Analysis of metabolites in plasma reveals distinct metabolic features between Dahl salt-sensitive rats and consomic SS.13(BN) rats

Abstract

Salt-sensitive hypertension is a major risk factor for cardiovascular disorders. Our previous proteomic study revealed substantial differences in several proteins between Dahl salt-sensitive (SS) rats and salt-insensitive consomic SS.13(BN) rats. Subsequent experiments indicated a role of fumarase insufficiency in the development of hypertension in SS rats. In the present study, a global metabolic profiling study was performed using gas chromatography/mass spectrometry (GC/MS) in plasma of SS rats (n=9) and SS.13(BN) rats (n=8) on 0.4% NaCl diet, designed to gain further insights into the relationship between alterations in cellular intermediary metabolism and predisposition to hypertension. Principal component analysis of the data sets revealed a clear clustering and separation of metabolic profiles between SS rats and SS.13(BN) rats. 23 differential metabolites were identified (P<0.05). Higher levels of five TCA cycle metabolites, fumarate, cis-aconitate, isocitrate, citrate and succinate, were observed in SS rats. Pyruvate, which connects TCA cycle and glycolysis, was also increased in SS rats. Moreover, lower activity levels of fumarase, aconitase, α-ketoglutarate dehydrogenase and succinyl-CoA synthetase were detected in the heart, liver or skeletal muscles of SS rats. The distinct metabolic features in SS and SS.13(BN) rats indicate abnormalities of TCA cycle in SS rats, which may play a role in predisposing SS rats to developing salt-sensitive hypertension.

Keywords: Dahl salt-sensitive rats; Hypertension; Mass spectrometry; Metabolomics; TCA cycle.

Fig. 1

SS and SS.13^BN rats exhibited distinct metabolic profiles. (A) PCA 3-D sores plot based on the metabolic profiling data obtained from plasma of SS rats and SS.13^BN rats. The black spheres indicate SS.13^BN rats, and blue spheres indicate SS rats. (B) PLS-DA 3-D sores plot based on the metabolic profiling data obtained from plasma of SS rats and SS.13^BN rats. The black squares indicate SS.13^BN rats, and blue triangles indicate SS rats. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Verification of GC/MS results with a second method. Plasma levels of pyruvate (A), citrate (B), and fumarate (C) were found to be higher in SS rats (n = 9) than in SS.13^BN rats (n = 8) using two analytical methods. *P < 0.05, **P < 0.01, based on two-tailed t-test; bars are means ± SD.

Fig. 3

TCA cycle enzyme activities in liver, skeletal muscle and heart from SS.13^BN rats and SS rats. A, ACON (µmol aconitate/min/mg); B, KGD (µmol NADH/min/mg); C, LDH (µmol NADH/min/mg); D, MDH (µmol NADH/min/mg); E, FH (µmol fumarate/min/mg); F, SCS (µmol succinyl-CoA/min/mg); G, NADP⁺-ICD (µmol NADPH/min/mg); H, NAD⁺-ICD (µmol NADH/min/mg). Enzyme activities were determined by triplicate assays per preparation (n = 4). Statistically significant alterations are indicated by an asterisk, *P < 0.05, **P < 0.01, based on a t-test.

Fig. 4

Alterations related to TCA cycle in SS rats compared with SS.13^BN rats. Red represents up-regulated metabolites or increased enzyme activities, and green represents down-regulated metabolites or decreased enzyme activities. The levels of citrate, aconitate, isocitrate, succinate, fumarate and pyruvate, lactate were increased in plasma of SS rats, whereas glyceric acid-3P decreased. The activities of ACON, KGD, SCS and FH were significantly decreased in the heart, live, skeletal muscle, or kidneys of SS rats. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)