NOS3 (eNOS) mRNA in Whole Blood of Untreated Grade 1 Hypertension: A Case–Control Study with Reference-Gene Sensitivity Analysis

Background: Reduced endothelial nitric oxide synthase (eNOS; gene NOS3) activity contributes to hypertension, but whether NOS3 transcripts in blood differ in early, untreated hypertension is unknown. Objective: To compare whole-blood NOS3 mRNA between adults with untreated grade 1 hypertension and normotensive adults and to examine its dependence on the reference gene. Methods: In this case–control study, 30 untreated adults with grade 1 hypertension and 30 normotensive adults aged 40–55 years were recruited in West Sumatra, Indonesia. NOS3 and GAPDH were quantified by SYBR Green qRT-PCR; ΔCq was compared by Mann–Whitney U test. Reference-gene dependence was assessed by covariance adjustment, slope and efficiency analyses and equivalence testing; age and sex adjustment was bounded over datasets consistent with group-level data. Results: ΔCq was higher in hypertension (median 4.05 vs 2.18; p = 0.046; Hodges–Lehmann fold change 0.47, 95% CI 0.18–0.99; Livak 0.46, 0.19–1.10; Welch p = 0.081; post hoc power 41%). NOS3 Cq did not differ (p = 0.383), whereas GAPDH Cq was 1.30 cycles lower in hypertension. The NOS3–GAPDH slope was 0.19 (errors-in-variables upper bound 0.58; significance required ≥0.81). The GAPDH-adjusted fold change was 0.95 (0.71–1.27), equivalent within two-fold (p < 0.001). Adjusted age and sex effects were not identifiable. Conclusion: Lower whole-blood NOS3 relative to GAPDH in untreated grade 1 hypertension tracked GAPDH Cq and was absent after GAPDH adjustment, so a change in NOS3 transcript abundance was not demonstrated. With a single reference gene, such measurements cannot be interpreted without multi-reference normalization.