynamic changes during pregnancy.5,16,17,18,29 Moreover, inhibition of NO synthesis in pregnant rats has been reported to produce hypertension and proteinuria.5 Since homocysteine has been shown to decrease NO in various animal models, we evaluated the effect of hyperhomocysteinemia on urinary nitrate/nitrite excretion which is a measure of whole body NO production. We found that nitrate/nitrite excretion in hyperhomocysteimic pregnant rats was significantly decreased compared to their respective controls. We also found that hyperhomocysteinemia had no effect on urinary excretion of endothelin, which is a measure of renal production of endothelin. Despite the fact that homocysteine decreased urinary excretion of nitrate/nitrite hyperhomocysteimia in pregnant rats did not alter blood pressure in the pregnant rats on a normal sodium intake. Thus, it is quite possible that the decrease in nitrate/nitrite excretion observed in our study reflects changes in nitric oxide production in tissues that are not involved in pressure regulation or that the level of nitric oxide synthesis inhibition produced by hyperhomocysteinemia was not sufficient to affect blood pressure.
Numerous studies have shown that long-term inhibition of NO synthesis results in a chronic rightward shift in the pressure natriuresis relationship and a sodium sensitive form of hypertension.19 We have also shown that placental ischemia leads to hypertension, a rightward shift in the pressure natriuresis relationship.30 To explore the possibility that hyperhomocysteinemia may affect chronic pressure natriuresis relationship and produce a sodium sensitive form of hypertension in pregnant animals, we examined the effect of homocysteine on the steady state blood pressure levels in control pregnant rats, pregnant homocyteine treated rats, virgin rats and homocyteine treated virgin rats maintained on a normal and high sodium diet. As we previously reported the chronic pressure natriuresis relationship was shifted leftward in control pregnant rats as compared to control virgin rats.30 Increased circulating homocysteine levels did not affect the chronic pressure natriuresis relationship in virgin or pregnant rats, nor did it produce a salt-sensitive form of hypertension.
In summary, although clinical studies have reported a 2-3 fold elevation in plasma levels of homocysteine in women who developed preeclampsia, the importance of hyperhomocysteinemia in causing increases in arterial pressure during pregnancy has been unclear. In this study, we determined the effects of a 2-3 fold elevation in plasma homocysteine levels on arterial pressure, chronic pressure natriuresis relationship and endothelial factors during pregnancy in the rat. Homocysteine treatment for 4 weeks was associated with a 2-3 fold increase in plasma homocysteine levels in pregnant rats. While homocysteine treatment decreased urinary nitrate/nitrite levels in pregnant rats, hyperhomocysteinemia had no effect on mean arterial pressure nor on the chronic pressure natriuresis relationship in pregnant rats. These results suggest that while clinical studies have reported elevations in plasma levels of homocysteine in women who developed preeclampsia, it is unlikely that a 2-3 fold increase in homocysteine contributes to the hypertension observed in preeclamptic women.
This work was supported by National Institute of Health Grants HL-51971 and HL-07635. We also thank Kathy Cockrell and Elizabeth Sullivan for their excellent technical assistance.
Disclosures: There are no financial disclosures and no conflict of interest to report from any of the authors.