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Effects of switching from lopinavir/ritonavir to atazanavir/ritonavir on muscle glucose uptake and visceral fat in HIV-infected patients
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AIDS:
17 July 2009
Stanley, Takara L; Joy, Tisha; Hadigan, Colleen M; Liebau, James G; Makimura,
Hideo; Chen, Cindy Y; Thomas, Bijoy J; Weise, Steven B; Robbins, Gregory K; Grinspoon, Steven K
aProgram in Nutritional Metabolism, USA
bDivision of Infectious Diseases, USA
cDepartment of Radiology, Massachusetts General Hospital and Harvard Medical
School, Boston, Massachusetts, USA
dNational Institutes of Health, Bethesda, Maryland, USA.
"We show that switching from LPV/r to ATV/r increased glucose uptake in the anterior thigh muscle as measured by PET, decreased triglyceride and total cholesterol, and significantly reduced visceral fat over 6 months. To our knowledge, this is the first study to demonstrate that switching to ATV/r from LPV/r increases glucose uptake into muscle in vivo using PET."......"Fasting serum triglyceride decreased significantly in the ATV/r group compared with the LPV/r group, P = 0.02, Fig. 2a (the net difference over time between treatment groups was -182 ± 64 mg/dl, ATV/r vs. LPV/r; Table 3). Similarly, fasting total cholesterol (P = 0.01) and non-high-density lipoprotein (HDL) cholesterol (P = 0.007) decreased significantly in the ATV/r group (Table 3). No significant change was observed in HDL or LDL (Table 3)."
Abstract
Objective: To determine the effects of switching from lopinavir/ritonavir (LPV/r) to atazanavir/ritonavir (ATV/r) on muscle glucose uptake, glucose homeostasis, lipids, and body composition.
Methods: Fifteen HIV-infected men and women on a regimen containing LPV/r and with evidence of hyperinsulinemia and/or dyslipidemia were randomized to continue LPV/r or to switch to ATV/r (ATV 300 mg and ritonavir 100 mg daily) for 6 months. The primary endpoint was change in thigh muscle glucose uptake as measured by positron emission tomography. Secondary endpoints included abdominal visceral adipose tissue, fasting lipids, and safety parameters. The difference over time between treatment groups (treatment effect of ATV/r relative to LPV/r) was determined by repeated measures ANCOVA.
Results: After 6 months, anterior thigh muscle glucose uptake increased significantly (treatment effect +18.2 ± 5.9 µmol/kg per min, ATV/r vs. LPV/r, P = 0.035), and visceral adipose tissue area decreased significantly in individuals who switched to ATV/r (treatment effect -31 ± 11 cm2, ATV/r vs. LPV/r, P = 0.047). Switching to ATV/r significantly decreased triglyceride (treatment effect -182 ± 64 mg/dl, ATV/r vs. LPV/r, P = 0.02) and total cholesterol (treatment effect -23 ± 8 mg/dl, ATV/r vs. LPV/r, P = 0.01), whereas high-density lipoprotein and low-density lipoprotein did not change significantly. Fasting glucose also decreased significantly following switch to ATV/r (treatment effect -15 ± 4 mg/dl, ATV/r vs. LPV/r, P = 0.002).
Conclusion: Switching from LPV/r to ATV/r significantly increases glucose uptake by muscle, decreases abdominal visceral adipose tissue, improves lipid parameters, and decreases fasting glucose over 6 months.
Introduction
HIV-infected patients receiving highly active antiretroviral (ARV) therapy often demonstrate changes in fat, characterized by central adiposity and/or loss of fat in the extremities and face, as well as dyslipidemia, insulin resistance, and increased cardiometabolic risk. A number of these abnormalities may be associated with protease inhibitor therapy. In-vitro studies have demonstrated that protease inhibitors reduce glucose uptake via direct binding to the GLUT4 transporter protein [1]. GLUT4 inhibition occurs in a dose-dependent manner and is evident within the range of the therapeutic concentrations achieved in HIV infection [1,2]. Both ritonavir (RTV) and lopinavir (LPV) are among the more potent inhibitors of GLUT4 [3,4]. Atazanavir (ATV), a relatively newer protease inhibitor, does not affect insulin-stimulated glucose uptake in vitro [3-5], but this has not been previously assessed in vitro with RTV boosting. In addition, a sensitive measure of in-vivo glucose uptake, positron emission tomography (PET), has not previously been assessed. Many protease inhibitors, including RTV and LPV, also impair adipocyte differentiation [5-9], likely by preventing maturation and nuclear localization of sterol-regulator-element-binding-protein-1 (SREBP1) [10,11]. In contrast, unboosted ATV does not have a significant effect on adipocyte differentiation [5,6].
Taken together, these studies suggest that ATV should have relatively less effect on insulin sensitivity and lipid metabolism than other protease inhibitors. Indeed, a study in healthy, non-HIV-infected men has confirmed that short-term treatment with ATV does not alter insulin-stimulated glucose uptake or glycogen storage rate, whereas LPV/r decreases both of these parameters [12]. Similarly, in HIV-infected individuals receiving ARV therapy, switching the protease inhibitor component to unboosted ATV decreased 2-h glucose level following oral glucose tolerance test (OGTT) and significantly improved lipid parameters [13].
There is little evidence to date regarding the effects of boosted ATV (ATV/r) on glucose and lipids. In healthy, HIV-negative individuals, LPV/r decreased insulin-stimulated glucose uptake to a significantly greater degree than ATV/r, but 2-h glucose following OGTT increased following both LPV/r and ATV/r [14]. The current study compares the effects of LPV/r and ATV/r on body composition, lipids, insulin-stimulated glucose uptake, and tissue-specific glucose uptake as measured by PET in HIV-infected individuals over a relatively long period of 6 months.
Discussion
Disorders of glucose homeostasis and dyslipidemia have been associated with use of specific protease inhibitors and may contribute to increased cardiovascular disease risk in HIV-infected patients receiving chronic HAART [24]. ATV has demonstrated reduced effects on glucose and lipids, but limited studies have been done in which patients have been switched to ATV in the context of continuing other ARVs and maintaining ongoing ritonavir at low boosting doses. In this study, NRTI backbones were similar between treatment groups and did not change during the study, isolating metabolic differences between LPV/r and ATV/r. We show that switching from LPV/r to ATV/r increased glucose uptake in the anterior thigh muscle as measured by PET, decreased triglyceride and total cholesterol, and significantly reduced visceral fat over 6 months. To our knowledge, this is the first study to demonstrate that switching to ATV/r from LPV/r increases glucose uptake into muscle in vivo using PET.
The findings that switching from LPV/r to ATV/r increases glucose uptake into muscle and decreases fasting glucose are consistent with studies showing that LPV and RTV inhibit GLUT4 in vitro [1-4], whereas ATV does not demonstrate an effect on glucose uptake [3,4]. Whole body glucose disposal by clamp approached, but did not reach, significance with a treatment effect favoring ATV/r vs. LPV/r. Limited sample size and variability may have contributed to this result. In addition, the clamp results might reflect ongoing hepatic insulin resistance in spite of improved muscle-specific glucose uptake, as shown specifically by PET. It is particularly significant that individuals in our study who switched to ATV/r demonstrated increased muscle glucose uptake in spite of continued RTV boosting. The RTV used for boosting, combined with the other ARVs in our individuals' treatment regimens, may have diminished the overall improvement in insulin sensitivity.
Our finding of a beneficial effect on lipids, equal to an improvement of approximately 180 mg/dl in triglyceride and 20 mg/dl in total cholesterol over 6 months following a switch to ATV/r, is consistent with the results of other studies comparing ATV/r to other regimens in both HAART-naive and treatment-experienced populations [25-27]. Low-dose ritonavir has itself been shown to contribute to dyslipidemia [28,29], and thus it is clinically relevant that we have shown a significant reduction in triglyceride levels in the context of ongoing ritonavir therapy.
To our knowledge, the significant decrease in visceral adipose tissue after switch to ATV/r has not been reported previously, and it is of a similar magnitude (-15%) to that observed in other studies with strategies such as growth hormone or growth hormone receptor hormone analogs [30-34]. Jemsek et al. [35] demonstrated that, in a cohort of ARV-naive individuals, both ATV and efavirenz increased VAT to a similar degree after 48 weeks of treatment. Likewise, Moyle et al. [36] did not demonstrate a difference in VAT or subcutaneous adipose tissue (SAT) 48 weeks after a switch from other protease inhibitors to ATV/r. The cohorts in these studies, however, may have had fewer metabolic abnormalities than individuals in our cohort. The eligibility criteria for Moyle's study specified waist circumference greater than 90 cm, whereas our study population had a mean waist circumference of 102 ± 4 cm and therefore may have had more visceral adiposity. Similarly, the median BMI in the Jemsek cohort was 23 kg/m2, compared to an average BMI in our cohort of 29 kg/m2. Further, the Jemsek cohort was ARV-naive, whereas our individuals were treatment-experienced and selected on the basis of hyperinsulinemia or hyperlipidemia secondary to HAART. Recent data from Pischon et al. [37] demonstrate that increased central adiposity poses a risk for increased mortality, independent of BMI among non-HIV patients. Though similar studies have not been done in the HIV population, reduction in visceral fat is nonetheless likely a useful treatment goal for HIV patients [38]. Moreover, the reduction in visceral fat with switch to ATV/r suggests that LPV/r was contributing to increased visceral adiposity in our patients, as no other element of the ARV regimen was changed.
In terms of safety, we did not see an overall change in immune function among patients completing the study, but one patient was discontinued for an increase in viral load on ATV/r and subsequently achieved virologic suppression after changing to another regimen. Switching to ATV/r from LPV/r should only be considered in those patients who have stable virologic suppression and no contraindications to atazanavir, including hypersensitivity and the use of proton pump inhibitors or certain statins. Moreover, any switch of ARV therapy carries a risk of virologic failure and requires careful, frequent monitoring. For those patients who are virologically stable and are experiencing significant metabolic complications from HAART, however, our data suggest that a switch to ATV/r may ameliorate cardiometabolic risk.
We did see anticipated increases in bilirubin, although none of the patients had clinically significant increases in magnitude to necessitate study discontinuation. There was also a small but statistically significant increase in ALT in the group that switched to ATV/r. No patient developed grade 2-4 ALT elevation in either the LPV/r or the ATV/r group, however, and no individual discontinued the study because of ALT elevation. Increase in transaminases after switch to ATV/r has not been reported in larger studies [27,39], and the finding of increased ALT in our smaller study requires further investigation.
Our study has a number of limitations. Our sample size was small due to the complexity of the study procedures, and thus may have been underpowered to detect changes in specific parameters. Although it is clear that ATV/r improved glucose uptake into the muscle and overall glucose levels, further studies characterizing hepatic glucose production during clamp will be needed to determine the direct effects of LPV/r switching to ATV/r on hepatic insulin resistance. Moreover, changes in glucose metabolism may have been greater without the use of ritonavir, but we felt that use of boosted atazanavir was more clinically relevant because recommendations for initial treatment of HIV specify protease inhibitor boosting with low-dose ritonavir [40].
In conclusion, this study demonstrates that switching from LPV/r to ATV/r increases muscle glucose uptake, reduces visceral adiposity, and decreases triglyceride and total cholesterol. Larger studies are needed to confirm whether the metabolic advantages we demonstrated from switching to ATV/r can be sustained longer than 6 months, in a larger population of HIV-infected patients, and whether the increases in glucose trafficking into muscle will eventually translate into improved overall insulin sensitivity. Moreover, it will be important to determine whether changes in visceral fat, glucose, and lipids translate into decreased cardiovascular disease risk, large enough to justify the risks associated with such a switch. However, our study at least suggests the potential utility of such a switch, with continued low-dose ritonavir boosting, for the HIV patient on long-term HAART with hyperinsulinemia, dyslipidemia, and increased central adiposity on LPV/r.
Results
Of 16 individuals who enrolled in the study, one discontinued prior to the end of his baseline visit and was not randomized or included in the analysis. Following randomization, one individual in the ATV/r group discontinued for personal reasons. Data from 14 individuals (six ATV/r and eight LPV/r) were available 1 month after randomization. Following the 1-month visit, one individual in the ATV/r group discontinued because of increased viral load following the protease inhibitor switch, and one individual in the LPV/r group who had low grade viremia at baseline discontinued because subsequent HIV-genotyping showed resistance to LPV, necessitating a change in ARV regimen. Data from 12 individuals (five ATV/r and seven LPV/r) were available 6 months after randomization. One person in each treatment group was receiving stable lipid-lowering therapy.
Baseline characteristics
Baseline clinical characteristics are shown in Table 1. Eighty percent of individuals were men, and 73% of individuals had undetectable HIV viral load (<50 copies/ml). Mean duration of HIV-infection was 14 ± 1 years. The components of individuals' ARV regimens are listed in Table 2. On average, individuals had been on their current ARV regimen for 3 ± 0.5 years; all individuals had been taking their current ARVs for at least 9 months. There were no significant differences between treatment groups in baseline use of specific Nucleoside Reverse Transcriptase Inhibitors (NRTIs), and each individual's NRTI backbone remained the same throughout the study. Mean body mass index (BMI) of the cohort was 29.3 ± 1.9 kg/m2. BMI and body composition measures were not different between the two study groups (Tables 1 and 3). Waist circumference was not different between the groups (104 ± 8 vs. 100 ± 4 cm, P = 0.69). Individuals randomized to ATV/r had higher low-density lipoprotein (LDL) values at baseline (128 ± 8 vs. 94 ± 12 mg/dl, P = 0.03) and a trend toward higher total cholesterol levels at baseline (215 ± 12 vs. 182 ± 10 mg/dl, P = 0.06). There was also a trend toward lower CD4+ cell counts in the ATV/r group at baseline (417 ± 70 vs. 623 ± 66, ATV/r vs. LPV/r, P = 0.05). There were no statistically significant baseline differences in aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, muscle glucose uptake measured by PET, or glucose uptake (M/I) during euglycemic clamp (Table 3). In addition, there were no baseline differences in total daily caloric intake (2059 ± 340 vs. 2373 ± 244 kcal/day, ATV/r vs. LPV/r, P = 0.47), macronutrient intake, or physical activity as measured by Modifiable Activity Questionnaire (data not shown).
Glucose uptake by thigh muscle (PET)
Insulin-mediated glucose uptake into anterior thigh muscle increased significantly in individuals who switched to ATV/r compared with those who remained on LPV/r (P = 0.035; Table 3).
Glucose homeostasis
Fasting glucose decreased significantly in the ATV/r compared with the LPV/r group (P = 0.002), with a net difference over time between the treatment groups of -15 ± 4 mg/dl in the ATV/r vs. LPV/r group (Table 3). For whole body insulin-stimulated glucose disposal as measured during euglycemic, hyperinsulinemic clamp, the treatment effect trended in a direction favoring ATV/r, but the P value was not significant (P = 0.12).
Body composition
Body weight did not change significantly in either group during the study (Table 3). Individuals who changed to ATV/r demonstrated a significant decrease in visceral adipose tissue (VAT) (change from baseline -18 ± 11 vs. +13 ± 3 cm2, net difference of -31 ± 11 cm2, ATV/r vs. LPV/r, P = 0.047, Fig. 1). Mean decrease in VAT from baseline was 15.5% in the ATV/r group, compared with a mean increase of 10.8% in the LPV/r group. No significant effects of switching to ATV/r were demonstrated on abdominal subcutaneous fat, thigh subcutaneous fat, or percentage total body fat (Table 3). Neither total fat, extremity fat, nor leg fat by DEXA changed significantly between the groups (data not shown).
Lipids
Fasting serum triglyceride decreased significantly in the ATV/r group compared with the LPV/r group, P = 0.02, Fig. 2a (the net difference over time between treatment groups was -182 ± 64 mg/dl, ATV/r vs. LPV/r; Table 3). Similarly, fasting total cholesterol (P = 0.01) and non-high-density lipoprotein (HDL) cholesterol (P = 0.007) decreased significantly in the ATV/r group (Table 3). No significant change was observed in HDL or LDL (Table 3).
Immune function
Neither CD4 nor viral load changed significantly between treatment groups over 6 months (Table 3).
Diet and exercise
Four-day food records collected at baseline and 6-month visits showed no significant changes between groups in total caloric intake (change from baseline -322 ± 78 vs. -273 ± 326 kcal/day, ATV/r vs. LPV/r, P = 0.89) or intake of carbohydrate, fat, or protein (data not shown). Metabolic equivalents (METs) of leisure-time activity also did not significantly change between groups (data not shown).
Adverse effects
Total bilirubin increased significantly during the study in the ATV/r relative to the LPV/r group (Table 3 and Fig. 2b). Two individuals in the ATV/r group had grade 3 elevation (bilirubin 2.6-5.0 mg/dl [23]), but bilirubin normalized in one of these individuals by the 2-month visit. Two ATV/r individuals had grade 4 elevation (bilirubin >5.0 mg/dl [23]); in both cases, this elevation persisted throughout the study. No individual developed symptoms or discontinued due to hyperbilirubinemia. Individuals in the ATV/r compared with the LPV/r group also demonstrated a mild but significant increase in serum ALT (P = 0.004; Table 3). No individual had ALT elevation of grade 2 or greater (≥2.6 times ULN [23]) throughout the study. There was no significant difference in AST between treatment groups.
Table 3. Treatment effect on body composition, metabolic, immune function, and safety parameters.
Methods
Individual selection
Sixteen men and women with HIV infection were recruited through informational mailings to HIV-care providers, postings in HIV community organizations, newspaper advertisements, and the Massachusetts General Hospital (MGH) HIV database. Recruitment began in March 2006, and the last patient was randomized in May 2008. Written, informed consent was obtained from each individual in accordance with the MGH and the Massachusetts Institute of Technology (MIT) institutional review boards. The study was approved by the MGH Institutional Review Board and by the MIT Committee on the Use of Humans as Experimental Subjects.
Inclusion criteria included age 18-65 years, previously diagnosed HIV infection, stable ARV regimen including LPV/r for at least 6 months prior to study entry, and one or more of the following metabolic complications: fasting insulin of at least 15 µU/ml, total cholesterol of at least 200 mg/dl, triglycerides of at least 150 mg/dl, or treatment with lipid-lowering medication. Exclusion criteria included hemoglobin less than 11.0 g/dl; pregnancy; history of diabetes mellitus or current therapy with an antidiabetic agent; therapy with glucocorticoid, growth hormone, or other anabolic agents in the 3 months prior to study entry; contraindication to ATV/r use, including use of proton pump inhibitors, lovastatin, or simvastatin; and new or serious opportunistic infection. In addition, individuals were enrolled only if their HIV-care providers agreed to their participation.
Study design
This was a 6-month, randomized, nonblinded comparison of continuing therapy with LPV/r vs. switching to ATV/r. All assessments were performed after a 12-h overnight fast. After a screening visit to determine eligibility, participants underwent baseline testing, including single-slice computed tomography (CT) scan of the abdomen and mid-thigh, PET scan of the thigh with 2-deoxy-[18F]fluoro-D-glucose (FDG) infusion conducted during a euglycemic hyperinsulinemic clamp, dual-energy X-ray absorptiometry (DEXA) scan, anthropometrics, and fasting laboratory evaluation. Four-day food records were collected, and a Modifiable Activity Questionnaire was administered.
Following the baseline evaluation, individuals were randomized in a 1: 1 ratio to continue their current ARV regimen including LPV/r or to switch from LPV/r to ATV/r (300 mg atazanavir and 100 mg ritonavir once daily). No other component of the ARV regimen was changed during the study. The randomization list was prepared by the MGH pharmacy and kept by a third party who provided the treatment assignment to the study investigator at the end of each individual's baseline visit. Randomization was not blinded to patients, primary HIV care providers, or investigators in order to facilitate clinical monitoring, including the likely increase in bilirubin in those assigned to ATV/r.
Individuals returned for safety visits 2 weeks, 1, 2, and 4 months following randomization. At the 1-month visit, individuals also underwent 2-h euglycemic hyperinsulinemic clamp. Six months following randomization, individuals returned for an evaluation identical to their baseline assessment.
Computed tomography of thigh and abdomen at L4
CT scans were performed with a LightSpeed 16-slice CT scanner (General Electric, Milwaukee, Wisconsin, USA) as previously described [15,16].
Hyperinsulinemic euglycemic clamp
Following a 12-h fast, individuals received an infusion of 40 mU/m2 per min regular insulin following a priming dose of 200 mU/m2 per min given over 2 min. A variable infusion of 20% dextrose maintained plasma glucose concentrations at a euglycemic value of 5 mmol/l (90 mg/dl). Blood glucose was determined every 5 min using a B-Glucose Analyzer (Hemocue, Lake Forest, California, USA). Insulin samples were collected every 20 min. At baseline and 6-month visits, the clamp procedure continued until the PET scan was complete. At the 1-month visit, the duration of the clamp was 120 min.
2-Deoxy-[18F]fluoro-D-glucose-positron emission tomography of mid-thigh
Individuals were placed in a supine position and had placement of a venous catheter for infusion and a catheter in the radial artery for blood sampling. The mid-thigh region was centered in the camera's field using a reference mark corresponding to the single-slice CT scan. Euglycemic hyperinsulinemic clamp was started as described above. Once the individual's glucose had achieved steady state, transmission images of 10 min duration were acquired with a rotating 68 Ge pin source. Following transmission, approximately 10 mCi of FDG were injected intravenously over 1.5 min, and sequential PET images were acquired. Arterial blood samples were obtained every 15 s for 3 min, every 30 s for 2 min, every 1 min for 5 min, every 5 min for 55 min, and at 75 and 90 min. Arterial FDG plasma radioactivity was measured using a calibrated Baird Atomic well counter.
Body composition analysis
Whole body DEXA was performed using a Hologic QDR-4500 densitometer (Hologic, Bedford, Massachusetts, USA) to determine total body and regional fat mass. Body fat was also measured using a Bioelectrical Impedance Analyzer (RJL Systems, Clinton Township, Michigan, USA).
Assays
Assays were performed at the MIT Clinical Research Center core laboratory and the Harvard CTSA Core Lab. Serum insulin was measured using either radioimmunoassay (RIA; Siemens Medical Solutions Diagnostics, Deerfield, Illinois; intra-assay and interassay coefficients of variation from 3.1 to 9.3% and from 4.9 to 10.0%, respectively) or chemiluminescence immunoassay (Ultra-sensitive Beckman Access-2 Chemiluminescence platform; Beckman Coulter, Chaska, Minnesota, USA; sensitivity 0.03 IU/ml, precision 3-5.6%). The correlation between the two assays was r = 0.99, P < 0.0001. HIV viral load (Ultrasensitive Roche Amplicor v1.5, lower limit of detection 50 copies/ml), CD4 cell count, serum glucose, lipids, and chemistries were measured using standard methodologies.
Calculations
Regional 2-deoxy-[18F]fluoro-D-glucose uptake
A General Electric/Scanditronix PC4096 15-slice whole body tomograph was used to produce sequential images over the thigh muscle over 90 min. Images were reconstructed using a filtered back-projection algorithm, and projection data were corrected for nonuniformity of detector response, dead time, random coincidences, and scattered radiation. Images were corrected for attenuation by a 68 Ge transmission source. Four to six irregular-shaped regions of interest (ROIs) were placed, taking particular caution to exclude bone and major vascular structures. A three-compartment kinetic model was used to estimate glucose kinetics [17,18]. A nonlinear least-squares fitting procedure was used to determine the rate constants (k1, k2, k3, k4) using the arterial input function and the composite tissue time-activity curves. FDG metabolic rate (K) was calculated according to the equation K = (k1k3)/(k2 + k3) [17,18]. Muscle glucose uptake was obtained by multiplying K by the plasma glucose concentration and then dividing by the lumped constant value of 1.2 [18-20].
Insulin-stimulated glucose disposal
The method of DeFronzo et al. [21] was used to determine insulin-stimulated glucose disposal (M) for the interval between 100 and 120 min. M was indexed to fat-free mass (M/LBM, µmol/kg FFM per min) and corrected for insulin (M/I).
Statistics
The primary endpoint was insulin-stimulated thigh muscle glucose uptake measured by PET. Initial sample size of N = 16 was calculated to provide 80% power to detect a 30% change in muscle glucose uptake between groups, using a SD of 19 µmol/kg per min [22]. Baseline characteristics in the two groups were compared using Student's t-test for normally distributed variables and Wilcoxon Rank Sum test for nonnormally distributed variables. For variables measured only at baseline and 6 months (PET, CT, DEXA, dietary intake, and physical activity data), Student's t-test was used to compare changes from baseline and determine the treatment effect (net difference over time between the ATVr vs. LPV/r groups) over 6 months. For all other variables, repeated measures ANCOVA, controlling for baseline values, was used to assess the treatment effect of the randomization over 6 months (net difference over time between the ATV/r vs. LPV/r groups). All available data were used. In all Student's t-test calculations, unequal variances were assumed. SAS was used for repeated measures analysis. All other statistical analyses were performed using SAS JMP software, version 5.0.1.2 (SAS Institute, Cary, North Carolina, USA). Statistical significance was defined as P less than 0.05. Results are mean ± SEM unless otherwise indicated.
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