Blood samples were collected in a plasma ethylenediamine tetra-acetic acid tube. Samples were centrifuged at 2,500g for 10 min, and plasma was collected and stored at — 80°C until it was assayed. Repeat freeze-thaw cycles were avoided. Spot urine specimens were obtained and stored at — 80°C until they were assayed. Measurements of VEGF and bFGF were performed, by batch, in duplicate, using an enzyme-linked immunoassay for VEGF and bFGF (Quantikine; R&D Systems; Minneapolis, MN). The coefficients of variation for bFGF were 10 to 14% for urine, and 9 to 11% for plasma. The coefficients of variation for VEGF were 4 to 7% for urine, and 5 to 7% for plasma. There was no significant cross-reactivity between the bFGF or VEGF antibodies and other known growth factors.
Given the nonnormal distribution of the growth factor levels, statistical analyses were performed using nonparametric methods, and summary measures are presented as medians and interquartile ranges (IQRs). The Wilcoxon rank-sum test was used to compare two-sample means, the Kruskal-Wallis test to compare means from multiple samples, and the Spearman rank correlation to examine correlation coefficients. Multiple comparisons were adjusted using the Bonferroni method. To perform regression analysis given the nonnormal distribution of the data, analysis of variance and regression on ranks of growth factor levels was used to adjust comparisons for multiple covariates. Analyses were performed using SAS (version 8.0; SAS Institute, Cary, NC) and STATA (version 7.0; Stata Corporation; College Station, TX) statistical software packages. Statistical significance was defined as p < 0.05. my canadian pharmacy
Urine bFGF Levels
The results of the growth factor levels by etiology of PAH are summarized in Table 2. Patients with PAH had significant elevations in median urine bFGF compared to control subjects (2,305 pg/L vs 1,111 pg/L, p < 0.0001). There was a difference in median urine bFGF level according to etiology of PAH: primary pulmonary hypertension (PPH), 2,741 pg/L; congenital heart disease (CHD), 2,330 pg/L; connective tissue disease (CTD), 1,493 pg/L; p = 0.15 (Fig 1). Significant pairwise comparisons in urine bFGF levels were observed for PPH vs control subjects (p < 0.0001) and for CHD vs control subjects (p < 0.05). There was no significant difference according to gender.
Figure 1. Urine bFGF levels (median, IQR) for control, CHD, CVD, and PPH. Urine bFGF levels are significantly higher in patients with PAH compared to control subjects (p < 0.0001). Among diagnostic groups, there is a significant difference between PPH and control subjects and between CHD and control subjects.
Table 2—Angiogenic Growth Factor Levels in PAH
|Etiology||Control Subjects||PPH||CHD||CTD||p Value t|
|Urine bFGF, pg/L||1,111 (592-1,834)||2,741$ (1,334-5,490)||2,330! (1,153-4,470)||1,493 (500-3,849)||< 0.0001|
|Plasma bFGF, pg/mL||0.5 (0.5-1.19)||2.15! (0.5-9.3)||1.74(1.1-3.1)||1.04 (0.5-3.2)||0.05|
|Urine VEGF, pg/mL||84 (59-163)||100 (49-157)||92 (39-115)||71 (46-153)||0.68|
|Plasma VEGF, pg/mL||13.2 (7.5-42.3)||41.4 (7.5-92)||22.3 (7.5-42.9)||21.8 (7.5-46.7)||0.14|